In silico approach towards lipase mediated chemoenzymatic synthesis of (S)-ranolazine, as an anti-anginal drug

Article abstract of DOI:10.1039/c6ra06879k

An in silico modelling based biocatalytic approach for the synthesis of drugs and drug intermediates in enantiopure forms is a rationalized methodology over the organo-chemical routes. In this study, enzyme-ligand based docking was carried out using (RS)-ranolazine, as the model drug for the screening of a suitable biocatalyst for the kinetic resolution of the racemic drug. The differential interaction of the two enantiomers with the lipase was analyzed on the basis of docking score and H-bond interaction with the amino acid residues, which helped to define the trans-esterification mechanism. Ranolazine [N-(2,6-dimethylphenyl)-2-[4-(2-hydroxy)-3-(2-methoxyphenoxy)propylpiperazin-1-yl]acetamide], an anti-anginal drug, significantly reduces the frequency of anginal attack and has also been used for the treatment of ventricular arrhythmias, and bradycardia. Various lipases were examined via computational as well as wet lab screening and Candida antartica lipase in the form of CLEA was the most efficient one for the (S)-selective kinetic resolution of (RS)-ranolazine, with highest conversion and enantiomeric excess. This is the first report of the chemo-enzymatic synthesis of (S)-ranolazine where the whole drug molecule was used for lipase catalysis. The present study showed that the combination of in silico studies and a classical wet lab approach could change the paradigm of biocatalysis.

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In-silico approach towards lipase mediated chemoenzymatic
synthesis of (S)-Ranolazine, as an anti-anginal drug
Ganesh Sawanta, Saptarshi Ghosha, Sooram Banesha, Jayeeta Bhaumika and Uttam Chand
Banerjeea*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In silico modelling based biocatalytic approach for the synthesis of drugs and drug intermediates in enantiopure forms is a
rationalized methodology over the organo-chemical routes. In this study, enzyme-ligand based docking was carried out
using (RS)-ranolazine, as the model drug for the screening of a suitable biocatalyst for the kinetic resolution of the racemic
drug. The differential interaction of the two enantiomers with the lipase was analyzed on the basis of docking score and H-
bond interaction with the amino acid residues, which helped to define the trans-esterification mechanism. Ranolazine [N-
(2,6-dimethylphenyl)-2-[4-(2-hydroxy-3-(2-methoxyphenoxy)propylpiperazin-1-yl]acetamide], an anti-anginal drug,
significantly reduces the frequency of anginal attack and has also been used for the treatment of ventricular arrhythmias,
and bradycardia. Various lipases were examined via computational as well as wet lab screening and Candida antartica
lipase in the form of CLEA was the most efficient one for the (S)-selective kinetic resolution of (RS)-Ranolazine, with
highest conversion and enantiomeric excess. This is the first report of chemo-enzymatic synthesis of (S)-Ranolazine where
the whole drug molecule was used for lipase catalysis. The present study showed that the combination of in silico studies
and classical wet lab approach could change the paradigm of biocatalysis.
www.rsc.org/
Keywords: In silico, Biocatalysis, Ranolazine, Lipase, Enantiopure, Chemo-enzymatic
1. Introduction
Computational approach for the exploration of suitable For small molecule docking, various softwares are available
biocatalyst candidates for the enantioselective synthesis of such as GOLD, MOE, Molegro Virtual Docking, Maestro Glide
drugs and drug intermediates is an emerging field in and each one of them has different scoring algorithm5.
chemoenzymatic synthesis1. Docking of small molecules with According to various reports, biocatalysts are used in most of
the enzymes/receptors reveals the information about the the biotransformation reactions with high regio- and
binding nature and interactions with the enzyme residues. In stereoselectivity6-9
.
Enzymes are environment friendly
chiral compound synthesis, enantioselectivity of the alternatives over conventional chemical catalysts as they can
biocatalyst (enzyme) is a crucial factor and screening of operate under ambient temperature and pressure10,11. In
catalysts in terms of selectivity is also a major concern2. The pharmaceutical sector, the new biocatalytic approaches for
combination of enzyme kinetics and molecular modelling was the synthesis of drugs and drug intermediates are particularly
used to construct a model of how the enantiomers of appealing due to low cost and minimal use of toxic
secondary alcohols bind in the active site of this enzyme during chemicals8,12,13,14. Lipases belonging to the class of serine
transesterfication reactions3. The two enantiomers of a hydrolases (α/β hydrolases super family) can act on the
secondary alcohol are shown to bind in two different organic–aqueous interface and their industrial potential is also
orientations in the active site. The enantioselectivity is caused well
by the difference in the free energy gaps between the ground transesterification reaction of lipase through catalytic triad has
states of the enantiomers and the corresponding transition been already described by various researchers18,19
states, which the enantiomers form with the enzyme. Incorporation of in silico studies along with the conventional
known15,16,17
.
Notably,
the
mechanism
of
.
The thermodynamic ground states of the enantiomers are the biocatalytic approach reduces both time and production
same. Therefore, the enantioselectivity is determined by the expenses significantly. It will help to select suitable enzyme
difference in free energy between the two transition states4. preparations prior to the wet laboratory screening and
importantly will give access to determine the mechanism of
biocatalysis.
Ranolazine has been approved by US FDA in 200620 for the
a.Department of Pharmaceutical Technology (Biotechnology), National Institute of
Pharmaceutical Education and Research, Sec-67, S. A. S. Nagar-160062, Punjab,
INDIA.
*Corresponding author
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
treatment of chronic angina as it significantly reduces frequent
anginal attack and replaces the use of short acting
nitroglycerine21. According to the report of the American Heart
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Association, cardiovascular disease is the leading cause of synthesis was described with 90% yield (Lu et al., 2004)28. An
global mortality, accounting for 17.3 million deaths per year, improved process for the synthesis of (RS)-ranolazine with 68%
which is expected to reach to more than 23.6 million by yield was reported by Aalla et al29. These chemical synthetic
203022. In this study, Ranolazine [N-(2, 6-dimethylphenyl)-2-[4- methods involve costly catalysts and harsh reaction conditions,
(2-hydroxy-3-(2-methoxyphenoxy)propyl)piperazin-1-yl]
therefore they are neither cost-effective nor environment
acetamide], member of the ß blocker family used as an anti- friendly. Biocatalysis can be the only alternative to make the
anginal agent23 was selected as the model drug for the process benign and economical. The first chemoenzymatic
computational catalysis. Unlike other drugs used to treat synthesis of both enantiomers of ranolazine was reported by
chronic angina, ranolazine shows negligible effect on heart Moen et al. (2005)30. A multistep synthetic procedure of
rate and blood pressure. The major mechanism is believed to ranolazine was also reported with 70% yield31. There was a
be its inhibitory effect on late sodium influx during the cardiac report on water based chemistry for the synthesis of
potential of cardiomyocytes, which is particularly important in (RS)/(R)/(S)-ranolazine by Kommi et al. (2013) with 93% yield32.
ischemic or hypoxic condition24. By altering the intracellular Regioselective ring opening by tangstate mediated catalysis for
sodium level, ranolazine can affect the sodium-dependent the synthesis of ranolazine was also reported by Pathare et al.
calcium channels during myocardial ischemia. Thus, ranolazine (2013) with 85% yield33.
indirectly prevents the calcium overload and prevent cardiac Here we report a retrosynthetic pathway for (S)-ranolazine as
ischemia25. Of the two enantiomers, (S)-enantiomer showed shown in Figure 1. Lipase mediated kinetic resolution of the
the anti-anginal activity whereas the (R)-form showed anti- entire drug (also synthesized chemically) was carried out. This
diabetic activity26. The (S)-enantiomer is potentially stronger is the first instance where the complete drug has been used
and better anti-arrhythmic drug than either the (R)- for the resolution. In-silico approach has been used for the
enantiomer or racemic ranolazine26. Several chemical methods screening of lipases and to determine the enantioselective
were reported for the synthesis of (RS)-ranolazine or reaction mechanism. Overall, a combination of in silico and
enantiomers. A synthetic procedure continuing a single step biocatalytic approach afforded the enantiopure ranolazine as a
was reported by Li et al. (2003) for the synthesis of (RS)- breakthrough in the treatment of cardiovascular diseases.
ranolazine with 96% yield27. In another report, one step
Figure 1. Retrosynthetic pathway for the synthesis of (S)-ranolazine
2. Results and Discussion
2.1 Chemical synthesis of (RS)-ranolazine as a standard compound
The first building block of ranolazine synthesis, 2-chloro-N-
(2,6-dimethylphenyl) acetamide
reaction of 2,6-dimethylaniline
97% yield (Scheme 1). Further, to incorporate the piperazine
ring, compound was treated with piperazine which resulted
into the formation of product with 70% yield (Scheme 1). To
introduce the chiral center, N-(2,6-dimethylphenyl)-2-
(piperazin-1-yl)acetamide was treated with (RS)-epichloro-
2
was synthesized by the
Prior to optimizing and synthesizing the enatiopure ranolazine
through biocatalytic route, it was necessary to prepare the
standard compound via a chemical route. Recently a research
group has developed a chemical route for the synthesis of
enatiopure ranolazine. However, the major pitfall of the
method was the use of expensive catalysts.32 We modified the
chemical pathway to synthesize racemic ranolazine as
described below.
1
and chloroacetylchloride with
2
3
3
hydrin in the presence of SDS in water as a solvent which
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afforded product
4
with 70% yield (Scheme 1). This reaction is as a solvent, in the 3rd and 4th step of chemical synthesis makes
a good example of water based chemistry which makes the it attractive in terms of industrial synthesis as it makes the
process green. Likewise, the racemic form of the drug process cost-effective and environment friendly. The current
molecule was synthesized by treating compound
methoxyphenol in the presence of K2CO3 in water to afford with comparable yield.32
(RS)-Ranolazine with 75% yield (Scheme 1). The use of water
4 with synthetic method is simpler than the previously reported one
5
Scheme 1. First step: Synthesis of 2-chloro-N-(2, 6-dimethylphenyl) acetamide 2; Second step: Synthesis of N-(2, 6-dimethylphenyl)-2-piperazin-1-yl)acetamide 3;
Third step: Synthesis of (RS)-4 i.e. introduction of chiral center; Fourth step: Synthesis of (RS)-ranolazine 5
2.2 Computational approach for the screening of biocatalysts
Being outstanding biocatalysts, enzymes play dominant role in interaction for the transesterification reaction (Figure 3).
most of the chemicals reactions.34 To understand the The docking score is more negative for (S)-5
57.28] than the
enzymatic reaction mechanism towards chemical reaction and other enantiomer (R)-5 40.67], which clearly indicates better
form and selectivity for
A CLEA to form H-bond (3.12 Å), which is not an ideal
[-
[-
screening of the suitable biocatalysts, in silico docking interaction of the lipase with the (S)
techniques play an important role.35 Computational screening acylation.
of enzymes for biocatalytic reactions can open up a new
-
avenue for green catalysis as it will provide a rational behind
the study. Among a variety of docking tools available36, the
Molegro Molecular Viewer 2.5.0 (CLC Bio, Qiagen) program
was used for the screening of lipase, as the biocatalyst towards
the enantiopure synthesis of ranolazine. Docking of various
lipases was carried out with (RS)-5 as the ligand (Zinc
21984084) and molecular docking score and H-bond lengths
between the ligand and the various amino acid residues of the
enzymes were analyzed. The consequences showed varied
docking scores, interaction with the amino acid residues of the
typical enzyme and difference in H-bond lengths. Interestingly,
the R and S forms of the substrate interacted differently with
the same enzyme.1,37.Only specifically oriented substrate (R or
S) can be interacted significantly with the amino acids present
in active site of the enzyme due to distereomeric product
formation.
Figure 2. Interaction of (S)-5 with CAL-A lipase CLEA. The interaction of amino acid
residues of enzyme with the ligand was shown by dotted circle.
In the present study, the interaction of OH group at the chiral
centre of (RS)-5 with the amino acid and the distance of the H-
bond was considered for the screening of the preeminent
apposite enzyme for the kinetic resolution of (RS)-5. Out of 11
enzymes screened, the S form of (RS)-5 interacted significantly
to the amino acid (Glu 67) of Candida antartica lipase A CLEA
only, with H-bond length of 3.07
Å (Table 1). The Figure 2
represents, -OH group of the ligand (S)-5, interacted with the
carboxyl oxygen of C. antartica lipase A showing H-bond but in
the opposite direction, here the -OH group of the ligand (R)-5
interacted with the -NH2 group of Phe 435 in C. antartica lipase
Figure 3. Interaction of (R)-5 with CAL-A lipase CLEA. The interaction of amino
acid residues of enzyme with the ligand was shown by dotted circle.
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Candida cylindricea, C. rugosa CRL L1754, C. rugosa CRL 62316,
C. rugosa CRL 90860, Aspergillus niger, immobilized lipase in
sol-gel-AK from Pseudomoas cepacia, lipase acrylic resin from
C. antartica lipase A, lipozyme of Mucor meihei lipase, cross
linked enzyme aggregate of C. antartica lipase, Amano-30
lipase, and lipase from porcine pancreas were used for the
screening. The biocatalytic reaction was carried out in toluene
using vinyl acetate as the acyl donor (Scheme 2).
Scheme 2. Lipase catalyzed kinetic resolution of (RS)-ranolazine (5)
Various lipases were screened and cross-linked enzyme
aggregates (CLEA) of C. antartica lipase showed 53.55%
conversion and enantioselectivity of substrate was very low
(31.83%), which is better than the other lipases. To increase
the enantiomeric excess of both substrate and product,
optimization of various reaction parameters were carried out
Figure 4. Docking result showing interaction of different lipases with (S)-5:
(a) 2NW6, (b) 1LPN, (c) 1LPO, (d) 1GZ7, (e) 1CLE, (f) 1UZA
using CLEA of C. antartica lipase as the biocatalyst.
Table 2. Screening of lipase for the kinetic resolution of (RS)-ranolazine 5a
Name of lipase
eeS
(%)b
0.7
32
eeP
(%)c
2.0
28
Conversion
Ee
(%)d
27
54
46
15
31
9.0
69
39
69
54
42
C. cylindricea lipase
C. antartica lipase A CLEA
Aspergillus niger lipase
C. rugosa 62316
1.0
2.3
1.1
1.1
1.1
1.1
1.0
1.1
1.5
1.1
1.1
3.1
1.0
0.1
0.5
5.2
2.3
5.2
4.2
0.7
4.0
5.5
19
C. rugosa 90860
C. rugosa L-1754
5.0
2.3
4.0
2.3
3.6
1.0
Pseudomoas cepacia PCL
C. antartica acrylic resin
Amano-30 lipase
Figure 5. Docking result showing interaction of different lipases with (S)-5: (a)
3LIP, (b) 1PCN, (c) 3TGL, (d) 3WNB.
Porcine pancreatic lipase
Mucor meihei lipase
a(RS)-5 (10 mM) in toluene (800 µL) was treated with vinyl acetate (200 µL,
2.5 mM) at 30 °C in presence of lipase (15 mg).
This shows that how the interaction between the chiral
substrate (ligand) and the lipase determines the
enantioselectivity towards the synthesis of pure enantiomer.
Among the other lipases, C. antartica lipase acrylic resin and
Amano lipase have shown no significant interaction with the
bEnantiomeric excess of substrate calculated by using formula = (R-S)/(R+S)*100
cEnantiomeric excess of product calculated by using formula = (S-R)/(S+R)*100
dConversions were calculated from enantiomeric excess of substrate (eeS) and
product (eeP) using the formula: Conversion (C) = eeS/(eeS + eeP)
eE values were calculated using the formula: E = [ln (1 – C (1 + eeP)]/ [ln (1 –C (1 -
eeP)].
substrate (RS)-5 (Table 1) (Figures 4, 5).
Docking with Mucor meihei lipase showed highly positive
docking score, indicating poor binding and interaction with the
substrate (Table 1). Most of the other lipases showed similar
docking score for both the enantiomers, indicating the non-
Lipase mediated biocatalytic reactions are highly affected by
different physico-chemical parameters such as reaction
medium, reaction time, temperature, enzyme and substrate
concentration, solvent and type of acyl donor. The enantio-
selectivity of the enzyme changes under different reaction
conditions. To obtain the ideal reaction condition, effect of
various reaction parameters were studied. Post optimization,
the most suitable reaction condition was determined i.e.
reaction time, 48 h; temperature, 30 °C; enzyme concen-
tration, 30 mg/mL; substrate concentration, 5 mM (Table 3).
selective binding with the substrate (RS)-
5 and resulting in
non-selective acylation and low enantiomeric excess.
2.3 Screening of lipases for the kinetic resolution of (RS)-5
To support the in-silico data, wet lab screening was performed
with various commercial lipases to select the best enzyme on
the basis of enantioselectivity, for the kinetic resolution of
(RS)-
5 (Table 2). Among the different sources, lipase from
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Table 1. In-Silico docking study showing the interaction of different lipases with (S)/(R)-5
(S)-Ranolazine
(R)-Ranolazine
PDB IDa
Docking
scoreb
Steric
interaction
score
Interacting
residues with H-
bond distancec
Docking
scoreb
Steric interaction
score
Interacting
residues with H-
bond distancec
Lipase
Amano-30
2NW6
3GUU
-54.73
-57.28
-64.17
No interaction
-67.14
-40.67
-76.35
-46.18
No interaction
Candida antartica
-59.02
Asn63-3.034 Å;
Glu67-3.071 Å
Phe435-3.12 Å
lipase A CLEA
Lipase from
1LPN
1LPO
1GZ7
1CLE
1UZA
3LIP
-36.0
-36.19
-51.96
-60.34
-47.56
-48.69
-52.99
297.39
-63.44
-44.63
-35.89
-52.15
-65.74
-53.62
-50.27
-56.41
280.62
-73.36
Arg183 2.465Å;
Asn222-2.113Å
-33.94
-40.56
-47.13
-57.98
-49.88
-51.46
-47.81
226.7
-41.61
-45.6
Gln187-3.05 Å
Asp40-3.170 Å
C. rugosa 62316
Lipase from
Asn222-3.119 Å;
Trp221-2.652 Å
C. rugosa 90860
Lipase from
Asn351-3.367 Å;
Tyr299- 2.659 Å
-50.41
-62.22
-59.29
-58.23
-58.67
204.99
-77.76
Tyr299-3.009 Å;
Arg303-3.149 Å
C. rugosa L-1754
Lipase from
Ser209-2.526 Å
Ser209-2.515 Å
C. cylindricea
Lipase from
His113-3.282 Å;
Tyr124-3.385 Å
No interaction
Aspergillus niger
Lipase from
Asn239-3.106 Å;
Thr231-2.728 Å
Leu234-3.106 Å;
Ala226-1.730 Å
P. cepacia
Porcine pancreatic
lipase
1PCN
3TGL
3W9B
Asn81-2.267 Å;
Asn83-2.914 Å
Asn83-2.542Å
No interaction
No interaction
Lipase from
Ile193-3.133 Å
Mucor meihei
C. antartica lipase
No interaction
-66.71
in acrylic resin
aProtein Data Bank identity number of the lipase
bMolecular docking simulation searches through numerous potential binding modes of the ligand (ZINC 21984084) in the pocket denoted
as the score, which is calculated by the following formula: Score = Starget-ligand + Sligand
cH-bond distance of interacting amino acid residue of the enzyme and the ligand (ZINC 21984084)
reaction with solvents of different logP values (See supporting
information S2). The result showed significant role of reaction
medium on the enantioselectivity and conversion of the lipase.
Amongst the six different solvents screened, diethyl ether was
found to be ideal with conversion of 48% and both high eeS
(90%) and eeP (98%). Also, the enantiomeric ratio (E) is
significantly higher (323) for diethyl ether than the other
solvents. Hence, further lipase mediated reactions were
carried out using diethyl ether as the solvent (See supporting
information S3). The role of acyl donor in the kinetic resolution
Table 3. Optimum reaction condition for the kinetic resolution of (RS)-ranolazine 5a
Physico-chemical
parameters
Optimum
value
eeS
(%)b
eeP
(%)c
Conversion
(%)d
Reaction time
48 h
30 °C
73
76
76
69
100
100
100
100
42
43
43
41
Temperature
Substrate concentration
Enzyme concentration
5 mM
30 mg/mL
aKinetic resolution of (RS)-5 was carried out with Candida antartica lipase A CLEA
bEnantiomeric excess of substrate calculated by using formula = (R-S)/(R+S)*100
cEnantiomeric excess of product calculated by using formula = (S-R)/(S+R)*100
dConversions were calculated from enantiomeric excess of substrate (eeS) and
product (eeP) using the formula: Conversion (C) = eeS/(eeS + eeP)
of (RS)-5 was also studied and vinyl acetate (2.5 mM) was
found to be the efficient one with 48% conversion (eeS 90%,
eeP 99%). The enantiomeric ratio (E) was also higher (588) with
vinyl acetate over other acyl donors used.
The effect of solvent on the kinetic resolution of (RS)-
ranolazine
5 was studied by carrying out the biocatalytic
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3.3
Synthesis
of
N-(2,6-dimethylphenyl)-2-piperazin-1-yl)-
2.4 Synthesis of (S)-ranolazine through deacylation
acetamide (3)
After successful synthesis of the enantiopure acylated
For the second step, 2-chloro-N-(2,6-dimethylphenyl)-
acetamide was reacted with piperazine in methanol at 60 °C
to obtain the product with 70% practical yield. Recovered
derivative
enantiopure ranolazine. To synthesize (S)-ranolazine
acylated derivative was isolated after the biocatalytic
reaction. The acylated derivative, (S)- was deacylated in
6
, we focused on the synthesis of our target drug i.e.
2
5, the
3
6
product was further analyzed by GC-MS and NMR
spectroscopy. 1H NMR (CDCl3, 400 MHz) δ: 2.25(s, 6H), 2.68-
2.69 (d, J= 4 Hz, 4H), 2.97-2.99 (t, 4H), 3.18 (s, 2H), 7.10-7.12
(dd, J= 8 Hz, 3H), 8.67 (s, 1H); m/z: 247.95 (M = C14H21N3O).
6
aqueous K2CO3 at room temperature for 2 h (Scheme 3) which
afforded (S)-ranolazine.
O
CH3
CH3
3.4 Synthesis of (RS)-2-(4-(3-chloro-2-hydroxyphenyl)piperazin-1-
yl)-N-(2,6-dimethylphenyl)acetamide 4
NH
NH
K2CO3 MeOH
O
O
OH
O
N
N
RT,2 h
N
O
CH3
N
O
O
O
25-30 oC, 2 h
CH3
N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide 3 was trea-
(S)-6
(
S
)-5
ted with (RS)-epichlorohydrin in the presence of SDS using
water as solvent to obtain (RS)-2-(4-(3-chloro-2-hydroxy-
Scheme 3. Deacylation of the acylated derivative 6 for the synthesis of (S)-
Ranolazine
phenyl)piperazin-1-yl)-N-(2,6-dimethylphenyl)acetamide
4
5
with 70% isolated yield. Recovered product was analyzed by
GC-MS and NMR spectroscopy. 1H NMR (CDCl3, 400 MHz) δ:
8.63 (s, 1H), 7.06-7.14 (m, 4H), 4.57 (s, 1H), 3.84-3.88 (dd, J=
16 Hz, 2H), 3.68-3.70 (d, J= 8 Hz, 1H), 3.59-3.61 (dd, J= 8 Hz,
2H), 3.26–3.32 (m, 4H), 2.88 (brs, 4H), 2.82 (s, 1H), 2.26 (brs,
6H); m/z: 340.35 (M = C17H26ClN3O2).
3. Experimental
3.1 General experimental details
3.1.1 Reagents
(RS)-glycidol, (R)-glycidol, (S)-glycidol, 2-methoxyphenol,
isopropyl amine and the lipase preparations from Candida
antartica (CAL) in acrylic resin, C. rugosa 62316 (CRL 62316), C.
rugosa 90860 (CRL 90860), C. rugosa L-1754 (CRL L1754), C.
cylindracea (CCL), Aspergillus niger (ANL), cross linked enzyme
aggregates (CLEA) of C. antartica, Pseudomonas cepacia (PCL),
immobilized lipase from Mucor meihei (MML), and lipase from
porcine pancreas (PPL) was purchased from Sigma-Aldrich
(USA) and FlukaTM. Lipase AY “Amano”30 were purchased from
Amano Chem Ltd (USA). The analytical and commercial grade
solvents such as hexane, ethyl acetate etc. was procured from
various commercial sources. Solvents of HPLC grade such as
hexane and 2-propanol were obtained from J. T. Baker.
3.5 Synthesis of (RS)-ranolazine (5)
To obtain the final drug molecule, (RS)-2-(4-(3-chloro-2-
hydroxyphenyl)piperazin-1-yl)-N-(2,6-dimethylphenyl)
acetamide
presence of K2CO3 in water which resulted the synthesis of
(RS)-Ranolazine with 75% yield. Recovered product was
4 was further treated with methoxy phenol in the
5
characterized by GC-MS and NMR spectroscopy. 1H NMR
(CDCl3, 400 MHz) δ: 6.98–6.96 (m,3H), 6.86–6.77 (m, 4H),
4.06–4.02 (m, 1H), 3.89–3.86 (m, 1H),3.82–3.78 (m, 1H), 3.71
(s, 3H), 2.58 (brs, 8H), 2.55–2.63 (m, 4H) 2.25 (s, 6H); 13C NMR
(CDCl3, 100 MHz) δ: 168.41, 149.4,148.3, 134.97, 133.61,
128.32, 127.21, 121.97, 120.95, 114.78, 111.9,72.3, 66.0,
61.66, 60.53, 55.8, 53.8, 53.58, 18.67; m/z: 428.38 (M=
C24H33N3O4). The product was further characterized by chiral
HPLC analysis using chiralcel OD-H column (hexane:2-
propanol:: 9:1), tR = 15 min, tS = 22.1 min
3.1.2 Analysis
Reactions were analyzed by 1H NMR and 13C NMR spectra,
obtained with Bruker DPX 400 (1H 400 MHz and 13C 100 MHz),
and chemical shifts were expressed in δ units relative to the
tetramethylsilane (TMS) signal as an internal reference in
CDCl3. IR spectra (wave number in cm-1) were recorded on
Nicolet FT-IR impact 400 instrument. Analytical TLC of all the
reactions were carried out on Merck plates. SRL silica gel (60-
120 mesh) was used in column chromatography. The
enantiomeric excess (ee) was determined by HPLC (Shimadzu
LC-10AT 'pump, SPD- 10A UV-VIS detector) using a Chiralcel
OD-H column (0.46 mm x 250 mm; 5 µm, Daicel, Japan) at
254 nm, with mobile phase, hexane:2-propanol (9:1); flow
rate, 1 mL/min and column temperature of 25 °C.
3.6 In-silico study of screening of lipases for the kinetic resolution
of (RS)-5
The docking study of (RS)-5 with different lipases was executed
in the MVD programme using Docking wizard38. In this process,
initially eleven different lipases (2NW6, 3GUU, 1LPN, 1LPO,
1GZ7, 1CLE, 1UZA, 3LIP, 1PCN, 3TGL, 3W9B) were imported
from PDB (Protein Data Bank) and complexed small molecules
were removed. Prior to docking, the docking cavities were
recognized using the ‘Cavity Prediction’ module which utilizes
probe (size 1.20
Å) in a grid resolution of 0.80 Å, which
3.2 Synthesis of 2-chloro-N-(2,6-dimethylphenyl) acetamide (2)
expanded van-der-waals molecular surface representation to
specify docking site. MolDock scoring function and MolDock
Optimizer genetic algorithm was employed to search and score
dock poses. MolDock is an extended PLP based docking scoring
function with newly added H-bonding and electrostatic energy
terms to improve docking accuracy39. The best dock poses
were retrieved based on the lowest binding energy among
multiple poses criteria and after graphical evaluations. The
graphical illustrations of protein-ligand interactions were made
In the first step, 2, 6-dimethylaniline
chloroacetyl chloride at 4 °C in dichloromethane to obtain 2-
chloro-N-(2,6-dimethylphenyl)acetamide with 97% practical
1 was treated with
2
yield. Recovered product was further analyzed by GC-MS and
1
NMR spectroscopy. H NMR (CDCl3, 400 MHz) δ: 2.26 (s, 6H),
4.27 (s, 2H), 7.11-7.19 (m, 3H), 7.28 (s, 1H); 13C NMR (CDCl3,
100 MHz) δ: 18.32, 42.8, 127.92, 128.39, 132.67, 135.36,
164.38; m/z: 197.66 (M= C10H12ClNO).
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using the Molegro Molecular Viewer (MMV) 2.5.0 programme 3.9 Deacylation of (S)-6 for the synthesis of (S)-5
(CLC Bio, Qiagen Inc.)40. Amino acid residues of lipases involved A solution of K2CO3 (0.27 g, 2 mmol) in distilled water (1 mL)
in the interaction with the ligand were determined and was added separately to (S)-
respective H-bond lengths were calculated. Also, this the reaction mixture was kept under stirring for 2 h at room
interaction with amino acid residues helped to explain the temperature (30 C). After completion, the reaction mixture
reaction mechanism. The differential interaction of (R)/(S)- was extracted with EtOAc and water. The combined organic
6 (1 mmol) in methanol (5 mL) and
°
5
with the lipases were shown through the Molegro Molecular layer was dried over Na2SO4 and concentrated under vacuum
Viewer and it reveals the basis for the enantioselectivity of the to
biocatalyst.
obtain the crude, which was purified by column chromato-
graphy (hexane:ethyl acetate:: 8:2) on silica gel (100-200
3.7 Enantioselective transesterification of (RS)-5
Commercial lipases from different sources such as Candida mesh) to obtain (S)-
5 (Scheme 6).
antartica, C. rugosa 90860, C. rugosa 62316, C. rugosa L-1754, (S)-
5:
1H NMR (CDCl3, 400 MHz) δ: 6.98–6.96 (m, 3H), 6.86–
C. cylindracea, Aspergillus niger, porcine pancreas, AY-Amano 6.77 (m, 4H), 4.06–4.02 (m, 1H), 3.89–3.86 (m, 1H), 3.82–3.78
30 and the immobilized lipases like sol-gel-Ak from (m, 1H), 3.71 (s, 3H), 2.58 (brs, 8H), 2.55–2.63 (m, 4H) 2.25 (s,
Pseudomonas cepacia, immobilized lipozyme from Mucor 6H); 13C NMR (CDCl3, 100 MHz) δ: 168.41, 149.4,148.3, 134.97,
meihei, lipase acrylic resin from C. antartica CLEA, were used 133.61, 128.32, 127.21, 121.97, 120.95, 114.78, 111.9,72.3,
for kinetic resolution of (RS)-5. All the lipases were individually 66.0, 61.66, 60.53, 55.8, 53.8, 53.58, 18.67; m/z: 428.38 (M=
) 5 mM in C24H33N3O4); [α]D20= -3.1 (c 4.0, CHCl3). The product was
taken into different conical flasks (5 mL). Substrate (
5
0.8 mL toluene along with 0.2 mL vinyl acetate (2.5 mM), as further characterized by chiral HPLC using chiralcel OD-H
acyl donor was added into each flask. The flasks were then column (hexane:2-propanol:: 9:1), tS-5 = 22.1 min.
capped and kept in an incubator shaker at 37
48 h. Reactions were worked up after 48 h and the conversion Computational catalysis has become a rationalized approach
and enantiomeric excess were analyzed by HPLC. for in silico biocatalysts screening for the synthesis of
°C (200 rpm) for 4. Conclusion
The effect of different physico-chemical parameters such as enantiopure drugs and drug intermediates, as exemplified
organic solvents, reaction time, temperature, enzyme and here using ranolazine as a model drug. The differential
substrate concentrations, and acyl donors was studied to interaction of the two enantiomers with lipase, as determined
optimize the reaction condition for achieving the best by the docking score and H-bonding pattern, proved to be
conversion and highest enantiomeric excess (See supporting advantageous to select the suitable enzyme preparation for
information S1, S2, S3).
the kinetic resolution of (RS)-Ranolazine. After the in silico and
wet lab based screening, Candida antartica lipase CLEA was
3.8 Preparative-scale transesterification reaction of (RS)-5
The resolution of (RS)-5 was carried out in preparative scale found to be the most efficient catalyst for the resolution of
under the optimized condition. The transesterification was (RS)-Ranolazine with higher conversion and enantiomeric
performed with 5 mM substrate and C. antartica lipase A CLEA excess. Under the optimized reaction conditions, 48.5%
at 30 °C using vinyl acetate (2.5 mM) as acyl donor in 50 mL conversion with 80% enantiomeric excess was achieved which
toluene. After 48 h, the reaction mixture was filtered and led towards the green synthesis of (S)-ranolazine. This
enzyme preparation was washed with toluene when the combinatorial approach of in silico studies and biocatalysis to
transformation was 48.5% with eeS of 80%. The solvent was synthesize enantiopure ranolazine, which avoids the use of
evaporated under vacuum and resulting dried residue was toxic metal catalysts, proved to be a game changer in
subjected to flash chromatography (hexane:ethyl acetate::8:2). biocatalysis.
1
: H NMR (CDCl3, 400 MHz) δ: 8.7-8.68 (s, 1H), 7.28 (brs, 5. Acknowledgements
(S)-6
3H), 7.0-6.91 (dd, 4H), 5.36 (s, 1H), 4.24-4.18 (m, 2H), 3.88- GS acknowledges Department of Biotechnology, Govt. of India
3.87 (d, J= 4 Hz, 3H), 3.24-3.20 (d, J= 16 Hz, 2H), 2.71-2.61 (m, for providing financial support to carry out the work. SG
4H), 2.24 (brs, 6H), 2.09 (s, 2H), 1.59 (brs, 6H); m/z: 470.32 (M= acknowledges Indian Council of Medical Research, Govt. of
C26H35N3O5). The product was further characterized by chiral India for this work.
HPLC using chiralcel OD-H column (hexane:2-propanol:: 9:1), 6. References
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Graphical abstract