Identification, Synthesis, and Control of Process-Related Impurities in

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Identification, Synthesis, and Control of Process-Related Impurities in Antipsychotic Drug Substance Brexpiprazole Rahul Tyagi, Harnam Singh, Jagat Singh, Himanshu Arora, Vijayalaxmi Yelmeli, Mohit Jain, Sathyanarayana Girigani, and Pramod Kumar Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00074 • Publication Date (Web): 05 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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Organic Process Research & Development

Identification, Synthesis, and Control of ProcessRelated Impurities in Antipsychotic Drug Substance Brexpiprazole Rahul Tyagi,* Harnam Singh, Jagat Singh, Himanshu Arora, Vijayalaxmi Yelmeli, Mohit Jain Sathyanarayana Girigani and Pramod Kumar* Micro Labs Ltd., Chemical Research Department, API-R&D Centre, Bommasandra-Jigini Link Road, KIADB INDL Area, Bommasandra, Bangalore 560105, Karnataka, India

ABSTRACT This article describes the identification, synthesis, and control of several unknown related substances present in the form of critical impurities which were observed during the process development of antipsychotic drug substance Brexpiprazole (BRX). On the basis of liquid chromatography-mass spectrometry (LC-MS) evidence of impurities and analysis of the employed synthetic route of BRX, the structures of unknown impurities were proposed. Many critical impurities were synthesized and their chemical structures were established by using different spectroscopy techniques. To the best of our knowledge, this is the first report where several new impurities were recognized, addressed and controlled effectively while developing a robust and efficient process for the BRX drug substance.

1 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

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KEY WORDS: Brexpiprazole, antipsychotic, Process development, impurities, active pharmaceutical ingredients, critical impurities INTRODUCTION Brexpiprazole (7-{4-[4-(1-Benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one) is a dopamine D2 receptor partial agonist drug, approved by the United States Food and Drug Administration (US FDA) on 10 July 2015. It is used for the treatment of schizophrenia and for adjunctive therapy to antidepressants for the treatment of the major depressive disorder (MDD). The

drug

was

by Otsuka and Lundbeck,

developed

and

it

is

available

as

REXULTI® (Brexpiprazole) tablets for oral administration.1

N O

N H

O

S

N

Figure-1: Brexpiprazole (BRX)

It is important to develop commercially viable processes for drug substance manufacture to allow greater and more affordable access in the health care sector. Regarding the process development of drug substances, it is essential to know the origin and method of control of any unwanted substances present in it. The limit should be controlled under the threshold of toxicological concern (TTC) for the purpose of ensuring safety and efficacy of the drug and to meet the requirements of various drug regulatory agencies.2,3 The impurities in drug substances mostly come from starting substrates, reagents, solvents and side reactions of the synthetic route employed. Therefore, assessment and control of the undesired substances is an essential aspect of the drug development journey, with special consideration of patient health risk.4,5 Herein, we report the isolation/synthesis and characterization of process related critical impurities (more difficult to control under desired regulatory limits) of BRX drug substance in 2 ACS Paragon Plus Environment

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order to evaluate their origin/fate and thereafter their control strategies in the developed process as per International Council for Harmonisation (ICH) guidelines. To the best of our knowledge, there is no easy access to these impurities. There are scattered literature reports, predominantly disclosing the synthesis of BRX in patents.6 However, no synthetic details of impurities have been reported. In this context, this is the first report where many critical process impurities of the BRX drug substance have been addressed along with their control strategies in the process.

RESULTS AND DISCUSSION We desired to develop a commercially viable and robust process for producing the Brexpiprazole drug substance, while conforming to drug regulatory requirements. In this process, we observed that there are many synthetic protocols reported in the prior art literature for the preparation of BRX drug substance. 7 However, most of the synthetic routes were found to have primarily academic value rather than commercial value as evaluated after our early stage feasibility studies in the lab. Further, the innovator protocol was found to be very promising and was considered further for the process development of BRX.6b,8,9 The innovator route has employed the use of three major key starting materials, namely: 7-Hydroxy-1H- quinolin-2-one (BRX-S1), 1-Bromo4-chlorobutane (BRX-S2), and 1‐(1‐Benzothiophen‐4‐yl)piperazine hydrochloride (BRX-S3). The synthetic pathways involve a two-step process as shown in Scheme-1. It involves the condensation of four-carbon chain i.e. BRX-S2 with BRX-S1 by nucleophilic substitution reaction to afford 7-(4-chlorobutoxy)-1H-quinolin-2-one (BRX-1) as an isolated intermediate. The resulting BRX-1 reacts with BRX-S3 by nucleophilic substitution reaction and leads to BRX crude compound BRX-2 (Scheme-1).



HO BRX-S1 [161.16]

N H

O

+

a b

Cl

Br

Cl

Page 4 of 25

O

BRX-S2 [171.46]

BRX-1 [251.71]

N H

O

S

S N

NH HCl

BRX-S3 [254.78]

N

d N

c

BRX-2 [433.57]

S

O

N H

O

S

e

N N HCl

O BRX-3 [470.03]

N H

O

N N BRX [433.57]

O

N H

O

Scheme-1: The innovator synthetic route for the preparation of Brexpiprazole (BRX) a) K2CO3, DMF, H2O, 30-40 C, 5 h, 79 % yield; b) Purification in MeOH, Reflux, 79 % yield; c) K2CO3, DMF, KI, 90-100 oC, 2 h d) EtOH, Acetic acid, reflux, Conc. HCl e) EtOH, H2O, NaOH,70 C, 83 % yield. At the outset of process development, BRX-1, and BRX active pharmaceutical ingredient (API) were obtained in 75-78% and 83-85 % isolated yield respectively by following the experimental and purification procedures as reported in the innovator’s literature reports.8 Even though the innovator process was quite capable to produce BRX effectively in lab-scale studies, it nevertheless resulted in formation of a number of undesired by-products (more than the threshold limits as per ICH guidelines) as analyzed by HPLC. Thus, there was an urgency of process improvement and control of unwanted substances under the desired limits. Further investigation was started for identifying the tentative structure of these process-related impurities of BRX-1 and BRX-2 in each chemical transformation by LC-MS technique for qualitative analysis. The LC-MS analysis data of BRX-1 (crude) sample revealed approximately eleven undesired substances at retention times (RT in minutes) 4.20, 6.45, 14.79, 17.14, 24.73, 25.15, 27.14 27.67, 29.85 31.48 and 31.85 having [M+H]+ m/z 162.10, 234.09, 377.19, 296.16, 467.17, 467.16, 342.17, 386.12, 467.22, 342.19 and 386.11 respectively (Table-1). 4 ACS Paragon Plus Environment

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Table-1: BRX-1 stage impurities LC-MS (RT/RRT) and LC-MS [M+H]+ (m/z) with proposed chemical structures code S.No.

LC-MS (~RT)

LC-MS (~RRT)

LC-MS [M+H]+ (m/z)

Proposed Chemical Structure (Code) *

1.

4.20

0.27

162.11

BRX-S1

2.

6.45

0.41

234.09

BRX-1-H

3.

14.79

0.93

377.19

BRX-1-I

4.

17.14

1.08

296.15

BRX-1-G

5.

24.73

1.56

467.17

BRX-1-J/BRX-1-K/BRX-1-L/BRX-1-M

6.

25.15

1.59

467.16


7.

27.14

1.71

342.11

BRX-1-A/BRX-1-B

8.

27.67

1.75

386.12

BRX-1-C/BRX-1-D/BRX-1-E/BRX-1-F

9.

29.85

1.89

467.22


10.

31.48

1.99

342.15

BRX-1-A/BRX-1-B

11.

31.85

2.01

386.15

BRX-1-C/BRX-1-D/BRX-1-E/BRX-1-F

*For chemical structures, consider Figure-2

Whereas LC-MS analysis of BRX-2 revealed eight impurities at RT 5.38, 7.25, 12.96, 13.64, 15.82, 17.38, 20.13 and 25.42 showing mass [M+H]+ of m/z 219.09, 466.16, 450.23, 649.34, 706.42, 649.32, 706.42 and 649.39 respectively (Table-2) and one carry over impurity of BRX-1 stage i.e. BRX-1-I at RT 14.86 of m/z 377.13 (LC-MS data is available in Supporting Information).



Page 6 of 25

Table-2: BRX-2 stage impurities LC-MS (RT/RRT) and LC-MS [M+H]+ (m/z) with proposed chemical structures code S.No.

LC-MS (~RT)

LC-MS (~RRT)

LC-MS [M+H]+ (m/z)

Proposed chemical structure(Code)*

1.

5.38

0.47

219.08

BRX-S3

2.

7.25

0.63

466.16

BRX-B

3.

12.96

1.13

450.25

BRX-A

4.

13.64

1.19

649.32

BRX-648A/BRX-648B/BRX-648C/BRX648D

5.

15.82

1.38

706.42

BRX-705A/BRX-705B

6.

17.38

1.52

649.32


7.

20.13

1.76

706.42

BRX-705A/BRX-705B

8.

25.42

2.22

649.39


*For chemical structures, consider Figure-2

Considering the HPLC, LC-MS results and process route for BRX (Scheme-1), the following structures were envisaged as shown in Figure-2.

Cl

O

N

Cl

O

O

N

O

Cl

Cl

O

N

Br

O

Cl

BRX-1-B [341.09]

BRX-1-A [341.09]

Br

O

N

Cl

O

O

N

O

BRX-1-C [385.04]

Br

O

BRX-1-D [385.04]

N H

O

BRX-1-G10 [295.02]

BRX-1-F [385.04]

BRX-1-E [385.04]

Br

O

Cl

O

Br

Cl

O

N

N H

O

OH

BRX-1-H11 [233.10]

O

N H

O

O

H N

O

6c

BRX-1-I [376.14]


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H N

O

O

N

O

O

H N

O

Cl

O

N

O

N

O

O

O

H N

O

Cl

O

Cl

BRX-1-K [466.16]

BRX-1-J [466.16] Cl

N

O

BRX-1-L [466.16]

O

N

+

O

H N

O

N H

O

S

N O

O

BRX-A [449.17]

BRX-1-M [466.16]

O

-

S

S

+

+

O

N H

N

S

N O

O

N

N N

O

BRX-B [465.17]

N

N

O

BRX-705A [705.31] S N O

N

S O

N

N

O

S

N

N

O

N

O

N

O

H N

N

O

H N

N

O

BRX-705B [705.31]

S

N

O

O

BRX-648B [648.27]

BRX-648A [648.27] S

O

N

O

O

H N

O

N N

N

N

O

O

N

O

S

BRX-648C [648.27]

H N

O

BRX-648D [648.27]

Figure-2: Proposed structures for process-related impurities of BRX by LC-MS Further, a systematic study was initiated to investigate the origin of each impurity in the BRX route of synthesis (Scheme-1) along with their control in the process to produce BRX (API) which meets quality standards. Many impurities were isolated, identified, synthesized (Scheme-2 & 3) and characterized for this purpose. To know the origin and fate of each impurity, the obtained samples of each stage of BRX process were examined by HPLC (Table-3 & Table-4) for quantitative analysis under the optimized analytical method. 7 ACS Paragon Plus Environment


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Table-3: HPLC analysis report of crude and purified samples for BRX-1 intermediate (As per innovator process) Sample

BRX-1-I RRT 0.81

BRX-1 2.59 % (Crude) BRX-1 0.42 % (Purified) UN: Unknown

BRX-1-G RRT 1.07

UN m/z 466 RRT 1.23

UN UN m/z 466 BRX-1-B m/z 466 RRT RRT 1.47 RRT 1.28 1.49

4.08 %

0.28 %

0.53 %

3.07 %

5.23 %

0.19 %

0.27 %

0.24 %

UN m/z 466 RRT 1.50

BRX-1-A RRT 1.72

UN RRT 1.77

0.23 %

0.37 %

3.18 %

0.43 %

0.01 %

0.15 %

0.27 %

0.02 %

Table-4: HPLC analysis report of crude and purified samples for BRX (As per innovator process): Sample

BRX-S3 RRT 0.66

BRX-1-I RRT 0.93

BRX-1 RRT 1.15

BRX-648B RRT 1.23

BRX-2 0.26 % 0.35 % 0.08 % 0.22 % (Crude) BRX ND 0.03 % ND 0.18 % (API) UNSHI*: Unknown Single Highest Impurity (for information)

BRX-705B RRT 1.34

BRX-705A RRT 1.41

BRX-648A RRT 1.43

UNSHI * RRT 1.11

0.31 %

0.71 %

0.57 %

0.93 %

0.12 %

0.27 %

0.26 %

0.01 %

In BRX-1 (crude) sample analysis, several impurities were observed such as BRX-1-I, BRX-1G, BRX-1-B, BRX-1-A along with four other unknown isomeric impurities of same molecular mass peak m/z 466 (Table-3). Simultaneously, the HPLC analysis of BRX-2 showed four critical impurities which were synthesized, characterized and identified as BRX-705A, BRX-705B, BRX-648A and BRX-648B (Table-4) and which couldn’t be controlled under ICH limits even after multiple purification attempts, thus leading us to focus on various reaction parameters studies to control them. Brief process optimization and control strategy details for process impurities formation: During process optimization studies for stage-1, it was understood and thereafter established that the stoichiometric ratio of BRX-S2 and K2CO3 with respect to starting material BRX-S1 is critical to control BRX-1 intermediate impurities. It was observed that the impurity BRX-1-I was the most critical undesired adduct, as it carried all the way to BRX (API) and was resistant to several purification attempts by different organic solvents. We accomplished this critical 8 ACS Paragon Plus Environment

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impurity contamination limit (< 0.1 %) by controlling its formation in step-1 with the use of 3.0 mole equivalents of BRX-S2 (in the process patent, 2.0 mole equivalents of BRX-S2 were used). However, the formation of non-polar impurities BRX-1-A and BRX-1-Bwas enriched up to 2.5% due to the nucleophilic substitution reaction at other nucleophilic sites of BRX-S1. Thereafter, both the impurities were controlled to the level of 0.1% with the implementation of ethyl acetate (against methanol in the process patent) purification. There were many isomeric impurities observed at stage-1, predominantly BRX-1-(C-F) and BRX-1-(J-M) as shown in Figure-2. Even these byproducts were well controlled under the limits of not more than 0.1% up to 6.0 hours reaction time, it amplified sharply by extending the reaction time as observed during inline reaction monitoring. These isomeric impurities were anticipated to turn into critical impurities in stage-2 by reacting with BRX-S3. To control them, the parameter of K2CO3 mole equivalents was studied and 1.1 mole equivalents (against 1.0 mole equivalents in the process patent) with respect to BRX-S1 achieved the best conversion to BRX-1 along with the controlled formation of isomeric impurities, irrespective of reaction time. Further their limit was controlled under 0.1% (except BRX-1-I and BRX-1-G) by introducing ethyl acetate purification at this stage. The impurity BRX-1-G was also observed to be amplified up to 5% with extended reaction time and didn’t washout in the purification by using ethyl acetate. However, BRX-1-G was effectively converted to BRX-2 in next stage of the process thus not harming the yield and quality of the drug substance. On the basis of the above studies and results, the precise control of mole equivalents of K2CO3 was well understood for the control of these critical impurities. Specifically, the use of 1.0 mole equivalent or less was not sufficient to achieve complete consumption of BRX-S1. However, several undesired impurities were formed (2-3 fold w.r.t to established limits) when K2CO3 was used at more than 1.2 mole equivalents in the process. With the optimized process parameters, BRX-1 increase of 10-12% yield from the existing patent process was achieved, along with control of contaminant of BRX-1-I to the level of not more than 0.8%. The HPLC analytical results for first stage samples with the improved optimized process are shown in Table-5 (BRX-1 crude and purified).



Page 10 of 25

Table-5: HPLC analysis report of crude and purified samples for BRX-1 intermediate (As per optimized process) Sample

BRX-1-I

BRX-1-G

BRX-1-B

BRX-1-A

UNSHI*

BRX-1 (Crude)

0.50 %

3.36 %

2.11 %

1.34 %

0.13 %

BRX-1 (Purified)

0.64 %

3.51 %

0.04 %

0.08 %

0.03 %

UNSHI*: Unknown Single Highest Impurity (for information)

With these changes, the quality/purity of isolated BRX-2 was improved significantly but still carrying critical impurities named BRX-705A, BRX-705B, BRX-648A, and BRX-648B within the limits of < 0.15%. These impurities (except BRX-648A) were controlled well within a limit of < 0.05% by introducing and optimizing the first purification of BRX-2 in a mixture of dichloromethane/ethyl acetate. To control the impurities like BRX-648A and BRX-1-I, one more purification (acid-base treatment) was employed which limited them to less than 0.05% (Table6) in the process. Accordingly, the desired quality of BRX (API) could be achieved consistently with purity above 99.8% and isolated yield 84%.

Table-6: HPLC analysis report of crude and purified samples for BRX (As per improved optimized process): Sample

BRX-S3

BRX-1-I

BRX-1

BRX648B

BRX-2 (Crude) 0.48 0.21 0.62 0.02 BRX-2(purified) 0.05 0.12 0.19 0.02 BRX (API) purified) ND 0.01 0.01 0.02 UNSHI*: Unknown Single Highest Impurity (for information)

BRX705B

BRX705A

BRX648A

UNSHI*

0.02 0.01 0.01

0.09 0.04 0.02

0.07 0.05 0.01

0.42 0.08 0.02

Justification of Impurities Formation in Process: The stage-1 impurities named BRX-1-(A-G) may be observed in the process due to overalkylation/halogen-exchange of BRX-1. The use of excess moles of BRX-S2 and in-situ generated KBr in the reaction at this stage are mainly responsible for these impurities. The impurity BRX-1-H may be formed due to nucleophilic substitution reaction of BRX-1-G with water. The BRX-1-I impurity is well known in the literature and it is known to form due to nucleophilic substitution reaction of BRX-S1 with BRX-1-G. Further, the isomeric impurities 10 ACS Paragon Plus Environment

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with proposed structures BRX-1-(J-M) may be observed due to over-alkylation of BRX-1-I with BRX-S2 or the reaction of BRX-S1 with BRX-1-A and BRX-1-B. The stage-2 impurities, particularly BRX-705A and BRX-705B may be formed due to the carryover of impurities BRX-1-A and BRX-1-B of stage-1 and their nucleophilic substitution reaction with BRX-S3. Impurity BRX-648A and BRX-648B may be formed by reaction of BRX-1 to BRX (API). Further, the impurities BRX-648C and BRX-648D may be developed by coupling of BRX-S3 with impurity BRX 1-L and BRX-1-M of stage-1 respectively. The BRX contains a piperazine moiety which may get oxidized under the reaction conditions employed and thus impurities BRX-A and BRX-B might be formed. Discussion of Impurities Synthesis: For the synthesis of stage-1 impurities of BRX, BRX-1 is a key starting substrate as shown in Scheme-2. The nucleophilic substitution reaction of BRX-1 with excess moles of BRX-S2 by using sodium hydride in acetonitrile at reflux produced BRX1-A and BRX-1-B impurities together. The BRX-1-dimer impurity i.e. BRX-1-I was achieved by the coupling of BRX-1 with BRX-S1 in DMF by using K2CO3 and KI. The impurity BRX-1-G was prepared by a nucleophilic substitution reaction of BRX-S1 with 1,4-dibromobutane. The BRX-1-H impurity was prepared by a nucleophilic substitution reaction of BRX-1 with sodium acetate followed by hydrolysis by using NaOH in THF/Water.

O

N H

BRX-1-H 233.26

O

O

N H

OH

O

N

N H

Cl

O

c

Cl

O

Cl

O BRX-1-A 342.26

+ O

N OH H BRX-S1 161.16

e

O

f H N

N

BRX-1 251.71

Cl

O BRX-1-I 376.41

O

d

O BRX-1-Bn 341.83

O

a b

O

O

N H

N

O

Cl

BRX-1-B 342.26 O BRX-1-G 296.16

Br

Cl

a) CH3COONa, DMF, 60−70 C; b) LiOH.H2O, THF, H2O; c) NaH, CH3CN, Br(CH2)4Cl, Reflux temperature; d) PhCH2Br, Ag2CO3, Acetone, Reflux temperature; e) BRX-S1, K2CO3, KI, DMF, 80−90C; f) Br(CH2)4Br, K2CO3, DMF, 30−40 C. Scheme-2: Synthetic scheme for stage-1 impurities BRX-1-A, BRX-1-B, BRX-1-G, BRX-1-H, BRX-1-I and impurity intermediate BRX-1-Bn 11 ACS Paragon Plus Environment


Page 12 of 25

Further, all the isolated impurities were co-spiked with isolated intermediate BRX-1, BRX-2 and BRX and confirmed their presence by LC-MS and HPLC analysis. S

N N

O

N

H N

O

O

O

c

BRX-648A 648.81

S

d

e N

N O

N

O O

BRX-648B 648.81

O

N H

BRX 433.57

H N

N H

O

BRX-A 449.57 +

N

+ N

N

+

S

O

O

O

O

N H

+

+

O

S

N O

N

S

N

S

N O

O

BRX-B 465.57

S

S

S

a N

N N

O

N

O

b

S

N

N

+ NH2

N Cl

-

BRX-S3 254.78

BRX-705A 705.97

N O

BRX-705B 705.97

N

O N

a) BRX-1-A, K2CO3, KI, DMF, 6 h, 80−90 C; b) BRX-1-B, K2CO3, KI, DMF, 6h, 80−90 C; c) BRX-1-Bn, K2CO3, KI, DMF, 6h, 100−110 C; d) HBr, 5h,60C; e) mCPBA, CH2Cl2, 2h, 20-25 °C. Scheme-3: Synthetic route for BRX stage-2 impurities BRX-705A, BRX-705B, BRX-A, BRXB, BRX-648A and BRX-648B Two of the impurities of stage-2 (BRX-705A and BRX-705B) were achieved by the coupling of key starting material BRX-S3 impurities carried over from stage-1 i.e. BRX-1-A and BRX-1-B respectively by using standard reaction conditions used for the preparation of BRX-2. For accomplishing BRX-648A & 648B impurities, the intermediate BRX-1-Bn (Scheme-2) was coupled with BRX by using CsCO3 and KI in DMF. The resultant compound on debenzylation by HBr gave a mixture of BRX-648A & BRX-648B. The other two impurities i.e. BRX-648C & BRX-648D were observed in LC-MS. The possible structures were suggested as shown in Figure-2. The attempts to isolate these two impurities to further confirm the proposed chemical structures were not fruitful. The two oxidized impurities of BRX i.e. BRX-A and BRX-B were synthesized by using mCPBA in CH2Cl2. EXPERIMENTAL SECTION General Information: All the used materials were purchased from commercial suppliers and used as supplied. Melting points were recorded (uncorrected) on a Buchi M-565 melting point 12 ACS Paragon Plus Environment

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monitor. IR spectra of samples were recorded on Shimadzu IR Affinity-I FT-IR spectrophotometer. 1H NMR spectra were recorded on Bruker AMX-400 spectrometer using tetramethylsilane as an internal standard.

13

C NMR spectrum were recorded using the same

spectrometer at 100 MHz. Chemical shifts are reported in parts per million (δ), coupling constants (J-values) are reported in hertz (Hz), and spin multiplicities are indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet), bt (broad triplet). The experiments to determine the molecular mass were performed on a Velos Pro ion trap mass spectrophotometer from Thermo Scientific (San Jose, CA, USA) equipped with electrospray ionization (ESI) source. Procedures and Characterizations: 7-(4-Chlorobutoxy)quinolin-2(1H)-one

(BRX-1):

To

a

stirred

suspension

of

7-

hydroxyquinolin-2(1H)-one BRX-S1 (5.0 g, 0.031 moles), potassium carbonate (4.8 g, 0.035 moles) in DMF (50.0 ml) and water (5.0 ml), and 1-Bromo-4-chlorobutane BRX-S2 (15.96 g, 0.093 moles) were added at room temperature. The reaction mass was stirred for 4 h at 30-40 °C. The reaction completion was observed by TLC/HPLC. The reaction was quenched by adding water (50 ml). The solid thus precipitated was filtered and washed with water. This crude product was dissolved in ethyl acetate (150 mL) at 70-80 °C and filtered through a celite bed. The obtained filtrate was distilled off partially at 70-80 °C followed by cooling to 5-10 °C. The solid precipitate was filtered. The isolated wet solid was dried under vacuum oven at 60 °C to afford 7-(4-Chlorobutoxy)quinolin-2(1H)-one BRX-1 (6.6 g, 85 % yield) as a white solid. 1HNMR (400 MHz, DMSO-d6): δ 11.63 (bs, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 6.81 (2H, m), 6.34 (d, J = 9.2 Hz, 1H), 4.05 (t, J = 5.6 Hz, 2H), 3.72 (t, J = 5.6 Hz, 2H), 1.89-1.88 (m, 4H).13C-NMR (100 MHz, DMSO-d6): δ 162.28, 160.32, 140.64, 140.00, 129.24, 118.54, 113.36, 110.75, 98.66, 67.00, 45.13, 28.84, 26.08. IR (KBr, cm-1): 3145.90, 3072.60, 2991.59, 2953.02, 2906.73, 2841.15, 1909.53, 1774.51, 1654.92, 1624.06, 1552.70, 1508.33, 1475.54, 1456.26, 1436.97, 1411.89, 1390.68, 1375.25, 1319.31, 1292.31, 1267.23 1219.01, 1201.65, 1155.36, 1138.00, 1114.86, 1043.49, 1012.63, 995.27, 954.76, 912.33, 846.75, 833.25, 777.31, 744.52, 727.16, 705.95, 624.94, 549.71, 520.78, 499.56, 468.70, 455.20 and 433.98. MS (ES+): calculated for C13H14ClNO2 251.71 and found 252.16. M.P: 127.7 °C.



Page 14 of 25

7-{4-[4-(1-Benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one (BRX-2): To a stirred suspension of 1‐(1‐Benzothiophen‐4‐yl)piperazinehydrochloride BRX-3 (10.12 g, 0.040 moles), potassium carbonate (6 g, 0.042 moles) and potassium iodide (7 g, 0.042 moles) in DMF (80 ml), 7-(4-Chlorobutoxy)quinolin-2(1H)-one BRX-1 (10.0 g, 0.040 moles) was added at room temperature. The reaction mass temperature was raised to 70 °C and maintained for 10 h. The reaction completion was observed by TLC/HPLC. The reaction was quenched by adding water (50 ml). The solid thus precipitated was filtered and washed with water. The crude product was purified in a mixture of dichloromethane and ethyl acetate and isolated solid was further purified by acid-base purification (Experimental section). The purified product was dried under vacuum oven at 60 °C to afford 7-{4-[4-(1-Benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)one BRX (14.9 g, 84 % yield) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ 11.60 (s, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.70 (d, J = 5.6 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 5.6 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.81 (m, 2H), 6.31 (d, J = 9.2 Hz, 1H), 4.05 (t, J = 6.4 Hz, 2H), 3.06 (bs, 4H), 2.61 (bs, 4H), 2.44 (t, J = 7.2 Hz, 2H), 1.83-1.76 (m, 2H), 1.67-1.62 (m, 2H). 13C-NMR (100 MHz, DMSO-d6): δ 162.26, 160.47, 148.27, 140.67, 140.41, 140.02, 133.38, 129.25, 125.82, 125.10, 121.90, 118.47, 116.63, 113.28, 112.03, 110.87, 98.61, 67.63, 57.38, 52.99, 51.74, 26.57, 22.72. IR (KBr, cm-1): 3311.78, 3143.97, 3066.82, 2949.16, 2816.07, 1940.39, 1896.03, 1653.00, 1622.13, 1556.55, 1508.33, 1479.40, 1465.90, 1446.61, 1415.75, 1371.39, 1315.45, 1263.37, 1238.30, 1220.94, 1195.87, 1132.21, 1097.50, 1039.63, 999.13, 966.34, 927.76, 864.11, 833.25, 813.96, 796.60, 769.60, 719.45, 696.30, 678.94, 623.01, 553.57, 530.42, 484.13 and 459.06. MS (ES+): calculated for C25H27N3O2S 433.57 and found 434.30. M.P.: 181.7–183.4 °C. BRX-2 Purification procedure: In a cleaned and dried RBF, methylene dichloride (300 ml) and BRX-2 (Crude, 15.0 g) were charged and heated at 35-40 °C. The hot reaction mass was filtered through a celite bed. The filtrate was charged in a cleaned and dried RBF and distilled off methylene dichloride partially at atmospheric pressure at 35-40 °C and ethyl acetate added (45 ml) at 30-40 °C. Reaction mass was heated at 40-45 °C and gradually allowed to cool to 20-30 °C. The precipitated solid was filtered and dried in oven to obtain BRX-2 (13.8 g). The isolated compound was further purified with acid-base treatment by using HCl (1.5 mole equivalent w.r.t. BRX-2) in methanol (20 volumes w.r.t BRX-2) followed by NaOH (1.05 mole equivalent w.r.t


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BRX-2) in MeOH (20 volumes w.r.t BRX-2) at 20-30 °C to afford BRX (13.2 g) in the form of white solid. 2,7-bis(4-chlorobutoxy)quinoline(BRX-1-A)

and

7-(4-Chlorobutoxy)-1-(4-chlorobutyl)-

quinolin-2(1H)-one (BRX-1-B): To a stirred suspension of 7-(4-Chlorobutoxy)quinolin-2(1H)one BRX-1(20.00 g, 0.079 moles) and sodium hydride (5.0 g, 60 %, 0.125 moles) in acetonitrile (400 ml), 1-Bromo-4-chlorobutane BRX-S2 (34 g, 0.198 moles) was charged at 25-30 °C. The reaction mass was refluxed for 6-7 hours. The reaction completion was observed by TLC. The reaction was quenched by adding water (500 ml) at room temperature and extracted with dichloromethane (2 x 200 ml). Solvent was evaporated under vacuum and isolated crude product was purified by column chromatography (0-5 % methanol in dichloromethane was used as an eluent) to isolate BRX-1-A (10.6 g, 39 % yield) & BRX-1-B (7.9 g, 29 % yield) impurities in the form of white and light yellow solid respectively. BRX-1-A: 1H-NMR (400 MHz, DMSO-d6): δ 8.13 (d, J = 8.8 Hz, 1H), 7.77 (d, , J = 8.8 Hz, 1H), 7.16 (s, 1H), 7.07 (dd, J = 10.8, 6.8 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.43 (bt, 2H), 4.15 (bt, 2H), 3.8-3.7 (m, 4H), 1.95-1.85 (m, 8H). 13C-NMR (100 MHz, DMSO-d6): δ 162.05, 159.87, 147.74, 138.86, 128.76, 119.47, 115.97, 110.05, 107.07, 66.95, 64.59, 45.19, 45.14, 28.97, 28.89, 26.07, 26.01. IR (KBr, cm-1): 3001.24, 2962.66, 2912.51, 2904.80, 2891.30, 2873.94, 2846.93, 1944.25, 1909.53, 1685.79, 1624.06, 1608.63, 1608.63, 1571.99, 1560.41, 1508.33, 1481.33, 1460.11, 1436.97, 1413.82, 1394.53, 1379.10, 1365.60, 1309.67, 1265.30, 1251.80, 1217.08, 1195.87, 1136.07, 1111.00, 1058.92, 1043.49, 1028.06, 1008.06, 991.41, 977.91, 974.05, 904.61, 844.82, 837.11, 808.17, 781.17, 769.60, 761.88, 742.59, 729.09, 707.88, 678.94, 648.08, 628.79, 565.14, 538.14, 511.14, 495.71, 474.49, 447.49 and 439.77. MS (ES+): calculated for C17H21Cl2NO2 342.26 and found 342.26. M.P: 73.3–74.9 °C. BRX-1-B: 1H-NMR (400 MHz, DMSO-d6): δ 7.83 (d, J = 9.2 Hz,1H), 7.65 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.43 (d, J = 9.6 Hz, 1H, d), 4.26 (t, J = 7.2 Hz, 2H), 4.16 (t, J = 5.6 Hz, 2H), 3.75-3.71 (m, 4H), 1.99-1.72 (m, 8H).

13

C-NMR (100 MHz, DMSO-d6): δ

161.36, 160.85, 140.45, 139.18, 130.49, 117.64, 114.47, 110.10, 99.14, 67.20, 67.08, 45.11, 45.07, 29.35, 29.85, 26.05, 24.25. IR (KBr, cm-1): 3097.68, 3030.17, 2970.38, 2951.09, 2922.16, 2902.87, 2879.72, 2843.07, 1880.60, 1654.92, 1639.49, 1627.92, 1587.42, 1554.63, 1512.19, 15 ACS Paragon Plus Environment


Page 16 of 25

1473.62, 1463.97, 1448.54, 1438.90, 1404.18, 1394.53, 1381.03, 1354.03, 1321.24, 1303.88, 1284.59, 1236.37, 1197.79. MS (ES+): calculated for C17H21Cl2NO2 342.26 and found 342.25. M.P: 75.1–77.2 °C.

7-(4-Bromobutoxy)quinolin-2(1H)-one (BRX-1-G): The synthesis procedure is same as of 7(4-Chlorobutoxy)quinolin-2(1H)-one BRX-1. 1-4 dibromobutane was used in place of 1-bromo4-chlorobutane BRX-S2. 7-(4-Bromobutoxy)quinolin-2(1H)-one (BRX-1-G) was obtained as a white solid with 82 % yield. 1

H-NMR (400 MHz, DMSO-d6): δ 11.60 (1H, bs), 7.80 (d, J = 9.6 Hz, 1H), 7.55 (d, J = 9.2 Hz,

1H), 6.78-6.76 (m, 2H), 6.31 (d, J = 9.6 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.60 (t, J = 6.4 Hz, 2H), 1.99-1.93 (m, 2H), 1.91-1.82 (m, 2H). 13C-NMR (100 MHz, DMSO-d6): δ 162.25, 160.30, 140.63, 140.00, 129.24, 118.54, 113.34, 110.74, 98.65, 67.04, 59.75, 29.03, 27.31. IR (KBr, cm1

): 3138.18, 3022.45, 2939.52, 2839.22, 1903.74, 1789.94, 1654.92, 1622.13, 1556.55, 1510.26,

1475.54, 1456.26, 1444.68, 1415.75, 1394.53, 1375.25, 1346.31, 1315.45, 1278.81, 1259.52, 1238.30, 1222.87, 1203.58, 1138.00, 1116.78, 1060.85, 1049.28, 1002.98, 970.19, 954.76, 941.26, 929.69, 898.83, 840.96, 833.25, 798.53, 775.38, 759.95, 696.30, 682.80, 632.65, 623.01, 549.71, 520.78, 478.35, 466.77, 443.63, 430.13, 412.77. MS (ES+): calculated for C13H14BrNO2 296.16 and found 296.16. M.P: 125.5–127.1 °C.

7-(4-Hydroxybutoxy)quinolin-2(1H)-one (BRX-1-H): To a stirred solution of BRX-1 (10 g) in DMF (100 ml), CH3COONa (8.0 g) was charged at room temperature. The reaction mass temperature was raised to 70 °C and maintained for 7 h. The reaction completion was observed by TLC. The reaction was quenched by adding water (200 ml) at room temperature and extracted with dichloromethane (2 x 100 ml). Solvent was evaporated under vacuum to isolate the compound BRX-1-OCOCH3 (9.2 g) as a white solid. The isolated compound BRX-1-OCOCH3 was charged in THF (100 ml) at room temperature. Aqueous LiOH.H2O solution (14.0 g in 100 ml water) was added to the reaction mass and stirred for 5-6 hours at room temperature. Organic solvent was evaporated on rotavapor and pH was adjusted to ~7 by hydrochloric acid. The solid thus precipitated was filtered, washed with water (2 x 50 ml) and dried under vacuum. The isolated crude product was purified by column chromatography to obtain BRX-1-H (5.5 g, 70 % yield) as a white solid.

1

H-NMR (400 MHz, DMSO-d6): δ 11.58 (s, 1H), 7.81 (d, J = 9.2 Hz, 16 ACS Paragon Plus Environment

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1H), 7.56 (d, J = 8.8 Hz, 1H), 6.81-6.78 (m, 2H), 6.31 (d, J = 9.2 Hz, 1H), 4.49 (t, J = 4.8 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 3.49-3.44 (q, J = 6.0 Hz, 2H), 1.81-1.74 (m, 2H), 1.61-1.54 (m, 2H).

13

C-NMR (100 MHz, DMSO-d6): δ 162.25, 160.46, 140.66, 140.02, 129.24, 118.47,

113.26, 110.84, 98.56, 67.69, 60.38, 28.91, 25.36. IR (KBr, cm-1): 3342.64, 3140.11, 3061.03, 2960.73, 2868.15, 2594.26, 1953.89, 1764.87, 1647.21, 1624.06, 1571.99, 1556.55, 1512.19, 1471.69, 1417.68, 1400.32, 1390.68, 1377.17, 1328.95, 1269.16, 1222.87, 1201.65, 1147.65, 1120.64, 1062.78, 1047.35, 1022.27, 1002.98, 987.55, 941.26, 893.04, 840.96, 829.39, 775.38, 713.66, 624.94, 555.50, 532.35, 501.49, 474.49, 464.84, 428.20 and 410.84. MS (ES+): calculated for C13H15NO3 233.26 and found 234.15. M.P: 187.9–193.8 °C. 7,7'-[butane-1,4-diylbis(oxy)]di(quinolin-2(1H)-one) (BRX-1-I): To a stirred suspension of 7(4-Chlorobutoxy)quinolin-2(1H)-one (3.0 g, 0.012 moles) and potassium iodide (2.17 g, 0.0131 moles) in DMF (15 ml), 7-hydroxyquinolin-2(1H)-one (2.0 g, 0.0124 moles) and potassium carbonate (3.8 g, 0.0275 moles) were charged at room temperature. The reaction mass temperature was raised to 80-90 °C and maintained for 20 h. The reaction completion was observed by TLC. The reaction was quenched by adding water (50 ml). The solid thus precipitated was filtered, washed with water and dried to obtain BRX-1-I (3.7 g, 83 % yield) as a cream color solid. 1

H-NMR (400 MHz, DMSO-d6): δ 11.59 (s, 2H), 7.82 (d, J = 9.6 Hz, 2H), 7.57 (d, J = 9.2 Hz,

2H), 6.81-6.79 (m, 4H), 6.31 (d, J = 9.2 Hz, 2H), 4.09 (bs, 4H), 1.92 (bs, 4H).

13

C-NMR (100

MHz, DMSO-d6): δ 162.25, 160.39, 140.66, 140.03, 129.27, 118.53, 113.33, 110.82, 98.62, 67.45, 25.34. IR (KBr, cm-1): 3271.27, 3138.18, 3078.39, 2951.09, 2879.72, 2848.86, 2598.12, 1909.53, 1674.21, 1625.99, 1556.55, 1512.19, 1487.12, 1471.69, 1411.89, 1396.46, 1377.17, 1323.17, 1267.23, 1238.30, 1222.87, 1203.58, 1182.36, 1157.29, 1141.86, 1062.78, 1020.34, 1012.63, 1002.98, 945.12, 937.40, 927.76, 898.83, 844.82, 835.18, 792.74, 781.17, 771.53, 761.88, 752.24, 727.16, 628.79, 565.14, 551.64, 532.35, 484.13, 459.06 and 430.13. MS (ES+): calculated for C22H20N2O4 376.42 and found 377.21. M.P: 310.7 °C.

2,7‐bis({4‐[4‐(1‐Benzothiophen‐4‐yl)piperazin‐1‐yl]butoxy})quinoline (BRX-705A): To a stirred suspension of 1‐(1‐Benzothiophen‐4‐yl)piperazinehydrochloride BRX-S3 (0.8 g, 0.0031 moles) and potassium carbonate (0.5 g, 0.0036 moles)

in DMF (10 ml), 2,7-bis(417

ACS Paragon Plus Environment


Page 18 of 25

chlorobutoxy)quinoline BRX-1-A (0.5 g, 0.0015 moles) and potassium iodide (0.55 g, 0.0033 moles) were added at room temperature. The reaction mass temperature was raised to 90 °C and maintained for 6 h. The reaction completion was observed by TLC. The reaction was quenched by adding water (60 ml) at room temperature and extracted with dichloromethane (2 x 50 ml). Solvent was evaporated under vacuum and isolated crude product was purified by column chromatography (1-5 % methanol in dichloromethane was used as an eluent) to isolate 2,7-bis{4[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinoline impurity BRX-705A (0.7 g, 68 % yield) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 7.89 (d, J = 8.8 Hz, 1H), 7.58-7.53 (m, 3H), 7.42-7.36 (m, 4H), 7.29-7.24 (m, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.03-7.00 (dd, J = 6.4, 2.4 Hz, 1H), 6.89 (d, J = 7.6 Hz, 2H), 6.75 (d, J = 8.8 Hz, 1H), 4.49 (t, J = 6.4 Hz, 2H), 4.15 (t, J = 6.4 Hz, 2H), 3.19 (bs, 8H), 2.71 (bs, 8H), 2.56-2.51 (m, 4H), 1.95-1.85 (m, 4H), 1.80-1.75 (m, 4H).

13

C-NMR (100 MHz, CDCl3): δ 162.94, 160.50, 148.64, 148.54, 141.22, 138.42, 134.18,

128.51, 125.14, 125.03, 122.04, 119.86, 117.08, 116.44, 112.28, 110.53, 107.32, 67.99, 65.74, 58.56, 58.42, 53.72, 52.27, 27.35, 27.28, 23.76, 23.66. IR (KBr, cm-1): 3101.54, 3066.82, 3026.31, 2941.44, 2873.94, 2812.21, 2771.71, 2677.20, 1618.28, 1608.63, 1577.77, 1560.41, 1535.34, 1508.33, 1490.97, 1473.62, 1450.47, 1438.90, 1411.89, 1363.67, 1305.81, 1261.45, 1238.30, 1215.15, 1195.87, 1132.21, 1056.99, 1033.85, 1014.56, 966.34, 889.18, 864.11, 833.25, 798.53, 786.96, 748.38, 700.16, 688.59, 669.30, 638.44, 626.87, 597.93, 557.43, 543.93, 474.49, 460.99 and 437.84. MS (ES+): calculated for C41H37N5O2S2 705.97 and found 706.41. M.P: 72.0–76.8 °C.

7‐{4‐[4‐(1‐Benzothiophen‐4‐yl)piperazin‐1‐yl]butoxy}‐1‐{4‐[4‐(1‐benzothiophen‐4‐yl)piperazin‐1‐yl]butyl}‐1,2‐dihydroquinolin‐2‐one (BRX-705B): To a stirred suspension of 1-1‐ (1‐Benzothiophen‐4‐yl)piperazinehydrochloride (0.8 g, 0.0031 moles) and potassium carbonate (0.5 g, 0.0036 moles) in DMF (10 ml), BRX-1-B (0.4 g, 0.0012 moles) and potassium iodide (0.5 g, 0.0030 moles) were added at room temperature. The reaction mass temperature was raised to 90 °C and maintained for 6 h. The reaction completion was observed by TLC. The reaction was quenched by adding water (60 ml) at room temperature and extracted with dichloromethane (2 x 50 ml). Solvent was evaporated under vacuum and isolated crude product was purified by column chromatography (1-5 % methanol in dichloromethane was used as an eluent) to isolate 1,7-bis{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-oneBRX-705B

(0.5 18


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g, 61 % yield) impurity as a white solid. 1H-NMR (400 MHz, CDCl3): δ7.59 (d, J = 9.6 Hz, 1H), 7.55-7.51 (dd, J = 8.0, 2.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.41-7.36 (m, 4H), 7.29-7.24 (m, 2H), 6.89-6.86 (m, 3H), 6.83-6.80 (dd, J = 8.4, 2.0 Hz, 1H), 6.55 (d, J = 9.6 Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 3.19 (bs, 8H), 2.70 (bs, 8H), 2.60-2.47 (m, 4H), 1.93-1.82 (m, 4H), 1.80-1.69 (m, 4H).

13

C-NMR (100 MHz, CDCl3): δ 162.69, 161.38, 148.58, 148.54,

141.25, 140.97, 138.92, 134.17, 130.39, 125.14, 125.11, 125.07, 122.04, 121.98, 118.71, 117.15, 117.09, 115.35, 112.27, 109.35, 100.04, 68.26, 58.33, 58.18, 53.72, 52.24, 52.22, 42.26, 27.39, 25.33, 24.33, 23.63.IR (KBr, cm-1): 3429.43, 3064.89, 2939.52, 2870.08, 2810.28, 2677.20, 2358.94, 1651.07, 1622.13, 1587.42, 1562.34, 1519.91, 1512.19, 1506.41, 1450.47, 1408.04, 1373.32, 1352.10, 1313.52, 1269.16, 1236.37, 1180.44, 1132.21, 1058.92, 1014.56, 966.34, 864.11, 835.18, 786.96, 748.38, 700.16, 688.59, 632.65, 596.00, 557.43, 543.93, 513.07, 470.63, 457.13, 443.63, 430.13, 426.27, 416.62, 408.91 and 401.19. MS (ES+): calculated for C41H37N5O2S2 705.97 and found 706.42. M.P: 86.3–95 °C. 7-{4-[(7-{4-[4-(1-Benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2-yl)oxy]butoxy}-1,2dihydroquinolin-2-one(BRX-648A)

&

7-{4-[4-(1-Benzothiophen-4-yl)piperazin-1-

yl]butoxy}-1-{4-[(2-oxo-1,2-dihydroquinolin-7-yl)oxy]butyl}-1,2-dihydroquinolin-2-one (BRX-648B): 2-(benzyloxy)-7-(4-chlorobutoxy)quinolone (Bn-BRX-1): To a stirred suspension of BRX-1 (12.0 g, 0.048 moles) and silver carbonate (15.0 g, 0.046 moles) in acetone (120 ml), benzyl bromide (16.5 g, 0.096 moles) was charged at room temperature. The reaction mass was refluxed for 6-7 hrs. The reaction completion was observed by TLC. The reaction was filtered, solvent was evaporated under vacuum and isolated crude product was purified by column chromatography (1-2 % methanol in dichloromethane was used as an eluent) to isolate 2(benzyloxy)-7-(4-chlorobutoxy)quinolone (Bn-BRX-1) as an oily mass. To a stirred suspension of BRX (15.0 g, 0.035 moles) and Cs2CO3 (20 g, 0.061 moles) in DMF (75 ml), Bn-BRX-1 (14.0 g, 0.040 moles) was charged at room temperature. The reaction mass temperature was raised to 110 °C and maintained for 6 hrs. The reaction completion was observed by TLC. The reaction was quenched by adding water (100 ml) at room temperature and extracted with dichloromethane (2 x 50 ml). Solvent was evaporated under vacuum to get an oily mass. The oily mass was further treated with hydrobromic Acid (47% Aq, 75.0 mL) for 5.0 hrs at 60 °C. Reaction mass was cooled to room temperature, water added (75 mL), and pH 9-10 was adjusted by using 20 % NaOH solution. Aqueous layer was extracted with THF (2 x 100 mL). 19 ACS Paragon Plus Environment


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The solvent was evaporated under vacuum and the oily mass crystallized in n-Hexane. BRX648A and BRX-648B impurities were separated and purified by using silica gel column chromatography in dichloromethane and methanol. BRX-648A (4.3 g, 19 % yield as an off white solid) and BRX-648B (3.2 g, 14 % yield as an off white solid). BRX-648A: 1H-NMR (400 MHz, CDCl3): δ 12.27 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 9.6, 1H), 7.59-7.53 (m, 2H), 7.43-7.37 (m, 3H), 7.31-7.24 (m, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.05-7.00 (dd, J = 8.8, 2.4 Hz, 1H), 6.88-6.86 (m, 2H), 6.80-6.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.74 (d, J = 9.6 Hz, 1H), 4.50 (bt, 2H), 4.12 (m, 4H), 3.20 (bs, 4H), 2.72 (bs, 4H), 2.52 (t, J = 7.2, 2H), 2.02 (bs, 4H), 1.91 (m, 2H), 1.77 (M, 2H). 13C-NMR (100 MHz, CDCl3): δ 164.94, 162.85, 161.53, 160.52, 148.61, 148.52, 141.23, 140.93, 140.46, 138.50, 134.20, 129.16, 128.52, 125.15, 125.06, 122.04, 119.90, 118.10, 117.11, 116.51, 114.28, 112.71, 112.31, 110.48, 107.34, 99.10, 68.13, 67.98, 65.39, 58.42, 53.72, 52.25, 27.35, 26.14, 25.83, 23.63. IR (KBr, cm-1): 3631.96, 3421.72, 3143.97, 3064.89, 3026.31, 2943.37, 2872.01, 2814.14, 1654.92, 1620.21, 1558.48, 1508.33, 1467.83, 1454.33, 1415.75, 1365.60, 1305.81, 1261.45, 1236.37, 1219.01, 1195.87, 1134.14, 1039.63, 1018.41, 966.34, 864.11, 835.18, 798.53, 771.53, 750.31, 700.16, 624.94, 597.93, 557.43, 545.85, 507.28, 474.49, 462.92 and 401.19. MS (ES+): calculated for C38H40N4O4S 648.81 and found 649.37. M.P: 96.2–102.6 °C. BRX-648B: 1H-NMR (400 MHz, CDCl3): δ 11.69 (s, 1H) 7.68 (d, J = 9.6 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.42-7.36 (m, 3H), 7.27-7.22 (m, 1H), 6.88-6.75 (5H, m), 6.56 (d, J = 9.2 Hz, 2H), 6.52 (d, J = 9.2 Hz, 2H), 4.37 (bt, 2H), 4.14 (bt, 2H), 4.05 (t, 6.56 (t, J = 6.0 Hz, 2H), 3.17 (bs, 4H), 2.72 (bs, 4H), 2.53 (t, 6.56 (d, J = 6.8 Hz, 2H), 1.98 (bs, 4H), 1.87-1.79 (m, 7H). 13C-NMR (100 MHz, CDCl3): δ 164.82, 162.76, 161.44, 161.32, 148.47, 141.23, 140.84, 140.47, 139.07, 134.14, 130.45, 129.19, 125.12, 121.95, 118.59, 118.24, 117.15, 115.32, 114.33, 112.56, 112.27, 109.91, 99.52, 99.17, 68.19, 68.05, 58.34, 53.69, 52.16, 41.95, 27.34, 26.55, 24.21, 23.54. IR (KBr, cm-1): 3421.72, 3151.69, 3066.82, 2943.37, 2872.01, 2818.00, 1869.02, 1772.58, 1653.00, 1624.06, 1583.56, 1558.48, 1508.33, 1450.47, 1411.89, 1375.25, 1317.38, 1265.30, 1234.44, 1222.87, 1197.79, 1178.51, 1138.00, 1122.57, 1058.92, 1022.27, 1002.98, 966.34, 837.11, 752.24, 624.94, 597.93, 559.36, 543.93, 462.92, 445.56, 437.84, 424.34, 418.55, 410.84 and 403.12. MS (ES+): calculated for C38H40N4O4S 648.81 and found 649.40. M.P: 93.6–97.9 °C.


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4-(1-Benzothiophen-4-yl)-1-{4-[(2-oxo-1,2-dihydroquinolin-7-yl)oxy]butyl}piperazin-1-ium1-olate(BRX-A

i.e.

BRX-N-Oxide)

and

1-(1-Benzothiophen-4-yl)-4-{4-[(2-oxo-1,2-

dihydroquinolin-7-yl)oxy]butyl}piperazine-1,4-diium-1,4-bis(olate)(BRX-B i.e. BRX-N-N'Dioxide): To a stirred suspension of BRX (5.0 g, 0.011moles) in dichloromethane (50 ml) mChloroperbenzoic acid (3.6 g, 0.011 moles) was charged at room temperature. The reaction mass was stirred for 1 h at room temperature. The reaction completion was observed by TLC. The reaction was quenched by adding water (50 ml) and pH 9-10 was adjusted with 10 % sodium hydroxide solution. The solid thus precipitated was filtered and washed with water. Crude product was slurried with n-Hexane. BRX-A and BRX-B impurities were separated and purified by using silica gel column chromatography in dichloromethane and methanol. BRX-A (1.6 g, 30 % yield as an off white solid) and BRX-B (2 g, 37 % yield as an off white solid). BRX-A: 1H-NMR (400 MHz, DMSO-d6): δ 11.71 (S, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.74 (d, J = 5.6 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H), 7.47 (d, J = 5.6 Hz, 1H), 7.32 (t, J= 7.6 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.83-6.79 (m, 2H), 6.31 (d, J = 9.6 Hz, 1H), 4.08 (t, J = 6.0 Hz, 2H), 3.3 – 3.0 (m, 6H), 2.12-2.04 (2H, m), 1.90-1.80 (m, 2H). 1

H-NMR (400 MHz, DMSO-d6 with CF3COOH): δ 7.83 (d, J = 9.6 Hz, 1H), 7.78 (d, J = 5.6 Hz,

1H), 7.72 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 5.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 6.83 (m, 2H), 6.32 (d, J = 9.6 Hz, 1H), 4.10 (t, J = 6.0 Hz, 2H), 4.01 (t, J = 11.2 Hz, 2H), 3.90-3.82 (m, 4H), 354-3.37 (m, 4H), 2.12-2.04 (2H, m), 1.90-1.80 (m, 2H). 13C-NMR (100 MHz, DMSO-d6 with CF3COOH): δ 162.32, 160.33, 159.03, 158.66, 158.30, 157.93, 146.71, 140.64. 140.12, 133.41, 129.35, 126.70, 125.10, 121.74, 119.88, 118.61, 117.96, 117.00, 114.11, 113.50, 112.86, 111.22, 110.87, 98.75, 67.44, 67.03, 62.19, 45.69, 25.48, 18.29. IR (KBr, cm-1): 3383.14, 3070.68, 2951.09, 2883.58, 1651.07, 1624.06, 1562.34, 1558.48, 1510.26, 1462.04, 1452.40, 1415.75, 1392.61, 1373.32, 1346.31, 1311.59, 1265.30, 1244.09, 1220.94, 1195.87, 1139.93, 1116.78, 1070.49, 1045.42, 1012.63, 972.12, 941.26, 839.03, 812.03, 788.89, 756.10, 702.09, 624.94, 599.36, 538.14, 470.63, 443.63, 428.20, 416.62 and 412.77. MS (ES+): calculated for C25H27N3O3S 449.57 and found 450.18. M.P: 135.6–143.0 °C. BRX-B: 1H-NMR (400 MHz, DMSO-d6): δ 12.03 (s, 1H), 8.49 (d, J = 7.6 Hz, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 5.2 Hz, 1H), 7.81 (d, J = 9.6 Hz, 1H), 7.567.50 (m, 2H), 6.86-6.79 (m, 2H), 6.30 (d, J = 9.2 Hz, 1H), 4.98 (t, J = 10.4 Hz, 2H), 4.29 (t, J = 10.8 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.49 - 3.40 (m, 2H), 3.14 (t, J = 8.8 Hz, 4H), 2.09 - 2.05 21 ACS Paragon Plus Environment


(m, 2H), 1.86 - 1.83 (m, 2H).

13

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C-NMR (100 MHz, DMSO-d6): δ 162.34, 160.37, 149.42,

141.65, 140.72. 139.99, 130.70, 129.24, 129.10, 124.11, 124.03, 122.52, 118.54, 117.25, 113.35, 110.79, 98.72, 69.06, 67.43, 61.65, 59.23, 26.11, 18.54. IR (KBr, cm-1): 3383.14, 3244.27, 3109.25, 2989.66, 2962.66, 1660.71, 1631.78, 1556.55, 1512.19, 1477.47, 1446.61, 1433.11, 1411.89, 1394.53, 1375.25, 1340.53, 1325.10, 1315.45, 1274.95, 1226.73, 1203.58, 1147.65, 1138.00, 1112.93, 1064.71, 1053.13, 1031.92, 1014.56, 981.77, 947.05, 912.33, 867.97, 846.75, 837.11, 813.96, 796.60, 781.17, 725.24, 702.09, 626.87, 590.22, 549.71, 520.78, 505.35, 486.06, 462.92, 455.20, 447.49, 428.20 and 403.12. MS (ES+): calculated for C25H27N3O4S 465.57 and found 466.25. M.P: 142.9–159.1 °C.

CONCLUSION For the process development of active pharmaceutical ingredients (API), it is of foremost importance to know the fate of related substances as per ICH recommendations. In this regard, a number of chemical structures of possible impurities were proposed, identified, synthesized and characterized in connection with the Brexpiprazole (API) drug substance. In addition, we have for the first time in the literature addressed the strategies to control the impurities for BRX (API) either by parametric optimization of the process or by purification methods. We believe that this study will be of immense help to organic/analytical chemists of the generics industry to produce and quantitatively study the BRX impurities in order to meet the various requirements of drug regulatory agencies. ACKNOWLEDGEMENTS The authors are thankful to the management of Micro Labs Ltd., Research & Development Centre (Bangalore) for providing necessary facilities. Authors would like to thank Analytical Research & Development Department for their co-operation in carrying out this work. I gratefully acknowledge the time devoted by Mr. Albert Pape (Yung Zip Pharmaceutical Taiwan) for editing the manuscript. ASSOCIATED CONTENT


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Supporting Information: It contains LC-MS methods, selected copies of LC-MS spectrum and HPLC analytical reports of intermediates BRX-1 (crude), BRX-1 (pure), BRX-2 crude, purified and BRX. 1H-NMR and 13C-NMR of BRX-1, BRX, BRX-1-A, BRX-1-B, BRX-1-G, BRX-1-H, BRX-1-I, BRX-705A, BRX-705B, BRX-648A, BRX-648B, BRX-A and BRX-B. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author *Pramod Kumar E-mail: [email protected] Micro Labs Ltd., API R&D Centre, Bangalore 560105, Karnataka, India *Rahul Tyagi

Email: [email protected] Micro Labs Ltd., API R&D Centre, Bangalore 560105, Karnataka, India References:

1 . Center For Drug Evaluation 205422orig1s000&205422orig2s000.

And

Research.

Application

Number:

2. Patil, G. D.; Kshirsagar, S. W.; Shinde, S. B.; Patil, P. S.; Deshpande, M. S.; Chaudhari, A. T.;

Sonawane, S. P.; Maikap, G. C.; Gurjar, M. K. Identification, Synthesis, and Strategy For Minimization of Potential Impurities Observed In Raltegravir Potassium Drug Substance, Org. Process Res. Dev. 2012, 16, 1422-1429. 3 . Huang, Y.; Ye, Q.; Guo, Z.; Palaniswamy V. A.; Grosso, J. A. Identification of Critical Process Impurities and Their Impact on Process Research and Development,Org. Process Res. Dev. 2008, 12,632-636.



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4 . International Conference on Harmonization (ICH) Q3A(R): Impurities in new drug substances; ICH: Geneva Switzerland 2002. 5. Mishra, B.; Thakur, A.; Mahata, P. P. Pharmaceutical impurities: A Review, Int. J. Pharma. Chem.2015, 05(07), 232-240. 6 . a. Yamashita, H.; Sakurai, Y.; Miyamoto, M.; Nakamura, Y.; Kuroda, H.; Minowa, T. Piperazine-Substituted Benzothiophene Derivatives As Antipsychotic Agents, US9260420B2, 2016. b. Yamashita, H.; Ito, N.; Miyamura, S.; Oshima, K.; Matsubara, J.; Kuroda, H.; Takahashi, H.; Shimizu, S.; Tanaka, T, Piperazine-substituted benzothiophenes for treatment of mental disorders, US20100179322 A1, 2010. c. Luo, X.; Fu, T.; Zhang, Y.; Zhang, H.; Zuo, X.; Lei, H.; Deng, J. Brexpiprazole impurity compound and preparation method thereof,CN106892909A, 2017. 7. Liu, Z.; W. U. C.;Liu, Y.; Zhang, R.; He, W.; Tian, G.; Shen, J. Methods for preparing brexpiprazole, key intermediates thereof and salts thereof, US20160272624A1, 2016.

8 . Koichi; S.; Naoto; U.; Masahiro, S.; Shigeo, F.; Shin, O. Method for Producing Benzo[b]thiophene Compound, WO2013015456A1, 2013. 9. Shinhama, K.; Utsumi, N.; Sota, M.; Fujieda, S.; Ogasawara, S. Method for Producing Benzo[b]thiophene Compound, US9206169B2, 2015. 10. Chen, X.; Sassano, M. F.; Zheng, L.; Setola, V.; Chen, M.; Bai,X.; Frye, S. V.; Wetse, W. C.; Roth, B. L.; Jin, J. Structure–Functional Selectivity Relationship Studies of β-Arrestin-Biased Dopamine D2 Receptor Agonists, J. Med. Chem. 2012, 55(16), 7141-7153. 11. Prasad, S.; V, Sahu, A.; Shimpi, N. K.; Ponnuru, Batharaju, R.; Phadhuri, N.K. Process for the Preparation of Brexpiprazole, WO2017025987A1, 2017.


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Graphical Abstract S

N N

O

N

H N

O

O

O

BRX-648A S

S S

N

N N

O

BRX-648B

N

O O

N

N

H N

O

N

O

Brexpiprazole (API)

N H

O

BRX-705B

O

N

N

O N

S

N

N N

O

N

O

S

N

BRX-705A

Brexpiprazole Critical Impurities Identification, Synthesis, Characterization and Control Strategy in Process Development


S

Identification, Synthesis, and Control of Process-Related Impurities in

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