AUTHOR OF THIS BLOG

DR ANTHONY MELVIN CRASTO, WORLDDRUGTRACKER

Preparation of Fine Particles with Improved Solubility Using a Complex Fluidized-Bed Granulator Equipped with a Particle-Sizing Mechanism

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Jun 062016
 


Fig. 1. Schematic Representation of a Complex Fluidized-Bed Granulator

1: Exhaust air, 2: bag filter, 3: partition tube, 4: impeller, 5: rotor disc, 6: inlet air, 7: screen, 8: spray nozzle.

 

Preparation of Fine Particles with Improved Solubility Using a Complex Fluidized-Bed Granulator Equipped with a Particle-Sizing Mechanism

Abstract

A new type of fluidized-bed granulator equipped with a particle-sizing mechanism was used for the preparation of fine particles that improved the solubility of a poorly water-soluble drug substance. Cefteram pivoxyl (CEF) was selected as a model drug substance, and its solution with a hydrophilic polymer, hydroxypropyl cellulose (HPC-L), was sprayed on granulation grade lactose monohydrate (Lac). Three types of treated particles were prepared under different conditions focused on the spraying air pressure and the amount of HPC-L. When the amount of HPC-L was changed, the size of the obtained particles was similar. However, particle size distribution was dependent on the amount of HPC-L. Its distribution became more homogenous with greater amounts of HPC-L, but the particle size distribution obtained by decreasing the spraying air pressure was not acceptable. By processing CEF with HPC-L using a complex fluidized-bed granulator equipped with a particle-sizing mechanism, the dissolution ratio was elevated by approximately 40% compared to that of unprocessed CEF. Moreover, in the dissolution profile of treated CEF, the initial burst was suppressed, and nearly zero order release was observed up to approximately 60% in the dissolution profile. This technique may represent a method with which to design fine particles of approximately 100 µm in size with a narrow distribution, which can improve the solubility of a drug substance with low solubility.

Conclusion

Three types of treated particles were prepared using a complex fluidized-bed granulator equipped with a particle-sizing mechanism under different conditions focused on the spraying air pressure and the amount of HPC-L. When the amount of HPC-L was changed, the size of the obtained particles was similar. However, particle size distribution was dependent on the amount of HPC-L. Its distribution became more homogenous with greater amounts of HPC-L, but the particle size distribution obtained by decreasing the spraying air pressure was not acceptable.

By processing CEF with HPC-L using this device, the dissolution ratio was elevated by approximately 40% compared to that of unprocessed one. Moreover, in the dissolution profile of treated CEF, the initial burst was suppressed, and nearly zero order release was observed up to approximately 60% in the dissolution profile.

The present method is applicable to the design of fine particles of approximately 100 µm in size with a narrow distribution, which improved the solubility of drug substance.

////////fine particle, particle size distribution, dissolution, complex fluidized-bed granulator

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Cyclopropylacetyl-(R)-carnitine

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Jun 062016
 
NMR Data of Cyclopropylacetyl-(R)-carnitine (4)

Position 1H-NMR (300 MHz, D2O) HOD=4.79 13C-NMR (75 MHz, D2O) DSSa)=−2.04 HMBC correlation
1 177.1 C2-Ha, Hb
2 a: 2.49 (1H, dd, J=8.0, 16.0 Hz) 40.9 C3-H, C4-Ha
b: 2.63 (1H, dd, J=5.6, 16.0 Hz)
3 5.63 (1H, m) 67.5 C2-Ha, Hb, C4-Hb
4 a: 3.60 (1H, d, J=14.0 Hz) 68.9 C2-Ha, Hb, C3-H, NMe
b: 3.86 (1H, dd, J=9.0, 14.0 Hz)
NMe3 3.18 (9H, s) 54.5 C4-Ha, Hb
1′ 175.6 C2′-Ha, Hb
2′ a: 2.27 (1H, dd, J=7.0, 16.0 Hz) 39.7 C4′-Ha, C5′-Ha
b: 2.36 (1H, dd, J=7.4, 16.0 Hz)
3′ 0.98 (1H, m) 6.7 C4′-Ha, C5′-Ha, C2′-Ha, Hb
4′ a: 0.15 (1H, m) 4.2 C2′-Ha, Hb
b: 0.52 (1H, m)
5′ a: 0.15 (1H, m) 4.4 C2′-Ha, Hb
b: 0.52 (1H, m)

a) DSS=sodium 2,2-dimethyl-2-silapentane-5-sulfonate.

To confirm the structure of 4, the identical carnitine ester was synthesized by condensation of (R)-carnitine and cyclopropylacetic acid using an acid chloride method (see Experimental). The 1H- and 13C-NMR data of the natural and synthetic samples were identical, and the absolute configuration was also determined to be R by comparing the specific rotation of the synthetic compound and that of the natural one. Thus, the isolated cyclopropane-containing new compound was confirmed to be cyclopropylacetyl-(R)-carnitine (4). Interestingly, carnitine esters, including a cyclopropylcarboxylic acid ester, were also isolated from a Boletaceae mushroom.9) We examined the toxicity of 4 in mouse by an intraperitoneal route; however, no toxicity was detected.

Cyclopropylacetyl-(R)-carnitine is specific to genuine R. subnigricans and sufficiently stable under ordinary experimental conditions. In addition, the upfield signals in the 1H-NMR spectrum corresponding to the cyclopropane core are easily recognizable in the 1H-NMR spectrum of crude mixtures of fruiting bodies; therefore, it would be a useful chemical marker for the identification of genuine R. subnigricans (Fig. 3).

Fig. 3. 1H-NMR Spectra (500 MHz, D2O, TSPa)=0.00 ppm)(A) Authentic sample of cyclopropylacetyl-(R)-carnitine (4). (B) Crude water extract of R. subnigricans collected in Kyoto. (C) Crude water extract of Russula sp. collected in Miyagi. (D) Crude water extract of Russula sp. collected in Saitama. a) TSP=3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt.

Cyclopropylacetyl-(R)-carnitine (4)

Isolation of Cyclopropylacetyl-(R)-carnitine (4) from the Russula subnigricans (Collected in Kyoto)

The fruiting bodies (500 g) of Russula subnigricans (collected in Kyoto) were cut into pieces and soaked in aqueous 0.3% AcOH (1.5 L) at 4°C overnight. The extract was filtered through filter paper under suction and then the filtrate was concentrated to about 100 mL under reduced pressure. The concentrated solution was dialyzed (relative molecular mass (Mr) 14000) against aqueous 0.3% AcOH (2.0 L×2) overnight. The dialyzate was concentrated to dryness and lyophilized to give a crude extract (27.1 g). A part (4.8 g) of the crude extract was dissolved in 1% AcOH in MeOH (48 mL), and then the soluble part was applied to an alumina column (aluminium oxide 90 standardized, Merck, 32 g), which was eluted with 1% AcOH in MeOH (300 mL). The eluate was concentrated to 5 mL, and this was diluted with aqueous 0.3% AcOH (10 mL) and chromatographed on ODS (Cosmosil 140C18 OPN, 16 g) which was eluted with H2O (300 mL) and 50% aqueous MeOH (100 mL). After removal of MeOH from the 50% MeOH fraction, the aqueous solution was washed with EtOAc (100 mL×3). The aqueous layer was concentrated in vacuo and then chromatographed on ODS by elution with 20% MeOH. The obtained fractions which contained a cyclopropane derivative were concentrated (16.2 mg) and purified by preparative TLC (ODS, 20% CH3CN) followed by HPLC (ODS ϕ6×250 mm, eluted with H2O for 10 min and then linear gradient from H2O to 20% CH3CN for 50 min at a flow rate of 1.5 mL/min with monitoring at 210 nm) to give 4 (3.4 mg, tR=20.9 min) as colorless syrup. Rf=0.31 (ODS, 1 : 4 MeOH–H2O); IR νmax (KBr): 3735, 3468, 1732, 1594, 1398, 1188, 1054 cm−1; LR-FAB-MS m/z 244 [M+H]+, 162 [M−C5H6O]+, HR-FAB-MS m/z 244.1572 [M+H]+; Calcd for C12H22NO4, 244.1549; [α]D31 −14.5 (H2O, c=0.96).

 

 

Synthesis of Cyclopropylacetyl-(R)-carnitine (4)

To a stirred cyclopropylacetic acid (0.098 mL, 1.05 mmol) was added thionyl chloride (0.080 mL, 1.10 mmol) and the mixture was stirred at room temperature (rt) for 1 h. To the crude acid chloride was directly added (R)-carnitine (86.0 mg, 0.533 mmol) and the mixture was stirred at rt for 1.5 h. After evaporation, the residue was dissolved into H2O and the aqueous layer was washed with EtOAc. The aqueous layer was applied to ODS column chromatography (H2O). The eluate was neutralized by saturated aqueous NaHCO3 solution and then concentrated to dryness. The resulting residue was extracted with MeOH and it was filtered through Celite. The filtrate and washings were concentrated in vacuo to afford cyclopropylacetyl-(R)-carnitine (4) (60.4 mg, 47% yield) as colorless foam. mp 180°C (decomp.);Rf=0.31 (ODS, 1 : 4 MeOH–H2O); IR νmax (KBr): 3735, 3433, 1734, 1592, 1392, 1179, 1034 cm−1; 1H-NMR (D2O, HOD=4.79) δ: 0.15 (2H, m, H-4′, 5′), 0.52 (2H, m, H-4′, 5′), 0.99 (1H, m, H-3′), 2.28, (1H, dd, J=7.0, 16.0 Hz, H-2′), 2.36 (1H, dd, J=7.4, 16.0 Hz, H-2′), 2.49 (1H, dd, J=8.0, 16.0 Hz, H-2), 2.63 (1H, dd, J=5.6, 16.0 Hz, H-2), 3.18 (9H, s, 4-N+Me3), 3.61 (1H, d, J=14.0 Hz, H-4), 3.86 (1H, dd, J=9.0, 14.0 Hz, H-4), 5.63 (1H, m, H-3); 13C-NMR (D2O, DSS=–2.04) δ: 4.2 (C4′, 5′), 4.4 (C4′, 5′), 6.7 (C3′), 39.7 (C2′), 40.9 (C2), 54.5 (4-N+Me3), 67.5 (C3), 68.9 (C4), 175.7 (C1′), 177.2 (C1); LR-FAB-MS m/z 244 [M+H]+, 162 [M−C5H6O]+, HR-FAB-MS m/z 244.1555 [M+H]+; Calcd for C12H22NO4, 244.1549; [α]D34 −16.6 (H2O, c=0.67).

 

Identification of Cyclopropylacetyl-(R)-carnitine, a Unique Chemical Marker of the Fatally Toxic MushroomRussula subnigricans

Abstract

A toxic mushroom, Russula subnigricans, causes fatal poisoning by mistaken ingestion. In spite of the potent bioactivity, the responsible toxin had not been identified for about 50 years since its first documentation. Recently, we isolated an unstable toxin and determined the structure. The slow elucidation was partly due to the instability of the toxin and also due to misidentification of R. subnigricans for similar mushrooms. To discriminate genuine Russula subnigricans from similar unidentified Russula species, we searched for a unique chemical marker contained in the mushroom. Cyclopropylacetyl-(R)-carnitine specific to R. subnigricans was identified as a novel compound whose1H-NMR signals appearing in the upfield region were easily recognizable among the complicated signals of the crude extract.

/////////cyclopropylacetyl-(R)-carnitine, cycloprop-2-ene carboxylic acid, russuphelin G, mushroom poisoning, Russula subnigricans,Russulaceae

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AM 2394

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Jun 062016
 

str1

AM 2394

1-(6′-(2-hydroxy-2-methylpropoxy)-4-((5-methylpyridin-3-yl)oxy)-[3,3′-bipyridin]-6-yl)-3-methylurea

Urea, N-​[6′-​(2-​hydroxy-​2-​methylpropoxy)​-​4-​[(5-​methyl-​3-​pyridinyl)​oxy]​[3,​3′-​bipyridin]​-​6-​yl]​-​N‘-​methyl-

CAS 1442684-77-6
Chemical Formula: C22H25N5O4
Exact Mass: 423.1907

Array Biopharma Inc., Amgen Inc. INNOVATORS

AM-2394 is a potent and selective Glucokinase agonist (GKA), which catalyzes the phosphorylation of glucose to glucose-6-phosphate. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes

Type 2 diabetes mellitus (T2DM) is a disease characterized by elevated plasma glucose in the presence of insulin resistance and inadequate insulin secretion. Glucokinase (GK), a member of the hexokinase enzyme family, catalyzes the phosphorylation of glucose to glucose-6-phosphate in the presence of ATP.

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Glucokioase i exok ase IV or D> is a glycolytic enssyiris that plays, an importaat. role irt blood sugar regulation .related to glucose utifeattoti a»d metabolism in the liver and pancreatic beta cells. Serving as a glucose sessor, gtoeokiuase controls lasma glucose, levels. Glucokinaae plays a doal rob in .reducing plasma glucose levels; glucose-mediated activation of the en¾ymc in hepatocytes facilitates hepatic giocose npiafcc aad glycogen synthesis, while that la pancreatic beta ceils ultimately induces ins lin seeretio«. Both of these effects in turn reduce plasma glucose levels.

Clinical evidence has shown that, glueokitiase variants with, decreased, and increased activities are associated with mature easel, diabetes of the y ung { O0Y2) and persistent: hyperinsul nemic hypoglycemia &( infancy (PHHI), respectively. lso, aoo n.sulin dependent diabetes rneilitos (NIDDM) patients have been reported to have inappropriately lo giueokaiase activity; Ftirtherrnare. overexpressioa of glucokiuase it* dietary or gesetie animal models of diabetes either prevents, aoKiiorafes, or reverses the progress of pathological. symptoms in the disease. For these reasons, compounds that activate gfecokiaase have been sought by the pitasaaceatjeai liidustry.

International patent application, Publication No. WO 2 7/OS3345, which was published on May 10, 200?, discloses as giocokinase act ators certain 2-an«.aopyridiiie derivatives bearing at the 3 -position a meihyieneoxy-dkrked aromatic group a d on. the ammo group a heteroaryl ring, such as dna/oly! or i A4-lmadiazoiyl

it has .now been found that pyridyl ureas are useful as glneokirtase activators. Cettain of these •compounds have been, found to have an outstanding combination of properties that especially adapts them, for oral use to control plasma glucose levels.

 

 

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394

Departments of Therapeutic Discovery, Metabolic Disorders, and Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
Departments of Metabolic Disorders, Comparative Biology and Safety Sciences and Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
§ Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, United States
ACS Med. Chem. Lett., Article ASAP
DOI: 10.1021/acsmedchemlett.6b00140

http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.6b00140

 

Abstract Image

Glucokinase (GK) catalyzes the phosphorylation of glucose to glucose-6-phosphate. We present the structure–activity relationships leading to the discovery of AM-2394, a structurally distinct GKA. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes.

PATENT

WO 2013086397

 http://www.google.com/patents/WO2013086397A1?cl=en

 COPYING ERROR

Example. 1734 t¾^Jtiyi¾rea

Figure imgf000643_0007

Step A: In 100 mL of DMA were corafeiaed 1 ^545miSO- -ll«omp ridinr2-yl)-3-i«e hir8a- (17.5 g, 70,5 ii!-!to!). 5-o:ieS:t}yI yiidlii~3- ). (9,24 g, S4.7 ΪΗΪΪΪΟ!}, sad CO · (10.1 g, 77.6 mmo!) mid heated to 90 *C for 5 days. After that time, the reaction was om lete a d to it was added water arid DCM and stirred vigorously for 3 hr. The resulting solid was isolated via vacuum .filtratiott nd the cake was wasted mill rater and DCM. The DCM in tli aqueous rime was dried vdth a stream of aidogeji aad vigorous sbrriug. Use resulting solid was then collected via vacuum filtration aad these solids were

Stirred vig rousl in f 0% MeOH irt EtOAc arid die res dtipg solid was colleeied. via vactiiars fiirfati m.

Trie two batches wen i coiiibiaed to yield I-(5-bmmo-4 5^»ie†fey pyiidin-3-yl xy)p Tidin-2- d 3~ metbySurea (I S J g, 5 3.7 om»)i, 76% yield).

S e .8: In 2 niL ofc ioxane

Figure imgf000644_0001

yI) iyridMJ-2-yios:y)pf¾ps3i-2-oI (0,098 g, 0.33 «ΜΠΟΪ), -i5-bs¾tao-4-{5-a3fidiy I py f idia-3 – ylosy)f5yridia-2-yl)-3-raethyl«rea (0.075 g, 0.22 tn ol.. t, and.2M poiass.ua» carbonate (0.33 ml, 0.67 m oi} artd tfets was s parged wi h At .for 10 mia before PdC§4dppl)*DCM (0.01 g g, 0.022 msttol) was added and dre reae!io a was sparged for aaotber 5 ma-, ir efore a was sealed and heated to 100 oversight The react! art was then loaded directly onto s ilica gel (50% acetone to PCM w4i. }%

MH40H) to afford i – (6′-(2diydioxy-2i-H5eth:ylpropCis:y) -4-{ 5″i:t re th y Ipy r i d i rt -3- io s y ) -3 ,3 : -bipyr id i rt -6- yl)-3-aie5¾ylt)rea φ.? 42 , 0.096 m ol, 43 % yield). !1 1 HMR (400 Mife, CDCij) 3 ppm 9.06 is,. !H),

S.33 is, 1H>, 8,27 (rs 2H), 8. Π (s, I H): K. (s, IHU 82 (dd, j-S.fi, 5.9 H HI), 1.21 (S !H), 6,«8

(d, Hz, i i i ). 6. ,4 (s:. m>, 4.25 (s, 2H), 2,87 (dj =4,3 Hz„ 3H) 2,37 (s, 3H>. 1 .33 is, <SH). Mass speetram (apci) tar/, : – 423.9 (M÷H).

REFERENCES

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394
Paul J. Dransfield, Vatee Pattaropong, Sujen Lai, Zice Fu, Todd J. Kohn, Xiaohui Du, Alan Cheng, Yumei Xiong, Renee Komorowski, Lixia Jin, Marion Conn, Eric Tien, Walter E. DeWolf Jr., Ronald J. Hinklin, Thomas D. Aicher, Christopher F. Kraser, Steven A. Boyd, Walter C. Voegtli, Kevin R. Condroski, Murielle Veniant-Ellison, Julio C. Medina, Jonathan Houze, and Peter Coward
Publication Date (Web): May 23, 2016 (Letter)
DOI: 10.1021/acsmedchemlett.6b00140

/////////Glucokinase activator,  GKA,  AM-2394, 1442684-77-6, AM 2394, Amgen

O=C(NC)NC1=CC(OC2=CC(C)=CN=C2)=C(C3=CC=C(OCC(C)(O)C)N=C3)C=N1

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JNJ-54257099

 PRECLINICAL, Uncategorized  Comments Off on JNJ-54257099
Jun 062016
 

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Abstract Image

JNJ-54257099,

1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione

MW 374.28, C14 H19 N2 O8 P

CAS 1491140-67-0

2,​4(1H,​3H)​-​Pyrimidinedione, 1-​[(2R,​2′R,​4aR,​6R,​7aR)​-​dihydro-​2-​(1-​methylethoxy)​-​2-​oxidospiro[4H-​furo[3,​2-​d]​-​1,​3,​2-​dioxaphosphorin-​7(6H)​,​2′-​oxetan]​-​6-​yl]​-

1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphos-phinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione

Janssen R&D Ireland INNOVATOR

Ioannis Nicolaos Houpis, Tim Hugo Maria Jonckers, Pierre Jean-Marie Bernard Raboisson, Abdellah Tahri

 

 

 

STR1

Tim Hugo Maria Jonckers

 

Tim Jonckers was born in Antwerp in 1974. He studied Chemistry at the University of Antwerp and obtained his Ph.D. in organic chemistry in 2002. His Ph.D. work covered the synthesis of new necryptolepine derivatives which have potential antimalarial activity. Currently he works as a Senior Scientist at Tibotec, a pharmaceutical research and development company based in Mechelen, Belgium, that focuses on viral diseases mainly AIDS and hepatitis. The company was acquired by Johnson & Johnson in April 2002 and recently gained FDA approval for its HIV-protease inhibitor PREZISTA™.

Abdellah TAHRI

Principal Scientist at Janssen, Pharmaceutical Companies of Johnson and Johnson

 

 

Pierre Raboisson

Pierre Raboisson

PhD, Pharm.D
Head of Medicinal Chemistry

DATA

Chiral SFC using the methods described(Method 1, Rt= 5.12 min, >99%; Method 2, Rt = 7.95 min, >99%).

1H NMR (400 MHz, chloroform-d) δ ppm 1.45 (dd, J = 7.53, 6.27 Hz, 6 H), 2.65–2.84 (m, 2 H), 3.98 (td, J = 10.29, 4.77 Hz, 1 H), 4.27 (t,J = 9.66 Hz, 1 H), 4.43 (ddd, J = 8.91, 5.77, 5.65 Hz, 1 H), 4.49–4.61 (m, 1 H), 4.65 (td, J = 7.78, 5.77 Hz, 1 H), 4.73 (d, J = 7.78 Hz, 1 H), 4.87 (dq, J = 12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J = 8.03 Hz, 1 H), 7.20 (d, J = 8.03 Hz, 1 H), 8.78 (br. s., 1 H);

31P NMR (chloroform-d) δ ppm −7.13. LC-MS: 375 (M + H)+.

 

HCV is a single stranded, positive-sense R A virus belonging to the Flaviviridae family of viruses in the hepacivirus genus. The NS5B region of the RNA polygene encodes a RNA dependent RNA polymerase (RdRp), which is essential to viral replication. Following the initial acute infection, a majority of infected individuals develop chronic hepatitis because HCV replicates preferentially in hepatocytes but is not directly cytopathic. In particular, the lack of a vigorous T-lymphocyte response and the high propensity of the virus to mutate appear to promote a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease, and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantations. There are six major HCV genotypes and more than 50 subtypes, which are differently distributed geographically. HCV genotype 1 is the predominant genotype in Europe and in the US. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, perhaps explaining difficulties in vaccine development and the lack of response to current therapy.

Transmission of HCV can occur through contact with contaminated blood or blood products, for example following blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has led to a downward trend in post-transfusion HCV incidence. However, given the slow progression to the end-stage liver disease, the existing infections will continue to present a serious medical and economic burden for decades.

Therapy possibilities have extended towards the combination of a HCV protease inhibitor (e.g. Telaprevir or boceprevir) and (pegylated) interferon-alpha (IFN-a) / ribavirin. This combination therapy has significant side effects and is poorly tolerated in many patients. Major side effects include influenza-like symptoms, hematologic

abnormalities, and neuropsychiatric symptoms. Hence there is a need for more effective, convenient and better-tolerated treatments.

The NS5B RdRp is essential for replication of the single-stranded, positive sense, HCV RNA genome. This enzyme has elicited significant interest among medicinal chemists. Both nucleoside and non-nucleoside inhibitors of NS5B are known. Nucleoside inhibitors can act as a chain terminator or as a competitive inhibitor, or as both. In order to be active, nucleoside inhibitors have to be taken up by the cell and converted in vivo to a triphosphate. This conversion to the triphosphate is commonly mediated by cellular kinases, which imparts additional structural requirements on a potential nucleoside polymerase inhibitor. In addition this limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays capable of in situ phosphorylation.

Several attempts have been made to develop nucleosides as inhibitors of HCV RdRp, but while a handful of compounds have progressed into clinical development, none have proceeded to registration. Amongst the problems which HCV-targeted

nucleosides have encountered to date are toxicity, mutagenicity, lack of selectivity, poor efficacy, poor bioavailability, sub-optimal dosage regimes and ensuing high pill burden and cost of goods.

Spirooxetane nucleosides, in particular l-(8-hydroxy-7-(hydroxy- methyl)- 1,6-dioxaspiro[3.4]octan-5-yl)pyrimidine-2,4-dione derivatives and their use as HCV inhibitors are known from WO2010/130726, and WO2012/062869, including

CAS-1375074-52-4.

There is a need for HCV inhibitors that may overcome at least one of the disadvantages of current HCV therapy such as side effects, limited efficacy, the emerging of resistance, and compliance failures, or improve the sustained viral response.

The present invention concerns HCV-inhibiting uracyl spirooxetane derivatives with useful properties regarding one or more of the following parameters: antiviral efficacy towards at least one of the following genotypes la, lb, 2a, 2b, 3,4 and 6, favorable

profile of resistance development, lack of toxicity and genotoxicity, favorable pharmacokinetics and pharmacodynamics and ease of formulation and administration.

Such an HCV-inhibiting uracyl spirooxetane derivative is a compound with formula I

including any pharmaceutically acceptable salt or solvate thereof.

PATENT

WO 2015077966

https://www.google.com/patents/WO2015077966A1?cl=en

Synthesis of compound (I)

(5) (6a)

Synthesis of compound (6a)

A solution of isopropyl alcohol (3.86 mL,0.05mol) and triethylamine (6.983 mL, 0.05mol) in dichloromethane (50 mL) was added to a stirred solution of POCI3 (5)

(5.0 mL, 0.055 lmol) in DCM (50 mL) dropwise over a period of 25 min at -5°C. After the mixture stirred for lh, the solvent was evaporated, and the residue was suspended in ether (100 mL). The triethylamine hydrochloride salt was filtered and washed with ether (20 mL). The filtrate was concentrated, and the residue was distilled to give the (6) as a colorless liquid (6.1g, 69 %yield).

Synthesis of compound (4):

CAS 1255860-33-3 is dissolved in pyridine and 1,3-dichloro-l, 1,3,3-tetraisopropyldisiloxane is added. The reaction is stirred at room temperature until complete. The solvent is removed and the product redissolved in CH2CI2 and washed with saturated NaHC03 solution. Drying on MgSC^ and removal of the solvent gives compound (2). Compound (3) is prepared by reacting compound (2) with p-methoxybenzylchloride in the presence of DBU as the base in CH3CN. Compound (4) is prepared by cleavage of the bis-silyl protecting group in compound (3) using TBAF as the fluoride source.

Synthesis of compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature. The reaction mixture was cooled to -20°C, and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of lOmin. The mixture was stirred at this temperature for 15min and then NMI was added (0.84 g, 10.23 mmol), dropwise over a period of 15 min. The mixture was stirred at -15°C for lh and then slowly warmed to room temperature in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum ether/EtOAc (10: 1 to 5: 1 as a gradient) to give (7a) as white solid (0.8 g, 32 % yield).

Synthesis of compound (I)

To a solution of (7a) in CH3CN (30 mL) and H20 (7 mL) was add CAN portion wise below 20° C. The mixture was stirred at 15-20° C for 5h under N2. Na2S03 (370 mL) was added dropwise into the reaction mixture below 15°C, and then Na2C03 (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH2C12

(100 mL*3). The organic layer was dried and concentrated to give the residue. The residue was purified by column chromatography to give the target compound (8a) as white solid. (Yield: 55%)

1H NMR (400 MHz, CHLOROFORM- ) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6 H), 2.65 -2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, J=9.66 Hz, 1 H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1 H), 4.49 – 4.61 (m, 1 H), 4.65 (td, J=7.78, 5.77 Hz, 1 H), 4.73 (d, J=7.78 Hz, 1 H), 4.87 (dq, J=12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J=8.03 Hz, 1 H), 7.20 (d, J=8.03 Hz, 1 H), 8.78 (br. s., 1 H); 31P NMR (CHLOROFORM-^) δ ppm -7.13; LC-MS: 375 (M+l)+

 

PATENT

https://www.google.co.in/patents/WO2013174962A1?cl=en

The starting material l-[(4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-l,6-dioxa- spiro[3.4]octan-5-yl]pyrimidine-2,4(lH,3H)-dione (1) can be prepared as exemplified in WO2010/130726. Compound (1) is converted into compounds of the present invention via a p-methoxybenzyl protected derivative (4) as exemplified in the following Scheme 1. cheme 1

Figure imgf000011_0001

Examples

Scheme 2

Synthesis of compound (8a)

Figure imgf000015_0001

Synthesis of compound (2)

Compound (2) can be prepared by dissolving compound (1) in pyridine and adding l,3-dichloro-l,l,3,3-tetraisopropyldisiloxane. The reaction is stirred at room temperature until complete. The solvent is removed and the product redissolved in CH2CI2and washed with saturated NaHC03 solution. Drying on MgSC^ and removal of the solvent gives compound (2).

Synthesis of compound (3)

Compound (3) is prepared by reacting compound (2) with p-methoxybenzylchloride in the presence of DBU as the base in CH3CN.

Synthesis of compound (4)

Compound (4) is prepared by cleavage of the bis-silyl protecting group in compound (3) using TBAF as the fluoride source.

Synthesis of compound (6a)

A solution of isopropyl alcohol (3.86 mL,0.05mol) and triethylamine (6.983 mL, 0.05mol) in dichloromethane (50 mL) was added to a stirred solution of POCl3 (5) (5.0 mL, 0.055 lmol) in DCM (50 mL) dropwise over a period of 25 min at -5°C. After the mixture stirred for lh, the solvent was evaporated, and the residue was suspended in ether (100 mL). The triethylamine hydrochloride salt was filtered and washed with ether (20 mL). The filtrate was concentrated, and the residue was distilled to give the (6) as a colorless liquid (6.1g, 69 %yield).

Synthesis of compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature. The reaction mixture was cooled to -20°C, and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of lOmin. The mixture was stirred at this temperature for 15min and then NMI was added (0.84 g, 10.23 mmol), dropwise over a period of 15 min. The mixture was stirred at -15°C for lh and then slowly warmed to room temperature in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum ether/EtOAc (10:1 to 5: 1 as a gradient) to give (7a) as white solid (0.8 g, 32 % yield).

Synthesis of compound (8a)

To a solution of (7a) in CH3CN (30 mL) and H20 (7 mL) was add CAN portion wise below 20°C. The mixture was stirred at 15-20°C for 5h under N2. Na2S03 (370 mL) was added dropwise into the reaction mixture below 15°C, and then Na2C03 (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH2C12

(100 mL*3). The organic layer was dried and concentrated to give the residue. The residue was purified by column chromatography to give the target compound (8a) as white solid. (Yield: 55%)

1H NMR (400 MHz, CHLOROFORM- ) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6 H), 2.65 – 2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, J=9.66 Hz, 1 H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1 H), 4.49 – 4.61 (m, 1 H), 4.65 (td, J=7.78, 5.77 Hz, 1 H), 4.73 (d, J=7.78 Hz, 1 H), 4.87 (dq, J=12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J=8.03 Hz, 1 H), 7.20 (d, J=8.03 Hz, 1 H), 8.78 (br. s., 1 H); 31P NMR (CHLOROFORM-^) δ ppm -7.13; LC-MS: 375 (M+l)+ Scheme 3

Synthesis of compound (VI)

Figure imgf000017_0001

Step 1: Synthesis of compound (9)Compound (1), CAS 1255860-33-3 ( 1200 mg, 4.33 mmol ) and l,8-bis(dimethyl- amino)naphthalene (3707 mg, 17.3 mmol) were dissolved in 24.3 mL of

trimethylphosphate. The solution was cooled to 0°C. Compound (5) (1.21 mL, 12.98 mmol) was added, and the mixture was stirred well maintaining the temperature at 0°C for 5 hours. The reaction was quenched by addition of 120 mL of tetraethyl- ammonium bromide solution (1M) and extracted with CH2CI2 (2×80 mL). Purification was done by preparative HPLC (Stationary phase: RP XBridge Prep CI 8 ΟΒϋ-10μιη, 30x150mm, mobile phase: 0.25% NH4HCO3 solution in water, CH3CN) , yielding two fractions. The purest fraction was dissolved in water (15 mL) and passed through a manually packed Dowex (H+) column by elution with water. The end of the elution was determined by checking UV absorbance of eluting fractions. Combined fractions were frozen at -78°C and lyophilized. Compound (9) was obtained as a white fluffy solid (303 mg, (0.86 mmol, 20%> yield), which was used immediately in the following reaction. Step 2: Preparation of compound (VI)

Compound (9) (303 mg, 0.86 mmol) was dissolved in 8 mL water and to this solution was added N . N’- D ic y c ! he y !-4- mo rph line carboxamidine (253.8 mg, 0.86 mmol) dissolved in pyridine (8.4 mi.). The mixture was kept for 5 minutes and then

evaporated to dryness, dried overnight in vacuo overnight at 37°C. The residu was dissolved in pyridine (80 mL). This solution was added dropwise to vigorously stirred DCC (892.6 mg, 4.326 mmol) in pyridine (80 mL) at reflux temperature. The solution was kept refluxing for 1.5h during which some turbidity was observed in the solution. The reaction mixture was cooled and evaporated to dryness. Diethylether (50 mL) and water (50 mL) were added to the solid residu. N’N-dicyclohexylurea was filtered off, and the aqueous fraction was purified by preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-ΙΟμιη, 30x150mm, mobile phase: 0.25% NH4HCO3 solution in water, CH3CN) , yielding a white solid which was dried overnight in vacuo at 38°C. (185 mg, 0.56 mmol, 65% yield). LC-MS: (M+H)+: 333.

1H NMR (400 MHz, DMSO-d6) d ppm 2.44 – 2.59 (m, 2 H) signal falls under DMSO signal, 3.51 (td, J=9.90, 5.50 Hz, 1 H), 3.95 – 4.11 (m, 2 H), 4.16 (d, J=10.34 Hz, 1 H), 4.25 – 4.40 (m, 2 H), 5.65 (d, J=8.14 Hz, 1 H), 5.93 (br. s., 1 H), 7.46 (d, J=7.92 Hz, 1 H), 2H’s not observed

Paper

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00382,

Discovery of 1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3′-5′-Cyclic Phosphate Ester Prodrug of 2′-Deoxy-2′-Spirooxetane Uridine Triphosphate Useful for HCV Inhibition

Janssen Infectious Diseases − Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium
J. Med. Chem., Article ASAP
DOI: 10.1021/acs.jmedchem.6b00382
Publication Date (Web): May 14, 2016
Copyright © 2016 American Chemical Society
*Phone: +32 014601168. E-mail: tjoncker@its.jnj.com.

JNJ-54257099 (9) is a novel cyclic phosphate ester derivative that belongs to the class of 2′-deoxy-2′-spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNA-dependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line 9 is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of 9 in primary human hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species reveals the formation of substantial levels of 2′-deoxy-2′-spirooxetane uridine triphosphate (8), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that 9 displays a superior profile compared to its phosphoramidate prodrug analogues (e.g., 4) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 days of oral administration of 9

////////////JNJ-54257099, 1491140-67-0, JNJ54257099, JNJ 54257099

O=C(C=C1)NC(N1[C@H]2[C@]3(OCC3)[C@H](O4)[C@@H](CO[P@@]4(OC(C)C)=O)O2)=O

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