Identification, Synthesis, and Control of Process-Related Impurities in the Antipsychotic Drug Substance Brexpiprazole

Article abstract of DOI:10.1021/acs.oprd.8b00074

This article describes the identification, synthesis, and control of several unknown related substances present in the form of critical impurities that were observed during the process development for the 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 the unknown impurities were proposed. Many critical impurities were synthesized, and their chemical structures were established 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 during the development of a robust and efficient process for the BRX drug substance.

Full text of DOI:10.1021/acs.oprd.8b00074

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Full Paper
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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Identification, Synthesis, and Control of Process
Related Impurities in Antipsychotic Drug
Substance Brexpiprazole
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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.
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KEY WORDS: Brexpiprazole, antipsychotic, Process development, impurities, active
pharmaceutical ingredients, critical impurities
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INTRODUCTION
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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 developed by Otsuka and Lundbeck, and it is available as
REXULTI® (Brexpiprazole) tablets for oral administration.1
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N
S
N
O
N
H
O
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
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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.
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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-Bromo-
4-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).
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a
b
Cl
Cl
O
N
H
O
+
Br
S
HO
S
N
H
O
BRX-S2
[171.46]
BRX-1
[251.71]
BRX-S1
[161.16]
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N
NH
HCl
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BRX-S3
[254.78]
N
d
N
O
N
H
O
c
BRX-2
[433.57]
S
S
e
N
N
N
N
O
N
H
O
O
N
H
O
HCl
BRX-3
[470.03]
BRX
[433.57]
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 C, 2 h d) EtOH, Acetic acid, reflux, Conc. HCl e) EtOH, H2O,
o
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).
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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
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S.No. LC-MS (~RT) LC-MS (~RRT) LC-MS [M+H]+ (m/z)
Proposed Chemical Structure (Code) *
BRX-S1
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1.
2.
4.20
6.45
0.27
0.41
0.93
1.08
1.56
1.59
1.71
1.75
1.89
1.99
2.01
162.11
234.09
377.19
296.15
467.17
467.16
342.11
386.12
467.22
342.15
386.15
BRX-1-H
3.
14.79
17.14
24.73
25.15
27.14
27.67
29.85
31.48
31.85
BRX-1-I
4.
BRX-1-G
5.
BRX-1-J/BRX-1-K/BRX-1-L/BRX-1-M
BRX-1-J/BRX-1-K/BRX-1-L/BRX-1-M
BRX-1-A/BRX-1-B
6.
7.
8.
BRX-1-C/BRX-1-D/BRX-1-E/BRX-1-F
BRX-1-J/BRX-1-K/BRX-1-L/BRX-1-M
BRX-1-A/BRX-1-B
9.
10.
11.
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).
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Table-2: BRX-2 stage impurities LC-MS (RT/RRT) and LC-MS [M+H]+ (m/z) with proposed
chemical structures code
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S.No.
1.
LC-MS (~RT)
5.38
LC-MS (~RRT)
LC-MS [M+H]+ (m/z)
219.08
Proposed chemical structure(Code)*
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0.47
0.63
1.13
1.19
BRX-S3
BRX-B
BRX-A
2.
7.25
466.16
3.
12.96
450.25
4.
13.64
649.32
BRX-648A/BRX-648B/BRX-648C/BRX-
648D
5.
6.
15.82
17.38
1.38
1.52
706.42
649.32
BRX-705A/BRX-705B
BRX-648A/BRX-648B/BRX-648C/BRX-
648D
7.
8.
20.13
25.42
1.76
2.22
706.42
649.39
BRX-705A/BRX-705B
BRX-648A/BRX-648B/BRX-648C/BRX-
648D
*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
Br
Cl
Cl
Cl
O
N
O
O
N
O
O
N
O
Cl
BRX-1-B
[341.09]
BRX-1-A
[341.09]
BRX-1-C
[385.04]
Br
Cl
O
N
O
Br
Cl
O
N
O
O
N
O
Br
Cl
BRX-1-F
[385.04]
BRX-1-E
[385.04]
BRX-1-D
[385.04]
H
O
N
O
O
N
O
Br
OH
O
N
O
O
N
O
H
H
H
BRX-1-G10
[295.02]
6c
BRX-1-H11
[233.10]
BRX-1-I
[376.14]
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H
N
H
N
O
O
Cl
O
N
O
O
N
O
O
O
O
N
O
6
7
8
9
H
N
O
O
Cl
Cl
BRX-1-K
[466.16]
BRX-1-L
[466.16]
BRX-1-J
[466.16]
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Cl
O
N
O
N
S
N+
O-
O
N
H
O
H
N
O
O
BRX-A
[449.17]
BRX-1-M
[466.16]
O-
N+
S
S
S
N+
O-
N
N
O
N
H
O
N
N
O
N
O
BRX-B
[465.17]
BRX-705A
[705.31]
S
N
S
N
S
N
O
N
O
S
N
N
O
O
N
O
N
O
N
O
H
N
N
O
O
H
N
N
O
BRX-705B
[705.31]
BRX-648B
[648.27]
BRX-648A
[648.27]
S
H
N
O
O
O
N
O
N
N
N
O
N
O
N
H
N
O
O
S
BRX-648D
[648.27]
BRX-648C
[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.
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Table-3: HPLC analysis report of crude and purified samples for BRX-1 intermediate (As per
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innovator process)
UN
UN
UN
UN
BRX-1-I BRX-1-G
BRX-1-A
RRT
UN
RRT
1.77
m/z 466 m/z 466 BRX-1-B m/z 466 m/z 466
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Sample
RRT
0.81
RRT
1.07
RRT
1.23
RRT
1.28
RRT 1.47
RRT
1.49
RRT
1.50
1.72
BRX-1
(Crude)
BRX-1
(Purified)
2.59 %
0.42 %
4.08 %
5.23 %
0.28 % 0.53 %
0.19 % 0.27 %
3.07 %
0.24 %
0.23 % 0.37 %
0.01 % 0.15 %
3.18 %
0.27 %
0.43 %
0.02 %
UN: Unknown
Table-4: HPLC analysis report of crude and purified samples for BRX (As per innovator
process):
UNSHI
BRX-S3
RRT
0.66
BRX-1-I BRX-1
RRT
0.93
BRX-648B
RRT
BRX-705B
RRT
BRX-705A BRX-648A
*
RRT
1.11
Sample
RRT
1.15
RRT
1.41
RRT
1.43
1.23
1.34
BRX-2
(Crude)
BRX
0.26 %
ND
0.35 %
0.03 %
0.08 %
ND
0.22 %
0.18 %
0.31 %
0.12 %
0.71 %
0.27 %
0.57 %
0.26 %
0.93 %
0.01 %
(API)
UNSHI*: Unknown Single Highest Impurity (for information)
In BRX-1 (crude) sample analysis, several impurities were observed such as BRX-1-I, BRX-1-
G, 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
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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.
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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).
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Table-5: HPLC analysis report of crude and purified samples for BRX-1 intermediate (As per
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optimized process)
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Sample
BRX-1-I
BRX-1-G
BRX-1-B
BRX-1-A
UNSHI*
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BRX-1 (Crude)
0.50 %
0.64 %
3.36 %
3.51 %
2.11 %
0.04 %
1.34 %
0.08 %
0.13 %
0.03 %
BRX-1 (Purified)
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% (Table-
6) 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):
BRX-
648B
BRX-
705B
BRX-
705A
BRX-
648A
Sample
BRX-S3 BRX-1-I
BRX-1
UNSHI*
BRX-2 (Crude)
BRX-2(purified)
0.48
0.05
ND
0.21
0.12
0.01
0.62
0.19
0.01
0.02
0.02
0.02
0.02
0.01
0.01
0.09
0.04
0.02
0.07
0.05
0.01
0.42
0.08
0.02
BRX (API) purified)
UNSHI*: Unknown Single Highest Impurity (for information)
Justification of Impurities Formation in Process:
The stage-1 impurities named BRX-1-(A-G) may be observed in the process due to over-
alkylation/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
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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.
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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.
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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 BRX-
1-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.
c
a
b
Cl
Cl
OH
Cl
O
N
O
O
N
H
O
O
N
H
O
BRX-1
251.71
BRX-1-H
233.26
BRX-1-A
342.26
d
+
O
N
H
OH
Cl
BRX-S1
161.16
O
N
O
O
Cl
e
O
N
O
BRX-1-Bn
341.83
f
BRX-1-B
342.26
H
N
Br
O
O
Cl
O
N
H
O
O
N
BRX-1-I
BRX-1-G
296.16
H
376.41
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
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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.
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S
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N
N
S
H
N
N+
O-
S
N
O
O
O
N
O
O
N
H
O
c
e
BRX-A
S
BRX-648A
648.81
+
N
449.57
+
d
O-
N+
N
O
N
H
O
N
S
BRX
433.57
N
N+
O-
O
N
O
O
N
H
O
H
N
O
O
BRX-648B
648.81
BRX-B
465.57
S
S
S
a
b
N
N
N
S
N
N
N
NH+2
O
N
O
N
N
S
Cl-
O
N
O
N
BRX-705B
BRX-S3
254.78
BRX-705A
705.97
705.97
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, BRX-
B, 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
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monitor. IR spectra of samples were recorded on Shimadzu IR Affinity-I FT-IR
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spectrophotometer. H NMR spectra were recorded on Bruker AMX-400 spectrometer using
tetramethylsilane as an internal standard. 13C 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.
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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
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afford 7-(4-Chlorobutoxy)quinolin-2(1H)-one BRX-1 (6.6 g, 85 % yield) as a white solid. H-
NMR (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.
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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,
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2H), 1.83-1.76 (m, 2H), 1.67-1.62 (m, 2H). C-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.
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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.
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BRX-1-A: H-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.
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BRX-1-B: H-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
13
(t, J = 5.6 Hz, 2H), 3.75-3.71 (m, 4H), 1.99-1.72 (m, 8H). 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,
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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.
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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-bromo-
4-chlorobutane BRX-S2. 7-(4-Bromobutoxy)quinolin-2(1H)-one (BRX-1-G) was obtained as a
white solid with 82 % yield.
1H-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,
13
2H), 1.99-1.93 (m, 2H), 1.91-1.82 (m, 2H). C-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, cm-
1): 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 %
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yield) as a white solid. H-NMR (400 MHz, DMSO-d6): δ 11.58 (s, 1H), 7.81 (d, J = 9.2 Hz,
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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). 13C-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.
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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.
1H-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,
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2H), 6.81-6.79 (m, 4H), 6.31 (d, J = 9.2 Hz, 2H), 4.09 (bs, 4H), 1.92 (bs, 4H). 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(4-
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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 %
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yield) as a white solid. H-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,
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4H). 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
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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
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(m, 4H), 1.80-1.69 (m, 4H). 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}-1,2-
dihydroquinolin-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).
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The solvent was evaporated under vacuum and the oily mass crystallized in n-Hexane. BRX-
648A 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).
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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.
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BRX-648B: H-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,
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2H), 1.98 (bs, 4H), 1.87-1.79 (m, 7H). C-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-ium-
1-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) m-
Chloroperbenzoic 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).
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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).
1H-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.
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BRX-B: H-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), 7.56-
7.50 (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
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(m, 2H), 1.86 - 1.83 (m, 2H). 13C-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.
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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
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HPLC analytical reports of intermediates BRX-1 (crude), BRX-1 (pure), BRX-2 crude, purified
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and BRX. 1H-NMR and C-NMR of BRX-1, BRX, BRX-1-A, BRX-1-B, BRX-1-G, BRX-1-H,
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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.
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AUTHOR INFORMATION
*Corresponding Author
*Pramod Kumar
E-mail: pramodkumar@microlabs.in
Micro Labs Ltd., API R&D Centre, Bangalore 560105, Karnataka, India
*Rahul Tyagi
Email: rahulttyagi26@gmail.com
Micro Labs Ltd., API R&D Centre, Bangalore 560105, Karnataka, India
References:
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Graphical Abstract
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N
H
N
N
O
O
O
N
O
BRX-648A
S
S
S
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H
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BRX-648B
BRX-705B
O
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H
O
Brexpiprazole (API)
S
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N
O
N
O
BRX-705A
Brexpiprazole Critical Impurities Identification, Synthesis, Characterization and Control Strategy
in Process Development
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