1Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth University, Maharashtra, Pune 411038, India
2Department of Pharmaceutical Chemistry, College of Pharmaceutical Sciences, RAK Medical & Health Sciences University, P.O. Box 11172, Ras Al-Khaimah, UAE
Academic Editors: J. N. Latosinska and K. Ohyama
Copyright © 2012 S. S. Havele and S. R. Dhaneshwar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The present study describes the degradation of dorzolamide HCl under different International Conference on Harmonization prescribed stress conditions (hydrolysis, oxidation, thermolysis, and photolysis) and application of a specific and selective stability-indicating chromatography assay. HPTLC was done on silica Gel 60 F254 TLC plate using chloroform: methanol in the ratio of 9.0 : 1.0 (v/v) as mobile phase and detection at 253 nm.
Dorzolamide hydrochloride (DORZO), (4S,6S)-4-ethylamino-5,6-dihydro-6-methyl-4H-thieno(2,3-b)thiopyran-2-sulfonamide 7,7-dioxide monohydrochloride (Figure 1), is a carbonic anhydrase inhibitor used in eye drops to treat increased pressure in the eye caused by open-angle glaucoma and to treat a condition called hypertension of the eye .
Figure 1: Chemical structure of dorzolamide hydrochloride.
The literature review reveals that several methods have been reported for the estimation of DORZO in biological fluids [2–5], and there are some methods reported by HPLC [6–9], spectroscopy [10, 11], and capillary electrophoresis , and there are some methods reported for estimation of DORZO in pharmaceutical formulation [13–15]. According to current good manufacturing practices, all drugs must be tested with a stability-indicating assay method before release. So far, to our present knowledge, no stability-indicating HPTLC assay method for the determination of DORZO is available in the literature.
Now a days, HPTLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, need for minimum sample preparation, and high sample throughput. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC—thus reducing the analysis time and cost per analysis.
It was felt necessary to develop stability-indicating HPTLC method for the determination of DORZO as bulk drug and pharmaceutical dosage form and separate the drugs from the degradation products under the ICH-suggested conditions. Therefore, the aim of the present study was to develop and validate stability-indicating HPTLC assay methods for DORZO as bulk drug and in pharmaceutical dosage form as per International Conference on Harmonisation guidelines [16–19].
2.1. Drug and Reagents
Pharmaceutical grade DORZO working standard was obtained as generous gift from FDC Ltd., Mumbai, India. The eye drop sample (Ocudor eye drops, manufactured by Lumina (FDC Limited)) was obtained commercially. The DORZO was labeled (per 1 mL) as 22.26 mg. All chemicals and reagents were of analytical grade and were purchased from Merck Chemicals, Mumbai, India.
2.2.1. For Stress Study
High-precision heating mantel (Remi, India) capable of controlling the temperature with in ±1°C was used for generating hydrolytic degradation products. The thermal degradation study was performed using a high-precision hot air oven (Kumar Scientific Works, Pune, India) capable of controlling the temperature within ±2°C. Photodegradation was carried out in a photostability chamber (Thermolab, Scientific Equipment Pvt. Ltd.) equipped with lighting system to comply with ICH guideline for photostability condition with white fluorescent exposure of 1.2 million lux hours and integrated near ultraviolet energy exposure of 200 watts hours/sq.mts (option 2 of the ICH guideline Q1B). At any given time, UV energy and visible illumination were tested using a calibrated lux meter (Lutron, LX-101A).
2.3. Chromatographic Conditions
The HPTLC system consisted of a Camag Linomat 5 semiautomatic spotting device (Camag, Muttenz, Switzerland), a Camag twin-trough chamber (10 cm×10 cm), Camag winCATS software 22.214.171.12437 and a 100 μL Hamilton syringe. Sample application was done on precoated silica gel 60 F254 TLC plates (10 cm×10 cm). TLC plates were prewashed with methanol and activated at 80°C for 5 min prior to the sample application. Densitometric analysis was carried out utilizing Camag TLC scanner 3.
2.4. Preparation of Standard Stock Solutions
A standard stock solution of 1 mg/mL DORZO was prepared in water.
Working standard solutions were prepared by dilution of the stock solution with water to reach a concentration range of 100–500 ng/spot.
2.5. Preparation of Analytical Samples
1 mL of the Ocudor eye drop was transferred into a 100 mL volumetric flask adjusted up to 100 mL with water and sonicated for 15 min. The resulting solutions (sample solutions) were then analyzed by HPTLC after filtration through Whatman filter paper 41.
2.6. Stress Degradation Studies
A standard stock solution was used for stress degradation to provide an indication of the stability-indicating property and specificity of the method. In all degradation studies, the average peak area of DORZO after application of six replicates was obtained.
Acidic decomposition studies were performed by heating the drug solution under reflux at 60°C for 2 h with 1 M hydrochloric acid. Studies under alkaline hydrolysis were performed in 0.2 M sodium hydroxide solution under reflux at 60°C for 4.5 h. The resulting solutions were applied to the plates for HPTLC. The degradation behavior of drug in neutral conditions was studied by mixing 5 mL of a standard stock solution (1 mg/mL) with 5 mL double-distilled water and heated at 80°C for 5 days, and subsequently for 10 days.
2.6.2. Hydrogen Peroxide-Induced Degradation
To study hydrogen peroxide-induced degradation, the drug solution was treated with 6% hydrogen peroxide at room temperature for 72 h. The solution was then heated in a boiling water bath for 10 min to completely remove excess hydrogen peroxide.
Photodegradation studies were carried out according to option 2 of Q1B in ICH guidelines. The stock solution as well as solid drug were exposed to light for an overall illumination of 1.2 million lux/h and an integrated near ultraviolet energy of 200 W hm−2 for 4 days by fluorescent near-UV and cool white light.
10 mg standard drug in solid form was placed in an oven at 50°C for 30 days to study dry heat degradation. To study wet heat degradation, the drug was kept in a humidity chamber at 50°C, 75% relative humidity for 3 months.
2.7. Optimization of the Method
The HPTLC procedure was optimized to establish a stability-indicating assay. Both pure and degraded drug solutions were applied to HPTLC plates and chromatographed with different mobile phases.
2.8. Analytical Method Validation
The developed chromatographic method was validated for linearity, precision, accuracy, sensitivity, robustness, and system suitability.
2.8.1. Linearity and Range
From the working standard solution, five different concentrations (100–500 ng/spot) were spotted on the TLC plate.
The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.
The precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analysis of three different concentrations of 100, 300, and 500 ng/spot six times on the same day. The intermediate precision of the method was checked by repeating studies on two different days.
Sensitivity was determined by establishing the limit of detection (LOD) and limit of quantitation (LOQ) which represent the concentration of the analyte that would yield signal-to-noise ratios of 3 for LOD and 10 for LOQ, respectively. The LOD and LOQ were determined by measuring the magnitude of analytical background by spotting a blank and calculating the signal-to-noise ratio for DORZO by spotting a series of solutions until the S/N ratio 3 was obtained for the LOD and 10 for the LOQ.
The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The parameters which included change in mobile-phase composition like chloroform:methanol in the ratio of (8.9 : 1.0 v/v), (9.0 : 0.9 v/v), (9.1 : 1.0 v/v), and (9.0 : 1.1 v/v) were varied and densitograms were run. The amount of mobile phase was varied over the range of ±0.1%. The plates were prewashed by methanol and activated at 110°C for 4, 5, and 6 min, respectively, prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from ±10 min. Robustness of the method was done at a concentration level of 400 ng/spot. Robustness of the method was done at three different concentration levels (100, 300, 500 ng/spot). These chromatographic variations were evaluated for resolution between DORZO and degradation products.
The specificity of the method was determined by analysis of drug standard and test samples. Identification of the DORZO spot from the samples was confirmed by comparison of its and spectrum with those from a standard. In addition, the peak purity of DORZO was assessed by acquiring spectra at the peak-start (S), peak-apex (M), and peak-end (E) positions of the spot and calculating the correlation between them. The specificity of the method was also checked by separation of DORZO in the presence of its degradation products.
Accuracy of the method was carried out by applying the method to preanalyzed drug sample to which known amounts of DORZO standard powder corresponding to 80, 100, and 120% of label claim had been added (standard addition method) mixed, and the powder was extracted and analyzed by running chromatograms in optimized mobile phase. These mixtures were analyzed by the proposed method. The experiment was performed in triplicate, and recovery (%) was calculated.
2.8.7. Analysis of Eye Drops
The contents of drug in eye drops were determined by the proposed method using the calibration curve.
2.8.8. Solution Stability
The solution stability of DORZO was carried out by leaving the test solution in tightly capped volumetric flasks at room temperature for 24 h and assayed at 6 h interval, against the freshly prepared standard solution. The % RSD of assay of DORZO was calculated for the study period during solution stability experiments.
3. Results and Discussion
In this work, analytical HPTLC method with UV detection was developed and validated for the determination of DORZO in bulk drug and pharmaceutical formulations. To prove the method was stability indicating, the drug was assayed in presence of its degradation products obtained under stress conditions like acidic, basic, neutral, and oxidative conditions, photodegradation, and thermal degradation.
3.1. Method Development and Optimization
The HPTLC procedure was optimized with a view to develop a suitable HPTLC method for the analysis of DORZO and its degradation products. Several trials were made by using different solvent systems containing nonpolar solvents and relatively polar solvents. Initially, chloroform and methanol were tried in different ratios. Finally, the mobile phase consisting chloroform:methanol (9.0 : 1.0 v/v) gave better resolution and sharper peaks with values of 0.28 ± 0.05 (Figure 2). Well-defined spots were obtained when the chamber was saturated with the mobile phase for 20 min. The analytical wavelength of 253 nm was chosen on the basis of the absorption spectrum recorded in the range of 200–400 nm.
Figure 2: Densitogram of standard dorzolamide hydrochloride (700 ng/spot).
3.2. Detection of Degradation Products
The drug solution refluxed with 1 M HCl at 60°C for 2 h. Degradation product was formed with of 0.33 in HPTLC (Figure 3). The drug solution in 0.2 M sodium hydroxide solution under reflux at 60°C showed degradation for 4.5 h associated with a major degradation product at of 0.32 (Figure 4), DORZO under neutral hydrolysis did not show degradants as the peak area remained constant which indicated drug stability under the conditions investigated.
Figure 3: Densitogram of the acid degradation product 300 ng/spot; condition: 1 M HCl at room temperature for 2 h; peak 1 (dorzolamide hydrochloride, : 0.29), peak 2 (degraded, : 0.33).
Figure 4: Densitogram of base-treated dorzolamide hydrochloride 300 ng/spot; condition: 0.2 M NaOH at 60°C for 4.5 h; peak 1 (dorzolamide hydrochloride, : 0.28), peak 2 (degraded, : 0.32).
The drug was unstable to oxidative degradation; 27.65% degradation was observed when it was reacted with 6% hydrogen peroxide at room temperature for 72 h. The chromatogram had degradation product peak at of 0.66 (Figure 5).
Figure 5: Densitogram of H2O2 (6%, reflux for 72 min) treated dorzolamide hydrochloride 700 ng/spot; peak 1 (degraded, : 0.28) and peak 2 (dorzolamide hydrochloride, : 0.66).
3.2.3. Dry and Wet Heat Degradation Product
There was no significant degradation of solid DORZO on exposure to dry heat at 50°C for 2 months, which indicated that drug was stable against thermal stress. However, the exposure of drug to 40°C/75% RH for 3 months resulted in slight degradation (less than 5%).
DORZO was degraded in photochemical degradation after exposing drug to a combination of white fluorescent and integrated near ultraviolet energy at 1.2 million lux hours and 200 watts hours/sq.mts, respectively, for 4 days forming single major degradation product at of 0.67 in HPTLC (Figure 6). The data of summary of degradation are listed in Table 8.
Figure 6: Densitogram of photodegradation (4 days) treated dorzolamide hydrochloride 700 ng/spot; peak 1 (degraded, : 0.26) and peak 2 (dorzolamide hydrochloride, 0.64).
3.3. Validation of the Chromatographic Method
Linearity was evaluated by analysis of working standard solutions of DORZO of five different concentrations. The range of linearity was from 100 to 500 ng/spot. The regression data obtained are represented in Table 1. The result shows that within the concentration range mentioned above, there was an excellent correlation between peak area and concentration of each drug.
Table 1: Linear regression data for the calibration curvesa.
The drug response was linear () over the concentration range between 100 to 500 ng/spot. The values of slope, intercept, and correlation coefficient were 2.073, 56.78, and 0.999 respectively.
The results of the repeatability and intermediate precision experiments are shown in Table 2. The developed methods were found to be precise, with RSD values for repeatability and intermediate precision <2%, as recommended by ICH guidelines. Separation of the drugs was found to be similar when analysis was performed on different chromatographic systems on different days.
Table 2: Repeatability and intermediate day precision of methodsa.
3.3.3. LOD and LOQ
The LOD and LOQ values were found to be 30 and 95 ng/spot.
The correlations between spectra acquired at the peak-start (S), peak-apex (M), and peak-end (E) positions of a spot were: (S,M) = 0.9992 and (M,E) = 0.9995. Good correlation () was also obtained between standard and sample spectra of DORZO. The specificity of the method is also apparent in Figure 3–6. In these figures, DORZO is completely separated from its degradation products. The peaks are sharp and clearly separated at baseline.
3.3.5. Robustness of the Method
The standard deviation of peak areas was calculated for each change of condition, and RSD was found to be <2%. Such low values of the RSD are indicative of the robustness of the method (Table 3).
Table 3: Robustness testing.
3.3.6. Solution Stability Studies
No additional peak was found in the densitogram of sample from solution stability. The results from solution stability experiments confirmed that standard solutions were stable up to 24 h for assay and related substances analysis as shown in Table 4.
Table 4: Stability of drugs in sample solutionsa.
3.3.7. Recovery Studies
Good recoveries of the DORZO were obtained at various added concentrations for Ocudor eye drops as shown in Table 5.
Table 5: Recovery studiesa.
3.3.8. Analysis of a Commercial Formulation
Experimental results of the amount of DORZO in eye drops, expressed as a percentage of label claims, were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present in eye drops. Commercially available eye drops were analyzed using the proposed procedure in Table 6.
Table 6: Applicability of the HPTLC method for the analysis of the pharmaceutical formulations.
The data of summary of validation parameters are listed in Table 7.
Table 7: Summary of validation parameters.
Table 8: Summary of stress study.
Stability-indicating HPTLC method was developed for DORZO and validated as per ICH guidelines. In this study, intrinsic stability of DORZO was established using various ICH-recommended stress conditions. The drug as such was very stable in solid form and in solution. In the latter case, unknown decomposition products were formed under stress condition. The drug was found to degrade more extensively in alkaline condition than in acidic condition. Mild degradation was also seen in oxidative stress conditions and photolytic degradation, but the drug was stable to thermal stress. The method was validated for parameters like linearity, precision, accuracy, specificity, robustness and so forth. and was also applied to marketed samples. Thus, the method can be employed for analysis of drug during stability studies.
Trusopt, 120279-96-1, 1cil, Trusopt (TN), Dorzolamide (DZA), Dorzolamide (INN), MK507
Molecular Weight:324.44004 g/mol
(4S,6S)-4-(ethylamino)-5,6-dihydro-6-methyl-4H- thieno[2,3-/?]thiopyran-2-sulfonamide 7,7-dioxide
Antiglaucoma Agents, OCULAR MEDICATIONS, Ophthalmic Drugs, Carbonic Anhydrase Inhibitors
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 an anti-glaucoma agent, and acts by decreasing the production of aqueous humour. It is optically applied in the form of a 2% eye drops.
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.
Dorzolamide hydrochloride is used to lower increased intraocular pressure in open-angle glaucoma and ocular hypertension.
It lowers IOP by about 20%.
Ocular stinging, burning, itching and bitter taste. it causes shallowing of the anterior chamber and leads to transient Myopia.
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.
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.
The dorzolamide hydrochloride product is prepared from the aminated intermediate of Formula IV by the following scheme.
 Preparation of dorzolamide hydrochloride product from the animated intermediate of Formula IV
 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.
 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).
 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).
 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).
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.,
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:
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.
(viii) (ix-a ) Trans and Cis (x)
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:
(xvi) (xvii ) Trans:Cis:: 95: 5 (xviii)
(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.
chlorinating agent, cyclinization
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.
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.
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
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).
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%
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.