Structural Correction and Process Improvement for ... - ACS Publications

Subscriber access provided by ALBRIGHT COLLEGE

Full Paper

Structural Correction and Process Improvement for Control of Critical Process Impurity of Ezetimibe Madhava Rao Mannam, Srimurugan Sankareswaran, venugopal Reddy Gaddam, Senthilkumar Natarajan, Rajasekhara Prasad Kottapalli, and Pramod Kumar Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00024 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Organic Process Research & Development

Structural Correction and Process Improvement for Control of Critical Process Impurity of Ezetimibe

Madhava Rao Mannam†,‡, Srimurugan Sankareswaran †*, Venugopal Reddy Gaddam†, Senthilkumar Natarajan†, Rajasekhara Prasad Kottapalli‡, and Pramod Kumar†* †Micro

Labs Ltd., API R&D Centre, Bommasandra-Jigini Link Road, KIADB INDL Area, Bommasandra, Bangalore 560105, Karnataka, India. ‡Department

of Chemistry, Koneru Lakshmaiah Education Foundation, Vaddeswaram –522 502, Andhra Pradesh, India

ABSTRACT A new process related impurity of ezetimibe is identified and characterized. The impurity is critical and common to most of the manufacturing routes of ezetimibe. Structural characterization using HMBC indicates the presence of a 6-membered ring rather than a 9membered ring as proposed by the innovator of ezetimibe. Prominently, the existing pharmacopoeial methods of ezetimibe are not capable of detecting this impurity. Control strategy is established by appropriate process control that is capable of purging the impurity to levels comfortably below the regulatory requirement. Formation of diastereomer impurity during the demonstration of scale-up batch under optimized condition is attributed to epimerization of ezetimibe induced by thermal degradation of the silylating agent. KEY WORDS: Ezetimibe, Process impurity, Improved process, Structure correction, Epimerization

ACS Paragon Plus Environment

Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION The control of pharmaceutical impurities is currently a critical and challenging issue to the pharmaceutical industry. Impurities in pharmaceuticals are unwanted components with unwanted pharmacological or toxicological effects that remain with the active pharmaceutical ingredients (APIs), or develop upon its aging and influence the efficacy and safety of the pharmaceutical products.1 Consequently, impurity profiling that describes detection, identification/structure elucidation and quantitative determination of impurities has become crucial as per various regulatory requirements.2 Knowing the structure of an impurity is essential for allowing assessment of its toxicological implications and for understanding its formation mechanisms, which is critical knowledge for improving the synthetic chemical process and optimizing the formulation.3 Recently regulatory agencies reported a major issue regarding the detection of a genotoxic impurity, NDMA (N-nitrosodimethylamine), and subsequently NDEA (Nnitrosodiethylamine), in valsartan, an API used to manufacture generic angiotensin receptor blockers (ARBs) that resulted in recalls from the market.4 Ezetimibe (marked as Zetia, Ezetrol or Ezedoc) is the first lipid-lowering drug that inhibits intestinal uptake of dietary and biliary cholesterol without affecting the absorption of fat-soluble nutrients.5 It may be used alone or together with statins for the treatment of primary hypercholesterolemia, homozygous sitosterolemia, homozygous familial hypercholesterolemia and mixed hyperlipidemia. Impurity profiling of ezetimibe is reported in a number of scientific articles that assist regulatory agencies in ensuring the availability of consistently high quality API in market.6 In an attempt to develop generic ezetimibe, following the most commonly adopted synthetic route, we observed the formation of a critical process impurity that was not disclosed in any of the prior art. The impurity is formed at the late stage of the process and carried forward to final API. More importantly, available pharmacopoeial methods for impurity profiling and assay determination using HPLC do not resolve this impurity from the main peak. A book chapter by Schering-Plough research institute discusses the same impurity formation during their product development.7 Structural elucidation of the impurity indicated that the correct structure of the impurity is different from the one reported in the article by ScheringPlough research institute.


Page 2 of 16

Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION Ezetimibe is made by a three stage process starting from 4-(4-fluorobenzoyl)butyric acid 2 as described earlier (Scheme 1).8 Attachment of chiral auxillary to carboxylic acid group of 2 was achieved in high yield without the necessity for protection/deprotection of aryl carbonyl group. This requires activation of carboxylic acid 2 using pivaloyl chloride followed by reaction with (S)-4-phenyl-2-oxazolidinone. Crystallization in methanol after acid-base work-up affords chiral auxillary tethered intermediate 3 in good quality and 80% isolation yield. O

O

O (i)

OH F

O

O NH

+

O

2

Ph

F

O

O

3

OH

O (iii)

N F

O

Ph

F

Ph

4

(S)

O N

(S)

O

(S)

O

(ii)

O

+

O

HO F

N

(S)

OH OTMS F

O

(iv) N H

(S)

(R)

N Ph

(S)

O

(S)

O

OTMS (v)

(R)

(S)

F

O

OH F

(vi) (S)

1

N

F

5

Scheme 1 Route of synthesis of ezetimibe: (i) TEA, Pivaloyl chloride, DMAP, DMF, Toluene, (ii) Con.H2SO4, NaHCO3, Methanol, 82% ; (iii) DCM, BH3.DMS, (R)-CBS, MeOH, H2O2, H2SO4, 98% ; (iv) DCM, DIPEA, TMSCl, TiCl4, Ti(OPr)4 , DCM, IPA/Methanol, aq. Tartaric acid, EtOAc, Na2CO3, 65% ; (v) DCM, BSA, TBAF, AcOH, citric acid, 85%; (vi) 2.5N H2SO4, IPA, Water, 95%. Asymmetric reduction of 3 using (R)-CBS/BDMS catalyst system affords (S)-alcohol 4 in high enantioselectivity. Subsequent silyl protection of 4 and its reaction with silyl protected imine 5 in the presence of mixed titanium catalyst generated in situ results in stereoselective aldol-type condensed product 5. The selectivity of desired anti isomer to undesired syn isomer was found in



the ratio 8:2, when performed the coupling reaction at -40oC. The selectivity and yield of the reaction strongly depends on the nature of titanium species generated in the reaction system. It was also noted that, the stereoselectivity was not improved further by performing the reaction at comparatively lower temperatures. Interestingly, the aldol-condensation product was isolated as free alcohol rather than protected trimethylsilyl derivative as reported by most of the manufacturers.9 This isolation strategy gives a stable intermediate as required by GMP and helps in eliminating the undesired diastereomer (syn isomer) completely during the isolation by filtration. The quality obtained was consistently above 99%. Final stage of synthesis is the cyclization of diol 5 to azetidinone under fluoride ion catalysis conditions reported in prior art. Having achieved high purity in the penultimate intermediate (5), the cyclization was expected to generate final API directly in high purity. However, in contrast to the expectation, this final stage proved to be troublesome and generated critical process impurities that are found to carryover to final API. Treatment of 5 with silylating agent, N,Obis(trimethylsilyl)acetamide resulted trisilylation of the molecule wherein both diol and amino groups are silylated as trimethylsilyl groups. Addition of fluoride ion induces cyclization of Nsilyl group to generate desired ezetimibe in the protected form. Acid-catalyzed deprotection of silyl groups using aqueous sulphuric acid in isopropyl alcohol followed by dilution with water precipitated ezetimibe as filterable solid. Among three chemical transformations (silyl group protection, cyclization and deprotection of silyl groups), only cyclization reaction could be monitored by HPLC. The inherent incompatibility of silyl derivatives with HPLC mobile phase conditions was a major challenge in development of analytical method. The reaction monitoring data indicated high conversions during cyclization reaction although the isolated yields were often lower. In order to understand the missing mass balance of reaction, complete precipitation of ezetimibe was achieved by addition of excess water to aqueous IPA reaction mass. The isolated yield was close to theoretical yield with HPLC purity over 99%. The material however gave lower assay against reference standard, when analyzed by pharmacopoeial HPLC method (100 min run time). No additional peaks could be observed by HPLC either in purity or assay method. Surprisingly, in-house assay method by HPLC developed with shorter run time (15 min) gave the clue for understanding the missing mass balance. A new peak well separated from main peak, identified as impurity-A and amounted to missing assay was observed during the analysis of the sample. LC-MS was immediate performed to gain more information on the impurity-A.


Page 4 of 16

Page 5 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Furthermore, attempts were made to isolate the impurity-A from enriched filtrate mother liquor employing column chromatography. The isolated impurity-A matched HPLC retention time and served as an analytical standard.

OH

O

H N

OH

OH

O N

F

O

N

HO

O F

(a)

N

O

NH O 6-Exo-Tet F

9-Exo-Tet F

O

N

O

OH

HO

HO (5) F

(b) F

Figure 1 Plausible structures of impurity-A with m/z 572 formed during cyclization reaction ESI-MS analysis of impurity-A showed a mass of m/z 572, identical to that of starting material 5. A different MS/MS pattern obtained during LC-MS analysis ruled out the possibility of impurity-A to be diastereomer of 5. Anticipating reorganization of 5 during the reaction, impurity disclosed by Schering-Plough research scientist for the same stage was compared.7 The article mentioned competitive cyclization involving endo-cyclic carbonyl group of oxazolidinone resulting in a 9-membered ring as impurity (Fig. 1a). Initially we assumed same structure to impurity-A and found that the final step of the process generated this impurity to the level as high as 20-30% and accounted for lower isolation yield. The formation of 9-membered cyclized impurity at significant levels was expected to be unusual on the basis of Baldwin’s rules for ring closure. Not many literatures are available that demonstrate facile 9-membered ring formation by 9-exo-tet pathway when more favored 4-exo-tet is possible. Characterization techniques were utilized to establish the structure of impurity-A. Preliminary NMR and MS analysis of impurity indicated the presence of ring structure formed by reorganization of 5 under experimental condition. HMBC together with COSY and HSQC was used to follow the carbon-carbon connectivity and understand the size of ring present in the structure of impurity. Interestingly, a triplet proton signal assigned to an exchangeable proton (confirmed by deuterium exchange with D2O as well as absence of carbon correlation in HSQC) correlated to methylene carbon C2´ in HMBC by a two bond coupling (marked in Fig. 2 by red arrow). Such a correlation was difficult to explain with 9-membered structure as the



exchangeable amide N-H proton gives a doublet correlating methylene carbon C2´ by a three bond coupling. Additionally, a three bond coupling observed for the same triplet proton signal with methine carbon C1´ in HMBC could not be correlated with reported structure (marked in Fig. 2 by blue arrow). In order to explain the missing spectral assignment, an alternative 6membered ring structure is proposed for impurity-A (Fig. 1b). A 6-membered ring is expected to form by the attack of nitrogen anion at the endo-cyclic carbonyl group followed by C-O bond cleavage rather than C-N bond.11 This revised structure with primary alcohol as leaving group explains all HMBC spectral behavior of the impurity. Since Baldwin’s rule for tetragonal system favors all 3 to 7-exo-tet process. Further, mass fragment analyses (Fig. 3) also support the favour of six membered ring structure, this re-assigned structure also explains the facile formation of impurity to the level of 20-30% during cyclization process.

OH

O N

F

N

C 1'

C2' OH

O

HO F

Figure 2: Zoomed HMBC spectrum of impurity-A. Arrows on structure indicates significant HMBC correlations. With HPLC method available at hand, optimization of process parameters and identification of purification methods to minimize and eliminate impurity-A was taken up. Various reaction parameters like nature of solvent, mode of addition of TBAF, quantity of TBAF and reaction temperatures were screened to identify the optimum condition for the control of impurity.


Page 6 of 16

Page 7 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


F

H+

HO O N

N

H+

F

O N

Ph

N O

O -H2O

HO

Ph

HO OH

OH F m/z: 555.26

F m/z: 573.26 Impurity-A

-H2O

HO

H+

F

-

O

Ph

N

N

Ph O

F

H+

O N

HO

NH F

O

m/z: 537.24

HO OH

-

F

F

m/z: 453.15 -H2O

Ph H+

O N

NH O

HO

F m/z: 435.19

Figure 3: MS spectra and fragmentation pathway of Impurity-A for structural confirmation DCM and toluene afford complete reaction conversion for silyl protection and cyclization. In both the cases, impurity-A was formed competitively although toluene formed only half the level compared to similar reaction performed in DCM (Table 1). Excess of TBAF was not beneficial in reducing the impurity. Dumping of solid TBAF to the reaction mass was adopted as it formed slurry in toluene. It was found that slow reaction rates at lower temperature resulted slightly



Page 8 of 16

elevated level of impurity-A compared to reaction performed at ambient temperature. Conversely, higher temperature accelerated reaction conversion and reduced impurity-A formation (Table 1, entry 21). Dilution was beneficial in retarding the undesired cyclization (Table 1, entry 27). Due to generation of diastereomer (vide infra) at higher temperatures, 2025oC with 14 volume dilution was identified as the best reaction condition for performing the cyclization reaction (Table 1, entry 18). Table 1 Screening and optimization of cyclization reaction condition Reaction Conditions Volume Reaction Equivalents of TBAF Solvent o (x) temperature ( C) (solvent) 1 Acetonitrile 7 40-45 10 mol% (solid) 2 TBME 7 40-45 10 mol% (solid) 3 n-Heptane 7 40-45 10 mol% (solid) 4 DCM 7 25-30 2.5 mol% (DCM) 5 DCM 7 25-30 2.5 mol% (toluene) 6 DCM 7 25-30 5 mol% (toluene) 7 DCM 7 25-30 10 mol% (toluene) 8 DCM 7 30-35 2.5 mol% (DCM) 9 Toluene 7 40-45 10 mol% (solid) 10 Toluene 7 40-45 10 mol% (solid) 11 Toluene 7 50-55 10 mol% (solid) 12 Toluene 7 60-65 2.5 mol% (solid) 13 Toluene 7 60-65 10 mol% (solid) 14 Toluene 7 80-85 2.5 mol% (solid) 15 Toluene 7 80-85 10 mol% (solid) 16 Toluene 12 20-25 2.5 mol% (solid) 17 Toluene 12 50-55 2.5 mol% (solid) 18 Toluene 14 20-25 2.5 mol% (solid) 19 Toluene 14 20-25 2.5 mol% (solid in lots) 20 Toluene 14 20-25 2.5 mol% (DCM) 21 Toluene 14 60-65 2.5 mol% (solid) 22 Toluene 15 0-5 2.5 mol% (solid) 23 Toluene 15 10-15 2.5 mol% (solid) 24 Toluene 15 25-30 2.5 mol% (solid) 25 Toluene 15 60-65 2.5 mol% (solid) 26 Toluene 15 95-100 2.5 mol% (solid) 27 Toluene 20 20-25 2.5 mol% (solid) * Incomplete reaction and insignificant conversion observed. Entry

Impurity-A (%) --* --* --* 13.9 15.32 14.15 13.49 18.73 7.41 7.41 7.05 6.46 7.54 5.4 6.06 5.90 4.45 5.18 5.67 5.78 4.13 7.73 6.47 5.06 3.56 4.14 3.09

Optimization study concluded that process parameters could not completely eliminate the impurity-A formation and the best condition restricts impurity-A formation to the level of 3-5%.


Page 9 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Attention was then focused on downstream isolation procedure to establish strong process control on the purge of impurity. The behavior of impurity-A removal was found to be critical with respect to crystallization conditions adopted. Due to high solubility of ezetimibe in organic solvents, it can be isolated only by precipitation technique induced by addition of an anti-solvent like water. The most successful condition, as followed by most of the prior art disclosure, was using IPA as solvent and water as anti-solvent. Table-2 lists the risk-assessment experiments performed to identify the robust process condition for isolating API in high purity. Slow and dropwise addition of 5 volumes of water to reaction mass dissolved in 5 volumes of IPA affords good yield with consistently high purity devoid of impurity-A (Table 2, entry 5). The identified crystallization condition is capable of removing impurity-A as high as 10%. Table 2 Optimization of crystallization solvent for effective removal of impurity-A

Entry 1 2 3 4 5 6 7 8 9 11 12

Impurity-A (%) content in Purification Conditions crude Ezetimibe 2.56 10 volumes IPA & 8 volumes water added dropwise 2.56 10 volumes IPA & 8.5 volumes water added dropwise 2.56 10 volumes IPA & 10 volumes water added dropwise 2.83 5 volumes IPA & 5 volumes water dumped 2.83 5 volumes IPA & 5 volumes water added dropwise 2.83 5 volumes IPA & 7.5 volumes water added dropwise 2.83 5 volumes IPA & 8 volumes water added dropwise 2.83 5 volumes IPA & 10 volumes water added dropwise 4.52 5 volumes IPA & 12 volumes water added dropwise 5.24 0.5% IPA in water 6 volumes 2.58 5 volumes Methanol & 5 volumes water


Yield (%)

Impurity-A (%) content in Ezetimibe

85.0

Not Detected

88.0

Not Detected

90.0

Not Detected

94.0

0.06

94.0

Not Detected

95.0

0.07

96.0

0.08

97.0

0.23

98.0

3.66

97.0 82.0

4.79 0.06


Page 10 of 16

The optimized process was transferred to production unit for execution of GMP batches. First scale-up batch displayed performance similar to R&D batches with respect to level of impurityA. Unexpectedly, the batch generated a chiral SSS diastereomer impurity to the level of 2.1% which was never observed in lab batches. A detailed investigation was immediately carried out to understand the root cause for the formation of chiral impurity. It was evident that, desired product underwent partial epimerization under the reaction condition to generate the epimer. Importantly, under identical operation conditions, R&D batches did not display any epimerization. The only difference observed in the plant batch was the additional hold up time of reaction mass at 50-55oC till the release of reaction monitoring results from QC. Upon reviewing various factors, we finally identified that the silylating agent BSA to be the reason for epimerization during the scale-up batch. BSA is reported to be thermally unstable and generate acetonitrile as the degradation product.10 During the additional hold up time (Table 3) at higher temperature, BSA, (6.0 mol equivalents) underwent gradual degradation to generate acetonitrile in the reaction mass. To understand the impact of acetonitrile in accelerating epimerization process, lab batch was performed and spiked with 8% acetonitrile before the addition of TBAF. The batch unexpectedly generated SSS diastereomer (4.6%) in higher amount. We therefore concluded, acetonitrile formed by degradation of BSA provided solvent polarity favoring epimerization process. The identification of valid root cause was further verified by conducting second trial batch of same size with no hold up time (cooling the reaction mass after the maintenance time). As expected no epimerization was observed and plant batch trend matched lab trend for the final stage. Table 3 Reaction mass holding time study for formation SSS isomer S.No

1 2 3 4 5

Reaction mass maintenance time (hr) at 52.52.5 °C 1.0 1.0 1.0 1.0 1.0

Reaction mass holding time (hr) at 52.52.5 °C 0 1 2 3 4

SSS isomer content in Reaction monitoring 1.02 1.37 1.52 2.10 3.27


SSS isomer content after isolation

SSS isomer after purification

0.18 0.35 0.44 0.62 0.77

0.04 0.07 0.13 0.19 0.25

Page 11 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION All materials were purchased from commercial suppliers. Unless specified otherwise, all reagents and solvents were used as supplied by manufacturers. Melting points were determined by open air capillary with Buchi M-565 and are uncorrected. IR spectra of samples were recorded on Shimadzu IR Affinity-I FT-IR spectrophotometer. 1H NMR spectra (400 MHz) and 13CNMR

spectra (100 MHz) were recorded in CDCl3, DMSO-d6, on Bruker 400 MHz NMR

(ASCEND, 5 mm PABBO) instrument and mass spectra were determined on Velos Pro Ion Trap Mass Spectrophotometer (Thermo Scientific) (3R,4S)-1-(4-Fluorophenyl)-3-[(S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) (1) Compound 5 (80g, 0.139 mol) was added to toluene (1120 mL) under nitrogen atmosphere in a 3-neck RB flask to obtain a slurry. The reaction mass was heated to 50-55oC and BSA (170.5 g, 0.838 mol) was added slowly over a period of 1.0-1.5 h under nitrogen atmosphere. The reaction was maintained for 1 h at the same temperature, cooled down to room temperature and monitored for reaction completion. Sodium sulfate (5.95 g, 0.041 mol) was added to the reaction mass and cooled down to 20-25oC. TBAF.3H2O (1.1 g, ) was added to the reaction mass as solid and stirred for a period of 1 h under nitrogen atmosphere. After the completion of reaction, citric acid solution (320 mL) was added slowly to the reaction mass and the layers were separated. The toluene layer was washed with water (160 mL) and distilled completely under vacuum to obtain viscous oil. IPA (400 mL) was added to the reaction mass followed by slow addition of dilute sulphuric acid (40 mL). The reaction mass was maintained at the same temperature for 1 h and then treated with slow addition of water (640 mL). The solid obtained was maintained for a period of 1 h, filtered and washed with aqueous IPA. Wet solid was further dried under vacuum to afford 46 g (81 %) of 1 as off-white to white solid. Characterization data of 1: MR: 164-166 1H

NMR (400 MHz, DMSO-D6) δ 9.54, (s, -OH), 7.29-7.33 (m, 2H), 7.20-7.24 (m, 4H),

7.09-7.16 (m, 4H), 6.75-6.78 (d, J=8.4Hz, 2H), 5.29-5.30 (d, J=4.8 Hz, 1H), 4.80-4.81



(d, J=2.4Hz, 1H), 4.48-4.52 (m, 1H), 3.07-3.10 (m, 1H), 1.71-1.87 (m, 4H);

Page 12 of 16

13C

NMR

(400 MHz, DMSO-D6) δ 167.41, 159.91-162.31 (d, J=240 Hz), 156.89-159.28 (d,

J=238.7 Hz), 157.50, 142.18-142.21 (d, J=2.8Hz), 134.05-134.07 (d, J=2.5Hz), 127.96, 127.54,127.62, 118.25-118.33 (d, J=7.9Hz), 115.79, 115.74-115.96 (d, 22.5 Hz), 114.61-114.82 (d, J=20.9Hz), 71.16, 59.69, 59.50, 36.45, 24.60 ; IR (KBr, cm-1): 3263, 2963, 2912, 1716, 1614, 1592, 1404, 1220, 1157, 1066; ESI-MS m/z 408.38.

Purification of ezetimibe Crude ezetimibe (100 g) having impurity-A 2.83% was dissolved in IPA (500 mL) to obtain clear solution at 25-30oC. Water (500 mL) was added slowly to the reaction mass during which solid was precipitated. The solid obtained was maintained for a period of 1 h, filtered and washed with 1:1 aqueous IPA. Wet solid was further dried under vacuum to afford 94 g (94 %) of pure ezetimibe as off-white to white anhydrous crystalline solid and found impurity-A not detected (5R,6S)-1-(4-Fluorophenyl)-5-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-3-[(1S)-2hydroxy-1-phenylethyl]-6-(4-hydroxyphenyl)-1,3-diazinane-2,4-dione (Impurity-A) Filtrate mother liquor obtained during the isolation of 6 was concentrated and extracted with DCM. The DCM layer was distilled under vacuum and the residue was chromatographed on silica gel and the pure fractions corresponding to impurity-A are collected and concentrated to generate a analytical standard of impurity-A. Characterization data of Impurity-A: MR: 144.7145.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, -OH), 7.12-7.33 (m, 15H), 6.66-6.68 (d, J=8.4 Hz, 2H), 5.73-5.77 (t, J=7.2Hz, 1H), 5.37-5.38 (d, J=4.4 Hz, 1H), 5.10-5.13 (t,

J=5.2Hz, 1H), 4.79(s, 1H), 4.57-4.58 (m, 1H), 4.03-4.22 (m, 1H), 2.68-2.69 (m, 1H), 1.62-1.81(m, 1H);

13C

NMR (100MHz, DMSO-d6) δ 171.42, 159.88-162.28(d, J=240

Hz), 159.07-161.49 (d, J=242 Hz), 156.99, 151.75, 142.06-142.09 (d, J=2.8 Hz),


Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


138.83, 138.17-138.20 (d, J=2.8Hz), 128.69, 128.91, 128.02, 127.37, 127.56, 126.94, 115.59-115.81 (d, J=22.5 Hz), 115.42, 114.56-114.77 (d, J=20.9 Hz), 71.26, 62.35, 60.68, 57.02, 49.98, 36.50, 27.46; IR (KBr, cm-1): 3391, 3069, 2949, 1713, 1659, 1603, 1435, 1435, 1220, 1155, 1045; ESI-MS m/z 573.28

CONCLUSION A critical process impurity of ezetimibe formed at the last chemical stage is identified and characterized. Structural characterization of impurity indicated 6-membered ring structure rather than 9-membered ring structure proposed in prior-art. Detection method by HPLC was developed as the impurity does not show specificity in the existing pharmacopoeia methods. Careful selection of process parameters minimize the competitive side reaction which in combination with improved isolation condition leads to complete purge of impurity to levels much below the regulatory requirement.

ASSOCIATED CONTENT Supporting information Copies of IR, ESI-MS, 1H-NMR and 13C-NMR of compounds 2, 5, 1 and impurity impurity-A

AUTHOR INFORMATION *Corresponding Authors Sankareswaran Srimurugan E-mail: [email protected], Tel.: 08110-415647, ext. 274

Pramod Kumar



E-mail: [email protected], Tel.: 08110-415647, ext. 245

Conflict of interest The author declared no conflict of interest.

ACKNOWLEDGMENT

The authors are thankful to the management of Micro Labs Ltd., API Division Centre, ML-27, Bangalore. We also wish to thank Mr. Vijay Kumar and Mr. Mohit Jain for their valuable analytical support and cooperation.

REFERENCES: 1 International Conference On Harmonization Guidelines, Q3A (R2): Impurities In New Drug Substances (Revised Guideline) 2006. 2 Warad, T.A., Bhusnure, O.G., Gholve S.B., “Impurity profile of pharmaceuticals ingradient,” J. Pharm. Res. 2016, 10, 523. 3 (a) Qiu, Fenghe, Daniel L. Norwood., “Identification of pharmaceutical impurities,” J. Liq. Chromatogr. Relat. Technol. 2007, 30, 877. (b) Görög, Sándor., “Drug safety, drug quality, drug analysis,” J. Pharm. Biomed. Anal. 2008, 48, 247. 4 FDA Press Release, “FDA announces voluntary recall of several medicines containing valsartan following detection of an impurity,” fda.gov, July 17, 2018.


Page 14 of 16

Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 (a) Bays, Harold., “Ezetimibe” Expert Opin. Investig. Drugs 2002, 11, 1587. (b) Van Heek, M., H. Davis., “Pharmacology of ezetimibe,” Eur Heart. J Suppl. 2002, 4, J5-J8. 6 (a) Gajjar, Anuradha K., Vishal D. Shah., “Isolation and structure elucidation of major alkaline degradant of Ezetimibe,” J. Pharm. Biomed. Anal. 2011, 55, 225. (b) Raman, Bhanu, Brajesh A. Sharma, Rahul Butala, Pradeep D. Ghugare, Ashok Kumar., “Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR,” J. Pharm. Biomed. Anal. 2010, 52, 73. (c) Guntupalli, Srikanth, Uttam Kumar Ray, N. Murali, P. Badrinadh Gupta, Vundavilli Jagadeesh Kumar, D. Satheesh, Aminul Islam., “Identification, isolation and characterization of process related impurities in ezetimibe,” J. Pharm. Biomed. Anal. 2014, 88, 385. (d) Ren, Yun, RenJun Li, Yong Deng, Mei Guan, Yong Wu, Li Hai., “First synthesis and characterization of SRR/RSS-ezetimibe,” Tetrahedron Lett. 2013, 54, 6443. (e) Ren, Yun, Yan-Jun Duan, Ren-Jun Li, Yong Deng, Li Hai, Yong Wu., “First synthesis and characterization of key stereoisomers related to ezetimibe,” Chin. Chem. Lett. 2014, 25, 1157. 7 Thiruvengadam, T. K., Sudhakar, A. R., Wu, G. Process Chemistry in the Pharmaceutical Industry, Vol.1, chapter 13, 221. 8 Thiruvengadam, T. K., Fu, X., Tann, C, H., McAllister, T. L., Chiu, J. S., Colon, C., “Process for the synthesis of azetidinones,” US6207822 B1, Mar.27, 2001 9 (a) Hesk, D., G. Bignan, J. Lee, J. Yang, K. Voronin, C. Magatti, P. McNamara., “Synthesis of 3H, 14C and 13C6 labelled Sch 58235,” J. Labelled comp. Radiopharm. 2002, 45, 145. (b) Yong, W., Lizhong, L., Zhiqiang, S., Jianqiang, Y., Jin, W., Heyun, Y., “Molecular sieve catalytic synthesis method of ezetimibe intermediate,” CN105622661, Jun. 1, 2016 (c) Baoquin, W., Lizhong, L., Zhiqiang, S., Jianqiang, Y., Jin, W., Heyun, Y., “Slurry bed continuous production method of ezetimibe intermediate,” CN105566374 , May.11, 2016 10 (a) Smith, Edgar D., “Gas Chromatographic Analysis of Bis (Trimethylsilyl) Acetamide,” J. Chromatogr. Sci. 1972, 10, 34. (b) Shinohara, T., Kudo, M., Ueno, S., Maruyama, M., N,O-Bis(Trimethylsilyl) Acetamide stabilization,” US5264601, Nov.23, 1993.



Page 16 of 16

11 Please refer supporting information for detailed assignment and spectral characterization of Impurity-A. GRAPHICAL ABSTRACT F

OH OH

NH O (S)

(R)

RO

(S)

N

O

O

(S)

R = Silyl, Benzyl protecting groups

F

O Ezetimibe OH

OR F

OH

OH F

O (S)

O

(S)

HO

(R)

(S)

Cyclization/Deprotection

(R) (S)

N

(S)

NH (S)

F

(R) (S)

N

N O

HO

O

O F Impurity-Reported structure

F Impurity- Reassigned structure


(S)

N

F

Structural Correction and Process Improvement for ... - ACS Publications

Recommend Documents