With Persistence And Phase 3 Win, Amicus Nears First Drug Approval …….Migalastat

Phase 3 drug, Uncategorized Comments Off

Aug 212014

Migalastat hydrochloride
CAS Number: 75172-81-5 hydrochloride

CAS BASE….108147-54-2

ABS ROT = (+)

+53.0 °

Conc: 1 g/100mL; Solv: water ; 589.3 nm; Temp: 24 °C

IN Van den Nieuwendijk, Adrianus M. C. H.; Organic Letters 2010, 12(17), 3957-3959

3,4,5-Piperidinetriol,2-(hydroxymethyl)-, hydrochloride (1:1), (2R,3S,4R,5S)-

Molecular Structure:

Formula: C6H14ClNO4
Molecular Weight:199.63
Synonyms: 3,4,5-Piperidinetriol,2-(hydroxymethyl)-, hydrochloride, (2R,3S,4R,5S)- (9CI);

3,4,5-Piperidinetriol,2-(hydroxymethyl)-, hydrochloride, [2R-(2a,3a,4a,5b)]-;

Migalastat hydrochloride;Galactostatin hydrochloride;

(2S,3R,4S,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol hydrochloride;

1-Deoxygalactonojirimycin
1-Deoxygalactostatin
Amigal
DDIG
Migalastat
UNII-C4XNY919FW

Melting Point:160-2 °C………http://www.google.com/patents/DE3906463A1?cl=de
Boiling Point:382.7 °C at 760 mmHg
Flash Point:185.2 °C

Amicus Therapeutics, Inc. innovator

Aug 2014

http://www.xconomy.com/new-york/2014/08/20/with-persistence-and-phase-3-win-amicus-nears-first-drug-approval/?utm_source=rss&utm_medium=rss&utm_campaign=with-persistence-and-phase-3-win-amicus-nears-first-drug-approval

Amicus Therapeutics was on the ropes in late 2012 when its pill for a rare condition called Fabry Disease108147-54-2 failed a late-stage trial. It had already put seven years of work into the drug, and the setback added even more development time and uncertainty to the mix. But the Cranbury, NJ-based company kept plugging away, and now it looks like all the effort could lead to its first approved drug.

Amicus (NASDAQ: FOLD) is reporting today that the Fabry drug, migalastat, succeeded in the second of two late-stage trials. It hit two main goals that essentially measured its ability to slow the decline of Fabry patients’ kidney function comparably to enzyme-replacement therapy (ERT)—the standard of care for the often-fatal disorder.

Amicus believes the results, along with those from an earlier Phase 3 trial comparing migalastat to a placebo, are good enough to ask regulators in the U.S. and Europe for market approval.

“These are the good days to be a CEO,” says Amicus CEO John Crowley (pictured above). “It’s great when a plan comes together and data cooperates.”

Crowley says Amicus will seek approval of migalastat first in Europe and is already in talks with regulators there. In the next few months, Amicus will begin talking with the FDA about a path for approval in the U.S. as well.

End feb 2013

About Amicus Therapeutics

Amicus Therapeutics is a biopharmaceutical company at the forefront of therapies for rare and orphan diseases. The Company is developing orally-administered, small molecule drugs called pharmacological chaperones, a novel, first-in-class approach to treating a broad range of human genetic diseases. Amicus’ late-stage programs for lysosomal storage disorders include migalastat HCl monotherapy in Phase 3 for Fabry disease; migalastat HCl co-administered with enzyme replacement therapy (ERT) in Phase 2 for Fabry disease; and AT2220 co-administered with ERT in Phase 2 for Pompe disease.

About Migalastat HCl

Amicus in collaboration with GlaxoSmithKline (GSK) is developing the investigational pharmacological chaperone migalastat HCl for the treatment of Fabry disease. Amicus has commercial rights to all Fabry products in the United States and GSK has commercial rights to all of these products in the rest of world.

As a monotherapy, migalastat HCl is designed to bind to and stabilize, or “chaperone” a patient’s own alpha-galactosidase A (alpha-Gal A) enzyme in patients with genetic mutations that are amenable to this chaperone in a cell-based assay. Migalastat HCl monotherapy is in Phase 3 development (Study 011 and Study 012) for Fabry patients with genetic mutations that are amenable to this chaperone monotherapy in a cell-based assay. Study 011 is a placebo-controlled study intended primarily to support U.S. registration, and Study 012 compares migalastat HCl to ERT to primarily support global registration.

For patients currently receiving ERT for Fabry disease, migalastat HCl in combination with ERT may improve ERT outcomes by keeping the infused alpha-Gal A enzyme in its properly folded and active form thereby allowing more active enzyme to reach tissues.2Migalastat HCl co-administered with ERT is in Phase 2 (Study 013) and migalastat HCl co-formulated with JCR Pharmaceutical Co. Ltd’s proprietary investigational ERT (JR-051, recombinant human alpha-Gal A enzyme) is in preclinical development.

About Fabry Disease

Fabry disease is an inherited lysosomal storage disorder caused by deficiency of an enzyme called alpha-galactosidase A (alpha-Gal A). The role of alpha-Gal A within the body is to break down specific lipids in lysosomes, including globotriaosylceramide (GL-3, also known as Gb3). Lipids that can be degraded by the action of α-Gal are called “substrates” of the enzyme. Reduced or absent levels of alpha-Gal A activity leads to the accumulation of GL-3 in the affected tissues, including the kidneys, heart, central nervous system, and skin. This accumulation of GL-3 is believed to cause the various symptoms of Fabry disease, including pain, kidney failure, and increased risk of heart attack and stroke.

It is currently estimated that Fabry disease affects approximately 5,000 to 10,000 people worldwide. However, several literature reports suggest that Fabry disease may be significantly under diagnosed, and the prevalence of the disease may be much higher.

1. Bichet, et al., A Phase 2a Study to Investigate the Effect of a Single Dose of Migalastat HCl, a Pharmacological Chaperone, on Agalsidase Activity in Subjects with Fabry Disease, LDN WORLD 2012

2. Benjamin, et al., Molecular Therapy: April 2012, Vol. 20, No. 4, pp. 717–726.

http://clinicaltrials.gov/show/NCT01458119

http://www.docstoc.com/docs/129812511/migalastat-hcl

Migalastat hydrochloride is a pharmacological chaperone in phase III development at Amicus Pharmaceuticals for the oral treatment of Fabry’s disease. Fabry’s disease occurs as the result of an inherited genetic mutation that results in the production of a misfolded alpha galactosidase A (alpha-GAL) enzyme, which is responsible for breaking down globotriaosylceramide (GL-3) in the lysosome. Migalastat acts by selectively binding to the misfolded alpha-GAL, increasing its stability and promoting proper folding, processing and trafficking of the enzyme from the endoplasmic reticulum to the lysosome.

In February 2004, migalastat hydrochloride was granted orphan drug designation by the FDA for the treatment of Fabry’s disease.

The EMEA assigned orphan drug designation for the compound in 2006 for the treatment of the same indication. In 2007, the compound was licensed to Shire Pharmaceuticals by Amicus Therapeutics worldwide, with the exception of the U.S., for the treatment of Fabry’s disease.

In 2009, this license agreement was terminated. In 2010, the compound was licensed by Amicus Therapeutics to GlaxoSmithKline on a worldwide basis to develop, manufacture and commercialize migalastat hydrochloride as a treatment for Fabry’s disease, but the license agreement terminated in 2013.

Chemical Name:	DEOXYGALACTONOJIRIMYCIN, HYDROCHLORIDE
Synonyms:	DGJ;Amigal;Unii-cly7m0xd20;GALACTOSTATIN HCL;DGJ, HYDROCHLORIDE;Migalastat hydrochloride;Galactostatin hydrochloride;DEOXYGALACTONOJIRIMYCIN HCL;1-DEOXYGALACTONOJIRIMYCIN HCL;1,5-dideoxy-1,5-imino-d-galactitol

DEOXYGALACTONOJIRIMYCIN, HYDROCHLORIDE Structure

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Links

http://www.google.co.in/patents/WO1999062517A1?cl=en

Example 1

A series of plant alkaloids (Scheme 1, ref. 9) were used for both in vitro inhibition and intracellular enhancement studies of α-Gal A activity. The results of inhibition experiments are shown in Fig. 1 A.

Among the tested compounds, 1-deoxy-galactonojirimycin (DGJ, 5) known as a powerful competitive inhibitor for α-Gal A, showed the highest inhibitory activity with IC₅₀ at 4.7 nM. α-3,4-Di-epi-homonojirimycin (3) was an effective inhibitor with IC₅₀ at 2.9 μM. Other compounds showed moderate inhibitory activity with IC₅₀ ranging from 0.25 mM (6) to 2.6 mM (2). Surprisingly, these compounds also effectively enhanced α-Gal A activity in COS-1 cells transfected with a mutant α-Gal A gene (R301Q), identified from an atypical variant form of Fabry disease with a residual α- Gal A activity at 4% of normal. By culturing the transfected COS-1 cells with these compounds at concentrations cat 3 – 10-fold of IC₅₀ of the inhibitors, α-Gal A activity was enhanced 1.5 – 4-fold (Fig. 1C). The effectiveness of intracellular enhancement paralleled with in vitro inhibitory activity while the compounds were added to the culture medium at lOμM

concentration (Fig. IB).

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Links

WO 2008045015

or http://www.google.com/patents/EP2027137A1?cl=en, http://www.google.com/patents/US7973157?cl=en

This invention relates to a process for purification of imino or amino sugars, such as D-1-deoxygalactonojirimycin hydrochloride (DGJ’HCl). This process can be used to produce multi-kilogram amounts of these nitrogen-containing sugars.

Sugars are useful in pharmacology since, in multiple biological processes, they have been found to play a major role in the selective inhibition of various enzymatic functions. One important type of sugars is the glycosidase inhibitors, which are useful in treatment of metabolic disorders. Galactosidases catalyze the hydrolysis of glycosidic linkages and are important in the metabolism of complex carbohydrates. Galactosidase inhibitors, such as D-I- deoxygalactonojirimycin (DGJ), can be used in the treatment of many diseases and conditions, including diabetes (e.g., U.S. Pat. 4,634,765), cancer (e.g., U.S. Pat. 5,250,545), herpes (e.g. , U.S. Pat. 4,957,926), HIV and Fabry Disease (Fan et al, Nat. Med. 1999 5:1, 112-5).

Commonly, sugars are purified through chromatographic separation. This can be done quickly and efficiently for laboratory scale synthesis, however, column chromatography and similar separation techniques become less useful as larger amounts of sugar are purified. The size of the column, amount of solvents and stationary phase (e.g. silica gel) required and time needed for separation each increase with the amount of product purified, making purification from multi-kilogram scale synthesis unrealistic using column chromatography.

Another common purification technique for sugars uses an ion- exchange resin. This technique can be tedious, requiring a tedious pre-treatment of the ion exchange resin. The available ion exchange resins are also not necessarily able to separate the sugars from salts (e.g., NaCl). Acidic resins tend to remove both metal ions found in the crude product and amino- or imino-sugars from the solution and are therefore not useful. Finding a resin that can selectively remove the metal cations and leave amino- or imino-sugars in solution is not trivial. In addition, after purification of a sugar using an ion exchange resin, an additional step of concentrating the diluted aqueous solution is required. This step can cause decomposition of the sugar, which produces contaminants, and reduces the yield.

U.S. Pats. 6,740,780, 6,683,185, 6,653,482, 6,653,480, 6,649,766, 6,605,724, 6,590,121, and 6,462,197 describe a process for the preparation of imino- sugars. These compounds are generally prepared from hydroxyl-protected oxime intermediates by formation of a lactam that is reduced to the hexitol. However, this process has disadvantages for the production on a multi-kg scale with regard to safety, upscaling, handling, and synthesis complexity. For example, several of the disclosed syntheses use flash chromatography for purification or ion-exchange resin treatment, a procedure that is not practicable on larger scale. One particularly useful imino sugar is DGJ. There are several DGJ preparations disclosed in publications, most of which are not suitable for an industrial laboratory on a preparative scale (e.g., >100 g). One such synthesis include a synthesis from D-galactose (Santoyo-Gonzalez, et al, Synlett 1999 593-595; Synthesis 1998 1787-1792), in which the use of chromatography is taught for the purification of the DGJ as well as for the purification of DGJ intermediates. The use of ion exchange resins for the purification of DGJ is also disclosed, but there is no indication of which, if any, resin would be a viable for the purification of DGJ on a preparative scale. The largest scale of DGJ prepared published is 13 g (see Fred-Robert Heiker, Alfred Matthias Schueller, Carbohydrate Research, 1986, 119-129). In this publication, DGJ was isolated by stirring with ion-exchange resin Lewatit MP 400 (OH^“) and crystallized with ethanol. However, this process cannot be readily scaled to multi- kilogram quantities.

Similarly, other industrial and pharmaceutically useful sugars are commonly purified using chromatography and ion exchange resins that cannot easily be scaled up to the purification of multi-kilogram quantities.

Therefore, there is a need for a process for purifying nitrogen- containing sugars, preferably hexose amino- or imino-sugars that is simple and cost effective for large-scale synthesis

FIG. 1. HPLC of purified DGJ after crystallization. The DGJ is over 99.5% pure.

FIG. 2A. ¹H NMR of DGJ (post HCl extraction and crystallization), from 0 – 15 ppm in DMSO.

FIG. 2B. ¹H NMR of DGJ (post HCl extraction and crystallization), from 0 – 5 ppm, in DMSO.

FIG. 3 A. ¹H NMR of purified DGJ (after recrystallization), from 0 – 15 ppm, in D₂O. Note OH moiety has exchanged with OD.

FIG. 3B. ¹H NMR of purified DGJ (after recrystallization), from 0 –

4 ppm, in D₂O. Note OH moiety has exchanged with OD.

FIG. 4. ¹³C NMR of purified DGJ, (after recrystallization), 45 – 76 ppm.

One amino-sugar of particular interest for purification by the method of the current invention is DGJ. DGJ, or D-l-deoxygalactonojirimycin, also described as (2R,3S,4R,5S)-2-hydroxymethyl-3,4,5-trihydroxypiperidine and 1- deoxy-galactostatin, is a noj irimycin (5-amino-5-deoxy-D-galactopyranose) derivative of the form:

Example 1: Preparation and Purification of DGJ

A protected crystalline galactofuranoside obtained from the technique described by Santoyo-Gonzalez. 5-azido-5-deoxy-l,2,3,6-tetrapivaloyl-α-D- galactofuranoside (1250 g), was hydrogenated for 1-2 days using methanol (10 L) with palladium on carbon (10%, wet, 44 g) at 50 psi of H₂. Sodium methoxide (25% in methanol, 1.25 L) was added and hydrogenation was continued for 1-2 days at 100 psi ofH₂. Catalyst was removed by filtration and the reaction was acidified with methanolic hydrogen chloride solution (20%, 1.9 L) and concentrated to give crude mixture of DGJ • HCl and sodium chloride as a solid. The purity of the DGJ was about 70% (w/w assay), with the remaining 30% being mostly sodium chloride.

The solid was washed with tetrahydrofuran (2 x 0.5 L) and ether (I x 0.5 L), and then combined with concentrated hydrochloric acid (3 L). DGJ went into solution, leaving NaCl undissolved. The obtained suspension was filtered to remove sodium chloride; the solid sodium chloride was washed with additional portion of hydrochloric acid (2 x 0.3 L). All hydrochloric acid solution were combined and slowly poured into stirred solution of tetrahydrofuran (60 L) and ether (11.3 L). The precipitate formed while the stirring was continued for 2 hours. The solid crude DGJ* HCl, was filtered and washed with tetrahydrofuran (0.5 L) and ether (2 x 0.5 L). An NMR spectrum is shown in FIGS. 2A-2B.

The solid was dried and recrystallized from water (1.2 mL /g) and ethanol (10 ml/1 ml of water). This recrystallization step may be repeated. This procedure gave white crystalline DGJ* HCl, and was usually obtained in about 70- 75% yield (320 – 345 g). The product of the purification, DGJ-HCl is a white crystalline solid, HPLC >98% (w/w assay) as shown in FIG. 1. FIGS. 3A-3D and FIG. 4 show the NMR spectra of purified DGJ, showing the six sugar carbons.

Example 2: Purification of 1-deoxymannojirimycin 1 -deoxymannojirimycin is made by the method described by Mariano

(J. Org. Chem., 1998, 841-859, see pg. 859, herein incorporated by reference). However, instead of purification by ion-exchange resin as described by Mariano, the 1-deoxymannojirimycin is mixed with concentrated HCl. The suspension is then filtered to remove the salt and the 1-deoxymannojirimycin hydrochloride is precipitated crystallized using solvents known for recrystallization of 1- deoxymannojirimycin (THF for crystallization and then ethanol/water.

Example 3: Purification of (+)-l-deoxynojirimycin

(+)-l-deoxynojirimycin is made by the method Kibayashi et al. (J. Org. Chem., 1987, 3337-3342, see pg. 334I₅ herein incorporated by reference). It is synthesized from a piperidine compound (#14) in HCl/MeOH. The reported yield of 90% indicates that the reaction is essentially clean and does not contain other sugar side products. Therefore, the column chromatography used by Kibayashi is for the isolation of the product from non-sugar related impurities. Therefore, instead of purification by silica gel chromatography, the (+)-l-deoxynojirimycin is mixed with concentrated HCl. The suspension is then filtered to remove the salt and the nojirimycin is crystallized using solvents known for recrystallization of nojirimycin.

Example 4: Purification of Nojirimycin

Nojirimycin is made by the method described by Kibayashi et al. (J.

Org. Chem., 1987, 3337-3342, see pg. 3342). However, after evaporating of the mixture at reduced pressure, instead of purification by silica gel chromatography with ammonia-methanol-chloroform as described by Kibayashi, the nojirimycin is mixed with concentrated HCl. The suspension is then filtered to remove the impurities not dissolved in HCl and the nojirimycin is crystallized using solvents known for recrystallization of nojirimycin.

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Links

Synthesis of (+)-1-deoxygalactonojirimycin and a related indolizidine
Tetrahedron Lett 1995, 36(5): 653

Synthesis of (+)-1-deoxygalactonojirimycin and a related indolizidine

Original Research Article
Pages 653-654
Carl R. Johnson, Adam Golebiowski, Hari Sundram, Michael W. Miller, Renee L. Dwaihy
Amido-alcohol 1 is transformed via aminal 2 into 1-deoxygalactonojirimycin (3) and indolizidine 4.

Amido-alcohol 1 is transformed via aminal 2 into 1-deoxygalactonojirimycin (3) and the structurally related indolizidine 4.

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Links

Synthesis of D-galacto-1-deoxynojirimycin (1,5-dideoxy-1,5-imino-D-galactitol) starting from 1-deoxynojirimycin
Carbohydr Res 1990, 203(2): 314

Synthesis of d-galacto-1-deoxynojirimycin (1,5-dideoxy-1,5-imino-d-galactitol) starting from 1-deoxynojirimycin
Pages 314-318
Fred-Robert Heiker, Alfred Matthias Schueller
- First page PDF

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Synthesis of (+)-1,5-dideoxy-1,5-imino-D-galactitol, a potent alpha-D-galactosidase inhibitor
Carbohydr Res 1987, 167: 305

Synthesis of (+)-1,5-dideoxy-1,5-imino-d-galactitol, a potent α-d-galactosidase inhibitor
Pages 305-311
Ronald C. Bernotas, Michael A. Pezzone, Bruce Ganem
- First page PDF

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Links

SEE

Monosaccharides containing nitrogen in the ring, XXXVII. Synthesis of 1,5-didexy-1,5-imino-D-galactitol
Chem Ber 1980, 113(8): 2601

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Links

Org. Lett., 2010, 12 (17), pp 3957–3959

DOI: 10.1021/ol101556k

http://pubs.acs.org/doi/abs/10.1021/ol101556k

+53.0 °

Conc: 1 g/100mL; Solv: water ; 589.3 nm; Temp: 24 °C

van den Nieuwendijk, Adrianus M. C. H.; Organic Letters 2010, 12(17), 3957-3959

The chemoenzymatic synthesis of three 1-deoxynojirimycin-type iminosugars is reported. Key steps in the synthetic scheme include a Dibal reduction−transimination−sodium borohydride reduction cascade of reactions on an enantiomerically pure cyanohydrin, itself prepared employing almond hydroxynitrile lyase (paHNL) as the common precursor. Ensuing ring-closing metathesis and Upjohn dihydroxylation afford the target compounds.

http://pubs.acs.org/doi/suppl/10.1021/ol101556k/suppl_file/ol101556k_si_002.pdf

COMPD 18

D-galacto-1-deoxynojirimicin.HCl (18).

D-N-Boc-6-OBn-galacto-1-deoxynojirimicin (159 mg, 0.450 mmol) was dissolved in a mixture of MeOH
(10 mL) and 6 M HCl (2 mL). The flask was purged with argon, Pd/C-10% (20 mg) was added and a balloon
with hydrogen gas was placed on top of the reaction. The mixture was stirred overnight at room temperature.
Pd/C was removed by filtration and the filtrate evaporated to yield the crude product (90 mg, 100%) as a
white foam that needed no further purification.
[α]24D = + 53.0 (c = 1, H2O);

[lit4a [α]24D = +44.6 (c = 0.9, H2O); lit4b [α]20D = +46.1 (c = 0.9, H2O)].
HRMS calculated for [C6H13NO4 + H]+164.09173; Found 164.09160.
1H NMR (400 MHz, D2O) δ 4.20 (dd, J = 2.7, 1.1 Hz, 1H), 4.11 (ddd, J = 11.4, 9.7, 5.4 Hz, 1H), 3.88 (ddd,
J = 20.9, 12.2, 6.8 Hz, 2H), 3.68 (dd, J = 9.7, 3.0 Hz, 1H), 3.55 (dd, J = 12.5, 5.4 Hz, 1H), 3.46 (ddd, J = 8.6,
4.8, 1.0 Hz, 1H), 2.97 – 2.86 (t, J = 12.0 Hz, 1H). [lit4c supporting information contains 1
H NMR-spectrumof an authentic sample].
13C NMR (101 MHz, D2O) δ 73.01, 66.97, 64.69, 60.16, 59.15, 46.15

4a) Ruiz, M.; Ruanova, T. M.; Blanco, O.; Núñez, F.; Pato, C.; Ojea, V. J. Org. Chem. 2008, 73, 2240
– 2255.

4b) Paulsen, H.; Hayauchi, Y.; Sinnwell, V. Chem. Ber. 1980, 113, 2601 – 2608. c)
McDonnell, C.; Cronin, L.; O’Brien, J. L.; Murphy, P. V. J. Org. Chem. 2004, 69, 3565 – 3568.

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(- ) FORM………… BE CAREFUL

Short and straightforward synthesis of (-)-1-deoxygalactonojirimycin
Org Lett 2010, 12(6): 1145

http://pubs.acs.org/doi/abs/10.1021/ol100037c

The mildness and low basicity of vinylzinc species functioning as a nucleophile in addition to α-chiral aldehydes is characterized by lack of epimerization of the vulnerable stereogenic center. This is demonstrated by a highly diastereoselective synthesis of 1-deoxygalactonojirimycin in eight steps from commercial starting materials with overall yield of 35%.

Figure 1. Structures of nojirimycin (1) and DGJ (2).

SEE SUPP INFO

http://pubs.acs.org/doi/suppl/10.1021/ol100037c/suppl_file/ol100037c_si_001.pdf

(-)-1-deoxygalactojirimycin hydrochloride as transparent colorless needles.
[α]D -51.4 (D2O, c 1.0)

1H-NMR (D2O) δ ppm 4.09 (dd, 1H, J 2.9 Hz, 1.3 Hz), 4.00 (ddd, 1H, J = 11.3 Hz, 9.7 Hz, 5.3 Hz),
3.80 (dd, 1H, J = 12,1 Hz, 8.8 Hz), 3.73 (dd, 1H, J = 12.1 Hz, 8.8 Hz), 3.56 (dd, 1H, J = 9.7 Hz, 2.9
Hz), 3.44 (dd, 1H, J = 12.4 Hz, 5.3 Hz), 3.34 (ddd, 1H, J = 8.7 Hz, 4.8 Hz, 1.0 Hz), 2.8 (app. t, 1H,
J = 12.0 Hz)
13C-NMR (D2O, MeOH iSTD) δ 73.6, 67.5, 65.3, 60.7, 59.7, 46.7
HRMS Measured 164.0923 (M + H – Cl) Calculated 164.0923 (C6H13NO4 + H – Cl)

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Links

Concise and highly stereocontrolled synthesis of 1-deoxygalactonojirimycin and its congeners using dioxanylpiperidene, a promising chiral building block
Org Lett 2003, 5(14): 2527

http://pubs.acs.org/doi/abs/10.1021/ol034886y

A concise and stereoselective synthesis of the chiral building block, dioxanylpiperidene 4 as a precursor for deoxyazasugars, starting from the Garner aldehyde 5 using catalytic ring-closing metathesis (RCM) for the construction of the piperidine ring is described. The asymmetric synthesis of 1-deoxygalactonojirimycin and its congeners 1−3 was carried out via the use of 4in a highly stereocontrolled mode.

mp 135-135.5 °C [lit.3mp 137-139 °C];

[α]D25 +27.8° (c 0.67, H2O)
[lit.3[α]D23 +28° (c 0.5, H2O)];

1H NMR (300 MHz, D2O) δ 2.59–2.65 (m, 1H), 2.81–2.87 (m, 1H),
3.02–3.08 (m, 1H), 3.46–3.48 (m, 2H), 3.59–3.66 (m, 3H); 13C NMR (75 MHz, D2O) δ 44.7, 57.1,

58.4, 70.9, 71.4, 73.3 [lit4 13C NMR (125 MHz, D2O) δ 44.5, 56.8, 58.3, 70.1, 70.7, 72.3];

HRMScalcd for C6H13NO4 (M+) 163.0855, Found 163.0843. Anal. calcd for C6H13NO4: C, 44.16; N,
8.58; H, 8.03. Found: C, 44.31; N, 8.55; H, 7.71.

3. Schaller, C.; Vogel, P.; Jager, V. Carbohydrate Res. 1998, 314, 25-35.
4. Lee, B. W.; Jeong, Ill-Y.; Yang, M. S.; Choi, S. U.; Park, K. H. Synthesis 2000, 1305-1309.

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Links

Applications and limitations of the I2-mediated carbamate annulation for the synthesis of piperidines: Five- versus six-membered ring formation
J Org Chem 2013, 78(19): 9791

http://pubs.acs.org/doi/abs/10.1021/jo401512h

A protecting-group-free synthetic strategy for the synthesis of piperidines has been explored. Key in the synthesis is an I2-mediated carbamate annulation, which allows for the cyclization of hydroxy-substituted alkenylamines into piperidines, pyrrolidines, and furans. In this work, four chiral scaffolds were compared and contrasted, and it was observed that with both d-galactose and 2-deoxy-d-galactose as starting materials, the transformations into the piperidines 1-deoxygalactonorjirimycin (DGJ) and 4-epi-fagomine, respectively, could be achieved in few steps and good overall yields. When d-glucose was used as a starting material, only the furan product was formed, whereas the use of 2-deoxy-d-glucose resulted in reduced chemo- and stereoselectivity and the formation of four products. A mechanistic explanation for the formation of each annulation product could be provided, which has improved our understanding of the scope and limitations of the carbamate annulation for piperidine synthesis.

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Links

Ruiz, Maria; Journal of Organic Chemistry 2008, 73(6), 2240-2255

http://pubs.acs.org/doi/abs/10.1021/jo702601z

ROT +44.6 ° Conc: 0.9 g/100mL; Solv: water ; 589.3 nm; Temp: 24 °C

A general strategy for the synthesis of 1-deoxy-azasugars from a chiral glycine equivalent and 4-carbon building blocks is described. Diastereoselective aldol additions of metalated bislactim ethers to matched and mismatched erythrose or threose acetonides and intramolecular N-alkylation (by reductive amination or nucleophilic substitution) were used as key steps. The dependence of the yield and the asymmetric induction of the aldol addition with the nature of the metallic counterion of the azaenolate and the γ-alkoxy protecting group for the erythrose or threose acetonides has been studied. The stereochemical outcome of the aldol additions with tin(II) azaenolates has been rationalized with the aid of density functional theory (DFT) calculations. In accordance with DFT calculations with model glyceraldehyde acetonides, hightrans,syn,anti-selectivitity for the matched pairs and moderate to low trans,anti,anti-selectivity for the mismatched ones may originate from (1) the intervention of solvated aggregates of tin(II) azaenolate and lithium chloride as the reactive species and (2) favored chair-like transition structures with a Cornforth-like conformation for the aldehyde moiety. DFT calculations indicate that aldol additions to erythrose acetonides proceed by an initial deprotonation, followed by coordination of the alkoxy-derivative to the tin(II) azaenolate and final reorganization of the intermediate complex through pericyclic transition structures in which the erythrose moiety is involved in a seven-membered chelate ring. The preparative utility of the aldol-based approach was demonstrated by application in concise routes for the synthesis of the glycosidase inhibitors 1-deoxy-d-allonojirimycin, 1-deoxy-l-altronojirimycin, 1-deoxy-d-gulonojirimycin, 1-deoxy-d-galactonojirimycin, 1-deoxy-l-idonojirimycin and 1-deoxy-d-talonojirimycin.

…………………..

Links

J. Org. Chem., 1991, 56 (2), pp 815–819

DOI: 10.1021/jo00002a057

http://pubs.acs.org/doi/abs/10.1021/jo00002a057

………………

Links

Hinsken, Werner; DE 3906463 A1 1990

http://www.google.com/patents/DE3906463A1?cl=de

Example 1 Preparation of 1,5-dideoxy-1,5-imino-D-glucitol hydrobromide

A suspension of 1,5-dideoxy-1,5-imino-D-glucitol (500 g) in isopropanol (2 l) with 48% hydrochloric acid, bromine (620 g). The suspension is stirred for 2 hours at 40 ° C, cooled to 0 ° C and the product isolated by filtration.

Yield: 700 g (93% of theory),
mp: 184 ° C.

Example 2 Preparation of 1,5-dideoxy-1,5-imino-D-mannitol hydrobromide

The prepared analogously to Example 1 from 1,5-dideoxy 1,5-imino-D-mannitol and 48% hydrobromic acid.

Yield: 89% of theory;

C₆H₁₄NO₄Br (244.1)
Ber .: C 29.5%; H 5.8%; N 5.7%; Br 32.7%;
vascular .: C 29.8%; H 5.8%; N 5.8%; Br 32.3%.

Example 3 Preparation of 1,5-dideoxy-1,5-imino-D-Galactitol- hydrochloride

The preparation was carried out analogously to Example 1 from 1,5-dideoxy-1,5-imino-D-galactitol and corresponding mole ratios of 37% hydrochloric acid.
yield: 91% of theory
, mp: 160-162 ° C.

Amat et al., “Eantioselective Synthesis of 1-deoxy-D-gluonojirimycin From A Phenylglycinol Derived Lactam,” Tetrahedron Letters, pp. 5355-5358, 2004.
2		Chernois, “Semimicro Experimental Organic Chemistry,” J. de Graff (1958), pp. 31-48.
3		Encyclopedia of Chemical Technology, 4th Ed., 1995, John Wiley & Sons, vol. 14: p. 737-741.
4		Heiker et al., “Synthesis of D-galacto-1-deoxynojirimycin (1, 5-dideoxy-1, 5-imino-D-galactitol) starting from 1-deoxynojirimycin.” Carbohydrate Research, 203: 314-318, 1990.
5		Heiker et al., 1990, “Synthesis of D-galacto-1-deoxynojirimycin (1,5-dideoxy-1, 5-imino-D-galactitol) starting from 1-deoxynojirimycin,” Carbohydrate Research, vol. 203: p. 314-318.
6	*	Joseph, Carbohydrate Research 337 (2002) 1083-1087.
7	*	Kinast et al. Angew. Chem. Int. Ed. Engl. 20 (1998), No. 9, pp. 805-806.
8	*	Lamb, Laboratory Manual of General Chemistry, Harvard University Press, 1916, p. 108.
9		Linden et al., “1-Deoxynojirimycin Hydrochloride,” Acta ChrystallographicaC50, pp. 746-749, 1994.
10		Mellor et al., Preparation, biochemical characterization and biological properties of radiolabelled N-alkylated deoxynojirimycins, Biochem. J. Aug. 15, 2002; 366(Pt 1):225-233.
11	*	Mills, Encyclopedia of Reagents for Organic Synthesis, Hydrochloric Acid, 2001 John Wily & Sons.
12		Santoyo-Gonzalez et al., “Use of N-Pivaloyl Imidazole as Protective Reagent for Sugars.” Synthesis 1998 1787-1792.
13		Schuller et al., “Synthesis of 2-acetamido-1, 2-dideoxy-D-galacto-nojirimycin (2-acetamido-1, 2, 5-trideoxy-1, 5-imino-D-galacitol) from 1-deoxynojirimycin.” Carbohydrate Res. 1990; 203: 308-313.
14		Supplementary European Search Report dated Mar. 11, 2010 issued in corresponding European Patent Application No. EP 06 77 2888.
15		Uriel et al., A Short and Efficient Synthesis of 1,5-dideoxy-1,5-imino-D-galactitol (1-deoxy-D-galactostatin) and 1,5-dideoxy-1,5-dideoxy-1,5-imino-L-altritol (1-deoxy-L-altrostatin) From D-galactose, Synlett (1999), vol. 5, pp. 593-595.

1-Deoxygalactonojirimycin:

(a) Liguchi, T.; Tajiri, K.; Ninomiya, I.; Naito, T. Tetrahedron2000, 56, 5819−5833.

(b) Mehta, G.; Mohal, N. Tetrahedron Lett. 2000, 41, 5741−5745.

(d) Johnson, C. R.; Golebiowsky, A.; Sundram, H.; Miller, M. W.; Dwaihy, R. L. TetraherdonLett. 1995, 36, 653−654.

(e) Uriel, C.; Santoyo-Gonzalez, F. Synlett 1999, 593−595.

(f) Ruiz, M.; Ruanova, T. M.; Ojea, V.; Quintela, J. M. Tetrahedron Lett. 1999, 40, 2021−2024.

(g) Shilvock, J. P.; Fleet, G. W. J. Synlett 1998, 554−556.

(h) Chida, N.; Tanikawa, T.; Tobe, T.; Ogawa, S. J. Chem. Soc., Chem. Commun. 1994, 1247−1248.

(i) Aoyagi, S.; Fujimaki, S.; Yamazaki, N.; Kibayashi, C. J. Org. Chem. 1991, 56, 815−819.

(j) Kajimoto, T.; Chen, L.; Liu, K. K. C.; Wong, C. H. J. Am. Chem. Soc. 1991, 113, 6678−6680.

(k) Bernotas, R. C.; Pezzone, M. A.; Ganem, B. Carbohydr. Res. 1987, 167, 305−311. 1-Deoxyidonojirimycin:

(l) Singh, O. V.; Han, H. Tetrahedron Lett. 2003, 44, 2387−2391.

(m) Schaller, C.; Vogel, P.; Jager, V. Carbohydr. Res. 1998, 314, 25−35.

(n) Fowler, P. A.; Haines, A. H.; Taylor, R. J. K.; Chrystal, E. J. T.; Gravestock, M. B. Carbohydr. Res. 1993,246 377−381.

(o) Liu, K. K. C.; Kajimoto, T.; Chen, L.; Zhong, Z.; Ichikawa, Y.; Wong, C. H.J. Org. Chem. 1991, 56, 6280−6289. 1-Deoxygulonojirimycin: ref 5l.

(p) Haukaas, M. H.; O’Doherty, G. A. Org. Lett. 2001, 3, 401−404.

(q) Ruiz, M.; Ojea, V.; Ruanova, T. M.; Quintela, J. M. Tetrahedron: Asymmetry 2002, 13, 795−799. (r) Liao, L.-X.; Wang, Z.-M.; Zhang, H.-X.; Zhou, W.-S. Tetrahedron: Asymmetry 1999, 10, 3649−3657.

Cortendo AB: First Patient Enrolled into NormoCort Phase 3 SONICS Trial Following a Successful EU Investigator Meeting

Phase 3 drug, Uncategorized Comments Off

Aug 132014

KETOCONAZOLE 2S 4R

ALSO

142128-57-2
228850-16-6 (tartrate)

(-)-cis-1-Acetyl-4-[4-[2(S)-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4(R)-ylmethoxy]phenyl]piperazine

531.431, C26 H28 Cl2 N4 O4

COR-003
DIO-902
LDKTZ

CORTENDO

licensee DiObex

Biological Role(s):	antifungal agent An antimicrobial agent that destroys fungi by suppressing their ability to grow or reproduce. Antifungal agents differ from industrial fungicides in that they defend against fungi present in human or animal tissues.

Application(s):	antifungal agent An antimicrobial agent that destroys fungi by suppressing their ability to grow or reproduce. Antifungal agents differ from industrial fungicides in that they defend against fungi present in human or animal tissues.

Ketoconazole, 1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3– dioxolan-4-yl]methoxy]phenyl]piperazine, is a racemic mixture of the cis enantiomers (-)-(2S,4R) and (+)-(2R,4S) marketed as an anti-fungal agent. Ketoconazole inhibits fungal growth through the inhibition of ergosterol synthesis.(-)-Ketoconazole, the (2S,4R) enantiomer contained in the racemate of ketoconazole, is in phase III clinical trials at Cortendo for the treatment of endogenous Cushing’s syndrome. The company and licensee DiObex had also been developing the drug candidate for the treatment of type 2 diabetes; however, no recent development has been reported for this research.Preclinical studies have demonstrated the drug candidate’s ability to inhibit the synthesis of cortisol, resulting in substantial clinical benefits including lowering both blood pressure and cholesterol in addition to controlling glucose levels. It has also been shown that (-)-ketoconazole is responsible for virtually all of the cortisol synthesis inhibitory activity present in the racemate. Rights to the compound are shared with Cortendo.In 2012, orphan drug designation was assigned in the U.S. for the treatment of endogenous Cushing’s syndrome.

August 12, 2014 02:30 AM Eastern Daylight Time

GÖTEBORG, Sweden.–(BUSINESS WIRE)–Cortendo AB (OSE:CORT) today announced that the first patient has been enrolled into the Phase 3 SONICS trial, i.e., “Study Of NormoCort In Cushing’s Syndrome.”

“The enrollment of the first patient into the SONICS trial represents a significant milestone for Cortendo”

The patient was enrolled by one of the trial’s lead principal investigators at a Pituitary Center from a prestigious institution in Baltimore, Maryland. “The enrollment of the first patient into the SONICS trial represents a significant milestone for Cortendo”, said Dr. Theodore R Koziol. ”The SONICS clinical trial team is acutely focused on the implementation of the trial following a successful EU Investigator’s meeting in Barcelona in July, which we believe further solidified the foundation for the trial.”

Cortendo successfully completed its European Investigator meeting supporting SONICS held in Barcelona, Spain on July 17-18. More than 35 investigators/study coordinators, including many of the world’s leading Cushing’s experts from 24 study sites, were in attendance and received training for the trial. Based on the positive feedback from the meeting, Cortendo has gained further confidence that NormoCort (COR-003) has the potential to be an important future treatment option for patients afflicted with Cushing’s Syndrome. A second US Investigator meeting is also being planned for later this year.

”It was gratifying to participate in the NormoCort SONICS trial investigator meeting in my home town of Barcelona with so many esteemed colleagues dedicated to treating patients with Cushing’s Syndrome”, said Susan Webb M.D. Ph.D. Professor of Medicine Universitat Autonoma de Barcelona. ”There remains a significant unmet medical need for patients, and I am delighted to be part of the development of this new therapy”.

Cortendo has also further strengthened its internal as well as external teams to support the study and to position the trial for an increased recruitment rate. In July, Cortendo added both an experienced physician and internal Clinical Operations Director to the NormoCort development team. Cortendo, working in concert with its CROs supporting the SONICS trial, now has a team of approximately 20 personnel on the NormoCort development program.

Cortendo has previously communicated its plan to meet the recruitment goal by increasing the number of study sites from 38 to 45 worldwide. The company is at various levels of activation with more than 30 study sites to date. Therein, Cortendo expects a large proportion of the sites to be activated by the end of the third quarter this year and remains confident that essentially all sites will be open by the end of 2014.

Risk and uncertainty

The development of pharmaceuticals carries significant risk. Failure may occur at any stage during development and commercialization due to safety or clinical efficacy issues. Delays may occur due to requirements from regulatory authorities not anticipated by the company.

About Cortendo

Cortendo AB is a biopharmaceutical company headquartered in Göteborg, Sweden. Its stock is publicly traded on the NOTC-A-list (OTC) in Norway. Cortendo is a pioneer in the field of cortisol inhibition and has completed early clinical trials in patients with Type 2 diabetes. The lead drug candidate NormoCort, the 2S, 4R-enantiomer of ketoconazole, has been re-focused to Cushing’s Syndrome, and has entered Phase 3 development. The company’s strategy is to primarily focus its resources within orphan drugs and metabolic diseases and to seek opportunities where the path to commercialization or partnership is clear and relatively near-term. Cortendo’s business model is to commercialize orphan and specialist product opportunities in key markets, and to partner non-specialist product opportunities such as diabetes at relevant development stages.

Cortendo AB (publ)

Sweden: Box 47 SE-433 21 Partille Tel. / Fax: +46 (0)31-263010

USA: 555 East Lancaster Ave Suite 510 Radnor, PA 19087 Tel: +1 610-254-9200 Fax: +1 610-254-9245

This information was brought to you by Cision http://news.cision.com

Contacts

Alexander Lindström
Chief Financial Officer Office
+1 610 254 9200
Mobile : +1 917 349 7210
E-mail : alindstrom@cortendo.com

Ketoconazole, 1-acetyl-4- [4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolan-4-yl] methoxy] phenyl] piperazine, is a racemic mixture of the cis enantiomers (-)-(2S, 4R) and (+)-(2R, 4S) marketed as an anti-fungal agent. Ketoconazole inhibits fungal growth through the inhibition of ergosterol synthesis. Ergosterol is a key component of fungal cell walls.
More recently, ketoconazole was found to decrease plasma cortisol and to be useful, alone and in combination with other agents, in the treatment of a variety of diseases and conditions, including type 2 diabetes, Metabolic Syndrome (also known as the Insulin Resistance Syndrome, Dysmetabolic Syndrome or Syndrome X), and other medical conditions that are associated with elevated cortisol levels. SeeU.S. Patent Nos. 5,584,790 ; 6,166,017 ; and 6,642,236 , each of which is incorporated herein by reference. Cortisol is a stress-related hormone secreted from the cortex of the adrenal glands. ACTH (adenocorticotropic hormone) increases cortisol secretion. ACTH is secreted by the pituitary gland, a process activated by secretion of corticotropin releasing hormone (CRH) from the hypothalamus.
Cortisol circulates in the bloodstream and activates specific intracellular receptors, such as the glucocorticoid receptor (GR). Disturbances in cortisol levels, synthetic rates or activity have been shown to be associated with numerous metabolic complications, including insulin resistance, obesity, diabetes and Metabolic Syndrome. Additionally, these metabolic abnormalities are associated with substantially increased risk of cardiovascular disease, a major cause of death in industrialized countries. See Mårin P et al., “Cortisol secretion in relation to body fat distribution in obese premenopausal women.” Metabolism 1992; 41:882-886, Bjorntorp, “Neuroendocrine perturbations as a cause of insulin resistance.” Diabetes Metab Res Rev 1999; 15(6): 427-41, and Rosmond, “Role of stress in the pathogenesis of the metabolic syndrome.” Psychoneuroendocrinology 2005; 30(1): 1-10, each of which is incorporated herein by reference.
While ketoconazole is known to inhibit some of the enzymatic steps in cortisol synthesis, such as, for example, 17α hydroxylase (Wachall et al., “Imidazole substituted biphenyls: a new class of highly potent and in vivo active inhibitors of P450 17 as potential therapeutics for treatment of prostate cancer.” Bioorg Med Chem 1999; 7(9): 1913-24, incorporated herein by reference) and 11b-hydroxylase (Rotstein et al., “Stereoisomers of ketoconazole: preparation and biological activity.” J Med Chem 1992; 35(15): 2818-25) and 11β-hydroxy steroid dehydrogenase (11β-HSD) (Diederich et al., “In the search for specific inhibitors of human 11β-hydroxysteroid-dehydrogenases (11β-HSDs): chenodeoxycholic acid selectively inhibits 11β-HSD-L” Eur J Endocrinol 2000; 142(2): 200-7, incorporated herein by reference) the mechanisms by which ketoconazole decreases cortisol levels in the plasma have not been reported. For example, there is uncertainty regarding the effect of ketoconazole on the 11β-hydroxy steroid dehydrogenase (11β-HSD) enzymes. There are two 11β-HSD enzymes. One of these, 11β-HSD-I, is primarily a reductase that is highly expressed in the liver and can convert the inactive 11-keto glucocorticoid to the active glucocorticoid (cortisol in humans and corticosterone in rats). In contrast, the other, 11β-HSD-II, is primarily expressed in the kidney and acts primarily as an oxidase that converts active glucocorticoid (cortisol in humans and corticosterone in rats) to inactive 11-keto glucocorticoids. Thus, the plasma concentration of active glucocorticoid is influenced by the rate of synthesis, controlled in part by the activity of adrenal 11β-hydroxylase and by the rate of interconversion, controlled in part by the relative activities of the two 11β-HSD enzymes. Ketoconazole is known to inhibit these three enzymes (Diederich et al., supra) and the 2S,4R enantiomer is more active against the adrenal 11β-hydroxylase enzyme than is the 2R,4S enantiomer (Rotstein et al., supra). However, there are no reports describing the effect of the two ketoconazole enantiomers on either of 11β-HSD-I or 11β-HSD-II, so it is not possible to predict what effects, if any, the two different ketoconazole enantiomers will each have on plasma levels of the active glucocorticoid levels in a mammal.
Ketoconazole has also been reported to lower cholesterol levels in humans (Sonino et al. (1991). “Ketoconazole treatment in Cushing’s syndrome: experience in 34 patients.” Clin Endocrinol (Oxf). 35(4): 347-52; Gylling et al. (1993). “Effects of ketoconazole on cholesterol precursors and low density lipoprotein kinetics in hypercholesterolemia.” J Lipid Res. 34(1): 59-67) each of which is incorporated herein by reference). The 2S,4R enantiomer is more active against the cholesterol synthetic enzyme 14 αlanosterol demethylase than is the other (2R,4S) enantiomer (Rotstein et al infra). However, because cholesterol level in a human patient is controlled by the rate of metabolism and excretion as well as by the rate of synthesis it is not possible to predict from this whether the 2S,4R enantiomer of ketoconazole will be more effective at lowering cholesterol levels.
The use of ketoconazole as a therapeutic is complicated by the effect of ketoconazole on the P450 enzymes responsible for drug metabolism. Several of these P450 enzymes are inhibited by ketoconazole (Rotsteinet al., supra). This inhibition leads to an alteration in the clearance of ketoconazole itself (Brass et al., “Disposition of ketoconazole, an oral antifungal, in humans.” Antimicrob Agents Chemother 1982; 21(1): 151-8, incorporated herein by reference) and several other important drugs such as Glivec (Dutreix et al., “Pharmacokinetic interaction between ketoconazole and imatinib mesylate (Glivec) in healthy subjects.” Cancer Chemother Pharmacol 2004; 54(4): 290-4) and methylprednisolone (Glynn et al., “Effects of ketoconazole on methylprednisolone pharmacokinetics and cortisol secretion.” Clin Pharmacol Ther 1986; 39(6): 654-9). As a result, the exposure of a patient to ketoconazole increases with repeated dosing, despite no increase in the amount of drug administered to the patient. This exposure and increase in exposure can be measured and demonstrated using the “Area under the Curve” (AUC) or the product of the concentration of the drug found in the plasma and the time period over which the measurements are made. The AUC for ketoconazole following the first exposure is significantly less than the AUC for ketoconazole after repeated exposures. This increase in drug exposure means that it is difficult to provide an accurate and consistent dose of the drug to a patient. Further, the increase in drug exposure increases the likelihood of adverse side effects associated with ketoconazole use.
[0008]

Rotstein et al. (Rotstein et al., supra) have examined the effects of the two ketoconazole cis enantiomers on the principal P450 enzymes responsible for drug metabolism and reported “…almost no selectivity was observed for the ketoconazole isomers” and, referring to drug metabolizing P450 enzymes: “[t]he IC50 values for the cis enantiomers were similar to those previously reported for racemic ketoconazole”. This report indicated that both of the cis enantiomers could contribute significantly to the AUC problem observed with the ketoconazole racemate.
One of the adverse side effects of ketoconazole administration exacerbated by this AUC problem is liver reactions. Asymptomatic liver reactions can be measured by an increase in the level of liver specific enzymes found in the serum and an increase in these enzymes has been noted in ketoconazole treated patients (Sohn, “Evaluation of ketoconazole.” Clin Pharm 1982; 1(3): 217-24, and Janssen and Symoens, “Hepatic reactions during ketoconazole treatment.” Am J Med 1983; 74(1B): 80-5, each of which is incorporated herein by reference). In addition 1:12,000 patients will have more severe liver failure (Smith and Henry, “Ketoconazole: an orally effective antifungal agent. Mechanism of action, pharmacology, clinical efficacy and adverse effects.” Pharmacotherapy 1984; 4(4): 199-204, incorporated herein by reference). As noted above, the amount of ketoconazole that a patient is exposed to increases with repeated dosing even though the amount of drug taken per day does not increase (the “AUC problem”). The AUC correlates with liver damage in rabbits (Ma et al., “Hepatotoxicity and toxicokinetics of ketoconazole in rabbits.” Acta Pharmacol Sin 2003; 24(8): 778-782 incorporated herein by reference) and increased exposure to the drug is believed to increase the frequency of liver damage reported in ketoconazole treated patients.
Additionally, U.S. Patent No. 6,040,307 , incorporated herein by reference, reports that the 2S,4R enantiomer is efficacious in treating fungal infections. This same patent application also reports studies on isolated guinea pig hearts that show that the administration of racemic ketoconazole may be associated with an increased risk of cardiac arrhythmia, but provides no data in support of that assertion. However, as disclosed in that patent, arrhythmia had not been previously reported as a side effect of systemic racemic ketoconazole, although a particular subtype of arrhythmia, torsades de pointes, has been reported when racemic ketoconazole was administered concurrently with terfenadine. Furthermore several published reports (for example, Morganroth et al. (1997). “Lack of effect of azelastine and ketoconazole coadministration on electrocardiographic parameters in healthy volunteers.” J Clin Pharmacol. 37(11): 1065-72) have demonstrated that ketoconazole does not increase the QTc interval. This interval is used as a surrogate marker to determine whether drugs have the potential for inducing arrhythmia. US Patent Number 6,040,307 also makes reference to diminished hepatoxicity associated with the 2S,4R enantiomer but provides no data in support of that assertion. The method provided in US Patent Number 6,040,307 does not allow for the assessment of hepatoxicity as the method uses microsomes isolated from frozen tissue.

…………………………

http://www.google.com/patents/EP1853266B1?cl=en

DIO-902 is the single enantiomer 2S,4R ketoconazole and is derived from racemic ketoconazole. It is formulated using cellulose, lactose, cornstarch, colloidal silicon dioxide and magnesium stearate as an immediate release 200 mg strength tablet. The chemical name is 2S,4R cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl] methoxyl]phenyl] piperazine, the formula is C₂₆H₂₈Cl₂N₄O₄, and the molecular weight is 531.44. The CAS number is 65277-42-1, and the structural formula is provided below. The chiral centers are at the carbon atoms 2 and 4 as marked.

[0132]

Ketoconazole is an imidazole-containing fungistatic compound. DIO-902 is an immediate release tablet to be taken orally and formulated as shown in the table below.

Component	Percentage
2S,4R ketoconazole; DIO-902	50%
Silicified Microcrystalline Cellulose, NF (Prosolv HD 90)	16.5
Lactose Monohydrate, NF (316 Fast-Flo)	22.4
Corn Starch, NF (STA-Rx)	10
Colloidal Silicon Dioxide, NF (Cab-O-Sil M5P)	0.5
Magnesium Stearate, NF	0.6

The drug product may be stored at room temperature and is anticipated to be stable for at least 2 years at 25° C and 50% RH. The drug is packaged in blister packs.

ketoconazole 2S,4R enantiomer

ketoconazole 2S,4S enantiomer

ketoconazole 2R,4R enantiomer

ketoconazole 2R,4S enantiomer

……………………..

Journal of Medicinal Chemistry (Impact Factor: 5.61). 08/1992; 35(15):2818-25. DOI: 10.1021/jm00093a015

http://pubs.acs.org/doi/abs/10.1021/jm00093a015

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Enantioselective separation of ketoconazole enantiomers by membrane extraction

http://www.sciencedirect.com/science/article/pii/S1383586611001638

A new process has been developed to separate ketoconazole (KTZ) enantiomers by membrane extraction, with the oppositely preferential recognition of hydrophobic and hydrophilic chiral selectors in organic and aqueous phases, respectively. This system is established by adding hydrophobic l-isopentyl tartrate (l-IPT) in organic strip phase (shell side) and hydrophilic sulfobutylether-β-cyclodextrin (SBE-β-CD) in aqueous feed phase (lumen side), which preferentially recognizes (+)-2R,4S-ketoconazole and (−)-2S,4R-ketoconazole, respectively. The studies performed involve two enantioselective extractions in a biphasic system, where KTZ enantiomers form four complexes with SBE-β-CD in aqueous phase and l-IPT in organic phase, respectively. The membrane is permeable to the KTZ enantiomers but non-permeable to the chiral selector molecules. Fractional chiral extraction theory, mass transfer performance of hollow fiber membrane, enantioselectivity and some experimental conditions are investigated to optimize the separation system. Mathematical model of I/II = 0.893e^0.039NTU for racemic KTZ separation by hollow fiber extraction, is established. The optical purity for KTZ enantiomers is up to 90% when 9 hollow fiber membrane modules of 30 cm in length in series are used.

Full-size image (10 K)

I, (−)-2S,4R-ketoconazole;
II, (+)-2R,4S-ketoconazole;
CDs, cyclodextrin derivatives;
l-IPT, l-isopentyl tartrate;
d-IPT, d-isopentyl tartrate;
HP-β-CD, hydroxypropyl-β-cyclodextrin;
Me-β-CD, methyl-β-cyclodextrin;
β-CD, β-cyclodextrin;
NTU, number of transfer units;
HTU, height of a transfer unit;
PVDF,polyvinylidene fluoride

…………………….

Stereoselective synthesis of both enantiomers of ketoconazole from (R)- and (S)-

Stereoselective synthesis of both enantiomers of ketoconazole from (R)- and (S)-epichlorohydrin

Original Research Article
Pages 1283-1294
Pelayo Camps, Xavier Farrés, Ma Luisa García, Joan Ginesta, Jaume Pascual, David Mauleón, Germano Carganico
Bromobenzoates (2R,4R)- and (2S,4S)-18, prepared stereoselectively from (R)- and (S)-epichlorohydrin, were transformed into (2R,4S)-(+)- and (2S,4R)-(−)-Ketoconazole, respectively, following the known synthetic protocols for the racemic mixture.

Tetrahedron Asymmetry 1995, 6(6): 1283

Stereoselective syntheses of both enantiomers of ketoconazole (1) from commercially available (R)- or (S)-epichlorohydrin has been developed. The key-step of these syntheses involves the selective substitution of the methylene chlorine atom by benzoate on a mixture of and or of their enantiomers, followed by crystallization of the corresponding cis-benzoates, (2S,4R)-18 or(2S,4S)-18, from which (+)- or (−)-1 were obtained as described for (±)-1. The ee’s of (+)- and (−)-ketoconazole were determined by HPLC on the CSP Chiralcel OD-H.

………………..

WO 1996029325

http://www.google.com/patents/WO1996029325A1?cl=en

The incidence of fungal infections has considerably increased over the last decades. Notwithstanding the utility of the antifungal compounds commercialized in the last 15 years, the investigation in this field is however very extensive. During this time, compounds belonging to the azole class have beer, commercialized for both the topical and oral administrations, such a class including imidazoles as well as 1,2,4-triazoles. Some of these compounds car. show m some degree a low gastrointestinal tolerance as well as hepatotoxycity.

A large number of pharmaceutically active compounds are commercialized as stereoisomeric mixtures. On the other hand, the case in which only one of said stereoisomers is pharmaceutically active is frequent.

The undesired enantiomer has a lower activity and it sometimes may cause undesired side-effects.

Ketoconazole (1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane-4-yl]methoxy]phenyl]piperazine), terconazole (1-[4-[[2(2,4-dichlorophenyl)-2-[(1H-1 , 2 ,4-triazol-1-yl)methyl]-1,3-dioxolane-4-yl]methoxy]phenyl]-4-(1-methylethyl)piperazine) and other related azole antifungal drugs contain in their structure a substituted 1,3-dioxolane ring, in which carbon atoms C2 and C4 are stereogenic centres, therefore four possible stereoisomers are possible. These compounds are commercialized in the form or cis racemates which show a higher antifungal activity than the corresponding trans racemates.

The cis homochiral compounds of the present invention, which are intermediates for the preparation of enantiomerically pure antifungal drugs, have been prepared previously in the racemic form and transformed into the different azole antifungal drugs in the racemic form [J. Heeres et al., J . Med . Chem . , 22 , 1003 (1979). J . Med . Chem . , 26, 611 (1983), J . Med . Chem . , 27 , 894 (1984) and US 4,144,346, 4,223,036, 4,358,449 and 4,335,125].

Scheme 1 shows the synthesis described for racemic ketoconazole [J. Heeres et al., J . Med . Chem . , 22 , 1003 (1979)]. Scheme 1

)

The synthesis of racemic terconazole [J. Heeres et al., J. Med . Chem . , 26 , 611 11983)] is similar. differing in the introduction of a 1 H- 1 , 2,4-triazol-1-yl substituent in place of 1H-imidazol-1-yl and in the nature of the phenol used in the last step of the synthetic sequence, which phenol is 1-methylethyl-4-(4- hydroxyphenyl)piperazme instead of 1-acetyl-4-(4-nydroxyphenyl)piperazine.

The preparation of racemic itraconazole [J. Heeres et al., J. Med . Chem. , 27 , 894 (1984)] is similar to that of terconazole, differing only in the nature of the phenol used in the last step of the synthetic sequence.

In the class of azoles containing a 1,3-dioxolane ring and a piperazine ring and moreover they are pure enantiomers, only the preparation of (+)- and (-)-ketoconazole has been described [D. M. Rotstein et al., J. Med . Chem . , 35, 2818 (1992)] (Scheme 2) starting from the tosylate of (+)- and (-) 2,2-dimethyl-1,3-dioxolane-4-methanol.

Scheme 2

This synthesis suffers from a series of drawbacks, namely: a) the use of expensive, high molecular weight starting products which are available only on a laboratory scale, and b) the need for several chromatographies during the process in order to obtain products of suitable purity, which maKes said synthesis economically unattractive and difficult to apply industrially.

Recently (N. M. Gray, WO 94/14447 and WO 94/14446) the use of (-)-ketoconazole and (+)-ketoconazole as antifungal drugs causing less side-effects than (±)-ketoconazole has been claimed.

The industrial preparation of enantiomerically pure antifungal drugs with a high antifungal activity and less side-effects is however a problem in therapy. The present invention provides novel homochiral compounds which are intermediates for the industrial preparation of already known, enantiomerically pure antifungal drugs such as ketoconazole enantiomers, or of others which have not yet been reported in literature, which are described first in the present invention, such as (+)-terconazole and (-)-terconazoie, which show the cited antifungal action, allowing to attain the same therapeutical effectiveness using lower dosages than those required for racemic terconazole

Example 14 : (2S,4R)-(-)-1-acetyl-4-[4-[ [2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)-methyl]-1,3-dioxolane-4-yl]methoxy]phenyl]piperazine, (2S,4R) -(- )-ketoconazole.

This compound is prepared following the process described above for (2R,4S)-(+)-ketoconazole. Starting from HNa (60-65% dispersion in paraffin, 32 mg, 0.80 mmol), 1-acetyl-4-(4-hydroxyphenyl)piperazine (153 mg, 0.69 mol) and (2S,4S)-(-)-IV (Ar = 2,4-dichlorophenyl, Y = CH, R = CH₃) (250 mg, 0.61 mmol), upon crystallization from an acetone:ethyl acetate mixture, (2S,4R) -(-)-ketoconazole is obtained [(2S,4R)-V Ar = 2,4-dichlorophenyl, Y = CH, Z = COCH₃] (196 mg, 61% yield) as a solid, m.p. 153-155ºC (lit. 155-157ºC); [α]_D ²⁰ = -10.50 (c = 0.4, CHCl₃) (lit. [α]_D ²⁵ = -10.58. c = 0.4, CHCl₃) with e.e. > 99% (determined by HPLC using the chiral stationary phase CHIRALCEL OD-H and ethanol:hexane 1:1 mixtures containing 0.1 % diethylamine as the eluent).

Figure imgf000007_0001

+ KETOCONAZOLE…. UNDESIRED

Example 7: (2 R ,4S)-(+)-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane-4-yl]methoxy]phenyl]piperazine (22, 4 S)-(+)-ketoconazole.

To a suspension of NaH (dispersed in 60-65% paraffin, 19.2 mg, 0.48 mmol) in anhydrous DMSO (3 ml),

1-acetyl-4-(hydroxyphenyl)piperazine (102 mg, 0.46 mmol) is added and the mixture is stirred for 1 hour at room temperature. Then, a solution of (2R,4R) – (+)-IV (Ar = 2,4-dichlorophenyl, Y = CH, R = CH₃) (160 mg, 0.39 mmol) in anhydrous DMSO (5 ml) is added, and the mixture is heated at 80ºC for 4 hours. The reaction mixture is allowed to cool to room temperature, diluted with water

(20 ml) and extracted with CH₂Cl₂ (3 × 25 ml). The combined organic phases are washed with water (3 × 25), dried with Na₂SO₄ and the solvent is evaporated off under vacuum. The oily residue thus obtained is crystallized from an acetone:ethyl acetate mixture to give (2R,4S)-(+)-ketoconazole ( (2R, 4 S) -V , Ar 2,4-dichlorophenyl, Y = CH , Z = COCH₃ ) ( 110 mg , 5 3 % yie ld ) as a white solid, m.p. 155-156°C (lit. 154-156ºC), [α]_D ²⁰ = + 8.99 (c = 0.4, CHCl₃) (lit. [α]_D ²⁵ = + 8.22, c = 0.4, CHCl₃), with e.e. > 99% (determined by HPLC using the chirai stationary phase CHIRALCEL OD-H and ethanol:hexane 1:1 mixtures containing 0.1% of diethylamine, as the eluent; (+)-Ketoconazole retention time 73,28 min. (-)-Ketoconazole, retention time 79.06 min).

IR (KBr), ʋ : 2875, 1645, 1584, 1511, 1462, 1425, 1250, 103S, 313 cm^-1.

¹H NMR (500 MHz, CDCl₃), δ : 2.12 (s, 3H, COCH₃),

3.02 (m, 2H, 3-H₂), 3.05 (m, 2H, 5-H₂), 3.27 (dd, J= 9.5

Hz, J’=7.0 Hz, 1H) and 3.70 (dd, J=9.5 Hz, J’=5.0 Hz, 1 H) (4″-CH₂), 3.60 (m, 2H, 6-H₂), 3.76 (m, 2H, 2-H₂), 3.73 (dd, J=8.0 Hz, J’=5.0 Hz, 1H) and 3.86 (dd, J=8.0 Hz, J’=6.5 Hz, 1H) (5″-H₂), 4.34 (m, 1H, 4″-H), 4.40 (d, J=15.0 Hz, 1H) and 5.00 (d, J=15.0 Hz, 1H) (CH₂-N), 4.34

(m, 1H, 4″-H), 6.76 [d, J = 9.0 Hz, 2H, 2′(C6′ )-H], 6.88

[d, J=9.0 Hz, 2H, C3′(C5)-H], 6.96 (s, 1H, imidazole 5- H), 6.99 (s, 1H, imidazole 4-H), 7.25 (dd, J=8.5 Hz, J’=2.0 Hz, 1H, 5″‘-H), 7.46 (d, J=2.0 Hz, 1H, 3″‘-H),

7.53 (s, 1H, imidazole 2-H), 7.57 (d, J=8.5 Hz, 1H,

6″‘-H).

¹³C NMR (75.4 MHz, CDCI₃), δ : 21.3 (CH₃, COCH₃), 41.4 (CH₂, C₂), 46.3 (CH₂, C6), 50.6 (CH₂, C3), 51.0 (CH₂, C5), 51.2 (CH₂, CH₂-N), 67.6 [CH₂, C5″ and 4″-CH₂), 74.7 (CH, C4″), 108.0 (C, C2″), 115.2 [CH, C2′(6′)], 118.8 [CH, C3′(5′)], 121.2 (CH, imidazole C5), 127.2 (CH, C5″‘), 128.5 (CH, imidazole C4), 129.5 (CH, C6′”), 131.3 (CH, C3″‘), 133.0 (C, C2″‘), 134.6 (C, C1′”), 135.8 (C, C4″‘), 138.8 (CH, imidazole C2), 145.6 (C, C1′), 152.8 (C, C4’), 168.9 (C, CO).

…………………………

Experimental and theoretical analysis of the interaction of (+/-)-cis-ketoconazole with beta-cyclodextrin in the presence of (+)-L-tartaric acid
J Pharm Sci 1999, 88(6): 599

Experimental and theoretical analysis of the interaction of (±)-cis-ketoconazole with β-cyclodextrin in the presence of (+)-l-Tartaric acid (pages 599–607)

Enrico Redenti, Paolo Ventura, Giovanni Fronza, Antonio Selva, Silvia Rivara, Pier Vincenzo Plazzi and Marco Mor

Article first published online: 12 JUN 2000 | DOI: 10.1021/js980468o

http://onlinelibrary.wiley.com/doi/10.1021/js980468o/pdf

¹H NMR spectroscopy was used for determining the optical purity of cis-ketoconazole enantiomers obtained by fractional crystallization. The chiral analysis was carried out using β-cyclodextrin in the presence of (+)-l-tartaric acid. The mechanism of the chiral discrimination process, the stability of the complexes formed, and their structure in aqueous solution were also investigated by ¹H and ¹³C chemical shift analysis, two-dimensional NOE experiments, relaxation time measurements, and mass spectrometry experiments. Theoretical models of the three-component interaction were built up on the basis of the available NMR data, by performing a conformational analysis on the relevant fragments on ketoconazole and docking studies on the components of the complex. The model derived from a folded conformation of ketoconazole turned out to be fully consistent with the molecular assembly found in aqueous solution, as inferred from NOE experiments. An explanation of the different association constants for the complexes of the two enantiomers is also provided on the basis of the interaction energies.

WO1993019061A1 *	Mar 10, 1993	Sep 30, 1993	Janssen Pharmaceutica Nv	Itraconazole and saperconazole stereoisomers
WO1994025452A1 *	Apr 28, 1994	Nov 10, 1994	Ashit K Ganguly	Process for preparing intermediates for the synthesis of antifungal agents
EP0050298A2 *	Oct 13, 1981	Apr 28, 1982	Hoechst Aktiengesellschaft	1-(1,3-Dioxolan-2-ylmethyl) azoles, process for their preparation and their use
EP0052905A1 *	Nov 19, 1981	Jun 2, 1982	Janssen Pharmaceutica N.V.	Novel (2-aryl-4-phenylthioalkyl-1,3-dioxolan-2-yl-methyl)azole derivatives
US5208331 *	Jun 18, 1992	May 4, 1993	Syntex (U.S.A.) Inc.	Process for preparing 1,3-dioxolane derivatives

AmVac begins Phase III trial of Gynevac vaccine for bacterial vaginosis

Phase 3 drug, Uncategorized, VACCINE Comments Off

Jun 192014

AmVac begins Phase III trial of Gynevac vaccine for bacterial vaginosis

AmVac has commenced a Phase III study of its Gynevac to assess the safety and efficacy of the vaccine in bacterial vaginosis (BV) treatment.

Manufactured in accordance with the current GMP standards, the therapeutic vaccine is based on a blend of inactivated lactobacilli strains.

http://www.marketwatch.com/story/amvac-initiates-phase-iii-trial-with-its-lead-vaccine-gynevac-for-the-treatment-of-bacterial-vaginosis-2014-06-17

Love, cast in stone: Temples in India depicting erotic art

India is dotted with many glorious temples, but erotica on the walls of some arouses curiosity and even puzzles tourists. There are various theories about the reason for such vivid depiction of erotica–mass sex education, warding off natural calamities and the devdasi system. Due to the presence of 64 Yogini temples near Khajuraho, Padawali, Konarak/Lingaraj etc., scholars also attribute the erotic art to Tantric practices, which revolve around the ultimate union of the male and the female energy and forms referred to as Maithuna. Whatever the reason be, the brazenness or ethereal beauty of temple erotica will never cease to amaze us.

Khajuraho, Madhya Pradesh

Built by the Chandela Kings who were greatly influenced by Tantric traditions, this temple is said to represent the ultimate seductress.

While the fine sandstone statues built earlier have a well rounded finish, the ones made later are more angular. In his history of the Kamasutra, Mc Connachie describes the amorous sculptures as “the apogee of erotic art”, where the twisting, broad hipped and high breasted nymphs, fleshy apsaras and extravagantly interlocked maithunas run riot along the surface of stone.

The various scenes of passionate love making, in acrobatic postures that sometimes border on the physically impossible, strike viewers. Look out for the bold panels of multiple partners engaged with each other. For an interesting perspective on Khajuraho, watch the Sound and Light show. The best time to visit is during the Khajuraho Dance Festival in the first week of February.

Markandeshwar Temple, Maharashtra

Near the naxal district of Gadchiroli, the Markandeshwar temple complex, by the River Wainganga, showcases a sprinkling of erotic art. A couple performing ‘fellacio’ will raise eyebrows. Know to be built by danavas (evil forces) in one night, the temple is made from stone, and follows Hemadpanth architecture. The annual fair during Mahashivratri attracts devotees from far and wide every year. Hiring a car from Nagpur is recommended, unless you fancy hitch-hiking with villagers past moonlit fields or changing several buses and autos. If you’re stranded, look for the dharamshala near the temple.

Padawali Temple, Madhya Pradesh

In Morena district near the Chambal Valley, once notorious for dacoits, lies the fortress of Padawali. Two stalwart lion statues greet you at its entrance. The temple inside has earned the reputation being a ‘Mini Khajuraho’ due to the prevalence of erotic art. The difference between big brother Khajuraho and Padawali Temple, is that the erotic art here seems less acrobatic and more ‘real life’ and ‘doable’. The carvings of maithunas in various positions, ranging from simple to difficult almost brings the Kamasutra to life.

Ranakpur Jain Temples, Rajasthan

This marble temple of superlative beauty is a ‘vision in white’ with its domes, shikharas and turrets. Over 1,444 intricately carved marble pillars hold up the temple and a monolithic marble rock depicting over 100 snakes catches the eye. Look out for a panel depicting several experimental love making scenes, in a line with a central queen-like figure seated on a throne, with an amorous midget on her lap. It’s interesting to note that not only Hindus, but even Jains decorated temples with erotic art. It hints at how nudity had a religious connect due to the ‘Digambara’ ideology or the Tantric cult.

Sun Temple, Orissa

When I first visited the Sun Temple at Konarak in Orissa, as a giggly 16-year-old , I was taken aback by how the panels revealed way more about the ‘birds and bees’ than our biology classes had taught us. My second visit recently, helped me appreciate the beautiful erotic art better. The brazenness of the sculptures here gives Khajuraho stiff competition; one of the most scandalous panels is of a dog licking a woman’s genital area. I overheard a guide say, “this was considered a cure for sex related infections, as the dog’s saliva has antibiotic properties.” Scenes of polygamy, polyandry and lesbian love are blissfully abundant.

An architectural genius, this temple shows the Sun God on a colossal chariot drawn by seven horses. The word Konarak is a combination of Kona (corner) and Arka (Sun). The temple was previously located closer to the sea, but the magnetic properties of its stone caused shipwrecks. This, along with the dark colour of its stones, earned it the tile of ‘The Black Pagoda’. An interesting study in contrast is the famous Jagannath Temple at Puri, also referred to as ‘The White Pagoda’ due to its whitewashed walls. If you are an art enthusiast you must visit the Konarak Archaeological Museum nearby that contains fallen sculptures from the temple.

Sun Temple, Gujarat

It is believed to be the place where Lord Rama conducted a yagna here to purify himself of the sin of killing a Brahmana-Ravana. Like Konarak, its architecture is such that the temple catches the first rays of the rising sun. The most striking feature of the temple is a perfectly designed Kama Kunda (water tank) meant for ablutions and for a reflection of the temple in the water. It has lateral stone steps leading down to the tank, allowing both direct and diagonal descent from all sides. Carvings of men and women in various acts of sex with small midget like creatures are prominent. However, due to erosion the detailing of the stone carvings is blurred in places.

Osian, Rajasthan

Amidst the sand dunes of Thar, Osian has a cluster of Hindu and Jain temples dating back to the 11 century AD. The Sachiya Mata temple dedicated to the resident Goddess has a gorgeous carved archway leading up to the shrine and has some beautiful depiction of erotic love locked couples, complete with details like the bed on which the couples lie.

Virupaksha Temple, Karnataka
On the banks of the Tungabhadra River, this temple with beautiful pillars and towered gateways dedicated to Lord Shiva in his avatar as Virupaksha. It is one of the oldest functioning temples since the 7th century AD. A panel that catches the eyes depicts a nude woman being ‘admired’ by men and women around her. It is best to visit the temple, during the Hampi festival in November. While in the area, also check out the erotic art on the pillars of the Achyutaraya temple.

Several other temples in South India like Belur, Halebidu, Somanathapura and Nugguhalli, the Badami and Banashankari temples of the Chalukya times and the Vijayanagar temples of Bhatkal and Lepakshi also have a profusion of erotic art. The Meenakshi temple of Madurai and Veeraranarayan temple of Gadag have erotic sculptures on their Gopuram. (Information about other temples with erotic art in South India taken from www.kamat.com)

No one has summed up the beauty of erotica on temple walls better than Tagore while he was referring to Konarak, ‘The language of man here is defeated by the language of stone.’

FINAFLOXACIN IN PHASE II for the treatment of ear infections

Phase 3 drug Comments Off

Mar 292014

FINAFLOXACIN

(S-cyano-1-cyclopropyl-ό-fluoro-T-^aS, 7aS)-hexahydropyrrolo [3,4- b]-1,4-oxazin-6(2H)-yl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid)

7-[(4aS,7aS)-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazin-6-yl]-8-cyano-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid |

BAY-35-3377
BY-377

CAS Registry Number: 209342-40-5

HYD SALT

(-)-(4aS,7aS)-8-Cyano-1-cyclopropyl-6-fluoro-4-oxo-7-(perhydropyrrolo[3,4-b]-1,4-oxazin-6-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride

209342-41-6,

C20 H19 F N4 O4 . Cl H
MW	434.849

Synonyms: Finafloxacin, UNII-D26OSN9Q4R,

MerLion Pharmaceuticals (Singapore)…POSTER…….http://www.merlionpharma.com/sites/default/files/file/PPS/F1-2036_Wohlert.pdf

H. pylori, Broad-Spectrum

Finafloxacin is a novel fluoroquinolone being developed by MerLion Pharmaceuticals. Under neutral pH conditions (pH 7.2–7.4), the compound has shown in vitro activity equivalent to that of ciprofloxacin. However, under slightly acidic pH5.8 the compound shows enhanced potency.

Other marketed fluoroquinolones, such as ciprofloxacin, levofloxacin and moxifloxacin, exhibit reduced activity at slightly acidic pH 5.0–6.5. This feature of finafloxacin makes the compound suitable for use in the treatment of infections in acidic foci of infections such as urinary tract infections

Finafloxacin hydrochloride, a novel highly potent antibiotic, is in phase III clinical trials at Alcon for the treatment of ear infections. MerLion Pharmaceuticals is evaluating the product in phase II clinical trials at for the treatment of Helicobacter pylori infection and for the treatment of lower uncomplicated urinary tract infections in females.

A quinolone, finafloxacin holds potential for the treatment of Helicobacter pylori infection and urinary tract infection. Unlike existing antibiotics, finafloxacin demonstrates a unique acid activated activity whereby it becomes increasingly active under acidic conditions.

In 2009, a codevelopment agreement was signed between Chaperone Technologies and MerLion Pharmaceuticals. In 2011, finafloxacin hydrochloride was licensed to Alcon by MerLion Pharmaceuticals in North America for the treatment of ear infections.

MerLion Pharmaceuticals has announced that the FDA has granted a Qualified Infectious Disease Product Designation and Fast Track Status for finafloxacin. The company is currently recruiting patients for the Phase II clinical trial of the compound for the treatment of complicated urinary tract infections (cUTI) and/or acute pyelonephritis compared to ciprofloxacin

Finafloxacin and derivatives thereof can be synthesized according to the methods described in U.S. Patent No. 6,133,260 to Matzke et al., the contents of which are herein incorporated by reference in their entirety. The compositions of the invention are particularly directed toward treating mammalian and human subjects having or at risk of having a microbial tissue infection. Microbial tissue infections that may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et al., “The Sanford Guide to Antimicrobial Therapy 2007” 37 Edition (Antimicrobial Therapy, Inc.). Particular microbial tissue infections that may be treatable by embodiments of the present invention include those infections caused by bacteria, protozoa, fungi, yeast, spores, and parasites.

SYNTHESIS

WO1998026779A1

http://www.google.sc/patents/WO1998026779A1 COPY PASTE ON BROWSER

8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-di-azabicyclo [4.3.0] non-8-yl)-l, 4-dihydro-4-oxo-3-quinolinecarboxylic acid.

The compounds, which are suitable for use in the invention are known already to some extent in EP-A-0350733, EP-A-0550903 as well as from DE-A-4329600 or can be prepared according to the processes described in .

If, for example 9,10-difluoro-3 ,8-dimethyl-7-oxo-2 ,3-dihydro-7H-pyrido [l ,2,3-d, e] [l, 3,4] benzoxadiazine-6 -carboxylic acid and 2-oxa-5 ,8-diazabicyclo [4.3.0] nonane, the reaction can be represented by the following equation:

The 7-halo-quinolonecarboxylic acid derivatives used for preparing the compounds of Fomel (I) of the invention are known or can be prepared by known methods. Thus, the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid, or of the 7-chloro-8-cyano-l-cyclopropyl-6-fluoro- l been ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester described in EP-A-0 276 700th The corresponding 7-fluoro derivatives can be, for example, via the following reaction sequence to build:

An alternative process for preparing the intermediate compound 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride as the starting material for the preparation of 7-chloro-

8-cyano-1-cyclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is used (EP-A-0276700) and in the 3-cyano-2 ,4,5-trifluoro- benzoyl can be converted, is based on 5-fluoro-l ,3-xylene, 5-fluoro-l ,3-xylene, in the presence of a catalyst under ionic conditions in the nucleus disubstituted to 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene, and this is subsequently chlorinated chlorinated under free radical conditions in the side chains of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methylbenzene. This is the 2,4-dichloro-5-fluoro-3-dichloromethyl-benzoic acid to give 2,4-dichloro-5-fluoro-3-formyl-benzoic acid, and then hydrolyzed to 2,4-dichloro-5-fluoro-3 N-hydroxyiminomethyl acid implemented. By treatment with thionyl chloride, 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride is obtained, which can still be ,4,5-trifluoro-ben-zoylfluorid converted by a chlorine / fluorine exchange on-3-cyano-2 .

The amines used for the preparation of compounds of formula (I) according to the invention are known from EP-A-0550903, EP-A-0551653 as well as from DE-A-4 309 964th

An alternative to the synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-dihydro-drobromid or the free base 1 S, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0 ] nonane and the corresponding IR, 6R enantiomer provides the following path represents:

Starting material for this synthesis is the cis-l ,4-dihydroxy-2-butene, which is converted to the bis-mesylate with mesylation tosylamide for 1-tosylpyrrolidine. This is converted into the epoxide m-chloroperbenzoic. The ring opening of the epoxide by heating in isopropanol with ethanolamine to trans-3-hydroxy-4 – (2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl)-pyrrolidine in 80% yield. Tetrahydrofuran is then in pyridine / reacted with tosyl chloride, with cooling to Tris-tosylate, which as a crude product in a mixture with some tetra-tosyl derivative with basichen reaction conditions to give the racemic trans-5 ,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane is cylisiert. At this stage occurs with high selectivity of a chromatographic resolution kieselgelgebundenem poly (N-methacryloyl-L-leucine-d menthylamide) as the stationary phase. The desired enantiomer, (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane, is of a purity of

> 99% ee. Cleavage of the p-tosyl protecting groups is carried out with HBr-acetic acid to the lS, 6S-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide, the one with a base such as sodium or potassium hydroxide or with the aid of ion exchanger can be converted into the free base. The analogous sequence may be used for the preparation of lR, 6R-2-Oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide.

HBr / AcOH

Synthesis of lS, 6S-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Examples of compounds of the invention are mentioned in addition to the compounds listed in the preparation examples, the compounds listed in Table 1 below, which can be used both in racemic form as well as enantiomerically pure or diastereomerically pure compounds. Table 1:

Example 1 Z

8-cyano-1-cyclopropyl-6 ,7-difluoro-1 ,4-dihydro-4-oxo-3-quinoline-carboxylic acid ethyl ester

a 3-bromo-2 ,4,5-trifluoro-benzoate

To a mixture of 1460 ml of methanol and 340 g of triethylamine, 772 g of 3-bromo-2 ,4,5-trifluoro-benzoyl fluoride was added dropwise under ice cooling. There is one

Stirred for an hour at room temperature. The Reaktionsgemsich is concentrated, the residue dissolved in water and methylene chloride, and the aqueous phase was extracted with methylene chloride. After drying the organic phase over sodium sulfate, concentrated, and the residue was distilled in vacuum. This gives 752.4 g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester of boiling point 122 ° C/20 mbar.

b. 3-Cyano-2 ,4,5-trifluoro-benzoic acid methyl ester:

269 g of 3-bromo-2 ,4,5-trifluoro-benzoic acid methyl ester and 108 g of copper cyanide are heated to reflux in 400 ml of dimethylformamide for 5 hours. , All volatile components of the reaction mixture are then distilled off in vacuo. The distillate was then fractionated on a column. This gives 133 g of 3-cyano-2 ,4,5-trifluoro-benzoate of boiling point 88-89 ° C / 0.01 mbar.

c. 3-Cyano-2 ,4,5-trifluoro-benzoic acid

A solution of 156 g of 3-cyano-2 ,4,5-trifluoro-benzoate in 960 ml of glacial acetic acid, 140 ml of water and 69 ml concentrated sulfuric acid is heated for 8 hours under reflux. Then the acetic acid is distilled off under vacuum and the residue treated with water. Of failed-ne solid is filtered off, washed with water and dried. Obtained

118.6 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid as a white solid, mp 187-190 ° C.

d 3-cyano-2 ,4,5-trifluoro-benzoyl chloride:

111 g of 3-cyano-2 ,4,5-trifluoro-benzoic acid and 84 g of oxalyl chloride are stirred in 930 ml of dry methylene chloride with the addition of a few drops of dimethylformamide for 5 hours at room temperature. The methylene chloride is evaporated and the residue distilled in vacuo. This gives 117.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl chloride as a yellow oil.

e 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester:

To a solution of 36.5 g of 3-dimethylamino-acrylate and 26.5 g of triethylamine in 140 ml toluene, a solution of 55 g 3-cyano-2, 4,5 – trifluoro-benzoyl chloride are added dropwise in 50 ml of toluene so that the temperature 50-55 ° C remains. Then stirred for 2 hours at 50 ° C.

The reaction mixture is concentrated in vacuo and used without further

Processing used in the next step. f 2 – (3-cyano-2 ,4,5-trifluoro-benzoyl)-3-cyclopropylamino-acrylic acid ethyl ester:

To the reaction product of step e 30 g of glacial acetic acid are added dropwise at 20 ° C. A solution of 15.75 g of cyclopropyl amine in 30 ml of toluene is added dropwise. The mixture is stirred at 30 ° C for 1 hour. Are then added 200 ml of water, stirred 15 minutes, the organic phase is separated off and shakes it again with 100 ml of water. The organic phase is dried over sodium sulfate and concentrated in vacuo. The crude product thus obtained is a set-without further purification in the next step.

g 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester:

The reaction product from stage f and 27.6 g of potassium carbonate are stirred in 80 ml dimethylformamide for 16 hours at room temperature. The reaction mixture is then poured into 750 ml ice water, the solid filtered off with suction and washed with 80 ml cold methanol. After drying, 47 g of 8 – cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinoline carboxylic acid ethyl ester, mp 209-211 ° C.

Example 2 Z

2,4-dichloro-5-fluoro-l ,3-dimethylbenzene

a solvent-free

In 124 g of 3,5-dimethyl-fluorobenzene 1 g of anhydrous iron (III) chloride are pre-loaded and launched with the speed of chlorine (about 4 h), with which the reaction. This is initially slightly exothermic (temperature increase from 24 to 32 ° C) and is maintained by cooling below 30 ° C. After addition of 120 g of chlorine, the mixture is determined. According to GC analysis are 33.4% monochloro compound, formed 58.4% desired product and 5%> overchlorinated connections. The hydrogen chloride is removed and the reaction mixture is then distilled in a column in a water jet vacuum:

In the run 49 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene obtained at 72-74 ° C/22 mbar. After 5 g of an intermediate fraction proceed at 105 ° C/22 mbar 75 g of 2,4 – dichloro-5-fluoro-l ,3-dimethylbenzene via, Melting range: 64 – 65 ° C.

b in 1,2-dichloroethane

1 kg of 3,5-dimethyl-fluorobenzene and 15 g of anhydrous iron (III) chloride are placed in 1 1 1 ,2-dichloroethane and chlorine is introduced in the same extent as the reaction proceeds (about 4 h). The reaction is initially exothermic (temperature rise from 24 to 32 ° C) and is kept below 30 ° C by cooling. After the introduction of 1200 g of chlorine are according to GC analysis 4% monochloro compound, 81.1% and 13.3% desired product overchlorinated connections emerged. After distilling off the solvent and the hydrogen chloride is distilled in a column in a water jet vacuum:

In the run 40 g of 2-chloro-5-fluoro-l ,3-dimethylbenzene receive. After some intermediate run going at 127-128 ° C/50 mbar 1115 g of 2,4-dichloro-5-fTuor-l ,3-dimethyl-ethylbenzene over.

Example 3 Z

2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloromethylbenzene

In a photochlorination using chlorine inlet and outlet for the hydrogen chloride to a scrubber and a light source in the vicinity of the chlorine inlet tube, 1890 g of 2,4-dichloro-5-fluoro-l ,3-dimethylbenzene pre-loaded and at 140 to 150 ° C. Chlorine metered. Within 30 hours 3850 g of chlorine are introduced. The content of the desired product according to GC analysis is 71.1% and the proportion of connections minderchlorierten 27.7%. The DestiUaton a 60 cm column with Wilson spirals provides a flow of 1142 g, which can be reused in the chlorination. The main fraction at 160-168 ° C / 0.2 mbar gives 2200 g of 2,4-dichloro-5-fluoro-3-dichloromethyl-l-trichloro-methyl benzene having a melting range of 74-76 ° C. After one recrystallization

Sample from methanol, the melting point 81-82 ° C.

Example Z 4

2,4-dichloro-5-fluoro-3-formyl-benzoic acid

In a 2500 ml stirred apparatus with gas discharge are presented 95% sulfuric acid at 70 ° C. and under stirring, 500 g of molten added dropwise 2,4-dichloro-5-fluoro-3-dichloromethyl-1 trichloromethylbenzene. It is after a short while hydrochloric development. Is metered during a 2 h and stirred until the evolution of gas after. After cooling to 20 ° C., the mixture is discharged ice to 4 kg and the precipitated solid is filtered off with suction. The product is after-washed with water and dried.

Yield: 310 g, melting range: 172-174 ° C

Example Z 5

2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl-benzoic acid

In a stirred reactor 80 g of hydroxylamine hydrochloride in 500 ml of ethanol are charged and added dropwise 200 ml of 45% strength sodium hydroxide solution and then with 40 – 200 g of 2,4-dichloro-5-fluoro-3-formyl-benzoic acid added 45.degree.The reaction is slightly exothermic and it is stirred for 5 h at 60 ° C. After cooling to

Room temperature is provided by the dropwise addition of hydrochloric acid to pH <3, the product taken up in tert-butyl methyl ether, the organic phase separated and the solvent distilled off. The residue obtained 185 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid, melting range: 190 – 194 ° C.

Example No. 6

2,4-dichloro-3-cyano-5-benzoyl-fιuor

In a stirred vessel with metering and gas outlet via a reflux condenser to a scrubber 600 ml of thionyl chloride are introduced and registered at 20 ° C. 210 g of 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl benzoic acid in the proportion as hydrochloric developed and sulfur dioxide. After the addition the mixture is heated until the gas evolution under reflux. Mixture is then distilled, and boiling in the range of 142-145 ° C/10 mbar, 149 g of 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride (98.1% purity by GC) Melting range: 73-75 ° C.

Example No. 7

3-Cyano-2 ,4,5-trifluoro-benzoyl

50 g of potassium fluoride are suspended in 120 ml of tetramethylene sulfone and at 15 mbar for drying distilled (ca. 20 mL).Then, 50.4 g of 2,4 – dichloro-3-cyano-5-fluoro-benzoyl chloride was added and stirred at an internal temperature with exclusion of moisture for 12 hours at 180 ° C. Are removed by vacuum distillation to 32.9 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride in the boiling range of 98 –

Obtain 100 ° C/12 mbar.

Example No. 8

3-Cyano-2 ,4,5-trifluoro-benzoyl chloride

76.6 g of 3-cyano-2 ,4,5-trifluoro-benzoyl fluoride together with 1 g of anhydrous

Aluminum chloride introduced at 60-65 ° C and then added dropwise 25 g of silicon tetrachloride gas in the course of development. After the evolution of gas at 65 ° C is distilled in a vacuum. Boiling range 120-122 ° C/14 mbar, 73.2 g of 3 – cyano-2 ,4,5-trifluoro-benzoyl chloride over.

Example No. 9

1 – (toluene-4-sulfonyl-pyrroline

In a 20 1 HC4-HWS boilers are 2.016 kg (17.6 mol)

Submitted methanesulfonyl chloride in dichloromethane and 12 1 at -10 ° C internal temperature under strong cooling (-34 ° C) solution of 705 g (8.0 mol) of 2-butene-l ,4-diol in 1.944 kg (2.68 1 , 19.2 mol) of triethylamine was added dropwise over 30 minutes. A yellow suspension stirred for 1 hour at -10 ° C and then treated with 4 1 of water, the temperature rises to 0 ° C.The suspension is warmed to room temperature, stirred for 10 minutes at room temperature and then fed in a 30 1 separating funnel. The phases are stirred separately (good phase separation) and the aqueous phase extracted with 2 1 of dichloromethane. The combined dichloromethane phases are presented in a pre-cooled 20 1 HC4 vessel and kept at 0 ° C.

In another 20-1 HC4 boiler distillation 1.37 kg (8.0 mol) toluenesulfonamide be submitted in 6 1 toluene. It is mixed with 3.2 kg of 45% sodium hydroxide solution, 0.8 1 of water and 130.5 g Tetrabutylammomiimhydrogensulfat, heated to 40 ° C maximum temperature inside and creates a vacuum. Then, the previously obtained

Dichloromethane (15.2 1) was added dropwise over 1.5 hours while the dichloromethane was removed by distillation at 450 mbar (bath temperature: 60 ° C). During the distillation is foaming. In the end, a solution is available at an internal temperature of 33-40 ° C. After the addition of dichloromethane is distilled off, until barely distillate is (duration: about 85 minutes; internal temperature 40 ° C at 60 ° C bath temperature at the end). The vessel contents will be warm transferred to a separating funnel and rinsed the tank with water and 5 1 2 1 toluene at 50 ° C. Before phase separation, the solids are extracted in the intermediate phase and washed with 0.5 1 of toluene. The organic phase is extracted with 2.4 1 of water, separated and evaporated to dryness on a rotary evaporator. The solid residue (1758 g) is suspended in 50 ° C bath temperature in 1.6 1 of methanol, the suspension is transferred into a 10 1-flanged flask and the flask rinsed with diisopropyl 2,4 1. The mixture is heated to reflux temperature (59 ° C) and stirred for 30 minutes under reflux. The suspension is cooled to 0 ° C., stirred at 0 ° C for 1 hour and extracted with 0.8 1 of a cold mixture of ether Methanol/Diisopropyl-: washed (1 1.5). The crystals are dried under a nitrogen atmosphere at 50 ° C/400 mbar.

Yield: 1456 g (81.5% of theory)

Example Z 10

3 – (toluene-4-sulfonylV6-oxa-3-aza-bicvclo [3.1.0] hexane

^{o “|” h “CH3}

334.5 g (1.5 mol) of l-(toluene-4-sulphonyl)-pyrroline are dissolved in 1.5 1 of dichloromethane at room temperature and over 15 minutes with a suspension of 408 g (approx. 1.65 to 1, 77 mol) of 70-75% m-chloroperbenzoic acid in 900 ml of dichloromethane (cools added in manufacturing from). The mixture is heated under reflux for 16 hr (test for

Peroxide with KI / starch paper shows yet to peroxide), the suspension was cooled to 5 ° C, sucks the precipitated m-chlorobenzoic acid and washed with 300 ml of dichloromethane (peroxide with Precipitation: negative; precipitate was discarded). The filtrate is to destroy excess peroxide with 300 ml of 10% sodium sulfite solution, washed twice (test for peroxide runs now negative), extracted with 300 ml of saturated sodium bicarbonate solution, washed with water, dried with sodium sulfate and about a quarter of the volume evaporated. Again on test peroxide: negative. The mixture is concentrated and the solid residue is stirred with ice cooling, 400 ml of isopropanol, the precipitate filtered off and dried at 70 ° C in vacuum.

Yield: 295 g (82.3%),

Mp: 136-139 ° C,

TLC (dichloromethane methanol 98:2): 1 HK (Jodkammer)

Example CLOSED

^{trans-3-Hydroxy-4-(2-hydroxy-ethylamino-l-(‘toluene-4-sulfonyl’)} pyrrolidine

643.7 g (2.65 mol) 3 – (Toluoι-4-sulfonyl)-6-oxa-3-aza-bicyclo [3.1.0] hexane to 318.5 ml with ethanolamine in 4 1 of isopropanol at reflux for 16 hours cooked. After TLC monitoring, further 35.1 ml (total 5.86 mol) of ethanolamine added to the mixture and boiled again until the next morning. The mixture is filtered hot with suction and the filtrate concentrated on a rotary evaporator to 3.5 ltr. After seeding and stirring at room temperature for 3.5 1 diisopropyl ether are added, and stirred at 0 ° C for 6 hours. The precipitated crystals are filtered off, with 250 ml of a mixture of isopropanol / diisopropyl ether (1: 1) and washed 2 times with 300 ml of diisopropyl ether and dried overnight under high vacuum.

Yield: 663.7 g (83% of theory), content: 96.1% (area% by HPLC). Example Z 12

trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl)-pyrrolidin-3-yl] – ftoluol-4-sulfonyl)-amino]-ethyl ester)

552 g (1.837 mol) of trans-3-hydroxy-4-(2-hydroxy-ethylamino)-l-(toluene-4-sulfonyl) – pyrrolidine are dissolved under argon in 1.65 1 tetrahydrofuran and 0.8 1 of pyridine dissolved and at -10 ° C in portions 700 g (3.675 mol) p-toluenesulfonyl chloride are added thereto. The mixture is then stirred at this temperature for 16 hours. The work is done by adding 4.3 18.5 1% aqueous hydrochloric acid, extraction twice with dichloromethane (3 1, 2 1), washing the combined organic phases with saturated Natriurnhydrogencarbonatlösung (3 1, 2 1), drying over sodium sulfate, extracting and distilling off the solvent in vacuo. The residue is dried overnight at the oil pump and crude in the next reaction. There were 1093 g as a hard foam (content [area% by HPLC]: 80% Tris-tosyl-product and 13% tetra-tosyl-product, yield see next step). Example Z 13

rac. trans-5 ,8-bis-tosyl-2-oxa-5 .6-diazabicyclor4 .3.01 nonane

1092 g of crude trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l-(toluene-4-sulfonyl) – pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino]-ethyl} were dissolved in tetrahydrofuran and 9.4 1 at 0-3 ° C with 1.4 1 of a 1.43 molar solution of sodium hydroxide in

Methanol reacted. After half an hour at this temperature, 2.1 1 of water and 430 ml of diluted (2:1) was added to the mixture and acetic acid with previously isolated crystals of trans-toluene-4-sulfonic acid {2 – [[4-hydroxy-l – (toluene-4-sulfo-phenyl)-pyrrolidin-3-yl] – (toluene-4-sulfonyl)-amino] ethyl}-seeded. The suspension is stirred overnight at 0 to -4 ° C. The next morning, the crystals are filtered off, washed twice with 400 ml of cold mixture of tetrahydrofuran / water (4:1) and dried at 3 mbar at 50 ° C overnight.

Yield: 503 g of white crystals (62.7%> of theory over 2 steps), content: 99.7% (area% by HPLC). Example Z 14

Preparative chromatographic resolution of racemic rac. trans-5.8-bis-tosyl-2-oxa-5.6-diazabicyclor4.3.0] nonane

The chromatography of the racemate at room temperature in a column (inner diameter 75 mm), which with 870 g of a chiral stationary phase (kie-selgelgebundenes poly (N-methacryloyl-L-leucine-d menthylamide) based on the mer captomodifizierten silica Polygosil 100 , 10 microns; see EP-A 0 379 917) is filled (bed height: 38 cm). Detection is carried out using a UV detector at 254 nm

For the sample application using a solution of a concentration of 100 g of rac. trans-5 ,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 3000 ml of tetrahydrofuran. A Trenncyclus is carried out under the following conditions: with the aid of a pump is required for 2 min at a flow of 50 ml / min, a part of the sample solution and the same time at a flow rate of 50 ml / min, pure n-heptane to the column.

Thereafter eluted at a flow rate of 100 ml / min 18 minutes with a mixture of n-Heptan/Tetrahydrofuran (3/2 vol / vol). This is followed for 3 minutes at a flow of 100 ml / min elution with pure tetrahydrofuran. Thereafter, further eluted with n-Heptan/Tetrahydro-furan (3/2 vol / vol). This cycle is repeated several times.

The first eluted enantiomer is the (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo-[4.3.0] nonane, which is isolated by concentration. The eluate of the more retarding enantiomers is largely evaporated in vacuo, and the precipitated crystals are filtered off with suction and dried. From the separation of 179 g of racemate in this

As 86.1 g (96.2% of theory) of the enantiomer (lS, 6S) -5,8-bis-tosyl-2-oxa-5, 6 – diazabicyclo [4.3.0] nonane having a purity of> 99 % ee. Example Z 15

(LR, 6R-2-oxa-5.6-diazabicvclo [4.3.0] nonane dihydrobromide

38.3 g (87 mmol) of (lS, 6R) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] nonane in 500 ml of 33 -% HBr / glacial acetic acid 10 g added anisole and heated for 4 hours at 60 ° C (bath). After standing overnight, the suspension is cooled, the precipitate filtered, with

100 ml of abs. Ethanol and dried at 70 ° C under high vacuum.

Yield: 23.5 g (93%) of white solid product, mp 309-310 ° C (dec.), DC (dichloromethane/methanol/17% aq ammonia 30:8:1.): 1 HK

[Α] _D: + 0.6 ° (c = 0.53, H ₂ O) (fluctuating).

Example Z 16

(LS.6S-2-oxa-5.6-diazabicvclor4.3.01nonan-Dihvdrobromid

Z is analogous to Example 15 from (lS, 6S) -5,8-bis-tosyl-2-oxa-5 ,6-diazabicyclo [4.3.0] no-nan (1S, 6S)-2-oxa-5, 6-diazabicyclo [4.3.0] nonane dihydrobromide receive. Example Z 17

(1 R.6R-2-oxa-5.8-diazabicvclo [4.3.Olnonan

1 Method: 5,8 g (20 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydro-drobromid are suspended in 100 ml of isopropanol at room temperature with 2.4 g ( 42.9 mmol) and powdered potassium hydroxide while leaving about 1 hour in an ultrasonic bath. The suspension is cooled in an ice bath, filtered, washed with isopropanol and the undissolved salt, the filtrate was concentrated and distilled in a Kugelrohr oven at 150-230 ° C oven temperature and 0.7 mbar. Obtained 2.25 g (87.9% of theory) of a viscous oil which crystallizes. [Α] _D -21.3 ° (c = 0.92, CHC1 ₃₎ Accordingly, this reaction can be carried out in ethanol.

2 Method: A homosexual genie catalyzed mixture of (lR, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane dihydrobromide and 620 mg (11 mmol) of powdered potassium hydroxide is dry in a Kugelrohr apparatus at 0.2 mbar and increasing oven temperature to 250 ° C distilled. Obtained 490 mg (76.6% of theory) of (lR, 6R) -2 – oxa-5 ,8-diazabicyclo [4.3.0] nonane as a viscous oil which slowly crystallized.

3 Method: 100 g of moist, pretreated cation exchanger (Dowex 50WX, H ⁺ – form, 100-200 mesh, capacity: 5.1 meq / g of dry or 1.7 meq / mL) are charged into a column with about 200 ml 1 N HC1 activated and washed neutral with water 3 1. A solution of 2.9 g (10 mmol) of (lS, 6R)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane

Dihydrobromide in 15 ml of water is added to the ion exchanger, and then washed with 2 1 water, and eluted with approximately 1 1 1 N ammonia solution. The eluate is evaporated. concentrated. Yield: 1.3 g of a viscous oil (quantitative), DC (dichloromethane/methanol/17% NH ₃ 30:8:1): 1 HK, GC: 99.6% (area).

Example Z 18

(LS.6SV2-oxa-5.8-diazabicvclor4.3.01nonan

Z is analogous to Example 17 from (lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane-di-hydrobromide the free base (lS, 6S)-2-oxa-5 ,8-diazabicyclo [ 4.3.0] nonane made.

Example Z 19

2 – (2,4-dichloro-3-cyano-5-fluoro-benzoyl)-3-dimethylamino-acrylic acid ethyl ester

To a solution of 626 g (4.372 mol) of 3-dimethylamino-acrylate and 591 g (4.572 mol) of ethyl-diisopropyl-amine (Hunigs base) in 1060 ml of dichloromethane, a solution of 1075 g starting at room temperature 2,4-dichloro -3-cyano-5-fluoro-benzoyl chloride (94% pure, corresponding to 1010.5 g = 4.00 mol) was dropped in 850 ml of dichloromethane. The temperature rises to 50-55 ° C (dropwise addition about 90 minutes). Then stirred for 2 hours at 50 ° C and the reaction mixture was used without further purification in the next step.

Example Z 20

2 – (2,4-dichloro-3-Cyano-5-fluoro-benzoyl-3-cvclopropylamino-acrylate

To the reaction mixture from the above step 306 g (5.1 mol) of glacial acetic acid are added dropwise under cooling at about 15 ° C. Then, with further cooling at 10-15 ° C. 267.3 g (4.68 mol) of cyclopropyl amine is added dropwise. Immediately after which the reaction mixture is mixed with 1300 ml of water under ice-cooling and 15 minutes stirred well. The dichloromethane layer was separated and used in the next step.

Example 21 Z

7-chloro-8-cyano-1-cyclopropyl-6-fluoro-1.4-dihydro-4-oxo-3-chinolincarbonsäureethyl ester

To a heated to 60-70 ° C suspension of 353 g (2.554 mol) of potassium carbonate in 850 ml of N-methylpyrrolidone, the dichloromethane phase is dropped from the precursor (about 90 minutes). During the addition of the dichloromethane at the same time

Reaction mixture was distilled off. Then the reaction mixture for 5 Vz hours at 60-70 ° C is well stirred. The mixture is cooled to about 50 ° C. and distilled under a vacuum of about 250 mbar residual dichloromethane from. At room temperature is added dropwise 107 ml 30% hydrochloric acid under ice cooling, then to obtain a pH of 5-6 is set. Then, 2,200 ml of water are added under ice cooling. The reaction mixture is thoroughly stirred for 15 minutes, the solid was then filtered off and washed on the filter twice with 1000 ml of water and extracted three times with 1000 ml of ethanol and then dried in a vacuum oven at 60 ° C.

Yield: 1200 g (89.6% of theory).

This product can be purified, if desired by, the solid is stirred in 2000 ml of ethanol for 30 minutes at reflux. You filtered hot with suction, washed with 500 ml of ethanol and dried at 60 ° C in vacuum. Melting point: 180-182 ° C.

Η-NMR (400 MHz, CDC1 _3): d = 1.2 to 1.27 (m, 2H), 1.41 (t, 3H), 1.5-1.56 (m, 2H), 4, 1 to 4.8 (m, 1H), 4.40 (q, 2H), 8.44 (d, J = 8.2 Hz, H), 8.64 (s, 1H) ppm.

Example Z 22

7-chloro-8-cyano-1-cvclopropyl-6-fluoro-1 ,4-dihydro-4-oxo-3-quinolinecarboxylic acid

33.8 g (0.1 mol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylate dissolved in a mixture of 100 ml of acetic acid, 20 ml water and 10 ml concentrated sulfuric acid was heated for 3 hours under reflux. After cooling, the mixture is poured onto 100 ml of ice water, the precipitate filtered off, washed with water and ethanol and dried at 60 ° C in vacuum.

Yield: 29.6 g (96% of theory),

Mp 216-21 C. (with decomposition)

Example 1

A 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS.6S-2-oxa-5.8-diazabicvclo [4.3.0] non-8-yl – 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid

1.00 g (3.26 mmol) of 7-chloro-8-cyano-l-cyclopropyl-6-fluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are heated with 501 mg (3.91 mmol) of ( lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] nonane and 0.9 ml of triethylamine in 30 ml of acetonitrile was stirred at 40-45 ° C under argon for 25 hours. All volatile components in vacuo. removed and the residue recrystallized from ethanol. Yield: 1.22 g (94%)

Melting point: 294 ° C. (with decomposition)

B) ^{8-Cyano-l-cyclopropyl-6-fluoro-7-(‘(lS.6S-2-oxa-5} ,8-diazabicvclo [4.3.01nonan-8-YLV 1.4-dihydro-4-oxo-3- quinoline carboxylic acid Hvdrochlorid

200 mg (0.63 mmol) of 8-cyano-l-cyclopropyl-6 ,7-difluoro-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid ethyl ester to be 97 mg (0.75 mmol) of (lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] nonane and 0.17 ml of triethylamine in 3 ml of acetonitrile was stirred at 40-45 ° C for 2 hours under argon. All volatile components in vacuo. removed, the residue treated with water, insolubles filtered off and the filtrate was extracted with dichloromethane. The organic phase is dried over sodium sulfate and then concentrated under reduced pressure. a. The resulting residue is dissolved in 6 ml of tetrahydrofuran and 2 ml of water and 30 mg (0.72 mmol) of lithium hydroxide monohydrate was added. After 16 hours of stirring at room temperature, acidified with dilute hydrochloric acid and the resulting precipitate was filtered off with suction and dried. Yield: 155 mg (57%) Melting point:> 300 ° C

C) 8-Cyano-l-cvclopropyl-6-fluoro-7-((lS, 6S-2-oxa-5.8-diazabicvclo [4.3.01non-8 yiyi.4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride

1 g (2.5 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5 ,8-diazabicyclo [4.3.0] non-8-yl )-l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid is suspended in 20 ml of water was added to the suspension, 10 ml hydrochloric acid and stirred for In at room temperature for 3 hours. The resulting precipitate is filtered off, washed with ethanol and dried at 80 ° C under high vacuum.

Yield: 987 mg (90.6% of theory), Melting point: 314-316 ° C. (with decomposition).

D) 8-Cyano-l-cvclopropyl-6-fluoro-7-(iS, 6S)-2-oxa-5.8-diazabicyclo [4.3.0] non-8-YLV 1 ,4-dihydro-4-oxo-3 -quinoline carboxylic acid hydrochloride

86.4 g (217 mmol) of 8-cyano-l-cyclopropyl-6-fluoro-7-((lS, 6S)-2-oxa-5, 8 – diazabicyclo [4.3.0] non-8-yl) – l ,4-dihydro-4-oxo-3-quinolinecarboxylic acid are dissolved at room temperature in 963 ml of water and 239 ml of 1 N aqueous sodium hydroxide solution. After filtration and washing with 200 ml of water is added to 477 ml in aqueous hydrochloric acid and the precipitated crystals placed at 95 ° C to 100 ° C in solution. The solution is cooled overnight, the precipitated crystals are filtered off with suction and washed three times with 500 ml of water and dried in vacuum.

Yield 90 g (94.7% of theory), content:> 99% (area% by HPLC) 99.6% ee. [] _D ^23: -112 ° (c = 0.29, N NaOH).

……………….

Tetrahedron Lett 2009, 50(21): 2525

A novel approach to Finafloxacin hydrochloride (BAY35-3377)

Pages 2525-2528
Jian Hong, Zonghua Zhang, Huoxing Lei, Haiying Cheng, Yufang Hu, Wanliang Yang, Yinglin Liang, Debasis Das, Shu-Hui Chen, Ge Li

Graphical abstract

Finafloxacin hydrochloride, an important clinical compound was synthesized by a novel synthetic approach. An active intermediate ethyl 7-chloro-8-cyano-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 19 was prepared by a new route. The chiral (S,S′)-N-Boc 10 was derived from protected pyrrolidine and the absolute stereochemistry was established by X-ray analysis.

http://www.sciencedirect.com/science/article/pii/S0040403909005875

……………….

Durata Therapeutics, Inc. Finafloxacin for the treatment of cUTI and/or acute pyelonephritis. Available online: http://www.clinicaltrials.gov/ct2/show/NCT01928433 (accessed on 28 September 2013).
Merlion Pharma. A multi-dose, double-blind, double-dummy, active control, randomized clinical (Phase II) study of two dosing regimens of finafloxacin for the treatment of cUTI and/or acute pyelonephritis.Available online: http://www.clinicaltrialsregister.eu/ctr-search/trial/2011–006041–14/PL/ (accessed on 14 April 2013).
Pharma, M. FDA Grants Qualified Infectious Disease Product Designation and Fast Track Status for MerLion Pharma’s Lead Antibacterial Candidate Finafloxacin; Merlion Pharma: Singapore, 2013; Volume 2013.
Lemaire, S.; van Bambeke, F.; Tulkens, P.M. Activity of finafloxacin, a novel fluoroquinolone with increased activity at acid pH, towards extracellular and intracellular Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila. Int. J. Antimicrob. Agents 2011, 38, 52–59, doi:10.1016/j.ijantimicag.2011.03.002.
Finafloxacin hydrochlorideDrugs Fut 2009, 34(6): 451
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WO2011003091A1 *	2 Jul 2010	6 Jan 2011	Alcon Research, Ltd.	Compositions comprising finafloxacin and methods for treating ophthalmic, otic, or nasal infections
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GB2289674A *				Title not available

SUROTOMYCIN for Clostridium difficile-associated diarrhea

Phase 3 drug Comments Off

Mar 272014

Surotomycin

http://www.ama-assn.org/resources/doc/usan/surotomycin.pdf

N-[(2E)-3-(4-Pentylphenyl)-2-butenoyl]-D-tryptophyl-D-asparaginyl-N-[(3S,6S,9R,15S,18R,21S,24S,30S,31R)-3-[2-(2-aminophenyl)-2-oxoethyl]-24-(3-aminopropyl)-15,21-bis(carboxymethyl)-6-[(2R)-1-carboxy-2 -propanyl]-9-(hydroxymethyl)-18,31-dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28-nonaazacyclohentriacontan-30-yl]-L-α-asparagine

MOLECULAR FORMULA C77H101N17O26

MOLECULAR WEIGHT 1680.7

SPONSOR Cubist Pharmaceuticals, Inc.

CODE DESIGNATION CB-183,315

CB-315, CB-183315, CB-183,315

CAS REGISTRY NUMBER 1233389-51-9

U.S. – Fast Track (Treat Clostridium difficile-associated diarrhea (CDAD));
U.S. – Qualified Infectious Disease Program (Treat Clostridium difficile-associated diarrhea (CDAD))

Company	Cubist Pharmaceuticals Inc.
Description	Oral antibacterial lipopeptide
Therapeutic Modality	Macrocycle

Latest Stage of Development	Phase III
Standard Indication	Diarrhea (infectious)
Indication Details	Treat Clostridium difficile-associated diarrhea (CDAD)

EMEA……..

Name
P/0096/2013: EMA decision of 29 April 2013 on the agreement of apaediatric investigation plan and on the granting of a deferral for surotomycin (EMEA-001226-PIP01-11)

Surotomycin is an investigational oral antibiotic. This antibiotic is under investigation for the treatment of life-threatening Diarrhea, commonly caused by the bacteria Clostridium difficile.^[1]

CB-183315 is an investigational antibacterial drug candidate in phase III clinical trials at Cubist for the treatment of Clostridium difficile-associated diarrhea. It is a potent, oral, cidal lipopeptide. In 2012, Qualified Infectious Disease Product Designation was assigned in the U.S. for the treatment of clostridium difficile-associated diarrhea (CDAD).

Surotomycin (CB-315)

Surotomycin Overview

Surotomycin Fact Sheet

Surotomycin is an antibacterial lipopeptide discovered by Cubist scientists in our research laboratories in Lexington, Massachusetts. Surotomycin is both bactericidal against Clostridium difficile and more potent than vancomycin in vitro. Surotomycin stays at the site of infection in the bowel, with minimal systemic absorption and it does not interfere with normal bowel flora. Based on its features and its preclinical safety profile, Cubist filed an Investigational New Drug (IND) Application for surotomycin in December 2008.

Following safety and pharmacokinetic studies in healthy human volunteers, Cubist began a Phase 2 study in April 2010 to assess the safety and efficacy of surotomycin in patients with CDAD, in particular to assess its ability to reduce relapse rates. In this trial of 209 patients, two different doses of surotomycin were studied and compared with oral vancomycin. The higher dose demonstrated a high clinical cure rate as evidenced by resolution of diarrhea, comparable to oral vancomycin. The most interesting results in this study, however, relate to recurrence rates. The percent of patients who had an initial response to treatment but who subsequently had a recurrence or relapse was 36 percent in the oral vancomycin arm and was 17 percent in the surotomycin 250mg treatment group — about a 50% reduction in relapse rate, which was statistically significant. In this trial, 32% of patients were infected with the hypervirulent NAP-1 strain of C. difficile. The clinical response rate in the subset of patients infected with the NAP-1 strain was comparable across the surotomycin and oral vancomycin groups. Though not statistically significant, there was a modest reduction in the relapse rates in the subset of surotomycin patients infected with NAP-1 strains.

The ability to reduce relapses is important to both patients and health care providers. In the Phase 2 study we assessed the impact of surotomycin and oral vancomycin on normal bowel flora. Treatment with surotomycin had a very minimal impact on levels of Bacteroides, a key normal bowel bacterial species, compared to oral vancomycin which resulted in a marked depletion of stool levels of these bacteria during treatment. Why does this matter? The reason is — bowel flora like Bacteroides are critical in providing a competitive environment in the bowel that prevents C. difficile overgrowth. We believe that it is this difference in impact on normal bowel flora that helps explain the differences seen in recurrence rates following treatment with Surotomycin versus oral vancomycin.

Surotomycin’s Phase 3 program includes two identical global, randomized, double-blind, active-controlled, multi-center trials. The primary objective is to demonstrate non-inferiority of surotomycin versus the comparator, oral vancomycin, in clinical response at the end of treatment in adult subjects with CDAD, using a non-inferiority margin of 10%. We also have designed this trial to allow us to demonstrate that sustained clinical response to surotomycin at the end of the study is superior to oral vancomycin. Also, we will fully evaluate the safety of surotomycin in the study subjects.

In late 2012 Cubist received from the FDA a Qualified Infectious Disease Product (QIDP) designation for surotomycin. Additionally, in early 2013 Cubist was granted Fast track status for surotomycin. The QIDP designation and subsequent granting of Fast Track status was made possible by the GAIN Act, Title VIII (Sections 801 through 806) of the Food and Drug Administration Safety and Innovation Act. The GAIN Act provides pharmaceutical and biotechnology companies with incentives to develop new antibacterial and antifungal drugs for the treatment of life-threatening infectious diseases caused by drug resistant pathogens. Qualifying pathogens are defined by the GAIN Act to include multi-drug resistant Gram-negative bacteria, including Pseudomonas, Acinetobacter, Klebsiella, and Escherichia coli species; resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; multi-drug resistant tuberculosis; and Clostridium difficile.

About CDAD

CDAD is a disease caused by an overgrowth of, and subsequent toxin production by, C. difficile, a resident anaerobic spore-forming Gram-positive bacterium of the lower gastrointestinal tract. This overgrowth is caused by the use of antibiotics for the treatment of common community and hospital acquired infections (HAIs). Although they treat the underlying infection, many antibiotics disrupt the natural gut flora and allow C. difficile to proliferate. C. difficile produces enterotoxin and cytotoxin, which can lead to severe diarrhea, sepsis and even death. While most types of HAIs are declining, the infection caused by C. difficile remains at historically high levels. According to the latest data from the Centers for Disease Control, C. difficile continues to be the leading cause of death associated with gastroenteritis in the US. For CDAD alone, there was more than a five-fold increase in deaths between 1999 and 2007. C. difficile causes diarrhea linked to 14,000 American deaths each year. About 25% of C. difficile infections first show symptoms in hospital patients; 75% first show in nursing home patients or in people recently cared for in doctors’ offices and clinics. C. difficile infections cost at least $1 billion in extra health care costs annually.

ChemSpider 2D Image | Surotomycin | C77H101N17O26 SUROTOMYCIN

CB-183,315 is a cyclic lipopeptide antibiotic currently in Phase III clinical trials for the treatment of Clostridium difficile-associated disease (CDAD). As disclosed in International Patent Application WO 2010/075215, herein incorporated by reference in its entirety, CB-183,315 has antibacterial activity against a broad spectrum of bacteria, including drug-resistant bacteria and C. difficile. Further, the CB-183,315 exhibits bacteriacidal activity.

CB-183,315 (Figure 1) can be made by the deacylation of BOC-protected daptomycin, followed by acylation and deprotection as described in International Patent Application WO 2010/075215.

During the preparation and storage of CB-183,315, the CB-183,315 molecule can convert to structurally similar compounds as shown in Figures 2-4, leading to the formation of anhydro-CB-183,315 (Figure 3) and beta-isomer of CB-183,315 (“B- isomer CB183,315” in Figure 2). Accordingly, one measure of the chemical stability of CB- 183 ,315 is the amount of CB- 183 ,315 (Figure 1 ) present in the CB- 183 ,315 composition relative to the amount of structurally similar compounds including anhydro-CB-183,315 (Figure 3) and beta-isomer of CB-1 83,315 (Figure 2). The amount of CB-183,315 relative to the amount of these structurally similar compounds can be measured by high performance liquid chromatography (FIPLC) after reconstitution in an aqueous diluent (e.g., as described in Example 10). In particular, the purity of CB-183,315 and amounts of structurally similar compounds (e.g., Figures 2, 3 and 4) can be determined from peak areas obtained from HPLC (e.g., according to Example 10 herein), and measuring the rate of change in the amounts of CB-183,315 over time can provide a measure of CB-183,315 chemical stability in a solid form.

There is a need for solid CB-183,315 compositions with improved chemical stability in the solid form (i.e., higher total percent CB-183,315 purity over time), providing advantages of longer shelf life, increased tolerance for more varied storage conditions (e.g., higher temperature or humidity) and increased chemical stability.

……………..

WO2010075215A1

http://www.google.com/patents/WO2010075215A1?cl=en ………… copy paste link

Example 1

Preparation of N-{1 -[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl-D- asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D-alanyl-L-α- aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

1003 1004

Step 1 : Preparation of (E)-ethyl 3-(4-pentylphenyl) but-2-enoate (1002).

A mixture of commercially available 1-(4-pentylphenyl)ethanone (5 g, 26.3 mmol) and (ethoxycarbonylmethylene)-triphenylphosphorane (18.3 g, 52.5 mmol) was stirred at 150 ⁰C for 48 hours under a nitrogen atmosphere. The reaction mixture was cooled to ambient temperature and diluted with ethyl acetate (50 ml_) and petroleum ether (200 ml_). The suspension was filtered through a fritted funnel. The concentrated filtrate was purified by flash column chromatography with silica gel (petroleum ether : ethyl acetate = 80:1 ) to give the title compound (1.6 g) having the following physical data: ¹H NMR (300 MHz, δ, CDCI₃) 0.90 (br, 3H), 1.36 (br, 7), 1.63 (br, 2H), 2.58 (s, 3H), 2.63 (br, 2H), 4.22 (q, 2H), 6.15 (s, 1 H), 7.20 (d, 2H), 7.41 (d, 2H).

Step 2: Preparation of (E)-3-(4-pentylphenyl) but-2-enoic acid (1003).

A solution of compound 1002 (1.5 g, 5.77 mmol) in ethanol (50 ml_) and 3N potassium hydroxide (25 ml_) was stirred at 45 ⁰C for 3 hours. The reaction mixture was concentrated and the resulting residue was diluted with water (50 ml_). The aqueous solution was acidified to pH 2 with 1 N hydrochloric acid and extracted with EtOAc (2 * 30 ml_). The combined organic layers were dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (silica gel, petroleum ether : ethyl acetate = 10:1) to afford the title compound (0.95 g) having the following physical data: 1 H NMR (300 MHz, δ, CDCI3) 0.90 (br, 3H), 1.33 (br, 4H), 1.62 (br, 2H), 2.60 (br, 5H), 6.18 (s, 1 H), 7.18 (d, 2H), 7.42 (d, 2H).

Step 3: Preparation of (E)-3-(4-pentylphenyl)but-2-enoyl chloride (1004).

Oxalyl chloride (3.2 mL, 36.60 mmol) and DMF (50 μl_) were added drop wise to a solution of compound 1003 (5.0 g, 21.52 mmol) in dichloromethane (100 mL) at 0 ⁰C. The reaction solution was warmed up to room temperature and stirred for 4 hours. The reaction mixture was concentrated in vacuum and the residue was dried under hi-vacuum for 3 hours. The crude product was used in the next step without further purification.

Step 4: Preparation of N-{1 -[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl-D- asparaginyl-L-α-aspartyl-L-threonylglycyl-L-[(N-tert-butoxycarbonyl)-ornithyl]-L-α- aspartyl-D-alanyl-L-α-aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2- diamino-γ-oxobenzenebutanoic acid (13-→4)-lactone (1005).

Deacylated BOC-protected daptomycin (3.5Og, 2.23 mmol) and sodium bicarbonate (1.13 g, 61.0 mmol) were dissolved in THF (130 mL) and water (50 mL). The deacylated BOC-protected daptomycin sodium bicarbonate solution was cooled to 0 ⁰C. and a solution of compound 1004 (1.96 g, 7.82 mmol) in THF (20 mL) was then introduced. The reaction mixture was warmed to room temperature and stirred for 4 hours. The mixture was concentrated in vacuum to remove THF. The remaining aqueous solution was loaded on a C18 flash chromatography column (35mηn^χ 300mm, Bondesil HF C18 resin purchased from Varian). The column was first washed with water to remove salt and then with methanol to wash out product. Crude compound 1005 (3.46 g) was afforded as a white solid after removal of methanol. MS m/z 1780.8 (M + H)⁺.

Steps 5-6: Preparation of N-{1-[(E)-3-(4-pentylphenyl)but-2-enoyl]}-L-tryptophyl- D-asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D-alanyl-L-α- aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

TFA (10 ml_) was added to a solution of compound 1005 (3.46 g) in DCM (50 mL) at room temperature. The reaction mixture was stirred vigorously for 45 minutes and added slowly to vigorously stirring diethyl ether (100 mL). The resulting yellow precipitation was collected by filtration. The crude product was purified by Preparative HPLC to afford the TFA salt of compound 6 (0.75 g). MP carbonate resin (purchased from Biotage) was added to the solution of compound 6 TFA salt (0.70 g, 0.39 mmol) in anhydrous methanol (30.0 mL). The mixture was stirred at room temperature for 4 hours. The resins were removed by filtration and rinsed with methanol. The methanol solution was concentrated under vacuum to give product as off-white solid (408 mg). MS m/z 1680.7 (M + H)⁺.

Example 1 b

Alternative preparation of N-{1-[(E)-3-(4-pentylphenyl)but-2-enoyl]}- L-tryptophyl-D-asparaginyl-L-α-aspartyl-L-threonylglycyl-L-ornithyl-L-α-aspartyl-D- alanyl-L-α-aspartylglycyl-D-seryl-(3R)-3-methyl-L-α-glutamyl-(αS)-α,2-diamino-γ- oxobenzenebutanoic acid (13→4)-lactone (49).

daptomycin,

1003

A solution of (E)-3-(4-pentylphenyl)but-2-enoic acid (1 100 g, 4.73 mol), Λ/-Ethyl-Λ/’-(3-dimethylaminopropyl)carbodiimide hydrochloride (907 g, 4.73 mol), HOBT (640 g, 4.73 mol) and 4-(dimethylamino)pyridine (22 g, 0.18 mol) in DMF (11 L) was stirred at room temperature for 4 hours at which point the activation of the (E)-3-(4-pentylphenyl)but-2-enoic acid was deemed complete by HPLC.

This reaction mixture was added to a suspension of Deacylated BOC- protected daptomycin (2600 g, 1.66 mol), sodium bicarbonate (804 g, 9.57 mol) in water (11.25 L) and 1 ,4-dioxane (33.75 L). The mixture was stirred at room temperature for 2.5 hours at which time HPLC indicated complete consumption of Deacylated BOC-protected daptomycin. The reaction mixture was diluted with water (22.5 L) and cooled with an ice bath. Concentrated hydrochloric acid (5.25 L) was added while maintaining the internal temperature below 30 ⁰C. After the addition, the solution was stirred at room temperature for 5 days at which time HPLC indicated complete consumption of the Boc protected intermediate.

The reaction mixture was washed with methyl terf-butyl ether (90 L then approximately 60 L then approximately 45 L then approximately 45 L) to remove 1 ,4-dioxane. The remaining solution (approximately 44 L) was adjusted to pH 2.69 with 2N sodium hydroxide (11.3 L) and water (53.4 L). This material was processed by Tangential Flow Filtration (TTF) with a 1 K membrane until the total volume was reduced to 54 L.Water (120 L) was added in two portions and the solution was concentrated to 52 L by continued TTF. The aqueous solution (30 L of 52 L) was purified by chromatography using the following protocol: The aqueous solution was brought to three times of its volume (30 L→90l_) with 20% IPA in aqueous ammonium acetate solution (50 mM). The diluted solution was applied to a 38 L HP20SS resin column at 1.5 L/min. The column was eluted with IPA solution in aqueous 50 mM ammonium acetate (25%→30%→35%, 60 L each concentration).

Fractions (approximately 11 L) were collected and analyzed by HPLC. The fractions with HPLC purity less than 80% were combined and purified again using the same method. The key fractions from both chromatographic separations (with HPLC purity >80%) were combined and acidified with concentrated HCI to pH 2-3. The resulting solution was desalted on an ion exchange column (HP20SS resin, 16 L) which was eluted with WFI (until conductivity = 4.8 μS) followed by IPA in WFI (36 L 10%→ 40 L 60%). The yellow band which was eluted with 60% IPA (approximately 19L) was collected, adjusted to pH 2-3 with concentrated HCI and lyophilized to yield 636.5 g of Compound 49 (HPLC purity of 87.0%). MS m/z 1680.7 (M + H)⁺.

……………………………..

see formulation

WO2012162567A1

May 24, 2012

Nov 29, 2012

Cubist Pharmaceuticals, Inc.

Cb-183,315 compositions and related methods

References

http://www.cubist.com/downloads/Surotomycin-Fact-Sheet-13013.pdf
1. Cubist Pharmaceuticals. Cubist products and pipeline. Available online: http://www.cubist.com/products/(accessed on 15 April 2013).
2. Cubist Pharmaceuticals. Study of CB-183,315 in patients with Clostridium difficile associated diarrhea.Available online: http://www.clinicaltrials.gov/ct2/show/NCT01597505 (accessed on 15 April 2013).
3. Cubist Pharmaceuticals. A study of CB-183,315 in patients with Clostridium difficile associated diarrhea.Available online: http://www.clinicaltrials.gov/ct2/show/NCT01598311 (accessed on 15 April 2013).
4. Mascio, C.T.M.; Mortin, L.I.; Howland, K.T.; van, P.A.D.G.; Zhang, S.; Arya, A.; Chuong, C.L.; Kang, C.; Li, T.; Silverman, J.A. In vitro and in vivo characterization of CB-183,315, a novel lipopeptide antibiotic for treatment of Clostridium difficile. Antimicrob. Agents Chemother. 2012, 56, 5023–5030, doi:10.1128/AAC.00057-12.
5. WO2012162567A1 May 24, 2012 Nov 29, 2012 Cubist Pharmaceuticals, Inc. Cb-183,315 compositions and related methods

WO2001097851A2 *	Jun 18, 2001	Dec 27, 2001	Cubist Pharm Inc	Compositions and methods to improve the oral absorption of antimicrobial agents
WO2010075215A1	Dec 18, 2009	Jul 1, 2010	Cubist Pharmaceuticals, Inc.	Novel antibacterial agents for the treatment of gram positive infections
WO2011063419A2 *	Nov 23, 2010	May 26, 2011	Cubist Pharmaceuticals Inc.	Lipopeptide compositions and related methods

LESINURAD

Phase 3 drug Comments Off

Mar 162014

ChemSpider 2D Image | Lesinurad sodium | C17H13BrN3NaO2S

Lesinurad

Acetic acid, 2-[[5-bromo-4-(4-cyclopropyl-1-naphthalenyl)-4H-1,2,4-triazol-3-yl]thio]-,
sodium salt (1:1)
Sodium 2-{[5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-
yl]sulfanyl}acetate

2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid

MOLECULAR FORMULA C17H13BrN3NaO2S

MOLECULAR WEIGHT 426.3

http://clinicaltrials.gov/show/NCT01508702

http://www.ama-assn.org/resources/doc/usan/lesinurad.pdf

Ardea Biosciences, Inc.

Lesinurad
RDEA 594
RDEA594
UNII-09ERP08I3W

Gout phase 3

Gout is associated with elevated levels of uric acid that crystallize and deposit in joints, tendons, and surrounding tissues. Gout is marked by recurrent attacks of red, tender, hot, and/or swollen joints.

This study will assess the serum uric acid lowering effects and safety of lesinurad compared to placebo in patients who are intolerant or have a contraindication to allopurinol or febuxostat.

http://euroscan.org.uk/technologies/technology/view/2386

Lesinurad (RDEA-594, lesinurad sodium) is a selective urate transporter-1 (URAT-1) inhibitor, which blocks the reabsorption of urate within the renal proximal tubule. It is intended for the treatment of gout after failure of first line therapy and is administered orally at 400mg once daily

A Phase 3 Randomized, Double-Blind, Multicenter, Placebo- Controlled Study to Assess the Efficacy and Safety of Lesinurad Monotherapy Compared to Placebo in Subjects With Gout and an Intolerance or Contraindication to a Xanthine Oxidase Inhibitor

AstraZeneca’s lesinurad (formerly known as RDEA-594) is a selective oral Uric Acid Transporter URAT1 inhibitor currently in Phase III development for the treatment of of gout. The regulatory filings for lesinurad in the US and Europe are expected for the first half of 2014.

Synthesis of Lesinurad (RDEA-594), AstraZeneca’s potential blockbuster drug for gout 阿斯利康痛风试验药物Lesinurad的合成

Gout (also known as podagra when it involves the big toe), while not life-threatening, is an excruciatingly painful condition caused by a buildup of a waste product in the blood called uric acid, which is normally eliminated from the body through urine. Excess Uric acid crystallizes and get deposited in the joints (usually the big toes), creating symptoms similar to an acute arthritis flare. Gout has seen a recent gradual resurgence as a result of rising obesity rates and poor diet according to a study in the journal Annals of the Rheumatic Diseases.

The current Standard treatment for gout works by inhibiting a protein called xanthine oxidase that helps in the formation of the uric acid. These therapies, some of which have been used for more than 50 years, are not effective in all patients. One is a generic drug called allopurinol that was approved in the U.S. in 1966. The other is febuxostat, marketed by Takeda Pharmaceutical Co. in the U.S. asUloric and by Ipsen SA and others in Europe as Adenuric and approved in the U.S. in 2009.

AstraZeneca’s new product Lesinurad, a selective uric acid re-absorption inhibitor (SURI), tackles gout by blocking a protein called Uric acid trasporter 1 (URAT1) that otherwise would cause the body to reabsorb the uric acid. AstraZeneca acquired lesinurad (aka RDEA-594) as part of its $1.26 billion takeouver of San Diego-based Ardea Biosciences in 2012. RDEA594 is a metabolite of RDEA806, a non-nucleoside reverse transcriptase inhibitor originally developed for HIV.

In top-line results from a Phase III LIGHT study released by AstraZeneca in December 2013 on gout patients who get no benefit from Zyloprim (allopurinol) and febuxostat, lesinurad alone significantly reduced serum levels of uric acid. The company has three other phase III studies ongoing that are testing the use of the drug alongside allopurinol and febuxostat, and these should generate results in the middle of 2014. Analysts at JPMorgan Chase forecast lesinurad alone may have peak sales of $1 billion a year. AstraZeneca also has a second, more potent drug called RDEA3179 to treat elevated levels of uric acid or hyperuricemia. Pfizer’s KUX-1151, licensed from Japan’s Kissei Phmarceuticals, is in early stage development.

Gout is not an automatic success indication of drugmakers. Savient Pharmaceuticals filed for Chapter 11 bankruptcy in October 2013 in the face of a severe cash crisis, having spent hundreds of millions of dollars on its would-be flagship — the gout-fighting drug Krystexxa (pegloticase) — with limited results. Krystexxa (pegloticase), a twice-monthly infusion designed to treat severe chronic gout that doesn’t respond to conventional therapy, was approved by the U.S. Food and Drug Administration in September 2010. Crealta Pharmaceuticals acquired Savient for $120.4 million in December 2013.

Synthesis of Lesinurad (RDEA-594), AstraZeneca’s potential blockbuster drug for gout 阿斯利康痛风试验药物Lesinurad的合成

Lesinurad
RDEA-594
2-{[5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetic acid
CAS number: 878672-00-5 (Lesinurad), 1151516-14-1 (Lesinurad sodium)
Mechanism of Action:once-daily inhibitor of URAT1, a transporter in the kidney that regulates uric acid excretion from the body
US patents:US8242154 , US8173690, US808448
Indication: Gout
Developmental Status: Phase III (US, UK, EU)
Originator: Ardea Biosciences (Acquired by AstraZeneca for $1.26 billion in 2012)
Developer: AstraZeneca

…………………………

http://www.google.co.in/patents/US8242154

Example 8 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid

Sodium hydroxide solution (2M aqueous, 33.7 mL, 67 mmol, 2 eq) was added to a suspension of 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)-N-(2-chloro-4-sulfamoylphenyl)acetamide (prepared by previously published procedures; 20 g, 34 mmol) in ethanol (200 mL) and the mixture heated at reflux for 4 hours. Charcoal (10 g) was added, the mixture stirred at room temperature for 12 hours and the charcoal removed by filtration. The charcoal was washed several times with ethanol and the filtrate then concentrated. Water (200 mL) was added and then concentrated to approx. one third volume, to remove all ethanol. Water (200 mL) and ethyl acetate (250 mL) were added, the mixture stirred vigorously for 15 mins and the organic layer removed. The aqueous layer was cooled to 0° C. and acidified by treatment with HCl (1N) resulting in the formation of a cloudy oily precipitate. The mixture was extracted with ethyl acetate (3×) and the combined organic extracts dried over sodium sulfate and concentrated to give 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid as an off white solid (11.2 g, 82%).

Example 102 Methyl 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetate

Cyclopropylmagnesium bromide (150 mL, 0.5M in tetrahydrofuran) was slowly added to a solution of 1-bromonaphthalene (10 g, 50 mmol) and [1,3-bis(diphenylphosphino)propane]dichloro nickel (II) in tetrahydrofuran (10 mL) stirred at 0° C., and the reaction mixture stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and ethyl acetate and aqueous ammonium chloride were added. After extraction, the organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-cyclopropylnaphthalene (6.4 g, 76%).

Sodium nitrite (30 mL) was slowly added (over 2 hours) to 1-cyclopropylnaphthalene (6.4 g, 38 mmol) stirred at 0° C. The reaction mixture was stirred at 0° C. for an extra 30 min and then slowly poured into ice. Water was added, followed by ethyl acetate. After extraction, the organic layer was washed with aqueous sodium hydroxide (1%) and water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-cyclopropyl-4-nitronaphthalene (5.2 g, 64%).

A solution of 1-cyclopropyl-4-nitronaphthalene (5 g, 23 mmol) in ethanol (200 mL) was stirred under hydrogen in the presence of Pd/C (10% net, 1.8 g). The reaction mixture was shaken overnight, filtered over celite, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-amino-4-cyclopropylnaphthalene (3.1 g, 73%).

Thiophosgene (1.1 g, 9.7 mmol) was added to a stirred solution of 1-amino-4-cyclopropylnaphthalene (1.8 g, 9.7 mmol) and diisopropylethylamine (2 eq) in dichloromethane (50 mL) at 0° C. The reaction mixture was stirred for 5 min at 0° C. and then aqueous HCl (1% solution) was added. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and the solvent removed under reduced pressure. Hexane was added, and the resulting precipitate was filtered. The solvent was evaporated to yield 1-cyclopropyl-4-isothiocyanatonaphthalene (1.88 g, 86%).

A mixture of aminoguanidine hydrochloride (3.18 g, 29 mmol), 1-cyclopropyl-4-isothiocyanatonaphthalene (3.24 g, 14 mmol) and diisopropylethylamine (3 eq) in DMF (20 mL) was stirred at 50° C. for 15 hours. The solvent was removed under reduced pressure, toluene added, and the solvent was evaporated again. Sodium hydroxide solution (2M, 30 mL) was added and the reaction mixture heated at 50° C. for 60 hours. The reaction mixture was filtered and the filtrate neutralized with aqueous HCl (2M). The mixture was re-filtered and the solvent removed under reduced pressure. The residue was purified by silica gel chromatography to yield 5-amino-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazole-3-thiol (2.0 g, 49%).

Methyl 2-chloroacetate (0.73 mL, 8.3 mmol) was added dropwise over 5 mins to a suspension of 5-amino-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazole-3-thiol (2.24 g, 7.9 mmol) and potassium carbonate (1.21 g, 8.7 mmol) in DMF (40 mL) at room temperature. The reaction was stirred at room temperature for 24 h and slowly poured into a stirred ice-cold water solution. The tan precipitate was collected by vacuum filtration and dried under high vacuum at 50° C. for 16 h in the presence of P₂O₅to yield methyl 2-(5-amino-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetate (2.24 g, 80%).

Sodium nitrite (2.76 g, 40 mmol) was added to a solution of methyl 2-(5-amino-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetate (0.71 g, 2 mmol) and benzyltriethylammonium chloride (1.63 g, 6 mmol) in bromoform (10 mL). Dichloroacetic acid (0.33 mL, 4 mmol) was then added and the reaction mixture stirred at room temperature for 3 h. The mixture was directly loaded onto a 7-inch column of silica gel, packed with dichloromethane (DCM). The column was first eluted with DCM until all bromoform eluted, then eluted with acetone/DCM (5:95) to give methyl 2-(5-bromo-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetate (713 mg, 85%).

Example 104 Sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetate

Aqueous sodium hydroxide solution (1M, 2.0 mL, 2.0 mmol) was added dropwise over 5 mins to a solution of 2-(5-bromo-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid (810 mg, 2.0 mmol) in ethanol (10 mL) at 10° C. The mixture was stirred at 10° C. for a further 10 mins. Volatile solvents were removed in vacuo to dryness to provide sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)acetate as a solid (850 mg, 100%).

Example 103 2-(5-Bromo-4-(1-cyclopropylnapthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid

A solution of lithium hydroxide (98 mg, 4.1 mmol) in water (10 mL) was added dropwise over 5 mins to a solution of methyl 2-(5-bromo-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetate (prepared as described in example 1 above; 1.14 g, 2.7 mmol) in ethanol (10 mL) and THF (10 mL) at 0° C. The mixture was stirred at 0° C. for a further 45 mins and then neutralized to pH 7 by the addition of 0.5N HCl solution at 0° C. The resulting mixture was concentrated in vacuo to ⅕th of its original volume, then diluted with water (˜20 mL) and acidified to pH 2-3 by the addition of 0.5N HCl to produce a sticky solid. (If the product comes out as an oil during acidification, extraction with DCM is recommended.) The tan solid was collected by vacuum filtration and dried under high vacuum at 50° C. for 16 h in the presence of P₂O₅to yield 2-(5-bromo-4-(1-cyclopropylnaphthalen-4-yl)-4H-1,2,4-triazol-3-ylthio)acetic acid (1.02 g, 93%).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.84-0.91 (m, 2 H) 1.12-1.19 (m, 2 H) 2.54-2.61 (m, 1 H) 3.99 (d, J = 1.45 Hz, 2 H) 7.16 (d, J = 7.88 Hz, 1 H) 7.44 (d, J = 7.46 Hz, 1 H) 7.59-7.70 (m, 2 H) 7.75 (td, J = 7.62, 1.14 Hz, 1 H) 8.59 (d, J = 8.50 Hz, 1 H) 12.94 (br. s., 1 H)

Mass found: 404.5 (M + 1)

……

POLYMORPHS AND SYNTHESIS

WO2011085009A2

Described herein are various polymorphic, crystalline and mesophase forms of sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4-triazol-3-ylthio)acetate which decreases uric acid levels, (see for example US patent publication 2009/0197825, US patent publication 2010/0056464 and US patent publication 2010/0056465). Details of clinical studies involving sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4- triazol-3-ylthio)acetate have been described in International patent application

PCT/US2010/052958.

Polymorph Form A

In one embodiment, sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H- l,2,4-triazol-3-ylthio)acetate polymorph Form A exhibits an x-ray powder diffraction pattern characterized by the diffraction pattern summarized in Table 1 A or Table IB. In some embodiments, provided herein is a polymorph of sodium 2-(5-bromo-4-(4- cyclopropylnaphthalen-l-yl)-4H-l,2,4-triazol-3-ylthio)acetate comprising at least 3 peaks of (±0.1°2Θ) of Table 1A or IB. In certain embodiments, provided herein is a polymorph of sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4-triazol-3-ylthio)acetate comprising at least 4 peaks of (±0.1°2Θ) of Table 1A or IB, at least 5 peaks of (±0.1°2Θ) of Table 1A or IB, at least 6 peaks of (±0.1°2Θ) of Table 1A or IB, at least 8 peaks of

(±0. Γ2Θ) of Table 1A or IB, at least 10 peaks of (±0. Γ2Θ) of Table 1A, at least 15 peaks of (±0. Γ2Θ) of Table 1A, at least 20 peaks of (±0. Γ2Θ) of Table 1A, at least 25 peaks of (±0.1 °2Θ) of Table 1A, or at least 30 peaks of (±0.1 °2Θ) of Table 1A.

Examples

I Preparation of compounds

Example 1: Preparation of sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4- triazol-3-ylthio)acetate

Sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen- 1 -yl)-4H- 1 ,2,4-triazol-3-ylthio)acetate was prepared according to previously described procedures (see US patent publication

2009/0197825) and as outlined below.

[00103] Aqueous sodium hydroxide solution (1M, 2.0 mL, 2.0 mmol) was added dropwise over 5 min to a solution of 2-(5-bromo-4-(l-cyclopropylnaphthalen-4-yl)-4H- l,2,4-triazol-3-ylthio)acetic acid (810 mg, 2.0 mmol) in ethanol (10 mL) at 10 °C. The mixture was stirred at 10 °C for a further 10 min. Volatile solvents were removed in vacuo to dryness to provide sodium 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4- triazol-3-ylthio)acetate as a solid (850 mg, 100%).

Example 2: Preparation of 2-(5-Bromo-4-(4-cyclopropylnaphthalen- 1 -yl)-4H- 1 ,2,4-triazol- 3-ylthio)acetic acid

2-(5-Bromo-4-(4-cyclopropylnaphthalen- 1 -yl)-4H- 1 ,2,4-triazol-3-ylthio)acetic acid was prepared according to previously described procedures (see US patent publication

2009/0197825) and as outlined below.

[00104] Route i:

Sodium hydroxide solution (2M aqueous, 33.7 mL, 67 mmol, 2 eq) was added to a suspension of 2-(5-bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4-triazol-3-ylthio)-N- (2-chloro-4-sulfamoylphenyl)acetamide (prepared by previously published procedures, see US 2009/0197825; 20 g, 34 mmol) in ethanol (200 mL) and the mixture heated at reflux for 4 hours. Charcoal (10 g) was added, the mixture stirred at room temperature for 12 hours and the charcoal removed by filtration. The charcoal was washed several times with ethanol and the filtrate then concentrated. Water (200 mL) was added and then concentrated to approx. one third volume to remove all ethanol. Water (200 mL) and ethyl acetate (250 mL) were added, the mixture stirred vigorously for 15 min and the organic layer removed. The aqueous layer was cooled to 0 °C and acidified by treatment with HCl (IN) resulting in the formation of a cloudy oily precipitate. The mixture was extracted with ethyl acetate (3x) and the combined organic extracts dried over sodium sulfate and concentrated to give 2-(5- bromo-4-(4-cyclopropylnaphthalen-l-yl)-4H-l,2,4-triazol-3-ylthio)acetic acid as an off white solid (11.2 g, 82%).

[00105] Route ii:

STEP A: 1-Cyclopropylnaphthalene

Cyclopropylmagnesium bromide (150 mL, 0.5M in tetrahydrofuran) was slowly added to a solution of 1-bromonaphthalene (10 g, 50 mmol) and [l,3-bis(diphenylphosphino)propane] dichloro nickel (II) in tetrahydrofuran (10 mL) stirred at 0 °C, and the reaction mixture stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and ethyl acetate and aqueous ammonium chloride were added. After extraction, the organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-cyclopropylnaphthalene (6.4 g, 76%). ] STEP B: l-Cyclopropyl-4-nitronaphthalene

Sodium nitrite (30 mL) was slowly added (over 2 hours) to 1-cyclopropylnaphthalene (6.4 g, 38 mmol) stirred at 0 °C. The reaction mixture was stirred at 0 °C for an extra 30 min and then slowly poured into ice. Water was added, followed by ethyl acetate. After extraction, the organic layer was washed with aqueous sodium hydroxide (1%) and water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield l-cyclopropyl-4-nitronaphthalene (5.2 g, 64%).

[00108] STEP C: l-Amino-4-cyclopropylnaphthalene

A solution of l-cyclopropyl-4-nitronaphthalene (5 g, 23 mmol) in ethanol (200 mL) was stirred under hydrogen in the presence of Pd/C (10% net, 1.8 g). The reaction mixture was shaken overnight, filtered over celite, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield l-amino-4-cyclopropylnaphthalene (3.1 g, 73%).

STEP D: l-Cyclopropyl-4-isothiocvanatonaphthalene

Thiophosgene (1.1 g, 9.7 mmol) was added to a stirred solution of l-amino-4- cyclopropylnaphthalene (1.8 g, 9.7 mmol) and diisopropylethylamine (2 eq) in

dichloromethane (50 mL) at 0 °C. The reaction mixture was stirred for 5 min at 0 °C and then aqueous HCl (1% solution) was added. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and the solvent removed under reduced pressure. Hexane was added, and the resulting precipitate was filtered. The solvent was evaporated to yield l-cyclopropyl-4-isothiocyanatonaphthalene (1.88 g, 86%>).

[00110] STEP E: 5-Amino-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazole-3- thiol

A mixture of aminoguanidine hydrochloride (3.18 g, 29 mmol), l-cyclopropyl-4- isothiocyanato naphthalene (3.24 g, 14 mmol) and diisopropylethylamine (3 eq) in DMF (20 mL) was stirred at 50 °C for 15 hours. The solvent was removed under reduced pressure, toluene added, and the solvent was evaporated again. Sodium hydroxide solution (2M, 30 mL) was added and the reaction mixture heated at 50 °C for 60 hours. The reaction mixture was filtered and the filtrate neutralized with aqueous HCl (2M). The mixture was re-filtered and the solvent removed under reduced pressure. The residue was purified by silica gel chromatography to yield 5-amino-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazole-3- thiol (2.0 g, 49%). [00111] STEP F: Methyl 2-(5-amino-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4- -3 -ylthio)acetate

Methyl 2-chloroacetate (0.73 mL, 8.3 mmol) was added dropwise over 5 min to a suspension of 5-amino-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazole-3-thiol (2.24 g, 7.9 mmol) and potassium carbonate (1.21 g, 8.7 mmol) in DMF (40 mL) at room

temperature. The reaction was stirred at room temperature for 24 h and slowly poured into a stirred ice-cold water solution. The tan precipitate was collected by vacuum filtration and dried under high vacuum at 50 °C for 16 h in the presence of P2O5 to yield methyl 2-(5- amino-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazol-3-ylthio)acetate (2.24 g, 80%).

[00112] STEP G: Methyl 2-(5-bromo-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4- triazol-3 -ylthio)acetate

Sodium nitrite (2.76 g, 40 mmol) was added to a solution of methyl 2-(5-amino-4-(l- cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazol-3-ylthio)acetate (0.71 g, 2 mmol) and benzyltriethylammonium chloride (1.63 g, 6 mmol) in bromoform (10 mL). Dichloroacetic acid (0.33 mL, 4 mmol) was then added and the reaction mixture stirred at room

temperature for 3 h. The mixture was directly loaded onto a 7-inch column of silica gel, packed with dichloromethane (DCM). The column was first eluted with DCM until all bromoform eluted, then eluted with acetone/DCM (5:95) to give methyl 2-(5-bromo-4-(l- cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazol-3-ylthio)acetate (713 mg, 85%).

[00113] STEP H: 2-(5-Bromo-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazol-3- )acetic acid

A solution of lithium hydroxide (98 mg, 4.1 mmol) in water (10 mL) was added dropwise over 5 min to a solution of methyl 2-(5-bromo-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4- triazol-3-ylthio)acetate (1.14 g, 2.7 mmol) in ethanol (10 mL) and THF (10 mL) at 0 °C. The mixture was stirred at 0 °C for a further 45 min and then neutralized to pH 7 by the addition of 0.5N HC1 solution at 0 °C. The resulting mixture was concentrated in vacuo to l/5th of its original volume, then diluted with water (~20 mL) and acidified to pH 2-3 by the addition of 0.5N HC1 to produce a sticky solid. (If the product comes out as an oil during acidification, extraction with dichloromethane is recommended.) The tan solid was

collected by vacuum filtration and dried under high vacuum at 50 °C for 16 h in the

presence of P₂O₅ to yield 2-(5-bromo-4-(l-cyclopropylnaphthalen-4-yl)-4H-l,2,4-triazol-3- ylthio)acetic acid (1.02 g, 93%).

………………………….

US20100081827

EXAMPLES

The following experiments are provided only by way of example, and should not be understood as limiting the scope of the invention.

COMPOUNDS OF THE INVENTION 2-[5-Bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide (Method A)

1-Cyclopropyl-naphthalene

Cyclopropylmagnesium bromide (150 mL, 0.5 M in tetrahydrofuran) was slowly added to a solution of 1-bromo-naphthalene (10 g, 50 mmol) and [1,3-bis(diphenylphosphino)propane]dichloronickel(II) in tetrahydrofuran (10 mL) stirred at 0° C. The reaction mixture was stirred at room temperature for 16 hours and the solvent was evaporated under reduced pressure. EtOAc and ammonium chloride in water were added. After extraction, the organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-cyclopropyl-naphthalene (6.4 g, 76%).

1-Cyclopropyl-4-nitro-naphthalene

Sodium nitrite (30 mL) was slowly added (over 2 hours) to 1-cyclopropyl-naphthalene (6.4 g, 38 mmol) stirred at 0° C. The reaction mixture was stirred at 0° C. for an extra 30 min and then was slowly poured into ice. Water was added, followed by EtOAc. After extraction, the organic layer was washed with a 1% aqueous solution of NaOH, then washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 1-cyclopropyl-4-nitro-naphthalene (5.2 g, 64%).

1-Amino-4-cyclopropyl-naphthalene

A solution of 1-cyclopropyl-4-nitro-naphthalene (5 g, 23 mmol) in ethanol (200 mL) was stirred under hydrogen in the presence of Pd/C (10% net, 1.8 g). The reaction mixture was shaken overnight, then filtered over celite. The solvent was evaporated, and the residue was purified by silica gel chromatography to yield 1-amino-4-cyclopropyl-naphthalene (3.1 g, 73%).

1-Cyclopropyl-4-isothiocyanato-naphthalene

Thiophosgene (1.1 g, 9.7 mmol) was added to a solution of 1-amino-4-cyclopropyl-naphthalene (1.8 g, 9.7 mmol) and diisopropylethylamine (2 eq) in dichloromethane (50 mL) stirred at 0° C. The reaction mixture was stirred for 5 min at this temperature, then a 1% solution of HCl in water was added and the organic layer was separated, washed with brine, dried over sodium sulfate, filtered and the solvent was evaporated under reduced pressure. Hexane was added, and the resulting precipitate was filtered. The solvent was evaporated to yield 1-cyclopropyl-4-isothiocyanatonaphthalene (1.88 g, 86%).

5-Amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazole-3-thiol

A mixture of aminoguanidine hydrochloride (3.18 g, 29 mmol), 1-cyclopropyl-4-isothiocyanato-naphthalene (3.24 g, 14 mmol) and diisopropylethylamine (3 eq) in DMF (20 mL) was stirred at 50° C. for 15 hours. The solvent was evaporated, toluene was added, and the solvent was evaporated again. A 2.0 M aqueous solution of sodium hydroxide (30 mL) was added and the reaction mixture was heated at 50° C. for 60 hours. The reaction mixture was filtered, and the filtrate was neutralized with a 2.0 M aqueous solution of HCl. New filtration, then evaporation of solvent and purification of the residue by silica gel chromatography to yield 5-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazole-3-thiol (2.0 g, 49%).

2-[5-Amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)Acetamide

In a solution of 5-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazole-3-thiol (708 mg, 2.5 mmol), K₂CO₃(380 mg, 2.5 mmol) in DMF (20 mL) was added 2-chloro-N-(2-chloro-4-sulfamoylphenyl)acetamide (710 mg, 2.5 mmol). The reaction mixture was stirred at room temperature overnight. Upon completion of the reaction, the solvent was evaporated. The residue was purified by silica gel chromatography to yield 2-[5-Amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide (1.26 g, 95%).

2-[5-Bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide

Dichloroacetic acid (180 uL, 2.2 mmol) was added to a suspension of 2-[5-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide (0.59 g, 1.1 mmol), sodium nitrite (1.5 g, 22 mmol) and BTEABr (0.91 g, 3.3 mmol) in dibromomethane (30 mL). The reaction mixture was stirred at room temperature for 4 hours, then extracted with dichloromethane and sodium bicarbonate in water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to yield 2-[5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide (224 mg, 31%).

2-[5-Bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazole-3-ylsulfanyl]-N-(2-chloro-4-sulfamoylphenyl)acetamide (Method B)

2-[5-Amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetic acid methyl ester


Materials	Amount	Mol. Wt.	mmoles


thiotriazole	2.24	g	282.36	7.9
methyl chloroacetate	0.73	ml	108.52	8.3 (1.05 eq)
potassium carbonate	1.21	g	138.21	8.7 (1.1 eq)
dimethylformamide	40	ml		(5 mL/mmol)

Procedure:

To a suspension of thiotriazole and potassium carbonate in DMF was added methyl chloroacetate dropwise at room temperature for 5 min. The reaction was stirred at room temperature for 24 h and slowly poured into a stirred ice-cold water solution. The tan precipitate was collected by vacuum filtration and dried under high vacuum at 50° C. for 16 h in the presence of P₂O₅to yield 2.24 g (80%) of the title compound.

2-[5-Bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetic acid methyl ester


Materials	Amount	Mol. Wt.	mmoles


thiotriazole L10183-58	709	mg	354.43	2.0
bromoform	10	ml		(5 ml/mmol)
sodium nitrite	2.76	g	69.00	40	(20 eq)
benzyltriethylammonium	1.63	g	272.24	6.0	(3 eq)
bromide
dichloroacetic acid	0.33	ml	128.94	4.0	(2 eq)

Procedure:

To a solution of 2-[5-amino-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetic acid methyl ester and benzyltriethylammonium chloride in bromoform was added sodium nitrite. To the mixture was added dichloroacetic acid and the reaction mixture was stirred at room temperature for 3 h. The mixture was directly loaded onto a 7-inch column of silica gel that was packed with CH₂Cl₂. The column was first eluted with CH₂Cl₂until all CHBr₃eluted, and was then eluted with acetone/CH₂Cl₂(5:95) to give 713 mg (85%) of the title compound.

2-[5-Bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetic acid


Materials	Amount	Mol. Wt.	mmoles


thiotriazole methyl ester	1.14	g	418.31	2.7
tetrahydrofuran	10	ml		(~3 ml/mmol)
ethanol	10	ml		(~3 ml/mmol)
water	10	ml		(~3 ml/mmol)
lithium hydroxide	98	mg	23.95	4.1 (1.5 eq)

Procedure:

To a solution of 2-[5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-[1,2,4]triazol-3-ylsulfanyl]acetic acid methyl ester, in a mixture of THF and EtOH at 0° C., was added a solution of LiOH in H₂O dropwise over 5 min. The reaction was complete after stirring at 0° C. for an additional 45 min. The reaction was neutralized to pH 7 by the addition of 0.5 N HCl solution at 0° C., and the resulting mixture was concentrated in vacuo to ⅕th of its original volume. The mixture was diluted with H₂O (˜20 mL) and acidified to pH 2-3 by the addition of 0.5 N HCl to produce sticky solid. (If the product comes out as an oil during acidification, extraction with CH₂Cl₂is recommended.) The tan solid was collected by vacuum filtration and dried under high vacuum at 50° C. for 16 h in the presence of P₂O₅to yield 1.02 g (93%) of the title compound.

REF:

Esmir Gunic, Jean-Luc Girardet, Jean-Michel Vernier, Martina E. Tedder, David A. Paisner;Compounds, compositions and methods of using same for modulating uric acid levels;US patent number US8242154 B2 ;Also published as US20100056465, US20130040907;Original Assignee: Ardea Biosciences, Inc

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Gunic, Esmir; Galvin, Gabriel;Manufacture of 2-[5-bromo-4-(cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio]acetic acid and related compounds;PCT Int. Appl., WO2014008295 A1

Zamansky, Irina et al;Process for preparation of polymorphic, crystalline, and mesophase forms of 2-[[5-bromo-4-(4-cyclopropyl-1-naphthalenyl)-4H-1,2,4-triazol-3-yl]thio]acetic acid sodium salt; PCT Int. Appl., WO2011085009

Gunic, Esmir et al;Preparation of naphthalene thio triazole derivatives and their use for modulating uric acid levels; U.S. Pat. Appl. Publ., 20100056465
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Quart, Barry D. et al;Preparation of azole carboxylates as modulators of blood uric acid levels;PCT Int. Appl., 2009070740, 04 Jun 2009

Girardet, Jean-Luc et al;Preparation of S-triazolyl α-mercaptoacetanilides as inhibitors of HIV reverse transcriptase;PCT Int. Appl., WO2006026356

US20100056465 * Sep 4, 2009 Mar 4, 2010 Ardea Biosciences Compounds, compositions and methods of using same for modulating uric acid levels
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BARDOXOLONE.. Upcoming blockbuster

Phase 3 drug Comments Off

Mar 042014

BARDOXOLONE METHYL

Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate

methyl 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oate

2-Cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid methyl ester
(6aR,6bS,8aR,12aS,14aR,14bS)-11-Cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxylic acid methyl ester

BARD
CDDO-Me
Methyl-CDDO
NSC-713200
RTA-402
TP-155C

218600-53-4^CAS

218600-44-3 (free acid)

Bardoxolone methyl (also known as “RTA 402” and “CDDO-methyl ester”) is an orally-available first-in-class synthetic triterpenoid. It is an inducer of the Nrf2 pathway, which can suppress oxidative stress and inflammation, and is undergoing clinical development for the treatment of advanced chronic kidney disease (CKD) in type 2 diabetes mellitus patients.

Bardoxolone methyl was previously being investigated by Reata Pharmaceuticals, Inc. in partnership with Abbott Laboratories and Kyowa Hakko Kirin, as an experimental therapy for advanced chronic kidney disease (CKD) in type 2 diabetes mellitus patients. Reata, in consultation with the BEACON Steering Committee, has decided to terminate the Phase 3 BEACON trial of bardoxolone methyl in patients with stage 4 chronic kidney disease and type 2 diabetes. This decision was made based upon a recommendation of the Independent Data Monitoring Committee (IDMC) to stop the trial “for safety concerns due to excess serious adverse events and mortality in the bardoxolone methyl arm.” ^[1]^[2]^[3]^[4]

RTA-402 is a triterpenoid anti-inflammatory agent in phase II trials at Reata Pharmaceuticals for the treatment of pulmonary arterial hypertension.

This company and M.D. Anderson Cancer Center had been evaluating clinically the product for the treatment of lymphoma. Reata had been evaluating the compound in combination with gemcitabine in patients with unresectable pancreatic cancer and melanoma. Preclinical studies were also being conducted by Reata for the treatment of inflammatory bowel disease (IBD) and autoimmune disease. Reata Pharmaceuticals and Kyowa Hakko Kirin had been conducting phase II clinical studies for the treatment of diabetic nephropathy. Reata and Abbott also had been conducting phase III clinical trials for delaying progression to end-stage renal disease in patients with chronic kidney disease and type 2 diabetes; however, in 2012 these trials were discontinued due to serious adverse events and mortality. Phase II clinical trials for this indication were discontinued by Kyowa Hakko Kirin in Japan. The compound had been in early clinical studies for the treatment of multiple myeloma; however, no recent development has been reported for this indication. Phase I clinical trials for the treatment of solid tumors have been completed.

RTA-402 has demonstrated a wide variety of potentially therapeutic mechanisms, including inhibition of inducible nitric oxide synthase and cyclooxygenase expression, stimulation of expression of cytoprotective enzymes such as NAD(P)H quinine oxidoreductase and hemeoxygenase-1, and reduction in pSTAT3 levels. In cancer patients, the drug candidate exploits fundamental physiological differences between cancerous and non-cancerous cells by modulating oxidative stress response pathways. Due to this mechanism, RTA-402 is toxic to cancer cells, but induces protective antioxidant and anti-inflammatory responses in normal cells. In previous studies, the compound was shown to inhibit growth and cause regression of cancerous tumors as a single agent and, in combination with radiation and chemotherapy, to suppress radiation and chemotherapy-induced toxicities in normal tissues and cause minimal toxicity in non-human primates when dosed orally at very high doses for 28 consecutive days.

An analog of RTA-401, RTA-402 is a compound found in medicinal plants with a greater potency than the natural product.

RTA-401 was originally developed at Dartmouth College and M.D. Anderson Cancer Center. In November 2004, Reata completed a license agreement with these organizations, and was granted exclusive worldwide rights to this new class of anticancer compounds. In 2008, orphan drug designation was assigned by the FDA for the treatment of pancreatic cancer. In 2010, the compound was licensed to Kyowa Hakko Kirin by Reata Pharmaceuticals in China, Japan, Korea, Thailand and Southeast Asian countries for the treatment of chronic kidney disease. Abbott acquired rights to develop and commercialize the drug outside US, excluding certain Asian markets.

Phase 1

Bardoxolone methyl was first advanced into the clinic to assess its anticancer properties. In two Phase 1 trials that included 81 oncology patients, bardoxolone methyl reduced serum creatinine levels, with a corresponding improvement in estimated glomerular filtration rate (eGFR). Improvements were more pronounced in a subset of patients with established CKD and were maintained over time in patients who continued on bardoxolone methyl therapy for 5 months. Based on these observed effects and the well-described role of oxidative stress and inflammation in CKD, especially in type 2 diabetes, it was hypothesized that bardoxolone methyl could improve renal function in CKD patients with type 2 diabetes.^[5]

Phase 2

A multi-center, double-blind, placebo-controlled Phase 2b clinical trial (BEAM) conducted in the US studied 227 patients with moderate to severe CKD (eGFR 20 – 45 ml/min/1.73m²) and type 2 diabetes. The primary endpoint was change in estimated GFR following 24 weeks of treatment. Following 24 weeks, patients treated with bardoxolone methyl experienced a mean increase in estimated GFR of over 10 ml/min/1.73m², compared with no change in the placebo group. Approximately three-quarters of bardoxolone methyl treated patients experienced an improvement in eGFR of 10 percent or more, including one-quarter who saw a significant improvement of 50% or more compared to less than 2% of patients on placebo. Adverse events were generally manageable and mild to moderate in severity. The most frequently reported adverse event in the bardoxolone methyl group was muscle spasm. Final data was published in The New England Journal of Medicine.

Concerns have been raised whether there is a true improvement in kidney function because of the significant weight loss of the patients in the active-treatment-group that ranged from 7.7-10.1 kg (7-10% of the initial body weight) and whether this weight loss in patients receiving bardoxolone included muscle wasting with a commensurate decrease in the serum creatinine level. In that case the decrease in creatinine would not necessarily be a true improvement in kidney function.^[6]^[7]^[8]^[9]^[10]

Phase 3

A multinational, double-blind, placebo-controlled Phase 3 outcomes study (BEACON) was started in June 2011, testing bardoxolone methyl’s impact on progression to ESRD or cardiovascular death in 1600 patients with Stage 4 CKD (eGFR 15 – 30 ml/min/1.73m²) and type 2 diabetes. This phase 3 trail was halted in October 2012 because of adverse effects (namely a higher cardiovascular mortality in the treatment arm).^[11]

Mechanism of action

Bardoxolone methyl is an inducer of the KEAP1–Nrf2 pathway.

………………

WO1999065478A1

In a preferred embodiment, such compounds include derivatives of ursolic acid and oleanoic acid. In a particularly preferred embodiment, derivatives of OA, e.g., 2-cyano-3,12-dioxoolean-l,9-dien-28oic acid (CDDO):

have been found to be effective in suppression of human breast cancer cell growth, and highly potent in many vitro assay systems such as: suppression of nitric oxide and prostaglandin production in macrophages, inhibition of growth of human breast cancer cells, suppression of nitric oxide formation in rat prostate cells, and suppression of prostaglandin formation in human colon fibroblasts, as detailed in the Figures.

Compounds were synthesized as below:

Scheme 1

Scheme 2

a: HCO₂Et/MeONa/THF,b: PhSeCl/AcOEt; 30%H₂0₂/THF,c: NH₂OH-HCI EtOH/H₂O, d: MeONa/MeOH/Et₂O,e: KOH/MeOH,f: Jones,g:HCO₂Et/MeONa/PhH,h: Lil/DMF Compound 10 was prepared by formylation of OA (Compound 9) (Simonsen and Ross, 1957) with ethyl formate in the presence of sodium methoxide in THF (Clinton et al., 1961). Compound 7 was obtained by introduction of a double bond at C-l of Compound 10 with phenylselenenyl chloride in ethyl acetate and sequential addition of 30%) hydrogen peroxide (Sharpless et al, 1973). Compound 11 was synthesized from Compound 10 by addition of hydroxylamine in aqueous ethanol; cleavage of Compound 11 with sodium methoxide gave Compound 12 (Johnson and Shelberg, 1945). Compound 14 was prepared from Compound 13 (Picard et al, 1939) by alkali hydrolysis followed by Jones oxidation. Compound 15 was prepared by formylation of Compound 14 with ethyl formate in the presence of sodium methoxide in benzene. Compound 16 was synthesized from Compound 15 by addition of hydroxylamine. Cleavage of 16 with sodium methoxide gave Compound 17. Compound 6 (CDDO) was prepared by introduction of a double bond at C-l of Compound 17 with phenylselenenyl chloride in ethyl acetate and sequential addition of 30% hydrogen peroxide, followed by halogenolysis with lithium iodide in DMF (Dean, P.D.G., 1965).

………………………………………………

WO2009/146216 A2,

Figure imgf000075_0001

Compounds 401, 402, 404, 402-04, 402-35 and 402-56 can be prepared according to the methods taught by Honda et al. (1998), Honda et al. (2000b), Honda et al. (2002), Yates et al. (2007), and U.S. Patent 6,974,801, which are all incorporated herein by reference. The synthesis of the other compounds are disclosed in the following applications, each of which is incorporated herein by reference: U.S. Application Nos. 61/046,332, 61/046,342, 61/046,363, 61/046,366, 61/111,333, 61/111,269, and 61/111,294. The synthesis of the other compounds are also disclosed in the following separate applications filed concurrently herewith, each of which is incorporated herein by reference in their entireties: U.S. Patent Application by Eric Anderson, Xin Jiang, Xiaofeng Liu; Melean Visnick, entitled “Antioxidant Inflammation Modulators: Oleanolic Acid Derivatives With Saturation in the C- Ring,” filed April 20, 2009; U.S. Patent Application by Eric Anderson, Xin Jiang and Melean Visnick, entitled “Antioxidant Inflammation Modulators: Oleanolic Acid Derivatives with Amino and Other Modifications At C-17,” filed April 20, 2009; U.S. Patent Application by Xin Jiang, Xioafeng Liu, Jack Greiner, Stephen S. Szucs, Melean Visnick entitled, “Antioxidant Inflammation Modulators: C-17 Homologated Oleanolic Acid Derivatives,” filed April 20, 2009.

………………………………………………

Chemical Communications, 2011 , vol. 47, 33 p. 9495 – 9497

http://pubs.rsc.org/en/Content/ArticleLanding/2011/CC/c1cc11633a#!divAbstract

http://www.rsc.org/suppdata/cc/c1/c1cc11633a/c1cc11633a.pdf NMR GIVEN

Graphical abstract: DDQ-promoted dehydrogenation from natural rigid polycyclic acids or flexible alkyl acids to generate lactones by a radical ion mechanism

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO)
A mixture of 1 (0.25 g, 0.51 mmol) and DDQ (0.12 g, 0.51 mmol) in anhydrous benzene (20 mL) was
refluxed for 15 min. After filtration, the filtrate was evaporated in vacuo to give a residue, which was
subjected to flash column chromatography (petroleum ether/EtOAc) to give CDDO as an amorphous
solid (0.23 g, 91%). The title compound was known as CAS 218600-44-3

m.p. 180-182 °C;
ESI-MS: 490 [M-H]-, 492 [M+H]+;

1H NMR (300M Hz, CDCl3, 25 °C, TMS): δ 8.05 (1H, s), 5.99 (1H, s), 3.03-2.98 (2H, m), 1.55,1.38,
1.34, 1.22, 1.00, 0.91, 0.85 (each 3H,s ,CH3) ppm.

………………………..

SYNTHESIS

Journal of Medicinal Chemistry, 2000 , vol. 43, 22 p. 4233 – 4246

http://pubs.acs.org/doi/full/10.1021/jm0002230

Abstract Image

BARDOXOLONE METHYL…………Methyl 2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (25). A mixture of 64 (1.51 g, 2.97 mmol) and DDQ (98%) (0.77 g, 3.32 mmol) in dry benzene (80 mL) was heated under reflux for 30 min. After insoluble matter was removed by filtration, the filtrate was evaporated in vacuo to give a solid. The solid was subjected to flash column chromatography [benzene−acetone (10:1)] to give 25 as an amorphous solid (1.38 g, 92%): [α]23D +33° (c 0.68, CHCl3). UV (EtOH) λmax (log ε): 244 (4.07) nm. IR (KBr): 2950, 2872, 2233, 1722, 1690, 1665 cm-1. 1H NMR (CDCl3): δ 8.04 (1H, s), 5.96 (1H, s), 3.68 (3H, s), 3.02 (1H, ddd, J = 3.4, 4.9, 13.4 Hz), 2.92 (1H, d, J = 4.9 Hz), 1.47, 1.31, 1.24, 1.15, 0.99, 0.98, 0.88 (each 3H, s). 13C NMR (CDCl3): δ 199.0, 196.8, 178.3, 168.6, 165.9, 124.2, 114.7, 114.6, 52.1, 49.8, 47.8, 47.3, 45.9, 45.2, 42.7, 42.2, 35.9, 34.6, 33.4, 32.9, 31.8, 31.6, 30.8, 28.1, 27.1, 26.8, 24.7, 23.2, 22.7, 21.8, 21.7, 18.4. EIMS (70 eV) m/z: 505 [M]+(100), 490 (81), 430 (42), 315 (47), 269 (40). HREIMS Calcd for C32H43O4N: 505.3192. Found: 505.3187. Anal. (Table 1).

FREE ACID

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic Acid (26). A mixture of 25 (612 mg, 1.21 mmol) and LiI (3.0 g) in dry DMF (10 mL) was heated under reflux for 4 h. To the mixture were added water and 5% aqueous HCl solution. The mixture was extracted with EtOAc (three times). The extract was washed with water (three times) and saturated aqueous NaCl solution (three times), dried over MgSO4, and filtered. The filtrate was evaporated in vacuo to give an amorphous solid. The solid was subjected to flash column chromatography [hexanes−EtOAc (1:1) followed by CH2Cl2−MeOH (15:1)] to give crude 26 (530 mg). The crude product was purified by recrystallization from benzene to give crystals. To remove benzene completely, the crystals were dissolved in CH2Cl2 (20 mL) and the solvent was evaporated in vacuo to give benzene-free26 as an amorphous solid (405 mg, 68%): [α]22D +33 ° (c 0.28, CHCl3). UV (EtOH) λmax (log ε): 240 (4.21) nm. IR (KBr): 2950, 2867, 2235, 1692, 1665 cm-1. 1H NMR (CDCl3): δ 8.05 (1H, s), 6.00 (1H, s), 3.06−2.98 (2H, m), 1.48, 1.34, 1.25, 1.16, 1.02, 1.00, 0.90 (each 3H, s). 13C NMR (CDCl3): δ 199.0, 196.8, 183.7, 168.8, 165.9, 124.2, 114.7, 114.5, 49.8, 47.8, 47.1, 45.9, 45.2, 42.7, 42.3, 35.8, 34.5, 33.3, 33.0, 31.8, 31.5, 30.8, 28.1, 27.1, 26.8, 24.8, 23.2, 22.6, 21.72, 21.71, 18.4. EIMS (70 eV) m/z: 491 [M]+ (100), 476 (62), 445 (29), 430 (27), 269 (94). HREIMS Calcd for C31H41O4N: 491.3036. Found: 491.3020. Anal. (Table 1).

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Bioorganic and Medicinal Chemistry Letters, 1998 , vol. 8, 19 p. 2711 – 2714

http://www.sciencedirect.com/science/article/pii/S0960894X9800479X

Full-size image (3 K)

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Bioorganic and Medicinal Chemistry Letters, 2005 , vol. 15, # 9 p. 2215 – 2219

http://www.sciencedirect.com/science/article/pii/S0960894X05003306

Full-size image (5 K)

………………..

WO2002047611A2

Method of synthesis of CDDO. CDDO may be synthesized by the scheme outlined below.

Methyl-CDDO. Methyl-CDDO (CDDO-Me), the C-28 methyl ester of CDDO, also exerts strong antiproliferative and apoptotic effects on leukemic cell lines and in primary AML samples in vitro as well as induces monocytic differentiation of leukemic cell lines and some primary AMLs. Thus, CDDO-Me provides chemotherapy for the treatment of leukemias. The present invention demonstrates that this effect is profoundly increased by combination of CDDO-Me with other chemotherapeutic agents. These include retinoids such as ATRA, 9-cis retinoic acid, , LG100268, LGD1069 (Targretin, bexarotene), fenretinide [N-(4- hydroxyphenyl)retinamide, 4-HPR], CD437 and other RXR and RAR-specific ligands. This combination also increases ara-C cytotoxicity, further reduces AML colony formation, inhibits ERK phosphorylation and promotes Bcl-2 dephosphorylation, and inhibits in vitro angiogenesis. The ability of CDDO-Me in combination with retinoids to induce differentiation in leukemic cells in vitro show that these compounds may have similar in vivo effects. The anti-angiogenic properties of CDDO-Me further increase its potent anti-leukemia activity in combination with retinoids. Furthermore, CDDO-Me was found to be more potent at lower concentrations than CDDO.

Method of synthesis of CDDO-Me.

CDDO-Me may be synthesized by the scheme outlined below.

The present invention provides combinations of CDDO-compounds and chemotherapeutic agents that are useful as treatments for cancers and hematological malignancies. In one embodiment, the chemotherapeutics are retinoids. As CDDO- compounds are PPARγ ligands and PPARγ is known to be altered in many types of cancers, the inventors contemplate, that ligation of PPARγ in combination with retinoids such as, RXR-specific ligands, provides a mechanistic basis for maximal increase in transcriptional activity of the target genes that control apoptosis and differentiation. The CDDO-compounds and retinoids in combination demonstrate an increased ability to induce differentiation, induce cytotoxicity, induce apoptosis, induce cell killing, reduce colony formation and inhibit the growth of several types of leukemic cells.

…………………..

Org Lett. 2013 Apr 5;15(7):1622-5. doi: 10.1021/ol400399x. Epub 2013 Mar 26.

Efficient and scalable synthesis of bardoxolone methyl (cddo-methyl ester).

http://pubs.acs.org/doi/pdf/10.1021/ol400399x

Bardoxolone methyl (2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid methyl ester; CDDO-Me) (1), a synthetic oleanane triterpenoid with highly potent anti-inflammatory activity (levels below 1 nM), has completed a successful phase I clinical trial for the treatment of cancer and a successful phase II trial for the treatment of chronic kidney disease in type 2 diabetes patients. Our synthesis of bardoxolone methyl (1) proceeds in ∼50% overall yield in five steps from oleanolic acid (2), requires only one to two chromatographic purifications, and can provide gram quantities of 1.

References

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ClinicalTrials.gov NCT01351675 Bardoxolone Methyl Evaluation in Patients With Chronic Kidney Disease and Type 2 Diabetes (BEACON)
Design and synthesis of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, a novel and highly active inhibitor of nitric oxide production in mouse macrophages
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Novel synthetic oleanate triterpenoids: A series of highly active inhibitors of nitric production in mouse macrophages
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WO 1999065478
WO 2013169553
CN 102875634
US 2012330050
US 2012071684
WO 2011130302
WO 2010093944
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Citing Patent	Filing date	Publication date	Applicant	Title
US8440854 *	Jan 23, 2012	May 14, 2013	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with amino acid and other modifications at C-17
US8513436	Dec 19, 2011	Aug 20, 2013	Reata Pharmaceuticals, Inc.	Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators

WO2002047611A2 *	Nov 28, 2001	Jun 20, 2002	Univ Texas	Cddo-compounds and combination therapies thereof
WO2008064132A2 *	Nov 16, 2007	May 29, 2008	Dartmouth College	Synthetic triterpenoids and tricyclic-bis-enones for use in stimulating bone and cartilage growth
WO2009118441A1 *	Feb 12, 2009	Oct 1, 2009	Consejo Superior De Investigaciones Cientifícas	Use of pentacyclic triterpene for the preparation of a pharmaceutical compound intended for the treatment of multiple sclerosis
WO2013083659A1	Dec 5, 2012	Jun 13, 2013	Cambridge Enterprise Limited	Combination treatment comprising ho – 1 inhibitor and immunotherapeutic agent
US7176237	Jan 15, 2003	Feb 13, 2007	The Trustees Of Dartmouth College	Tricyclic-bis-enone derivatives and methods of use thereof
US7435755	Nov 28, 2001	Oct 14, 2008	The Trustees Of Dartmouth College	CDDO-compounds and combination therapies thereof
US7678830	Feb 7, 2007	Mar 16, 2010	Trustees Of Dartmouth College	Tricyclic-bis-enone derivatives and methods of use thereof
US7714012	Nov 16, 2007	May 11, 2010	Trustees Of Dartmouth University	Synthesis and biological activities of new tricyclic-bis-enones (TBEs)
US7795305	Oct 10, 2008	Sep 14, 2010	Board Of Regents, The University Of Texas System	CDDO-compounds and combination therapies thereof
US7863327	May 3, 2005	Jan 4, 2011	Trustees Of Dartmouth College	Therapeutic compounds and methods of use
US7915402	Apr 20, 2009	Mar 29, 2011	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with saturation in the C-ring
US7943778	Apr 20, 2009	May 17, 2011	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: C-17 homologated oleanolic acid derivatives
US8034955	Oct 29, 2007	Oct 11, 2011	Trustees Of Dartmouth College	Therapeutic compounds and methods of use
US8067394	May 10, 2010	Nov 29, 2011	Trustees Of Dartmouth College	Synthesis and biological activities of new tricyclic-bis-enones (TBEs)
US8067465	Mar 11, 2010	Nov 29, 2011	The Trustees Of Dartmouth College	Tricyclic-bis-enone derivatives and methods of use thereof
US8071632	Apr 20, 2009	Dec 6, 2011	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: novel derivatives of oleanolic acid
US8124656	Feb 23, 2011	Feb 28, 2012	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with saturation in the C-ring
US8124799	Apr 20, 2009	Feb 28, 2012	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with amino and other modifications at C-17
US8129429	Jan 12, 2009	Mar 6, 2012	Reata Pharmaceuticals, Inc.	Synthetic triterpenoids and methods of use in the treatment of disease
US8258329	Apr 20, 2009	Sep 4, 2012	Reata Pharmaceuticals, Inc.	Dehydroandrosterone analogs including an anti-inflammatory pharmacore and methods of use
US8299046	Nov 16, 2007	Oct 30, 2012	Trustees Of Dartmouth College	Synthetic triterpenoids and tricyclic-bis-enones for use in stimulating bone and cartilage growth
US8314137	Jul 22, 2009	Nov 20, 2012	Trustess Of Dartmouth College	Monocyclic cyanoenones and methods of use thereof
US8338618	Nov 11, 2011	Dec 25, 2012	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: novel derivatives of oleanolic acid
US8394967	Feb 23, 2011	Mar 12, 2013	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: C-17 homologated oleanolic acid derivatives
US8440820	Jan 11, 2012	May 14, 2013	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with saturation in the C-ring
US8440854	Jan 23, 2012	May 14, 2013	Reata Pharmaceuticals, Inc.	Antioxidant inflammation modulators: oleanolic acid derivatives with amino acid and other modifications at C-17
US8455544	Jan 26, 2012	Jun 4, 2013	Reata Pharmaecuticals, Inc.	Synthetic triterpenoids and methods of use in the treatment of disease
US8513436	Dec 19, 2011	Aug 20, 2013	Reata Pharmaceuticals, Inc.	Pyrazolyl and pyrimidinyl tricyclic enones as antioxidant inflammation modulators
US8586775	Aug 24, 2011	Nov 19, 2013	Trustees Of Dartmouth College	Therapeutic compounds and methods of use

Professor Honda received his B.S. degree in Chemistry in 1974, his M.S. degree in Organic Chemistry in 1976, and his Ph.D. in Organic Chemistry in 1979 from the University of Tokyo. In 1979, he joined the Department of Drug Discovery Chemistry at Suntory Institute for Biomedical Research in Japan and worked there as a drug synthetic chemist (finally senior researcher) for 13 years. In 1991, he joined the Central Pharmaceutical Research Institute at Japan Tobacco Inc. and worked as a chief senior researcher for 3 years. In 1995, he joined Dr. Gribble’s laboratory at Dartmouth College as a research associate. In 1998, he joined the research faculty of Dartmouth College. In 2005, he was promoted to Research Associate Professor.http://www.dartmouth.edu/~chem/faculty/th.html

Dr. Honda and his collaborators have further explored new structures based on CDDO and different five-ringed triterpenoids.

During the course of these investigations, Dr. Honda has designed three-ringed compounds with similar enone functionalities in rings A and C to those of CDDO, but having a much simpler structure than five-ringed triterpenoids. He and his collaborators have found that they are also a novel class of potent anti-inflammatory, cytoprotective, growth suppressive, and pro-apoptotic compounds. Amongst such three-ringed compounds, TBE-31 with the C-8a ethynyl group is much more potent than CDDO in various bioassays in vitro and in vivo. Thus, further investigation (design, synthesis, biological evaluation, etc.) of new TBE-31 analogues is currently being performed in order to discover analogues having different and/or better features than TBE-31, for example, higher potency and lower toxicity, better bioavailability and different distributions in organs, high water-solubility and so on.

Mechanism studies suggest that CDDO regulates various molecules regarding inflammation, differentiation, apoptosis, and proliferation by reversible Michael addition between the cyano enone functionality of CDDO and the sulfhydryl groups of cysteine moieties on these molecules. Based on this fact and the structure of TBE-31, Dr. Honda has designed single-ringed compounds, which represent the ideal simple structure. The synthesis of these new compounds is currently in progress.

Sonidegib/Erismodegib..Novartis Cancer Drug LDE225 Meets Primary Endpoint in Phase 2

Phase 3 drug Comments Off

Feb 202014

Sonidegib/Erismodegib

CODE DESIGNATION ..LDE225, NVP-LDE-225

Treatment of medulloblastoma PHASE3 2014 FDA FILING

Treatment of advanced basal cell carcinoma PHASE3 2014 FDA FILING

Treatment of SOLID TUMORS..PHASE1 2017 FDA FILING

READMalignant Solid Tumors of Childhood

THERAPEUTIC CLAIM Oncology, Antineoplastics & Adjunctive Therapies

CHEMICAL NAMES

1. [1,1′-Biphenyl]-3-carboxamide, N-[6-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-3-pyridinyl]-2-
methyl-4′-(trifluoromethoxy)-, rel-

2. N-{6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl}-2-methyl-4′-
(trifluoromethoxy)biphenyl-3-carboxamide

N-[6-[(2S,6R)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl]-2-methyl-3-[4-(trifluoromethoxy)phenyl]benzamide

N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-(trifluoromethoxy)biphenyl-3-carboxamide

MOLECULAR FORMULA C26H26F3N3O3

MOLECULAR WEIGHT 485.5

SPONSOR Novartis Pharma AG

CAS REGISTRY NUMBER 956697-53-3 free form

NOTE… DIPHOSPHATE SALT IS THE DRUG WITH CAS 1218778-77-8

sonidegib – European Medicines Agency READ THIS..

Summary EudraCT Number: 2012-004022-21 Sponsor’s Protocol … READ THIS

Novartis announced that the pivotal trial of the investigational oral compound LDE225 (sonidegib) in advanced basal cell carcinoma met its primary endpoint of demonstrating an objective response rate among patients within six months of treatment. Objective response included complete response (clinically significant tumor response with complete absence of disease) and partial response (clinically significant tumor shrinkage).

Basal cell carcinoma is the most common form of skin cancer, accounting for more than 80% of non-melanoma skin cancers, and can be highly disfiguring and life-threatening if it grows. Worldwide incidence of basal cell carcinoma is rising by 10% each year due to factors such as an aging population and increased ultraviolet exposure. Although basal cell carcinoma rarely metastasizes, once it does, it can be associated with significant morbidity.

“For people living with advanced basal cell carcinoma there are currently limited treatment options,” said Alessandro Riva, president, Novartis Oncology ad interim and global head, Oncology Development and Medical Affairs. “These results demonstrate the potential for LDE225 to offer a treatment option for this patient population, and we look forward to sharing these data with regulatory authorities worldwide.”

Full study results will be presented at a future scientific meeting.

About the Study

The Phase II, randomized, double-blind BOLT (Basal cell carcinoma Outcomes in LDE225 Trial) study was designed to assess the safety and efficacy of two oral dose levels of LDE225 (200 mg and 800 mg) in patients with locally advanced or metastatic basal cell carcinoma[4], which are subtypes of advanced basal cell carcinoma.

The primary endpoint was the proportion of patients achieving an objective response rate, defined as a confirmed complete response and partial response as their best overall response per modified RECIST criteria, within six months of starting treatment with LDE225. Key secondary endpoints of the study included assessing the duration of tumor responseand the rate of complete response. Other secondary endpoints included progression-free survival, time to tumor response and overall surviva

Date: February 19, 2013

Source: Novartis

MORE ABOUT SONIDEGIB

Sonidegib (INN) or Erismodegib (USAN), also known as LDE225 is a Hedgehog signalling pathway inhibitor (via smoothened antagonism) being developed as an anticancer agent by Novartis.[1][2] It has been investigated as a potential treatment for:

Pancreatic cancer [3][4][5][6]
Breast cancer [7][8]
Basal cell carcinoma of the skin[9][10][11]
Small cell lung cancer [12]
Medulloblastoma [13][14]
Advanced solid tumours (including ovarian, breast, pancreatic, stomach, oesophageal cancers and glioblastoma multiforme)[15][16][17]
Acute leukaemia [18]
Chronic myeloid leukaemia [19]
Myelofibrosis and Essential thrombocythaemia [20]

NVP-LDE-225, a product candidate developed by Novartis, is in phase III clinical trials for the treatment of medulloblastoma and basal cell carcinoma. Phase II trials are in progress for the treatment of adult patients with relapsed or refractory or untreated elderly patients with acute leukemia.

Early clinical trials are ongoing for the oral treatment of advanced solid tumors, for the treatment of myelofibrosis in combination with ruxolitinib and for the treatment of small cell lung cancer. A phase II clinical trial for the treatment of basal cell carcinomas in Gorlin’s syndrome patients with a cream formulation of NVP-LDE-225 was discontinued in 2011 since the formulation did not demonstrate tumor clearance rate sufficient to support further development.

Dana-Farber Cancer Institute and the Massachusetts General Hospital are conducting phase I clinical trials for the treatment of locally advanced or metastatic pancreatic cancer in combination with chemotherapy. In 2009, orphan drug designation was assigned in the E.U. for the treatment of Gorlin syndrome.

It has demonstrated significant efficacy against melanoma in vitro and in vivo.[21] It also demonstrated efficacy in a mouse model of pancreatic cancer.[22]

NVP-LDE225 Diphosphate salt (Erismodegib, Sonidegib)

Formula Image

Synonym:Erismodegib, Sonidegib

CAS Number:1218778-77-8

Mol. Formula:C26H26F3N3O3 ∙ 2H3PO4

MW:681.5
nmr.http://www.chemietek.com/Files/Line2/Chemietek,%20NVP-LDE225%20[02],%20NMR.pdf
hplc–http://www.chemietek.com/Files/Line3/Chemietek,%20NVP-LDE225%20[02],%20HPLC.pdf

Brief Description:

A potent, selective, and orally bioavailable Smoothened (Hedgehog Signaling Pathway) antagonist, currently in clinical trials. Diphosphate salt offers a much better bioavailability than free base (Ref. a)

a. Pan, S., et al, Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist, ACS Med. Chem. Lett., 2010, 1 (3), pp 130–134.

About LDE225

LDE225 (sonidegib) is an oral, investigational, selective smoothened inhibitor being studied in a variety of cancers. Smoothened (SMO) is a molecule that regulates the hedgehog (Hh) signaling pathway, which plays a critical role in stem cell maintenance and tissue repair. LDE225 is currently in clinical development for a variety of diseases including myelofibrosis, leukemia and solid tumors.

Given that LDE225 is an investigational compound, the safety and efficacy profile has not yet been fully established. Access to this investigational compound is available only through carefully controlled and monitored clinical trials. These trials are designed to better understand the potential benefits and risks of the compound. Given the uncertainty of clinical trials, there is no guarantee that LDE225 will ever be commercially available anywhere in the world.

Possibility (LDE225) is effective in medulloblastoma relapsed or refractory hedgehog pathway inhibitor sonidegib has been revealed. That the anti-tumor effect was observed in some patients and tolerability in 1/2 test phase.

4th Quadrennial Meeting of the World Federation of Neuro-Oncology in conjunction with the 18th Annual Meeting of the Society for Neuro-Oncology, which was held in San Francisco November 21 to 24 in (WFNO-SNO2013), rice Dana-Farber It was announced by Mark Kieran Mr. Children’s Hospital Cancer Center.

The research group, announced the final results of the Phase 1 trial that target advanced solid cancer in children of sonidegib. 　1 dose increased multi-test phase, was initiated from 372mg/m2 once-daily dosing to target children under the age of 18 more than 12 months. (233mg/m2 group 11 people, 16 people 372mg/m2 group, 11 people group 425mg/m2, 680mg/m2 group 21 women) who participated 59 people, including medulloblastoma 38 patients. 12 median age was (2-17).

Creatine phosphokinase elevation of grade 4 only were seen at 372mg/m2 as dose-limiting toxicity only, and became two recommended dose phase and 680mg/m2. 　Nausea muscle pain creatine kinase rise malaise (22.0%) (15.3%) (15.3%), (13.6%), vomiting side effects were many, was (13.6%). Hypersensitivity vomiting creatine kinase increased (3.4%) (1.7%) (1.7%), rhabdomyolysis side effects of grade 3/4 was (1.7%). 　(One group 372mg/m2, 425mg/m2 group one) complete response was obtained in two people, a strong correlation was found between the activation of the hedgehog pathway and effect.

Phase III clinical trials that target medulloblastoma the activated hedgehog pathway currently are underway.

About Novartis

Novartis provides innovative healthcare solutions that address the evolving needs of patients and societies. Headquartered in Basel, Switzerland, Novartis offers a diversified portfolio to best meet these needs: innovative medicines, eye care, cost-saving generic pharmaceuticals, preventive vaccines and diagnostic tools, over-the-counter and animal health products. Novartis is the only global company with leading positions in these areas. In 2013, the Group achieved net sales of USD 57.9 billion, while R&D throughout the Group amounted to approximately USD 9.9 billion (USD 9.6 billion excluding impairment and amortization charges). Novartis Group companies employ approximately 136,000 full-time-equivalent associates and operate in more than 140 countries around the world.

Increased levels of Hedgehog signaling are sufficient to initiate cancer formation and are required for tumor survival.

These cancers include, but are not limited to, prostate cancer (“Hedgehog signalling in prostate regeneration, neoplasia and metastasis”, Karhadkar S S, Bova G S, Abdallah N, Dhara S, Gardner D, Maitra A, Isaacs J T, Berman D M, Beachy P A., Nature. 2004 Oct. 7; 431(7009):707-12;

“Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling”, Sanchez P, Hernandez A M, Stecca B, Kahler A J, DeGueme A M, Barrett A, Beyna M, Datta M W, Datta S, Ruiz i Altaba A., Proc Natl Acad Sci USA. 2004 Aug. 24; 101(34):12561-6),

breast cancer (“Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer”, Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, Kuroki S, Katano M., Cancer Res. 2004 Sep. 1; 64(17):6071-4),

medulloblastoma (“Medulloblastoma growth inhibition by hedgehog pathway blockade”, Berman D M, Karhadkar S S, Hallahan A R, Pritchard J I, Eberhart C G, Watkins D N, Chen J K, Cooper M K, Taipale J, Olson J M, Beachy P A., Science. 2002 Aug. 30; 297(5586):1559-61),

basal cell carcinoma (“Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions”, Williams J A, Guicherit O M, Zaharian B I, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter J A, Rubin L L, Wang F Y., Proc Natl Acad Sci USA. 2003 Apr. 15; 100(8):4616-21;

“Activating Smoothened mutations in sporadic basal-cell carcinoma”, Xie J, Murone M, Luoh S M, Ryan A, Gu Q, Zhang C, Bonifas J M, Lam C W, Hynes M, Goddard A, Rosenthal A, Epstein E H Jr, de Sauvage F J., Nature. 1998 Jan. 1; 391(6662):90-2),

pancreatic cancer (“Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis”, Thayer S P, di Magliano M P, Heiser P W, Nielsen C M, Roberts D J, Lauwers G Y, Qi Y P, Gysin S, Fernandez-del Castillo C, Yajnik V, Antoniu B, McMahon M, Warshaw A L, Hebrok M., Nature. 2003 Oct. 23; 425(6960):851-6;

“Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours”, Berman D M, Karhadkar S S, Maitra A, Montes De Oca R, Gerstenblith M R, Briggs K, Parker A R, Shimada Y, Eshleman J R, Watkins D N, Beachy P A., Nature. 2003 Oct. 23; 425(6960):846-51),

and small-cell lung cancer (“Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer”, Watkins D N, Berman D M, Burkholder S G, Wang B, Beachy P A, Baylin S B., Nature. 2003 Mar. 20; 422(6929):313-7).

PATENTS

1 WO 2007131201

2 WO 2008154259

3 WO 2010033481

4 WO 2011009852

5 WO 2011062939

………………………………………

SYNTHESIS

WO2011009852A2

2-Methyl-4′-tr{fluoromethoxy-biphenyl-3-carboxylic acid {6-(cis-2,6-dimethyl- morpholin-4-yl)-pyrid»n-3-yl|-amide:

The following Examples serve to illustrate the invention without limiting the scope thereof, it is understood that the invention is not limited to the embodiments set forth herein, but embraces ali such forms thereof as come within the scope of the disclosure,

Step 1:

To a solution of 2-chloro-5-nitro-pyridine 1 (5.58 g, 35.2 mmoL) and c/s-2,6- dimethylmorpholine (4.05 g, 35.2 mmoL) in anhydrous DMF (30 mi.) was added K2CO3 (9.71 g, 70.4 mnrtoL). The mixture was heated at 50ºC overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na2SO4 and concentrated to give crude product 3 as a yellow solid, after purification by silica gel chromatography, obtained pure product (7.80 g, 93.2%). LC-MS m/z: 238.2 (M+ 1).

Step 2:

The above material 3 (7.3Og. 30.8 mmoL) was hydrogenated in the presence of 10% Pd-C (1.0 g) in MeOH (120 ml) under hydrogen overnight. The suspension was filtered through celite and the filtrate was concentrated to give the crude product 4 (5.92 g) as a dark brown oil which was used directly in the next step without further purification. LC-MS m/z. 208.2 (M+1).

Step 3:

To a solution of 3-bromo-2-methyl benzoic acid (2.71 g, 12.6 mmoL), 6-((2S,6R)-2,6- dimethylmorpholino)pyridin-3-arnine 4 (2.61 g, 12.6 mmoL), and HATU (4.80 g, 12.6 mmoL) in anhydrous DMF (30 mL) was added diisopropylethylamine (6.58 mL, 37.8 mmoL) dropwise. The resulting mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (50 mL), and then extracted with EtOAc (3×120 mL). The organic layer was dried and concentrated to give the crude product. This crude product was then purified by flash column chromatography using 30% EtOAc in hexane as eiuent to give 5 as a white solid (4.23 g, 83.0%). LC-MS m/z: 404.1 (M+1).

Step 4:

A mixture of 4-(trif!uoromethoxy)phenylboronic acid (254 mg, 1.24 mmol), 3-bromo- N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-ylJ-4-methyl-benzamide 5 (250 mg, 0.62mmol), Pd(PPh3)4 (36 mg, 0.03 mmol), Na2CO3 (2.0M aqueous solution, 1.23 mL, 2.4 mmol) and DME (4.5 mL) in a sealed tube was heated at 130ºC overnight. The reaction mixture was diluted with EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine and concentrated to give the crude product which was then purified by preparative mass triggered HPLC (C18 column, etuted with CH3CN-H2O containing 0.05% TFA) to give N-(6-((2S,6R)-2,6-dimethyfmorpholino)pyridin-3-yl)-2-rnethyl- 4′-(trifluoromethoxy)biphenyi-3-carboxamide (183.5 mg, 61.1% yield). LC-MS m/z: 486.2 (M+1).

The resultant crystalline product (Form A) was converted to the amorphous form by dissolving in 3% w/w aqueous ethanol, and the resultant solution spray dried at about 150ºC.

Form B was prepared by heating the amorphous form in an oven at 110ºC for 2 hours. In a further embodiment, the invention relates to a process step or steps, or an intermediate as described herein.

……………………

PAPER

ChemMedChem, 2013 , vol. 8, 8 p. 1261 – 1265

http://onlinelibrary.wiley.com/doi/10.1002/cmdc.201300217/full

http://onlinelibrary.wiley.com/store/10.1002/cmdc.201300217/asset/supinfo/cmdc_201300217_sm_miscellaneous_information.pdf?v=1&s=702e175bfb44b5a61c41e750695f7f7cd20be83d

Continued optimization provided a novel type of Smoothened (Smo) antagonist based on a pyridazine core. The compound, NVP-LEQ506, currently in phase I clinical trials, combines high intrinsic potency and good pharmacokinetic properties resulting in excellent efficacy in rodent tumor models of medulloblastoma. Activity against a Smo mutant conferring resistance observed in a previous clinical trial with a competitor compound suggests additional therapeutic potential.

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Links

SYNTHESIS

US20120196849, ENTRY…..95

LC-MS m/z 486.2 (M + 1)

USE SIMILAR METHODOLOGY

EXAMPLESThe present invention is further exemplified, but not limited, by the following example that illustrates the preparation of compounds of Formula I according to the invention.Example 1 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide

Step 1: To a solution of 3-iodo-4-methyl-benzoic acid (10.0 g, 38.2 mmol) in methanol (70 ml) is added concentrated sulfuric acid (0.5 ml). The reaction mixture is heated at 70° C. for 48 hours, cooled to room ambient temperature and then concentrated. After that, ethyl acetate (100 ml) and aqueous NaHCO3 (saturated, 100 ml) solution are added to the residue. The organic layer is separated and washed again with aqueous NaHCO3 (saturated, 100 ml) solution. The organic layer is separated, dried over anhydrous Na2SO4 and concentrated to yield 3-iodo-4-methyl-benzoic acid methyl ester 1. It is used without further purification in the next step. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.87 (d, 1H, J=8.4 Hz), 7.48 (d, 1H, J=8.4 Hz), 3.85 (s, 3H), 3.35 (s, 3H); LC-MS m/z: 277.0 (M+1).

Step 2: To a round-bottom flask containing 3-iodo-4-methyl-benzoic acid methyl ester (1.38 g, 5.00 mmol), 4-cyanophenylboronic acid (1.10 g, 7.48 mmol), palladium acetate (168 mg, 0.748 mmol), 2-(dicyclohexylphosphino)biphenyl (0.526 g, 1.50 mmol) and potassium fluoride (0.870 g, 15.0 mmol) is added anhydrous 1,4-dioxane (15 ml). The flask is purged with argon and sealed. The mixture is stirred at 130° C. for 18 hours, cooled to ambient temperature and then water (20 ml) and ethyl acetate (20 ml) are added. Solid is removed under vacuum filtration. The filtrate is extracted with EtOAc (20 ml×2). The organic layers are combined, washed with aqueous HCl (5%, 20 ml) and saturated NaHCO3 (20 ml). It is dried over MgSO4, and concentrated. The residue is purified by silica gel column chromatography (EtOAc/Hexane, gradient) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2; LC-MS m/z: 252.1 (M+1).

Step 3: To a solution of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2 (2.56 g, 10.3 mmol) in 1,4-dioxane-H2O (1:1 mixture, 20 ml) is added NaOH (1.22 g, 30.2 mmol)). The reaction is stirred at ambient temperature for 24 hours. To this mixture is added aqueous HCl (1 N, 36 ml) and it is then extracted with ethyl acetate (40 ml×3). The organic layers are combined, dried over anhydrous Na2SO4. The solver is removed. The solid obtained is washed with small amount of acetonitrile and air dried to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3: 1H NMR (DMSO-d6) δ 7.94 (d, 2H, J=8.0 Hz), 7.84 (dd, 1H, J1=8.4 Hz, J2=1.2 Hz), 7.75 (d, 1H, J=1.2 Hz), 7.61 (d, 2H, J=8.0 Hz), 7.48 (d, 1H, J=8.4 Hz), 2.29 (s, 3 H); LC-MS m/z 238.1 (M+1).

Step 4: To a suspension of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3 (40 mg, 0.17 mmol) in anhydrous methylene chloride (5 ml) is added 2 drops of DMF. Then oxalyl chloride (32 mg, 22 μl, 0.25 mmol) is added. The mixture is stirred at ambient temperature until it turns clear. After that, it is concentrated, re-dissolved in anhydrous methylene chloride (3 ml), and added to a solution of 4-(morpholine-4-sulfonyl)-phenylamine (61 mg, 0.25 mmol) and triethylamine (34 mg, 47 μl, 0.33 mmol) in methylene chloride (2 ml). The mixture is stirred for 2 hours, concentrated and the residue is purified by preparative mass triggered HPLC (C18 column, eluted with CH3CN—H2O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide: 1H NMR (DMSO-d6) δ 10.64 (s, 1H), 8.07 (d, 2H, J=8.8 Hz), 7.97 (d, 2H, J=8.4 Hz), 7.95 (d, 1H, J=8.8 Hz), 7.89 (s, 1H), 7.43 (d, 2H, J=8.4 Hz), 7.67 (d, 2H, J=8.8 Hz), 7.53 (d, 2H, J=8.8 Hz), 3.63 (m, 4H), 2.84 (m, 4H) 2.32 (s, 3H); LC-MS m/z: 462.1 (M+1).

Example 2 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide

Step 1: To a solution of 2-chloro-5-nitro-pyridine 4 (2.38 g, 15 mmol.) and cis-2,6-dimethylmorpholine (1.73 g, 15 mmol.) is added K2CO3 (4.14 g, 30 mmol.). The mixture was heated at 50° C. overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na2SO4 and concentrated to give crude product 6 as a yellow solid. The crude product is used directly in next step without further purification. LC-MS m/z: 238.1 (M+1).

Step 2: The above crude material 6 is hydrogenated in the presence of Pd—C (0.2 g) in MeOH (100 mL) under hydrogen over 10 h. The suspension is filtered through celite and the filtrate is concentrated to give the crude product 7 as a dark brown oil which is used directly in the next step without further purification. LC-MS m/z: 208.1 (M+1).

Step 3: To a solution of 3-bromo-4-methyl benzoic acid (108 mg, 0.5 mmol.), 6-(2,6-Dimethyl-morpholin-4-yl)-pyridin-3-ylamine 7 (104 mg, 0.5 mmol.), amd HATU (190 mg, 0.5 mmol.) in dry DMF (5 mL) is added triethylamine (139 uL, 1.0 mmol.) dropwise. The resulting mixture is stirred at room temperature for 2 h. After concentration, the residue is partitioned between EtOAc and water. The organic layer is dried and concentrated to give the crude product. The final compound is purified by flash column chromatography using 50% EtOAc in hexane as eluent to give 8 as a white solid. LC-MS m/z: 404.1 (M+1).

Step 4: A mixture of 4-cyanophenyl boronic acid (18 mg, 0.12 mmol), 3-bromo-N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamide 8 (40 mg, 0.1 mmol), Pd(PPh3)4 (11 mg, 0.01 mmol), and Na2CO3 (42 mg, 0.4 mmol) in a combined solvent system of toluene (0.2 mL) and water (0.2 mL) and ethanol (0.05 mL) is heated at 140° C. under microwave irradiation for 30 min. The reaction mixture is diluted with EtOAc and water. The aqueous layer is extracted with EtOAc. The combined organic layer is washed with brine and concentrated to give the crude product which is purified by preparative mass triggered HPLC (C18 column, eluted with CH3CN—H2O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide. LC-MS m/z: 427.2 (M+1).

USE THIS COMPD IN ABOPVE AND YOU WILL GET SONIDEGIB

4-(Trifluoromethoxy)phenylboronic acid

CAS Number 139301-27-2
Linear Formula CF3OC6H4B(OH)2
Molecular Weight 205.93

CONDENSE WITH …3-bromo-N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamideACS Medicinal Chemistry Letters, 2010 , vol. 1, 3 p. 130 – 134

……………………………………………….

PAPER

ACS Medicinal Chemistry Letters, 2010 , vol. 1, 3 p. 130 – 134

http://pubs.acs.org/doi/full/10.1021/ml1000307

ENTRY 5m

A mixture of 4-(trifluoromethoxy)phenylboronic acid (254 mg, 1.24 mmol), 3-bromo-N-[6-(2,6-
dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamide E (250 mg, 0.62mmol), Pd(PPh3)4
(36 mg, 0.03 mmol), Na2CO3 (2.0M aqueous solution, 1.23 mL, 2.4 mmol) and DME (4.5 mL)
in a sealed tube was heated at 1300C overnight. The reaction mixture was diluted with EtOAc
and water. The aqueous layer was extracted with EtOAc. The combined organic layer was
washed with brine and concentrated to give the crude product which was then purified by
preparative mass triggered HPLC (C18 column, eluted with CH3CN-H2O containing 0.05% TFA)
to give N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-
(trifluoromethoxy)biphenyl-3-carboxamide (5m, 183.5 mg, 61.1% yield). LC-MS m/z: 486.2 (M+1).
HRMS (m/z): [M+H]+
calcd for C26H27N3O3F3 486.2005; found 486.1986,
1H-NMR (500 MHz, DMSO-d6): δ (ppm) 10.15 (s, 1H), 8.43 (d, 1H), 7.94 (dd, 1H), 7.52-7.43
(m, 5H), 7.38 (m, 1H), 7.33 (m, 1H), 6.86 (d, 1H), 4.06 (d, 2H), 3.62 (m, 2H), 2,34 (m, 2H), 2.22
(s, 3H), 1.16 (d, 6H).

http://pubs.acs.org/doi/suppl/10.1021/ml1000307/suppl_file/ml1000307_si_001.pdf

Reference

“LDE225 – PubChem”. PubChem. National Institutes of Health. Retrieved 16 February 2014.
Pan, S; Wu, X; Jiang, J; Gao, W; Wan, Y; Cheng, D; Han, D; Liu, J; Englund, NP; Wang, Y; Peukert, S; Miller-Moslin, K; Yuan, J; Guo, R; Matsumoto, M; Vattay, A; Jiang, Y; Tsao, J; Sun, F; Pferdekamper, AC; Dodd, S; Tuntland, T; Maniara, W; Kelleher, JF; Yao, Y; Warmuth, M; Williams, J; Dorsch, M (10 June 2010). “Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist”. ACS Medicinal Chemistry Letters 1 (3): 130–134. doi:10.1021/ml1000307.
“A Biomarker Study to Identify Predictive Signatures of Response to LDE225 (Hedgehog Inhibitor) In Patients With Resectable Pancreatic Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Gemcitabine + Nab-paclitaxel With LDE-225 (Hedgehog Inhibitor) as Neoadjuvant Therapy for Pancreatic Adenocarcinoma”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Dose-escalation, and Safety Study of LDE225 and Gemcitabine in Locally Advanced or Metastatic Pancreatic Cancer Patients”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Pilot Study of a Hedgehog Pathway Inhibitor (LDE-225) in Surgically Resectable Pancreas Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Study With LDE225 in Combination With Docetaxel in Triple Negative (TN) Advanced Breast Cancer (ABC) Patients (EDALINE)”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014.
“LDE225 in Treating Patients With Stage II-III Estrogen Receptor- and HER2-Negative Breast Cancer”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase II Study of Efficacy and Safety in Patients With Locally Advanced or Metastatic Basal Cell Carcinoma (BOLT)”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“To Evaluate the Safety, Local Tolerability, PK and PD of LDE225 on Sporadic Superficial and Nodular Skin Basal Cell Carcinomas(sBCC)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Trial to Evaluate the Safety, Local Tolerability, Pharmacokinetics and Pharmacodynamics of LDE225 on Skin Basal Cell Carcinomas in Gorlin Syndrome Patients”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Combination of the Hedgehog Inhibitor, LDE225, With Etoposide and Cisplatin in the First-Line Treatment of Patients With Extensive Stage Small Cell Lung Cancer (ES-SCLC)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase III Study of Oral LDE225 Versus (vs) Temozolomide (TMZ) in Patients With Hedge-Hog (Hh)-Pathway Activated Relapsed Medulloblastoma (MB)”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase I Dose Finding and Safety Study of Oral LDE225 in Children and a Phase II Portion to Assess Preliminary Efficacy in Recurrent or Refractory MB”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Phase Ib, Dose Escalation Study of Oral LDE225 in Combination With BKM120 in Patients With Advanced Solid Tumors”.ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Dose Finding and Safety of Oral LDE225 in Patients With Advanced Solid Tumors”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“LDE225 and Paclitaxel in Solid Tumors”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Study of Efficacy and Safety of LDE225 in Adult Patients With Relapsed/Refractory Acute Leukemia”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“Nilotinib and LDE225 in the Treatment of Chronic or Accelerated Phase Myeloid Leukemia in Patients Who Developed Resistance to Prior Therapy”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
“A Phase Ib/II Dose-finding Study to Assess the Safety and Efficacy of LDE225 + INC424 in Patients With MF”. ClinicalTrials.gov. National Institutes of Health. 13 February 2014. Retrieved 16 February 2014.
Jalili, A; Mertz, KD; Romanov, J; Wagner, C; Kalthoff, F; Stuetz, A; Pathria, G; Gschaider, M; Stingl, G; Wagner, SN (30 July 2013). “NVP-LDE225, a potent and selective SMOOTHENED antagonist reduces melanoma growth in vitro and in vivo.” (PDF). PloS one 8 (7): e69064. doi:10.1371/journal.pone.0069064. PMC 3728309.PMID 23935925.
Fendrich, V; Wiese, D; Waldmann, J; Lauth, M; Heverhagen, AE; Rehm, J; Bartsch, DK (November 2011). “Hedgehog inhibition with the orally bioavailable Smo antagonist LDE225 represses tumor growth and prolongs survival in a transgenic mouse model of islet cell neoplasms.”. Annals of Surgery 254 (5): 818–23.doi:10.1097/SLA.0b013e318236bc0f. PMID 22042473.
ChemMedChem, 2013 , vol. 8, 8 p. 1261 – 1265
ACS Med. Chem. Lett., 2010, 1 (3), pp 130–134.
MORE REF

sonidegib

Skin Cancer Foundation. “Skin Cancer Facts.” Available at:http://www.skincancer.org/skin-cancer-information/skin-cancer-facts . Accessed on February 14, 2014.

Rubin AI, Chen EH, Ratner D (2005). Current Concepts: Basal-Cell Carcinoma. N Engl J Med; 353:2262-9.

ClinicalTrials.gov. “A Phase II Study of Efficacy and Safety in Patients With Locally Advanced or Metastatic Basal Cell Carcinoma (BOLT)” Available at:http://clinicaltrials.gov/ct2/show/NCT01327053?term=%22LDE225%22+and+%22BOLT%22&rank=1. Accessed on February 14, 2014.

National Cancer Institute Dictionary of Cancer Terms. “Complete Response.” Available at: http://www.cancer.gov/dictionary?CdrID=45652 . Accessed on February 14, 2014.

National Cancer Institute Dictionary of Cancer Terms. “Partial Response.” Available at: http://www.cancer.gov/dictionary?CdrID=45819 . Accessed on February 14, 2014.

Wong C S M, Strange R C, Lear J T (2003). Basal cell carcinoma. BMJ; 327:794-798.

Copcu E, Aktas A. Simultaneous two organ metastases of the giant basal cell carcinoma of the skin. Int Semin Surg Oncol. 2005;2:1-6. Available at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544837/ . Accessed on February 14, 2014.

Skin Cancer Foundation. “Basal Cell Carcinoma Treatment Options.” Available athttp://www.skincancer.org/skin-cancer-information/basal-cell-carcinoma/bcc-treatment-options . Accessed on February 14, 2014.

Stuetz A, et al. LDE225, a specific smoothened inhibitor, for the topical treatment of nevoid basal cell carcinoma syndrome (Gorlin’s syndrome). Melanoma Research. 2010; 20:e40. Available at:http://journals.lww.com/melanomaresearch/Fulltext/2010/06001/FC24_LDE225,_a_specific_smoothened_inhibitor,_for.87.aspx#FC24_LDE225%2C_a_specific_smoothened_inhibitor%2C_for.87.aspx?s=2&_suid=139234380607909969110518506816.

Novartis.com. “The Pipeline of Novartis Oncology: LDE225.” Available at:http://www.novartisoncology.com/research-innovation/pipeline.jsp #. Accessed on February 14, 2014.

Children’s Medical Research Center, Children’s Memorial Hospital/Northwestern University Feinberg School of Medicine. “The Sonic hedgehog/patched/gli signal transduction pathway.” Available at http://www.childrensmrc.org/iannaccone/gli/ . Accessed on February 14, 2014.

Gupta S, Takebe N, LoRusso P. Targeting the Hedgehog pathway in cancer. Ther Adv Med Oncol. 2010 July; 2(4): 237-250. Available at:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126020/ . Accessed on February 14, 2014.

SONIDEGIB

Links

WO2004078163A2	Feb 26, 2004	Sep 16, 2004	Oern Almarsson	Pharmaceutical co-crystal compositions of drugs such as carbamazepine, celecoxib, olanzapine, itraconazole, topiramate, modafinil, 5-fluorouracil, hydrochlorothiazide, acetaminophen, aspirin, flurbiprofen, phenytoin and ibuprofen
WO2007113120A1	Mar 22, 2007	Oct 11, 2007	Frank Hoffmann	Stamping apparatus with feed device
WO2007131201A2 *	May 4, 2007	Nov 15, 2007	Irm Llc	Compounds and compositions as hedgehog pathway modulators
WO2008154259A1	Jun 4, 2008	Dec 18, 2008	Irm Llc	Biphenylcarboxamide derivatives as hedgehog pathway modulators

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IDRAPARINUX… Sanofi (PHASE III)

Phase 3 drug Comments Off

Feb 072014

IDRAPARINUX

Nonasodium (2S,3S,4S,5R,6R)-6-[(2R,3R,4S,5R,6R)-6-[(2R,3S,4S,5R,6R)-2-carboxy-4,5-dimethoxy-6-[(2R,3R,4S,5R,6S)-6-methoxy-4,5-disulfooxy-2-(sulfooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-disulfooxy-2-(sulfooxymethyl)oxan-3-yl]oxy-4,5-dimethoxy-3-[(2R,3R,4S,5R,6R)-3,4,5-trimethoxy-6-(sulfooxymethyl)oxan-2-yl]oxyoxane-2-carboxylic acid |

CAS number	149920-56-9

Formula	C₃₈H₅₅Na₉O₄₉S₇
Mol. mass	1727.17683 g/mol

CAS 162610-17-5 (free acid)

SANORG34006, SR-34006, SanOrg 34006, SanOrg-34006, UNII-H84IXP29FN, AC1MJ0N4, Org-34006

Methyl O-2,3,4-tri-O-methyl-6-O-sulfo-alpha-D-glucopyranosyl-(1–4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1–4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1–4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1–4)-2,3,6-tri-O-sulfo-alpha-D-glucopyran

Sanofi-Syn(Originator), Organon (Codevelopment), PHASE 3

methyl O-2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-O-di-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]D²⁰ = +46.2° (c=1; water). Anomeric protons chemical shifts: 5.43; 5.37; 5.16; 5.09; and 5.09 ppm.

Idraparinux sodium, or methyl O-2,3,4-tri-O-methyl-6-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronate sodium-(1→4)-O-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranose, is a pentasaccharide with antithrombotic activity.

The preparation of idraparinux by sulfatation of a deprotected pentasaccharide is described in Bioorganic & Medicinal Chemistry, 1994, Vol. 2, No. 11, pp. 1267-1280, and also in patent EP 0 529 715 B1.

Idraparinux sodium is an anticoagulant medication in development by Sanofi-Aventis.^[1]

It has a similar chemical structure and the same method of action as fondaparinux, but with an elimination half-life about five to six times longer (an increase from fondaparinux’s 17 hours to approximately 80 hours), which means that the drug should only need to be injected once a week.

As of July 2007, it has completed the Phase III clinical trial AMADEUS.

Idraparinux selectively blocks coagulation factor Xa.^[2]

See Heparin: Mechanism of anticoagulant action for a comparison of the mechanism of heparin, low-molecular-weight heparins, fondaparinux and idraparinux.

Idraparinux sodium is a synthetic pentasaccharide with indirect coagulation factor Xa inhibitor activity. The drug candidate had been in phase III clinical development at Sanofi (formerly known as sanofi-aventis) for the once-weekly long-term treatment and secondary prevention of venous thromboembolic events in patients with pulmonary embolism (PE) and deep vein thrombosis (DVT), as well as for the prevention of thromboembolic complications related to atrial fibrillation (AF).

However, no recent development has been reported for this research. The oligosaccharide is delivered by subcutaneous injection. Unlike other products, idraparinux is administered once weekly rather than daily, thereby increasing patient convenience.

Originally developed under a collaboration between sanofi-sventis and Akzo Nobel’s human healthcare business Organon, all rights to idraparinux were transferred to Sanofi in January 2004 in exchange for revenues based on future sales.

IDRAPARINUX

Several synthetic pentasaccharides have been developed, such as Idraparinux, where all hydroxyl groups are methylated or sulphated, as illustrated below:

Initially, the firm Organon developed a way of synthesis for the preparation of the “active pentasaccharide”. This synthesis, using the 3-0-benzyl-1 ,2-0-isopropylidene-a-D- glucofuranose as substrate (Van Boeckel et al., J. Carbohydr. Chem. 1985, 4, p.293-321 ), comprises more than 50 steps, and the inversion of configuration of the C5 carbon is carried out by the opening of an epoxide. After a step of protection followed by a bromination, the G unit is thus obtained. It is well known that the synthesis of said G unit is very tedious, due to the number of steps for obtaining such unit and the known tendency of L-idose derivatives to exist as furanoses. After being coupled to the H unit, successive steps of protection-deprotection then an oxidation reaction carried out on C6 carbon, lead to the GH disaccharide.

In the preparation of Idraparinux, the synthesis of the disaccharide GH is nearly similar to the above synthesis of early synthetic pentasaccharides. The major innovation lies in the obtaining of disaccharide EF by epimerization of disaccharide GH. The coupling of both disaccharides leads to the tetrasaccharide EFGH, which is further coupled to the D unit for obtaining said pentasaccharide. The preparation of the disaccharide EF from GH allows notably the decrease of the total number of the steps to approximatively 25 (Petitou, M.; Van Boeckel, C.A. Angew. Chem., Int.Ed. 2004, 43, p.31 18-3133).

Hence, all current syntheses of the “active pentasaccharide” comprise a large number of steps and more particularly involves the complex synthesis of key L-iduronic acid derivative (G unit). Indeed, the preparation of the G unit of the “active pentasaccharide” of heparin has always been a limiting step in the synthesis of antithrombotic heparin derivatives.

Thus, there is still a need for a new efficient process of preparation of L-iduronic acid derivative, which would not possess the drawbacks established above and would be compatible with industrial scales. Besides, there is a need for such process which would in addition lead to an improved process of preparation of the “active pentasaccharide” constituting the heparin derivatives.

Idrabiotaparinux, developed by sanofi-aventis, is the biotinylated pentasaccharide corresponding to the structure depicted below. The pentasaccharide structure of idrabiotaparinux is the same as idraparinux, another antithrombotic agent developed by sanofi-aventis (see structure below). However in idrabiotaparinux, the presence of a biotin hook covalently linked to the first saccharidic unit enables the compound to be neutralized by avidin or streptavidin, as described in the international patent application WO 02/24754 .
In the EQUINOX trial, which enrolled patients with DVT treated for 6 months with equimolar doses of either idrabiotaparinux or idraparinux, idrabiotaparinux, with the same anti-activated factor X pharmacological activity (hereafter “anti-Xa activity”) as idraparinux, was shown to have a similar efficacy, but, surprisingly, a better safety with less observed bleedings, in particular major bleedings.
Therefore, the subject-matter of the invention is the use of idrabiotaparinux for the manufacture of a medicament useful for the treatment and secondary prevention of thrombotic pathologies, wherein the use of idrabiotaparinux involves a decrease in the incidence of bleedings during said treatment.
In other words, the invention relates to the use of idrabiotaparinux as an antithrombotic treatment, wherein said use minimizes the risk of bleedings during the antithrombotic treatment. Indeed, idrabiotaparinux enables to increase the benefit-risk ratio during the antithrombotic treatment.

The L-ioduronic acid methyl ester derivative (XII) is then converted into its D-glucuronic acid methyl ester counterpart (XIII) by epimerization with NaOMe in refluxing MeOH, followed by esterification with MeI and KHCO3 in DMF.

Protection of the ester (XIII) with levulinic acid (IX) by means of DCC and DMAP in dioxane, followed by acetolysis of the anomeric center with sulfuric acid in acetic anhydride furnishes the disaccharide (XIV), which is then saponified with piperidine and subjected to reaction with trichloroacetonitrile and Cs2CO3 in THF to yield the imidate (XV).

Glycosylation of the disaccharide (XII) with the imidate (XV) by means of trimethylsilyl triflate in CH2Cl2, followed by removal of the levulinoyl group by means of hydrazine acetate, furnishes the tetrasaccharide (XVI), which is coupled with the glucosyl trichloroacetimidate (XVIII) by means of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 providing the pentasaccharide (XVII).

Glucosyl imidate (XVIII) is prepared by methylation of 1,6-anhydroglucose (XIX) with MeI and NaH in DMF, followed by acetolysis with Ac2O/TFA to give compound (XX), which is treated with piperidine in THF and finally with trichloroacetonitrile in dichloromethane in the presence of Cs2CO3.

The pentasaccharide (XVII) is deprotected by saponification with LiOH in THF/H2O2, and then hydrogenated over Pd/C in tert-butanol/water to provide a fully deprotected pentamer, which is finally subjected to sulfation with triethylamine sulfur trioxide complex in DMF and converted into the corresponding sodium salt by elution in a Dowex 50 XW4-Na+ or a Mono-Q anion-exchange column.

……………..

Glycosylation of sugar (I) with the idopyranosyl fluoride (II) by means of BF3.Et2O and molecular sieves in dichloromethane gives the disaccharide fragment (III), which is then converted into acetonide (V) by saponification of the ester functions with t-BuOK, followed by reaction with 2,2-dimethoxypropane (IV) in DMF and acidification with p-toluensulfonic acid. Methylation of acetonide (V) with MeI and NaH in DMF/MeOH provides the disaccharide (VI), which is then treated with HOAc to yield the 4′,6′-diol (VII). Selective silylation of the diol (VII) with tert-butyldimethylsilyl chloride (TBDMSCl) in pyridine leads to the 6′-O-TBDMS derivative (VIII), which is condensed with levulinic acid (IX) by means of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in dioxane to give the ester (X). Compound (X) is then submitted to simultaneous Jones oxidation and TBDMS removal with CrO3 and H2SO4/H2O in acetone to provide the iduronic acid derivative (XI), which is converted into the key intermediate (XII), first by esterification with MeI and KHCO3 in DMF and then by removal of the 4′-O-levulinoyl protecting group with HOAc and hydrazine hydrate in pyridine.

………………………

US20120041189

A crystalline form of the pentasaccharide methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose has now been isolated. This compound in its crystalline form has proven to be very useful for the preparation of idraparinux, since it makes it possible to obtain this product in a particularly interesting chemical yield and with a significant gain in quality, the purity being improved as regards the crude product obtained, as will be detailed hereinbelow. These gains in reaction yield and in purity for the production of idraparinux are considerable advantages from an industrial viewpoint, since improving the robustness of a process is a constant cause for concern, especially in the case of large-scale syntheses.

One subject of the invention is thus the compound methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic glucopyranose in crystalline form.

Methyl O-2,3,4-tri-O-methyl-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-β-D-glucopyranosyluronic acid-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranosyluronic acid-(1→4)-O-α-D-glucopyranose, referred to hereinbelow as the compound of formula (I), corresponds to the following formula:

The compound of formula (I) in crystalline form according to the invention has a powder X-ray diffractogram whose characteristic lines are approximately at 12.009; 7.703; 7.300; 7.129; 5.838; 4.665; 4.476 and 3.785 angströms (interplanar distances). It also has a melting point of about 203° C. (203° C.±1° C.).

EXAMPLE 1 Preparation of the Compound of Formula (I) in Crystalline Form (Scheme 1)

1.1: Preparation of the Compound of Formula (I′)

The compound of formula (I″) is obtained, for example, according to the teaching of patent EP 0 529 715 B1 or of the articles “Bioorg. Med. Chem.” (1994, Vol. 2, No. 11, pp. 1267-1280), “Bioorg. Med. Chem. Letters” (1992, Vol. 2, No. 9, pp. 905-910) or “Magnetic Resonance in Chemistry” (2001, Vol. 39, pp. 288-293). The compound of formula (I″) (5 g, 3.06 mmol) is dissolved in acetonitrile (10 mL). Deionized water (12.2 mL) and aqueous 30% sodium hydroxide solution (4.1 g) are then added. The mixture is heated to 40° C. and maintained at this temperature for 5 hours. The reaction medium is then cooled to 20° C. and acidified to pH 6.25 with aqueous 1N hydrochloric acid solution (about 17.7 g) before extraction with MTBE of certain impurities, the saponified product remaining in the aqueous phase. The residual acetonitrile, contained in the aqueous phase, is then removed by concentration, followed by diluting with deionized water (125 mL). The saponified product is finally precipitated at pH 1.5 by adding aqueous 1N hydrochloric acid solution (about 17.6 g) at 20° C. The suspension is maintained for 4 hours at 20° C. before filtration. The wet solid is finally dried in a vacuum oven at 30° C. to give 2.93 g (93.6%) of compound of formula (I).

NMR (anomeric protons of the saccharide units D, E, F, G, H): 5.79, 5.14, 5.55, 5.92, 4.94 ppm.

1.2 Preparation of the Crude Compound of Formula (I)

The compound of formula (I′) obtained after the preceding step is dissolved in tetrahydrofuran (18 mL). Palladium-on-charcoal (0.3 g) is added. The reaction medium is hydrogenated at 0.3 bar of hydrogen (relative pressure) for 4 hours. After filtering and evaporating, 2.12 g (99%) of the crude compound of formula (I) are obtained.

1.3: Preparation of the Compound of Formula (I) in Crystalline Form Using an Isopropanol/MTBE Mixture

The crude hydrogenated product obtained after the preceding step is dissolved in isopropanol (13 mL) at 65° C., and then crystallized at room temperature. The suspension is then cooled to 40° C., followed by addition of MTBE (13 mL), and is then cooled slowly to 10° C. After maintenance at 10° C. for 2 hours, the crystalline hydrogenated product is filtered off, washed and dried. 1.66 g of the compound of formula (I) in crystalline form are thus obtained, in the form of a cream-white powder. The reaction yield for the production of the compound of formula (I) in crystalline form, from the compound of formula (I′), is 92.5%. When expressed relative to the starting compound (I″), the reaction yield for the production of the compound of formula (I) in crystalline form is 86.6%.

NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (I) in crystalline form: 5.77, 5.11, 5.51, 5.84, 5.01 ppm.

1.4: Preparation of the Compound of Formula (I) in Crystalline Form Using Isopropanol

The crude hydrogenated product obtained after step 1.2 is dissolved in isopropanol (5 volumes) at 75° C. The medium is then cooled slowly until crystals appear, according to the known standard techniques for crystallization. The process is performed, for example, by a first step of cooling at 65° C. for 1 hour, and than a second step of cooling to a final temperature of 25° C. over 4 hours or of 5° C. over 6 hours, and finally maintenance at this final temperature for 30 minutes. The suspension is then filtered and rinsed with isopropanol (2×0.1 V) and compound (I) is isolated in the form of white crystals, which appear under a microscope in the form of needles. The ¹H NMR analysis of these crystals is identical to that described after step 1.3 above.

EXAMPLE 4 Preparation of Idraparinux from the Compound of Formula (I) in Crystalline Form (Scheme 2)

The preparation of idraparinux (II) from the compound of formula (I) is summarized in Scheme 2.

The compound of formula (I) in crystalline form, as obtained according to Example 1.3, is dissolved in N,N′-dimethylformamide (6.6 mL) and then heated to 30° C. Under an inert atmosphere, 3.8 g of pyridine-sulfur trioxide complex are added slowly, followed by maintenance at 30° C. for 4 hours. The reaction medium is then poured into aqueous 23.8% sodium hydrogen carbonate solution (16.3 g) maintained at a maximum of 25° C., to obtain the compound of formula (II). The reaction medium is kept stirring for hours. The solution of sulfated product is then poured onto an MTBE/isopropanol/ethanol mixture (171 mL/70 mL/70 mL). Precipitation of the product is observed, and, after filtering off, washing and drying the cake, 4.99 g (96.8%) of compound of formula (II) are obtained, and are then purified by anion-exchange chromatography according to the usual techniques.

NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (II): 5.48, 4.68, 5.44, 5.08, 5.18 ppm.

It thus appears that the process according to the invention makes it possible to obtain idraparinux (compound of formula (II)) in a chemical yield of about 84% (precisely 83.8% according to the protocols described above) starting from the compound of formula (I″), i.e. a gain in yield of about 30% relative to the process described in patent EP 0 529 715 B1.

………………..

EP0529715A1

methyl O-2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-di-O-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-2,3-O-di-methyl-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]_D²⁰ = +46.2° (c=1; water). Anomeric protons chemical shifts: 5.43; 5.37; 5.16; 5.09; and 5.09 ppm.

WAS PREPARED AS PER

Example 3

methyl O-4-O-(4-sulfoaminophenyl)-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt.

NOTE THIS IS ANALOGOUS PROCEDURE AND NOT SIMILAR

Methyl O-4-O-(4-nitrophenyl)-6-O-acetyl-2,3-O-di-phenylmethyl-α-D-glucopyranosyl-(1→4)-O-(methyl 3-O-methyl-2-O-acetyl-α-L-idopyranosyluronate)-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranoside (100 mg, 0.09 mmol), obtained by the known imidate coupling of the trichloroacetimidate of O-4-O-(4-nitrophenyl)-6-O-acetyl-2,3-O-di-phenylmethyl-α-D-glucopyranoside and methyl O-(methyl 3-O-methyl-2-O-acetyl-α-L-idopyranosyluronate)-(1→4)-O- 2,3,6-tri-O-acetyl-α-D-glucopyranoside, was dissolved in tetrahydrofuran (9 ml) and cooled to -5 °C. At this temperature a 30% aq. solution of hydrogen peroxide (4.5 ml) was added to the reaction mixture, and after 10 min a 1.25 M lithium hydroxide solution (4.7 ml) was added. The mixture was stirred for 1 h at -5 °C, after which time the temperature was raised to 0 °C and the mixture was stirred overnight. The reaction mixture was acidified with 6N hydrogen chloride at 0 °C to pH 1.5, after which the saponified compound was extracted with ethyl acetate. The organic layers were pooled, dried over magnesium sulfate, and evaporated to give 63 mg (84%) of methyl O-4-O-(4-nitrophenyl)-2,3-O-di-phenylmethy1-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside, which was dissolved in methanol (8 ml). 10% Pd on charcoal (63 mg) was added and the mixture hydrogenolyzed overnight. After filtration and evaporation 27 mg (50%) of methyl O-4-O-(4-aminophenyl)-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside were obtained.
13 mg of methyl O-4-O-(4-aminophenyl)-O-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-α-L-idopyranuronosyl-(1→4)-O-α-D-glucopyranoside were dissolved in 2 ml of dry N,N-dimethylformamide, and under an atmosphere of nitrogen 148 mg of triethylamine sulfurtrioxide complex were added. The mixture was stirred overnight at 50 °C, after which an aq. solution of sodium hydrogen carbonate was added under ice cooling. The mixture was stirred for 1 h at room temperature, concentrated to a small volume and desalted on a Sephadex G-10 column with water. The crude product obtained was purified by HPLC using a Mono-Q anion exchange column to give 11 mg (37%) of methyl O-4-O-(4-sulfoaminophenyl)-2,3,6-tri-O-sulfo-α-D-glucopyranosyl-(1→4)-O-3-O-methyl-2-O-sulfo-α-L-idopyranuronosyl-(1→4)-O-2,3,6-tri-O-sulfo-α-D-glucopyranoside nonakis sodium salt. [α]_D²⁰ = +52.2° (c=0.67; water). Anomeric protons chemical shifts: 5.5; 5.17; and 5.15 ppm.

………………………………..

BMCL Volume 19, Issue 14, 15 July 2009, Pages 3875–3879

http://www.sciencedirect.com/science/article/pii/S0960894X0900482X

Full-size image (16 K)

Full-size image (18 K)

Final elaboration of the pentasaccharide 1. Reagents and conditions: (a) TMSOTf, Et₂O, 4 Å MS, rt, 66% (28α), 15% (28β); (b) CAN, CH₃CN, toluene, H₂O, rt, 72%; (c) CCl₃CN, DBU, CH₂Cl₂, rt, 98%; (d) TMSOTf, 4 Å MS, CH₂Cl₂, rt, 51% (73% based on recovery of 4); (e) Pd/C (10%), H₂, t-BuOH, H₂O, rt; (f) SO₃·Et₃N, DMF, 50 °C, 93% (2 steps).

The final elaboration of the pentasaccharide 1 was illustrated in IN ABOVE SCHEME Coupling of the glucopyranosyl trichloroacetimidate 6 with disaccharide acceptor 5 in the presence of trimethylsilyl trifluoromethylsulfonate and powdered 4 Å molecular sieves at room temperature in diethyl ether afforded the desired α-coupled trisaccharide 28α in a yield of 66%, together with 15% of the separable β-coupled product 28β. The anomeric 4-methoxyphenyl group in trisaccharide 28α was removed with CAN, and the resulting lactol was readily converted into the trisaccharide trichloroacetimidate 3. Coupling of donor 3 with the disaccharide acceptor 4 in the presence of trimethylsilyl trifluoromethylsulfonate and powdered 4 Å molecular sieves at room temperature in dichloromethane afforded the fully protected pentasaccharide 2 in 51% yield (73% based on recovery of 4). Finally, pentasaccharide 2 was subject to hydrogenolysis of the benzyl protecting groups. The highly polar product without purification was O-sulfated directly with triethylamine-sulfur trioxide complex to afford the sulfated pentasaccharide 1 in an excellent yield of 93% (for two steps).

Summarizing, the potent anti-thromboembolic pentasaccharide Idraparinux (1) was synthesized in total 51 steps and in 4% overall yield from d-glucose and methyl α-d-glucopyranoside.¹⁸ The synthetic route is convergent with a linear sequence of 27 steps, and the transformations are scalable. The 4-methoxyphenol glycoside intermediates are easy to be purified by crystallization.

Compound 1: 54.2 (c 1.0, H₂O);

¹H NMR (400 MHz, D₂O) δ 3.27 (t, J = 8.4 Hz, 1H), 3.30–3.38 (m, 2H), 3.47 (s, 3H), 3.53 (s, 3H), 3.56 (s, 6H), 3.58 (s, 3H), 3.62 (s, 3H), 3.63 (s, 3H), 3.64 (s, 6H), 3.75 (d, J = 10.0 Hz, 1H), 3.83–3.97 (m, 4H), 3.98 (t, J = 8.8 Hz, 1H), 4.06–4.18 (m, 3H), 4.19–4.45 (m, 8H), 4.56 (br t, J = 9.6 Hz, 1H), 4.65 (t, J = 9.2 Hz, 1H), 4.66 (d, J = 7.6 Hz, 1H), 5.00 (br s, 1H), 5.11 (br s, 1H), 5.17 (d, J = 3.6 Hz, 1H), 5.43 (d,J = 3.2 Hz, 1H), 5.47 (d, J = 4.0 Hz, 1H);

ESI-MS m/z 774.1 [M−8Na+6H]²⁻, 763.0; [M−9Na+7H]²⁻, 508.5 [M−9Na+6H]³⁻.

………………….

WO2013050497A1

The process of preparation of Idraparinux having the following formula:

may comprise the following steps :

1 ) preparation a compound of formula (IXB)

(IXB) wherein R_a is methyl, R_b is methyl, R_c is methyl, T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl, by the process according to the invention;

2) epimerisation of the disaccharide (IXB) so as to form disaccharide D of formula :

3) protection of the 4′-OH of D with a levulinoyl ester;

4) acetolysis of the disaccharide resulting from step 3), followed by preparation of the corresponding imidate;

5) coupling the disaccharide imidate resulting from step 4) with (IXB) obtainable by the process of the invention, wherein R_a is methyl, R_b is methyl, R_c is methyl, T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl, to obtain a tetrasaccharide;

6) coupling the fully protected tetrasaccharide with a monosaccharide glycosyl imidate;

7) deprotection of the protecting groups by the successive saponification and hydrogenolysis;

8) sulfation of the hydroxyl groups.

In one embodiment, the present invention concerns a process of preparation of Idraparinux:

said process comprising the following steps:

preparation of a compound of formula (VI) such as defined above, from a compound of formula (V) such as defined above; preparation of a compound of formula (VII) such as defined above, from a compound of formula (VI) such as defined above;

preparation of a compound of formula (VIII) such as defined above, from a compound of formula (VII) such as defined above;

– preparation of a compound of formula (IX) such as defined above, from a compound of formula (VIII) such as defined above;

wherein in compounds of formulae (V), (VI), (VII), (VIII) and (IX), R-i , R₂, R3 and X are as defined above, R_a is methyl, R_b is methyl, R_c is methyl, R_d is methyl and R’ is the monosaccharide of formula :

wherein T-i is benzyl, T₂ is benzyl, T₃ is benzyl and T is methyl. The inventors advantageously found that the process of preparation of Idraparinux comprising the decarboxylation/intramolecular cyclisation tandem reaction, which allows the inversion of configuration of C5 carbon of the compound of formula (VI), is more efficient than the processes previously described in the literature. Indeed, the process according to the invention allows advantageously a significant decrease of the number of steps and thus an improvement of the overall yield. Thus, the process of preparation of Idraparinux may be carried out in industrial scales. The inventors found an efficient process of preparation of Idraparinux.

According to another object, the present invention concerns the use of compounds of formulae (V), (VI), (VI I), (VIII) and (IX), as intermediates for the preparation of Idraparinux. In particular, the present invention concerns the use of a compounds of formulae (VB), (VI B), (VI IB), (VI I IB) and (IXB), as intermediates for the preparation of Idraparinux The invention is further illustrated but not restricted by the description in the following examples. Example 1 :

Preparation of Methyl-4,6-0-benzylidene-a-D-glucopyranoside (la)

CHC1₃)

T_f = 166-167°C (litt. 165-166°C) To a solution of benzaldehyde (400 mL, 3.94 mol, 5.9 eq.) was added zinc chloride (100.3 g, 0.74 mol, 1 .1 eq.) under vigorous stirring. After homogenization of the solution methyl- a-D-glucopyranoside (129.6 g, 0.67 mol, 1.0 eq.) was added portionwise. After 16 hours stirring at room temperature the reaction mixture was diluted with diethyl ether (100 mL). The mixture was then poured dropwise and under vigorous stirring in a solution containing ice water (1 .5 L) and hexane (350 mL). The precipitate was filtered, washed with diethyl ether (3 x 300 mL) and dried under vacuum over KOH. The product was then recrystallised from CH₂CI₂ (720 mL) and washed with a Et₂0/CH₂CI₂ solution (75:25, 2 x 200 mL). The filtrate was repeatedly recrystallised five times from CH₂CI₂ to afford compound la as white crystals (136.97 g, 0.49 mol, 72%).

¹H NMR (CDCI3, 250 MHz): δ 2.35 (d, J_CH-O_H = 9.2 Hz, 1 H, OH), 2.83 (d, J_CH-O_H = 2.2 Hz, 1 H, OH), 3.46 (s, 3H, -OCH₃), 3.43-3.46 (m, 1 H, H-4), 3.63 (td, J_CH–_OH = ^_2,3 = 9.2 Hz, J_{1 2} = 3.9 Hz, 1 H, H-2), 3.70-3.81 (m, 2H, H-5, H-6), 3.93 (td, J = 9.2 Hz, J_CH–_OH = 2.2 Hz, 1 H, H- 3), 4.29 (m, 1 H, H-6 ), 4.79 (d, J_1i2 = 3.9 Hz, 1 H, H-1 ), 5.54 (s, 1 H, Ph-CH), 7.35-7.38 (m, 3H, H_Ar), 7.47-7.51 (m, 2H, H_Ar).

¹³C NMR (CDCI₃, 62.9 MHz): δ 55.6 (-OCH₃), 62.5 (C-5), 69.0 (C-6), 71.1 (C-3), 72.9 (C- 2), 81 .0 (C-4), 99.9 (C-1 ), 102.0 (Ph-CH), 126.4, 128.5, 129.4, 137.1 (6xC_Ar). IR (film) v (cm^“1): 3369 (O-H). Preparation of Methyl-2,3-di-0-methyl-4,6-0-benzylidene-a-D-glucopyranoside (Ma)

1₃)

°C)

To a solution of compound la (47.60 g, 0.17 mol, 1.0 eq.) in anhydrous THF (750 mL) under an argon atmosphere and cooled to 0°C, was added portionwise sodium hydride (60%, 16.93 g, 0.42 mol, 2.5 eq.). After 20 minutes methyl iodide was added dropwise (30 mL, 0.48 mol, 2.8 eq.) and the reaction mixture was allowed to reach room temperature. After 16 hours, methanol was added portionwise (75 mL) and the solution was stirred for another 15 minutes before being concentrated. The resulting residue was dissolved in EtOAc (400 mL) and washed with water (2 x 250 mL). The organic layer was dried (MgS0₄), filtered and concentrated. The resulting solid was dissolved in diethyl ether (1000 mL), hexane was added (400 mL) and the solvent was partially evaporated at low temperature. The crystals obtained were washed with hexane and the filtrate once again partially evaporated, filtered and the precipitate washed with hexane. The combined precipitates afforded compound Ma as white crystals (46.10 g, 0.15 mol, 88%).

¹H NMR (CDCI₃, 250 MHz): δ 3.30 (dd, J_2-3 = 9.1 Hz, J_1-2 = 3.7 Hz, 1 H, H-2), 3.44 (s, 3H, – OCH₃), 3.49-3.87 (m, 4H, H-4, H-5, H-6, H-3), 3.55 (s, 3H, -OCH₃), 3.64 (s, 3H, -OCH₃), 4.28 (dd, J_6-6. = 9.1 Hz, J_5-6 = 3.7 Hz, 1 H, H-6 ), 4.85 (d, J_1-2 = 3.7 Hz, 1 H, H-1 ), 5.54 (s, 1 H, Ph-CH), 7.36-7.41 (m, 3H, H_Ar), 7.48-7.52 (m, 2H, H_Ar).

¹³C NMR (CDCI₃, 62.9 MHz): δ 55.4, 59.5, 61.1 (3x-OCH₃), 62.3 (C-5), 69.1 (C-6), 79.9 (C-3), 81 .5 (C-4), 82.2 (C-2), 98.5 (C-1 ), 101.4 (Ph-CH), 126.2, 128.3, 129.0, 137.4 (6xC_Ar).

Preparation of Methyl-2,3-di-0-methyl-a-D-glucopyranoside (Ilia)

1₃)

To a suspension of compound Ma (10.33 g, 33.29 mmol, 1.0 eq.) in methanol (150 mL) was added para-toluenesulfonic acid monohydrate (322 mg, 1 .69 mmol, 0.05 eq.). After 4 hours stirring at room temperature, sodium carbonate (300 mg) was added and the reaction mixture was stirred an additional 15 minutes before filtration through a pad of Celite^®. Then the filtrate was concentrated and the residue obtained was dissolved in a mixture of distilled water/diethyl ether (3:1 , 150 mL). The organic layer was extracted with water (2 x 50 mL) then the combined aqueous phases were concentrated and dried one night under vacuum over KOH. The resulting residue was recrystallised from toluene using petroleum ether as a co-solvent. The crystals obtained were washed with hexane and dried under vacuum over KOH to obtain compound Ilia as white crystals (6.63 g, 29.83 mmol, 90%).

¹H NMR (DMSO, 400 MHz): δ 3.03 (dd, J_2-3 = 9.3 Hz, J_1-2 = 3.5 Hz, 1 H, H-2), 3.12-3.20 (m, 2H, H-3, H-4), 3.27 (s, 3H, -OCH₃), 3.32 (s, 3H, -OCH₃), 3.30-3.33 (m, 1 H, H-5), 3.44 (s, 3H, -OCH3), 3.40-3.46 (m, 1 H, H-6), 3.62 (ddd, J_6-6‘ = 1 1.6 Hz, J_CH-OH = 5.7 Hz, J_5-6 = 1 ,9 Hz, 1 H, H-6′), 4.52 (t, J = 5.7 Hz, 1 H, OH), 4.78 (d, Ji_-2 = 3.5 Hz, 1 H, H-1 ), 5.09 (d,

¹³C NMR (DMSO, 100.6 MHz): δ 54.1 , 57.4, 60.0 (3x-OCH₃), 60.6 (C-6), 69.5 (C-3), 72.5 (C-5), 80.9 (C-2), 82.8 (C-4), 96.4 (C-1 ).

IR (film) v (cm^“1): 3419 (O-H).

Preparation of Methyl methyl-2,3-di-0-methyl-a-D-glucopyranosiduronate (IVa)

C1₃)

To a solution of compound Ilia (500 mg, 2.25 mmol, 1.0 eq.) in distilled water (15 mL) were successively added NaBr (50 mg, 0.49 mmol, 0.2 eq.) and TEMPO (7 mg, 0.05 mmol, 0.02 eq.). The reaction mixture was cooled with the aid of an ice bath then a solution of NaOCI (13% v/v, 5.2 mL, 9.1 mmol, 4.0 eq.) was added. After 5 hours stirring at 0°C ethanol was added (96% v/v, 8 mL), then the pH was reduced to 2-3 by addition of HCI (10 % v/v). The solvent was evaporated and the residue obtained was suspended in methanol, filtered in order to remove the remaining salts and washed several times with dichloromethane and methanol. The filtrate was concentrated then dissolved, under an argon atmosphere, in dry methanol (40 mL). para-toluenesulfonic acid (85 mg, 0.45 mmol, 0.2 eq.) was added then the reaction mixture was heated under reflux overnight. The solvent was evaporated and the residue obtained was dissolved in EtOAc (60 mL). The organic layer was washed with a 5% aqueous NaHC0₃ solution (2 ^χ 20 mL) and with brine (1 ^χ 20 mL). The aqueous phase was extracted with dichloromethane (3 ^χ 20 mL). The combined organics were dried (MgS0₄), filtered and evaporated. Column chromatography (hexane/ethyl acetate 50:50) gave compound IVa as a colourless oil (503 mg, 2.00 mmol, 89%).

¹H NMR (CDCIs, 400 MHz): δ 3.10 (d, J_CH-OH = 3.0 Hz, 1 H, OH), 3.26 (dd, J_2-3 = 9.3 Hz, J_{1 -2} = 3.4 Hz, 1 H, H-2), 3.47 (s, 3H, -OCH₃), 3.49-3.52 (m, 1 H, H-3), 3.50 (s, 3H, -OCH₃), 3.62 (s, 3H, -OCH₃), 3.74 (td, J = 9.5 Hz, J_CH-OH = 3.0 Hz, 1 H, H-4), 3.82 (s, 3H, -OCH₃), 4.14 (d, J4.5 = 9.6 Hz, 1 H, H-5), 4.91 (d, Ji_-2 = 3.4 Hz, 1 H, H-1 ).

¹³C NMR (CDCI₃, 100.6 MHz): δ 52.9, 56.0, 59.1 , 61 .3 (4x-OCH₃), 70.6 (C-5), 71.7 (C-4), 80.9 (C-2), 81.8 (C-3), 98.1 (C-1 ), 170.9 (C=0).

IR (film) v (cm^“1): 3475 (O-H), 1750 (C=0).

Preparation of Methyl methyl^-O-il’-ethoxy^’-propyn-l’-ylJ^.S-di-O-methyl-a-D- glucopyranosiduronate (Va)

To a solution of compound IVa (4.56 g, 18.21 mmol, 1.0 eq.) in chloroform (stabilised with amylene, 200 mL) were added, under an argon atmosphere, P₂0₅ (5.31 g, 36.29 mmol, 2.0 eq.) and propargylaldehyde diethylacetal (5.2 mL, 36.27 mmol, 2.0 eq.), then the reaction mixture was heated at 60°C. After 4 hours stirring, the reaction mixture was filtered through a pad of Celite^® then the solvent was removed under vacuum. The crude mixture was suspended in EtOAc (300 mL), washed with a 5% NaHC0₃ aqueous solution (1 x 30 mL) and brine (1 x 30 ml_). The organic layer was dried (MgS0₄), filtered, and evaporated. Column chromatography (gradient hexane/ethyl acetate 80:20 – 20:80) afforded compound Va as a colourless oil (4.07 g, 12.24 mmol, 67%) in a diastereomeric mixture (64:36) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons EtO-CH), along with some unreacted compound IVa (1.17 g, 4.68 mmol, 26%).

¹H NMR (CDCI₃, 400 MHz): δ 1.18-1.25 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 2.56 (m, 1 H, H-C≡C-) (mixture), 3.26-3.31 (m, 1 H, H-2) (mixture), 3.43 (s, 3H, -OCH₃) (major), 3.44 (s, 3H, -OCH₃) (minor), 3.50 (s, 3H, -OCH₃) (mixture), 3.59 (s, 3H, -OCH₃) (minor), 3.62 (s, 3H, -OCH₃) (major), 3.47-3.62 (m, 2H, H-3, -OCH_aH_bCH₃) (mixture), 3.65-3.73 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.78 (s, 3H, -OCH₃) (major), 3.80 (s, 3H, -OCH₃) (minor), 3.78-3.86 (m, 1 H, H-4) (mixture), 4.15 (d, J_4-5 = 10.0 Hz, 1 H, H-5) (major), 4.18 (d, J_4-5 = 10.0 Hz, 1 H, H-5) (minor), 4.86-4.88 (m, 1 H, H-1 ) (mixture), 5.35 (d, J = 1.7 Hz, 1 H, EtO- CH) (minor), 5.58 (d, J = 1.7 Hz, 1 H, EtO-CH) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 15.0 (-OCH₂CH₃) (mixture), 52.6 (-OCH₃) (major), 52.7 (- OCH₃) (minor), 55.8 (-OCH₃) (mixture), 59.2 (-OCH₃) (major), 59.3 (-OCH₃) (minor), 60.4 (-OCH₂CH₃) (major), 61 .3 (-OCH₂CH₃) (minor), 61.4 (-OCH₃) (mixture), 70.1 (C-5) (minor), 70.2 (C-5) (major), 74.0 (H-C≡C-) (major), 74.2 (H-C≡C-) (minor), 76.4 (C-4) (minor), 76.7 (C-4) (major), 78.6 (H-C≡C-) (minor), 78.9 (H-C≡C-) (major), 81 .5 (C-2) (major), 81 .8 (C-2) (minor), 81.9 (C-3) (minor), 82.9 (C-3) (major), 92.6 (EtO-CH) (mixture), 97.9 (C-1 ) (minor), 98.0 (C-1 ) (major), 169.6 (C=0) (major), 169.9 (C=0) (minor). Elemental analysis: Calculated: C: 54.21 ; H: 7.28. Found: C: 54.17 ; H: 7.13.

ESI-MS (pos. mode): m/z = 355 [M+Na]⁺.

IR (film) v (cm^“1): 1752 (C=0), 3266 (≡C-H). Preparation of 4-0-(1′-ethoxy-2′-propyn-1^,-yl)-1 ,2,3-tri-0-methyl-a-D-gluco- pyranosiduronic acid (Via)

To a solution of compound Va (1.12 g, 3.37 mmol, 1.0 eq.) in EtOH/H₂0 (3:1 , 100 mL) was added sodium hydroxide (156 mg, 3.90 mmol, 1 .3 eq.). After 5 hours stirring at room temperature the solvent was evaporated. The residue obtained was dissolved in water (50 mL). The pH of the aqueous layer was reduced to 2-3 with a 5% citric acid aqueous solution, then the layer was saturated with sodium chloride before extraction with dichloromethane (10 x 20 mL). If necessary the pH was adjusted by addition of more citric acid aqueous solution. The combined organics were dried (MgS0₄), filtered and removed under vacuum. Compound Via was obtained without further purification as a colourless oil (1.020 g, 3.20 mmol, 95%), in a mixture of diastereomers (75:25) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons EtO-CH).

¹H NMR (CDCIs, 400 MHz): δ 1.16-1.24 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 2.59 (d, J = 1 .6 Hz, 1 H, H-C≡C-) (major), 2.62 (s I, 1 H, H-C≡C-) (minor), 3.25-3.33 (m, 1 H, H- 2), 3.44 (s, 3H, -OCH₃) (mixture), 3.51 (s, 3H, -OCH₃) (mixture), 3.62 (s, 3H, -OCH₃) (mixture), 3.54-3.62 (m, 2H, H-3, -OCH_aH_bCH₃) (mixture), 3.68-3.77 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.81-3.87 (m, 1 H, H-4) (mixture), 4.13-4.18 (m, 1 H, H-5) (mixture), 4.88-4.90 (m, 1 H, H-1 ), 5.45 (s I, 1 H, EtO-CH) (minor), 5.63 (d, J = 1.6 Hz, 1 H, EtO-CH) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 14.9 (-OCH₂CH₃) (mixture), 55.9 (-OCH₃) (mixture), 59.2 (-OCH₃) (minor), 59.3 (-OCH₃) (major), 60.7 (-OCH₂CH₃) (mixture), 61 .2 (-OCH₃) (minor), 61.3 (-OCH₃) (major), 70.1 (C-5) (mixture), 74.3 (H-OC-) (major), 74.8 (H-OC-) (minor), 75.7 (C-4) (minor), 76.4 (C-4) (major), 78.5 (H-C≡C-) (minor), 78.8 (H-C≡C-) (major), 81.4 (C-2) (major), 81 .7 (C-2) (minor), 81 .8 (C-3) (minor), 82.9 (C-3) (major), 92.5 (EtO-CH) (mixture), 97.8 (C-1 ) (minor), 98.0 (C-1 ) (major), 173.8 (C=0) (major), 174.0 (C=0) (minor).

ESI-MS (pos. mode): m/z = 341 [M+Na]⁺. IR (film) v (cm^“1): 1751 (C=0), 3268 (≡C-H).

Preparation of Methyl-4,7-anhydro-6-deoxy-6-methylene-7-ethoxy-2,3-di-0-methyl- a-L-/^‘d -heptopyranoside (Vila)

To a solution of compound Via (1.89 g, 5.92 mmol, 1 .0 eq.) in anhydrous THF (40 mL) and cooled to 0°C, were added IBCF (0.84 mL, 6.48 mmol, 1.1 eq.) and N- methylmorpholine (0.72 mL, 6.55 mmol, 1.1 eq.). After 20 minutes stirring the flask was covered with aluminium foil, 2-mercaptopyridine /V-oxide sodium salt (1 .77 g, 1 1.80 mmol, 2.0 eq.) was added and the reaction mixture was stirred at ambient temperature. After 2 hours, anhydrous THF (80 mL) then ie f-butylthiol (0.28 mL, 2.61 mmol, 1.6 eq.) were added. The aluminium foil was removed and the reaction mixture was irradiated and heated 30 minutes with a UV lamp (300W). The thiol excess was neutralized with a NaOCI aqueous solution (13% v/v, 10 mL). The reaction mixture was concentrated then dissolved in EtOAc (100 mL), washed successively with a 5% NaHC0₃ aqueous solution (2 x 15 mL), a 5 % citric acid aqueous solution (1 x 15 mL) and brine (1 x 25 mL), then the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (gradient dichloromethane/ethyl acetate 95:5 – 75:25) afforded compound Vila as a colourless oil (218 mg, 0.79 mmol, 48%), in a mixture of diastereomers (67:33) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons H-2).

¹H NMR (CDCIs, 400 MHz): δ 1.23 (t, J = 7.1 Hz, 3H, -OCH₂CH₃) (diastereomeric mixture), 3.13 (dd, J_2-3 = 9.6 Hz, J_1-2 = 3.0 Hz, 1 H, H-2) (minor), 3.30 (dd, J_2-3 = 5.0 Hz, J_1-2 = 1.6 Hz, 1 H, H-2) (major), 3.41 (s, 3H, -OCH₃) (minor), 3.47 (s, 3H, -OCH₃) (major), 3.50 (s, 3H, -OCH₃) (mixture), 3.53 (s, 3H, -OCH₃) (major), 3.55-3.61 (m, 1 H, -OCH_aH_bCH₃) (mixture), 3.72 (dd, J_2-3 = 5.0 Hz, J_3-4 = 2.8 Hz, 1 H, H-3) (major), 3.78-3.90 (m, 1 H, – OCH_aH_bCH₃) (mixture), 3.93-4.07 (m, 2H, H-3, H-4) (mixture), 4.59 (d I, J_4-5 = 4.0 Hz, 1 H, H-5) (major), 4.62 (d, J_1-2 = 1.7 Hz, 1 H, H-1 ) (major), (td, J_4-5 = 7.9 Hz, J = 2.6 Hz, 1 H, H- 5), (minor), 4.79 (d, J_1-2 = 3.0 Hz, 1 H, H-1 ) (minor), 5.35-5.57 (m, 3H, H-7, -C=CH₂) (mixture). ¹³C NMR (CDCI3, 100.6 MHz): δ 15.3 (-OCH₂CH₃) (minor), 15.4 (-OCH₂CH₃) (major), 56.5 (-OCH3) (minor), 56.8 (-OCH₃) (major), 58.6 (-OCH₃) (major), 59.1 (-OCH₃) (minor), 59.9 (- OCH3) (major), 60.3 (-OCH₃) (minor), 63.3 (-OCH₂CH₃) (minor), 63.8 (-OCH₂CH₃) (major), 74.2 (C-5) (minor), 74.7 (C-5) (major), 76.4 (C-3) (major), 77.0 (C-4) (major), 77.7 (C-2) (major), 79.0 (C-3) (minor), 79.7 (C-4) (minor), 80.1 (C-2) (minor), 99.3 (C-1 ) (major), 99.5 (C-1 ) (minor), 102.2 (C-7) (major), 103.0 (C-7) (minor), 1 1 1.6 (-C=CH₂) (minor), 1 15.2 (- C=CH₂) (major), 147.4 (C-6) (minor), 148.1 (C-6) (major).

ESI-MS (pos. mode): m/z = 297 [M+Na]⁺.

Preparation of Methyl-4,7-anhydro-7-ethoxy-2,3-di-0-methyl-a-L-/ o-hepto- pyranosid-6-ulose (Villa)

Through a solution of compound Vila (449 mg, 1 .64 mmol, 1.0 eq.) in anhydrous dichloromethane (10 ml_), under an argon atmosphere and cooled to -78°C, was bubbled ozone (0.2 L/min, 1 10 V). When the solution had turned dark blue, oxygen was bubbled through in order to remove the excess ozone. When the solution became colorless dimethylsulfide (5 drops) was added and the solution was brought to room temperature. After 1 h15 the reaction mixture was concentrated. Column chromatography (gradient dichloromethane/ethyl acetate 95:5 – 80:20) afforded compound Villa as a white solid (364 mg, 1.32 mmol, 80%), in a mixture of diastereomers (79:21 ) (the relative composition of the mixture was determined by ¹H NMR from integrations of protons H-2).

¹H NMR (CDCI₃, 400 MHz): δ 1.24-1 .28 (m, 3H, -OCH₂CH₃) (diastereomeric mixture), 3.10 (dd, J_2-3 = 10.2 Hz, J_1-2 = 2.9 Hz, 1 H, H-2) (minor), 3.17 (dd, J_2-3 = 9.4 Hz, J_1-2 = 2.8 Hz, 1 H, H-2) (major), 3.38 (s, 3H, -OCH₃) (major), 3.42 (s, 3H, -OCH₃) (minor), 3.50 (s, 3H, – OCH₃) (mixture), 3.63 (s, 3H, -OCH₃) (major), 3.66 (s, 3H, -OCH₃) (minor), 3.48-3.73 (m, 3H, H-3 major, -OCH_aH_bCH₃ major, -OCH_aH_bCH₃ minor), 3.77-3.95 (m, 2H, -OCH_aH_bCH₃ minor, -OCH_aH_bCH₃ major), 4.07 (dd, J_2-3 = 10.2 Hz, J_3-4 = 7.7 Hz, 1 H, H-3) (minor), 4.34 (d, J4-₅ = 9.1 Hz, 1 H, H-5) (minor), 4.39-4.44 (m, 2H, H-4 minor, H-5 major), 4.50 (dd, J_3-4 = 9.5 Hz, J_4-5 = 6.2 Hz, 1 H, H-4) (major), 4.76 (d, Ji_-2 = 2.8 Hz, 1 H, H-1 ) (major), 4.79 (d, J_1-2 = 2.9 Hz, 1 H, H-1 ) (minor), 4.89 (d, J = 1 ,1 Hz, 1 H, H-7) (minor), 4.93 (s I, 1 H, H-7) (major).

¹³C NMR (CDCI₃, 100.6 MHz): δ 15.1 (-OCH₂CH₃) (minor), 15.2 (-OCH₂CH₃) (major), 56.7 (-OCH₃) (minor), 57.2 (-OCH₃) (major), 59.3 (-OCH₃) (mixture), 59.8 (-OCH₃) (major), 60.6 (-OCH₃) (minor), 65.0 (-OCH₂CH₃) (minor), 65.5 (-OCH₂CH₃) (major), 70.2 (C-5) (major), 72.4 (C-5) (minor), 75.9 (C-4) (major), 79.2 (C-4) (minor), 79.4 (C-3) (major), 79.8 (C-2 major, C-3 minor), 80.2 (C-2) (minor), 96.1 (C-7) (major), 97.2 (C-7) (minor), 98.7 (C-1 ) (major), 99.0 (C-1 ) (minor), 205.3 (C-6) (minor), 205.6 (C-6) (major).

IR (film) v (cm^“1): 1783 (C=0).

ESI-MS (pos. mode): m/z = 299 [M+Na]⁺, 331 [M+Na+MeOH]⁺.

Preparation of Methyl methyl-2,3-di-0-methyl-a-L-idopyranosiduronate (IXa)

; CHCI3)

To a solution of compound Villa (50 mg, 0.18 mmol, 1 .0 eq.) in dichloromethane (3 mL), under an argon atmosphere and cooled to 0°C, were added m-CPBA (77%, 120 mg, 0.54 mmol, 3.0 eq.) and NaHC0₃ (20 mg, 0.23 mmol, 1 .3 eq.). After 3 hours stirring the solvent was removed under vacuum. The resulting residue was dissolved in EtOAc (30 mL), extracted with distilled water (2 x 10 mL), and the aqueous phase was concentrated. The crude mixture was dissoved in methanol (10 mL), para-toluenesulfonic acid monohydrate was added (4 mg, 0.02 mmol, 0.1 eq.) then the reaction mixture was heated to reflux and the reaction monitored by ¹H NMR in deuterated methanol. After 8 hours the solvent was evaporated. The residue obtained was dissolved in DMF (5 mL) then triethylamine (28 μί, 0.20 mmol, 1 .1 eq.) and methyl iodide (56 μί, 0.90 mmol, 5 eq.) were added. After 3h30 the reaction mixture was concentrated, dissolved in EtOAc (30 mL) and the organic phase was washed with a 5% NaHC0₃ aqueous solution (2 x 10 mL), a 5% citric acid aqueous solution (2 x 10 mL) and brine (1 x 10 mL). The aqueous phase was extracted with dichloromethane (5 x 10 mL) and the combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (dichloromethane/ethyl acetate 85:15) afforded compound Xla as a colourless oil (25 mg, 0.10 mmol, 56%). ¹H NMR (CDCI3, 400 MHz): δ 3.41 (d I, J_2-3 = 3.5 Hz, 1 H, H-2), 3.47 (s, 3H, -OCH3), 3.56 (s, 3H, -OCH3), 3.57 (s, 3H, -OCH3), 3.69 (t, J_2-3 = J3-4 = 3.5 Hz, 1 H, H-3), 3.75-3.78 (m, 1 H, OH), 3.80 (s, 3H, -OCH₃), 3.97 (m, 1 H, H-4), 4.42 (d, J_4-5 = 1 .6 Hz, 1 H, H-5), 4.61 (d,

¹³C NMR (CDCI₃, 100.6 MHz): δ 52.4, 57.5, 58.4, 60.8 (4x-OCH₃), 67.7 (C-4), 74.8 (C-5), 77.2 (C-2), 77.5 (C-3), 100.9 (C-1 ), 169.6 (C=0).

Elemental analysis: Calculated: C: 48.00 ; H: 7.25. Found: C: 47.62 ; H: 7.15.

ESI-MS (pos. mode): m/z = 272 [M+Na]⁺.

IR (film) v (cm^“1): 3491 (O-H), 1765 (C=0). Example 2 :

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(2′,3′-di-0-methyl-p-D-glucopyranosyl- uronate)-a-D-glucopyranoside (IVb)

To a solution of the co

(3.279 g, 5.00 mmol, 1.0 eq.) in a water/acetonitrile mixture (1 :1 , 300 mL) were added NaBr (105 mg, 1 .02 mmol, 0.2 eq.) and TEMPO (33 mg, 0.21 mmol, 0.04 eq.). The reaction mixture was cooled with the aid of an ice bath then a solution of NaOCI (13% v/v, 1 1.5 mL, 20.08 mmol, 4.0 eq.) was added. After 3 hours stirring at 0°C, NaOCI was added anew (13% v/v, 1 1 .5 mL, 20.08 mmol, 4.0 eq.). After two more hours ethanol was added (96% v/v, 20 mL), then the pH was reduced to 2-3 by addition of HCI (10% v/v). The solvent was evaporated and the residue obtained was suspended in DMF (40 mL) then triethylamine (2.8 mL, 2.032 g, 20.0 mmol, 4.0 eq.) and methyl iodide (6.2 mL, 14.136 g, 99.6 mmol, 20.0 eq.) were added. After 4 hours stirring at room temperature the solvent was evaporated and the residue obtained was dissolved in EtOAc (200 mL). The organic layer was washed with a 5% citric acid aqueous solution (1 χ 20 mL) and brine (1 χ 20 mL). The aqueous layer was extracted with dichloromethane (2 χ 20 mL). The combined organics were dried (MgS0₄), filtered and evaporated. The residue obtained was dissolved in DMF (20 mL), then triethylamine (1.4 mL, 1 .016 g, 10.0 mmol, 2.0 eq.) and methyl iodide (3.1 mL, 7.068 g, 49.8 mmol, 10.0 eq) were added. After 60 hours stirring at room temperature the solvent was evaporated and the residue obtained was dissolved in EtOAc (200 mL). The organic layer was washed with a 5% citric acid aqueous solution (2 x 20 mL) and brine (1 χ 20 mL). The organic layer was dried (MgS0₄), filtered and evaporated. Column chromatography (gradient hexane/ethyl acetate 80:20 – 50:50) gave compound IVb as a colourless oil which was dissolved in a diethyl ether/hexane mixture and evaporated at room temperature to afford a white solid (2.500 g, 3.66 mmol, 73%).

¹H NMR (CDCI₃, 400 MHz): δ 2.92 (d, 1 H), 2.93-2.98 (m, 2H), 3.41 (s, 3H), 3.48-3.76 (m, 5H), 3.51 (s, 3H), 3.60 (s, 3H), 3.63 (s, 3H), 3.87-3.98 (m, 3H), 4.36-4.41 (m, 1 H), 4.49- 5.08 (m, 6H), 4.61 (d, 1 H), 7.24-7.42 (m, 15H).

Elemental analysis: Calculated: C: 65.09 ; H: 6.79. Found: C: 65.29 ; H: 6.96.

ESI-MS (pos. mode): m/z = 705 [M+Na]⁺.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′-0-(1 “-ethoxy-2”-propyn-1 “-yl)-2′,3′- di-0-methyl-p-D-glucopyranosyluronate)-a-D-glucopyranoside (Vb)

E F To a solution of compound IVb (385 mg, 0.56 mmol, 1 .0 eq.) in chloroform (stabilised with amylene, 30 mL) were added, under an argon atmosphere, P₂0₅ (410 mg, 2.80 mmol, 5.0 eq.) and propargylaldehyde diethylacetal (0.4 mL, 2.79 mmol, 5.0 eq.), then the reaction mixture was heated at reflux. After 5 hours stirring, the reaction mixture was filtered through a pad of Celite^® then the solvent was removed under vacuum. The crude mixture was suspended in EtOAc (60 mL), washed with a 5% NaHC0₃ aqueous solution (1 x 15 mL) and brine (1 x 15 mL). The organic layer was dried (MgS0₄), filtered, and evaporated. Column chromatography (gradient hexane/ethyl acetate 90:10 – 70:30) afforded compound Vb as a colourless oil (275 mg, 0.36 mmol, 64%) in a diastereomeric mixture (64:36).

¹H NMR (CDCI₃, 250 MHz): δ 1.17-1 .27 (m, 3H), 2.55 (d, 0.36H), 2.57 (d, 0.64H), 2.92- 3.08 (m, 2H), 3.38 (s, 3H), 3.49 (s, 1.92H), 3.50 (s, 1.08H), 3.57 (s, 1.08H), 3.59 (s, 1.92H), 3.60 (s, 1.92H), 3.62 (s, 1.08H), 3.44-3.97 (m, 10H), 4.35 (t, 1 H), 4.46-4.76 (m, 6H), 5.03 (d, 1 H), 5.32 (d, 0.36H), 5.56 (d, 0.64H), 7.21-7.42 (m, 15H).

Elemental analysis: Calculated: C: 65.95 ; H: 6.85. Found: C: 65.92 ; H: 6.75.

ESI-MS (pos. mode): m/z = 787 [M+Na]⁺.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′-0-(1 “-ethoxy-2″-propyn-1 ” 1 ‘,2′,3’-tri-0-methyl-a-D-glucopyranosiduronic acid)-a-D-glucopyranoside (Vlb)

E F

To a solution of compound Vb (1.02 g, 1.33 mmol, 1.0 eq.) in EtOH/H₂0 (1 :1 , 100 mL) was added sodium hydroxide (82 mg, 2.05 mmol, 1.5 eq.). After 3 hours stirring at room temperature sodium hydroxide was added anew (27 mg, 0.68 mmol, 0.5 eq.). After an additional hour stirring the solvent was evaporated. The residue obtained was dissolved in water (40 mL). The pH of the aqueous layer was reduced to 2-3 with a 10% HCI aqueous solution then the layer was saturated with sodium chloride before extraction with dichloromethane (3 x 20 mL). The combined organics were dried (MgS0₄), filtered and removed under vacuum. Compound VIb was obtained without further purification as a white solid (930 mg, 1.24 mmol, 93%), in a diastereomeric mixture (63:37).

¹H NMR (CDCIs, 400 MHz): δ 1.17-1.28 (m, 3H), 2.65 (d, 0.63H), 2.68 (d, 0.37H), 2.90- 3.09 (m, 2H), 3.37 (s, 3H), 3.46 (s, 1.1 1 H), 3.57 (s, 1.89H), 3.58 (s, 1.1 1 H), 3.60 (s, 1.89H), 3.42-3.90 (m, 10H), 4.29 (d, 1 H), 4.46-4.97 (m, 7H), 5.44 (d, 0.37H), 5.61 (d, 0.63H), 7.28-7.44 (m, 15H).

ESI-MS (pos. mode): m/z = 773 [M+Na]⁺ , 795 [M-H+2Na]⁺ . ESI-MS (neg. mode): m/z = 749 [M-H]\

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′,7′-anhydro-6′-deoxy-6′-methylene-7′- ethoxy-2 3′-di-0-methyl-α-L-/^‘ o-heptopyranosyl)-α-D-glucopyranoside (Vllb)

To a solution of compound VIb (647 mg, 0.86 mmol, 1.0 eq.) in anhydrous THF (20 mL) and cooled to 0°C, were added IBCF (0.1 1 mL, 0.85 mmol, 1.0 eq.) and N- methylmorpholine (0.10 mL, 0.91 mmol, 1.1 eq.). After 10 minutes stirring, the flask was covered with aluminium foil, 2-mercaptopyridine /V-oxide sodium salt (512 mg, 3.43 mmol, 4.0 eq.) was added and the reaction mixture was stirred at ambient temperature. After 20 minutes anhydrous THF (100 mL) then ie f-butylthiol (0.18 mL, 1 .68 mmol, 2.0 eq.) were added. The aluminium foil was removed and the reaction mixture was irradiated and heated 15 minutes with a UV lamp (300W). The thiol excess was neutralized with a NaOCI aqueous solution (13%, 10 mL). The reaction mixture was concentrated then dissolved in EtOAc (100 mL), washed successively with a 5% NaHC0₃ aqueous solution (1 x 15 mL), a 5 % citric acid aqueous solution (1 x 15 mL) and brine (1 x 15 mL), then the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organics were dried (MgS0₄), filtered and concentrated. Column chromatography (gradient hexane/ethyl acetate 90 :10 – 70:30) afforded compound Vllb as a colourless oil (251 mg, 0.36 mmol, 42%) in a mixture of diastereomers (61 :39).

¹H NMR (CDCI₃, 250 MHz): δ 1.18-1.29 (m, 3H), 2.90-3.07 (m, 1 H), 3.37 (s, 1.17H), 3.38 (s, 1 .83H), 3.46 (s, 3H), 3.54 (s, 1.83H), 3.60 (s, 1 .17H), 3.29-4.00 (m, 10H), 4.1 1 -4.26 (m, 1 H), 4.50-4.96 (m, 8H), 5.09-5.48 (m, 3H), 7.23-7.39 (m, 15H).

ESI-MS (pos. mode): m/z = 720 [M+Na]⁺ _.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(4′,7′-anhydro-7′-ethoxy-2′,3′-di-0- methyl-a-L-/^‘ o-heptopyranosid-6′-ulosyl)-a-D-glucopyranoside (Vlllb)

Through a solution of compound Vllb (145 mg, 0.21 mmol, 1 .0 eq.) in anhydrous dichloromethane (10 mL), under an argon atmosphere and cooled to -78°C, was bubbled ozone (0.2 L/min, 1 10 V). When the solution had turned dark blue, oxygen was bubbled through in order to remove the excess ozone. When the solution became colorless dimethylsulfide (4 drops) was added and the solution was brought to room temperature. After 30 min stirring the reaction mixture was concentrated. Column chromatography (gradient hexane/ethyl acetate 90:10 – 60:40) afforded compound Vlllb as a white solid (100 mg, <67%) in a mixture of diastereomers (67:33).

¹H NMR (CDCI₃, 400 MHz): δ 1.21 -1 .28 (m, 3H), 2.93-3.07 (m, 1 H), 3.31-4.26 (m, 20H), 4.52-5.02 (m, 9H), 7.20-7.45 (m, 15H).

ESI-MS (pos. mode): m/z = 731 [M+Na]⁺ _.

Preparation of Methyl-2,3,6-tri-0-benzyl-4-0(methyl 2′,3′-di-0-methyl-a-L- idopyranosiduronate)-a-D-glucopyranoside (IXb)

To a solution of compound Vlllb (62 mg, 87 μηηοΙ, 1 .0 eq.) in dichloromethane (5 mL), under an argon atmosphere and cooled to 0°C, were added m-CPBA (77%, 58 mg, 259 μηηοΙ, 3.0 eq.) and NaHC0₃ (1 1 mg, 130 μηηοΙ, 1 .5 eq.). After 5 hours stirring the solvent was removed under vacuum. The reaction mixture was then dissolved in EtOAc (50 mL) and washed successively with a 5% NaHC0₃ aqueous solution (1 x 10 mL), a 5 % citric acid aqueous solution (1 x 10 mL) and brine (1 x 10 mL). The organic layer was dried (MgS0₄), filtered and concentrated. The crude mixture was dissolved in anhydrous methanol (10 mL) and sodium methoxide was added to reach pH = 10. After 30 minutes stirring at room temperature the reaction mixture was neutralized with Dowex^®, filtered through a pad of Celite^®, and concentrated. The residue obtained was dissolved in DMF (10 mL) then triethylamine (13 μί, 93 μηηοΙ, 1.1 eq.) and methyl iodide (27 μί, 434 μηηοΙ, 5.0 eq.) were added. After 2h30 stirring the reaction mixture was concentrated, dissolved in EtOAc (40 mL) and washed with a 5% citric acid aqueous solution (2 x 10 mL), a 5% NaHC0₃ aqueous solution (2 x 10 mL), and brine (1 x 10 mL). The organic layer was dried (MgS0₄), filtered and concentrated. Column chromatography (gradient hexane/ethyl acetate 60:40-50:50) afforded compound IXb as a colourless oil (12 mg, 18 μηηοΙ, 20% over two steps).

¹H NMR (CDCIs, 400 MHz): δ 3.23 (s, 3H), 3.20-3.25 (m, 1 H), 3.36 (s, 3H), 3.43 (s, 3H), 3.46 (s, 3H), 3.33-3.58 (m, 3H), 3.60-3.69 (m, 2H), 3.72-3.95 (m, 4H), 4.53-4.60 (m, 4H), 4.68-4.97 (m, 4H), 5.14 (s, 1 H), 7.24-7.37 (15H).

ESI-MS (pos. mode): m/z = 705 [M+Na]⁺ _.

……………

Tetrahedron

Volume 69, Issue 15, 15 April 2013, Pages 3149–3158

http://www.sciencedirect.com/science/article/pii/S0040402013003025

Abstract

Idraparinux, the fully O-sulfated, O-methylated, heparin-related pentasaccharide possessing selective factor Xa inhibitory activity, was prepared by two novel synthetic pathways. Each route was based on a 2+3 block synthesis utilizing the same l-iduronic acid-containing trisaccharide acceptor, which was glycosylated with either a glucuronide disaccharide donor or its non-oxidized precursor. The latter route, involving the oxidation of the glucose unit into d-glucuronic acid at a pentasaccharide level proved to be much more efficient, providing the target pentasaccharide in a reasonable overall yield.

Graphical abstract





……………………………

SYNTHESIS

US 20120041189 A1,

http://www.patexia.com/us-publications/20120041189
EXAMPLE 1Preparation of the Compound of Formula (I) in Crystalline Form (Scheme 1) 1.1: Preparation of the Compound of Formula (I′) The compound of formula (I″) is obtained, for example, according to the teaching of patent EP 0 529 715 B1 or of the articles “Bioorg. Med. Chem.” (1994, Vol. 2, No. 11, pp. 1267-1280), “Bioorg. Med. Chem. Letters” (1992, Vol. 2, No. 9, pp. 905-910) or “Magnetic Resonance in Chemistry” (2001, Vol. 39, pp. 288-293). The compound of formula (I″) (5 g, 3.06 mmol) is dissolved in acetonitrile (10 mL). Deionized water (12.2 mL) and aqueous 30% sodium hydroxide solution (4.1 g) are then added. The mixture is heated to 40° C. and maintained at this temperature for 5 hours. The reaction medium is then cooled to 20° C. and acidified to pH 6.25 with aqueous 1N hydrochloric acid solution (about 17.7 g) before extraction with MTBE of certain impurities, the saponified product remaining in the aqueous phase. The residual acetonitrile, contained in the aqueous phase, is then removed by concentration, followed by diluting with deionized water (125 mL). The saponified product is finally precipitated at pH 1.5 by adding aqueous 1N hydrochloric acid solution (about 17.6 g) at 20° C. The suspension is maintained for 4 hours at 20° C. before filtration. The wet solid is finally dried in a vacuum oven at 30° C. to give 2.93 g (93.6%) of compound of formula (I). NMR (anomeric protons of the saccharide units D, E, F, G, H): 5.79, 5.14, 5.55, 5.92, 4.94 ppm. 1.2 Preparation of the Crude Compound of Formula (I) The compound of formula (I′) obtained after the preceding step is dissolved in tetrahydrofuran (18 mL). Palladium-on-charcoal (0.3 g) is added. The reaction medium is hydrogenated at 0.3 bar of hydrogen (relative pressure) for 4 hours. After filtering and evaporating, 2.12 g (99%) of the crude compound of formula (I) are obtained. 1.3: Preparation of the Compound of Formula (I) in Crystalline Form Using an Isopropanol/MTBE Mixture The crude hydrogenated product obtained after the preceding step is dissolved in isopropanol (13 mL) at 65° C., and then crystallized at room temperature. The suspension is then cooled to 40° C., followed by addition of MTBE (13 mL), and is then cooled slowly to 10° C. After maintenance at 10° C. for 2 hours, the crystalline hydrogenated product is filtered off, washed and dried. 1.66 g of the compound of formula (I) in crystalline form are thus obtained, in the form of a cream-white powder. The reaction yield for the production of the compound of formula (I) in crystalline form, from the compound of formula (I′), is 92.5%. When expressed relative to the starting compound (I″), the reaction yield for the production of the compound of formula (I) in crystalline form is 86.6%. NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (I) in crystalline form: 5.77, 5.11, 5.51, 5.84, 5.01 ppm. 1.4: Preparation of the Compound of Formula (I) in Crystalline Form Using Isopropanol The crude hydrogenated product obtained after step 1.2 is dissolved in isopropanol (5 volumes) at 75° C. The medium is then cooled slowly until crystals appear, according to the known standard techniques for crystallization. The process is performed, for example, by a first step of cooling at 65° C. for 1 hour, and than a second step of cooling to a final temperature of 25° C. over 4 hours or of 5° C. over 6 hours, and finally maintenance at this final temperature for 30 minutes. The suspension is then filtered and rinsed with isopropanol (2×0.1 V) and compound (I) is isolated in the form of white crystals, which appear under a microscope in the form of needles. The 1H NMR analysis of these crystals is identical to that described after step 1.3 above. EXAMPLE 4 Preparation of Idraparinux from the Compound of Formula (I) in Crystalline Form (Scheme 2) The preparation of idraparinux (II) from the compound of formula (I) is summarized in Scheme 2. The compound of formula (I) in crystalline form, as obtained according to Example 1.3, is dissolved in N,N’-dimethylformamide (6.6 mL) and then heated to 30.degree. C. Under an inert atmosphere, 3.8 g of pyridine-sulfur trioxide complex are added slowly, followed by maintenance at 30.degree. C. for 4 hours. The reaction medium is then poured into aqueous 23.8% sodium hydrogen carbonate solution (16.3 g) maintained at a maximum of 25.degree. C., to obtain the compound of formula (II). The reaction medium is kept stirring for hours. The solution of sulfated product is then poured onto an MTBE/isopropanol/ethanol mixture (171 mL/70 mL/70 mL). Precipitation of the product is observed, and, after filtering off, washing and drying the cake, 4.99 g (96.8%) of compound of formula (II) are obtained, and are then purified by anion-exchange chromatography according to the usual techniques. NMR (anomeric protons of the saccharide units D, E, F, G, H) of the compound of formula (II): 5.48, 4.68, 5.44, 5.08, 5.18 ppm. It thus appears that the process according to the invention makes it possible to obtain idraparinux (compound of formula (II)) in a chemical yield of about 84% (precisely 83.8% according to the protocols described above) starting from the compound of formula (I”), i.e. a gain in yield of about 30% relative to the process described in patent EP 0 529 715 B1. IDRAPARINUX

References

Bousser MG, Bouthier J, Büller HR, et al. (January 2008). “Comparison of idraparinux with vitamin K antagonists for prevention of thromboembolism in patients with atrial fibrillation: a randomised, open-label, non-inferiority trial”. Lancet 371 (9609): 315–21. doi:10.1016/S0140-6736(08)60168-3.PMID 18294998.
Buller HR, Cohen AT, Davidson B, et al. (September 2007). “Idraparinux versus standard therapy for venous thromboembolic disease”. N. Engl. J. Med. 357 (11): 1094–104. doi:10.1056/NEJMoa064247. PMID 17855670.
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Chemistry – A European Journal, 2012 , vol. 18, 34 p. 10643 – 10652
Magnetic Resonance in Chemistry, 2001 , vol. 39, 5 p. 288 – 293
Tetrahedron, 2013 , vol. 69, 15 p. 3149 – 3158…….. MP 210-15 DEG CENT

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WO2002024754A1	Sep 20, 2001	Mar 28, 2002	Akzo Nobel Nv	Polysaccharides with antithrombotic activity comprising at least a covalent bond with biotin or a biotin derivative
WO2006030104A1 *	Sep 7, 2005	Mar 23, 2006	Sanofi Aventis	Biotinylated hexadecasaccharides, preparation and use thereof
WO2007042469A2 *	Oct 6, 2006	Apr 19, 2007	Organon Nv	Anticoagulant antithrombotic dual inhibitors comprising a biotin label

EP0230023A2 *	Dec 19, 1986	Jul 29, 1987	Marion Merrell Dow Inc.	Pharmaceutical compositions for the enhancement of wound healing
EP0300099A1 *	Jul 20, 1987	Jan 25, 1989	Akzo N.V.	New pentasaccharides
EP0301618A2 *	Jul 4, 1988	Feb 1, 1989	Akzo N.V.	New pentasaccharides
EP0454220A1 *	Apr 16, 1991	Oct 30, 1991	Akzo Nobel N.V.	Carbohydrate derivatives comprising a trisaccharide unit
GB1110939A *				Title not available
US3017407 *	Aug 18, 1958	Jan 16, 1962	Riker Laboratories Inc	Process for producing polysulfuric acid esters of polysaccharides

Lodenafil Carbonate … an Erectile Dysfunction Drug in Phase III

Phase 3 drug, Uncategorized 1 Response »

Jan 312014

Lodenafil carbonate

UNII-29X84F932D, CRIS-031

bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonate

5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one. IS THE NAME OF MONOMER

398507-55-6 CAS

Cristalia (Originator)

C47 H62 N12 O11 S2= MF
Molecular Weight	1035.199

Lodenafil is a drug belonging to a class of drugs called PDE5 inhibitor, which many other erectile dysfunction drugs such as sildenafil, tadalafil, and vardenafil also belong to. Like udenafil and avanafil it belongs to a new generation of PDE5 inhibitors.

Sildenafil, tadalafil, vardenafil, and the newer udenafil and avanafil selectively inhibit PDE5, which is cGMP-specific and responsible for the degradation of cGMP in the corpus cavernosum. These phosphodiesterase inhibitors are used primarily as remedies for erectile dysfunction, as well as having some other medical applications such as treatment of pulmonary hypertension.

Lodenafil is formulated as a dimer, lodenafil carbonate, which breaks down in the body to form two molecules of the active drug lodenafil. This formulation has higher oral bioavailability than the parent drug.^[1]

It is manufactured by Cristália Produtos Químicos e Farmacêuticos in Brazil and sold there under the brand-name Helleva.^[2]

Helleva (Lodenafil Carbonate) - 80mg (4 Tablets)

Helleva (Lodenafil Carbonate) is an oral PDE5 inhibitor prescribed to treat men suffering from erectile dysfunction. It operates by relaxing muscles and dilating blood vessels in the penis to increase circulation making it easier to attain and maintain an erection.

It has undergone Phase III clinical trials,^[3]^[4]^[5] but is not yet approved for use in the United States by the U.S. Food and Drug Administration.

lodenafil

………..

SYNTHESIS

WO 2002012241 OR US7148350

MONOMER synthesis

PIPERAZINE

AND

ETHYL CHLORO ACETATE

WILL GIVE

Ethyl 1-piperazinylacetate ChemSpider 2D Image | Ethyl 1-piperazinylacetate | C8H16N2O2

SEE RXN 1 BELOW

Reaction 1:

Synthesis of Piperazine Ethyl Acetate

To a reaction blend containing 100 g (3 Eq, 0.515 mol, MW=194) of piperazine, 26.3 mL (1.1 Eq, 0.189 mol, MW=101, d=0.726) of triethylamine in 200 mL of isopropanol, add to a solution previously prepared of 18.4 mL (1 Eq., 0.172 mol, MW=122.55, d=1.15) of chloroacetate of ethyl in 140 mL of isopropanol under stirring, at room temperature. Keep the reaction medium under stirring, monitoring the reaction termination by means of a chromatography of the thin layer (about 2–3 hours). Add a solution of 40.6 g (0.344 mol) of succinic acid in 140 mL of isopropanol. Keep the system under stirring for about 30 minutes to assure total precipitation of the succinate salt of piperazine formed. Filter this salt and concentrate the filtrate containing the mono and dialkyled derivatives. We obtain a slightly yellowish oil, which is used in later phases without purification.

Mass obtained=33 g

GC/MS: Monoalkylated derivative 72%, and dialkylated 22%.

ChemSpider 2D Image | Ethyl 1-piperazinylacetate | C8H16N2O2 Piperazine Ethyl Acetate

AND

5-(5-Chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one Structure

5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one

WILL REACT TO GIVE… 5-{2-ethoxy-5-[(4-ethyl acetate 1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-di-hydro-7H-pyrazole[4,3-d]pyrimidin-7-one AS IN RXN 4 BELOW

Reaction 4:

Synthesis of 5-{2-ethoxy-5-[(4-ethyl acetate 1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-di-hydro-7H-pyrazole[4,3-d]pyrimidin-7-one.

Suspend 24.6 g (60 mmol, MW=410.9) of 5-(5-chlorosulfonyl-2-etoxyphenyl)-1-methyl-3n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one in 900 mL of ethanol absolute. Under stirring and at room temperature, add at only one time, a solution containing 31.0 g (3 Eq., 180 mmol MW=172) of N-piperazine ethyl acetate (Reaction 1) dissolved in 150 mL of ethanol absolute. In an interval of 2–10 minutes, all solid is consumed, forming a clean and homogeneous solution, and after that starts the precipitation of the expected product. At the end of the reaction, which lasts 2–3 hours (monitored by chromatography of thin layer), the product is vacuum filtered and the solid is washed with two portions of 50 mL of iced absolute ethanol. 29 g are obtained (yielding=89%) from the product as a white solid of MP=165.5–166.5° C.

Reaction 7:

Intermediate 1

5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one. IS MONOMER

please note during LAH redn …………. the PIP CH2-C=O-O CH2 CH3 BECOMES PIP-CH2CH2-OH

To a suspension of lithium aluminum hydride (0.74 g 2.2 Eq. MW=37.9) in 25 mL of THF, slowly add, under stirring and at room temperature, a suspension of 5.0 g (9.1 mmol, MW=546.6) of 5-{2-ethoxy-5-[(4-ethyl acetate 1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-di-hydro-7H-pyrazole[4,3-d]pyrimidin-7-one in 50 mL of THF. The system is maintained under stirring, monitoring the consumption of the product by chromatography of thin layer, until the complete consumption of the starting reagent (about 5–6 hours). Slowly add water to the reaction medium and, when there is no longer release of H₂, add HCl 1M regulating pH for 7. Extract the product with 3 200 mL-portions of chloroform, dry with anhydrous sodium sulfate and vacuum concentrate the product. It is obtained 3.8 g of the product as a cream solid MP=183–187° C. yielding 83%. The same was crystallized from methanol and DMF yielding a slightly yellowish solid with melting point at 189–192° C.

note …………. the PIP CH2-C=O-O CH2 CH3 BECOMES PIP-CH2CH2-OH

HOMODIMER CARBONATE

EXAMPLE 1B

Homodimer Carbonate of Intermediate 1—Alternative Method

A phosgene solution (3.5 g, 35 mmol) dissolved in 20 mL of toluene was added dropwise to a solution of 2.02 g (4 mmol) of 5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one, suspended in 44 mL of toluene. The reaction mixture resulting is stirred and followed by chromatography analysis of thin layer every hour until the reagent conversion in its chloroformate was completed. When the analysis indicates the complete consumption of 5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one, the volatile compounds of the reaction are vacuum removed (solvents and phosgene), yielding the esther chloroformate raw derivative of 5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one.

The raw chloroformate obtained above (4.0 mmol, 2.27 g) is dissolved in about 30 mL of dichloromethane, to which is added 2.07 g (4.1 mmol) of 5-{2-ethoxy-5-[(4-hydroxyethyl-1-piperazinyl)sulfonyl]phenyl}-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one, followed by the addition of 4 mL of dichloromethane containing 450 mg of triethylamine. The reaction mixture is maintained under stirring, being followed by chromatography of thin layer every hour until this indicates the end of the reaction (disappearing of chloroformate derivative). The reaction mixture is then diluted with 60 mL of dichloromethane, washed with NaCl saturated solution, after with sodium bicarbonate saturated solution and again with NaCl saturated solution. Organic phase is separated and dry with anhydrous sodium sulfate. The solvent is then evaporated to dry, yielding the dimer carbonate as a slightly yellowish solid.

This compound is re-crystallized from ethanol:DMF, yielding a pale white solid. Yielding m=3.2 g (76%)

Microanalysis: Theoretical C, (54.53%); H, (6.04%); N, (16.24%);

Obtained C, (54.45%); H, (6.02%); N, (16.17%).

INFO ABOUT INTERMEDIATE

5-(5-Chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one Structure

5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3n-propyl-1,6-dihydro-7H-pyrazole[4,3-d]pyrimidin-7-one

CAS No.	139756-22-2
Chemical Name:	5-(5-Chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
Synonyms:	Sildenafil Chlorosulfone IMpurity;Sildenafil Chlorosulfonyl IMpurity;5-(5-CHLOROSULFONYL-2-ETHOXY PHENYL)-1-METHYL-3-N-PROPYL-1;3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1 H-pyrazolo-(4-3-d)-pyrimidine-5;5-(5-Chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one;3-(4,7-Dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxy-benzenesulfonyl Chloride;4-Ethoxy-3-(1-Methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyriMidin-5-yl)benzene-1-sulfonyl chloride
CBNumber:	CB11175931
Molecular Formula:	C17H19ClN4O4S

http://www.chemicalbook.com/ChemicalProductProperty_EN_CB11175931.htm

…………..

SYNTHESIS OF

http://www.google.co.in/patents/US6362178

2-butyrylamino-propionic acid

EXAMPLE 1A 2-Butyrylaminopropionic acid

22.27 g (250 mmol) of D,L-alanine and 55.66 g (550 mmol) of triethylamine are dissolved in 250 ml of dichloromethane, and the solution is cooled to 0° C. 59.75 g (550 mmol) of trimethylsilyl chloride are added dropwise, and the solution is stirred for 1 hour at room temperature and for 1 hour at 40° C. After cooling to −10° C., 26.64 g (250 mmol) of butyryl chloride are added dropwise, and the resulting mixture is stirred for 2 hours at −10° C. and for one hour at room temperature.

With ice-cooling, 125 ml of water are added dropwise and the reaction mixture is stirred at room temperature for 15 minutes. The aqueous phase is evaporated to dryness, the residue is titrated with acetone and the mother liquor is filtered off with suction. The solvent is removed and the residue is chromatographed. The resulting product is dissolved in 3N aqueous sodium hydroxide solution and the resulting solution is evaporated to dryness. The residue is taken up in conc. HCl and once more evaporated to dryness. The residue is stirred with acetone, precipitated solid is filtered off with suction and the solvent is removed under reduced pressure. This gives 28.2 g (71%) of a viscous oil which crystallizes after some time.

200 MHz ¹H-NMR (DMSO-d6): 0.84, t, 3H; 1.22, d, 3H; 1.50, hex, 2H; 2.07, t, 2H; 4.20, quin., 1H; 8.09, d, 1H.

EXAMPLE 3A 2-Ethoxybenzonitrile

25 g (210 mmol) of 2-hydroxybenzonitrile are refluxed with 87 g of potassium carbonate and 34.3 g (314.8 mmol) of ethyl bromide in 500 ml of acetone overnight. The solid is filtered off, the solvent is removed under reduced pressure and the residue is distilled under reduced pressure. This gives 30.0 g (97%) of a colourless liquid.

200 MHz ¹H-NMR (DMSO-d6): 1.48, t, 3H; 4.15, quart., 2H; 6.99, dt, 2H; 7.51, dt, 2H.

2-ethoxybenzamidine hydrochloride

EXAMPLE 4A 2-Ethoxybenzamidine hydrochloride

21.4 g (400 mmol) of ammonium chloride are suspended in 375 ml of toluene, and the suspension is cooled to 0° C. 200 ml of a 2M solution of trimethylaluminium in hexane are added dropwise, and the mixture is stirred at room temperature until the evolution of gas has ceased. After addition of 29.44 g (200 mmol) of 2-ethoxybenzonitrile, the reaction mixture is stirred at 80° C. (bath) overnight.

With ice-cooling, the cooled reaction mixture is added to a suspension of 100 g of silica gel and 950 ml of chloroform, and the mixture is stirred at room temperature for 30 minutes. The mixture is filtered off with suction, and the filter residue is washed with the same amount of methanol. The mother liquor is concentrated, the resulting residue is stirred with a mixture of dichloromethane and methanol (9:1), the solid is filtered off with suction and the mother liquor is concentrated. This gives 30.4 g (76%) of a colourless solid.

200 MHz ¹H-NMR (DMSO-d6): 1.36, t, 3H; 4.12, quart., 2H; 7.10, t, 1H; 7.21, d, 1H; 7.52, m, 2H; 9.30, s, broad, 4H.

EXAMPLE 10A 2-(2-Ethoxy-phenyl)-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one

7.16 g (45 mmol) of 2-butyrylamino-propionic acid and 10.67 g of pyridine are dissolved in 45 ml of THF and, after addition of a spatula tip of DMAP, heated to reflux. 12.29 g (90 mmol) of ethyl oxalyl chloride are slowly added dropwise, and the reaction mixture is refluxed for 3 hours. The mixture is poured into ice-water and extracted three times with ethyl acetate and the organic phase is dried over sodium sulphate and concentrated using a rotary evaporator. The residue is taken up in 15 ml of ethanol and refluxed with 2.15 g of sodium bicarbonate for 2.5 hours. The cooled solution is filtered.

With ice-cooling, 2.25 g (45 mmol) of hydrazine hydrate are added dropwise to a solution of 9.03 g (45 mmol) of 2-ethoxybenzamidine hydrochloride in 45 ml of ethanol, and the resulting suspension is stirred at room temperature for another 10 minutes. The ethanolic solution described above is added to this reaction mixture, and the mixture is stirred at a bath temperature of 70° C. for 4 hours. After filtration, the mixture is concentrated, the residue is partitioned between dichloromethane and water, the organic phase is dried over sodium sulphate and the solvent is removed under reduced pressure.

This residue is dissolved in 60 ml of 1,2-dichloroethane and, after addition of 7.5 ml of phosphorus oxychloride, refluxed for 2 hours. The mixture is diluted with dichloromethane and neutralized by addition of sodium bicarbonate solution and solid sodium bicarbonate. The organic phase is dried and the solvent is removed under reduced pressure. Chromatography using ethyl acetate and crystallization afford 4.00 g (28%) of a colourless solid, R_f=0.42 (dichloromethane/methanol=95:5)

200 MHz ¹H-NMR (CDCl₃): 1.02, t, 3H; 1.56, t, 3H; 1.89, hex, 2H; 2.67, s, 3H; 3.00, t, 2H; 4.26, quart., 2H; 7.05, m, 2H; 7.50, dt, 1H; 8.17, dd, 1H; 10.00, s, 1H.

EXAMPLE 15A 4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-imidazo[5,1-f][1,2,4]triazin-2-yl)-benzenesulphonyl chloride

At 0° C., 2.00 g (6.4 mmol) of 2-(2-ethoxy-phenyl)-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one are slowly added to 3.83 ml of chlorosulphonic acid. At room temperature, the reaction mixture is stirred ovemight, and then poured into ice-water and extracted with dichloromethane. This gives 2.40 g (91%) of a colourless foam.

200 MHz ¹H-NMR (CDCl₃): 1.03, t, 3H; 1.61, t, 2H; 1.92, hex, 2H; 2.67, s, 3H; 3.10, t, 2H; 4.42, quart., 2H; 7.27, t, 1H; 8.20, dd, 1H; 8.67, d, 1H; 10.18, s, 1H.

Example 22 2-[2-Ethoxy-5-(4-hydroxyethyl-1-amino-piperazine-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one

By the same method, starting with 0.04 g (0.097 mmol) of 4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-imidazo[5,1-f][1,2,4]triazin-2-yl)-benzenesulphonyl chloride and 0.04 g (0.29 mmol) of 1-amino-4-hydroxyethylpiperazine, 46 mg (91%) of 2-[2-ethoxy-5-(4-hydroxyethyl-1-amino-piperazine-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one are obtained.

R_f=0.08 (dichloromethane/methanol=19:1)

200 MHz ¹H-NMR (CDCl₃): 1.02, t, 3H; 1.59, t, 3H; 1.90, sex., 2H; 2.49, m, 6H; 2.62, s, 3H; 2.71, m, 4H; 3.00, t, 2H; 3.55, t, 2H; 4.31, quart., 2H; 7.14, d, 1H; 8.05, dd, 1H; 8.60, d, 1H.

…………..

Methods of analysis

The development of lodenafil carbonate was reported by Toque et al. (2008). They observed the effects of lodenafil carbonate on rabbit and human corpus cavernosum relaxation, activity of PDE5 in human platelets, stability and metabolic studies in comparison with sildenafil and lodenafil, as well as the pharmacological evaluation of lodenafil carbonate after intravenous and oral administration in male beagles.

The determination of PDE activity, stability of lodenafil carbonate in human, dog and rat plasma and the pharmacokinetic parameters after a single intravenous or oral dose was carried out by LC-MS/MS analysis

Codevilla et al. (2011a) developed a stability-indicating reversed-phase liquid chromatography method using ultraviolet (UV) detection for the quantitative determination of lodenafil carbonate in tablets. The method can be useful for routine quality control assay and stability studies.

Another study for the determination of lodenafil carbonate in tablets was developed by Codevilla et al. (2011b). As an alternative to the LC method the authors suggested a UV-spectrophotometric method for the analysis of lodenafil carbonate in pharmaceutical form. The UV method offers advantages over other analytical methods due to its rapidity, simplicity, and lower cost. Recently, Codevilla et al. (2012) developed and validated a capillary zone electrophoresis (CZE) method for determination of lodenafil carbonate in drug products. There are some advantages to use the CZE method, such as rapid analysis, small sample and reagent consumption, high separation efficiency (Furlanetto et al., 2001; Yang et al., 2010). The results obtained from the UV-spectrophotometric method and CZE method were compared statistically with the LC method (Codevilla et al., 2011a) and the results showed no significant difference between these methods.

References

Toque HA, Teixeira CE, Lorenzetti R, Okuyama CE, Antunes E, De Nucci G (September 2008). “Pharmacological characterization of a novel phosphodiesterase type 5 (PDE5) inhibitor lodenafil carbonate on human and rabbit corpus cavernosum”. European Journal of Pharmacology 591 (1–3): 189–95. doi:10.1016/j.ejphar.2008.06.055. PMID 18593576.
Cristália Product page. Retrieved on September 16, 2009.
ukmedix Lodenafil article. Retrieved on September 16, 2009.
Glina S, Toscano I, Gomatzky C, de Góes PM, Júnior AN, Claro JF, Pagani E (February 2009). “Efficacy and tolerability of lodenafil carbonate for oral therapy in erectile dysfunction: a phase II clinical trial”. The Journal of Sexual Medicine 6 (2): 553–7. doi:10.1111/j.1743-6109.2008.01079.x.PMID 19040623.
Glina S, Fonseca GN, Bertero EB, Damião R, Rocha LC, Jardim CR, Cairoli CE, Teloken C, Torres LO, Faria GE, da Silva MB, Pagani E (February 2010). “Efficacy and Tolerability of Lodenafil Carbonate for Oral Therapy of Erectile Dysfunction: A Phase III Clinical Trial”. The Journal of Sexual Medicine 7 (5): 1928–1936. doi:10.1111/j.1743-6109.2010.01711.x. PMID 20214718.
Toque H A et al., (2008) European Journal of Pharmacology, 591(1-3):189-95.
Exploring the role of PDE5 inhibition in the treatment of muscular dystrophy
Drugs Fut 2011, 36(4): 321