Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of their inhibitory activity against autotaxin enzyme

Article abstract of DOI:10.1016/j.bmc.2019.115216

Autotaxin (ATX), a glycoprotein (~125 kDa) isolated as an autocrine motility factor from melanoma cells, belongs to a seven-membered family of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP), and exhibits lysophospholipase D activity. ATX is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to produce the bioactive lipid lysophosphatidic acid (LPA), which is upregulated in a variety of pathological inflammatory conditions, including fibrosis, cancer, liver toxicity and thrombosis. Given its role in human disease, the ATX-LPA axis is an interesting target for therapy, and the development of novel potent ATX inhibitors is of great importance. In the present work a novel class of ATX inhibitors, optically active derivatives of 2-pyrrolidinone and pyrrolidine heterocycles were synthesized. Some of them exhibited interesting in vitro activity, namely the hydroxamic acid 16 (IC₅₀ 700 nM) and the carboxylic acid 40b (IC₅₀ 800 nM), while the boronic acid derivatives 3k (IC₅₀ 50 nM), 3l (IC₅₀ 120 nM), 3 m (IC₅₀ 180 nM) and 21 (IC₅₀ 35 nM) were found to be potent inhibitors of ATX.

Full text of DOI:10.1016/j.bmc.2019.115216

Journal Pre-proofs
Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of
their inhibitory activity against autotaxin enzyme
Dimitrios Triantafyllos Gerokonstantis, Aikaterini Nikolaou, Christiana
Magkrioti, Antreas Afantitis, Vassilis Aidinis, George Kokotos, Panagiota
Moutevelis-Minakakis
PII:
S0968-0896(19)31728-6
https://doi.org/10.1016/j.bmc.2019.115216
BMC 115216
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2019
11 November 2019
13 November 2019
Please cite this article as: D. Triantafyllos Gerokonstantis, A. Nikolaou, C. Magkrioti, A. Afantitis, V. Aidinis, G.
Kokotos, P. Moutevelis-Minakakis, Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of
their inhibitory activity against autotaxin enzyme, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.115216
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1
Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and
study of their inhibitory activity against autotaxin enzyme
Dimitrios Triantafyllos Gerokonstantis,† Aikaterini Nikolaou†, Christiana Magkrioti,§
Antreas Afantitis,# Vassilis Aidinis,§ George Kokotos,† Panagiota Moutevelis-
Minakakis†*
†Laboratory of Organic Chemistry, Department of Chemistry, National and
Kapodistrian University of Athens, Panepistimiopolis, Athens 15771, Greece
§Division of Immunology, Biomedical Sciences Research Center “Alexander
Fleming”, Athens 16672, Greece
#NovaMechanics Ltd, Nicosia 1065, Cyprus
*To whom correspondence should be addressed. P.M.M.; (phone) (30210) 7274484;
(fax) (30210) 7274761; (e-mail) pminakak@chem.uoa.gr.
Keywords: 2-pyrrolidinones; pyrrolidines; autotaxin; inhibitor
Abbreviations: DCU, dicyclohexylurea; DCC, dicyclohexylcarbodiimide; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; DCM, dichloromethane; THF,
tetrahydrofuran; TLC, thin-layer chromatography; EtOAc, ethyl acetate; EDC.HCl, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; HOBt, 1-
hydroxybenzotriazole; NHS, N-hydroxysuccinimide; Boc, t-butoxycarbonyl; Thr, S-
threonine; Asn, S-asparagine; TBDMS, t-butyldimethylsilyl.

2
Abstract
Autotaxin (ATX), a glycoprotein (~ 125 kDa) isolated as an autocrine motility factor
from melanoma cells, belongs to a seven-membered family of ectonucleotide
pyrophosphatase / phosphodiesterase (ENPP), and exhibits lysophospholipase D
activity. ATX is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to
produce the bioactive lipid lysophosphatidic acid (LPA), which is upregulated in a
variety of pathological inflammatory conditions, including fibrosis, cancer, liver
toxicity and thrombosis. Given its role in human disease, the ATX-LPA axis is an
interesting target for therapy, and the development of novel potent ATX inhibitors is
of great importance. In the present work a novel class of ATX inhibitors, optically
active derivatives of 2-pyrrolidinone and pyrrolidine heterocycles were synthesized.
Some of them exhibited interesting in vitro activity, namely the hydroxamic acid 16
(IC50 700 nM) and the carboxylic acid 40b (IC50 800 nM), while the boronic acid
derivatives 3k (IC50 50 nM), 3l (IC50 120 nM), 3m (IC50 180 nM) and 21 (IC50 35
nM) were found to be potent inhibitors of ATX.
1. Introduction
Autotaxin (ATX, also known as ENPP2) is an enzyme originally isolated in 1992 as
an autocrine motility factor from A2058 melanoma cells and is characterized as a 125
kDa glycoprotein.1 It belongs to a seven-membered family of ectonucleotide
pyrophosphatases/ phosphodiesterases (ENPPs), which are characterized by their
ability to catalyze the hydrolysis of pyrophosphate or phosphodiester bonds in
nucleotides.2 ATX is unique among the ENPPs since it acts as a lysophospholipase D
(lysoPLD), catalyzing the hydrolysis of lysophosphatidylcholine (LPC) into the
signaling lysophosphatidic acid (LPA) and choline.3, 4 The bioactive lipid mediator
LPA binds to specific G-protein-coupled receptors and activates the migration,

3
proliferation and survival of various cell types.5, 6 ATX is produced in various tissues
and is essential for vascular development, but it has also been involved in a variety of
chronic inflammatory conditions, such as arthritis and cancer, as summarized in a
number of review articles.7-12 ATX consists of two N-terminal somatomedin B-like
domains (SMB1 and SMB2), a central catalytic phosphodiesterase (PDE) domain, and
a C-terminal nuclease-like domain (NUC). A threonine (T210) residue in the active
site located in the PDE domain, along with two zinc ions, are responsible for its
hydrolytic catalytic activity.13 The crystal structure of mouse ATX was presented in
2011,14 while in the same year the structural basis of substrate discrimination and
integrin binding by ATX was reported.15 Several synthetic ATX inhibitors have been
developed both in academia and pharmaceutical industry, the majority of which are
derivatives of various heterocycles and have been summarized in a number of review
articles.16-19 Recently, a comprehensive overview of the most important series of
ATX inhibitors appeared in the literature,20 in which the structure activity relationship
and the mode of binding studies was carried out for the identification of advanced
leads. Among the most active ATX inhibitors, we refer to the boronic acid derivatives
and especially the most representative member (HA155),21-23 piperidine and
piperazine derivatives,24-27 2-oxo-pyrrolidine derivatives28 as well as hydroxamic acid
derivatives.29 Lastly, it was reported that the ATX inhibitor (GLPG1690) was well
tolerated in the phase I clinical trials and the results support its continued clinical
development as a novel treatment for idiopathic pulmonary fibrosis (IPF).30
The aim of our work was to develop novel ATX inhibitors, optically active
derivatives of 2-pyrrolidinone and pyrrolidine heterocycles, taking into consideration
the generally accepted standards that should be fulfilled for potentially
pharmacologically interesting compounds.31-33 The 2-pyrrolidinone template

4
considered as an essential pharmacophore group is present in known drugs for
patients with seizures, Alzheimer’s, senile dementia and other neurological problems
(piracetam), 34 for patients with respiratory failure (doxapram) 35 and other conditions.
Moreover, 2-pyrrolidinones incorporated in more complicated chemical structures
have shown significant pharmacological properties, including those associated with
the central nervous system.36 In recent years, we have designed and synthesized
optically active 2-pyrrolidinones, based on natural S-pyroglutamic acid, some of
which exhibited antihypertensive and anti-inflammatory activity. 37-40 S-pyroglutamic
acid is considered a unique chiral synthon and its asymmetric use is based on its two
differentiated carbonyls, the properties of which allow an extended derivatization on
the 5-membered ring of the starting compound and lead to a plethora of natural
products, e. g. domoic acid,41 an inhibitor of angiotensin converting enzyme (ACE)
for the treatment of hypertension,42 as well as to chemical auxiliaries in asymmetric
synthesis.43 The properties and applications of pyroglutamic acid as a versatile
building block in asymmetric synthesis have been extensively reviewed in the
literature.44-46
2. Results and Discussion
2.1. Chemistry
In this paper we present the synthesis of twenty nine, optically active compounds
derivatives of 2-pyrrolidinone, starting from the naturally derived S pyroglutamic acid
(2-oxotetrahydro pyrrol-5S-carboxylic acid) or its enantiomer (Table 1), as well as the
synthesis of six optically active compounds derivatives of pyrrolidine, starting from
the natural amino acid S proline or its enantiomer (Table 2). In these derivatives, the

5
2-pyrrolidinone heterocycle ring of the (S/R)-pyroglutamic acid or the pyrrolidine
heterocycle ring of the (S/R)-proline possess a benzyl type substituent at the N atom.
In most cases, the carboxylic group of the 5 position is coupled to an amidic bond
with a chain containing a benzyl- ether type substituent to which carboxylic groups
(11a-e, 40a-c), methyl ester (3a), sulfonamides (3f-h), boronic esters (3k-m),
imidazoles (13a, b), hydroxamic group (16), tetrazoles (17a,b, 41), triazole (19),
pyridine (20) and boronic acid (21) are attached, since several chemical structures, in
which these groups are incorporated, have shown appreciable inhibitory activity
against ATX.16-19 Our approach towards the synthesis of these compounds is depicted
in Schemes 1-16. In Scheme 1 the synthesis of the amides 3a-q is depicted. The N-
benzylated derivatives of S/R-pyroglutamic acid, prepared according to the literature,
were coupled using EDC.HCl, HOBt and the appropriate amount of Et3N with the
benzyl amines 2a-e, to afford the amides 3a-n and with benzyl amine to afford the
amides 3o, 3p. Under the same conditions the free S-pyroglutamic acid was coupled
with the amine 2a to afford the amide 3q.

6
O
i
NH2
N
O
HN
O
3a-n
a
+
+
R1
O
O
i
2a-d
, 2e
(as HCl salts)
N
OH
O
R2
O
R2
NH2
a
N
NH
O
R1
1a-f
4
R1
3o, 3p
O
N
O
H
a
NH
+
2a
O
N
O
OH
88%
O
5
3q
H
O
O
Reagents and Conditions: a) Et3N, HOBt, EDC.HCl in dry DCM, 0 oC to rt.
Scheme 1
Compound*
i
R1
H
R2
Yield %
1a47
1b48
1c49
1d50
S
R
S
S
H
Br
O
1e51
1f 51
3a
S
S
S
R
S
S
S
S
S
S
S
F
CF3
H
COOMe
COOMe
COOMe
COOMe
COOMe
SO2NH2
SO2NH2
SO2NH2
CN
91
76
78
97
87
93
75
72
90
3b
3c
H
Br
F
3d
3e
CF3
H
3f
3g
3h
3i
F
CF3
H

7
3j
S
S
F
CN
78
68
3k
H
O
B
B
B
O
O
3l
S
F
62
60
O
O
3m
R
H
O
3n
3o
3p
S
S
S
H
H
H
95
93
72
O
*References regarding the known compound
The synthesis of 4-benzylether benzyl amine derivatives 2a-d is depicted in Scheme
2.
NHBoc
NH2
NHBoc
a
b
O
97%
HO
7
HO
6
8a:
8b:
8c:
8d:
R2=COOMe, 91%
R2=SO2NH2, 95%
R2=CN, 97%
R2
Cl
NH3
R2=H, 88%
c
2a:
2b:
2c:
2d :
R2=COOMe, 97%
R2=SO2NH2, 97%
R2=CN, 99%
O
R2
R2=H, 98%
Scheme 2
Reagents and Conditions: a) Boc-OSu in DMF; b) methyl 4-(bromomethyl)benzoate or 4-(bromomethyl)
,
benzenesulfonamide or 4-(bromomethyl)benzonitrile or (bromomethyl)benzene K2CO3, 18-c-6, reflux in acetone,
overnight; c) 4N HCl in 1,4 Dioxane, in MeOH.
The 4-(aminomethyl)phenol (6) was selectively N-protected to the compound 7 using
Boc-OSu.52 The phenolic group of compound 7 was subsequently converted to benzyl
ether group using the appropriate benzyl bromide, K2CO3, 18-crown-6 as catalyst
with reflux in acetone overnight.50 More specifically, the commercially available
bromides methyl 4-(bromomethyl) benzoate, 4-(bromomethyl)benzonitrile and
bromomethyl benzene afforded the compounds 8a, 8c, 8d. 4-(bromomethyl)

8
benzenesulfonamide was prepared from the commercially available 4-methylbenzene
sulfonyl chloride according to the literature 53 and was used for the preparation of 8b.
After removal of the Boc protecting group under known acidic conditions, the desired
amines 2a-d were obtained as hydrochlorides.
The synthesis of free amine 2e was achieved as depicted in Scheme 3. The phenolic
group of compound 9 was converted to the corresponding benzyl ether, using the
appropriate commercially available bromide under the same conditions as described
in Scheme 2. The benzyl alcohol 10, was converted to the corresponding azide 54
which, without being isolated, was further converted to the amine 2e by the
Staudinger reaction.55
NH2
OH
b, c, d, e
a
OH
O
O
50%
O
O
HO
B
B
2e
10
9
O
O
Scheme 3
Reagents and Conditions: a) 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, K2CO3, 18-c-6, reflux
in acetone, overnight; b) MeSO2Cl, Et3N in DCM, 0 oC to rt; c) NaN3 in dry DMF, 55 oC, overnight; d) Ph3P in dry THF,
3 h; e) H2O overnight.
The carboxylic acids 11a-e were obtained after hydrolysis of the corresponding
methylesters (3a-e) and the acid 11f was obtained from 3q under standard alkaline
conditions (Scheme 4).

9
O
i
O
N
11a:
11b:
11c:
11d:
11e:
i:S, R1=H, 98%
i:R, R1=H, 100%
i:S, R1=Br, 90%
i:S, R1=F, 94%
i:S, R1=CF3, 85%
HN
a
3a-e
R1
11a-e
O
O
COOH
N
a
3q
H
NH
80%
O
O
11f
Scheme 4
Reagents and Conditions: a) 2N NaOH in MeOH.
COOH
The synthesis of derivatives 13a, b in which the imidazole moiety has been
incorporated is depicted in Scheme 5. The carboxylic acids 11a, d were converted to
the corresponding active esters using N-OH succinimide (NHS) and dicyclohexyl
carbodiimide (DCC) in dry THF. The intermediate active ester was further coupled
with histamine dihydrochloride in DMF using 0.3 M solution of Na2CO3 as base,
without being isolated. For purification, the crude product was directly protected with
Boc protecting group and then subjected to column chromatography according to the
literature.56 Deprotection of the compounds 12 a,b in acidic conditions afforded the
desired products 13a, b. For the synthesis of hydroxamic acid 16, the carboxylic acid
11a was coupled with the, commercially available O-(tetrahydro-2H-pyran-2-
yl)hydroxylamine (14) and the resulting compound 15 was further deprotected in
acidic conditions.

10
O
O
N
O
HN
N
HN
O
a, b, c
d
11a, d
O
R1
O
R1
13a:
13b:
R1=H, 95%
R1=F, 96%
12a:
12b:
R1=H, 76%
R1=F, 72%
O
HN
O
HN
N
N
Boc
Cl
HN
NH
O
O
N
N
O
HN
O
N
H
e
d
+
11a
H2N
O
94%
82%
O
14
O
15
16
O
O
HN
O
O
O
HN
OH
Scheme 5
Reagents and Conditions: a) NHS, DCC in dry THF, 2 h; b) Histamine dihydrochloride, 0.3 M Na2CO3 in DMF, 20
min; c) Boc2O, Et3N in DMF, overnight; d) 4N HCl in 1,4-dioxane, in MeOH; e) Et3N, HOBt, EDC.HCl, in dry DCM,
0 oC to rt.
For the insertion of the bio-isosteric tetrazole group of carboxylic group, the aromatic
nitriles 3i, j were reacted under conditions shown in Scheme 6, using microwave
irradiation.57
O
N
HN
O
a
3i, j
O
R1
17a:
17b:
R1=H, 91%
R1=F, 76%
N
N
N
N
H
Scheme 6
Reagents and Conditions: a) NaN3, NH4Cl in dry DMF, 20W, 90 oC, 1.5-2h.

11
In Scheme 7 the synthesis of triazole derivative 19, the pyridine derivative 20 as well
as the boronic acid derivative 21 are depicted. The compound 3n was catalytically
hydrogenated, and the resulting phenol 18 was converted to ethers 19, 20 and 21 using
the appropriate bromide. The 4-(bromomethyl)-1-phenyl-1H-1,2,3-triazole was
prepared from prop-2-yn-1-ol and azidobenzene, followed by PBr3 according to the
literature,58 while the 4-(bromomethyl) pyridin-1-ium bromide as well the (4-
(bromomethyl)phenyl)boronic acid were commercially available.
N
N
19:
20:
R=
R=
65%
O
O
N
Ph
N
N
b
HN
HN
O
O
N
70%
a
3n
21
c for
100%
18
19, 20, 21
OH
OH
21:
R=
72%
B
OH
OR
Scheme 7
Reagents and Conditions: a) H2, 10% Pd/C in MeOH, 3h; b) 4-(bromomethyl)-1-phenyl-1H-1,2,3-triazole or 4-
(bromomethyl)pyridin-1-ium bromide, K2CO3, 18-c-6, reflux in acetone, overnight; c) (4-(bromomethyl)phenyl)
boronic acid, K2CO3, reflux in acetone, overnight.
In derivatives 24 and 25, the substituents of positions 1 and 5 of pyroglutamic acid
were interchanged with respect to derivatives 11a and 17a, and their synthesis is
depicted in Scheme 8. The compound 3p was catalytically hydrogenated and the
phenolic derivative 22 was converted to the benzyl ethers 23a, b under conditions
previously described. Basic hydrolysis of compound 23a afforded acid 24, while its
bioisosteric tetrazole analog 25 was obtained from the aromatic nitrile 23b as shown
in Scheme 8.

12
O
O
O
O
O
O
N
N
N
b
a
HN
HN
22
HN
88%
3p
O
HO
O
O
23a:
23b:
R: COOMe, 92%
R: CN, 89%
R
O
O
O
N
N
d
HN
HN
23b
c
91%
23a
86%
O
O
24
25
N
HOOC
N
N
HN
Scheme 8
Reagents and Conditions: a) H2, 10% Pd/C in MeOH, 1.5 h; b) methyl 4-(bromomethyl)benzoate or 4-
(bromomethyl)benzonitrile, K2CO3, 18-c-6, reflux in acetone, overnight; c) 2N NaOH in MeOH; d) NaN3,
NH4Cl in dry DMF, 20W, 90 oC, 2 h.
The sulfonamide 28 was obtained by coupling the commercially available 4-
sulfamoyl benzoic acid (27) with amine 26. The latter was prepared from S-
pyroglutamic acid as previously reported.59
COOH
O
a
+
N
HN
N
NH2
O
O
83%
28
26
27
SO2NH2
SO2NH2
Scheme 9
Reagents and Conditions: a) HOBt, EDC.HCl, Et3N in dry DCM, 0 oC to rt.
The synthesis of compound 35 is depicted in Scheme 10. The O-TBDMS protected S-
pyroglutaminol derived from S-pyroglutamic acid 60 was N-benzylated using the
bromide 30.54 Product 31 was deprotected in acidic conditions and the resulting

13
alcohol 32 was converted to the tosylester 33. Nucleophilic attack of 33 by the
imidazole moiety together with Cs2CO3 in dry DMF at 55 oC afforded the product 34
in excellent yield,61 from which the desired compound 35 was obtained after basic
hydrolysis.
Br
O
O
a
b
N
N
H
29
+
N
OTBDMS
MeOOC
O
68%
92%
OTBDMS
O
OH
30
31
32
O
O
MeOOC
MeOOC
N
O
O
O
d
e
N
c
N
92%
88%
N
OTos
68%
N
O
O
O
N
N
33
34
35
HOOC
MeOOC
MeOOC
Scheme 10
Reagents and Conditions: a) NaH (60% in mineral oil) in dry THF, 0 oC to rt, overnight; b) 4N HCl in 1,4-dioxane in
AcOEt, 30 min; c) Tos-Cl, Et3N in DCM, 24 h; d) imidazole, Cs2CO3 in dry DMF, 55 oC, overnight; e) 2N NaOH in
MeOH.
Synthesis of the pyrrolidine derivatives in Table 2 is depicted in Schemes 11-16. The
secondary amino group of the S/R-proline (36a, b) methylester was benzylated using
bromomethyl benzene or 1-(bromomethyl)-4-fluorobenzene and Et3N as base to
afford the methylesters 37a-c. The carboxylic acids 38a-c obtained after basic
hydrolysis of these methylesters were coupled with the amines 2a or 2c to afford
compounds 39a-d. Data for the two known compounds, 37a62 and 38a63 are in
agreement with previously published data.

14
O
i
i
O
O
i
N
N
N
H
OH
c
O
i
O
N
O
a
b
N
Cl
H2
O
R1
R2
R1
36a, b
38a:
i: S, R1=H, 68%
37a:
37b:
37c:
i: S, R1=H, 78%
i:S, R1=F, 95%
i: R, R1=F, 72%
38b: :
i S, R1=F, 55%
38c:
39a:
39b: :
i: S, R1=H, R2=COOMe, 94%
i S, R1=F, R2=COOMe, 84%
R1
i: R, R1=F, 50%
39c:
39d:
i: R, R1=F, R2=COOMe, 86%
i:S, R1=H, R2=CN, 97%
Scheme 11
Reagents and Conditions: a) bromomethyl benzene or 1-(bromomethyl)-4-fluorobenzene, Et3N in DCM, 0 oC, 2-5 h; b)
or , Et3N, HOBt, EDC.HCl in dry DCM, 0 oC to rt.
2a 2c
4N NaOH in MeOH; c)
The desired carboxylic acids 40a-c were obtained by basic hydrolysis of the
corresponding methylesters 39a-c (Scheme 12) and the tetrazole derivative 41 from
the aromatic nitrile 39d under conditions already discussed (Scheme 13).
O
i
N
a
H
39a-c
N
O
COOH
40a:
40b:
40c:
i:S, R1=H, 65%
i:S, R1=F, 84%
i:R, R1=F, 90%
R1
Scheme 12
Reagents and Conditions: a) 4N NaOH in MeOH.
O
N
a
H
39d
N
O
55%
N
41
N
N
NH
Scheme 13
Reagents and Conditions: a) NaN3, NH4Cl in dry DMF, 20W, 90 oC, 2h.
The alternation of positions 1 and 5 of the two substituents in compounds 44a, b was
achieved by benzylating the amino group of (S)-N-benzylpyrrolidine-2-carboxamide

15
64 (42) using bromide 30 or 43 respectively (Scheme 14). The bromide 43 was
prepared from 4-(hydroxymethyl)phenol (Scheme 15).
N
Br
O
O
O
N
HN
a
H
+
HN
R2
O
42
44a:
44b:
R2=COOMe, 56%
R2=CN, 64%
R
30:
43:
R=COOMe
R=CN
Scheme 14
Reagents and Conditions: a) Et3N in DCM, 0 oC to rt, overnight.
OH
Br
OH
a
b
O
O
HO
88%
96%
45
43
9
NC
NC
Scheme 15
Reagents and Conditions: a) 4-(bromomethyl)benzonitrile, K2CO3, 18-c-6, reflux in acetone, overnight; b) PBr3 in dry
DCM, 0 oC to rt, 3h.
The desired carboxylic acid 46 was obtained after basic hydrolysis of 44a and its
bioisosteric analog 47 from the corresponding nitrile 44b as depicted in Scheme 16.
O
O
N
N
HN
HN
a
b
44a
44b
47
72%
91%
O
O
46
HOOC
N
N
N
N
H
Scheme 16
Reagents and Conditions: a) 4N NaOH in DCM:MeOH 9:1; b) NH4Cl, NaN3 in dry DMF,
20W, 90 oC, 2h.

16
2.2. Biology
All synthesized pyrrolidinone and pyrrolidine derivatives were tested for their in vitro
activity on recombinant mouse ATX using 65, 66 LPC (16:0) as a substrate and PF-
8380 as a well-established ATX inhibitor.24 The method employed the fluorogenic
H2O2 probe Amplex Red reagent (ThermoFischerScientific) and a tri-partite
enzymatic reaction, as explained in Materials and Methods. The calculated IC50s are
presented in Tables 1 and 2. The molecular weights of the new synthetic compounds
as well as the ClogP values, calculated using ChemOffice Ultra 11.00, are included in
Tables 1 and 2 since reasonable molecular weights (<500) and lipophilicity (ClogP<5)
are important physicochemical properties which indicate the potential of an inhibitor
as a possible drug.31
Table 1. In Vitro Potency of 2-Pyrrolidinones Based on (S) or (R)- Pyroglutamic acida
16:0
Compound
Structure
IC50
ClogPb
MW
(μΜ)
O
N
H
O
N
COOH
COOH
11a
4.5-5
3.77
458.51
O
O
N
H
O
N
11b
11c
>5
3.77
4.64
458.51
537.41
O
O
N
H
O
N
COOH
>5
O
Br

17
O
N
N
H
O
COOH
11d
11e
>5
>5
3.92
4.66
476.50
526.51
O
F
O
N
H
O
N
COOH
O
F3C
NH
O
N
H
11f
3a
>5
>5
1.62
4.00
368.39
472.54
O
O
COOH
O
N
N
H
O
O
O
O
O
N
H
N
3f
>5
>5
2.19
2.34
493.58
511.57
O
O
O
S
NH2
O
O
N
H
O
O
N
S
3g
O
NH2
O
F
O
N
H
O
O
N
S
NH2
3h
>5
3.08
561.58
O
O
F3C

18
O
N
O
O
H
B
N
3k
0.05
0.12
0.18
5.56
5.70
5.56
540.47
558.46
540.47
O
O
O
N
H
O
O
B
N
O
3l
O
F
O
N
H
N
O
3m
O
B
O
O
O
N
H
O
N
3n
>5
>5
4.04
2.45
414.50
588.10
O
O
H
N
H
Cl
NH
N
H
N
O
N
13a
O
O
O
N
H
H
H
N
O
Cl
N
N
NH
13b
>5
1.95
2.14
606.09
473.53
O
O
F
O
N
H
H
O
N
16
0.70
N
OH
O
O

19
O
N
N
N
N
O
H
NH
N
17a
17b
>5
>5
3.53
3.68
482.54
500.53
O
O
N
H
N
O
N
O
N
N
NH
F
O
N
N
H
O
N
N
N
19
20
21
>5
>5
3.58
2.53
3.48
481.56
415.49
458.32
O
O
N
O
N
H
N
O
O
N
OH
O
H
B
OH
N
0.035
O
O
N
O
O
HOOC
HN
24
25
>5
>5
3.77
3.53
458.51
482.54
O
N
N
N
O
O
HN
N
HN

20
O
N
O
H
S
N
NH2
28
>5
1.00
387.45
O
O
O
O
N
34
35
>5
>5
2.59
2.37
419.48
405.45
N
N
O
N
N
O
O
N
O
HOOC
O
N
H
N
3o
>5
>5
2.34
1.17
308.38
324.38
O
O
N
H
OH
N
18
O
aAmplex Red PLD assay.
bCalculated using ChemDraw.
The optically active carboxylic acid 11a, derivative of 2-pyrrolidinone, was tested
first and presented a modest inhibitory activity (IC50 4.5-5 μΜ). Comparison with its
enantiomer 11b (IC50 > 5μM) clearly indicates that the S- absolute configuration is
preferable for inhibitory activity. By insertion of para-bromo, fluoro and
trifluoromethyl benzyl moieties as N-substituents, compounds 11c, 11d, 11e,
respectively, were obtained with enhanced lipophilicity, but none of them presented
any appreciable inhibitory activity. The same result was obtained by compound 3a,
the methyl ester of 11a. In order to investigate the structure-activity relationship, the
carboxylic moiety was replaced with other functionalities including sulfonamides (

21
3f-h), boronic esters (3k-m), imidazoles (13a, b), hydroxamic acid (16), the bio-
isosteric group of tetrazole (17a, b), triazole (19), pyridine (20) and boronic acid (21).
Among all these derivatives of 2-pyrrolidinone, the boronic esters 3k (IC50 0.05 μΜ),
3l (IC50 0.12 μΜ), 3m (IC50 0.18 μΜ) and especially the less lipophilic derivative of
boronic acid 21 (IC50 0.035 μΜ), proved to be potent inhibitors of ATX. It should be
mentioned again that a series of boronic acid-based derivatives belong to the first
ATX inhibitor classes identified and have extensively been studied. The lower IC50
value of compound 3m, the enantiomer of 3k, again indicated the significance of the
S-configuration of the stereogenic center for inhibitory activity. By interchanging the
position of two substituents of the 2–pyrrolidinone ring, namely the benzylic group of
N-position and the carboxylic chain linked by an amidic bond at the 5 position (11a)
or its bio-isosteric tetrazole (17a), compounds 24, 25 were obtained respectively
(Table 1). Compound 24 showed decreased inhibitory activity compared to that of
11a, while compound 25 was inactive. In the case of compounds 11f, 3n, 3o, 18 with
reduced structure compared to 11a, as well as of sulfonamide 28 and of imidazole
derivatives 34, 35 in which the amidic bond at position 5 was omitted, no inhibitory
activity was observed. Interestingly, the hydroxamic acid 16 showed appreciable
inhibitory activity (IC50 0.70 μM). It should be mentioned that hydroxamic acids as
potent ATX inhibitors with very promising in vivo efficacy were recently reported in
an IPF model.29 Among the derivatives of pyrrolidine based on R/S proline,
compound 40b exhibited interesting inhibitory activity (IC50 0.80 μΜ) (Table 2),
while its enantiomer 40c was inactive. This result is in accordance with other
compounds mentioned above, once again indicating the importance of absolute
configuration of the stereogenic center of inhibitors.
Table 2. In Vitro Potency of Pyrrolidines Based on (S) or (R)- Prolinea

22
16:0
IC50
Compound
Structure
ClogPb
MW
(μΜ)
O
N
H
O
COOH
444.5
3
N
40a
>5
0.80
>5
2.75
O
N
H
O
N
462.5
2
COOH
40b
40c
2.89
F
O
N
H
O
N
462.5
2
COOH
2.89
F
O
N
H
468.5
6
O
N
N
N
NH
41
46
47
>5
>5
>5
4.78
2.75
4.78
N
N
O
O
444.5
3
HN
HOOC
N
O
N
O
468.5
6
HN
HN
N
N
aAmplex Red PLD assay.
bCalculated using ChemDraw.
2.3. Binding Mode of Inhibitor 21 to ATX

23
Molecular Docking studies were performed to elucidate the binding mode and
interactions of the most potent inhibitor 21. The nanomolar boronic-type ATX
inhibitor HA155 with the crystal structure of ATX (PDB code 2XRG) was employed
as a template for our docking studies. The functionality of inhibitor 21 interacts with
Thr209 and Asn230 (Figure 1). The structure based simulations suggest a similar
binding energy for 21 (-118.674) compared to HA155 (-105.036).15
Figure 1. Hydrogen bond interactions between ATX and inhibitor 21.
3. Conclusions
In this study we describe the synthesis of a new class of optically active compounds
that possess a 2-pyrrolidinone and pyrrolidine template and assess their in vitro
inhibitory activity against ATX. Among the 2-pyrrolidinone derivatives, three
compounds bearing the acidic 4,4,5,5 tetramethyl-1,2,3-dioxoborolane moiety, namely
3k (IC50 0.05 μΜ), 3l (IC50 0.12 μΜ), 3m (IC50 0.18 μΜ) and especially the boronic
acid derivative 21(IC50 0.035 μΜ), presented inhibitory potency for ATX at the
nanomolar level. Importantly, the most active compound 21 has beneficial
physicochemical properties in comparison to inhibitors 3k, 3l and 3m, with
lipophilicity ClogP 3.48 (<5) and molecular weight 458.32 (<500), thus fulfilling
Lipinski’s rules.31 These four inhibitors have a number of groups that can act as

24
hydrogen bond donors (<5), but also groups that can act as hydrogen bond acceptors
(<10). Molecular docking studies showed similar binding energy for 21 compared to
the known ATX-inhibitor, HA155. Furthermore, SAR studies revealed that
replacement of the boronic acid moiety of 21 by carboxylic group led to the compound
11a with modest inhibitory activity (IC50 4.5-5 μΜ), while the hydroxamic acid 16 (IC50
0.70 μΜ) was appreciably inhibitory. Among the pyrrolidine derivatives, compound
40b also presented interesting inhibitory activity (IC50 0.80 μΜ).
4. Experimental
4.1. Instruments and materials
All chemicals and solvents were reagent grade and used without purification. Dry
THF and extra dry DMF (99.8%) over molecular sieves were purchased from Acros.
Chromatographic purification of products was accomplished using Merck Silica Gel
60 (70−230 or 230−400 mesh). Thin layer chromatography (TLC) was performed on
Silica Gel 60 F254 aluminum plates. TLC spots were visualized with UV light or
phosphomolybdic acid in EtOH. Melting points were determined on a Büchi 530
apparatus and are uncorrected. 1H, 13C and 19F NMR spectra were recorded on a
Varian Mercury (200, 50 and 188 MHz respectively) in CDCl3, CD3OD, D2O and
DMSO-d6. Chemical shifts are given in ppm and coupling constants (J) in Hz. Peak
multiplicities are described as follows: s, singlet, d, doublet, t, triplet, and m,
multiplet. Specific rotations were measured at 25 °C on a Perkin–Elmer polarimeter
using a 10 cm cell. Electron spray ionization (ESI) mass spectra were recorded on a
Finnigan, Surveyor MSQ Plus spectrometer. HRMS spectra were recorded on a
Bruker Maxis Impact QTOF Spectrometer. For microwave irradiation reactions, a
CEM-Discover device was used. Dichloromethane was dried by standard procedures
and stored over molecular

25
sieves. All other solvents and chemicals were reagent grade and used without further
purification.
4.2. Synthesis
Carboxylic acids 1a-f, 38a, 4-(bromomethyl)benzenesulfonamide, 4-(bromomethyl)-
1-phenyl-1-H-1,2,3-triazole, 29, 30 and Boc-OSu were synthesized as described
elsewhere, and their analytical data are in accordance with literature. Benzyl bromide,
methyl 4-(bromomethyl)benzoate, 4-(bromomethyl)benzonitrile, 2-(4-(bromomethyl)
phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 4-(bromomethyl) pyridine-1-ium
bromide, (4-(bromomethyl)phenyl)boronic acid as well as compound 14 are
commercially available.
4.3. Compounds 3o-q, 15, 28, 39a-d: General procedure
To a stirred solution of the carboxylic acid (1.0 mmol) in anhydrous CH2Cl2 (5 mL),
the appropriate amine or its hydrochloric acid (1 mmol), Et3N (1 eq, 0.14 mL, 1 mmol
or 2 eq, 0.28mL, 2 mmol in case of hydrochloric acid), HOBt (1 eq, 140 mg, 1 mmol)
and EDCI (1.1 eq, 211 mg, 1.1 mmol) were added at 0 oC. The reaction mixture was
stirred overnight at room temperature and then the solvent was then removed under
reduced pressure. The residue was dissolved in AcOEt and washed with H2O, 5%
H2SO4, H2O, 5% NaHCO3, brine and H2O. In the case of proline washing with 5%
H2SO4, was omitted. The product was obtained after drying the organic phase over
anhydrous Na2SO4, evaporation of the solvent under reduced pressure, and
purification by column chromatography.
4.3.1. (S)-methyl 4-((4-((1-benzyl-5-oxopyrrolidine-2-
carboxamido)methyl)phenoxy)methyl) benzoate (3a). Prepared from 1a and 2a.
Eluent: AcOEt-petroleum ether (40-60 oC) 9:1. White solid; yield 91%; mp: 137.6-
138.4 oC; [α]D +11.8o (c 1.01, MeOH); Rf (AcOEt) 0.24; 1H NMR (CDCl3) δ 8,04-

26
8,00 (d, J = 8.2 Hz, 2H, Ar-H), 7.49-7.45 (d, J = 8,2 Hz, 2H, Ar-H), 7.26-7.09 (m, 7H,
Ar-H), 6.90-6.86 (d, J = 8.4 Hz, 2H, Ar-H), 6.53 (br s, 1H, NH), 5.08 (s, 2H, OCH2),
5.00-4.93 (d, J = 14.8 Hz, 1H, NCHH), 4.42-4.19 (m, 2H, NHCH2), 3.93-3.75 (m, 5H,
CH3 & CH & NCHH), 2.51-2.15 (m, 3H, COCH2 & CHH), 2.05-1.98 (m, 1H, CHH);
13C NMR (CDCl3) δ 175.86, 170.97, 166.88, 157.99, 142.10, 135.75, 130.61, 129.95,
129.73, 129.35, 128.89, 128.45, 127.96, 126.99, 115.10, 69.36, 60.68, 52.27, 45.76,
43.01, 29.81, 23.56; HRMS exact mass calculated for [M+Na]+ (C28H28N2NaO5)
requires m/z 495.1890, found m/z 495.1888; Anal. Calcd for C28H28N2O5: C, 71.17; H,
5.97; N, 5.93. Found: C, 71.49; H, 5.76; N, 6.07.
4.3.2. (R)-methyl 4-((4-((1-benzyl-5-oxopyrrolidine-2-carboxamido)methyl)
phenoxy)methyl) benzoate (3b). Prepared from 1b and 2a. Eluent: gradient from
AcOEt-petroleum ether (40-60 oC) 9:1 to AcOEt 100%. White solid; yield 76%; mp:
138.2-139.6 oC; [α]D -12.6o (c 1.04, MeOH); Rf (AcOEt) 0.24; 1H NMR (CDCl3) δ
8.01-7.98 (d, J = 8.4 Hz, 2H, Ar-H), 7.46-7.42 (d, J= 8.4 Hz, 2H, Ar-H), 7.25-7.04
(m, 7H, Ar-H), 6.94 (br s, 1H, NH), 6.88-6.83 (d, J= 8,8 Hz, 2H, Ar-H), 5.04 (s, 2H,
OCH2), 4.97-4.89 (d, J = 14.8 Hz, 1H, NCHH), 4.39-4.21 (m, 2H, NHCH2), 3.85-3.70
(m, 5H, CH3 & CH & NCHH), 2.45-2.13 (m, 1H, COCH2 & CHH), 2.04-1.93 (m, 1H,
CHH); 13C NMR (CDCl3) δ 175.72, 170.97, 166.79, 157.81, 142.07, 135.73, 130.74,
129.83, 129.59, 129.20, 128.74, 128.31, 127.79, 126.89, 114.94, 69.22, 60.42, 52.15,
45.52, 42.83, 29.73, 23.39; MS (ESI) m/z (%) 473.16 [ (M+H+), 100]; Anal. Calcd for
C28H28N2O5: C, 71.17; H, 5.97; N, 5.93. Found: C, 71.32; H, 5.86; N, 5.98.
4.3.3 (S)-methyl 4-((4-((1-(4-bromobenzyl)-5-oxopyrrolidine-2-carboxamido) methyl)
phenoxy)methyl)benzoate (3c). Prepared from 1c and 2a. Eluent: AcOEt-MeOH 9:1.
White solid; yield 78 %, mp: 146.1-148.3 oC, [a]D +2,1 ο (c 1.10, CHCl3); Rf
(AcOEt-MeOH 9:1) 0.53; 1H-NMR (CDCl3) δ 8.05-8.01 (2H, d, J = 8.2 Hz, Ar-H),

27
7.50-7.46 (2H, d, J = 8.2 Hz, Ar-H), 7.39-7.34 (2H, d, J = 8.4 Hz, Ar-H), 7.15-7.11
(2H, d, J = 8.6 Hz, Ar-H), 7.00-6.96 (2H, d, J = 8.0 Hz, Ar-H), 6.93-6.88 (2H, d, J =
8.4 Hz, Ar-H), 6.27 (1H, br s , NH), 5.11 (2H , s, OCH2), 4.90-4.83 (1H, d, J =14.8
Hz, NCHH), 4.43-4.19 (2H, m, NHCH2), 3.89 (3H, s, CH3), 3.83-3.76 (2H, m, NCHH
& CH), 2.52-2.17 (3H, m, COCH2 & CHH), 2.07-1.97 (1H, m, CHH); 13C-NMR
(CDCl3) δ 176.52, 171.40, 167.54, 158.79, 142.75, 135.57, 132.67, 131.14, 130.88,
130.64, 130.47, 130.05, 127.67, 122.64, 115.88, 70.10, 61.44, 52.91, 45.91, 43.80,
30.39, 24.28; ΜS (ESI) m/z (%) 549.11 [(M-H-), 100]; Anal. Calcd for C28H27BrN2O5:
C, 60.99; H, 4.94; N, 5.08. Found: C, 60.72; H, 5.02; N, 4.97.
4.3.4. (S) –methyl 4-((4- ((1-(4-fluorobenzyl) -5- oxopyrrolidine -2- carboxamido)
methyl)phenoxy) methyl) benzoate (3d).Prepared from 1e and 2a. Recrystallized from
MeOH. White solid; yield 84%; mp: 149-151 oC; [α]D +4.6 (c 1.00, DMF); Rf
(AcOEt) 0.59; 1H NMR (DMSO-d6) δ 8.57 (1H, br s, NH), 8.00-7.96 (2H, d, J = 8.0
Hz, 2H, Ar-H), 7.60-7.56 (2H, d, J = 8.2 Hz, Ar-H), 7.17-6.97 (8H, m, Ar-H), 5.21
(2H, s, OCH2), 4.83-4.75 (1H, d, J = 15.2 Hz, NCHH), 4.29-4.09 (2H, m, NHCH2),
3.94-3.85 (4H, m, CH & CH3), 3.74-3.67 (1H, d, J = 15.2 Hz, NCHH), 2.39-2.11 (3H,
m, COCH2 & CHH), 1.87-1.74 (1H, m, CHH); 13C NMR (DMSO-d6) δ 174.82,
170.74, 166.03, 161.47 (d, J = 241.45 Hz), 157.12, 142.80, 132.71 (d, J = 3.00 Hz),
131.47, 129.81 (d, J = 8.2 Hz), 129.37, 128.92, 128.72, 127.44, 115.31 (d, J = 21.15
Hz), 114.75, 68.46, 59.16, 52.20, 43.78, 41.62, 29.36, 22.85; 19F NMR (DMSO-d6) δ -
115.88; MS (ESI) m/z 491.36 [(M+H+), 100]; Anal. Calcd for C28H27FN2O5: C, 68.56;
H, 5.55; N, 5.71. Found: C, 68.72; H, 5.64; N, 5.38.
4.3.5. (S)-methyl 4-((4-((5-oxo-1-(4-(trifluoromethyl)benzyl)pyrrolidine-2-
carboxamido) methyl)phenoxy)methyl)benzoate(3e). Prepared from 1f and 2a. Eluent:
AcOEt-MeOH 9:1. White solid; yield 87%; mp: 169.2-171.4 oC; [α]D +3.6 (c 1.10,

28
CHCl3); Rf (AcOEt) 0.39; 1H NMR (CDCl3) δ 7.99-7.95 (2H, d, J = 7.8 Hz, Ar-H),
7.50-7.42 (4H, m, Ar-H), 7.20-7.16 (2H, d, J = 8.0 Hz, Ar-H), 7.13-7.09 (2H, d, J =
8.2 Hz, Ar-H), 6.88-6.84 (3H, m, Ar-H & NH), 5.05 (2H, s, OCH2), 4.98-4.91 (1H, d,
J = 15.0 Hz, NCHH), 4.31-4.26 (2H, m, NHCH2), 3.84-3.77 (5H, m, CH3 & CH &
NCHH), 2.53-2.40 (1H, m, COCHH), 2.35-2.16 (2H, m, COCHH & CHH), 2.07-1.97
(1H, m, CHH); 13C NMR (CDCl3) δ 175.87, 170.67, 166.87, 157.95, 142.07, 140.00,
130.00 (q, J = 23.3 Hz), 130.58, 129.87, 129.64, 129.22, 128.61, 126.92, 125.69 (q, J
= 3.5 Hz), 121.27, 115.04, 69.26, 60.50, 52.18, 45.12, 42.96, 29.58, 23.46; 19F NMR
(CDCl3) δ -62.88; MS (ESI) m/z (%) 541.38 [(M+H+), 100]; Anal. Calcd for
C29H27F3N2O5: C, 64.44; H, 5.03; N, 5.18. Found: C, 64.56; H, 4.91; N, 5.23.
4.3.6.(S)-1-benzyl-5-oxo-Ν-(4-(4-sulfamoylbenzyloxy)benzyl)pyrrolidine-2-
carboxamide (3f). Prepared from 1a and 2b. Eluent: AcOEt-MeOH 9:1. White solid;
yield 93 %; mp: 182.2-184.6 oC; [a]D -5.8 ο (c 1.00, DMF); Rf (AcOEt-MeOH 9:1)
1
0.57; H NMR (DMSO-d6) δ 8.57 (1H, br s, NH), 7,85-7,81 (2H, d, J = 8.2 Hz, Ar-
H), 7.63-7.60 (2H, d, J = 7.8 Hz, Ar-H), 7.36-6.97 (9H, m, Ar-H), 6.74 (2H, br s,
NH2), 5.20 (2H, s, OCH2), 4.90-4.82 (1H, d, J = 15.8 Hz, NCHH), 4.28-4.12 (2H, m,
NHCH2), 3.95-3.88 (1H, m, CH), 3.73-3.65 (1H, d, J = 15.4 Hz, NCHH), 2.36-2.11
(3H, m, COCH2 & CHH), 1.90-1.78 (1H, m, CHH); 13C NMR (DMSO-d6) δ 174.84,
170.83, 157.05, 143.45, 141.27, 136.53, 131.52, 128.69, 128.63, 127.75, 127.38,
125.87, 115.14, 114.81, 68.33, 59.17, 44.48, 41.63, 29.40, 22.90; HRMS exact mass
calculated for [M+Na]+ (C26H27N3NaO5S) requires m/z 516.1564, found m/z
516.1558; Anal. Calcd for C26H27N3O5S: C, 63.27; H, 5.51; N, 8.51. Found: C, 63.05;
H, 5.67; N, 8.60.
4.3.7. (S)-1-(4-fluorobenzyl)-5-oxo-N-(4-(4-sulfamoylbenzyloxy)benzyl) pyrrolidine-2-
carboxamide (3g).Prepared from 1e and 2b. Eluent: AcOEt-MeOH 9:1. White solid;

29
yield 75%; mp: 198.2-200.0 oC; [a]D +4.3 ο (c 1.00, DMF); Rf (AcOEt) 0.42; 1H
NMR (DMSO-d6) δ 8.58 (1H, br s, NH), 7.85-7.81 (2H, d, J = 7.8 Hz, Ar-H), 7.63-
7.59 (2H, d, J = 8.2 Hz, Ar-H), 7.38 (2H, br s, NH2), 7.18-7.04 (6H, m, Ar-H), 7.00-
6.96 (2H, d, J = 8.2 Hz, Ar-H), 5.20 (2H, s, OCH2), 4.83-4.75 (1H, d, J = 15.2 Hz,
NCHH), 4.20-4.11 (2H, m, NHCH2), 3.94-3.88 (1H, m, CH), 3.76-3.68 (1H, d, J =
15.2 Hz, NCHH), 2.39-2.11 (3H, m, COCH2 & CHH), 1.90-1.76 (1H, m, CHH); 13C
NMR (DMSO-d6) δ 174.82, 170.76, 160.27 (d, J = 241.45 Hz), 157.05, 156.40,
143.44, 141.22, 132.74 (d, J = 2.95 Hz), 131.46, 129.80 (d, J = 8.15 Hz), 129.15,
128.68, 127.70, 125.83, 115.31 (d, J = 21.05 Hz), 114.79, 68.34, 59.21, 43.82, 41.64,
29.35, 22.87; 19F NMR (DMSO-d6) δ -115.85; HRMS exact mass calculated for
[M+Na]+ (C26H26FN3NaO5S) requires m/z 534.1469, found m/z 534.1469; Anal. Calcd
for C26H26FN3O5S: C, 61.04; H, 5.12; N, 8.21. Found: C, 61.32; H, 5.28; N, 8.17.
4.3.8.(S)-5-oxo-Ν-(4-(4-sulfamoylbenzyloxy)benzyl)-1-(4-(trifluoromethyl)benzyl)
pyrrolidine-2-carboxamide (3h).Prepared from 1f and 2b. Eluent: AcOEt-MeOH 9:1.
White solid; yield 72%; mp: 202.2-204.8 oC; [a]D +5.3 ο (c 1.10, DMF); Rf (AcOEt-
MeOH 9:1) 0.51; 1H NMR (DMSO-d6) δ 8.56 (1H, br s, NH), 7.85-6.68 (14H, m,
ArH & NH2), 5.19 (2H, s, OCH2), 4.90-4.83 (1H, d, J = 15.8 Hz, NCHH), 4.19-4.10
(2H, m, NHCH2), 3.99-3.93 (1H, m, CH), 3.91-3.83 (1H, d, J = 16.0 Hz, NCHH),
2.42-2.11 (3H, m, COCH2 & CHH), 1.91-1.78 (1H, m, CHH); 13C NMR (DMSO-d6)
δ 175.07, 170.69, 157.06, 143.45, 141.55, 141.20, 131.37, 129.00 (q, J = 30.7 Hz),
128.68, 128.41, 127.68, 125.84, 125.46 (q, J = 3.45 Hz), 115.10, 114.77, 68.34, 59.42,
44.26, 41.66, 29.27, 22.91;19F NMR (DMSO-d6) δ -61.29; HRMS exact mass
calculated for [M-H]- (C27H25F3N3O5S) requires m/z 560.1472, found m/z 560.1477;
Anal. Calcd for C27H26F3N3O5S: C, 57.75; H, 4.67; N, 7.48. Found: C, 58.03; H, 4.58;
N, 7.65.