Dorzolamide Hydrochloride

Trusopt, 120279-96-1, 1cil, Trusopt (TN), Dorzolamide (DZA), Dorzolamide (INN), MK507

Molecular Formula:C10H16N2O4S3

Molecular Weight:324.44004 g/mol

(4S,6S)-4-(ethylamino)-6-methyl-7,7-dioxo-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide

(4S,6S)-4-(ethylamino)-5,6-dihydro-6-methyl-4H- thieno[2,3-/?]thiopyran-2-sulfonamide 7,7-dioxide

(4S-trans)-4-(ETHYLAMINO)-5,6-dihydro-6-methyl-4H-thieno(2,3-b)thiopyran-2-sulfonamide-7,7-dioxide

Antiglaucoma Agents, OCULAR MEDICATIONS, Ophthalmic Drugs, Carbonic Anhydrase Inhibitors

HS CODE:	2935009090

120279-96-1

130693-82-2..HCL

Laszlo Kovacs, Csaba Szabo, Erika Molnarne, Adrienne Kovacsne-Mezei, Claude Singer, Judith Aronhime, “Method of making dorzolamide hydrochloride.” U.S. Patent US20060155132, issued July 13, 2006.

 Dorzolamide is a carbonic anhydrase (CA) inhibitor. It is used in ophthalmic solutions (Trusopt) to lower intraocular pressure (IOP) in open-angle glaucoma and ocular hypertension.

Dorzolamide (trade name Trusopt) is a carbonic anhydrase inhibitor. It is ananti-glaucoma agent, and acts by decreasing the production of aqueous humour.[1] It is optically applied in the form of a 2% eye drops.[2]

History

This drug, developed by Merck, was the first drug in human therapy (market introduction 1995) which resulted from structure-baseddrug design. It was developed to circumvent the systemic side effects of acetazolamide which has to be taken orally.[2]

Uses

Dorzolamide hydrochloride is used to lower increased intraocular pressure in open-angle glaucoma and ocular hypertension.

Pharmacodynamics

It lowers IOP by about 20%.[2]

Side effects

Ocular stinging, burning, itching and bitter taste.[2] it causes shallowing of the anterior chamber and leads to transient Myopia.

Title: Dorzolamide

CAS Registry Number: 120279-96-1

CAS Name: (4S,6S)-4-(Ethylamino)-5,6-dihydro-6-methyl-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide

Molecular Formula: C10H16N2O4S3

Molecular Weight: 324.44

Percent Composition: C 37.02%, H 4.97%, N 8.63%, O 19.73%, S 29.65%

Derivative Type: Hydrochloride

CAS Registry Number: 130693-82-2

Manufacturers’ Codes: MK-507

Trademarks: Trusopt (Merck & Co.)

Molecular Formula: C10H16N2O4S3.HCl

Molecular Weight: 360.90

Percent Composition: C 33.28%, H 4.75%, N 7.76%, O 17.73%, S 26.65%, Cl 9.82%

Properties: mp 283-285°. [a]D24 -8.34° (c = 1 in methanol). Sol in water.

Melting point: mp 283-285°

Optical Rotation: [a]D24 -8.34° (c = 1 in methanol)

Dorzolamide Hydrochloride and its derivatives is known. U.S. Pat. No. 5,688,968 describes preparation of Dorzolamide HCl starting from chiral 5,6-dihydro-4-(S)-hydroxy-6-(S)-methyl-4H-thiopyran-7,7-dioxide, as depicted in scheme 1:

The process described in BP 0 296 879 (equivalent of U.S. Pat. No. 4,797,413) is of particular relevance. EP 0 296 879 describes the synthesis of Dorzolamide Hydrochloride starting from thiophene-2-thiol as depicted in scheme 2 and 3

The process described in EP 0,296,879 (scheme 2) has the following disadvantages: (a) The starting material Thiophene-2-thiol is unstable and undergoes oxidation to form disulfide, leading to lower yield of viii; (b) the yield of sulfonamide (xii) from sulphonic acid (x) is very poor (35%) and requires use of 18-crown-6 ether, which is expensive; (c) oxidation of alcohol (xiii) to sulfone is carried out using oxone which is expensive and hazardous; and separation of cis/trans isomer is done by column chromatography which is industrially inconvenient.

SYSTEMATIC (IUPAC) NAME
(4S,6S)-2-ethylamino-4-methyl-5,5-dioxo- 5λ6,7-dithiabicyclo[4.3.0]nona-8,10-diene-8-sulfonamide
CLINICAL DATA
TRADE NAMES	Trusopt
AHFS/DRUGS.COM	monograph
MEDLINEPLUS	a602022
PREGNANCY CATEGORY	US: C
LEGAL STATUS	US: ℞-only
ROUTES	Topical (eye drops)
PHARMACOKINETIC DATA
PROTEIN BINDING	~33%
HALF-LIFE	4 months
IDENTIFIERS
CAS NUMBER	130693-82-2  120279-96-1
ATC CODE	S01EC03
PUBCHEM	CID 5284549
DRUGBANK	DB00869
CHEMSPIDER	4447604
UNII	9JDX055TW1
KEGG	D07871
CHEBI	CHEBI:4702
CHEMBL	CHEMBL218490
CHEMICAL DATA
FORMULA	C10H16N2O4S3
MOLECULAR MASS	324.443 g/mol

TRUSOPT® (dorzolamide hydrochloride ophthalmic solution) is a carbonic anhydrase inhibitor formulated for topical ophthalmic use.

Dorzolamide hydrochloride is described chemically as: (4S-trans)-4-(ethylamino)-5,6-dihydro-6­methyl-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide monohydrochloride. Dorzolamide hydrochloride is optically active. The specific rotation is

Its empirical formula is C10H16N2O4S3•HCl and its structural formula is:

Dorzolamide hydrochloride has a molecular weight of 360.9 and a melting point of about 264°C. It is a white to off-white, crystalline powder, which is soluble in water and slightly soluble in methanol and ethanol.

TRUSOPT Sterile Ophthalmic Solution is supplied as a sterile, isotonic, buffered, slightly viscous, aqueous solution of dorzolamide hydrochloride. The pH of the solution is approximately 5.6, and the osmolarity is 260-330 mOsM. Each mL of TRUSOPT 2% contains 20 mg dorzolamide (22.3 mg of dorzolamide hydrochloride). Inactive ingredients are hydroxyethyl cellulose, mannitol, sodium citrate dihydrate, sodium hydroxide (to adjust pH) and water for injection. Benzalkonium chloride 0.0075% is added as a preservative.

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http://www.google.com/patents/EP1753769A2?cl=en

The dorzolamide hydrochloride product is prepared from the aminated intermediate of Formula IV by the following scheme.

[00056] Preparation of dorzolamide hydrochloride product from the animated intermediate of Formula IV

[00057] Fuming sulfuric acid (20%, 5 1) is cooled to -7°±2°C and the aminated intermediate of Formula IV (2.5 Kg) is added to it in portions during stirring. The temperature of the reaction mixture is increased to 20°+5°C during addition of the aminated intermediate of Formula IV. The reaction mixture is stirred for 22 hours at 20°±5°C. Thionyl chloride (20 1) is added to the stirred reaction mixture at 20±5°C. The reaction mixture is heated to 60°-65°C and stirred for 24 hours at this temperature. The mixture is cooled back to 40°±2°C and the excess amount of thionyl chloride is evaporated at this temperature under vacuum. (The volume of the residue: ~9 1.) The residue is cooled to -5°+2°C.

[00058] Ethyl acetate (75 1) is cooled to -10°±5°C and the residue is added to it at this temperature. The temperature of the diluted solution: 10°-25°C. Aqueous ammonia (25%, 75 1) is cooled to -10°±5°C and the residue is added to it at this temperature during effective stirring, while maintaining the temperature below 300C. The final pH: ~11. The slurry is cooled to 0°+2°C and stirred for 14 hours at this temperature. The formed ammonium sulfate is filtered and the cake is washed with ethyl acetate (2x 20 1 and 10 1). Ethyl acetate is evaporated from the filtrate at 38°±2°C under vacuum. The residue is heated to 38°±2°C, washed with toluene (3×37.5 1) at this temperature. Water (25 1) is added to the aqueous phase, cooled to 20°-25°C and extracted with ethyl acetate (3x 75 1, 37.5 1, and 37.5 1). The collected ethyl acetate phase is concentrated to ~ 100 1 at 38°±2°C under vacuum. The residue is cooled to 20°-25°C and hydrogen chloride in ethanol (5%, 10.8 1) is added to it during stirring. The formed slurry is stirred for 1 hour at 20°-25°C then cooled to 0°-4°C and stirred for 5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with ethyl acetate (2×20 1) and dried at 55°-60°C under vacuum for 4-8 hours to give Dorzolamide hydrochloride salt (~2 Kg).

[00059] Crude Dorzolamide hydrochloride salt (9 Kg) is solved in water (225 1) at 20°-25°C and the pH is set to 8.0-8.5 by addition of 25% of aqueous ammonia (2 1). The formed slurry is extracted with ethyl acetate (5×72 1). The collected ethyl acetate phase is concentrated to 180 1 by vacuum distillation. The residue is cooled to 20°-25°C, ethyl acetate (45 1) and hydrogen chloride in ethanol (5%, 22.5 1) are added to it during stirring (pH:~1.0). The formed slurry is stirred for 1 hour at 20°-25°C then cooled to 0°-4°C and stirred for 5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with ethyl acetate (2×30 1), and dried at 55°-60°C under vacuum for 4-8 hours to give purified Dorzolamide hydrochloride salt (~8.2Kg).

[00060] Purified Dorzolamide hydrochloride salt (8 Kg) dissolved in water

(24 1) at 95°-105°C and treated with active carbon (80 g). After filtration, the water solution is cooled gradually to 0°-4°C and stirred for 3-5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with cooled water (2×5 1) and dried at 55°-60°C under vacuum for 4-8 hours to give crystallized DRZ HCl salt (~6.6 Kg).

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http://www.google.com/patents/US20060142595

The invention provides a process for preparing 5,6-dihydro-4-(S)-(ethylamino)-6-(S)methyl-4H-thieno[2,3b]thiopyran-2-sulphonamide-7,7-dioxide hydrochloride of formula (I), comprising of nine steps, as depicted in scheme 4 below:

Example 8Preparation of Trans 5,6 dihydro-4H-4-ethylamino-6-methylthieno[2,3-b]thiopyran-2-sulfonamide-7,7 dioxide (X)A solution of product from example 7 (39.5 gm, 0.132 mole) in ethyl acetate (426 ml) was cooled to 0 to 5° C. and ethanolic HCl (20 ml) was added and stirred for 3 hrs at 0 to 5° C. The product was precipitated out, filtered and washed with chilled ethyl acetate. The cake was sucked to remove as much ethyl acetate as possible, and dried to get compound (21 gm) The product was suspended into ethyl acetate (210 ml), refluxed for 1 hr, then cooled to 10° C. The product was filtered and washed with chilled ethyl acetate. The cake was sucked to remove as much ethyl acetate as possible, and dried to hydrochloride salt of title compound (18 gm). The salt was then treated with saturated solution of sodium bicarbonate and mixture extracted with ethyl acetate. The organic extract were dried, filtered and concentrated to dryness to yield title compound (X) (15 gm, 37.98%).

Example 9Preparation of 5,6 dihydro-4H-4-(S)-ethylamino-6-(S)-methylthieno[2,3-b]thiopyran-2-sulfonamide-7,7 dioxide Hydrochloride (I)

A mixture of compound from example 8 (15 gm0.0462 mole) and di-p-toluyl-D-tartaric acid monohydrate (4.55 gm, 0.01125 mole) in n-propanol (1600 ml) was heated to boiling and hot solution filtered through a filter-aid pad with a layer of charcoal. The filtrate was concentrated by boiling to a volume of about (400 ml) and then allowed to crystallize. After standing overnight the crystals were filtered off and material recrystallized twice more from n-propanol (400 ml) to yield a 2:1 salt of free base to acid. Combined mother liquors from this recrystallization were saved for stage B. The salt was then treated with a saturated solution of sodium bicarbonate and mid extracted with ethyl acetate. The organic extract were dried, filtered and concentrated to dryness to yield (3.2 gm) of freebase. The hydrochloride salt was prepared from 5,6 N HCl ethanol and crystallized from methanol-isopropanol to yield (2.83 gm) of (+) isomer, SOR 8.23 (C 0.9 methanol) M.P. 283-285° C. The combine mother liquor was treated with saturated solution of sodium bicarbonate and mixture extracted with ethyl acetate. The organic exacts were dried, filtered and concentrated to dryness. The residue was treated with di-p-toluyl-L-tartaric acid monohydrate (4.55 gm, 0.01125 mole) in n-propanol (1600 ml) and the isomer separated by the process described previously to give title compound (I) (3.75 gm, 22.48%) SOR=−8.34 (C 1, Methaol) M.P. 283 to 285° C.,

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http://www.google.com/patents/WO2008135770A2?cl=en

Dorzolamide is chemically termed as (4S,6S)-4-(ethylamino)-5,6-dihydro-6-methyl-4H- thieno[2,3-/?]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride. Dorzolamide hydrochloride is represented by following structural Formula I:

HN ‘CH,

Formula I

Dorzolamide hydrochloride is known to be a carbonic anhydrase inhibitor useful in the treatment of ocular hypertension.

A process for the preparation of dorzolamide and its derivatives was first described in EP 0296879. The process of particular relevance is depicted in scheme 1. Scheme 1

(viϋ) (ix) Trans and Cis (x)

Trans (xi) Trans(+) (xii) ( I )

The process disclosed in scheme 1 has following disadvantages.

(a) The reduction of the ketone of sulfonamide (vi) using absolute ethanol is carried out at reflux and then stirred at room temperature for several hours to complete the reaction. This longer duration of reaction produces many impurities.

(b) Oxidation of alcohol (vii) to sulfone (viii) is carried out using oxone. The oxone has many disadvantages such as it is irritating to the eyes, skin, nose and throat. It should be used with adequate ventilation and exposure to its dust should be minimized. Traces of heavy metal salts catalyze the decomposition of oxone. It is practically insoluble in all organic solvents hence a phase transfer catalyst is required.

(c) Activation of the 4-hydroxy group of the sulfoaminated hydroxysulfone (viii) and nucleophilic substitution by desired ethylamine, results in all diastereomeric products (x) i.e. trans and cis isomers, which must be separated by column chromatography and resolved, further using resolving agent. As a result, product loss is greater when the desired product is the more active enantiomer.

An alternate route for the preparation of dorzolamide hydrochloride by the Ritter reaction is disclosed in EP0296879 and consists of the treatment of a aliphatic hydroxyl with a nitrile and a strong acid to form an amide. The process disclosed is as depicted in Scheme 2.

Scheme 2

(viii) (ix-a ) Trans and Cis (x)

Trans(+) (xii)

Trans (+/-) (xi) ( I )

The reaction involves conversion of hydroxysulfones (viii) to the corresponding acetoamidosulfones (ix-a) with retention of configuration followed by reduction of the amido group, chromatographic separation and resolution to obtain the desired trans isomer (I).

The prior art teaches the use of an excess quantity of sulfuric acid to carry out the Ritter reaction and hence a large quantity of ice is required for quenching the reaction mass. When the reaction mass in concentrated sulfuric acid comes into contact with ice, a large amount of localized heat is generated causing decomposition of material. Since a huge amount of water is required for quenching the reaction mass, the amount of ethyl acetate required for extraction is also substantially large. The work-up using water is not advisable nor applicable industrially.

United States Patent 5688968 describes an alternative route of preparation of dorzolamide hydrochloride starting from chiral 5,6-dihydro-4-(S)-hydroxy-6-(S)-methyl-4H-thiopyran-7,7- dioxide, as depicted in Scheme 3:

Scheme 3

(xiv) (XV)

(xiii)

(xvi) (xvii ) Trans:Cis:: 95: 5 (xviii)

HN CH,

(xix) ( I )

The process described in Scheme 3 has the following disadvantages: (a) Use of expensive chiral hydroxysulfone starting material. The process for the preparation of the chiral hydroxysulfone starting material is disclosed in U.S. Patents Nos. 5,157,129, 5,474,919 and 5,760,249. In these processes, the chiral hydroxysulfone is obtained by the asymmetric enzymatic reduction of the corresponding ketosulfone, or by cyclization of the chiral thienyl thiobutyric acid, obtained, in turn, from a chiral hydroxyester or lactone, and the subsequent stereospecific reduction of the resulting ketone, (b) The process according to this patent uses maleic acid to separate the undesired cis- isomer from dorzolamide. However this maleate salt formation to remove the cis isomer is only suitable when the ratio of trans/cis is greater than 95:5. That means, the maleate salt formation of dorzolamide does not the remove cis isomer exclusively when the cis isomer content is more than 5%. It sometimes requires repeated purification to achieve the desired chiral purity.

Another alternate route for the preparation of dorzolamide hydrochloride is disclosed in United States patent no.7109353 which involves the use of sodium perborate as an oxidant, as depicted in Scheme 4.

Scheme 4

chlorinating agent, cyclinization

Vl IV

VIl VlIl IX

The process disclosed in Scheme 4 has following disadvantages (a) Conversion of (i) to (ii) requires the mixture to be refluxed for 18-20 hrs which is time consuming and may cause impurity in the product.

(b) As the process uses the Ritter reaction to convert (vi) to (vii), a large amount of water is required to quench the hot mass of reaction which is not practical in an industrial set-up. (c) Sodium perborate is used as an oxidizing agent to convert (v) to (vi), which has got bleaching properties, and the handling of it may be injurious when done so for a prolonged period.

Yet another process for the preparation of dorzolamide is disclosed in United States publication no. 20060155132 which involves protecting the chiral 5,6-dihydro-4-(R)- hydroxy-6-(S)-methyl-4H-thieno-[2,3-b]thiopyran-7,7-dioxide as depicted in Scheme 5.

Scheme 5

protected amination benzyl sulphonyl chloride

The process disclosed in Scheme 5 has the following disadvantages, (a) The conversion process of compound (II) to (III) requires a very low temperature which ranges from -30° to 00C. (b) The amination process requires 16- 20 hrs, which is time consuming and may cause impurity in the product. All these disadvantages of the prior art are overcome by the process in accordance with the present invention.

Scheme 8

Example 4

Preparation of 5,6-Dihydro-4H-4-ethylamino-6-methylthieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide

A suspension of 5,6-dihydro-4H-4-acetylamino-6-methylthieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide (83.25 gms, 0.24 moles) in THF (832 ml) was cooled to 00C and sodium borohydride (49.11 gms, 1.29 moles) was added in lots maintaining temperature below 5°C. Reaction mass was stirred for 15 minutes at 5°C and boron trifluoride diethyl- etherate (249.75 ml, 287.2 gms, 2.02 moles) was added below 5°C. The reaction mass was stirred for 5 hours at 0°C to 5°C. Temperature of the reaction mass was raised to 25°C to 300C and stirred for 18 hours. The reaction mass was quenched in 1M sulphuric acid solution (1082 ml) below 5°C, temperature raised to 25°C to 30°C and stirred for 1 hour. The solvent was distilled under reduced pressure at 800C. The reaction mass was cooled to 100C and p H adjusted to 7 – 8 using 50% sodium hydroxide solution. Material was extracted in 1665 ml ethyl acetate once and 832 ml twice. The combined organic layers were washed with saturated sodium chloride solution, dried over sodium sulphate, charcoalised, filtered on hyflo, distilled to get title compound (77.42 gms). HPLC: 80:20::Trans:Cis

Example 7

Preparation of 5,6-Dihydro-4H-4-ethylamino-6-methylthieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide hydrochloride

(a) Dorzolamide di-p-toluyl-L-tartrate salt as prepared in example 6 (44.26 gms, 0.085 moles) was taken in ethyl acetate (557.0 ml), basified with saturated sodium bicarbonate solution. Reaction mass was stirred for 15 minutes at 25°C to 3O0C and aqueous layer was extracted with ethyl acetate (278 ml X 2). The organic layers were combined, washed with brine solution, dried over sodium sulphate, and charcoalized. To the clear solution, IPA + HCL (16.35 ml, 0.089 moles) was added, stirred for 30 minutes and ethyl acetate was removed by distillation at atmospheric pressure at 85°C to about 280 ml volume, cooled to 25-3O0C, stirred for 12 hours at same temperature and filtered to get 26.0 gms of dorzolamide hydrochloride. Trans (-) dorzolamide hydrochloride > 99.5% Trans (+) dorzolamide hydrochloride < 0.5% Cis Isomer <0.1%

(b) Dorzolamide hydrochloride was obtained in a similar manner in quantitative yield from the salt of example 6(b).

(c) Dorzolamide hydrochloride was obtained in a similar manner in quantitative yield from the salt of example 6(c).

Example 8

Preparation of 5,6-Dihydro-4H-4-ethylamino-6-methylthieno[2,3-b]thiopyran-2- sulfonamide -7,7-dioxide hydrochloride without isolation of base

Dorzolamide di-p-toluyl-L-tartrate (50 gms, 0.096 moles) prepared as per example 6, was charged in a round bottom flask along with isopropanol (1000 ml). The reaction mass was heated to 800C and charged with IPA-HCI (20 ml) dropwise to pH 3 to 4. The reaction mass was heated to reflux for 5-10 minutes. The clear solution obtained was concentrated to 100 ml. The reaction mass was charged with 300 ml ethyl acetate, cooled to 25°C, stirred for 12 to 14 hours at same temperature. The resulting dorzolamide hydrochloride was isolated by filtration and washed with ethyl acetate (50 ml), dried under vacuum at 60- 65 0C for 5-6 hours. Yield- 30 gms.

Trans (-) dorzolamide hydrochloride > 99.5% Trans (+) dorzolamide hydrochloride < 0.5% Cis Isomer <0.1%

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http://www.google.com/patents/EP0453288A1

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http://www.google.com/patents/US20060155132

Dorzolamide hydrochloride, known chemically as 5,6-dihydro-4-(S)-ethylamino-6-(S)-methyl-4H-thieno-[2,3-b]thiopyran-2-sulfonamide-7,7-dioxyde hydrochloride, is a topically effective carbonic anhydrase inhibitor useful in the treatment of ocular hypertension.

Dorzolamide hydrochloride has the structure of Formula I:

U.S. Pat. Nos. 4,677,155 and 4,797,413 disclose Dorzolamide. In the prior art synthesis of dorzolamide, a chiral hydroxysulfone is used as a starting material. The chiral hydroxysulfone starting material can be obtained using the processes disclosed in U.S. Pat. Nos. 5,157,129, 5,474,919, and 5,760,249. In the disclosed processes, the chiral hydroxysulfone is obtained by the asymmetric enzymatic reduction of the corresponding ketosulfone, or by cyclization of the chiral thienyl thiobutyric acid, obtained, in turn, from a chiral hydroxyester or lactone, and the subsequent stereospecific reduction of the resulting ketone.

Processes for the preparation of dorzolamide hydrochloride are described in U.S. Pat. Nos. 4,797,413, 5,157,129, and 5,688,968 and in U.S. patent application Publication Ser. No. 2003/0220509. The disclosed processes involve conversion of a hydroxysulfone to the corresponding acetamidosulfone by a Ritter reaction with retention of configuration, followed by introduction of a sulfonamido group, and the subsequent reduction of the amido group to an amine, providing the desired product.

The process disclosed in U.S. Pat. No. 4,797,413 includes activation of the 4-hydoxy group of the sulfonaminated hydroxysulfone with tosyl chloride and the introduction of the desired alkylamino group by nucleophilic substitution, resulting in all diastereomeric products, which must be separated and resolved. As a result, at least 75 percent of the product is lost when the desired product is the more active enantiomer.

EXAMPLE 2

Preparation of 5,6-dihydro-4-(S)-ethylamino-6-(S)-methyl-4H-thieno-[2,3-b]thiopyran 7,7-dioxide hydrochloride salt (Formula IV)

Tetrahydrofuran (50 l) and triethyl amine (4.8 l) are added to 4-(R)-hydroxy-5,6-dihydro-6-(S)-methyl-4H-thieno[2,3b]thiopyran-7,7-dioxide (5 Kg) and stirred under a nitrogen atmosphere at room temperature. The solution is cooled to −10° C. Benzylsulfonyl chloride (5.4 Kg) solved in THF (15 l) is added to the DRZ-19 THF solution in portions while maintaining the temperature below 0° C. The feeding funnel is washed with THF (2 l). The reaction mixture is stirred at 0° C. for 2-4 hours. The formed TEA HCl is filtered and the cake is washed with THF (2×10 l) Ethylamine in THF (30%, 63.7 l) is added to the filtrate and the reaction mixture is stirred at 20°-25° C. for 16 hours. Ethylamine gas prepared by heating of 70% EtNH2water solution (50 l) is absorbed in cooled THF (30 l). Water (20 l) is added to the reaction mixture and THF is evaporated from the filtrate at 40°±5° C. under vacuum. The residue is cooled to 20°-25° C., ethyl acetate (60 l) is added to it and stirred vigorously. After phase separation, the organic phase is washed with water (20 l). The ethyl acetate phase is heated to 40°±2° C. and hydrochloric acid (4M, ˜8-10 l) is added to it during stirring to set pH 2.0-2.5. The formed slurry is cooled to −8°±2° C. and stirred for 3 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with ethyl acetate (30 l) and dried at 55°-60° C. under vacuum for 4-8 hours to give the desired salt (˜5 Kg).

Preparation of dorzolamide hydrochloride product from the aminated intermediate of Formula IV

Fuming sulfuric acid (20%, 5 l) is cooled to −7°±2° C. and the aminated intermediate of Formula IV (2.5 Kg) is added to it in portions during stirring. The temperature of the reaction mixture is increased to 20°±5° C. during addition of the aminated intermediate of Formula IV. The reaction mixture is stirred for 22 hours at 20°±5° C. Thionyl chloride (20 l) is added to the stirred reaction mixture at 20°±5° C. The reaction mixture is heated to 60°-65° C. and stirred for 24 hours at this temperature. The mixture is cooled back to 40°±2° C. and the excess amount of thionyl chloride is evaporated at this temperature under vacuum. (The volume of the residue: ˜9 l.) The residue is cooled to −5°±2° C.

Ethyl acetate (75 l) is cooled to −10°±5° C. and the residue is added to it at this temperature. The temperature of the diluted solution: 10°-25° C. Aqueous ammonia (25%, 75 l) is cooled to −10°±5° C. and the residue is added to it at this temperature during effective stirring, while maintaining the temperature below 30° C. The final pH: ˜11. The slurry is cooled to 0°±2° C. and stirred for 14 hours at this temperature. The formed ammonium sulfate is filtered and the cake is washed with ethyl acetate (2×20 l and 10 l ). Ethyl acetate is evaporated from the filtrate at 38°±2° C. under vacuum. The residue is heated to 38°±2° C., washed with toluene (3×37.5 l) at this temperature. Water (25 l) is added to the aqueous phase, cooled to 20°-25° C. and extracted with ethyl acetate (3×75 l, 37.5 l, and 37.5 l). The collected ethyl acetate phase is concentrated to ˜100 l at 38°±2° C. under vacuum. The residue is cooled to 20°-25° C. and hydrogen chloride in ethanol (5%, 10.8 l) is added to it during stirring. The formed slurry is stirred for 1 hour at 20°-25° C. then cooled to 0°-4° C. and stirred for 5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with ethyl acetate (2×20 l) and dried at 55°-60° C. under vacuum for 4-8 hours to give Dorzolamide hydrochloride salt (˜2 Kg).

Crude Dorzolamide hydrochloride salt (9 Kg) is solved in water (225 l) at 20°-25° C. and the pH is set to 8.0-8.5 by addition of 25% of aqueous ammonia (2 l). The formed slurry is extracted with ethyl acetate (5×72 l). The collected ethyl acetate phase is concentrated to 180 l by vacuum distillation. The residue is cooled to 20°-25° C., ethyl acetate (45 l) and hydrogen chloride in ethanol (5%, 22.5 l) are added to it during stirring (pH:˜1.0). The formed slurry is stirred for 1 hour at 20°-25° C. then cooled to 0°-4° C. and stirred for 5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with ethyl acetate (2×30 l), and dried at 55°-60° C. under vacuum for 4-8 hours to give purified Dorzolamide hydrochloride salt (˜8.2 Kg).

Purified Dorzolamide hydrochloride salt (8 Kg) dissolved in water (24 l) at 95°-105° C. and treated with active carbon (80 g). After filtration, the water solution is cooled gradually to 0°-4° C. and stirred for 3-5 hours at this temperature. The slurry is filtered, the precipitated HCl salt is washed with cooled water (2×5 l) and dried at 55°-60° C. under vacuum for 4-8 hours to give crystallized DRZ HCl salt (˜6.6 Kg).

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Reaction of (I) with acetic anhydride-sulfuric acid in methylene chloride provided the sulfonic acid in 98% yield. Conversion to the sulfonyl chloride with phosphorous pentachloride in methylene chloride followed by treatment with aqueous ammonia gave the sulfonamide (II). Reduction of the carbonyl function with sodium borohydride and oxidation of the thiopyran sulfur with Oxone(R) yielded (IV). The 4-hydroxy substituent was converted to the acetylamino functionality under Ritter conditions. Reduction of (V) with borane-dimethylsulfide complex yielded (VI) as a mixture of diasteriomers. Chromatography on silica gel gave the trans-racemate, which was resolved into its individual enantiomers through the di-p-toluoyl-L-tartaric acid salt. The absolute configuration of the S,S-enantiomer, MK-507, was established by single crystal X-ray analysis.

……………………………………………………….

http://bonanzasite.com/synthesis-of-dorzolamide-hydrochloride

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//////////A new synthesis of MK-0507 has been described: The condensation of 3(R)-(tosyloxy)butyric acid methyl ester (I) with lithium 2-thienylmercaptide (II) in formamide-THF gives 3(S)-(2-thienylthio)butyric acid methyl ester (III), which is hydrolyzed with aqueous HCl to the corresponding free acid (IV). The intramolecular Friedel-Crafts’cyclization of (IV) with trifluoroacetic anhydride yields 6(S)-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-4-one (V), which is reduced with LiAlH4 in toluene to afford 4(R)-hydroxy-6(S)-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran (VI). Epimerization of (VI) with sulfuric acid gives the alcohol (VII) in a cis:trans ratio of 24:76%. Oxidation of (VII) with H2O2 and sodium tungstate yields the 7,7-dioxide (VIII; cis-trans mixture), which is acetylated with acetic anhydride to the acetate (IX). The reaction of (IX) with acetonitrile and sulfuric acid affords N-[6(S)-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-4-yl]acetamide 7,7-dioxide (X; cis-trans mixture), which is sulfonated with chlorosulfonic acid and then treated with SOCl2 to give 4-acetamide-6(S)-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonyl chloride 7,7-dioxide (XI; cis-trans mixture). The reaction of (XI) with concentrated aqueous NH4OH in THF yields the corresponding sulfonamide (XII), which by reduction with BH3-dimethylsulfide in THF affords 4-(ethylamino)-6(S)-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide (XIII; cis-trans mixture). Finally, this mixture is treated with maleic acid in acetone and the resulting maleates are submitted to fractionated crystallization, giving the maleate of the (4S,6S)-isomer, which is treated first with NaHCO3 and then with HCl to give MK-0507; [alpha](25)589 -17.1 C (c 1, H2O).

H-NMR spectral analysis

CAS NO. 130693-82-2, DORZOLAMIDE HCL H-NMR spectral analysis

C-NMR spectral analysis

CAS NO. 130693-82-2, DORZOLAMIDE HCL C-NMR spectral analysis

References

1.  Dorzolamide at Drugs.com. Revised: 12/2011
2. KD Tripari MD. Essentials of Medical Pharmacology (5 ed.). Jaypee Brothers Medical Publishers(P) Ltd. p. 88. ISBN 81-8061-187-6.

Further reading

• Kubinyi H (1999). “Chance favors the prepared mind–from serendipity to rational drug design”. J Recept Signal Transduct Res19 (1–4): 15–39.doi:10.3109/10799899909036635. PMID 10071748.
• Plummer C, MacKay E, Gelatt K (2006). “Comparison of the effects of topical administration of a fixed combination of dorzolamide-timolol to monotherapy with timolol or dorzolamide on IOP, pupil size, and heart rate in glaucomatous dogs”. Vet Ophthalmol 9 (4): 245–9.doi:10.1111/j.1463-5224.2006.00469.x. PMID 16771760.
• Grover S, Apushkin M, Fishman G (2006). “Topical dorzolamide for the treatment of cystoid macular edema in patients with retinitis pigmentosa”.Am J Ophthalmol 141 (5): 850–8. doi:10.1016/j.ajo.2005.12.030.PMID 16546110.
• Almeida G, Faria e Souza S (2006). “Effect of topical dorzolamide on rabbit central corneal thickness”. Braz J Med Biol Res 39(2): 277–81.doi:10.1590/S0100-879X2006000200015. PMID 16470316.

Reference:

CIPLA LIMITED; CURTIS, Philip, Anthony Patent: WO2008/135770 A2, 2008 ; Location in patent: Page/Page column 21-22 ;

RAGACTIVES, S.L. Patent: US2003/220509 A1, 2003 ; Location in patent: Page/Page column 12 ;

WO2011/101704 A1, ;

Literature References:

Carbonic anhydrase inhibitor. Prepn: J. J. Baldwin et al., EP 296879; eidem,US 4797413 (1988, 1989 both to Merck & Co.). Mechanism of action study: R.-F. Wang et al., Arch. Ophthalmol. 109, 1297 (1991).

HPLC determn in plasma and urine: B. K. Matuszewski, M. L. Constanzer,Chirality 4, 515 (1992).

Clinical evaluations in glaucoma and ocular hypertension: E. A. Lippa et al.,Ophthalmology 98, 308 (1991); E. A. Lippa et al., Arch. Ophthalmol. 110, 495 (1992).

REFERENCE
1	*	KAMEI K. ET AL.: ‘Chemical structure, physico-chemical properties and stability of dorzolamide hydrochloride‘ IYAKUHIN KENKYU vol. 25, no. 6, 1994, pages 438 – 452, XP008040715
2	*	QUINT M.-P. ET AL.: ‘Dorsolamide hydrochloride‘ ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS vol. 26, 1999, pages 283 – 316, XP008040718

EP2128161A1 *	May 30, 2008	Dec 2, 2009	Ragactives, S.L.	Process for obtaining 4-hydroxy-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-7,7-dioxide and its enantiomers, and applications thereof
WO2008135770A2*	May 7, 2008	Nov 13, 2008	Cipla Ltd	Process for preparing dorzolam ide
WO2009144263A2*	May 28, 2009	Dec 3, 2009	Ragactives, S.L.U.	PROCESS FOR OBTAINING 4-HYDROXY-6-METHYL-5, 6-DIHYDRO-4H-THIENO [2,3-b] THIOPYRAN-7, 7-DIOXIDE AND ITS ENANTIOMERS, AND APPLICATIONS THEREOF

WO2014005943A1*	Jun 28, 2013	Jan 9, 2014	Zach System S.P.A.	Process for preparing enantiomerically enriched oxamides
US8263787	May 7, 2008	Sep 11, 2012	Cipla Limited	Process for preparing dorzolamide

WO1994021645A1*	Mar 16, 1994	Sep 29, 1994	Thomas J Blacklock	ENANTIOSELECTIVE SYNTHESIS OF 5,6-DIHYDRO-(S)-4-(ETHYLAMINO)-(S)-6-METHYL-4H-THIENO[2,3-b]THIOPYRAN-2-SULFONAMIDE 7,7-DIOXIDE AND RELATED COMPOUNDS
EP0296879A1 *	Jun 23, 1988	Dec 28, 1988	Merck & Co., Inc.	Substituted aromatic sulfonamides as antiglaucoma agents
US5474919 *	Sep 13, 1994	Dec 12, 1995	Merck & Co., Inc.	Bioconversion process for the synthesis of transhydroxy sulfone by Rhodotorula rubra or Rhodotorula piliminae
US5760249 *	Aug 28, 1996	Jun 2, 1998	Merck & Co., Inc.	Synthesis of hydroxysulfone and related compounds
US20060142595 *	Dec 28, 2004	Jun 29, 2006	Council Of Scientific & Industrial Research	Starting by reacting a 2-halothiophene with a Grignard reagent in a solvent in situ with sulfur, triethylamine hydrochloride, crotonic acid and a base; product is chlorinated, cyclized, chlorosulfonated and aminated, reduced, oxidized, amidated, hydrogenated, neutralized, recrystallized and resolved

US5157129	Apr 18, 1990	Oct 20, 1992	Merck & Co., Inc.	Enantiospecific synthesis of s-(+)-5,6-dihydro-4-(r-amino)-4h-thieno(2,3-b)thiopyran-2-sulfonamide-7,7-dioxide
US5474919	Sep 13, 1994	Dec 12, 1995	Merck & Co., Inc.	Bioconversion process for the synthesis of transhydroxy sulfone by Rhodotorula rubra or Rhodotorula piliminae
US5688968	Jan 6, 1995	Nov 18, 1997	Merck & Co., Inc.	Enantioselective synthesis of 5,6-dihydro-(S)-4-(ethylamino)-(S)-6-methyl-4H-thieno 2,3-B!thiopyran-2-sulfonamide 7,7-dioxide
US5760249	Aug 28, 1996	Jun 2, 1998	Merck & Co., Inc.	Synthesis of hydroxysulfone and related compounds
US7109353	Dec 28, 2004	Sep 19, 2006	Council Of Scientific And Industrial Research	Process for preparing 5,6-dihydro-4-(S)-(ethylamino)-6-(S) methyl-4H-thieno[2,3b]thiopyran-2-sulphonamide-7,7-dioxide HCl
US20060155132	Jan 6, 2006	Jul 13, 2006	Kovacs Laszlo Z	Method of making dorzolamide hydrochloride
EP0296879A1	Jun 23, 1988	Dec 28, 1988	Merck & Co., Inc.	Substituted aromatic sulfonamides as antiglaucoma agents
WO1994021645A1	Mar 16, 1994	Sep 29, 1994	Thomas J Blacklock	ENANTIOSELECTIVE SYNTHESIS OF 5,6-DIHYDRO-(S)-4-(ETHYLAMINO)-(S)-6-METHYL-4H-THIENO[2,3-b]THIOPYRAN-2-SULFONAMIDE 7,7-DIOXIDE AND RELATED COMPOUNDS
WO2008135770A2	May 7, 2008	Nov 13, 2008	Cipla Ltd	Process for preparing dorzolam ide

http://www.pharmacopeia.cn/v29240/usp29nf24s0_m28035.html

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KAE 609, NITD 609, Cipargamin

Cipargamin, NITD 609

IUPAC Name: (3R,3'S)-5,7'-dichloro-6'-fluoro-3'-methylspiro[1H-indole-3,1'-2,3,4,9-tetrahydropyrido[3,4-b]indole]-2-one |

CAS Registry Number: 1193314-23-6
Synonyms: NITD609, NITD 609, NITD-609, GNF-609
KAE-609
NITD-609  

 390.238, C19 H14 Cl2 F N3 O

(1'R,3'S)-5,7'-Dichloro-6'-fluoro-3'-methyl-1,2,2',3',4',9'-hexahydrospiro[indole-3,1'-pyrido[3,4-b]indole]-2-one

(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one

Novartis Ag    innovator

NITD609 is an experimental synthetic antimalarial molecule belonging to the spiroindolone class.^[1][2] The compound was developed at the Novartis Institute for Tropical Diseases in Singapore, through a collaboration with the Genomics Institute of the Novartis Research Foundation (GNF), the Biomedical Primate Research Centre and the Swiss Tropical Institute. NITD609 is a novel, synthetic antimalarial molecule belonging to the spiroindolone class, awarded MMV Project of the Year 2009.

It is structurally related to GNF 493, a compound first identified as a potent inhibitor of Plasmodium falciparum growth in a high throughput phenotypic screen of natural products conducted at the Genomics Institute of the Novartis Research Foundation in San Diego, California in 2006. NITD609 was discovered by screening the Novartis library of 12,000 natural products and synthetic compounds to find compounds active against Plasmodium falciparum. The first screen turned up 275 compounds and the list was narrowed to 17 potential candidates.

KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum.http://www.nejm.org/doi/full/10.1056/NEJMoa1315860

KAE609 shows promise as next generation treatment for malaria

http://www.novartis.com/newsroom/media-releases/en/2014/1843976.shtml

• KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  
• KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  
• For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

KAE609 shows promise as next generation treatment for malaria

• KAE609 is the first antimalarial drug candidate with a novel mechanism of action to achieve positive clinical proof-of-concept in over 20 years
  
• KAE609 was tested in adult patients with uncomplicated malaria and showed a median parasite clearance time of 12 hours, including in patients with resistant infections[1]
  
• For more than a decade, Novartis has been a leader in the fight against malaria, setting the current gold standard for treatment and building one of the strongest malaria pipelines in the industry

The digital press release with multimedia content can be accessed here:

Basel, Switzerland, July 30, 2014 - Today, Novartis published clinical trial results in the New England Journal of Medicine showing that KAE609 (cipargamin), a novel and potent antimalarial drug candidate, cleared the parasite rapidly in Plasmodium falciparum (P. falciparum) and Plasmodium vivax (P. vivax) uncomplicated malaria patients[1]. Novartis currently has two drug candidates in development. Both KAE609 and KAF156 are new classes of anti-malarial compounds that treat malaria in different ways from current therapies, important to combat emerging drug resistance. Novartis has also identified PI4K as a new drug target with potential to prevent, block and treat malaria.

"Novartis is in the fight against malaria for the long term and we are committed to the continued research and development of new therapies to eventually eliminate the disease," said Joseph Jimenez, CEO of Novartis. "With two compounds and a new drug target currently under investigation, Novartis has one of the strongest malaria pipelines in the industry."

Malaria is a life-threatening disease primarily caused by parasites (P. falciparum and P. vivax) transmitted to people through the bites of infected Anopheles mosquitoes. Each year it kills more than 600,000 people, most of them African children[2].

"KAE609 is a potential game-changing therapy in the fight against malaria," said Thierry Diagana, Head of the Novartis Institute for Tropical Diseases (NITD), which aims to discover novel treatments and prevention methods for major tropical diseases. "Novartis has given KAE609 priority project status because of its unique potential of administering it as a single-dose combination therapy."

In June 2012, 21 patients infected by one of the two main malaria-causing parasite types took part in a proof-of-concept clinical study conducted in Bangkok and Mae Sot near the Thailand/Burma border where resistance to current therapies had been reported. Researchers saw rapid parasite clearance in adult patients (median of 12 hours)[2] with uncomplicated P. vivax or P. falciparum malaria infection including those with resistant parasites. No safety concerns were identified, however the study was too small for any safety conclusions.

"The growing menace of artemisinin resistance threatens our current antimalarial treatments, and therefore our attempts to control and eliminate falciparum malaria," said Nick White, Professor of Tropical Medicine at Mahidol University in Thailand and lead author of the NEJM article. "This is why we are so enthusiastic about KAE609; it is the first new antimalarial drug candidate with a completely novel mechanism of action to reach Phase 2 clinical development in over 20 years."

KAE609, the first compound in the spiroindolone class of treatment, works through a novel mechanism of action that involves inhibition of a P-type cation-transporter ATPase4 (PfATP4), which regulates sodium concentration in the parasite. Because KAE609 also appears to be effective against the sexual forms of the parasite, it could potentially help prevent disease transmission. The clinical trial was done in collaboration with the Wellcome Trust-Mahidol University - Oxford Tropical Medicine Research Programme. Research was supported by the Wellcome Trust, Singapore Economic Development Board, and Medicines for Malaria Venture.

KAE609 represents one of two new classes of antimalarial compounds that Novartis has discovered and published in the last four years.[3],[4] This drug candidate has shown potent in vitro activity against a broad range of parasites that have developed drug resistance against current therapies. KAE609 is currently being planned for Phase 2b trials.

References
[1] http://www.nejm.org/doi/full/10.1056/NEJMoa1315860
[2] World Health Organization, http://www.who.int/mediacentre/factsheets/fs094/en/
[3] Spiroindolones, a Potent Compound Class for the Treatment of Malaria, KAE609, Science, Sept. 2010
[4] Imaging of Plasmodium liver stages to drive next generation antimalarial drug discovery. Science Express, Nov. 17, 2011

http://www.ukmi.nhs.uk/applications/ndo/record_view_open.asp?newDrugID=6368

The current spiroindolone was optimized to address its metabolic liabilities leading to improved stability and exposure levels in animals. As a result, NITD609 is one of only a handful of molecules capable of completely curing mice infected withPlasmodium berghei (a model of blood-stage malaria).

Given its good physicochemical properties, promising pharmacokinetic and efficacy profile, the molecule was recently approved as a preclinical candidate and is now entering GLP toxicology studies with the aim of entering Phase I studies in humans in late 2010. If its safety and tolerability are acceptable, NITD609 would be the first antimalarial not belonging to either the artemisinin or peroxide class to go into a proof-of-concept study in malaria.

If NITD609 behaves similarly in people to the way it works in mice, it may be possible to develop it into a drug that could be taken just once - far easier than current standard treatments in which malaria drugs are taken between one and four times a day for up to seven days. NITD609 also has properties which could enable it to be manufactured in pill form and in large quantities. Further animal studies have been performed and researchers have begun human-stage trials.

NITD609
IUPAC name[hide] (1R,3S)-5’,7-Dichloro-6-fluoro-3-methyl-spiro[2,3,4,9-tetrahydropyrido[3,4-b]indole-1,3’-indoline]-2’-one
Identifiers
ChemSpider	24662493
Jmol-3D images	Image 1
SMILES [show]
InChI [show]
Properties
Molecular formula	C19H14Cl2FN3O
Molar mass	390.24 g mol−1

Malaria is an old infectious disease caused by four protozoan parasites, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. These four parasites are typically transmitted by the bite of an infected female Anopheles mosquito. Malaria is a problem in many parts of the world, and over the last few decades the malaria burden has steadily increased. An estimated 1 to 3 million people die every year from malaria - mostly children under the age of 5. This increase in malaria mortality is due in part to the fact that Plasmodium falciparum, the deadliest malaria parasite, has acquired resistance against nearly all available antimalarial drugs, with the exception of the artemisinin derivatives.
Leishmaniasis is caused by one of more than twenty (20) varieties of parasitic protozoa that belong to the genus Leishmania, and is transmitted by the bite of female sandflies. Leishmaniasis is endemic in some 90 countries, including many tropical and sub-tropical areas.
There are four main forms of leishmaniasis. Visceral leishmaniasis, also called kala-azar, is the most serious form and is caused by the parasite Leishmania donovani. Patients who develop visceral leishmaniasis can die within months unless they receive treatment. The two main therapies for visceral leishmaniasis are the antimony derivatives sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantim®). Sodium stibogluconate has been used for about 70 years and resistance to this drug is a growing problem. In addition, the treatment is relatively long and painful, and can cause undesirable side effects. Human African Trypanosomiasis, also known as sleeping sickness, is a vector-bome parasitic disease. The parasites concerned are protozoa belonging to the Trypanosoma Genus. They are transmitted to humans by tsetse fly {Glossina Genus) bites which have acquired their infection from human beings or from animals harbouring the human pathogenic parasites.
Chagas disease (also called American trypanosomiasis) is another human parasitic disease that is endemic amongst poor populations on the American continent. The disease is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking insects. The human disease occurs in two stages: the acute stage, which occurs shortly after the infection, and the chronic stage, which can develop over many years. Chronic infections result in various neurological disorders, including dementia, damage to the heart muscle and sometimes dilation of the digestive tract, as well as weight loss. Untreated, the chronic disease is often fatal.
The drugs currently available for treating Chagas disease are nifurtimox and benznidazole. However, problems with these current therapies include their adverse side effects, the length of treatment, and the requirement for medical supervision during treatment. Furthermore, treatment is really only effective when given during the acute stage of the disease. Resistance to the two frontline drugs has already arisen. The antifungal agent amphotericin b has been proposed as a second-line drug, but this drug is costly and relatively toxic.

SYNTHESIS..........WILL BE UPDATED
...........................................
http://www.google.com/patents/WO2009132921A1?cl=en

SCHEME G: Preparation of (lR,3S)-5',7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3'-indol-2'(l'iϊ)-one (35) and (lR,3S)-5'-chloro-6-fluoro-3- methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indoI-2'(l'H0-one (36)

Step 1 : POCl₃ (2.43 mL, 26.53 mmol) was added dropwise to N, N-dimethylformamide (15.0 mL) at -20 °C and stirred below -5 ⁰C for one hour. A solution of 6-chloro-5-fluoroindole (3.0 g, 17.69 mmol) in dimethylformamide (5.0 mL) was added dropwise to the above reaction mixture at -20 °C. The salt-ice bath was removed and the reaction mixture was warmed to 35 ⁰C, After one hour, the reaction was poured onto ice and basified by solid sodium bicarbonate and extracted with ethyl acetate. The combined organic layer was washed with water and then concentrated to give 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (3.4 g, 97 %) as a light brown solid. ¹H ΝMR (500 MHz, CDCl₃): δ 10.02 (s, 1 H), 8.10 (d, IH, J = 9.5 Hz), 7.87 (s, 1 H), 7.49 (d, IH, J= 5.5 Hz).
Step 2: The solution (0.2 M) of 6-chloro-5-fluoro-1H-indole-3-carbaldehyde (4.0 g, 20.24 mmol) in nitroethane (100 mL) was refluxed with ammonium acetate (1.32 g, 0.85 mmol) for 4 hours. The reaction mixture was concentrated under vacuum to remove nitroethane, diluted with ethylacetate and washed with brine. The organic layer was concentrated to give 6-chloro-5- fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 97 %) as a reddish orange solid. ¹H ΝMR (500 MHz, CDCl₃): δ 8.77 (s, IH), 8.32 (s, IH), 7.58 (d, IH, J= 2.5 Hz), 7.54 (d, IH, J = 9 Hz), 7.50 (d, IH, J= 5.9 Hz), 2.52 (s, 3H). Step 3: A solution of 6-chloro-5-fluoro-3-(2-nitro-propenyl)-1H-indole (5.0 g, 19.63 mmol) in tetrahydrofuran (10 mL) was added to the suspension of lithium aluminium hydride (2.92 g, 78.54 mmol) in tetrahydrofuran (20 mL) at 0 ⁰C and then refluxed for 3 hours. The reaction mixture was cooled to 0 °C, and quenched according to the Fischer method. The reaction mixture was filtered through celite and the filtrate concentrated to give 2-(6-chloro-5-fluoro-1H-indol-3- yl-1-methyl-ethylamine (4.7 g crude) as a viscous brown liquid. The residue was used without further purification. ¹H NMR (500 MHz, CDCl₃): δ 8.13 (s, IH), 7.37 (d, IH, 6.Hz), 7.32 (d, IH, J = 10 Hz), 7.08 (s, IH), 3.23-3.26 (m, IH), 2.77-2.81 (m, IH), 2.58-2.63 (m, IH), 1.15 (d, 3H, J= 6.5 Hz).
Step 4: A mixture of 2-(6-chloro-5-fluoro-1H-indol-3-yl-l-methyl-ethylamine (4.7 g, 20.73 mmol), 5-chloroisatin (3.76 g, 20.73 mmol) and p-toluenesulphonic acid (394 mg, 2.07 mmol) in ethanol (75 mL) was refluxed overnight. The reaction mixture was concentrated to remove ethanol, diluted with ethyl acetate and washed with saturated aqueous NaHCO₃. The organic layer was concentrated to give a brown residue, which was purified by silica gel chromatography (20 % ethyl acetate in hexane) to provide the corresponding racemate (4.5 g, 56 %) as a light yellow solid. The racemate was separated into its enantiomers by chiral chromatography to provide 35.
Compound 36 can be obtained in a similar fashion from 5-fluoroindole.
Alternatively 35 and 36 were be prepared in enantiomerically pure form by the following scheme.
SCHEME H: Alternative preparation of (lR,3S)-5',7-dichloro-6-fluoro-3-methyl-2,3,4,9- tetrahydrospiro[β-carboline-l,3'-indol-2'(1'H)-one (35)

Step 1 : To a solution of 6-chloro-5-fluoroindole (1.8 g, 10.8 mmol) and Ac₂O (10 niL) in AcOH (3OmL) was added L-serine (2.2 g, 20.9 mmol), the mixture was heated to 80 °C. After TLC indicated the reaction was complete, the mixture was cooled to 0 °C, neutralized to pH 11 , and washed with MTBE. The aqueous phase was acidified to pH 2 and extracted with EtOAc. The combined organic layers were washed with water and bπne, dπed with Na₂SO₄, filtered, and concentrated. The residue was purified with chromatography (Petroleum ether /EtOAc 1:1) to give 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 37% yield).
Step 2: 2-Acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (2.5g, 8.4mmol) was dissolved in aqueous NaOH (IN, 10 niL) and water added (70 mL). The mixture was heated to 37-38⁰C and neutralized with HCl (IN) to pΗ 7.3-7.8. L-Aminoacylase (0.5 g) was added to the mixture and allowed to stir for 2 days, maintaining 37-38⁰C and pΗ 7.3-7.8. The mixture was heated to 60 °C for another hour, concentrated to remove part of water, cooled and filtered. The filtrate was adjusted to pΗ 5.89 and filtered again. The filtrate was adjusted to pΗ 2.0 and extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc 1 : IEtOAc) to give R- 2-acetylamino-3-(6-chloro-5-fluoro-1H-mdol-3-yl)-propπonic acid as a light yellow solid (1.2 g, 48% yield). Step 3: R-2-acetylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-proprionic acid (1.2 g, 4.0 mmol) was dissolved in HCl (6N, 10 mL) and the mixture heated to reflux for 4 hours, and then concentrated to dryness. Toluene (50 mL) was added to the residue and concentrated to dryness to remove water and HCl. The residue was dried under vacuum and then dissolved in MeOH (20 mL). To the solution was added dropwise SOCl₂ (0.5 mL, 6.8 mmol) at 0 °C, and the mixture was stirred overnight. After removal of solvent, the residue was dissolved in THF/water (40/10 mL) and NaHCO₃ (1.0 g, 11.9 mmol) was added portionwise. Upon basifϊcation, BoC₂O (1.2 g, 5.5 mmol) added at 0 °C and allowed to stir at room temperature. After TLC indicated the reaction was finished, EtOAc was added and separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether /EtOAc: 5/1) to give R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l/-/-indol-3-yl)-proprionic acid methyl ester 460 g, 31% yield for 3 steps).
Step 4: To a solution of R-2-tert-butoxycarbonylamino-3-(6-chloro-5-fluoro-l//-indol-3-yl)- proprionic acid methyl ester (460mg, 1.2mmol) in dry ether (20 mL) was added portionwise LiAlH₄ (92 mg, 2.4 mmol) at 0 °C. The mixture was heated to reflux for 2 hours. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na₂SO₄. The mixture was filtered and the filtrate was washed with saturated aqueous NH₄Cl and water, dried with Na₂SO₄, filtered, concentrated to give a crude product (400 mg), which was used without further purification.
Step 5: To a solution of the crude product (400 mg, 1.2mmol) and Et₃N (0.3 mL, 2.2 mmol) in CH₂Cl₂ (5 mL) was added MsCl (160 mg, 1.4 mmol) dropwise at 0 °C. The mixture was stirred for 2 hours at room temperature. After TLC indicated the reaction was completed, the mixture was washed with water and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give methansulfonic acid (R)-2- ?ert-butoxycarbonylamino-3-(6-chloro-5-fluoro-1H-indol-3-yl)-propyl ester as a light yellow solid (300 mg, 57% yield, 2 steps)
Step 6: To a solution of mesylate (300 mg, 0.7mmol) in dry ether (20 mL) was added portionwise LiAlH₄ (55 mg, 1.4 mmol) at 0 °C. The mixture was stirred at room temperature overnight. After TLC indicated the reaction was finished, the mixture was cooled and carefully quenched with Na₂SO₄. The mixture was filtered and the filtrate was washed with saturated aqueous NH₄Cl and water, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 10: 1) to give [(5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)- 1 -methyl-ethyl] -carbamic acid tert-butyl ester as a light yellow solid (200 mg, 87% yield).
Step 7: A solution of [(S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethyl]-carbamic acid tert-butyl ester (200 mg, 0.6 mmol) in HCl/MeOH (10 mL) was stirred at room temperature. After TLC indicated the reaction was finished, the mixture was concentrated to remove the solvent. To the residue was added EtOAc (5OmL), and the mixture was neutralized with saturated NaHCO₃ to pH 8~9, and then extracted with EtOAc. The combined organic phases were dried with Na₂SO₄, filtered, concentrated to give a crude (S)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l- methyl-ethylamine which was used without further purification.
Step 8: To a solution of (5)-2-(6-chloro-5-fluoro-1H-indol-3-yl)-l-methyl-ethylamine (120 mg, 0.5 mmol) in EtOH (1OmL) was added 5-chloroisatin (90 mg, 0.5 mmol) and p-TsOΗ (8 mg, 0.04 mmol). The mixture was heated in a sealed tube at 110⁰C for 16 hours. After TLC indicated the reaction was finished, the mixture was cooled and concentrated. The residue was dissolved in EtOAc (2OmL) and washed with NaOH (IN) and brine, dried with Na₂SO₄, filtered, concentrated and the residue was purified with chromatography (petroleum ether/EtOAc 5:1) to give 36 (150mg, 64% yield over two steps).

Example 48 (15,3R)-5'-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indol]-2'(l'_JH)-one
(35)

35
Compound 35 may be prepared according to Scheme F using the same or analogous synthetic techniques and/or substituting with alternative reagents.
(lS^RVS'-Chloro-S-methyl-l^^^-tetrahydrospirotβ-carboline-l.S'-indoll-l^l'ZO-one: ¹H NMR (300 MHz, DMSO-^₆): δ 10.45 (s, IH), 10.42 (s, IH), 7.43 (d, J= 7.5 Hz, IH), 7.31 (dd, J = 2.1, 8.4 Hz, IH), 7.16 (d, J = 7.2 Hz, IH), 7.05-7.02 (m, 2H), 7.00-6.96 (m, IH), 6.92 (d, J = 8.1 Hz, IH), 3.98-3.86 (m, IH), 2.78 (dd, J= 3.6, 14.9 Hz, IH), 2.41 (dd, J= 4.5, 25.5 Hz, IH), 1.18 (d, J= 6.3 Hz, 3H); MS (ESI) m/z 338.0 (M+H)⁺.
Chiral compounds such as 36 and 37 can be prepared according to Scheme G or H using the same or analogous synthetic techniques and/or substituting with alternative reagents. Example 49
(IR^^-S'.T-Dichloro-ό-fluoro-S-methyl-l^^^-tetrahydrospiroIβ-carboline-l^'-indol]- 2\VH)-one (36)

36
35: ¹H NMR (500 MHz, DMSO-Jd) δ 10.69 (s, IH), 10.51 (s, IH), 7.43 (d, J = 10.0 Hz, IH), 7.33 (dd, J= 8.4, 2.2 Hz, IH), 7.27 (d, J= 6.5 Hz, IH), 7.05 (d, J= 2.3, IH), 6.93 (d, J= 8.5 Hz, IH), 3.91 (m, IH), 3.13 (bd, J= 6.2 Hz, IH), 2.74 (dd, J= 15.0 , 3.0 Hz, IH), 2.35 (dd, J= 15.0, 10.3, IH), 1.15 (d, J= 6.0, 3H);
MS (ESI) m/z 392.0 (M+2H)⁺;
[α]²⁵ _D = + 255.4°
Example 50
(lS,3R)-5',7-Dichloro-6-fluoro-3-methyI-2,3,4,9-tetrahydrospiro[β-carboline-l,3'-indol]- 2'(l'H)-one (37)

37
(lS^^-S'^-Dichloro-o-fluoro-S-methyl^jS^^-tetrahydrospirojP-carboline-l-S'-indol]- 2'(l'H)-one: ¹H NMR (500 MHz, CDCl₃) δ 8.49 (s, IH), 7.54 (s, IH), 7.24 (d, J= 9.7 Hz, IH), 7.21 (dd, J = 8.6, 2.0 Hz, IH), 7.14 (d, J= 6.0 Hz, IH), 7.11 (d, J= 1.8, IH), 6.77 (d, J= 8.3 Hz, IH), 4.14 (m, IH), 2.89 (dd, J = 15.4, 3.7 Hz, IH), 2.49 (dd, J = 15.3, 10.5, IH), 1.68 (bs, IH), 1.29 (d, J= 6.4 Hz, 3H); MS (ESI) m/z 392.0 (M+2H)⁺; [α]²⁵ _D -223.3°
.............................................
US 2011275613
http://www.google.com/patents/WO2013139987A1?cl=en

Prior art:
(1 'R, 3'S)-5, 7'-dichloro-6'-fIuoro-3'-methyl-2', 3',4', 9'-tetrahydrospiro[indoline-3, 1 - pyrido[3,4-b]indol]-2-one (eg. a compound of formula (IV), which comprises a spiroindolone moiety) and a 6-steps synthetic method for preparing, including known chiral amine intermediate compound (MA) are known (WO 2009/132921 ):

he present invention relates to processes for the preparation of spiroindolone compounds, such as (1'R,3'S)-5, 7'-dichloro-6'-fIuoro-3'-methyl-2',3',4',9'- tetrahydrospiro[indoline-3, 1 '-pyhdo[3.4-b]indol]-2-one.
(1 'R, 3'S)-5, 7'-dichloro-6'-fluoro-3'-methyl-2', 3',4 9'-tetrahydrospiro[indoline-3, 1 '- pyrido[3, 4-b]indol]-2-one is useful in the treatment and/or prevention of infections such as those caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Trypanosoma cruzi and parasites of the Leishmania genus such as, for example, Leishmania donovani., and it has the following structure:

(IVA)
(1 'R, 3'S)-5, 7'-dichloro-6'-fluoro-3'-methyl-2 3', 4', 9'-tetrahydrospiro[indoline-3, 1 - pyhdo[3, 4-b]indol]-2-one and a synthesis thereof are described in WO 2009/132921 Al in particular in Example 49 therein.

Example 10: Process for Conversion of Compound (IA) to Compound (IIA) in 30g Scale

458.97
152.48g /so-propylamine hydrochloride and 0.204g pyridoxalphosphate monohydrate were dissolved in 495ml water while stirring. To this yellow clear solution a solution of 30. Og ketone in 85ml poly ethylene glycol (average mol weight 200) within 15 minutes. Upon addition the ketone precipitates as fine particles which are evenly distributed in the reaction media. To the suspension 180ml triethanolamine buffer (0.1 mol/l, pH 7) were added and the pH was adjusted to 7 by additon of aqueous sodium hydroxide solution (1 mol/l). The reaction mixture is heated to 50°C and a solution of 1.62g transaminase SEQ ID NO: 134 dissolved in 162ml triethanolamine buffer (0 1 mol/l, pH 7) is added. The reaction mixture is continiously kept at pH 7 by addition of 1 mol/l aqueous sodium hydroxide solution. The reaction mixture is stirred 24h at 50°C and a stream of Nitrogen is blown over the surface of the reaction mixture to strip off formed acetone. The reaction mixture is then cooled to 25°C and filtered over a bed of cellulose flock. The pH of the filtrate is adjusted to «1 by addition of concentrated sulfuric acid. The acidified filtrated is extracted with 250 ml /so-Propyl acetate. The layers are separated and the pH of the aqueous phase is adjusted to ¾10 by additon of concentrated aqueous sodium hydroxide solution. The basified aqueous phase is extracted with /^'so-propyl acetate. The layers are seperated and the organic phase is washed with 100 ml water. The organic phase is concentrated by distillation to 2/3 of its origin volume. In a second reactor 33.98g (+)- camphor sulfonic acid is dissolved in 225 ml /^'so-propyl acetate upon refluxing and the concentrated organic phase is added within 10 minutes. After complete addition the formed thin suspension is cooled to 0°C within 2 hours and kept at 0°C for 15 hours. The precipitated amine-(+)-camphor sulfonate salt is filtered, washed with 70 ml /so-propyl acetate and dried at 40°C in vaccuum yielding 51.57g of colourless crystals (84.5% yield t.q.)
Analytical Data
IR:
v (crn ¹)=3296, 3061 , 2962, 2635, 2531 , 2078, 1741 , 1625, 1577, 1518, 1461 , 1415, 1392, 1375, 1324, 1302, 1280, 1256, 1226, 1 170, 1 126, 1096, 1041 , 988, 966, 937, 868, 834, 814, 790, 766, 746, 719, 669, 615.
LC-MS (ESI +):
Ammonium ion: m/z =227 ([M+H]), 268 ([M+H+CH₃CN]), 453 ([2M+H]).
Camphorsulfonate ion: m/z =250 ([M+NH₄]), 482 ([2M+NH4]).
LC-MS (ESI -):
Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H]).
1H-NMR (DMSO-d6, 400 MHz):
1 1.22 (br. s., 1 H), 7.75 (br. s., 3H), 7.59 (d, J = 10.3 Hz, 1 H), 7.54 (d, J = 6.5 Hz, 1 H), 7.36 (d, J = 2.3 Hz, 1 H), 3.37 - 3.50 (m, 1 H), 2.98 (dd, J = 14.3, 5.8 Hz, 1 H), 2.91 (d, J = 14.8 Hz, 1 H), 2,81 (dd, J = 14.3, 8.0 Hz, 1 H), 2.63 - 2.74 (m, 1 H), 2.41 (d, J = 14.6 Hz, 1 H), 2.24 (dt, J = 18.3, 3.8 Hz, 1 H), 1 .94 (t, J = 4.4 Hz, 1 H), 1.86 (dt, J = 7.4, 3 6 Hz, 1 H), 1.80 (d, J = 18.1 Hz, H), 1.23 - 1 .35 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H), 1.05 (s, 3H), 0.74 (s, 3H)
Free Amine (obtained by evaporatig the iso-Propylacetate layer after extraction of the basified aqueous layer):
¹H NMR (400MHz, DMSO-d₆): 11 .04 (br. s., 1 H), 7.50 (d, J = 10.5 Hz, 1 H), 7.48 (d, J = 6.5 Hz, 1 H), 7.25 (s, 1 H), 3.03 (sxt, J = 6.3 Hz, 1 H), 2.61 (dd, J - 14.3, 6.5 Hz, 1 H), 2.57 (dd, J = 14.1 , 6.5 Hz, 1 H), 1.36 (br. s., 2H), 0.96 (d, J = 6.3 Hz, 3H)
Example 11: Process for Conversion of Compound (HA) to Compound (IVB)

3. solvent exchange to TP
13.62 g 5-chloroisatin is suspended in 35 ml /so-propanol and 2.3 g triethyl amine is added. The suspension is heated to reflux and a solution of 34.42g amine-(+)-camphor sulfonate salt dissolved in 300 ml /^'so-propanol is added within 50 minutes. The reaction mixture is stirred at reflux for 17 hours. The reaction mixture is cooled to 75°C and 17.4g (+)-camphorsulfonic acid are added to the reaction mixture. Approximately 300 ml /so- propanol are removed by vacuum distillation. Distilled off /so-propanol is replaced by iso- propyl acetate and vacuum distillation is continued. This is distillation is repeated a second time. To the distillation residue 19 ml ethanol and 265 ml ethyl acetate is added and the mixture is heated to reflux. The mixture is cooled in ramps to 0°C and kept at 0°C for 24 hours. The beige to off white crystals are filtered off, washed with 3 portions (each 25 ml) precooled (0°C) ethylacetate and dried in vacuum yielding 40.3 g beige to off white crystals. (86.3% yield t.q.)
IR:
v (crrr)= 3229, 3115, 3078, 3052, 2971 , 2890, 2841. 2772. 2722, 2675, 2605, 2434. 1741 , 1718, 1621 , 1606, 1483, 1460, 1408, 1391 , 1372, 1336, 1307, 1277, 1267, 1238, 1202, 1 184, 1 162, 1 149, 1 128, 1067, 1036, 987, 973, 939, 919, 896, 871 , 857, 843, 785, 771 , 756, 717, 690, 678, 613.
LC-MS (ESI +):
Ammonium ion: m/z =390 ([M+H]), 431 ([M+H+CH₃CN]) Camphorsulfonate ion: m/z =250 ([M+NH₄]), 482 ([2M+NH4])
LC-MS (ESI -):
Camphorsulfonate ion: m/z=231 ([M-H]), 463 ([2M-H])
1H NMR (DMSO-d₆, 600 MHz):
11.49 (s, 1 H), 1 1.23 (s, 1 H), 10.29 - 10.83 (m, 1 H), 9.78 - 10.31 (m, 1 H), 7.55 - 7.60 (m, 2H), 7.52 (s, 1 H), 7.40 (d, J = 6.2 Hz, H), 7.16 (d, J = 8.8 Hz, 1 H), 4.52 - 4.63 (m, 1 H). 3.20 (dd, J = 16.3, 4.2 Hz, 1 H), 2.96 (dd, J = 16.1 , 11.3 Hz, 1 H), 2.90 (d, J = 15.0 Hz, 1 H), 2.56 - 2.63 (m, 1 H), 2.39 (d, J = 14.6 Hz, 1 H), 2.21 (dt, J = 18.0, 3.8 Hz, 1 H), 1.89 - 1.93 (m, 1 H), 1.81 (ddd, J = 15.3, 7.8, 3.7 Hz, 1 H), 1.76 (d, J = 18.3 Hz, 1 H), 1 .53 (d, J = 6.6 Hz, 3H), 1.20 - 1.33 (m, 2H), 0.98 (s, 3H), 0.70 (s, 3H)
Example 12: Process for Preparing a Compound of formula (IVA) ¹/z Hydrate

mw622.54 .............................................................................mw399.25
In a 750ml reactor with impeller stirrer 50g of compound (IVB) salt were dissolved in 300ml Ethanol (ALABD) and 100 ml deionised Water (WEM). The clear, yellowish sollution was heated to 58°C internal temperature. To the solution 85 g of a 10% aqueous sodium carbonate solution was added within 10 minutes. The clear solution was particle filtered into a second reaction vessel. Vessel and particle filter were each rinsed with 25 ml of a mixture of ethanohwater (3:1 v/v) in the second reaction vessel. The combined particle filtered solution is heated to 58°C internal temperature and 200ml water (WEM) were added dropwise within 15 minutes. Towards the end of the addition the solution gets turbid.
The mixture is stirred for 10 minutes at 58°C internal temperature and is then cooled slowely to room temperature within 4hours 30 minutes forming a thick, well stirable white suspension. To the suspension 200 ml water are added and the mixture is stirred for additional 15hours 20 minutes at room temperature. The suspension is filtered and the filter cake is washed twice with 25 ml portions of a mixture of ethanohwater 9: 1 (v/v). The colourless crystals are dried at 60°C in vacuum yielding 26.23g (=91.2% yield). H NMR (400 MHz, DMSO-d₆)
0.70 (s, 1H), 10.52 (s, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H),.26 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.83 - 4.00 (m,H), 3.13 (d, J = 6.0 Hz, 1H), 2.77 (dd, J = 15.1, 3.8 Hz, 1H), 2.38 (dd, J = 15.1, 10.5 Hz,H), 1.17 (d, J = 6.3 Hz, 3H).

 ......................................................

 Journal of Medicinal Chemistry, 2010 ,  vol. 53,   14  p. 5155 - 5164

http://pubs.acs.org/toc/jmcmar/53/14

(1R,3S)-5′,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,3′-indol]-2′(1′H)-one (19a)

¹H NMR (500 MHz, DMSO-d₆): δ 10.69 (s, 1H), 10.51 (s, 1H), 7.43 (d, J = 10.0 Hz, 1H), 7.33 (dd, J = 8.0, 2.2 Hz, 1H), 7.27 (d, J = 6.5 Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.13 (bd, J = 6.2 Hz, 1H), 2.74 (dd, J = 15.0, 3.0 Hz, 1H), 2.35 (dd, J = 15.0, 10.3 Hz, 1H), 1.15 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 392.0 (M + 2H)⁺; [α]_D²⁵ = +255.4° (c = 0.102 g/L, methanol).

......................................

http://www.google.com/patents/CN102432526A?cl=en&dq=%22cipargamin%22+OR+%22cipargamina%22+OR+%22cipargamine%22+OR+%22cipargaminum%22+OR+%22NITD609%22&source=uds

Z.Zhang, WO 2007 / 104714,2007).
[0007]

[0008] (2) year 2008 Roche pharmaceutical company disclosed a spiro [oxindole - cyclohexenone] skeleton biomedicine, PCT International Application No. W02008 / 055812. It also announced the preparation of anti-cancer agents and antagonists of the application of the compound is used as the interaction with MDM2 (reference:. Liu, J.-J; Zhang, Z; (Hoffmann-LaRoche AG), PCT Int App 1. . W02008 / 055812, 2008), its structural formula is as follows:
[0009]

[0010] (3) Melchiorre research group abroad chiral amines and o-fluoro-3-benzyl benzoate as catalyst methylene-indole-2-one (3-benzylideneindolin-2-one, CAS Number: 3359-49- 7) with α, β - unsaturated ketone synthesis of chiral spiro [cyclohexane _1,3'- indole] _2,4 '- dione [s pir0 [cycl0hexane-l, 3' -indoline] - 2 ', 4-diones] compounds (see:.. Bencivenni, G; ffu, LY; Mazzanti, A .; Giannichi, B.; Pesciaioli, F; Song, Μ P.; Bartoli, G.; Melchiorre, P .... .Angew Chem Int Ed 2009,48,7200), the structure of the total formula is as follows:
[0011]

[0012] (4) Gong Flow column team found to cyclohexanediamine derived Bronsted acid - a bifunctional catalyst Lewis base catalysis of 3-benzyl-methylene-indole-2-one and α, β- unsaturated 1,3 tandem reaction dicarbonyl compound (Nazarov reagent) can be obtained with high stereoselectivity chiral spiro [cyclohexane _1,3'- indol] -2 ', 4-dione [spiro [cyclohexane-l, 3 '-indoline] -2', 4-diones] compounds; and by this method successfully synthesized 7 Roche pharmaceutical companies to develop chiral anti-tumor agents (see: Q Wei, L -Z Gong, Org Lett 2010..... , 12, 1008.).
[0013] (5) Wang Lixin research group recently reported that primary amines derived from cinchona alkaloids and Bronsted acid as catalyst N- protected indolone compounds and double Michael addition reaction of diketene generate hand spiro [cyclohexane-1, 3'-indol] -2 ', 4-dione [spiro [cyclohexane-l, 3' -indoline] -2 ', 4-diones] type of tx ^ (: L. -L. Wang, L. Peng, J. -F. Bai, L. -N. Jia, X. -Y. Luo, QC Huang, L. -X. Wang, Chem. Commum. 2011,47, 5593.).

References

1.  "NITD 609". Medicines for Malaria Venture.
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WO2009132921A1 *	Apr 1, 2009	Nov 5, 2009	Novartis Ag	Spiro-indole derivatives for the treatment of parasitic diseases
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Рисунок из Science 2010, 329, 1175

Исследовательская группа Элизабет Винцелер (Elizabeth A. Winzeler) разработала новый препарат, первоначально проведя скрининг библиотеки, состоящей из 12000 соединений, а затем получив производные наиболее перспективных кандидатов. В результате долгой работы исследователи отобрали единственное соединение спироиндолоновой структуры, получившее регистрационный номер NITD609. В случае успешного прохождения экспертизы фармакологических и токсикологических свойств нового соединения исследователи надеются приступить к первой фазе его клинических испытаний уже в конце этого года.
Было обнаружено, что NITD609 быстро останавливает белковый синтез в организме возбудителя малярии, ингибируя ген аденозинтрифосфатазы, ответственной за транспорт катионов через мембрану клетки возбудителя. То, что механизм действия нового соединения отличается от механизма, характерного для других средств лечения малярии, объясняет причины успешного действия нового препарата в том числе и против штаммов малярии, выработавших резистентность.

 HPLC

Analyte quantization was performed byLC/MS/MS. Liquid chromatography was performed using an Agilent

1100 HPLC system(Santa Clara, CA), with the Agilent Zorbax XDB Phenyl (3.5μ, 4.6 x75 mm) column at

an oven temperature of 35 °C, coupled with a QTRAP4000 triple quadruple mass

spectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: acetonitrile:aceticacid (99.8:0.2, v/v), using a gradient, with flow rate of 1.0 mL/min, and run time of 5minutes. Under these conditions the retention time of9a

was 3.2 minutes. Compounddetection on the mass spectrometer was performed in electrospraypositive ionizationmode and utilized multiple reaction monitoring (MRM) for specificity (9atransitions338.3/295.1, 338.3/259.2) together with their optimized MS parameters. The lower limitof quantification for9awas 70 ng/mL.

Extraction and LCMS analysis of 20a.Plasma samples were extracted withacetonitrile:methanol-acetic acid (90:9.8:0.2 v/v) for the analyte and internal standard(17a) using a 3.6 to 1 extractant to plasma ratio. Analyte quantitation was performed by

LC/MS/MS. Liquid chromatography was performed using an Agilent1100 HPLC systemS7(Santa Clara, CA), with the Agilent Zorbax XDB-Phenyl (3.5μ, 4.6x75mm) column atan oven temperature of 45 °C coupled with a QTRAP 4000 triple quadruple massSpectrometer (Applied Biosystems, Foster City, CA). Instrumentcontrol and dataacquisition were performed using Applied Biosystems software Analyst 1.4.2. Themobile phases used were A: water:acetic acid (99.8:0.2, v/v) and B: methanol:acetic acid

(99.8:0.2, v/v), using gradient elution conditions with a flow rate of 1.0 mL/min and a runtime of 6 minutes