Synthesis, evaluation and molecular docking of prolyl-fluoropyrrolidine derivatives as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1111/cbdd.12142

A series of prolyl-fluoropyrrolidine derivatives were designed, synthesized and screened for in vitro inhibition of dipeptidyl peptidase IV. The SAR study revealed the influence of substituted chemical modifications on dipeptidyl peptidase IV inhibitory activity. Among all the compounds screened, compound 9 (IC₅₀ = 0.83 μm) and 10 (IC₅₀ = 0.43 μm) possessing aryl substituted piperazine with acetamide linker resulted as most potent dipeptidyl peptidase IV inhibitors. Both the compounds 9 and 10 resulted significant reduction in glucose excursion during oral glucose tolerance test in streptozotocin-induced diabetic rat model at single dose of 10 mg/kg. Molecular docking studies were performed to illustrate the probable binding mode and interactions of prolyl-fluoropyrrolidine nucleus and its derivatives at binding site of receptor. The fluoropyrrolidine moiety of prolyl-fluoropyrrolidine derivatives occupied S1 pocket as observed in the crystal structure (PDB id: 2FJP). The compounds 9 and 10 were observed to occupy S2 binding pocket and were observed to have interaction with Arg125, Tyr547 and Ser630 acquired through hydrogen bond. The aryl moiety at piperazine ring was found to extend into the cavity and interacted with Arg358. The observed interactions signalled that occupancy of the highly hydrophobic S2 pocket is very crucial for dipeptidyl peptidase IV inhibitory activity. A series of prolyl-fluoropyrrolidine derivatives were synthesized and evaluated for inhibition of dipeptidyl peptidase IV (DPP IV) for treatment of Type 2 diabetes. The binding position of docked compounds (stick rendering) in the binding pocket of DPP IV.

Full text of DOI:10.1111/cbdd.12142

Chem Biol Drug Des 2013; 82: 156–166
Research Article
Synthesis, Evaluation and Molecular Docking of
Prolyl-Fluoropyrrolidine Derivatives as Dipeptidyl
Peptidase IV Inhibitors
Mani Sharma1,*, Monica Gupta1, Divya Singh1,
peptidase IV/CD26 is a cell-surface protease belongs to
the prolyloligopeptidase family. It exists as both a mem-
Manoj Kumar2 and Punit Kaur2
brane-bound protein and a soluble protein in plasma. Dip-
eptidyl peptidase IV is a ubiquitously distributed serine
1Department of Pharmaceutical Chemistry, Delhi Institute
of Pharmaceutical Sciences and Research, Pushp Vihar,
Sector-3, M B Road, New Delhi, 110017, India
2Department of Biophysics, All India Institute of Medical
Sciences, New Delhi, 110029, India
*Corresponding author: Mani Sharma,
mani120484@gmail.com
protease that mediates the activities of a number of regu-
latory peptides (1,2). The two important endogenous pep-
tide
substrates
GLP-1
and
glucose-dependent
insulinotropic polypeptide (GIP) are considered as major
targets of DPP IV inhibitors. Incretin hormones as gluca-
gon-like peptide 1 (GLP-1) and GIP stimulate pancreatic
beta cells, which allows 50–70% of postprandial insulin
release (3). However, its rapid degradation in plasma by
serine protease DPP IV due to its short half-life limits their
therapeutic efficacy (2). Dipeptidyl peptidase IV inhibitors
stimulate insulin secretion, inhibit glucagon releases and
maintain glucose homoeostasis by extending the duration
of action of GLP-1 (4). Therefore, DPP IV inhibitor repre-
sents a novel class of antidiabetic drugs that prevent pro-
teolytic degradation of incretin hormones and thus
stimulates glucose-dependent insulin secretion (2,5).
A
series of prolyl-fluoropyrrolidine derivatives were
designed, synthesized and screened for in vitro inhibi-
tion of dipeptidyl peptidase IV. The SAR study
revealed the influence of substituted chemical modifi-
cations on dipeptidyl peptidase IV inhibitory activity.
Among all the compounds screened, compound
9
(IC50 = 0.83 lM) and 10 (IC50 = 0.43 lM) possessing aryl
substituted piperazine with acetamide linker resulted
as most potent dipeptidyl peptidase IV inhibitors. Both
the compounds 9 and 10 resulted significant reduction
in glucose excursion during oral glucose tolerance test
in streptozotocin-induced diabetic rat model at single
dose of 10 mg/kg. Molecular docking studies were
performed to illustrate the probable binding mode and
interactions of prolyl-fluoropyrrolidine nucleus and its
derivatives at binding site of receptor. The fluoropyrr-
olidine moiety of prolyl-fluoropyrrolidine derivatives
occupied S1 pocket as observed in the crystal struc-
ture (PDB id: 2FJP). The compounds 9 and 10 were
observed to occupy S2 binding pocket and were
observed to have interaction with Arg125, Tyr547 and
Ser630 acquired through hydrogen bond. The aryl moi-
ety at piperazine ring was found to extend into the
cavity and interacted with Arg358. The observed inter-
actions signalled that occupancy of the highly hydro-
phobic S2 pocket is very crucial for dipeptidyl
peptidase IV inhibitory activity.
Many reported DPP IV inhibitor possesses substituted pyr-
rolidine, cyanopyrrolidine and thiazolidines as proline
mimetic in P-1 site and showed potent DPP IV inhibitory
action (6–8). Dipeptidyl peptidase IV inhibitors having cy-
anopyrrolidine moiety enhances the potency due to strong
interaction of nitrile group by the active site Ser630, lead-
ing to delayed dissociation and slow tight binding of cer-
tain DPP IV inhibitors. The presence of cyanopyrrolidine
moiety is associated with chemical unstability due to intra-
molecular cyclization between the electrophilic nitrile of the
(S)-2-cyanopyrrolidine moiety and secondary amine of pro-
line (9). Although, prolyl-(S)-2-cyanopyrrolidine represents
the exceptionally potent fragment but chemical unstability,
short half-life and inadequate in vivo efficacy still encour-
ages to develop more potent and chemically stable com-
pounds (10,11). The structurally distinguished class as
dipeptide inhibitors resembles substrate analogs of P-2-P-
1 fragment as represented by most of the available DPP IV
inhibitors (11,12) (Figure 1).
Key words: diabetes, dipeptidyl peptidase IV, molecular
docking, prolyl-fluoropyrrolidine
Received 10 January 2013, revised
accepted for publication 25 March 2013
7 March 2013 and
Dipeptidyl peptidase IV enzyme is capable of recognizing
alanine or proline residues at P-1 position. Previous stud-
ies show that compounds having rigid conformation bound
to have interaction with S-2 pocket of DPP IV, thus
Dipeptidyl peptidase IV (DPP IV, also known as CD26) is a
novel therapeutic target for type II diabetes. Dipeptidyl
156
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12142

Prolyl-fluoropyrrolidine derivatives as DPP IV inhibitors
Figure 1: Design of prolyl-fluoropyrrolidine-based dipeptidyl peptidase (DPP) IV inhibitors.
making them exceptionally potent (6,11). As shown in pub-
lished reports, a-branched amino acid type and N-substi-
tuted glycine type present in clinical trial having efficacy for
diabetic patients (13–15). Despite the existence of a num-
ber of potent inhibitors of DPP IV, it was thought worth-
while to report compounds as prolyl-fluoropyrrolidine-
based derivatives that were based on neither cyanopyrroli-
dine nor thiazolidine moieties. Thus, we focused our atten-
tion towards a-substituents of proline moiety at P-2
position, and further exploration in an effort to optimize
incorporating fluoropyrrolidine resulting structurally similar
pharmacophore may led to improved DPP IV inhibitory
activity. In addition, necessary interactions between key
residues were explored using the prolyl-fluoropyrrolidine
derivatives, which could further be applied to modify future
novel chemical series.
are reported as s (singlet), d (doublet), t (triplet), q (quartet)
and m (multiplet). Mass spectra were measured on a
Waters-3100QSD. All the Fourier transform infrared (FT-IR)
spectra were recorded on Jasco-410 spectrophotometer.
Elemental analysis was carried out using Vario EL III ele-
mental analyzer. All reactions were carried out under dried
condition and performed using oven-dried glassware.
Step I: synthesis of 1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxylic acid (1)
BOC anhydride (1.02 mol) was added at rt to a stirring solu-
tion of L-proline (0.86 mol) and triethylamine (4.3 mol) in tet-
rahydrofuran (15 mL). After 4 h, the reaction mixture was
concentrated under reduced pressure. The residue
obtained was partitioned between water and ethyl acetate.
The aqueous layer was washed with ethyl acetate twice.
The organic layer was dried over sodium sulphate and evap-
orated under reduced pressure. The compound obtained
was recrystalized using mixture of dichloromethane and
hexane to afford the pure compound in excellent yield.
Thus, we report the chemical synthesis, structure–activity
relationship of compounds synthesized, in vitro DPP IV
inhibitory activity, in vivo antihyperglycaemic activity along
with molecular modelling studies as discussed below.
White solid; Yield: 89%; mp: 145–147 °C; Rf: 0.71; 1H
NMR (300 MHz, CDCl3) d ppm: 1.38 (s, 9H), 1.80–1.76
(m, 4H), 3.05 (t, J = 24, 4H); MS: 216 [M + H]+; Anal.
Calcd. For C10H17NO4: C, 55.80; H, 7.96; N, 6.51; Found:
C, 55.85; H, 7.90; N, 6.46.
Methods and Materials
Experimental
All commercial chemicals and solvents were purchased
from Sigma-Aldrich Chemical Company, St. Louis, MO,
USA and Spectrochem Pvt. Ltd. (Mumbai, India). Chemi-
cals and solvents were used as such without further purifi-
cation. Melting points of compounds were determined in
open capillary tubes using Hicon melting point instrument
and are uncorrected. 1H spectra were recorded on Bruker
NMR spectrometer at 300 MHz in CDCl3 or DMSO-d6 with
tetramethylsilane as an internal standard. Chemical shifts
are expressed as parts per million (ppm, d), and signals
Step II: synthesis of tert-butyl 2-[(3-
fluoropyrrolidin-1-yl)carbonyl]pyrrolidine-1-
carboxylate (2)
To a stirred solution of 1 (0.46 mol) in dichloromethane
(10 mL) were added 3-fluropyrrolidine, (0.46 mol),
1-hydroxybenzotriazole (0.51 mol), 1-(3-dimethyl aminopro-
pyl)-3-ethylcarbodiimide hydrochloride (0.51 mol) and
Chem Biol Drug Des 2013; 82: 156–166
157

Sharma et al.
triethylamine (1.39 mol) at 0 °C. After being stirred for 2 h
on ice bath, the reaction mixture was stirred at rt for 24 h.
The reaction mixture was poured into water and extracted
with dichloromethane. The organic layer was washed with
10% aqueous citric acid, saturated bicarbonate solution
and brine, then dried over anhydrous sodium sulphate
and evaporated to give white solid. The compound obtained
was recrystalized using mixture of dichloromethane and
hexane to afford the pure compound in excellent yield.
2-chloro-1-[4-(pyridin-2-yl) piperazin-1-yl]ethanone
(5)
Brown oil; Yield: 87%; Rf: 0.70; H NMR (300 MHz, CDCl3)
d ppm: 3.71–3.66 (m, 4H), 3.91–3.73 (m, 4H), 4.43 (s, 2H),
6.73 (t, J = 12 Hz, 1H), 6.99 (d, J = 9, 1H), 7.71 (t,
J = 15.3, 1H), 8.17 (s, 1H); FT-IR (mmax; per cm, neat): 3004
(C-H stretch), 2920 (C-H stretch), 1647 (C=O), 1541 (C=N), 775
(C-Cl stretch); Anal. Calcd. For C11H14ClN3O: C, 55.12; H,
5.89; N, 17.53; Found: C, 55.17; H, 5.85; N, 17.58.
1
White solid; Yield: 87%; mp: 153–155 °C; Rf: 0.67; 1H
NMR (300 MHz, CDCl3) d ppm: 1.4 (s, 9H), 1.92–1.87 (m,
4H), 2.36–2.33 (m, 2H), 3.50–3.42 (m, 2H), 3.64–3.56 (m,
2H), 3.85–3.76 (m, 2H), 5.4 (m, 1H); MS: 287 [M + H]+;
Anal. Calcd. For C14H23FN2O3: C, 58.72; H, 8.10; N,
9.78; Found: C, 58.67; H, 8.14; N, 9.82.
1-(azepan-1-yl)-2-chloroethanone (6)
Light brown oil; Yield: 78%; Rf: 0.69; 1H NMR (300 MHz,
CDCl3) d ppm: 1.79–1.73 (m, 4H), 1.87–1.82 (m, 4H),
3.58–3.42 (m, 4H), 4.11 (s, 2H); FT-IR (mmax; per cm, neat):
1647 (C=O), 1099 (C-N stretch), 651 (C-Cl stretch); Anal.
Calcd. For C8H14ClNO: C, 54.70; H, 8.03; N, 7.97; Found:
C, 54.74; H, 8.05; N, 7.99.
Step III: synthesis of (3-fluoropyrrolidin-1-yl)
(pyrrolidin-2-yl)methanone (3)
BOC protected amine 2 (0.57 mol) was dissolved in
10 mL DCM and added trifluoroacetic acid (1.7 mol) at
0 °C. The reaction was stirred at 0 °C for 12 h. The sol-
vent was removed under reduced pressure and product
obtained as corresponding amine TFA salt. The oily com-
pound obtained was used for the next reaction without
further purification.
2-chloro-1-(piperidin-1-yl) ethanone (7)
Yellow oil; Yield: 77%; Rf: 0.69; 1H NMR (300 MHz,
CDCl3) d ppm: 2.00–1.89 (m, 4H), 3.48 (t, J = 18 Hz, 4H),
4.0 (s, 2H); FT-IR (mmax; per cm, neat): 1647 (C=O), 1096
(C-N stretch), 802 (C-Cl stretch); MS: 162 [M + H]+; Anal.
Calcd. For C7H12ClNO: C, 52.02; H, 7.48; N, 8.67; Found:
C, 52.07; H, 7.52; N, 8.71.
Step IV: General procedure for synthesis of N-
substituted chloracetamides (4–8)
2-chloro-1-(pyrrolidin-1-yl) ethanone (8)
Yellow oil; Yield: 64%; Rf: 0.69; 1H NMR (300 MHz,
CDCl3-d6, TMS) d ppm: 1.89–2.00 (m, 4H), 3.42–3.48 (t,
4H, J = 18), 4.2 (s, 2H); FT-IR (mmax; per cm, neat): 1647
(C=O) 1096 (C-N stretch), 802 (C-Cl stretch); MS: 148
[M + H]+; Anal. Calcd. For C6H10ClNO: C, 48.82; H, 6.83;
N, 9.49; Found: C, 54.75; H, 8.08; N, 7.99.
Secondary amine (2.9 mol) was taken in 1, 4-dioxan
(10 mL) maintaining temperature 50–60 °C. The solution
of chloroacetyl chloride (3.33 mol) in 1, 4-dioxan (2 mL)
was added drop wise under continuous stirring. After
reflux of 2 h, cooled mixture to room temperature and stir-
red for 30 min. The completion of reaction was monitored
by thin-layer chromatography (TLC) using mobile phase,
chloroform/methanol (9.5:0.5). The reaction mixture was
concentrated under reduced pressure, and the resulting
mixture was diluted with water and neutralized using satu-
rated bicarbonate solution. The solution was further diluted
and partitioned between water and chloroform. The
organic layer was washed twice with water, brine and
dried over anhydrous sodium sulphate. The organic layer
was evaporated to the dryness under reduced pressure,
and resultant intermediates (4–8) were obtained in excel-
lent yields.
Step V: General procedures for synthesis of 9–16
A solution of (3-fluoropyrrolidin-1-yl) (pyrrolidin-2-yl)metha-
none 3 (0.02 mol), diisopropylethyl amine (0.06 mol) in
dry tetrahydrofuran was stirred on rt. A solution of N-
substituted acetamide/alkyl chloride (0.02 mol) in dry tet-
rahydrofuran (3 mL) was then added to the reaction mix-
ture. The reaction mixture was stirred on rt for 12 h. The
completion of reaction was confirmed through TLC, ethy-
lacetate/hexane (0.2:0.8) visualized under iodine vapours.
The reaction mixture was concentrated under reduced
pressure. The resulting mixture was diluted with cold
water and extracted with chloroform. The organic layer
was washed with water and brine. The extraction was
repeated twice, and combined organic layers were dried
over anhydrous sodium sulphate. The organic layers were
concentrated under reduced pressure using rotary evapo-
rator. Compounds obtained as oil were purified by col-
umn chromatography (5% MeOH in DCM). The final
desired synthetic compounds were obtained in good
yields.
2-chloro-1-[4-(pyrimidin-2-yl) piperazin-1-yl]
ethanone (4)
Yellow oil; Yield: 87%; Rf: 0.71; 1H NMR (300 MHz,
CDCl3) d ppm: 3.70–3.64 (m, 4H), 3.91–3.73 (m, 4H), 4.28
(s, 2H), 6.66 (t, J = 9, 1H), 8.38 (d, J = 9, 2H); FT-IR (mmax
;
per cm, neat): 2931 (C-H stretch), 1629 (C=O), 1493 (C=N),
640 (C-Cl stretch); Anal. Calcd. For C10H13ClN4O: C, 49.90;
H, 5.44; N, 23.28; Found: C, 49.94; H, 5.39; N, 23.24.
158
Chem Biol Drug Des 2013; 82: 156–166

Prolyl-fluoropyrrolidine derivatives as DPP IV inhibitors
2-[2-(3-Fluoro-pyrrolidine-1-carbonyl)-pyrrolidin-1-
yl]-1-(4-pyridin-2-yl-piperazin-1-yl)-ethanone (9)
Yellow solid; Yield: 68%; mp: 149–151 °C; Rf: 0.58; 1H
NMR (300 MHz, CDCl3) d ppm: 3.73–3.69 (m, 4H), 3.91–
3.88 (m, 4H), 4.45 (s, 2H), 6.65 (t, J = 12 Hz, 1H), 6.93
(d, J = 9, 1H), 7.7 (t, J = 15.3, 1H), 8.17 (s, 1H); FT-IR
(mmax; per cm, KBr): 2356 (C-H stretch), 1671 (C=O), 1478
(C=N), 1198 (C-F), 764 (Aromatic); MS: 390 [M + H]+;
Anal. Calcd. For C20H28FN5O2: C, 61.68; H, 7.25; N,
17.98; Found: C, 61.72; H, 7.27; N, 17.91.
Anal. Calcd. For C15H24FN3O2: C, 60.58; H, 8.13; N,
14.13; Found: C, 60.53; H, 8.09; N, 14.18.
(3-Fluoro-pyrrolidin-1-yl)-[1-(2-piperidin-1-yl-ethyl)-
pyrrolidin-2-yl]-methanone (14)
Yellow solid; Yield: 68%; mp: 149–151 °C; Rf: 0.55; 1H
NMR (300 MHz, CDCl3) d ppm: 1.09–1.21 (m, 4H), 1.35–
1.45 (m, 4H), 1.5–1.6 (m, 4H), 1.87–1.89 (m, 2H), 2.10–2.17
(m, 2H), 3.09–3.16 (q, 4H), 3.40–3.49 (m, 4H), 3.55–3.61
(m, 4H), 3.70–3.76 (m, 2H), FT-IR (mmax; per cm, KBr): 2973
(C-H stretch), 1688 (C=O), 1403 (C=N), 1167 (C-F); MS: 298
[M + H]+; Anal. Calcd. For C16H28FN3O: C, 64.61; H, 9.49;
N, 14.13; Found: C, 64.56; H, 9.53; N, 14.17.
2-[2-(3-Fluoro-pyrrolidine-1-carbonyl)-pyrrolidin-1-
yl]-1-(4-pyrimidin-2-yl-piperazin-1-yl)-ethanone
(10)
Yellow solid; Yield: 68%; mp: 149–151 °C; Rf: 0.58; 1H
NMR (300 MHz, CDCl3) d ppm: 1.91–1.85 (m, 4H), 2.32–
2.29 (m, 2H), 3.48–3.44 (m, 2H), 3.61–3.59 (m, 2H), 3.70–
3.64 (m, 4H), 3.83–3.81 (m, 2H), 3.96–3.93 (m, 4H), 4.28
[1-(2-Diethylamino-ethyl)-pyrrolidin-2-yl]-(3-fluoro-
pyrrolidin-1-yl)-methanone (15)
Yellow solid; Yield: 68%; mp: 149–151 °C; Rf: 0.53; 1H
NMR (300 MHz, CDCl3) d ppm: 1.18–1.24 (m, 4H), 1.31
(t, J = 6, 6H), 1.37–1.42 (m, 4H), 1.75–1.89 (m, 2H), 3.08
(q, J = 15, 4H), 3.38–3.58 (m, 4H), 4.23–4.26 (m, 4H); FT-
IR (mmax; per cm, KBr): 2974 (C-H stretch), 1695 (C=O),
1401 (C=N), 1166 (C-F); MS: 286 [M + H]+; Anal. Calcd.
For C15H28FN3O: C, 63.13; H, 9.89; N, 14.72; Found: C,
64.17; H, 9.87; N, 14.75.
(s, 2H), 6.62 (t, J = 9, 1H), 8.38 (d, J = 9, 2H); FT-IR (mmax
;
per cm, KBr): 2928 (C-H stretch), 1653 (C=O), 1473 (C=N),
1172 (C-F), 748 (Aromatic); MS: 391 [M + H]+; Anal.
Calcd. For C19H27FN6O2: C, 58.45; H, 6.97; N, 21.52;
Found: C, 58.48; H, 6.92; N, 21.48.
1-Azepan-1-yl-2-[2-(3-fluoro-pyrrolidine-1-
carbonyl)-pyrrolidin-1-yl]-ethanone (11)
{1-[2-(dimethylamino)ethyl]pyrrolidin-2-yl}(3-
fluoropyrrolidin-1-yl)methanone (16)
Yellow solid; Yield: 65%; mp: 149–151 °C; Rf: 0.57; 1H
NMR (300 MHz, CDCl3) d ppm: 1.33–1.25 (m, 4H), 1.71–
1.57 (m, 4H), 1.94–2.0 (m, 4H), 2.31–2.26 (m, 2H), 3.74–
3.70 (m, 4H), 3.85–3.79 (m, 4H), 4.70 (s, 2H) 5.37–5.19
(m, 1H); FT-IR (mmax; per cm, KBr): 2928 (C-H stretch), 1646
(C=O), 1392 (C=N), 1198 (C-F); MS: 326 [M + H]+; Anal.
Calcd. For C17H28FN3O2: C, 62.74; H, 8.67; N, 12.91;
Found: C, 62.71; H, 8.69; N, 12.96.
White solid; Yield: 63%; mp: 143–145 °C; Rf: 0.55; 1H
NMR (300 MHz, CDCl3) d ppm: 1.32–1.35 (t, J = 9, 6H),
1.38 (1.39–1.41, s, 4H), 1.83–1.89 (m, 2H), 3.01–3.05 (q,
4H), 3.34–3.47 (m, 4H), 4.21–4.24(m, 4H); FT-IR (mmax; per
cm, KBr): 2930 (C-H stretch), 1692 (C=O), 1402 (C=N),
1168 (C-F); MS: 258 [M + H]+; Anal. Calcd. For
C
13H24FN3O: C, 60.67; H, 9.40; N, 16.33; Found: C,
60.63; H, 9.38; N, 16.30.
2-{2-[(3-fluoropyrrolidin-1-yl)carbonyl]pyrrolidin-1-
yl}-1-(piperidin-1-yl)ethanone (12)
Pharmacological activity
Brown oil; Yield: 62%; Rf: 0.58; 1H NMR (300 MHz,
CDCl3) d ppm: 1.0–0.89 (m, 2H), 1.33–1.25 (m, 4H), 1.63–
1.36 (m, 4H), 2.38–2.31 (m, 2H), 3.78–3.74 (m, 4H), 3.85–
3.83 (m, 2H), 3.89–3.87 (m, 4H), 4.70 (s, 2H) 5.37–5.19
(m, 1H); FT-IR (mmax; per cm, neat): 2930 (C-H stretch),
1660 (C=O), 1387 (C=N), 1190 (C-F); MS: 312 [M + H]+;
Anal. Calcd. For C16H26FN3O2: C, 61.71; H, 8.42; N,
13.49; Found: C, 61.75; H, 8.38; N, 13.45.
The Institutional Animal Ethical Committee (IAEC) approved
the experiments for oral glucose tolerance test (OGTT).
Healthy Wistar rats used for the study were obtained from
Animal House of Delhi Institute of Pharmaceutical Sciences
and Research. Rats were housed in colony cages (three
female and one male rats per cage for breeding), at an
ambient temperature of 25 °C with 12 h light: 12 h dark
cycle. Animals were kept in polypropylene cages and were
fed with rat food as pellets and water ad libitum unrestrict-
edly. All experimental procedures were followed according
to IAEC (Protocol No.1/DIPSAR/IAEC/2010) and CPCSEA
guidelines.
2-{2-[(3-fluoropyrrolidin-1-yl)carbonyl]pyrrolidin-1-
yl}-1-(pyrrolidin-1-yl)ethanone (13)
Brown oil; Yield: 62%; Rf: 0.53; 1H NMR (300 MHz,
CDCl3) d ppm: 1.45–1.43 (m, 2H), 1.58–1.52 (m, 4H),
1.98–2.0 (m, 4H), 2.31–2.31 (m, 2H), 3.74–3.70 (m, 4H),
3.83–3.79 (m, 4H), 3.89–3.87 (m, 2H), 4.70 (s, 2H) 5.36–
5.19 (m, 1H); FT-IR (mmax; per cm, neat): 2936 (C-H stretch),
1644 (C=O), 1380 (C=N), 1190 (C-F); MS: 298 [M + H]+;
Induction of experimental diabetes
The experimental diabetes was induced using freshly
prepared solution of streptozotocin (STZ; Sigma-Aldrich)
at the dose level of 90 mg/kg (I.P.) in 0.1 M freshly pre-
Chem Biol Drug Des 2013; 82: 156–166
159

Sharma et al.
pared citrated buffer pH 4.5 to 2-day-old neonatal pups
(16,17). A total of 24 diabetic and six non-diabetic male
rats were used in the study. After 6 weeks of injection,
screening of animal was carried out for fasting blood
glucose level. Animals showing fasting glucose levels
>150 mg/dL were considered diabetic and selected for
screening of compounds. Diabetic rats were further
divided into four groups with six rats per group.
analysed by computational modelling. All the computa-
tional studies were performed in the modelling environ-
ment of Discovery Studio (DS) 2.0a. The reasonable
three-dimensional conformations of these derivatives were
developed from sketched molecule by energy minimization
and simulated annealing methods. The heavy atoms of
each of the four derivatives were individually sketched,
and their geometry was cleaned using Biopolymer module
of DS 2.0. The hydrogen atoms were added, force filed
parameters of CHARMm (20,21) were assigned, and par-
tial charges on the atoms were calculated by MMFF94
(22). It was followed by energy minimization for the optimi-
zation of geometry of compound using conjugate gradient
algorithm with the convergence criteria of root mean
Antihyperglycaemic activity
Male Wistar rats weighing 150–200 g having blood glucose
level >150 mg/dL were selected for OGTT. Animals of dia-
betic control group received vehicle (0.5% w/v methylcellu-
lose) and standard group received vildagliptin (10 mg/kg)
orally. The animals of experimental group were administered
single dose of test compounds (suspension in 0.5% w/v
methylcellulose) of 10 mg/kg body weight (18,19). After
30 min, glucose load (2 g/kg) was given orally to overnight
fasting rats prior to administration of the test compound/vehi-
cle. Blood samples were collected from tail vein at regular
intervals of 0, 30, 60, 90, 120 min after glucose load. The
blood glucose level was monitored using glucometer (ACCU-
CHEK, Active; Roche Diagnostics, Mannheim, Germany).
ꢀ
squared (r.m.s) gradient <0.05 kcal/mol/A in the presence
of distance-dependent dielectric implicit solvent model.
The energy-minimized conformation of each derivative was
subjected to conformational search using a simulated
annealing molecular dynamics approach. The compound
was first heated to a temperature of 700 K and then
annealed to 200 K for 5 ps. Thirty cycles were carried out
sequentially using the output of the previous cycle. The
conformation obtained at the end of each cycle was
further subjected to local energy minimization by employ-
ing Adopted basis Newton Raphson algorithm with the
ꢀ
convergence criteria of r.m.s gradient <0.01 kcal/mol/A in
Statistical evaluation
the presence of distance-dependent dielectric. The 30
energy-minimized structures were then superimposed, and
the lowest energy conformer in the major cluster was
taken to be the most probable conformation. The opti-
mized conformation of fluoropyrrolidine derivatives was
used for docking studies in the binding pocket of DPP IV.
The statistical data were expressed as mean Æ SEM. IC50
values were determined using non-linear regression analy-
sis. Statistical evaluation was performed by one-way ANOVA
followed by Dunnett’s posttest. Statistical studies and data
analyses were performed using GRAPHPAD PRISM Version 5.0
(San Diego, CA, USA).
The crystal structure of DPP IV complexed with a structur-
ally similar compound (PDB Id: 2FJP) was available in
dimeric state (23). As both the monomeric unit was essen-
tially the same in terms of structural parameters, the chain
A was taken as receptor model for docking of derivative
compounds. N-acetyl glucosamines present in the com-
plex were removed. The water molecules were also
removed as none of them were structurally conserved or
making any hydrogen bond with the ligand present. The
CHARMm force field parameters and partial charges were
assigned to the receptor model. It was subjected to
energy minimization to relax the structure and remove any
short contacts using conjugate gradient algorithm. The
binding site for docking studies was defined around the
active site region (catalytic triad of Ser630, Asp708 and
His740) that also includes the S1 and S2 subpocket. The
LigandFit (24) docking protocol was used for docking
studies. It combines a shape comparison filter with a
Monte Carlo conformational search to generate docked
poses consistent with the binding site shape. The side
chains of the binding site residues were kept flexible dur-
ing docking process to allow the local perturbations for
the better fit of the ligand, and rest of the receptor was
kept rigid. The docked complex was further refined using
conjugate gradient minimization algorithm on fully flexible
In vitro DPP IV enzyme inhibition assay
The compounds synthesized were evaluated for in vitro
DPP IV enzyme inhibition. The enzyme assay was per-
formed using DPP IV drug discovery kit (BML-AK 499; Enzo
Life Sciences, Plymouth Meeting, PA, USA). The activity of
test compounds was assayed using human recombinant
DPP IV enzyme, chromogenic substrate (H-Gly-Pro-AMC,
Km 114 lM), DPP IV inhibitor (P32/98), assay buffer and
calibration standard as provided in the kit. The assay was
performed using 96-well flat-bottomed microtitre plate fol-
lowed by addition of assay buffer, DPP IV enzyme and chro-
mogenic substrate (HGly-Pro-pNA). The assay principle and
procedure was followed as per manufacturer’s guidelines.
Solutions of the test compounds were made in dimethyl sul-
phoxide (DMSO) at different concentrations of 25, 50, 100,
200 lg/mL, and 20 lL were added to each well after fur-
ther dilutions. The plate was incubated at 37 °C for 10 min
to allow enzyme-inhibitor reaction and read continuously at
A-405 nm using Bio-Rad Elisa Plate Reader.
Modelling studies
The binding mode of the four fluoropyrrolidine derivatives
and their interactions in the binding pocket of DPP IV were
160
Chem Biol Drug Des 2013; 82: 156–166

Prolyl-fluoropyrrolidine derivatives as DPP IV inhibitors
Table 1: Dipeptidyl peptidase IV inhibitory effect of prolyl-fluoro-
pyrrolidide derivatives 3, 9–16
receptor and ligand molecules to relax the complex as the
receptor molecule, except side chains around the binding
pocket, was treated rigid during docking process. This
refined complex was analysed for binding mode as well as
interactions of fluoropyrrolidine derivatives, and their inter-
action energy was calculated.
O
R
N
N
F
The docking protocol of LigandFit was first verified by
docking the known inhibitor, biarylphenylalanine fluoropyrr-
olidine amide compound (compound 17), present in the
binding pocket of DPP IV (PDB Id: 2FJP, Chain A). The
ligand was extracted, and its bond order was corrected.
The force field parameters of CHARMm and partial
charges based on MMFF 94 were assigned. Then, ligand
was docked, and its docked conformation was compared
with the crystal conformation.
Compound
R
IC50 (lM)a
3
4.27
H
9
0.83
0.43
N
N
N
N
N
N
O
O
10
N
Results and Discussion
Chemistry
A series of prolyl-fluoropyrrolidine derivatives as DPP IV
inhibitors were designed, and synthetic strategy followed is
outlined in Scheme 1. The unsubstituted parent nucleus
prolyl-fluoropyrrolidine was prepared by coupling of car-
boxylic group of BOC-proline and 3-fluoropyrrolidine via
peptide coupling. Removal of BOC group was performed
using trifluoroacetic acid resulting in liberation of free
amine, which was simultaneously reacted with N-substi-
tuted chloroacetamides to give N-substituted 2-{2-[(3-flu-
oropyrrolidin-1-yl)carbonyl] pyrrolidin-1-yl}-acetamide. The
synthesis of targeted compounds was performed following
nucleophilic displacement of chlorine using several synthe-
sized intermediates and few commercially available alkyl
chlorides in the presence of triethylamine and THF as sol-
vent at room temperature. After completion of the reaction,
desired compounds were obtained in good yield and pur-
ity. The structures of the titled analogs were confirmed by
spectral analysis.
11
12
3.47
7.88
N
O
N
O
13
16.73
6.49
N
O
14
N
15
23.84
30.21
2.5
N
16
N
P 32/98
Biological activity
O
N
S
In vitro DPP IV activity
NH2
The synthesized prolyl-fluoropyrrolidine derivatives (9–16)
were evaluated in vitro for DPP IV enzyme inhibitory
activity, and results obtained are reported as micromolar
inhibitory concentration, IC50 (lM). In the present study,
2-substituted-1-(2-{[(3S)-3-fluoropyrrolidin-1-yl]carbonyl}
pyrrolidin-1-yl)ethanone derivatives were synthesized and
were evaluated against their DPP IV inhibitory activity.
Table 1 summarizes the DPP IV inhibitory activity of prol-
yl-fluoropyrrolidine derivatives-based DPP IV inhibitors
bearing a representative proline group. The unsubstituted
prolyl-fluoropyrrolidine scaffold 3 (IC50 = 4.27 lM) showed
weak micromolar inhibition. The optimization strategy
was focused to examine effect of various substitutions
on ring. Substitution using a 6-membered cyclic amino
aIC50 represents inhibitory concentration determined by non-linear
regression analysis using GRAPHPAD PRISM software. Values are
expressed as mean of three independent experiments.
group subsequently increased the activity compared with
the non-substituted parent compound. Of the synthe-
sized compounds, pyrimidyl piperazine derivative 10
(IC50 = 0.43 lM) having pyrimidyl moiety exhibited most
potent compound of the series. The pyridyl piperazine
analog 9 (IC50 = 0.83 lM) showed comparatively weak
inhibition but provided an inhibitor with rise in potency
as compared with non-substituted parent scaffold 3. The
Chem Biol Drug Des 2013; 82: 156–166
161

Sharma et al.
A
B
Figure 2: Effect of compounds 9
and 10 on blood glucose level in
an oral glucose tolerance test
(OGTT) using streptozotocin-
induced diabetic rats: (A) time–
course of changes in blood
glucose level and (B) area under
the blood glucose concentration–
time curve (AUC) for the period of
0–2 h during OGTT. Data are
expressed as mean Æ SEM for six
animals in each group.
***p < 0.0001 versus diabetic
group.
effect of 6- or 7-membered cyclic amines as azepanyl
derivative 11 (IC50 = 3.47 lM) preserved the activity as
compared with less bulky group as piperidine 12
(IC50 = 7.88 lM) and pyrrolidine 13 (IC50 = 16.73 lM).
The cyclic analogs were found to have good correlation
with variation in ring size was observed, indicating
minimum activity for pyrrolidine analog and maximum
for azepanyl derivative. Thus, it was indicated that bulk-
ier group can be well tolerated, and reduction in
bulkiness may be responsible for weakened inhibitory
activity.
fluoropyrrolidine derivatives led to improved in vitro activity
against DPP IV.
Antihyperglycaemic activity
Two compounds 9 and 10 were chosen for in vivo study
as a result of strong in vitro DPP IV enzyme inhibitory
potential. Both compounds were administered orally at a
dose of 10 mg/kg. The compounds lowered blood glu-
cose levels and significantly suppressed hyperglycaemia at
single dose of 10 mg/kg as compared with the control
group beginning 30 min after glucose loading. The results
indicate that the area under blood glucose concentration–
time curve and OGTT significantly reduced (<0.0001) glu-
cose excursion during 0–2 h as shown in Figure 2A, B.
Vildagliptin showed 41.0% reduction in blood glucose at
time of OGTT (at 1.5 h) at 10 mg/kg. Compounds 9 and
10 also showed glucose lowering up to 37.06% and
38.99%, respectively, at same dose and at the same time.
These results showed that the in vivo efficacy of com-
pounds 9 and 10 was comparable to vildagliptin at
10 mg/kg as shown in Figure 2A, B. Thus, oral dosing of
both compounds significantly improved glycaemic control
and decreased elevated glucose challenge.
Further, effect of alkyl chain length using alkyl chloride lin-
ker was assessed to understand the alterations in activity
as a replacement for an acetamide linker. The SAR
revealed that incorporation of ethylpiperidine 14
(IC50 = 6.49 lM) showed comparable potency with that of
compound 3. The inhibitory potency was retained with
introduction of N,N-diethylethamine 15 (IC50 = 23.84 lM).
The inhibition was decreased sharply when N,N-dimethy-
lethamine 16 (IC50 = 30.21 lM) was substituted, which
suggest that decrease in alkyl chain length and replace-
ment by methyl group may lead to decrease in inhibitory
activity. The efforts to optimize the substituted prolyl-
162
Chem Biol Drug Des 2013; 82: 156–166

Prolyl-fluoropyrrolidine derivatives as DPP IV inhibitors
Modelling studies
cent to catalytic triad of active site. The active site is
located in the hydrolase domain and comprised of cata-
lytic triad residues Ser630, Asp708 and His740.
Dipeptidyl peptidase IV enzyme consists of two domains,
a eight-stranded beta-propeller domain at the N-terminus
and an a/b-hydrolase domain at the C-terminus. It has a
very large cavity surrounded by two domains. This
includes a hydrophobic pocket, S1, comprising residues
Tyr631, Val656, Trp659, Tyr662, Tyr666 and Val711 adja-
The docking protocol was first validated by docking the
known inhibitor, biarylphenylalanine fluoropyrrolidine amide
compound (compound 17), present in the binding pocket
of DPP IV (PDB Id: 2FJP]. A deviation observed in the con-
formation of the docked ligand on superimposition with
that from the crystal conformation was used to verify the
docking protocol. The two conformation of compound
S14 overlapped very well and calculated r.m.s deviation
ꢀ
ꢀ
was found to be 0.8 A. The r. m. s. deviation of <2 A
between docked pose and crystal conformation of the
ligand is considered as successful docking in the light of
resolution of crystal structure of the complex under con-
sideration. This control docking experiment verified the Li-
gandFit docking protocol and hence used for docking of
fluoropyrrolidine derivatives.
The fluoropyrrolidine derivatives docked in a manner similar
to biarylphenylalanine fluoropyrrolidine amide compound
(compound 17; PDB id: 2FJP; Figure 3). The fluoropyrroli-
dine nucleus of their derivatives occupied S1 pocket simi-
lar to fluoropyrrolidine group of compound 17 observed in
the crystal structure (PDB id: 2FJP). The second pyrroli-
dine moiety occupies the position of dimethyl amide frag-
ment of compound 17 and forms hydrogen bond with
Tyr547 similar to compound 17.
Figure 3: The binding position of docked compounds (stick
rendering) in the binding pocket of dipeptidyl peptidase (DPP) IV
(GRASP surface rendering). The selected side chains of DPP IV
have been represented in stick rendering in grey and docked
compound 9 in cyan, 10 in magenta, 11 in yellow and compound
17 in orange.
A
B
C
D
Figure 4: The docked complex
of (A) compound 3, (B) compound
9, (C) compound 10 and (B)
compound 11 in the binding
pocket of dipeptidyl peptidase
(DPP) IV. The DPP IV is shown in
cartoon rendering with selected
binding site residues in (stick
rendering in green) and
compounds in (stick rendering in
cyan).
Chem Biol Drug Des 2013; 82: 156–166
163

Sharma et al.
O
O
NH
a
NH
N
N
b
c
O
O
O
O
O
O
HO
N
N
HO
1
2
3
F
F
O
O
b
R1
H
Cl
Cl
R1
+
Cl
- HCl
e
f
4-8
O
R2
R1
N
N
N
O
O
N
9-13
14-16
F
F
14, R2
15, R2
=
=
9, R1 =
10, R1=
(piperidin-yl-)ethyl
N,N - diethylethanamino
(pyridin-2-yl)piperazin -1-yl
(pyrimidyl-2-yl)piperazin-1-yl
16, R2 =N,N - dimethylethanamino
11, R1
12, R1
13, R1
azepanyl
=
piperidynyl
pyrrolidinyl
=
=
Scheme 1: Reagents and Conditions: (a) BOC anhydride, triethylamine, THF, (b) 3-Fluoropyrrolidine, 1-(3-dimethyl aminopropyl)-3-
ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, diisopropyl ethylamine, DCM (c) TFA, DCM, 0 C (d) H-R1 (secondary amines),
diisopropylethylamine, 1,4-dioxan, reflux, 2 h, (e) 4–8, dry tetrahydrofuran, 12 h, rt, (f) RCl, dry tetrahydrofuran, 12 h, rt.
Table 2: Docking results of compounds (3, 9, 10 and 11) with dipeptidyl peptidase IV
Docking energy (kcal/mol)
Interacting residues with docked ligand (hydrogen bonded residues are highlighted in
Ligand
Steric
Electrostatic Total
bold)
Compound 3
Compound 9
Compound 10 À31.08 À51.09
Compound 11 À31.54 À40.90
À25.36 À15.48
À31.12 À47.21
À41.36 R125, E205, E206, Y547, S630, Y631, Y662, Y666, N10
À79.33 R125, V207, S209, F357, R358, Y547, S630, Y631, V656, W659, Y662, Y666, V711
À82.17 R125, V207, S209, F357, R358, Y547, S630, Y631, V656, W659, Y662, Y666, V711
À72.44 R125, E205, E206, S209, F357, Y547, S630, V656, W659, Y662, Y666, V711
The compound 3, parent nucleus, binds to the binding
pocket near catalytic triad residues in a manner similar to
peptide-based Val-Pyr inhibitor (PDB id: 1N1M) (25). The
fluoropyrrolidine moiety occupies the S1 hydrophobic
pocket and stack with aromatic ring of Tyr666. The fluo-
compound 9 binds similar to compound 11 and forms
hydrogen bond with Arg125, Tyr547 and Ser630 (Fig-
ure 4B). The piperazine ring of compound 9 occupies the
position similar to seven-membered heterocyclic ring of
compound 11 and has van der waal interactions with phe-
nyl ring of Phe357. The pyridine moiety at piperazine ring
further extends into the cavity and interacts with Arg358.
ꢀ
rine atom is 2.8 A away from side chain hydroxyl group of
Ser630, the catalytic triad residues and hence inhibits the
DPP IV activity. The amide oxygen and tertiary amine
group of pyrrolidine ring forms hydrogen bond with side
chain of Arg125 and Glu205, respectively (Figure 4A).
The compound 10 has pyrimidine ring in place of pyri-
dine ring in compound 9. The two compounds binds in
an identical manner. The pyrimidine ring of compound
10 forms hydrogen bond with hydroxyl group of Ser209
in addition to all the hydrogen bonds observed between
The compound 9 has even larger substituent and carries a
pyridyl-substituted piperazine moiety. The nucleus of bulky
164
Chem Biol Drug Des 2013; 82: 156–166

Prolyl-fluoropyrrolidine derivatives as DPP IV inhibitors
compound
9 and DPP IV (Figure 4C). It was more
Conflict of Interest
potent than compound 9, and it might be because of
additional hydrogen bond between pyrimidine ring and
side chain of Ser209 according to modelling study. This
is also reflected by more negative electrostatic interac-
tion energy, which includes hydrogen bond energy as
compared with compound in spite of having equal steric
energy (Table 2).
None.
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Acknowledgment
The funding of this study was supported by Government
of NCT Delhi, India. The support from the Indian council of
Medical Research in the form of the Bio-Medical Informat-
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