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TR 700, TR 701FA, Tedizolid phosphate

September 30, 2015 3:40 am / Leave a comment

“TR-700”

5R)-3-{3-Fluoro-4-[6-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)-pyridin-3-yl]-phenyl}-5-hydroxymethyl-1,3-oxazolidin-2-one

US Patent Publication No. 20070155798, which is hereby incorporated by reference in its entirety, recently disclosed a series of potently anti-bacterial oxazolidinones including

wherein R═H, PO(OH)₂, and PO(ONa)₂.

(R)-3-(4-(2-(2-methyltetrazol-5-yl)pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-one dihydrogen phosphate, CAS 856867-55-5

DISODIUM SALT

CAS 856867-39-5

C₁₇ H₁₆ F N₆ O₆ P . 2 Na
2-Oxazolidinone, 3-[3-fluoro-4-[6-(2-methyl-2H-tetrazol-5-yl)-3-pyridinyl]phenyl]-5-[(phosphonooxy)methyl]-, sodium salt (1:2), (5R)-
- DA 7218, Tedizolid phosphate disodium salt

In addition, improved methods of making the free acid are disclosed in U.S. patent application Ser. No. 12/577,089, which is assigned to Trius Therapeutics, Inc., and which is incorporated herein by reference

crystalline (R)-3-(4-(2-(2-methyltetrazol-5-yl)pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-one dihydrogen phosphate 1 (R═PO(OH)₂), was more stable and non-hygroscopic than the salt forms that were tested. In addition, unlike typical crystallizations, where the crystallization conditions, such as the solvent and temperature conditions, determine the particular crystalline form, the same crystalline form of 1 (R═PO(OH)₂) was produced using many solvent and crystallization conditions. Therefore, this crystalline form was very stable, was made reproducibly, and ideal for commercial production because it reduced the chances that other polymorphs would form contaminating impurities during production. However, in all preliminary testing, the free acid crystallized as fine particles, making filtering and processing difficult.

To overcome difficulties in filtering and processing crystalline (R)-3-(4-(2-(2-methyltetrazol-5-yl)pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-one dihydrogen phosphate 1 (R═PO(OH)₂), processes described herein result in significantly reduced filtering time, avoid more toxic solvents, and significantly increased ease of preparing dosage forms such as tablets. It has been found that implementing various processes can control the particle size distribution of the resulting material, which is useful for making the crystalline form, and for commercial production and pharmaceutical use. Surprisingly, the process for increasing the particle size reduces the amount of the dimer impurity, in comparison to the process for making the free acid disclosed in U.S. patent application Ser. No. 12/577,089. Thus, various methods of making and using the crystalline form are also provided.

In addition, by using methods of making the free acid disclosed in U.S. patent application Ser. No. 12/577,089, which is assigned to the same assignee as in the present application, and by using the crystallization methods described herein, a crystalline free acid having at least 96% purity by weight may be formed that comprises a compound having the following formula:

(hereinafter “the chloro impurity”), i.e., (R)-5-(chloromethyl)-3-(3-fluoro-4-(6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl)phenyl)oxazolidin-2-one in an amount less than 1%.

Similarly, by using methods of making the free acid disclosed in U.S. patent application Ser. No. 12/577,089, which is assigned to the same assignee as in the present application, and by using the crystallization methods described herein, a crystalline free acid having at least 96% purity by weight may be formed that comprises a compound having the following formula:

(hereinafter “TR-700”), i.e., 5R)-3-{3-Fluoro-4-[6-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)-pyridin-3-yl]-phenyl}-5-hydroxymethyl-1,3-oxazolidin-2-one, in an amount less than 1%.

The crystalline free acid may have one or more of the attributes described herein.

In some aspects, a purified crystalline (R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-one dihydrogen phosphate, i.e., the free acid, has a purity of at least about 96% by weight. In some embodiments, the crystalline free acid has a median volume diameter of at least about 1.0 μm.

BRIEF DESCRIPTION OF THE DRAWINGS……http://www.google.com/patents/US8426389

FIG. 1 the FT-Raman spectrum of crystalline 1 (R═PO(OH)₂).

FIG. 2 shows the X-ray powder pattern of crystalline 1 (R═PO(OH)₂).

http://www.google.com/patents/US8426389

FIG. 3 shows the differential scanning calorimetry (DSC) thermogram of crystalline 1 (R═PO(OH)₂).

http://www.google.com/patents/US8426389

FIG. 4 shows the ¹H NMR spectrum of 1 (R═PO(OH)₂).

FIG. 5 depicts the TG-FTIR diagram of crystalline 1 (R═PO(OH)₂).

http://www.google.com/patents/US8426389

FIG. 6 is a diagram showing the dynamic vapor sorption (DVS) behavior of crystalline 1 (R═PO(OH)₂).

FIG. 7 is a manufacturing process schematic for 1 (R═PO(OH)₂) (TR-701 FA) in a tablet dosage form.

FIG. 8 is a manufacturing process schematic for 1 (R═PO(OH)₂) (TR-701 FA) Compounding Solution for Lyophilization.

FIG. 9 is a manufacturing process schematic for 1 (R═PO(OH)₂) (TR-701 FA) for Injection, 200 mg/vial: sterile filtering, filling, and lyophilization.

FIG. 10 is a representative particle size distribution of crystalline free acid without regard to controlling particle size distribution as also described herein.

FIG. 11 is a representative particle size distribution of crystalline free acid made using laboratory processes to control particle size described herein.

FIG. 12 is a representative particle size distribution of crystalline free acid made using scaled up manufacturing processes to control particle size described herein.

These impurities include

i.e., 5R)-3-{3-Fluoro-4-[6-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)-pyridin-3-yl]-phenyl}-5-hydroxymethyl-1,3-oxazolidin-2-one (“TR-700”) and/or

i.e., (R)-5-(chloromethyl)-3-(3-fluoro-4-(6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl)phenyl)oxazolidin-2-one (“chloro impurity”).

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Wockhardt, WO 2007023507, N-[[3-[3,5-difluoro-4-[4-(tetrazol-2-yl)piperidin-1-yl]phenyl]-2-oxo-1,3-oxazolidin-5-yl]methyl]acetamide

September 29, 2015 4:18 am / Leave a comment

Cas 928156-95-0,

Acetamide, N-[[(5S)-3-[3,5-difluoro-4-[4-(2H-tetrazol-2-yl)-1-piperidinyl]phenyl]-2-oxo-5-oxazolidinyl]methyl]-

C₁₈H₂₁F₂N₇O₃
Molecular Weight:	421.401246 g/mol

N-[[3-[3,5-difluoro-4-[4-(tetrazol-2-yl)piperidin-1-yl]phenyl]-2-oxo-1,3-oxazolidin-5-yl]methyl]acetamide

http://www.google.com.ar/patents/WO2007023507A2?cl=en

Example- 14 and 15

(S)-N- { 3- [4-(4-(2H-tetrazol-2-yl)-piperidin- 1 -yl)-3 , 5-difluorophenyl] -2-oxo-oxazolidin-

5-ylmethyl }-acetamide and

(S)-N- { 3- [4-(4-(l H-tetrazol- 1 -yl)-piperidin- 1 -yl)-3 , 5-difluorophenyl] -2-oxo-oxazolidin-

5-ylmethyl }-acetamide

and

A mixture of (S)-N-{3-[4-methanesulphonyloxy piperidin-l-yl)-3,5-difluorophenyl]-2- oxo-oxazolidin-5-ylmethyl}-acetamide (1.12 mM), tetrazole (1.68 mM), and K₂CO₃ (1.68 mM) in DMF (6 ml) was heated for 22 hrs at 85⁰C. The resulting mixture was poured into ice-water mixture, stirred for 30 min. And the separated solid was purified by column chromatography to obtain two isomeric products in 18% and 12% yields respectively. Isomer A: M.P. 234-237⁰C; MS(M+1)- 422 ; M.F. C₁₈H₂₁F₂N₇O₃ Isomer B: M.P. 214-217⁰C; MS(M+1)- 422 ; M.F. C₁₈H₂JF₂N₇O₃

WOCKHARDT LIMITED [IN/IN]; D-4, MIDC Area, Chikalthana, Aurangabad 431006 (IN)

Our New Drug Discovery team has developed a number of lead molecules, mainly in the area of anti-infectives; these are currently at various stages of development.

Of these molecules, the most advanced of the New Chemical Entities (NCE) is WCK 771, which has commenced Phase II human clinical trials.

WCK 771 is a broad-spectrum antibiotic, which has proven effective in treating diverse staphylococcal infections like MRSA and VISA.

Other lead molecules at various stages of pre-clinical trials are: WCK 2349, WCK 4873 and WCK 4086.

http://www.wockhardt.com/how-we-touch-lives/new-drug-discover.aspx

Evidence of water found on Mars

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Wockhardt, WO 2015136473, sodium (2S, 5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate

September 29, 2015 4:17 am / Leave a comment

WO-2015136473

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015136473&redirectedID=true

WOCKHARDT LIMITED [IN/IN]; D-4, MIDC Area, Chikalthana, Aurangabad 431006 (IN)

Our New Drug Discovery team has developed a number of lead molecules, mainly in the area of anti-infectives; these are currently at various stages of development.

Of these molecules, the most advanced of the New Chemical Entities (NCE) is WCK 771, which has commenced Phase II human clinical trials.

WCK 771 is a broad-spectrum antibiotic, which has proven effective in treating diverse staphylococcal infections like MRSA and VISA.

Other lead molecules at various stages of pre-clinical trials are: WCK 2349, WCK 4873 and WCK 4086.

http://www.wockhardt.com/how-we-touch-lives/new-drug-discover.aspx

WO-2015136473

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015136473&redirectedID=true
Process for the synthesis of sodium (2S, 5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (disclosed in WO2014135929) is claimed. Used as an intermediate in the synthesis of several antibacterial compounds. For a concurrent filing see WO2015136387, claiming the combination of an antibacterial agent with sulbactam.

In September 2015, Wockhardt’s pipeline lists several antibacterial programs, including WCK-771 and WCK-2349 (both in phase II), WCK-5107 (phase I), and also investigating iv and oral second generation oxazolidinones, WCK-4873, and iv and oral formulation of WCK-4086 (in preclinical stage) for treating the bacterial infection.

For a prior filing see WO2015125031, claiming the combination of an antibacterial agent (eg cefepime or cefpirome) and nitrogen containing bicyclic compound, useful for treating bacterial infection.

A compound of Formula (I), chemically known as sodium (25, 5i?)-6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxylate, can be used as an intermediate in the synthesis of several antibacterial compounds and is disclosed in PCT International Patent Application No. PCT/IB2013/059264. The present invention discloses a process for preparation of a compound of Formula (I).

Scheme 1

Example 1

Synthesis of sodium (25, 5R)-6-(benzyloxy)-7-oxo-l,6-diazabicvclor3.2.11octane-2- carboxylate

Step 1; Preparation of -Γl-Γ(feΓt-butyldimethylsilyl -oxymethyll-5-Γdimethyl(oxido -λ-4-sulfanylidenel-4-oxo-pentyll-carbamic acid tert-butyl ester (III):

To a suspension of trimethylsulfoxonium iodide (180.36 gm, 0.819 mol) in tetrahydrofuran (900 ml), sodium hydride (32.89 g, 0.819 mol, 60% in mineral oil) was charged in one portion at 30°C temperature. The reaction mixture was stirred for 15 minutes and then dropwise addition of dimethylsulphoxide (1.125 ml) was done over a period of 3 hours at room temperature to provide a white suspension. The white suspension was added to a pre-cooled a solution of 2-(feri-butyldimethylsilyl-oxymethyl)-5-oxo-pyrrolidine-l-carboxylic acid tert-buty\ ester (II) (225 g, 0.683 mol, prepared as per J. Org Chem.; 2011, 76, 5574 and WO2009067600) in tetrahydrofuran (675 ml) and triethylamine (123.48 ml, 0.887 mol) mixture at -13°C by maintaining the reaction mixture temperature below -10°C. The resulting suspension was stirred for additional 1 hour at -10°C. The reaction mixture was carefully quenched by addition of saturated aqueous ammonium chloride (1.0 L) at -10°C to 10°C. The reaction was extracted by adding ethyl acetate (1.5 L). The layers were separated and aqueous layer was re-extracted with ethyl acetate (500 ml x 3). The combined organic layer was washed successively with saturated aqueous sodium bicarbonate (1.0 L), water (2.0 L) followed by saturated aqueous sodium chloride solution (1.0 L). Organic layer was dried over sodium sulfate and evaporated under vacuum to provide 265 g of 5-[l-[(ieri-butyldimethylsilyl)-oxymethyl]-5-[dimethyl(oxido)- -4-sulfanylidene]-4-oxo-pentyl]-carbamic acid tert-buty\ ester (III) as an yellow oily mass.

Analysis:

Mass: 422.3 (M+l); for Molecular weight: 421.68 and Molecular Formula:

1H NMR (CDC1₃): δ 4.77 (br d, 1H), 4.38 (br s, 1H), 3.58 (br s, 3H), 3.39 (s, 3H), 3.38 (s, 3H), 2.17-2.27 (m, 2H), 1.73-1.82 (m, 2H), 1.43 (s, 9H), 0.88 (s, 9H), 0.01 (s, 3H), 0.04 (s, 3H).

Step 2: Preparation of 5-r4-benzyloxyimino-l-(fert-butyldimethylsilyl-oxymethyl)-5-chloro-pentyll-carbamic acid tert- butyl ester (IV):

To a suspension of 5-[l-[(ieri-butyldimethylsilyl)-oxymethyl]-5-[dimethyl(oxido)- -4-sulfanylidene]-4-oxo-pentyl]-carbamic acid tert-butyl ester (III) (440.0 g, 1.045 mol) in tetrahydrofuran (6.6 L), O-benzhydroxylamine hydrochloride (200.0 g, 1.254 mol) was charged. The reaction mixture was heated to 50°C for 2.5 hours. The reaction mixture was filtered through pad of celite and filtrate was concentrated to provide a residue. The residue was dissolved in ethyl acetate (5.0 L) and washed successively with saturated aqueous sodium bicarbonate (1.5 L), water (1.5 L) and saturated aqueous sodium chloride (1.5 L). Organic layer was dried over sodium sulfate. Solvent was evaporated under vacuum to yield 463.0 g of 5-[4-benzyloxyimino-l-(tert-butyldimethylsilyl-oxymethyl)-5-chloro-pentyl]-carbamic acid tert-butyl ester (IV) as an oily mass.

Analysis:

Mass: 486.1 (M+l); for Molecular weight: 485.4 and Molecular Formula:

1H NMR (CDCI₃): δ 7.26-1 6 (m, 5H), 5.10 (s, 2H), 4.66 (br d, 1H), 3.58-4.27 (m, 2H), 3.56-3.58 (m, 3H), 2.40-2.57 (m, 2H), 1.68-1.89 (m, 2H), 1.44 (s, 9H), 0.89 (s, 9H), 0.02 (s, 3H), 0.04 (s, 3H).

Step 3: Preparation of 5-5-benzyloxyimino-2-(fert-butyldimethylsilyl-oxymethyl)-piperidine-l-carboxylic acid tert-butyl ester (V):

To a solution of 5-[4-benzyloxyimino-l-(tert-butyldimethylsilyl-oxymethyl)-5-chloro-pentyl]-carbamic acid tert-butyl ester (IV) (463.0 g 0.954 mol) in tetrahydrofuran (6.9 L), was charged potassium feri-butoxide (139.2 g, 1.241 mol) in portions over a period of 30 minutes by maintaining temperature -10°C. The resulting suspension was stirred for additional 1.5 hours at -10°C to -5°C. The reaction mixture was quenched by addition of saturated aqueous ammonium chloride (2.0 L) at -5°C to 10°C. The organic layer was separated and aqueous layer was extracted with ethyl acetate (1.0 L x 2). The combined organic layer was washed with saturated aqueous sodium chloride solution (2.0 L). Organic layer was dried over sodium sulfate, and then evaporated under vacuum to yield 394.0 g of 5-5-benzyloxyimino-2-(ieri-butyldimethylsilyl-oxymethyl)-piperidine- 1 -carboxylic acid tert-butyl ester (V) as an yellow oily mass.

Analysis:

Mass: 449.4 (M+l) for Molecular weight: 448.68 and Molecular Formula: C₂₄H4oN₂0₄Si;

1H NMR (CDC1₃): δ 7.25-1 3 (m, 5H), 5.04-5.14 (m, 2H), 4.35 (br s, 1H), 3.95 (br s, 1H), 3.63-3.74 (br d, 2H), 3.60-3.63 (m, 1H), 2.70-2.77 (m, 1H), 2.33-2.41 (m, 1H), 1.79-1.95 (m, 2H), 1.44 (s, 9H), 0.88 (s, 9H), 0.03 (s, 3H), 0.04 (s, 3H).

Step 4: Preparation of (25,5R5)-5-benzyloxyamino-2-(tert-butyldimethylsilyl-oxymethyl)-piperidine-l-carboxylic acid tert-butyl ester (VI):

To a solution of 5-5-benzyloxyimino-2-(feri-butyldimethylsilyl-oxymethyl)-piperidine-l-carboxylic acid tert-butyl ester (V) (394.0 g, 0.879 mol) in dichloromethane (5.0 L) and glacial acetic acid (788 ml), was charged sodium cyanoborohydride (70.88 g, 1.14 mol) one portion. The resulting reaction mixture was stirred at temperature of about 25 °C to 30°C for 2 hours. The mixture was quenched with adding aqueous solution of sodium bicarbonate (1.3 kg) in water (5.0 L). The organic layer was separated and aqueous layer was extracted with dichloromethane (2.0 L). The combined organic layer washed successively with water (2.0 L), saturated aqueous

sodium chloride (2.0 L) and dried over sodium sulfate. Solvent was evaporated under vacuum to provide a residue. The residue was purified by silica gel column chromatography to yield 208 g of (25,5i?5)-5-benzyloxyamino-2-(ieri-butyldimethylsilyl-oxymethyl)-piperidine- 1 -carboxylic acid tert-buty\ ester (VI) as pale yellow liquid.

Analysis:

Mass: 451.4 (M+l); for Molecular weight: 450.70 and Molecular Formula: C₂4H42N₂0₄Si;

1H NMR (CDC1₃): δ 7..26-7.36 (m, 5H), 4.90-5.50 (br s, 1H), 4.70 (dd, 2H), 4.09-4.25 (m, 2H), 3.56-3.72 (m, 2H), 2.55-3.14 (m, 2H), 1.21-1.94 (m, 4H), 1.45 (s, 9H), 0.89 (s, 9H), 0.05 (s, 6H).

Step 5: Preparation of (25,5R5)-5-benzyloxyamino-2-(tert-butyldimethylsilyl-oxymethyl)-piperidine (VII):

To a solution of 5-5-benzyloxyamino-2-(feri-butyldimethylsilyl-oxymethyl)-piperidine-l-carboxylic acid tert-butyl ester (VI) (208 g, 0.462 mol) in dichloromethane (3.0 L), boron trifluoride diethyletherate complex (114.15 ml, 0.924 mol) was charged in one portion. The resulting reaction mixture was stirred at temperature of about 25°C to 35°C temperature for 2 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate (2.0 L). The organic layer was separated and aqueous layer was extracted with dichloromethane (1.5 L x 2). The combined organic layer was washed with saturated aqueous sodium chloride (1.0 L) and dried over sodium sulfate. Solvent was evaporated under vacuum to yield 159 g of (25,5i?5)-5-benzyloxyamino-2-(feri-butyldimethylsilyl-oxymethyl)-piperidine (VII) as a yellowish syrup.

Analysis:

Mass: 351.3 (M+l); for Molecular weight: 350.58 and Molecular Formula: C₁₉H₃₄N₂0₂Si.

Step-6: Preparation of (25,5R)-6-benzyloxy-2-(fert-butyl-dimethylsilyl-oxymethyl)-7-oxo-l,6-diaza-bicyclo-r3.2.11octane (VIII):

Part 1; Preparation of (2S,5RS)-6-benzyloxy-2-(fert-butyl-dimethylsilyl-oxymethyl)-7-oxo-l,6-diaza-bicvclo-r3.2.11octane:

To a solution of (25,5i?5)-5-benzyloxyamino-2-(feri-butyldimethylsilyl-oxymethyl)-piperidine (VII) (159.0 g, 0.454 mol) in a mixture of acetonitrile (2.38 L) and diisopropylethylamine (316.5 ml, 1.81 mol) was added triphosgene (59.27 gm, 0.199 mol) dissolved in acetonitrile (760 ml) at -15°C over 30 minutes under stirring. The resulting reaction mixture was stirred for additional 1 hour at -10°C. The reaction mixture was quenched by addition of saturated aqueous sodium bicarbonate (2.0 L) at -5°C to 10°C. Acetonitrile was evaporated from the reaction mixture under vacuum and to the left over aqueous phase, dichloromethane (2.5 L) was added. The organic layer was separated and aqueous layer extracted with dichloromethane (1.5 L x 2). The combined organic layer was washed successively with water (2.0 L), saturated aqueous sodium chloride (2.0 L) and dried over sodium sulfate. Solvent was evaporated under vacuum and the residue was passed through a silica gel bed to yield 83.0 g of diastereomeric mixture (25, 5i?5)-6-benzyloxy-2-(feri-butyl-dimethylsilyl-oxymethyl)-7-oxo-l,6-diaza-bicyclo-[3.2.1]octane in 50:50 ratio as a yellow liquid.

Part-2: Separation of diastereomers to prepare (25,5R)-6-benzyloxy-2-(fert-butyl-dimethylsilyl-oxymethyl)-7-oxo-l,6-diaza-bicvclo-r3.2.11octane:

A mixture of diastereomers (2S,5Z?S)-6-benzyloxy-2-(teri-butyl-dimethylsilyl-oxymethyl)-7-oxo-l,6-diaza-bicyclo-[3.2.1]octane in 50:50 ratio (47.0 gm, 0.125 mol), was dissolved in n-hexane (141 ml) and stirred at temperature of about 10°C to 15°C for 1 hour. Precipitated solid was filtered and washed with n-hexane (47 ml) to provide 12.0 g of diastereomerically pure (25,5i?)-6-benzyloxy-2-(tert-butyl-dimethylsilyl-oxymethyl)-7-oxo- 1,6-diaza-bicyclo-[3.2.1] octane (VIII) as a white crystalline material.

Analysis:

Mass: 377.3 (M+l); for Molecular weight: 376.58 and Molecular Formula:

1H NMR (CDCI₃): δ Ί -Ί.ΑΑ (m, 5H), 4.95 (dd, 2H), 3.76-3.85 (ddd, 2H), 3.37-3.40 (m, 1H), 3.28-3.31 (m, 2H), 2.89 (brd, 1H), 1.90-2.02 (m, 2H), 1.62- 1.74 (m, 2H), 1.56 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H).

Diastereomeric purity as determined by HPLC: 99.85%

Step-7: Preparation of (25,5R)-6-benzyloxy-2-hvdroxymethyl)-7-oxo-l,6-diaza-bicvclo-r3.2.11octane (IX):

To a solution of (25,5i?)-6-benzyloxy-2-(ieri-butyl-dimethylsilyl-oxymethyl)-7-oxo- l,6-diaza-bicyclo-[3.2.1]octane (VIII) ( 12.0 g, 31.9 rnmol) in tetrahydrofuran (180 ml) was charged tetra 7? -butyl ammonium fluoride (38.0 ml, 38 mmol, 1 M in tetrahydrofuran) at room temperature. The reaction mixture was stirred for 2 hours. It was quenched with saturated aqueous ammonium chloride ( 100 ml). The organic layer was separated and aqueous layer extracted with dichloromethane (150 ml x 3). The combined organic layer was washed with saturated aqueous sodium chloride (150 ml), dried over sodium sulfate and evaporated under vacuum to yield 24.0 g of (25,5i?)-6-benzyloxy-2-hydroxymethyl)-7-oxo-l ,6-diaza-bicyclo-[3.2.1]octane (IX) as a yellow liquid. The compound of Formula (IX) was purified by silica gel (60-120 mesh) column chromatography using a mixture of ethyl acetate and hexane as an eluent.

Analysis:

Mass: 263.1 (M+l); for Molecular weight: 262.31 and Molecular Formula: C₁₄H₁₈N₂0₃

1H NMR (CDCb): δ 7.34-7.42 (m, 5H), 4.95 (dd, 2H), 3.67-3.73 (m, 1H), 3.53-3.60 (m, 2H), 3.32-3.34 (m, 1H), 2.88-3.01 (m, 2H), 2.09 (brs, 1H), 1.57-2.03 (m, 2H), 1.53- 1.57 (m, 1H), 1.37- 1.40 (m, 1H).

Step 8: Preparation of sodium salt of (25, 5R)-6-benzyloxy-7-oxo-l,6-diaza-bicvclor3.2.11-octane-2-carboxylic acid (I):

Step I:

Compound of Formula (IX) obtained in step 8 above was used without any further purification. To the clear solution of (25,5i?)-6-benzyloxy-2-hydroxymethyl)-7-oxo-l,6-diaza-bicyclo-[3.2.1]octane (IX) (24.0 g, 31.8 mmol) (quantities added based upon theoretical basis i.e 8.3 g ) in dichloromethane (160 ml), was added Dess-Martin reagent (24.1 g, 57.24 mmol) in portions over 15 minutes. The resulting suspension was stirred for 2 hours at 25°C. The reaction was quenched by adding a solution, prepared from saturated aqueous sodium hydrogen carbonate solution (160 ml) and 72.0 g of sodium thiosulfate. Diethyl ether (160 ml) was added to the reaction mixture and it was stirred for 5-10 minutes and filtered through celite. Biphasic layer from filtrate was separated. Organic layer was washed with saturated aqueous sodium hydrogen carbonate solution (160 ml) followed by saturated aqueous sodium chloride solution (160 ml). Organic layer was dried over sodium sulfate and evaporated to dryness at 30°C to obtain 20.0 g of intermediate aldehyde, which was used immediately for the next reaction.

Step II:

To the crude intermediate aldehyde (20.0 g, 31.6 mmol) (quantities added based upon theoretical yield i.e. 8.2 g) obtained as above, was charged i-butyl alcohol (160 ml) and cyclohexene (10.8 ml, 110.6 mmol). The reaction mixture was cooled to temperature of about 10°C to 15°C. To this mixture was added clear solution prepared from sodium hypophosphate (14.8 g, 94.8 mmol) and sodium chlorite (5.7 g, 63.2 mmol) in water (82.0 ml) over a period of 30 minutes by maintaining temperature between 10°C to 15°C. The reaction mixture was further stirred for 1 hour and was quenched with saturated aqueous ammonium chloride solution. The reaction mixture was subjected to evaporation under vacuum at 40°C to remove i-butyl alcohol. Resulting mixture was extracted with dichloromethane (3 x 150 ml). Layers were separated. Combined organic layer was washed with aqueous brine solution, dried over sodium sulfate and evaporated to dryness under vacuum to obtain 16.0 g of crude residue. To this residue was added acetone (83 ml) to provide a clear solution and to it was added dropwise a solution of sodium 2-ethyl hexanoate (4.5 g) in acetone (24 ml). The reaction mixture was stirred for 15 hours at 25°C to 30°C to provide a suspension. To the suspension was added diethyl ether (215 ml) and stirred for 30 minutes. Resulting solid was filtered over suction, and wet cake was washed with cold acetone (83 ml) followed by diethyl ether (83 ml). The solid was dried under vacuum at 40°C to provide 3.6 g of off-white colored, non-hygroscopic sodium salt of (25, 5i?)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1]-octane-2-carboxylic acid (I).

Analysis:

Mass: 275.2 as M-1 (for free acid) for Molecular Weight: 298 and Molecular Formula:

NMR (DMSO-d₆): δ 7.43-7.32 (m, 5H), 4.88 (q, 2H), 3.48 (s, IH), 3.21 (d, IH), 2.73 (d, IH), 2.04-2.09 (m, IH), 1.77-1.74 (m, IH), 1.65-1.72 (m, IH), 1.55-1.59 (m, IH);

Purity as determined by HPLC: 97.47%;

[a]_D²⁵: -42.34° (c 0.5, water).

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GSK2334470

September 28, 2015 7:39 am / Leave a comment

GSK2334470

GSK2334470; 1227911-45-6; GSK-2334470; GSK 2334470;

(3S,6R)-1-[6-(3-Amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide

(3S.6/?V1-r6-(3-Amino-1 H-indazol-6-ylV2-(methylaminoV4-pyrimidinyll-Λ/-cvclohexyl-6- methyl-3-piperidinecarboxamide

Molecular Weight	462.59
Formula	C₂₅H₃₄N₈O
CAS Number	1227911-45-6

Glaxosmithkline Llc

Phosphoinositide Dependent Kinase (PDK) 1 Inhibitors

[α]²⁰_D = – 32.6 ^o (c 1.17, MeOH)

[α] D = -27.6 (Concentration = 1.16, Solvent = Methanol)

SOL………DMSO to 100 mM

ethanol to 100 mM

nmr……http://www.chemietek.com/Files/Line2/Chemietek,%20GSK2334470%20(1),%20NMR-DMSO.pdf

http://file.selleckchem.com/downloads/nmr/S708702-GSK2334470-HNMR-Selleck.pdf

GSK2334470 Structure

GSK2334470 is a potent and selective PDK1 (3-Phosphoinositide dependent protein kinase-1) inhibitor. GSK2334470 blocks the phosphorylation of known PDK1 substrates, but surprisingly find that the potency and kinetics of inhibition vary for different PDK1 targets. GSK2334470 subsequent activation of PDK1 substrates S6K1, SGK and RSK in HEK293, U87 and mouse embryonic fibroblast cell lines.

GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway.

GSK2334470 is a highly specific and potent inhibitor of PDK1 (3-Phosphoinositide dependent protein kinase-1) with IC50 of 10 nM. It does not suppress activity on other 96 kinases, including Aurora, ROCK, p38 MAPK and PI3K. GSK2334470 has been used in cells to ablate T-loop phosphorylation and activate SGK, S6K1 and RSK as well as suppress the activation of Akt.

PATENT

WO 2010059658

http://www.google.com/patents/WO2010059658A1?cl=en

Example 78

(3S.6/?V1-r6-(3-Amino-1 H-indazol-6-ylV2-(methylaminoV4-pyrimidinyll-Λ/-cvclohexyl-6- methyl-3-piperidinecarboxamide

To (3S,6R)-1-[6-(4-cyano-3-fluorophenyl)-2-(methylamino)-4-pyrimidinyl]-Λ/-cyclohexyl-6- methyl-3-piperidinecarboxamide (260 mg, 0.58 mmol) in EtOH (10 ml.) as a suspension at room temperature in a microwave vial was added hydrazine monohydrate (807 uL, 16.7 mmol, 30 equiv) in one portion. The mixture was capped and heated at 100 ⁰C for 48 hours. A duplicate run was performed. The crude reactions from both runs were combined, and concentrated in vacuo. The residue was taken up in 10 ml. of water. The resulting suspension was sonicated briefly, and filtered. The solids collected were dried under vacuum at room temperature over P₂O₅ for 18 hours, and then at 65 ⁰C under vacuum for another 18 hours to afford the title compound (410 mg) as a cream-colored solid. LC-MS (ES) m/z = 463 [M+H]⁺. ¹H NMR (400 MHz, CD₃OD): δ 1.16 – 1.32 (m, 3H),1.29 (d, J = 6.8 Hz, 3H), 1.34 – 1.45 (m, 2H), 1.65 – 1.68 (m, 1 H), 1.76 – 1.81 (m, 5H), 1.85 – 1.92 (m, 2H), 1.97 – 2.05 (m, 1 H), 2.35 – 2.42 (m, 1 H), 2.97 (s, 3H), 3.1 1 – 3.15 (m, 1 H),3.64 – 3.70 (m, 1 H), 4.45 – 4.65 (bs, 1 H), 4.72 – 4.92 (bs, 1 H), 6.45 (s, 1 H), 7.52 (dd, J =8.5, 1.14 Hz, 1 H), 7.75 (d, J = 8.3 Hz, 1 H), 7.85 (s, 1 H).

ntermediate 112

Cis- methyl-6-methyl-3-piperidinecarboxylate

A solution of cis-3-methyl 1-(phenylmethyl)-6-methyl-1 ,3-piperidinedicarboxylate (69 g, 237 mol) in EtOH (50 mL) and EtOAc (300 mL) was added to a slurry of 10% Pd/C (3.7 g) in EtOAc (30 mL) and EtOH (10 mL) EtOH under nitrogen in a Parr Shaker bottle. The mixture was hydrogenated under 65 psi at room temperature for 4 hours. The mixture was filtered through celite, and washed with EtOAc. The filtrate was concentrated in vacuo to give 37 g of the title compound as a liquid. LC-MS (ES) m/z = 158 [M+H]⁺.

Intermediate 113

Methyl (3S,6f?)-6-methyl-3-piperidinecarboxylate L-(+)-tartaric acid salt

L-(+)-Tartaric acid salt A suspension of L-(+)-tartaric acid (39 g, 260 mmol, 1.05 equiv) in IPA (200 ml.) and water (13 mL) water was heated in a water bath at 60⁰C until all dissolved. To this hot stirred solution was added neat racemic methyl (3S,6R)-6-methyl-3-piperidinecarboxylate (39 g, 248 mmol), followed by addition of 25 mL of IPA rinse. The resulting mixture was heated to 60 ⁰C, resulting in a clear solution, and then cooled to room temperature, while the hot water bath was removed. This hot solution was seeded with a sample of methyl (3S,6R)-6-methyl-3-piperidinecarboxylate L-(+)-tartaric acid salt that had a chiral purity of 98% ee, and aged at ambient temperature (with the water bath removed) for 20 minutes. The mixture turned into an oily texture with seeds still present. To the mixture was added 5 mL of water, and heated in the warm water bath at 43 ⁰C. The mixture became clear with the seeds still present. The heating was stopped, and the mixture was stirred in the warm water bath. After 20 minutes, the mixture gradually turned into a paste. After another 10 min, the water bath was removed, and the mixture was stirred at ambient temperature for another 1 hour. The resulting paste was filtered. The cake was washed with 50 mL of IPA, giving 62 g of wet solids. This cake was taken up in 150 mL of IPA and 8 mL of water, and stirred as a slurry while being heated in a water bath to 60 ⁰C (internal temp 55 ⁰C) for 5 minutes. The heating was turned off while the mixture was still stirred in the warm water bath. After 30 min, the mixture was filtered. The cake was washed with 100 mL of IPA. Drying under house vacuum at room temperature for 48 hours gave 46.7 g of solids. An analytical sample was derivatised to the corresponding N-Cbz derivative (as in the preparation of intermediate 1 11 ), which was determined by chiral HPLC (methods used to analyze the resolution of intermediate 11 1 above) to have 85% ee. This material was taken up in IPA (420 mL) and water (38 mL) as a suspension. The mixture was heated in a water bath to 65 ⁰C, at which time the mixture became a clear solution. The heating bath was removed. The mixture was seeded and aged at ambient temp for 20 hours. The solids formed were filtered, and washed with 100 mL of IPA. The solids collected were dried under house vacuum at room temperature for 24 h, and then under vacuum at room temperature for another 24 hours to give 28.5 g of the title compound. An analytical sample was converted to the N-Cbz derivative. The ee was determined to be 97.7%. LC-MS (ES) m/z = 158 [M+H]⁺.

Intermediate 114 4,6-Dichloro-Λ/-methyl-2-pyrimidinamine

Methylamine (2M solution, 113 ml_, 217 mmol, 2.05 equiv) was charged to a 1 L 3-neck flask fitted with a magnetic stirrer and a thermometer. The mixture was chilled in an ice bath. To this stirred solution was added via addition funnel a solution of 4,6-dichloro-2-(methylsulfonyl)pyrimidine (25 g, 1 10 mmol) in EtOAc (250 ml.) portionwise over a 25 minutes period. The temp was between 5-10 ⁰C. After completion of addition, the ice bath was removed, and the mixture was stirred for 1 hour at ambient temperature. LCMS showed conversion complete. The suspension was filtered, and washed with EtOAc. The filtrate was concentrated in vacuo. The residue was partitioned between water (100 ml.) and EtOAc (450 ml_). The organic was washed with brine, dried over MgSO₄, filtered and concentrated in vacuo to give white solids, which were triturated in 150 ml. of CH₂CI₂. These solids were collected by filtration and washing with cold CH₂CI₂ (50 ml_). Drying under house vacuum at room temperature for 20 hours, and then high vacuum at room temperature for 3 hours gave 9.31 g of the title compound as a solid. LC-MS (ES) m/z = 179 [M+H]⁺.

Intermediate 121 (3S,6/?)-1-r6-Chloro-2-(methylamino)-4-pyrimidinyll-Λ/-cvclohexyl-6-methyl-3-piperidinecarboxamide

To a suspension of (3S,6/?)-1-[6-chloro-2-(methylamino)-4-pyrimidinyl]-6-methyl-3-piperidinecarboxylic acid (3.05 g, 10.71 mmol) in CH₂CI₂ (50 ml.) at room temperature was added Hunig’s base (2.70 ml_, 15.43 mmol, 1.3 equiv) and cyclohexylamine (1.60 ml_, 14.2 mmol, 1.2 equiv), and the resulting mixture was chilled in an ice bath. To this stirred solution was added HATU (4.96 g, 13.1 mmol, 1.1 equiv) in one portion, and the resulting suspension was stirred in the ice bath for 30 minutes. LCMS showed conversion complete. The mixture was diluted with CH₂CI₂ (50 ml.) and filtered through celite. The filtrate was washed water (2 X 25 ml.) and then brine. The organic was dried over Na₂SO₄, filtered, and concentrated in vacuo. Silica gel column chromatography using gradient elution of 1 % EtOAc in CHCI₃ to 50% EtOAc in CHCI₃ afforded the title compound (4.26 g) as a foam. LC-MS (ES) m/z = 366 [M+H]⁺.

PAPER

Journal of Medicinal Chemistry (2011), 54(6), 1871-1895.

http://pubs.acs.org/doi/full/10.1021/jm101527u

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

REFERENCES

Najafov, et al., Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem.J. (2011), 433 (2) 357.

For a PDK1 inhibitor, the substrate matters.
Knight ZA. Biochem J. 2011 Jan 15;433(2):e1-2. PMID: 21175429.

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1.
Najafov A, et al. Biochem J. 2011 Jan 15;433(2):357-69. PMID: 21087210.

Jeffrey Axten

Jeffrey Michael Axten

Director, Medicinal Chemistry, Virtual Proof of Concept DPU at GlaxoSmithKline

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Improved one-pot synthesis of N, N-diisopropyl-3-(2-Hydroxy-5-methylphenyl)-3-phenyl propanamide; a key intermediate for the preparation of racemic Tolterodine

September 27, 2015 12:57 pm / 1 Comment on Improved one-pot synthesis of N, N-diisopropyl-3-(2-Hydroxy-5-methylphenyl)-3-phenyl propanamide; a key intermediate for the preparation of racemic Tolterodine

Tolterodine is chemically known as (R)-N,N-disiopropyl-3-(2-hydroxy-5-methyl phenyl)-3-phenyl propyl amine. Tolterodine acts as a muscarinic receptor antagonist. It is useful in the treatment of urinary incontinence [1]. Tolterodine tartrate acts by relaxing the smooth muscle tissues in the walls of the bladder by blocking cholinergic receptors[2]. Tolterodine tartrate [3] is marketed by Pharmacia & Upjohn in the brand name of Destrol®.

The present invention relates to a novel process for the preparation of N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4); a key intermediate for the preparation of Tolterodine (1). Some different approaches have been published [4–8] for the preparation of N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4). These methods involve multistep synthesis using hazardous, expensive reagents and some of the methods [6] involve activators like Grignard reagents, LDA, n-butyl lithium, Lewis acids. Hence there is a need to develop an alternative, plant friendly procedure for the preparation of N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4) from 3,4-dihydro-6-methyl-4-phenylcoumarin (2) (Fig1).

Tolterodine (1), Methyl 3-(2-hydroxy-5-methylphenyl)-3-phenylpropanoate (3) and N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4).

Improved one-pot synthesis of N, N-diisopropyl-3-(2-Hydroxy-5-methylphenyl)-3-phenyl propanamide; a key intermediate for the preparation of racemic Tolterodine

Ring opening reactions of dihydrocoumarins are well known in literature[9–11]. But in the present invention, we have described a new methodology (Scheme 1 & Scheme2) for the preparation ofN,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4) by using inexpensive and commercially vailable starting materials like 3, 4-dihydro-6-methyl 4-phenylcoumarin (2), which was synthesized from p-cresol and trans-cinnamic acid [12].

Scheme 1

N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide 4.

Scheme 2

N-Isopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide 5.

3,4-Dihyhydro-6-methyl 4-phenylcoumarin (2) reacts with diisopropylamine (6) in presence of acetic acid gives N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (4) at room temperature. This process of compound 4 is very useful for commercialization of Tolterodine 1 in plant.

General procedure for the synthesis of compounds 4-4c & 5-5c

To a solution of 3,4-dihyhydro-6-methyl 4-phenylcoumarin 2 (10 g, 42 mmol) in diisopropylether (200 mL), N,N-diisopropylamine (33.95 g, 336 mmol) and acetic acid (10 g, 168 mmol) were added at room temperature. The suspension was stirred for 16 h at room temperature. The reaction mass was concentrated, the resulting residue was crystallized with D.M.Water (50 mL) and diisopropyl ether (50 mL) mixture to gave N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide 4 (10.6 g, 75% yield).

N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide 4

IR (KBr) cm^-1: 3024 (Aromatic C-H, str.), 2949, 2904, 2869 (Aliphatic C-H, str.), 1630 (C═O, str.), 1609, 1555, 1510 (C═C, str.), 1469, 1459 (CH₂ bending), 1270 (C-N, str.), 1072 (C-O, str.), 788, 769 (Aromatic CH Out-of-plane bend). ¹H NMR (300 MHz, DMSO-d₆) δ 1.04 (d, 12H), 2.089 (s, 3H), 2.79 (m, 2H), 3.037 (m, 2H), 4.702 (t, 1H), 6.6 (d, 1H), 6.75 (d, 2H), 7.127-7.246 (m, 5H). ¹³C NMR (125 MHz, DMSO-d₆) δ 19.39, 20.36, 45.69, 115.33, 125.70, 127.20, 128.15, 130.60, 144.43, 152.23, 173.37. MS m/z: 340 [(M + H)⁺].

Improved one-pot synthesis of N, N-diisopropyl-3-(2-Hydroxy-5-methylphenyl)-3-phenyl propanamide; a key intermediate for the preparation of racemic Tolterodine

Garaga Srinivas¹²^*, Ambati V Raghava Reddy¹, Koilpillai Joseph Prabahar¹, Korrapati venkata vara Prasada Rao¹, Paul Douglas Sanasi² and Raghubabu Korupolu²

*Corresponding author: Garaga Srinivas srinivasgaraga@yahoo.com

¹Chemical Research and Development Department, Aurobindo Pharma Ltd, Survey No:71&72, Indrakaran Village, Sangareddy Mandal, Medak district, Hyderabad 502329, Andhra Pradesh, India

²Engineering Chemistry Department, AU college of Engineering, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India

Sustainable Chemical Processes 2014, 2:2 doi:10.1186/2043-7129-2-2

The electronic version of this article is the complete one and can be found online at:http://www.sustainablechemicalprocesses.com/content/2/1/2

http://www.sustainablechemicalprocesses.com/content/2/1/2/additional

srinivas garaga

Srinivas garaga

scientist at Aurobindo Pharma

Chemical Research and Development Department, Aurobindo Pharma Ltd

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Chlorzoxazone

September 24, 2015 2:02 pm / Leave a comment

Chlorzoxazone

	Chlorzoxazone; Paraflex; Chlorzoxazon; Myoflexin; Solaxin; 95-25-0;

5-chloro-3H-1,3-benzoxazol-2-one

A centrally acting central muscle relaxant with sedative properties. It is claimed to inhibit muscle spasm by exerting an effect primarily at the level of the spinal cord and subcortical areas of the brain. (From Martindale, The Extra Pharmacopoea, 30th ed, p1202)

Property	Value	Source
melting point	191-191.5	Marsh, D.F.; US. Patent 2,895,877; July 21, 1959; assigned to McNeil Laboratories, Inc.

Marsh, D.F.; US. Patent 2,895,877; July 21, 1959; assigned to McNeil Laboratories, Inc.

Chlorzoxazone (Paraflex) is a centrally acting muscle relaxant used to treat muscle spasm and the resulting pain or discomfort. It acts on the spinal cord by depressing reflexes. It is sold as Muscol or Parafon Forte, a combination of chlorzoxazone and acetaminophen (Paracetamol). Possible side effects include dizziness, lightheadedness, malaise, nausea, vomiting, and liver dysfunction. Used with acetaminophen it has added risk of hepatoxicity, which is why the combination is not recommended. It can also be administered for acute pain in general and for tension headache (muscle contraction headache).

Synthesis

Chlorzoxazone synthesis: Mcneilab Inc, David F Marsh. U.S. Patent 2,895,877

Chlorzoxazone, 5-chloro-2-benzoxazolione, is synthesized by a hetercyclization reaction of 2-amino-4-chlorophenol with phosgene.

2

The Chlorzoxazone with CAS registry number of 95-25-0 is also known as 2-Benzoxazolol, 5-chloro-. The IUPAC name is 5-Chloro-3H-1,3-benzoxazol-2-one. It belongs to product categories of Oxazole&Isoxazole; Intermediates & Fine Chemicals; Pharmaceuticals. Its EINECS registry number is 202-403-9. In addition, the formula is C₇H₄ClNO₂ and the molecular weight is 169.57. This chemical should be stored in sealed containers in cool, dry place and away from oxidizing agents.

Physical properties about Chlorzoxazone are: (1)ACD/LogP: 2.19; (2)# of Rule of 5 Violations: 0; (3)ACD/LogD (pH 5.5): 2.19; (4)ACD/LogD (pH 7.4): 2.15; (5)ACD/BCF (pH 5.5): 27.16; (6)AACD/KOC (pH 7.4): 340.79; (7)#H bond acceptors: 3; (8)#H bond donors: 1; (9)#Freely Rotating Bonds: 0; (10)Index of Refraction: 1.603; (11)Molar Refractivity: 39.18 cm³; (12)Molar Volume: 114 cm³; (13)Surface Tension: 50 dyne/cm; (14)Density: 1.486 g/cm³; (15)Flash Point: 157.5 °C; (16)Enthalpy of Vaporization: 60.3 kJ/mol; (17)Boiling Point: 336.9 °C at 760 mmHg; (18)Vapour Pressure: 5.58E-05 mmHg at 25 °C.

Preparation of Chlorzoxazone: it is prepared by reaction of 5-chloro-2-hydroxy-benzamide. The reaction needs reagents iodobenzene diacetate, KOH and solvent methanol at the temperature of 0 °C. The yield is about 68%.

References

D.F. Marsh, U.S. Patent 2,895,877 (1959).
Dong DL, Luan Y, Feng TM, Fan CL, Yue P, Sun ZJ, Gu RM, Yang BF. (2006). “Chlorzoxazone inhibit contraction of rat thoracic aorta”. Eur J Pharmacol 545 (2–3): 161–6. doi:10.1016/j.ejphar.2006.06.063. PMID 16859676.
Park J, Kim K, Park P, Ha J (2006). “Effect of high-dose aspirin on CYP2E1 activity in healthy subjects measured using chlorzoxazone as a probe”. J Clin Pharmacol 46 (1): 109–14. doi:10.1177/0091270005282635. PMID 16397290.
Wan J, Ernstgård L, Song B, Shoaf S (2006). “Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats”. J Pharm Pharmacol 58 (1): 51–61. doi:10.1211/jpp.58.1.0007. PMC 1388188. PMID 16393464.
PARAFON DSC (chlorzoxazone) tablet, Daily Med, U.S. National Library of Medicine

Chlorzoxazone
Systematic (IUPAC) name

5-chloro-3H-benzooxazol-2-one
Clinical data
Trade names	Parafonforte
AHFS/Drugs.com	monograph
MedlinePlus	a682577
Routes of administration	oral
Pharmacokinetic data
Bioavailability	well absorbed
Protein binding	13–18%
Metabolism	hepatic
Biological half-life	1.1 hr
Excretion	urine (<1%)
Identifiers
CAS Registry Number	95-25-0
ATC code	M03BB03
PubChem	CID: 2733
IUPHAR/BPS	2322
DrugBank	DB00356
ChemSpider	2632
UNII	H0DE420U8G
KEGG	D00771
ChEBI	CHEBI:3655
ChEMBL	CHEMBL1371
Chemical data
Formula	C₇H₄Cl N O₂
Molecular mass	169.565 g/mol

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As of September 2015, updated Requirements apply to the Application of a CEP!

September 24, 2015 3:25 am / Leave a comment

DRUG REGULATORY AFFAIRS INTERNATIONAL

As of September 2015, updated Requirements apply to the Application of a CEP!

The EDQM recently revised its certification policy. Read more here about what you now need to consider when applying for a Certificate of Suitability (CEP).

http://www.gmp-compliance.org/enews_05034_As-of-September-2015–updated-Requirements-apply-to-the-Application-of-a-CEP!_9159,9255,9299,9300,S-WKS_n.html

The EDQM recently published a revised version of its certification policy document titled “Content of the dossier for chemical purity and microbiological quality“. The revision takes into account the new regulatory developments in Europe that are reflected in many revised and, to some extent, new guidelines of the EMA, ICH as well as in some revised general chapters and monographs of the European Pharmacopoeia (see the summary of these guidance documents under “References” at the end of the policy document).

The aim of the policy document is to provide CEP applicants with a guideline for preparing the authorisation dossier and for compiling all the documents required for this…

View original post 850 more words

Gnidia glauca

September 22, 2015 3:45 am / Leave a comment

Gnidia glauca

Introduction

Search of complementary and alternative medicine has gained a thrust in the recent decade due to the pronounced side effects and health hazards of the chemically synthesized drugs. Hereby, a comprehensive knowledge about the traditionally used medicinal plants is indispensable for exploration of its novel bioactive components. One of such comparatively less explored medicinal plant is Gnidia glauca. Although, it has folkloric, traditional phytomedicinal and agrochemical applications in various parts of the world, still there are no available scientific validations or evidences to support the fact. In African medicine it is used for treatment of abdominal pain, cancers, wounds, snake bites, sore throat and burns. It is also well known for its piscicidal, insecticidal, molluscicidal and even homicidal activity for its use as arrow poisons. Similarly, its antineoplastic activity is reported to be remarkably superior [1]. However, till date there is no comprehensive information on the plant.

In view of the background, herein we present the first commentary on complete research carried out till date on G. glauca and its promises as complementary and alternative medicine (Figure 1).

Antimicrobial Activity

Plant pathogenic fungi are major cause of heavy losses in the crop yield as well as the economic turnover of the farmers. Hereby, development of eco-friendly herbal and cheap antifungal agents is of utmost importance. Aqueous extracts of various parts of G. glaucaexhibited variable mycelia inhibition against Phytophthora parasitica, a plant pathogenic fungi causing heart rot in pineapple. At a concentration of 5% the G. glauca seeds, leaves and barks showed an inhibition upto 19.16, 15.90 and 23.46%, respectively. Similarly, an enhanced activity was observed with a higher concentration at 10%, equivalent to 28.47, 34.59, 33.60% for seed, leaves and bark respectively [2]. A significant anticariogenic activity against Streptococcus mutans by the methanolic extract of G. glauca leaves was reported recently. The active extracts showed a high total phenolic (126.25 ± 0.20 μg GAE/ mg) and flavonoid (25.75 ± 0.10 μg CE/mg) content [3]. G. glauca bark extract is reported to have superior antibacterial activity against urinary tract infection causing pathogens likeEscherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalisas compared to leaf and flower [4].

Back Ache and Joint Ache

According to ethnobotanical information, roots of G. glauca are widely used as a traditional medicine in Embu and Mbeere districts, Eastern Province of Kenya for treatment of back ache and joint ache [5].

Insecticidal and Larvicidal Activity

Leaves of G. glauca are used in Kenya as insecticidal agent [5]. Sequentially extracted hexane and chloroform extracts of dried bark ofG. glauca exhibited moderate mosquito larvicidal activity, whereas hexane, choloroform and MeOH extracts of fresh bark of the plant showed superior larvicidal activity against second instar larvae of Aedes aegypti. Maximum activity upto 100 % mortality was exhibited by the chloroform extract of fresh bark within a few minutes. Bioassay guided fractionation confirmed that compounds like bicoumarin and Pimelea factor P2 are mostly responsible for larvicidal activity [6]. Aqueous extract of G. glauca leaf and bark showed a notable ovicidal activity against the eggs of teak defoliator, Hyblaea puera Cramer upto 44.4 and 45.7 %, respectively [7]. In order to check the antileukemic and piscicidal activity of G. glauca, dried ground roots were extracted at room temperature with 95% ethanol under stirring condition for 24 h. The extract was further partitioned in various proportion of chloroform – water mixture to yield the gold fish piscitoxic fraction identified as gnidiglaucin (C₃₂H₄₆O₁₀ ). However, the isolated compound failed to show inhibitory activity in in-vivo assay for antileukemic activity (P- 388) [8].

Antiviral

A recent ethnobotanical study on medicinal plants used by people in Zegie, Peninsula, Northwestern Ethiopia revealed that the root powder of G. glauca mixed with skimmed milk is taken orally for seven days for treatment of rabies [9].

Antioxidant Activity

The methanolic extract of G. glauca leaf with high antioxidant activity showed major phenolic content of 203.3 GAE/g. It could scavenge both ABTS (IC₅₀ = 16.3 μg/mL) and nitric oxide (IC₅₀ = 360.8 μg/mL) radicals. Further, FRAP value of 993.7 μm TE/mg was recorded at 30 min and 142.5 mg AAE/g of total antioxidant activity was evaluated [1]. In our previous report as well, we observed similar trend where the alcoholic extracts of G. glauca leaf showed high phenolic and flavonoid content. In case of pulse radiolysis generated hydroxyl radical scavenging second order rate constants of ethanolic extracts of G. glauca flower (4×10⁶) was found to be very high indicating superior activity, followed by its leaf (3.73×10⁶) and stem (3.66×10⁶). Methanol extract of leaf showed efficient scavenging activity against DPPH radical, super oxide and nitric oxide radicals [10].

Antidiabetic Activity

Metabolic enzymes, like α-amylase and α-glucosidase are considered as key targets for discovery of antidiabetic drugs. Ethanolic, methanolic and ethyl acetate extracts of G. glauca flowers showed an excellent inhibitory potential (~70 % and above) against α-amylase while only methanol extract of leaf showed high inhibition against α-glucosidase [11].

see….http://www.hindawi.com/journals/ecam/2012/929051/

Nanobiotechnology

The higher content of phenolics and flavonoids is responsible for the synthesis of gold nanoparticles by G. glauca flower extract. It showed one of the most rapid routes for synthesis to be completed entirely within just 20 min. The resulting AuNPs were small spheres with a diameter of 10 nm in majority. Exotic shapes like nanotriangles were also observed employing high resolution transmission electron microscopy along with other characterization tools. These AuNPs exhibited excellent catalytic properties in a reaction where 4-nitrophenol is reduced to 4-aminophenol by NaBH₄ [12].

Toxicology Study

Toxicology studies to establish the safety of methanolic extract of G. glauca barks and roots involved the evaluation of acute oral toxicity in female rats. Neither mortality, nor morbidity was observed at administered dosages of 175, 550 and 2000 mg/kg body wt., which reveal the safety of these extracts in the doses up to 2000 mg/kg body weight. This study establishing that an LD₅₀ value of G. glauca bark and root extracts, higher than 2000 mg/kg body weight is definitely advantageous for its clinical studies [13]. Thus it provided the scientific rationale supporting the wide usage of G. glauca for diverse therapeutic purposes [14].

Conclusion

G. glauca being one of the very important ethnomedicinal plant, will continue to be explored by researchers from various disciplines. In near future scientific discoveries, adding newer attributes to its therapeutic spectrum will surely enable it to emerge as one of the very vital model system, pivotal to many field of research like, pharmacognosy, pharmacy, phytochemistry, drug discovery and nanobiotechnology.

References

Rao SB, Jayanthi M, Yogeetha R, Ramakrishnaiah H, Nataraj J (2013) Free radical scavenging activity and reducing power of Gnidia glauca (Fresen.) Gilg. J App Pharm Sci 3: 203-207.

Naik ST, Maheswarappa V (2007) Prospects of using plant extracts in management of pineapple heart rot. Karnataka J AgricSci 20: 180-182.

Junaid S, Dileep N, Rakesh KN, Pavithra GM, Vinayaka KS, et al. (2013) Anticariogenic activity of Gnidia glauca (Fresen.) Gilg, Pothosscandens L. and ElaegnuskologaSchlecht. J App Pharm Sci 3: 020-023.

Prashith KTR, Vivek MN, Junaid S, Rakesh KN, Dileep N, et al. (2014) Inhibitory activity of Polyalthialongifolia, Anaphalislawii andGnidia glauca against Colletotrichumcapsici and urinary tract pathogens. SciTechnol Arts Res J 3: 26-30.

Kareru PG, Kenji GM, Gachanja AN, Keriko JM, Mungai G (2006) Traditional medicines among the Embu and Mbeere peoples of Kenya. Afr J Tradit Complement Altern Med 4: 75-86.

Amarajeewa BWRC, Mudalige AP, Kumar V (2007) Chemistry and mosquito larvicidal activity of Gnidia glauca. Proceedings of the Peradeniya University Research Sessions, Sri Lanka, 12: 101-102.

Javaregowda, Naik LK (2007) Ovicidal properties of plant extracts against the eggs of teak defoliator, Hyblaeapuera Cramer. Karnataka J AgricSci 20: 291-293.

Kupchan SM, Shizuri Y, Sumner WC Jr, Haynes HR, Leighton AP, et al. (1976) Isolation and structural elucidation of new potent antileukemicditerpenoid esters from Gnidia species. J Org Chem 41: 3850-3853.

Teklehaymanot T,Giday M (2007) Ethnobotanical study of medicinal plants used by people in Zegie Peninsula, Northwestern Ethiopia. J EthnobiolEthnomed 3: 12.

Ghosh S,Derle A,Ahire M, More P,Jagtap S, et al. (2013) Phytochemical analysis and free radical scavenging activity of medicinal plants Gnidia glauca andDioscoreabulbifera. PLoS One 8: e82529.

Ghosh S,Ahire M, Patil S, Jabgunde A, BhatDusane M, et al. (2012) Antidiabetic Activity of Gnidia glauca and Dioscoreabulbifera: Potent Amylase and Glucosidase Inhibitors. Evid Based Complement Alternat Med 2012: 929051.

Ghosh S, Patil S, Ahire M, Kitture R, Gurav DD, et al. (2012) Gnidia glauca flower extract mediated synthesis of gold nanoparticles and evaluation of its chemocatalytic potential. J Nanobiotechnology 10: 17.

Khadke SS, Pachauri DR, Mahajan SD.(2011) An acute oral toxicity study of Gnidia glauca (Fresen.) Gilg. in albino rats as per OECD guideline 425. Int J PharmTech Res 3: 787-791.

Kharat SS, Kumkar PB, Rajpure SR, Sonawane KS (2013) Qualitative phytochemical screening of Gnidia glauca (Fresen) Gilg. plant extract. Int J Pharm Bio Sci 4: 144-148.

Professor B.A. Chopade

M.Sc., Ph.D. Nottingham University, England
Fogarty Fellow Illinois University, Chicago, USAVice- Chancellor

Dr. Babasaheb Ambedkar Marathwada University
Aurangabad-431004
Maharashtra State, India
Ph.No. : (office) (0240)-2403111 Fax No. (0240)-2403113/335

E-mail : vc@bamu.ac.in

Balu A Chopade

Professor B.A. Chopade has been working as a Vice-Chancellor of Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra from 04/06/2014. He has been working as Professor of Microbiology and Coordinator of University of Potential Excellence Programme (UPE Phase I & II) of UGC in Biotechnology at University of Pune. He was Director of Institute of Bioinformatics and Biotechnology (IBB), University of Pune from 2006 to 2012. He has established and developed IBB as a unique national institute and as a centre of excellence in research, innovation and teaching in biotechnology in India. He has successfully established an innovative benchmarking of publications in peer reviewed international journals of repute by undergraduate students at IBB. He was Head of the Department of Microbiology, University of Pune from 1994, 1996-2000 and 2003-2006. He has 35 years of experience in research, innovation, teaching and administration at the University of Pune.

Professor Chopade has several national and international academic honors and professional distinctions to his credits. He was the Government of India Scholar at the University of Nottingham, England and obtained a Ph.D. degree in microbiology and molecular genetics (1983-1986). He was also the recipient of the most prestigious Fogarty International NIH Research Fellowship Award from Govt. of USA for Post-Doctoral Research at the University of Illinois at Chicago (1994-1996) in genetic engineering. He is also recipient of International Award in Microbiology from International Union of Microbiological Societies (IUMS) in 1986. He has had very distinguished academic career and has carved his career entirely on the basis of merit and academic excellence.He was also coordinator of ALIS link programme between British Council London and Department of Microbiology, University of Pune (1994-1997).

He has published more than 100 research papers in peer reviewed international and national journals with high impact factor. The total impact factor of his research is more than 260, with h-index 26 and i10-index 52. His work is cited more than 2002 times (www.scholar.google.com). He has obtained 2 USA and 8 Indian patents. His research work has been cited by Nobel Laureate Professor Arthur Kornberg from University of Stanford, California, USA. His pioneering work on e-DNA and Acinetobacter vesicles is also cited by “Nature” journal from England. His work also has been cited in 3 textbooks of microbiology published from USA and Europe. He has presented more than 150 papers in International and National Conferences and has given large number of plenary lectures. He has successfully supervised 27 Ph.D., 4 M.Phil. and 10 Post Doctoral scholars for their research. Currently 2 Post-Doctoral Fellows and 4 Ph.D. students are working with him. His 3 students had obtained Young Scientist Awards in 1993 at Stockholm, from International Congress of Chemotherapy (ICC), Europe. His research area includes microbial and molecular genetics, biotechnology and nanomedicine. He is on editorial board of Wealth of India Publication series, from CSIR New Delhi, as well as number of research journals. He has obtained research grants and funding of more than rupees 10 crores from national and international funding agencies. He has successfully completed 32 major research projects from various National and International funding agencies. He has developed a new herbal medicine “Infex” which is manufactured by Shrushti Herbal Pharma Ltd., Bangalore. He is a pioneer in the area of Industry-Academia interactions and entrepreneurship in biotechnology and microbiology at IBB, University of Pune.

He was a visiting scientist at the Pasteur Institute, Paris, France and King’s College, University of London in 1990. He has received number of awards and most notable are: Pradnya Bhushan Dr. Babasaheb Ambedkar Award(2014) Aurangabad. Bronze Medal, International Genetically Engineered Machines (iGEM), Massachusetts Institute of Technology (MIT), USA (2009), Pradnyavant Award (2011) by Undalkar Foundation, Karad. Maharashtra, Best teacher award by Pune Municipal Corporation (1993); Best research paper awards in microbial and molecular genetics (1988 & 2002) by Association of Microbiologist of India; He was recipient of Wadia Oration award (2008) by Institution of Engineers, India. Best research paper award in Bioinformatics (2009) by SBC, India. Summer Fellowship of Indian Academy of Sciences, Bangalore (2001). His biography is published by American Biographical Institute, USA (2000) and International Biographical Centre Cambridge (1991). He is member of American Society for Microbiology, USA and Society for General Microbiology, England since 1984. He is also a life member of number of national organizations like Association of Microbiologist of India (AMI) and Biotechnology Society of India (BSI), Society of Biological Chemists of India (SBC) Indian Science Congress (ISC). He is recipient of Marcus’s Who’s Who in Science and Engineering U.S.A. (2001), Marcus’s Who’s Who of the World, U.S.A. (2000), Marcus’s Who’s Who in Medicine, U.S.A. (2002), Marcus’s Who’s Who in Education, U.S.A. (2002).

He has been working on various authorities of University of Pune, as well as many State and Central Universities in India. Such as, Chairman, Board of Studies in Microbiology from 1997-2000 & 2005-2007. Member, BOS in Biotechnology (2005-2006, 2012-2017), Member of Academic Council (1997-2000 & 2000-2005) and Board of College and University Development (BCUD) of University of Pune from 1997-2000 & 2000-2005. Member, Faculty of Science, University of Pune (1997-2000, 2003-2005) and Member, Board of Teaching and Research (BUTR), (1997-2002). Member, Board of studies in Biochemistry and Molecular Biology, Central University Pondichery (2001-2003). Member, Board of Studies in Biochemistry and Molecular Biology, Shivaji University Kolhapur (2009-2014). Member, Board of Studies in Life Sciences, North Maharashtra University, Jalgaon (1994-1999). Member, Faculty of Science, Bharti Vidyapeeth Pune (2013-2018). Member, Faculty of Science, North Maharashtra University, Jalgaon (1990-1999).

He was chairman of large number of committees of UGC, New Delhi such as 11th Plan Research Committee, Research Projects and Deemed University Status since 2008. Chairman, International Travel Grants, (2008-2013). He was Chairman of State Eligibility Test (SET) in Microbiology for Govt. of Maharashtra and Goa from (1997-2000). He has active an involvement in the national and international scientific organizations. He has been involved in University administration in the various capacities for more than 33 years, as a chairman and member of large number of development, finance, examination and administration committees of University of Pune.

He is member of research and recognition committees of numerous state and central universities in India. He also worked as a coordinator of DBT Potential Excellence Programme at the Department of Microbiology, University of Pune (1994-1998). He is nominee of Department of Biotechnology, Government of India for Reliance Industries limited Mumbai, Biorefinery of Somaiya Group of Industries in Karnataka and Agharkar Research Institute (ARI) Pune.

His vision for Dr.Babasaheb Ambedkar Marathwada University (BAMU), Aurangabad is to transform it as one of the best research and innovation Universities in India and subsequently develop as a world class University.

///////Gnidia glauca, Phytochemistry, Ethnomedicine, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, india, Balu A Chopade

Beijing Shenogen Granted Fast Track Status for Novel Cancer Drug, Icaritin

September 21, 2015 3:14 am / Leave a comment

Icaritin; 118525-40-9; AC1NSXIV; UNII-UFE666UELY;

3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one

C₂₁H₂₀O₆
Molecular Weight:	368.3799 g/mol

The roots of Epimedium brevicornu Maxim

Beijing Shenogen Granted Fast Track Status for Novel Cancer Drug

Written by Richard Daverman, PhD, Executive Editor, Greg B. Scott.

Beijing Shenogen Biomedical announced that Icaritin, a China Class I cancer drug, was granted Fast Track Review status after the company filed its New Drug Approval submission to the Beijing Food & Drug Administration. Icaritin is an oral traditional Chinese medicine, derived from barrenwort, which targets the estrogen receptor α36. Shenogen has conducted clinical trials of Icaritin in patients with liver cancer, though it expects the drug will also prove effective in breast cancer and other estrogen-related cancers as well. More details…. http://www.chinabiotoday.com/articles/20150917

Antiproliferative agent (IC₅₀values are 8,13 and 18 μM for K562, CML-CP and CML-BC cells respectively). Inhibits H/R-induced PTK activation. Induces G(2)/M cell cycle arrest and mitochondrial transmembrane potential drop. Modulates MAPK/ERK/JNK and JAK2/STAT3 /AKT signaling. Inhibits PPAR-g. Modulates differentiation. Inhibits cytochrome P450 in vivo. Orally active.

Cardiovascular function improvement, hormone regulation and antitumor activity.
2. The anti-MM activity of Icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling.
3. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis.
4. The Icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of Icaritin on rat chondrocyte at high concentration was reversed.
5. Icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
6. Icaritin dose-dependently inhibits ENKL cell proliferation and induces apoptosis and cell cycle arrest at G2/M phase. Additionally, Icaritin upregulates Bax, downregulates Bcl-2 and pBad, and activates caspase-3 and caspase-9.

What is Epimedium ?

Herba epimedii (Epimedium, also called bishop’s hat, horny goat weed or yin yang huo), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. It is a genus of about 60 flowering herbs, cultivated as a ground cover plant and an aphrodisiac. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Most of them bloom in the early spring, and the leaves of some species change colors in the fall, while other species retain their leaves year round.

Figure 1 Epimedium

Epimedium Raw Material

The herbs we used to extract icariin is one species of Epimedium, which name is Epimedium brevicornum Maxim. This kind of epimedium only can be abundantly found in Gansu province of China. And because of the growth habit of this kind of herb, which only grows under trees, it can’t to be planted, only can harvest the wild one.

This wild epimedium contains quite a bit of active components, depending on its long growth time and rich nutrient. Usually the content of the icariin is not lower than 1%.

Below photo is the herb specimen which we use. Picking in the epimedium full-bloom stage. And the medicinal value of the herb is the best at this time. The herb we select contains roots, stems, leaves and flowers. And we extract with the whole herb.

Figure 2 Epimedium for extract

Epimedium Extract

Epimedium extract is a herbal supplement claimed to be beneficial for the treatment of sexual problems such as impotence. It is believed to contain a number of active components, including plant compounds that may have antioxidant activity and estrogen-like compounds. The major components of Epimedium brevicornum are icariin, epimedium B and epimedium C. It is reported to have anti-inflammatory, anti-proliferative, and anti-tumor effects. It is also reported to have potential effects on the management of erectile dysfunction.

Figure 3 HPLC spectrum of icariin

Our specification available is Icariin HPLC 50%- 98%. Below please see the the information for reference:

Figure 4 Epimedium Extract(Icariin)

Derivatives

The plant extracts of epimedium traditionally used for male impotence, and the individual compounds is icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. The E. brevicornum extract and its active principle icariin were active. To improve its inhibitory activity, some derivatives ware subjected to various structural modifications, which include icaritin, icariside II and 3,7-bis(2-hydroxyethyl) icaritin. There have some scientific papers report that the improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make such compounds a promising candidate for further development. We hope that our new products can help you to find more commercial opportunity.

In this way, we can introduce those products as below, and we can also provide more details about the products according to your demand. The 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin is as below.

Product Name	Specification	CAS No.
Icariin	HPLC 50%-98%	489-32-7
icaritin	HPLC 98%	118525-40-9
icariside II	HPLC 98%	113558-15-9
3,7-bis(2-hydroxyethyl) icaritin	HPLC 98%	1067198-74-6

Figure 4 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin

Main Function of Epimedium Extract

horny goat weed; epimedium; Icariin; penis medicine;epimedium p.e;epimedium brevicornum; shorthorned epimedium herb; Icariins; Icaritin; 3,7-Bis(2-Hydroxyethyl)Icaritin; icariin 60%; icariin 98%; epimedium graepimedium; icarisides II;epimedium sagittatum;epimedium leaf; barrenwort.powder extract

Epimedium has been used to treat male erectile dysfunction in Traditional Chinese Medicine for many centuries. The main functions of Epimedium brevicornum in ancient Chinese books focused on the nourishment of kidney viscera and reinforcement of ‘yang’, resulting in the restoration of erectile function in males.

Epimedium contains chemicals which might help increase blood flow and improve sexual function. It also contains phytoestrogens, chemicals that act somewhat like the female hormone estrogen that might reduce bone loss in postmenopausal women.

Figure 5 some products from epimedium extract

………..

PAPER

The novel total synthesis of icaritin (1), naturally occurring with important bioactive 8-prenylflavonoid, was performed via a reaction sequence of 8 steps including Baker-Venkataraman reaction, chemoselective benzyl or methoxymethyl protection, dimethyldioxirane (DMDO) oxidation, O-prenylation, Claisen rearrangement and deprotection, starting from 2,4,6-trihydroxyacetophenone and 4-hydroxybenzoic acid in overall yields of 23%. The key step was Claisen rearrangement under microwave irradiation. MS, ¹H and ¹³C NMR techniques have been used to confirm the structures of all synthetic compounds. – See more at: http://www.eurekaselect.com/124334/article

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PAPER

Figure 1: Structures of icariin (1), icariside I (2) and icaritin (3).

Synthesis of icariin from kaempferol through regioselective methylation and para-Claisen–Cope rearrangement

Qinggang Mei^1,2, Chun Wang¹, Zhigang Zhao³, Weicheng Yuan² and Guolin Zhang¹

¹Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
²Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China
³College of Chemistry and Environmental Protection Engineering, Southwest University for Nationalities, Chengdu 610041, China…http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-11-135

http://www.beilstein-journals.org/bjoc/content/supplementary/1860-5397-11-135-S1.pdf

Scheme 1: Reagents and conditions: (a) Ac₂O, pyridine, 94%; (b) BnBr, KI, K₂CO₃, acetone, 85%; (c) Me₂SO₄, K₂CO₃, acetone, MeOH, 82%; (d) MOMCl, N,N-diisopropylethylamine (DIPEA), CH₂Cl₂, 93%; (e) 3,3-dimethylallyl bromide, 18-crown-6, K₂CO₃, acetone, 86%; (f) Eu(fod)₃, NaHCO₃, PhCl, 85 °C, 61%; (g) MeOH, 3 M HCl (aq), reflux, 95%; (h) Pd/C, 1,4-cyclohexadiene, MeOH, 84%.

Scheme 2: Decomposition of 8.

Scheme 3: Claisen rearrangement of flavonol 8.

Scheme 4: Reagents and conditions: (a) 15, DMF/CHCl₃, Ag₂CO₃, molecular sieves (4 Å, powder); (b) 16, CH₂Cl₂, Ag₂O, molecular sieves (4 Å powder), 31% for 2 steps; (c) NH₃ (g), MeOH, 94%; (d) NH₃ (g), MeOH, 63% for 2 steps.

3 Nguyen, V.-S.; Shi, L.; Li, Y.; Wang, Q.-A. Lett. Org. Chem. 2014, 11, 677–681.

4. Dell’Agli, M.; Galli, G. V.; Dal Cero, E.; Belluti, F.; Matera, R.; Zironi, E.; Pagliuca, G.; Bosisio, E. J. Nat. Prod. 2008, 71, 1513–1517.

1H NMR

13C NMR

HMBC

NOESY

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The present invention relates to compositions comprising icariside I, and to a novel, one step method of preparing such compositions, comprising converting specific prenylated flavonol glycosides such as epimedium A, epimedium B, epimedium C, icariin, and their corresponding acetate derivatives contained in an Epimedium plant extract to a single compound, namely icariside I shown below as compound I, which was surprisingly discovered to be a strong PDE-5 inhibitor.

This invention further comprises compositions enriched for anhydroicaritin, and to methods of preparing such compositions. One method of this invention for preparing compositions enriched for anhydroicaritin comprises a one-step method of converting prenylated flavonol glycosides, specifically the sagittatoside compounds A, B, and C, and the corresponding acetate derivatives, present in Epimedium plant extracts to a single compound, namely anhydroicaritin shown below as compound II, which was also discovered to be a strong PDE-5 inhibitor.

http://www.google.com/patents/US6399579

EXAMPLES Example 1 Acid Hydrolysis of a 50% EtOH Extract and Purification by Reversed Phase ChromatographyWhole Epimedium grandiflorum leaves were extracted with a 1:1 mixture of ethanol and water at 55° C. The resulting extract (referred to as a “50% EtOH extract”) was filtered and the filtrate concentrated at 40-50° C. under vacuum and then dried under vacuum at 60° C. to a dry solid. The dried extract (131 g) containing approximately 5.8 g of total PFG’s was placed in a 2 liter round bottom flask and 1 L of 90% ethanol was added. The mixture was heated to reflux to help dissolve the solids. Concentrated sulfuric acid (28 mL) was added. The mixture refluxed for 2 hr, cooled to room temperature, and 900 mL of water added with stirring. Next the mixture was filtered using vacuum to remove insoluble sulfate salts and other solids and loaded on a 2.5×56 cm (275 mL) column packed with 250-600 micron divinylbenzene cross-linked polystyrene resin (Mitsubishi Chemical). The column was washed with 2 column volumes (CVs) of 60% ethanol and the icariside I was eluted with 2 CVs of 95% ethanol. The product pool was air-dried producing 11.3 g of brown solids. HPLC analysis (FIG. 5) showed that the solids contained 18% icariside I (peak 15.27 min) and 12% anhydroicaritin (peak 25.15 min). The recovery of the icariside I in the product pool was 87% of the amount present in the hydrolyzate.

Example 2 Purification of a Hydrolyzate by Liquid/liquid ExtractionThe ethanolic hydrolyzate (25 mL) prepared in Example 1 was mixed with 62.5 mL of de-ionized water and the pH was adjusted to 7.0 using 50% (w/w) sodium hydroxide solution. The resulting mixture was extracted with three 25 mL portions of ethyl acetate and the combined ethyl acetate extracts were back extracted with 150 mL of water. The ethyl acetate layers were combined, dried, and assayed for icariside I. HPLC analysis (FIG. 6) showed that the dried EtOAc fractions contained 22% icariside I (peak 15.29 min) and 11% anhydroicaritin (peak 25.27 min), and icariside I recovery into the ethyl acetate was 97% of the amount present in the hydrolyzate. The partition coefficient for icariside I between ethyl acetate and water was found to be 16, indicating that the icariside I has a high affinity for ethyl acetate over water.

Example 3 Acid Hydrolysis of a 50% EtOH Extract and Purification by PrecipitationThe dried extract (204 g) described in Example 1 was mixed with 1 L of 90% EtOH and then heated to reflux to help dissolve the solids. Sulfuric acid (25 ML) was added slowly with swirling. The mixture was refluxed 90 minutes and immediately chilled to stop the reaction. After cooling to room temperature, the mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials, and the cake was washed with about 350 mL of 90% ethanol. The resulting ethanolic hydrolyzate (1.34 L) contained 4.1 g of icariside I.

The ethanolic hydrolyzate prepared above (1.32 L) was placed in a 10 L container and 40 g of 50% (w/w) sodium hydroxide solution was added followed by 20 mL of phosphoric acid. Next 3.3 L of deionized water was added with stirring. The pH of this mixture was 2.4. Sodium hydroxide solution (50% w/w ) was added until the pH was 8.25. The mixture was heated to 65° C. to assist with the coagulation of the precipitate. The mixture was cooled to room temperature and stirred for 0.5 hr at room temperature before filtering through a cellulose filter using vacuum. The resulting brown solids were washed with 715 mL of 10% ethanol and dried either under vacuum at room temperature or in air at 55° C. to yield brown solids. HPLC analysis (FIG. 7) showed the solids contained 20% icariside I (peak 15.27 min) and 10% anhydroicaritin. Recovery of icariside I using this precipitation procedure was 94% of the amount present in the hydrolyzate.

Example 4 Acid Hydrolysis of a Water Extract and Purification by PrecipitationGround Epimedium grandiflorum leaves (0.40 kg) were mixed with 5 L water in a 10 L round bottom flask. The flask was placed on a rotary evaporator for two hours at a rotation speed of 120 rpm and a water bath temperature of 90° C. The extract was filtered under reduced pressure through cellulose paper. The resulting filtrate (3.2 L) was evaporated using the rotary evaporator to a volume of 100 mL and dried under vacuum at 50° C.

The dark brown solids prepared above (40.4 g) were mixed with 200 mL of 90% ethanol and 6.0 mL of sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with 15 mL of 90% ethanol. The resulting ethanolic hydrolyzate (215 mL) contained 0.53 g of icariside I.

The hydrolyzate prepared above (50 mL) was transferred to a 250 mL beaker and 2.5 mL of 50% (w/w) sodium hydroxide solution was added with stirring to adjust the pH of the solution to pH 9, followed by 1.5 mL of concentrated phosphoric acid. Deionized water (125 mL) was added, and the mixture was adjusted to pH 8.2 using 1.5 mL of 50% sodium hydroxide solution. The mixture was heated to 65° C. to assist with coagulation of the precipitate and cooled to room temperature. The mixture was allowed to sit undisturbed at room temperature for 30 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 25 mL of de-ionized water and dried under vacuum at room temperature or in air at 80° C. to produce olive-green solids. HPLC analysis (FIG. 8) showed the solids contained 60% icariside I (peak 15.33 min) and 2.4% anhydroicaritin (peak 25.40 min). Recovery of icariside I using this precipitation procedure was 92% of the amount present in the hydrolyzate.

Example 5 Enzymatic Hydrolysis of Icariside Ia) The substrate was a partially purified icariside I product with 20% icariside I and 11% anhydroicaritin. About 50 mg was dissolved in 10 mL of ethanol, and water or buffer was added until the mixture became cloudy (about 20% ethanol). The following dry enzymes were added to separate samples: α-amylase, α-glucosidase, β-amylase, β-glucosidase, hesperidinase, lactase, and pectinase. The samples were incubated overnight at 40 ° C. and analyzed by HPLC. The results were only semi-quantitative due to the difficulty in dissolving the anhydroicaritin that precipitated from the samples. However, several of the chromatograms did show a definite reduction in icariside I and increase in the ratio of anhydroicaritin to icariside I. The best results were obtained using hesperidinase, lactase, β-glucosidase and pectinase.

A larger scale experiment was done using hesperidinase in order to isolate pure anhydroicaritin for characterization. Pure icariside I (20 mg )was dissolved in 10 mL of ethanol and 50 mL of water and 200 mg of hesperidinase enzyme was added and the mixture was incubated for 24 hr at 40 ° C. Crude anhydroicaritin was collected via filtration and purified on a 2.5×30 cm semi-prep C-18 HPLC column using a gradient of 50:50 (MeCN/H₂O) to 80:20 (MeCN/H₂O) in 20 min. The pure anhydroicaritin was analyzed by LC/MS and proton NMR.

b) Enzymatic Hydrolysis of PFG’s: The purified PFG solids (55.3%, purified by reversed-phase chromatography of a 50% EtOH extract) were subjected to enzymatic hydrolysis with the same enzymes and conditions described in part (a). Hesperidinase, lactase, β-glucosidase and pectinase appeared to convert the mixture of PFG’s to a mixture of sagittatosides, but no icariside I or anhydroicaritin were observed. This indicated that these enzymes were specific for the 7-β-glucosyl group and did not hydrolyze the 3-position sugar(s).

Example 6 Preparation of a High Anhydroicaritin-containing ProductA high sagittatosides Epimedium sagittatum extract containing 24.7% total sagittatosides (assayed as icariin) and 8.1% icariin and other expected prenylated flavonol glycosides was obtained from China. A 50 g portion of this extract was mixed with 250 mL of 90% ethanol and 7.5 mL of concentrated sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes, then allowed to cool to room temperature. The hydrolyzed mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 20 mL of 90% ethanol. The resulting filtered ethanolic hydrolyzate (305 mL) contained 3.75 g of anhydroicaritin and 2.50 g of icariside I.

The filtered hydrolyzate prepared above (200 mL) was transferred to a 1000 mL container and 8.0 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 4.0 mL of phosphoric acid. De-ionized water (500 mL) was then added. This mixture was adjusted to pH 4.9 using 50% sodium hydroxide solution. The mixture was allowed to sit undisturbed at room temperature for 24 hours prior to decanting off the liquid. The resulting solids were macerated using de-ionized water and filtered under reduced pressure through cellulose paper. The resulting dark brown solids (11.9 g) were washed with de-ionized water and dried in air overnight. The dark brown solids contained 20% anhydroicaritin and 12% icariside I and an anhydroicaritin/icariside I ratio of 1.66. The recovery of anhydroicaritin in the precipitation procedure was 94% from the hydrolyzate.

Example 7 Recrystallization of Icariside IIcariside 1 (30 mg) obtained by a method described in Example 1 was dissolved in a minimum of hot tetrahydrofuran (THF). Hot methanol (approximately 10 mL) was then added. The hot THF/MeOH solution was filtered through a PTFE filter into a vial and allowed to evaporate at room temperature to about 5 mL, whereupon crystals began to form, and then placed in a 4° C. refrigerator for 24 hours. The crystals were filtered and washed with cold methanol and dried in a vacuum. Icariside I (21 mg) was isolated as yellow crystals and had a chromatographic purity of 97.4%.

Example 8 Large Scale Acid Hydrolysis of an Epimedium extractAn 800 g portion of an Epimedium sagittatum powder extract obtained from China containing about 13% total prenylflavonol glycosides as icariin was mixed with 4.0 L of 90% ethanol and 120 mL of sulfuric acid in a 10 L round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 200 mL of 90% ethanol. The resulting ethanolic hydrolyzate (4.0 L) contained 33.7 g of icariside I.

The ethanolic hydrolyzate prepared above was transferred to a 34 L container and 200 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 120 mL of phosphoric acid. De-ionized water (10 L) was then added. This mixture was adjusted to pH 8.2 using 120 mL of 50% sodium hydroxide solution. The mixture was stirred for 10 minutes and allowed to sit undisturbed at room temperature for 60 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 750 mL of de-ionized water and dried under vacuum at 50° C. or in air at 80° C. The olive-green solids contained 44.6% icariside I. Recovery of icariside I in the precipitation procedure was 96% from the hydrolyzate.

Example 9 Large Scale Purification of an Epimedium Extract Containing Prenylflavonoid GlycosidesA 3.7 kg portion of an Epimedium sagittatum powdered extract obtained from China containing approximately 10% total prenylflavonol glycosides (PFG’s) assayed as icariin was stirred with 35 L of 85/15 acetone/water (v/v) in a 50 L mixing tank. The mixture was stirred vigorously for 30 minutes and allowed to sit for 5 minutes. The acetone extract layer (36 L) was decanted from the tank and contained 362 g of PFG’s. Recovery of the PFG’s in this extraction procedure was 96%.

A portion (about 500 mL) of the acetone extract was dried under reduced pressure at 50° C. or less, providing 16.1 g of brown solids which were analyzed to contain 28.6% total PFG’s when assayed as icariin.

TABLE 1

			PDE-5
			IC₅₀
Entry	Sample description	% PFG’s	(μg/mL)


1	Vat extraction of Epimedium leaves,	8.0	5.78
	refluxing for 17 hours with methanol
2	Extract prepared by extracting Epimedium	7.2	4.24
	leaves with 50% ethanol
3	Extract prepared by extracting Epimedium	10.2	12.50
	leaves with 90% ethanol
4	Extract prepared by extracting Epimedium	16.30	5.27
	leaves with 50% EtOH and then purifying
	the extract (after removal of EtOH) by
	liq/liq extraction with butanol. Sample
	tested was the butanol fraction.
5	Extract prepared by extracting Epimedium	19.3	3.97
	leaves with 50% EtOH and purifying by
	liquid/liquid extraction. Sample tested was
	the aqueous fraction of the liq/liq extraction.
6	Purification of a 90% ethanol extract on	65.60	1.87
	a HP-20 reversed phase column

TABLE 2

			PDE-5
		%	IC₅₀
Entry	Sample description	icarside I	(μg/mL)


7	Crude hydrolyzate composition obtained	2.1	24.30
	from a 50% EtOH extract of Epimedium
	leaves
8	Crude hydrolyzate composition obtained	5.3	9.39
	from a 90% EtOH extract of Epimedium
	leaves
9	Icariside I fraction obtained from	21.4	1.50
	purifying hydrolyzate Sample No. 7 on a
	SP-70 reversed-phase column and
	eluting icariside I with alcohol
10	Pure (recrystallized) icariside I	100	0.33
11	Pure anhydroicaritin	0	1.50
12	icariside I hydrate	0	21.50
13	sildenafil	0	0.031

Liang DL & Zheng SL Effects of icaritin on cytochrome P450 enzymes in rats. Pharmazie 69:301-5 (2014).Read more (PubMed: 24791596) »
Guo Y et al. An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells. Eur J Pharmacol 658:114-22 (2011). Read more (PubMed: 21376032) »
Zhu Jf et al. Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings. PLoS One 6:e23720 (2011). Read more (PubMed: 21887305) »
The roots of Epimedium brevicornu Maxim

Patent	Submitted	Granted
Compositions comprising icariside I and anhydroicaritin and methods for making the same [US6399579]		2002-06-04
COSMETIC COMPOSITION CONTAINING HYDROLYSATES OF ICARIIN [US2009170787]	2009-07-02
COMPOUNDS AND METHODS FOR TREATING ESTROGEN RECEPTOR-RELATED DISEASES [US8252835]	2008-06-19	2012-08-28

/////////Beijing Shenogen, Granted Fast Track Status, Novel Cancer Drug, Icaritin, New Drug Approval submission, Beijing Food & Drug Administration, oral traditional Chinese medicine, barrenwort

ARTEMISININ

September 18, 2015 8:06 am / Leave a comment

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Artemisinin is a sesquiterpene lactone with an endoperoxide function. It was first isolated from the Chinese traditional herb—Artemisia annua L. and its structure was first confirmed by Chinese scientists in the 1970s. Artemisinin and its derivatives or analogues are currently regarded as the most promising weapons against multidrug-resistant malaria . Its unique 1,2,4-trioxane structure is entirely incompatible with the traditional antimalarial structure-activity theory, which attracted the interest of many researchers

(+) Artemisinin is a sesquiterpene endoperoxide lactone with an unprecedented structure is a natural medicine for the treatment of malaria in particular drug against drug resistant malaria and cerebral malaria. The total synthesis of this novel sesquiterpene is described using an intermolecular radical reaction on important intermediate iodolactone starting from terpene (+) isolimonene.

Malaria is probably as old as mankind and continues to affect millions of people throughout the world. Today some 500 million people in Africa, India, South East Asia and South America are exposed to endemic malaria and it is estimated to cause two and half million deaths annually, one million of which are children. Certainly malaria is a serious problem all over the globe. As a consequence, effective therapeutic agents against malaria are continuously being sought, especially against those strains of Plasmodium falciparum, which are resistant to conventional quinine and acridine based drugs. Artemisinin, which has been isolated from Artemisia Annua L. Compositae (Qinghao), is an active constituent of traditional Chinese herbal medicine which is used for the treatment of malaria in China for more than 1000 years.

a sesquiterpene endoperoxide lactone with an unprecedented structure is a natural medicine for the treatment of malaria, in particular drug against drug resistant and cerebral malaria. The exceptional pharmacological potential and extreme scarcity of the natural material together with its complex structure prompted us to study the total synthesis of (+) Artemisinin. The architectural complexity is attributed to the presence of 7 chiral centers with tetracyclic framework with an endoperoxide unit. Though many valuable contributions5-9 have been made towards the total synthesis of this unique structurally complex molecule, the need for a simple strategic route still remains, encouraging us to take up the total synthesis of this potent antimalarial drug.

Schimid, G.; Hofheinz, W. J. Am. Chem. Soc. 1983, 105, 624. 6. Xu, X. X.; Zhu, J.; Huang, D. Z.; Zhou, W. S. Tetrahedron 1986, 42, 819. 7. (a) Avery, M. A.; Chong, W. K. M.; White, C. J. J. Am. Chem. Soc. 1992, 114, 974. (b) Avery, M. A.; White, C. J.; Chong, W. K. M. Tetrahedron Lett. 1987, 28, 4629. 8. Ravindranathan, T.; Kumar, M. A.; Menon, R. B.; Hiremath, S. V. Tetrahedron Lett. 1990, 31, 755. 9. Liu, H. J.; Yeh, W. L.; Chew, S. Y. Tetrahedron Lett. 1993, 34, 4435.

IUPAC	(3R,5aS,6R,8aS,9R,12S,12aR)-octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano(4,3-j)-1,2-benzodioxepin-10(3H)-one
Structure	C15H22O5
CAS #	63968-64-9
Mol. Mass	282.33 g/mol
Density	1.24 ± 0.1 g/cm³
Melting Point	151-154 °C

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http://www.mdpi.com/1422-0067/13/4/5060/htm

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1,5,9-Trimethyl-(1R,4S,5R,8S,9R,12S,13R)-11,14,15,16-tetraoxatetracyclo [10.3.1.O4,13.O8,13] hexadecan-10-one (Artemisinin)

purified on preparative TLC (eluent petroleum ether/ethyl acetate, 90/10) to give 1 (6 mg) in 10% yield.

1 H NMR (500MHz, CDCl3): δ 1.00 (d, J = 6.0 Hz, 3H), 1.01-1.13 (m, 2H), 1.21 (d, J = 7.4 Hz, 3H), 1.34-1.43 (m, 3H), 1.44 (s, 3H), 1.74-1.79 (m, 2H), 1.86-1.90 (m, 1H), 1.97-2.07 (m, 2H), 2.40-2.46 (qxd, J = 3.8, 8.9 Hz, 1H), 3.36-3.41 (qxd, J = 1.7, 5.3, 5.4 Hz, 1H), 5.84 (s, 1H).

MS (FAB): m/z 283 (M+1).

IR (KBr): 1740 (δ-lactone) cm-1.

Optical rotation [α]D : (+) 87.94 (c=0.1, Dioxane).

http://www.arkat-usa.org/get-file/18950/

ARKIVOC 2003 (iii) 125-139

Total synthesis of (+) Artemisinin J. S. Yadav,

* R. Satheesh Babu and G. Sabitha Organic Chemical Sciences, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500 007, India E-mail: yadav@iict.ap.nic.in

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Total Synthesis

In 1982, G. Schmid and W. Hofheinz published a paper showing the complete synthesis of artemisinin. Their starting material was (-)-Isopulegol (2) which is then converted to methoxymethyl ether (3). The ether is hydroborated and then undergoes oxidative workup to give (4). The primary hydroxyl group was then benzylated and the methoxymethyl ether was cleaved resulting in (5) which then is oxidized to (6). Next, the compound was protonated and treated with (E)-(3-iodo-1-methyl-1-propenyl)-trimethylsilane to give (7). This resulting ketone was reacted with lithium methoxy(trimethylsily)methylide to obtain two diastereomeric alcohols, (8a) and (8b). 8a was then debenzylated using (Li, NH3) to give lactone (9). The vinylsilane was then oxidized to ketone (10). The ketone was then reacted with fluoride ion that caused it to undergo desilylation, enol ether formation and carboxylic acid formation to give (11). An introduction of a hydroperoxide function at C(3) of 11 gives rise to (12). Finally, this underwent photooxygenation and then treated with acid to produce artemisinin.⁶

6 G. Schmid, W. Hofheinz. “Total Synthesis of qinghaosu” J. Am. Chem. Soc.; 1983; 105 (3); 624-625

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Produce artemisinin with biosynthesis and chemical synthesis. The World Health Organization estimates that in 2010 there were >200 million cases of malaria worldwide that accounted for >650,000 deaths. Many promising strategies to combat malaria require use of artemisinin-based combination therapies, but artemisinin production—from natural sources or laboratory biosynthesis—is insufficient and expensive.

C. J. Paddon and J. D. Newman at Amyris (Emeryville, CA) and almost 50 colleagues in the United States, Canada, and China engineered a new strain ofSaccharomyces cerevisiae (baker’s yeast) to improve the production of artemisinic acid (1, a precursor for artemisinin) from glucose. This research was sponsored by the Institute for OneWorld Health with the support of the Bill & Melinda Gates Foundation.

The authors studied the biochemical pathway to 1 in S. cerevisiae. They then overexpressed the genes involved in artemisinin production and suppressed those related to other products. They also added isopropyl myristate oil to solubilize 1 and drive the equilibrium toward the product. They produced 1 in 25 g/L concentration.

The authors then developed a synthesis of artemisinin (2) from 1 that is suitable for large-scale production (see figure). Among the improvements are

the use of hydrogen to reduce the double bond in artemisinic acid,
esterification of the carboxylic acid group to avoid side reactions,
chemical generation of singlet oxygen (¹O₂) from H₂O₂, and
in the last step, the use of air, a safer and less expensive source of triplet oxygen (³O₂) than pure oxygen.

Artemisinin was obtained in 50% overall yield with higher purity than is usually found in commercial samples. This process is simple, scalable, and economically viable. It can potentially supply worldwide requirements of artemisinin to combat malaria. The process is not patented and is therefore freely available. (Nature 2013, 496, 528–532; José C. Barros)

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Friedrich Wöhler’s early syntheses of oxalic acid and urea heralded the age of synthetic organic chemistry. These reactions demonstrated the potential for man to generate compounds that had previously only been obtained from the extraction of biological substances. Remarkably, despite huge advances in chemical synthesis, almost all natural products synthesised to date have relied upon similar apparatus and techniques to those used by Wöhler in the 1820s. Steve Ley and his group are among the pioneers of the use of flow chemistry in synthesis, and have demonstrated the use of machines in place of the antiquated round-bottomed flasks still used in chemistry labs the world over.

GA?id=C3CS60246J The number of sequential operations required in traditional approaches to making molecules can make synthesis time-consuming. In particular, downstream processes such as purification of the desired compound from waste products can take much longer than the actual reaction. Importantly, flow chemistry can also offer significant improvements to work health and safety as hazardous chemicals can be manipulated in a closed system and therefore, risks associated with exposure are reduced.

In flow chemistry (at its most basic), a reaction is performed in a continuous flowing stream where substrates and reagents are combined inside inert tubing and pumped around a coil of tubing before being quenched or treated with the chemical required for the next stage of the transformation.

Ley and coworkers have recently published a review that presents some highlights from the use of flow chemistry in natural product synthesis. One of the notable examples featured in this review is the continuous flow, semi-synthesis of artemisinin bySeeberger and Lévesque. Artemisinin is a sesquiterpene that represents the frontline treatment for plasmodium falciparummalaria when used in combination with other therapeutics. The supply of artemisinin from natural sources is problematic as is the scalability of existing synthetic approaches.

Dihydroartemisinic acid 2, (derived from artemesinic acid 1) represents the starting point for this flow synthesis and first undergoes photooxidation to yield hydroperoxide 3. Subsequent treatment of 3 with strong acid, followed by oxidation provided hydroperoxide 5, which underwent a spontaneous cycloaddition sequence, leading to the generation of artemisinin6.

The use of a continuous flow reactor particularly enhanced the challenging photochemical transformations associated with the synthesis. Issues such as low mass transfer of oxygen gas into solution and low penetration of light were resolved by coiling the reaction tubing around a lamp to enabled effective generation of the singlet-oxygen required for the reaction. Additionally, improved mixing and temperature control could also be achieved. Crucially, this synthesis provides a low cost method to meet the escalating demand for artemisinin at affordable prices for patients in the developing world.

The elegant syntheses described in this review span a range of natural product classes and highlight the advantages that mechanisation of chemical processes can offer. As chemists seek to address medicinal and environmental challenges, perhaps greater emphasis should be placed on rational design rather than labour-intensive and repetitive tasks. The effective implementation of flow systems and technology could revolutionise the chemical sciences, and this review provides some exciting food for thought.

For more, read this Chem Soc Rev article in full:

Flow chemistry syntheses of natural products

Julio C. Pastre, Duncan L. Browne and Steven V. Ley

Chem. Soc. Rev., 2013, Advance Article

DOI: 10.1039/C3CS60246J

….

Although photocatalytic chemistry has been the subject of intense interest recently, the rate of these reactions is often slow due to the limited penetration of light into typical reaction media. Peter H. Seeberger at the Max-Planck Institute for Colloids and Surfaces in Potsdam and the Free University of Berlin showed (Chem. Sci. 2012, 3, 1612. DOI: 10.1039/C2SC01016J) that Ru(bpy)₃²⁺ catalyzed reactions such as the reduction of azide 1 to 2 can be achieved in as little as 1 min residence time using continuous flow, as opposed to the 2 h batch reaction time previously reported. The benefits of flow on a number of strategic photocatalytic reactions, including the coupling of 3 and 4 to produce 5, was also demonstrated (Angew. Chem. Int. Ed. 2012, 51, 4144. DOI: 10.1002/anie.201200961) by Corey R. J. Stephenson at Boston University and Timothy F. Jamison at MIT. In this case, a reaction throughput of 0.914 mmol/h compares favorably with 0.327 mmol/h for the batch reaction.

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ACTs (Artemisinin) drugs to treat malaria .

Earlier this year Francois Levesque and Peter Seeberger laid out their plans for scaling up the production of the important anti-malarial drug artemisinin (DOI). Their vision: the industrial production from dihydroartemisinic acid in a single continuous flow reaction. This month in Science, science writer Kai Kupferschmidt is not so sure.

Current artemisinin industrial production completely relies on extraction from thesweet wormwood plant. But help is on the way. Biotech company Amyris has trained special yeast cells to produce a precursor called artemisinic acid. The dihydro acid can then be obtained from artemisinic acid via reduction with hydroxylamine-O-sulfonic acid / MeOH (diazene).

In the Levesque/Seeberger procedure the next step to artemisinin is a photochemical reaction with singlet oxygen forming a hydroperoxide using teraphenylporphyrin asphotosensitizer followed by an ene reaction. This step is then followed by a thermal Hock rearrangement initiated by trifluoroacetic acid. Another round of oxygen adds another hydroperoxide unit and another rearrangement forms artemisinin itself. This sequence takes place in a continuous flow reactor and in the photochemical step all the tubing is wrapped around the lamp for maximum exposure to light.

So far so good but as Kupferschmidt found out, Amyris with backing from several charities and non-profits exclusively licensed the yeast cells to chemical company Sanofi. This company has decided the final chemical steps will take place via old-fashioned batch chemistry not flow chemistry. This is bad news for Seeberger but the man is not going to give up that easily. He is looking at two alternative ways to lay his hands on artemisinic acid: it is present in waste from sweet wormwood cultivation or better still, the plant can be engineered to produce it in larger quantities than artemisinin itself.

………………

As reported back in 2012 here chemical company Sanofi and the Bill and Melinda Gates Foundation have joined forces (Sanofi the know-how and Bill the money) to increase production of the important antimalarial drugartemisinin. In a recent OPRD publication Sanofi chemists present a commercial-scale (no-loss no profit) production line with a capacity of 60 tonnes, starting from yeast-produced artemisinic acid. Here is the summary.
In step one from artemisinic acid to dihydroartemisinic acid (a dehydrogenation) the Wilkinson catalyst was deemed too expensive and replaced by ruthenium chloride (R)-DTBM-Segphis (a modified segphos). Scale: 600 Kg, 90% diastereoselectivity. The compound was next activated with ethylchloroformate and potassium carbonate in dichloromethane to the anhydride. The photochemical step consisted of addingtetraphenylporphyrin as a sensitizer and trifluoroacetic acid in dichloromethane. The subsequent Schenck ene reaction / Hock rearrangement requires two equivalents of singlet oxygen. Where the prior art yielded 41% of product, this photochemical solution pushes out 55%. Side note: the article does not really explain why the acid was activated, the Seeberger procedure does not include this step. Remaining challenge: product isolation was accomplished by simultaneous DCM distillation – solvent replacement with n-heptane and crystallisation. Pretty amazing when considering this is still industrial production at the hundreds of kilogram scale and the final product is a labile peroxide!

Nature2013, 496 ( 7446) 528– 532

J. Am. Chem. Soc., 2012, 134 (33), pp 13577–13579

DOI: 10.1021/ja3061479

sicook@indiana.edu

http://pubs.acs.org/doi/abs/10.1021/ja3061479

Abstract

Malaria represents one of the most medically and economically debilitating diseases present in the world today. Fortunately, there exists a highly effective treatment based on the natural product artemisinin. Despite the development of several synthetic approaches to the natural product, a streamlined synthesis that utilizes low-cost chemical inputs has yet to materialize. Here we report an efficient, cost-effective approach to artemisinin. Key to the success of the strategy was the development of mild, complexity-building reaction cascades that allowed the use of readily available, affordable cyclohexenone as the key starting material.

Rf = 0.2 (hexanes/ethyl acetate, 5/1).

IR (film) ν/cm-1 2956 (m), 2933 (m), 2884 (m), 2861 (m), 1739 (s), 1201 (m), 1114 (s), 1033 (m), 1028 (m), 995 (s), 883 (m).

[α]D 20 = +64.0 (c 1.20, CHCl3) (nat. [α]D 20 = +66.6 (c 0.90, CHCl3)).

1H NMR (400 MHz, CDCl3) δ 5.84 (s, 1H), 3.38 (dq, J = 7.4, 5.5 Hz, 1H), 2.41 (ddd, J = 14.4, 12.9, 3.9 Hz, 1H), 2.06-1.92 (m, 2H), 1.90-1.82 (m, 1H), 1.79-1.70 (m, 2H), 1.52-1.31 (m, 3H), 1.42 (s, 3H), 1.18 (d, J = 7.4 Hz, 3H), 1.10-1.00 (m, 2H), 0.98 (d, J = 5.9 Hz, 3H).

13C NMR (100 MHz, CDCl3) δ 172.7, 106.0, 94.3, 80.1, 50.7, 45.6, 38.2, 36.5, 34.2, 33.5, 25.8, 25.5, 24.0, 20.5, 13.2.

HRMS calcd. for C15H22O5Na [M+ Na] 305.1365, found 305.1356.

………….

http://pubs.acs.org/doi/abs/10.1021/op4003196

Org. Process Res. Dev., 2014, 18 (3), pp 417–422

DOI: 10.1021/op4003196

*E-mail: Andreas.Burgard@sanofi.com, *E-mail: Joel.Turconi@sanofi.com

Abstract

A new commercial-scale alternative manufacturing process to produce a complementary source of artemisinin to supplement the plant-derived supply is described by conversion of biosynthetic artemisinic acid into semisynthetic artemisinin using diastereoselective hydrogenation and photooxidation as pivotal steps. This process was accepted by Prequalification of Medicines Programme (PQP) in 2013 as a first source of nonplant-derived-artemisinin in industrial scale from Sanofi production facility in Garessio, Italy.

Analytical Data of Semisynthetic Artemisinin

Optical Rotation: [α]²⁰_D = +74–78 [10 mg/mL in ethanol].

The melting point of the crystalline artemisinin was found to be about 159 °C.

The theoretical mass of [M + H]⁺ is 283.1545 amu. The high-resolution mass spectrum shows the [M + H]⁺ at m/z = 283.1557 amu. This measured mass is consistent with the [M + H]⁺formula C₁₅H₂₂O₅ within an deviation of 4.2 ppm. (amu: atomic mass unit)

Scheme 5. Manufacturing of semisynthetic artemisinin in production scale

……………..

http://pubs.acs.org/doi/abs/10.1021/op200373m

Org. Process Res. Dev., 2012, 16 (5), pp 1039–1042

DOI: 10.1021/op200373m

Publication Date (Web): February 21, 2012

*Email: a.lapkin@warwick.ac.uk. Fax: (+44) 24764 18922.

This article is part of the Continuous Processes 2012 special issue.

Abstract

Stoichiometric reduction of artemisinin to dihydroartemisinin (DHA) has been successfully transferred from batch to continuous flow conditions with a significant increase in productivity and an increase in selectivity. The DHA space-time-yield of up to 1.6 kg h^–1 L^–1 was attained which represents a 42 times increase in throughput compared to that of conventional batch process.

World Drug Tracker: Antimalarial flow synthesis closer to …

worlddrugtracker.blogspot.com630 × 280Search by image

The processes yields several artemisinin-derived APIs that are key components in Artemisinin Combination Therapies

Artemisinin (Cook, 2012).

(+)-Artemisinin (41) is currently the most effective drug against Plasmodium falciparum malaria as part of an artemisinin-based combination therapy (ACT). Although it can be isolated on an industrial scale from Artemisia annua, the market price of artemisinin (41) has fluctuated widely and traditional extraction does not provide enough material to meet the worldwide demand. Interestingly, recent efforts towards a cheaper and more efficient production of artemisinin (41) have mainly taken place in the areas of synthetic biology, semisynthesis and plant engineering, while there has been a lack of practical approaches using a straightforward total synthesis. Despite the fact that all the total syntheses of artemisinin, until 2010, were impressive from a feasibility point of view, none of them provided a solution for the low-cost synthesis of 41. This changed when Cook’s group recently published a scalable synthesis of artemisinin (41), which provides a blueprint for the cost-effective production of 41 and its derivatives below Key to their successful strategy was the use of reaction cascades that rapidly built complexity, starting from the cheap feedstock chemical, cyclohexenone (42). The latter was first subjected to a one-pot conjugate addition/alkylation sequence, to give ketone 43. A three-step sequence consisting of formylation, cycloaddition and a Wacker-type oxidation, yielded 9.4 g of methyl ketone 44. The challenging formation of the unusual peroxide bridge was initially met with failure, but was eventually realized by a reaction with singlet oxygen to give 41 amongst other oxidized intermediates. The entire synthetic sequence was conducted on a gram scale, required only three chromatographic purifications and was carried out in only five flasks. Considering the low cost of the commodity chemicals used and the conciseness of Cook’s synthesis, it is certainly worth being further investigated.

2015 January » All About Drugs

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7 Semi-synthesis of artemisinin using continuous flow. The Seeberger group has recently developed a continuous flow approach to the production of …

…………

http://pubs.acs.org/doi/abs/10.1021/ol2015434

http://pubs.acs.org/doi/suppl/10.1021/ol2015434/suppl_file/ol2015434_si_001.pdf

Org. Lett., 2011, 13 (16), pp 4212–4215

DOI: 10.1021/ol2015434

Publication Date (Web): July 15, 2011

yikangwu@sioc.ac.cn

Abstract

Attachment of H₂O₂ onto the highly hindered quaternary C-12a in an advanced qinghaosu (artemisinin) precursor has been achieved through a facile perhydrolysis of a spiro epoxy ring with the aid of a previously unknown molybdenum species without involving any special equipment or complicated operations. The resultant β-hydroxyhydroperoxide can be further elaborated into qinghaosu, illustrating an entry fundamentally different from the existing ones to this outstanding natural product of great importance in malaria chemotherapy.

QHS: M.p. 153-155 ºC (nat. m.p. 154-156 ºC).

[α]D 25 +67.6 (c 1.75, CHCl3), (nat. [α]D 24 +66.6 (c 1.57, CHCl3)).

1 H NMR (400 MHz, CDCl3) δ 5.83 (s, 1H), 3.36 (br dq, J = 7.2, 5.5 Hz, 1H), 2.40 (br ddd, J = 14.8, 13.8, 3.9 Hz, 1H), 2.06-1.93 (m, 2H), 1.90-1.82 (m, 1H), 1.78-1.67 (m, 2H), 1.50-1.30 (m, 3H), 1.41 (s, 3H), 1.17 (d, J = 7.3 Hz, 3H), 1.09-1.00 (m, 2H), 0.97 (d, J = 5.7 Hz, 3H);

13C NMR (100 MHz, CDCl3) δ 171.9, 105.3, 93.6, 79.4, 49.9, 44.8, 37.4, 35.8, 33.5, 32.8, 25.1, 24.7, 23.3, 19.7, 12.4.

FT-IR (film) 2959, 2933, 2884, 2861, 1738, 1450, 1378, 1212, 1201, 1114, 1033, 997, 882, 831 cm–1.

ESI-MS 283.1 ([M+H]+ ), 305.0 ([M+Na]+ ), 337.0 ([M+MeOH+Na]+ ); EI-HRMS: calcd for C15H22O5 (M+ ) 282.1467, found 282.1461.

………….

Ind. Eng. Chem. Res., 2013, 52 (22), pp 7157–7169

DOI: 10.1021/ie302495w

Publication Date (Web): March 26, 2013

*Tel.: +45 6550 7481. E-mail: bgr@kbm.sdu.dk.

This article is part of the PSE-2012 special issue..http://pubs.acs.org/doi/abs/10.1021/ie302495w

Abstract

A systematic method of conceptual process synthesis for recovery of natural products from their biological sources is presented. This methodology divides the task into two major subtasks namely, isolation of target compound from a chemically complex solid matrix of biological source (crude extract) and purification of target compound(s) from the crude extract. Process analytical technology (PAT) is used in each step to understand the nature of material systems and separation characteristics of each separation method. In the present work, this methodology is applied to generate process flow sheet for recovery of artemisinin from the plant Artemisia annua (A. annua). The process flow sheet is evaluated on the basis of yield and purity of artemisinin obtained in bench scale experiments. Yields of artemisinin obtained in individual unit operations of maceration, flash column chromatography, and crystallization are 90.0%, 87.1% and 47.6%, respectively. Results showed that the crystallization step is dominant to the overall yield of the process which was 37.3%.

update……..

Amalgamation of Synthetic Biology and Chemistry for High-Throughput Nonconventional Synthesis of the Antimalarial Drug Artemisinin

Dharmendra Singh^†, Derek McPhee^‡, Christopher J. Paddon^‡, Joel Cherry ^*^‡, Ghanshyam Maurya^†, Ganesh Mahale^†, Yogesh Patel^†, Neeraj Kumar^†, Subhash Singh^†, Brajesh Sharma^†, Lavkesh Kushwaha^†, Satinder Singh^†, and Ashok Kumar ^*^†

^† Chemistry Research and Development, Plot Number 123-AB, Ipca Laboratories Limited, Kandivali Industrial Estate, Kandivali West, Mumbai 400067, India

^‡ Research and Development, Amyris Inc., 5885 Hollis Street, Suite 100, Emeryville, California 94608, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00414

*E-mail: ashok.kumar@ipca.com., *E-mail: cherry@amyris.com.

(3R,5aS,6R,8aS,9R,12S,12aR)-Octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one (Artemisinin, 1)

The melting point of artemisinin batch 1 (AM2/AM2/16260 P2) was found to be 152.6–153.7 °C, and that of batch 2 (AM2/AM2/16260 Crop II) was 153–154 °C.(53)

The specific optical rotation of artemisinin batch 1 was [α]_D²⁰ = +76.55 [10 mg/mL in ethanol], and that of artemisinin batch 2 was [α]_D²⁰ = +76.51 [10 mg/mL in ethanol].(54)

53 Monographs for pharmaceutical substances (A–O). The International Pharmacopoeia, 4th ed. Vol. 1; World Health Organization: Geneva, 2006.
54.Monographs for pharmaceutical substances (A–O). The International Pharmacopoeia, 6th ed.; World Health Organization: Geneva, 2016.

The development of a cost-effective process for the production of artemisinin, the precursor of all artemisinin-derived drugs, the first-line treatment for malaria, has been a long-pursued endeavor. The breakthrough achievement of coaxing genetically engineered yeast to express Artemisia annua genes for the commercial production of artemisinic acid, an advanced intermediate in the synthesis of artemisinin, has yet to fully realize an affordable malaria treatment for the poor because of the lack of a cost-effective chemical conversion into artemisinin. We describe herein a commercially feasible and pragmatic synthesis of artemisinin from amorpha-4,11-diene, an early-stage intermediate produced in 2-fold higher molar yield than engineered yeast cells can process into artemisinic acid. The key to this novel approach is an exceedingly effective functionalization of the isopropenyl group of amorphadiene via endo-epoxyamorphadiene to give dihydroartemisinic acid, which upon esterification followed by oxidation and cyclicization furnishes pure artemisinin in approximately 60% yield.

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00414

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General procedure for the synthesis of compounds 4-4c & 5-5c

N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide 4

Improved one-pot synthesis of N, N-diisopropyl-3-(2-Hydroxy-5-methylphenyl)-3-phenyl propanamide; a key intermediate for the preparation of racemic Tolterodine

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Synthesis of icariin from kaempferol through regioselective methylation and para-Claisen–Cope rearrangement

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Total Synthesis

6 G. Schmid, W. Hofheinz. “Total Synthesis of qinghaosu” J. Am. Chem. Soc.; 1983; 105 (3); 624-625

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Amalgamation of Synthetic Biology and Chemistry for High-Throughput Nonconventional Synthesis of the Antimalarial Drug Artemisinin

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