Inhibition of protein glycation by urea and thiourea derivatives of glycine/proline conjugated benzisoxazole analogue-Synthesis and structure-activity studies

Article abstract of DOI:10.1016/j.ejmech.2012.12.029

Synthesis of a new series of urea/thiourea derivatives of Gly/Pro conjugated benzisoxazole has been reported. Structure of the compounds was characterized by physical and spectroscopical data and has been screened for their in vitro antiglycation activity. Several compounds showed promising activity with IC₅₀ < 5 μM compared to standard rutin (IC ₅₀ = 41.9 μM). Further, it was found that compounds containing methoxy and bromine substituents have exerted highly potent activity. Thus, the title compounds represent novel class of potent antiglycating agents.

Full text of DOI:10.1016/j.ejmech.2012.12.029

European Journal of Medicinal Chemistry 60 (2013) 325e332
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibition of protein glycation by urea and thiourea derivatives of
glycine/proline conjugated benzisoxazole analogue e Synthesis and
structureeactivity studies
*
C.S. Shantharam, D.M. Suyoga Vardhan, R. Suhas, M.B. Sridhara, D. Channe Gowda
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore e 570 006, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a new series of urea/thiourea derivatives of Gly/Pro conjugated benzisoxazole has been
reported. Structure of the compounds was characterized by physical and spectroscopical data and has
been screened for their in vitro antiglycation activity. Several compounds showed promising activity with
Received 21 July 2012
Received in revised form
8 December 2012
Accepted 11 December 2012
Available online 20 December 2012
IC50 < 5
m
M compared to standard rutin (IC50 ¼ 41.9
mM). Further, it was found that compounds con-
taining methoxy and bromine substituents have exerted highly potent activity. Thus, the title compounds
represent novel class of potent antiglycating agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Amino acids
Conjugation
Urea/thiourea
Antiglycation
1. Introduction
Urea is a functional moiety that is commonly found in natural
products and often displays a wide range of biological activities [5].
Diabetes is a very common endocrine disorder, which results
from an absolute or relative deficiency of insulin or insulin resis-
tance and is characterized by hyperglycemia. Diabetes predisposes
patients to chronic complications, and affects multiple parts of the
body. Life expectancy of diabetic patients is only two thirds of that
of the normal population. This chronic disorder affects 1e2% of the
world population [1].
Glycation (non-enzymatic glycosylation) results in Amadori
product which accumulates in living organisms that leads to struc-
tural and functional modifications of tissue proteins [2]. Many
studies have shown a significant role of glycation in the progress of
normal ageing and the pathogenesis of age-related diseases, such as
diabetes, atherosclerosis, end-stage renal disease, rheumatoid
arthritis, and neurodegenerative diseases. Therefore, targeting gly-
cation should have a broad and beneficial effect on ageing and age-
related diseases [3]. One can consider that AGEs inhibitors are the
Urea and thiourea compounds are known to possess antidiabetic
and antimicrobial activities [6]. Ureas and thioureas have been
studied for the systematic control of tuberculosis also [7]. A few
compounds of thiourea derivatives bearing benzothiazole moiety
have shown antimicrobial and anticancer activities [8]. A series of
diaryl substituted heterocyclic urea which have been reported to
inhibit cholesterol O-acy1 transferase (ACAT) as hypo-
cholestrolemic [9]. N,N-Disubstituted cyclic urea-3-benzamide was
found to be HIV protease inhibitor; which may be useful in the
treatment of AIDS [10]. Some of the urea compounds are multi-
stage glycation inhibitors with the highest post-Amadori activi-
ties. Some of these inhibited AGEeprotein cross-linking including
AGEecollagen cross-linking from the reaction of bovine serum
albumin with glucose [11].
The therapeutic applications of amino acids have received
a considerable attention in respiratory physiology, cardiology, renal
failure, neurological disorders, and congenital defects. Moreover
some of the amino acids are used as antiglycating agents also
[12,13]. One of the most frequently encountered heterocycle in
medicinal chemistry is benzisoxazole and its derivatives which are
reported to have diverse biological applications including antihis-
taminic, antifungal, antitumour, antiulcer, anticonvulsant, hypo-
glycemic, anti-inflammatory and antitubercular activities [14,15].
compoundswhichareable to cleavethe a-dicarbonyl species andact
as potent chelators of Cu2þ and inhibit the oxidation process [4].
Abbreviations: Boc, t-butoxycarbonyl; EDCI, 1-(3-dimethylaminopropyl)-3-
ethyl-carbodiimide; HOBt, 1-hydroxybenzotriazole; NMM, N-methyl morpholine;
TFA, trifluoroacetic acid.
* Corresponding author. Tel.: þ91 821 2419664 (O).
E-mail address: dchannegowda@yahoo.co.in (D.C. Gowda).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.029

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C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
Furthermore, benzisoxazole and its analogues constitute an active
class of compounds possessing antidiabetic activity [16].
The IC50 values are shown in Table 1. It is clear from the results that
all the synthesized ureido and thioureido derivatives of the
heterocyclic conjugates have shown enhanced activity than their
counter parts. Most of them are more potent than the standard,
rutin. Further, to the best of our knowledge, our synthesized
compounds have exhibited high activity than the compounds
documented in the literature [17e20].
Among the series, methoxy (3e8, 24e29) and bromo (18e21,
39e42) containing compounds have exhibited high activity. The
high activity of methoxy derivatives may be due to the electron
donating nature of methoxy group and that of bromo derivatives
may be due to less electron withdrawing (F > Cl > Br) and also due
to inductive and resonance effect. This may also be due to the fact
that the halogens particularly bromine is not involved in resonance
stabilization.
Jung et al. have synthesized methoxy derivatives of benzoqui-
nones and naphthoquinones and identified them as inhibitors of
glycation [17]. Later Khan et al. [18,19] have synthesized various
urea and isatin derivatives and evaluated their antiglycation
activity. Recently the same research group [20] has synthesized
benzoxazole derivatives and found that they act as possible anti-
glycating agents. It is evident from the results obtained by them
that the synthesized compounds have exhibited moderate or
equipotent activity as that of the standard. Prompted by the above
observations and in continuation of our research programme [21e
26], we envisioned our approach towards the synthesis of a new
series of ureido and thioureido derivatives of Gly/Pro conjugated
benzisoxazole followed by the antiglycation evaluation.
Among the two series of compounds tested, Gly containing
analogues have exhibited increased activity over the Pro containing
compounds. This may be due to the small size and simple nature of
Gly which would enhance the binding abilities. Between urea and
thiourea analogues, compounds with thiourea moiety have shown
increased activity over the former. This may probably be due to
more nucleophilic character of sulphur compared to oxygen. The
plausible mechanism of action involves the trapping of dicarbonyl
species and preventing oxidation using transition metal chelators
(Cu2þ) or free radical scavengers [4].
2. Results and discussion
2.1. Chemistry
The heterocycle, [3-(4-piperidyl)-6-fluoro-1,2-benzisoxazole]$
HCl was synthesized by following the previously reported method
[27]. This was further conjugated to Boc-Gly/Pro-OH using EDCI/
HOBt as coupling agent and NMM as base. Boc group of the
conjugates was removed using TFA and reacted with various
substituted phenyl isocyanates/isothiocyanates to obtain urea/
thiourea derivatives respectively (Scheme 1). Synthesis was
confirmed by IR, 1H NMR, HRMS and elemental analysis. In IR
spectra stretching frequencies appeared at 1615e1644 cmꢀ1 (CO),
w2040 cmꢀ1 (CS) and w3300 cmꢀ1 (NH) and in 1H NMR spectra
For the first time we have replaced ‘O’ by ‘S’ and succeeded in
obtaining highly potent molecules. Initially, we have chosen simple
amino acids like glycine and proline. Further, the work with other
amino acids and substituents is in progress.
signals appeared at
for thiourea compounds whereas for the urea derivatives signals
were observed at w8.05 (1H, t, eNH) and w8.95 (1H, s, eNH).
Further, the presence of all requisite peaks and the absence of
extraneous peaks confirm the synthesis.
d w9.95 (1H, s, eNH) and w10.36 (1H, s, eNH)
3. Conclusion
d
We have successfully synthesized a series of urea/thiourea
derivatives of Gly/Pro conjugated benzisoxazole with different
functionalities. Some of the representatives of the series were
identified as highly potent antiglycating agents. The antiglycating
activity of the synthesized compounds showed that urea and
thiourea moieties play a major role in enhancing the activity.
Further, it is interesting to note that OCH3 and Br act as active
moieties in inhibiting the glycation. Thus, nature of the substituent
2.2. Antiglycation activity (in vitro)
Evaluation of antiglycation activity of the synthesized
compounds was examined by the method of Nakagawa et al. [28].
Scheme 1. Synthesis of urea/thiourea derivatives of amino acids conjugated heterocycle.

C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
327
Table 1
Antiglycation activity of the synthesized compounds.
Entry
Compounds
IC50 ꢃ SEM (
m
M)a
Entry
Compounds
IC50 ꢃ SEM(
m
M)a
R
Z
R
Z
1
2
3
4
5
6
7
8
H
H
O
S
O
S
O
S
O
S
O
S
O
O
S
O
S
O
S
390.4 ꢃ 0.4
380.5 ꢃ 0.8
20.2 ꢃ 0.3
4.1 ꢃ 0.6
21.1 ꢃ 0.1
15.3 ꢃ 0.8
19.5 ꢃ 0.4
3.6 ꢃ 0.3
55.5 ꢃ 0.7
9.1 ꢃ 0.5
100.2 ꢃ 0.6
75.8 ꢃ 0.2
70.3 ꢃ 0.7
143.5 ꢃ 0.5
Inactive
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
II
H
H
O
S
O
S
O
S
O
S
O
S
O
O
S
O
S
O
S
340.1 ꢃ 0.5
295.1 ꢃ 0.3
30.6 ꢃ 0.7
5.2 ꢃ 0.4
2-OCH3
2-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
2-F
2-F
3-F
4-F
4-F
3-Cl
3-Cl
4-Cl
4-Cl
2-Br
3-Br
3-Br
4-Br
2-OCH3
2-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
2-F
2-F
3-F
4-F
4-F
3-Cl
3-Cl
4-Cl
4-Cl
2-Br
3-Br
3-Br
4-Br
33.4 ꢃ 0.2
20.5 ꢃ 0.1
22.1 ꢃ 0.7
3.9 ꢃ 0.4
9
54.8 ꢃ 0.9
7.8 ꢃ 0.8
10
11
12
13
14
15
16
17
18
19
20
21
I
116.7 ꢃ 0.1
76.1 ꢃ 0.4
70.9 ꢃ 0.6
145.4 ꢃ 0.7
125.7 ꢃ 0.8
60.3 ꢃ 0.3
59.4 ꢃ 0.8
7.8 ꢃ 0.2
83.9 ꢃ 0.9
Inactive
S
O
S
6.3 ꢃ 0.8
9.3 ꢃ 0.4
4.9 ꢃ 0.7
8.5 ꢃ 0.5
Inactive
S
O
S
6.9 ꢃ 0.4
4.7 ꢃ 0.1
O
O
6.8 ꢃ 0.6
Boc-Gly-
Heterocycle
Heterocycle
Boc-Pro-
Heterocycle
Rutin (Std)
Inactive
Inactive
41.9 ꢃ 0.7
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases.
was found to be crucial to improve the activity. This study extends
the knowledge of different substituents at phenyl ring and also
various amino acids which might be of interest for the identifica-
tion of more antiglycation agents.
4.2. General procedure for the conjugation of Boc-Gly/Pro-OH to
heterocycle (I and II)
Boc-Gly/Pro-OH (0.002 mol) and HOBt (0.306 g, 0.002 mol)
dissolved in DMF (10 mL/g of compound) and cooled to 0 ꢁC was
added NMM (0.22 mL, 0.002 mol). EDCI (0.458 g, 0.002 mol) was
added under stirring while maintaining the temperature at 0 ꢁC
and stirred for 15 min. To this a pre-cooled solution of [3-(4-
piperidinyl)-6-fluoro-1,2-benzisoxazole]$HCl (0.514 g, 0.002 mol)
and NMM (0.22 mL, 0.002 mol) in DMF (5 mL) was added slowly.
After 20 min, pH of the solution was adjusted to 8 by the addition of
NMM and the reaction mixture was stirred overnight at room
temperature. DMF was removed under reduced pressure and the
residue was poured into about 100 mL ice-cold 90% saturated
KHCO3 solution and stirred for 30 min. The precipitated product
was taken into CHCl3 and washed sequentially with 5% NaHCO3
solution (2 ꢂ 20 mL), water (2 ꢂ 20 mL), 0.1 N cold HCl solution
(2 ꢂ 20 mL) and finally brine solution (2 ꢂ 20 mL). The CHCl3 layer
was dried over anhydrous Na2SO4 and the solvent was removed
under reduced pressure. The products so obtained were recrystal-
lized from ether/petroleum ether to get desired products, I and II.
4. Experimental
4.1. Materials
Boc-amino acids, EDCI, HOBt and TFA were purchased from
Advanced Chem. Tech. (Louisville, Kentucky, USA). NMM and
substituted phenyl isocyanates/isothiocyanates were purchased
from SigmaeAldrich Co., USA. All solvents and reagents used for the
synthesis were of analytical grade. Silica gel for TLC was purchased
from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Progress
of the reaction was monitored by TLC using silica gel coated on glass
plates with the solvent system comprising chloroform/methanol/
acetic acid in the ratio 98:2:3 ðRaf Þ and 95:5:3 ðRfbÞ and the
compounds on TLC plates were detected by iodine vapours. Melting
points were determined on Superfit (India) melting point appa-
ratus and are uncorrected. Elemental analysis was performed by
using VARIO EL III CHNS Elementar. 1H NMR spectra were obtained
on VARIAN 400 MHz instrument using DMSO-d6 and the chemical
4.3. General procedure for the synthesis of ureido and thioureido
derivatives 1e42
shifts are reported as parts per million (d ppm) using TMS as an
internal standard. Electrospray ionization mass spectrometry (ESI-
MS) was performed on a Bruker electrospray mass spectrometer.
FT-IR was performed in Jasco spectrometer. Bovine serum albumin
(BSA) was purchased from Research Organics, Cleveland, while
other chemicals used for inhibition studies were purchased from
Sigma Aldrich.
Boc-Gly/Pro-Heterocycle (0.150 g) was deprotected by stirring in
TFA (1.5 mL) for 45 min. TFA was removed under reduced pressure,
triturated with ether, filtered, washed with ether and dried to
obtain TFA$H-Gly/Pro-Heterocycle (100%).
A solution of TFA$H-Gly/Pro-Heterocycle (0.001 mol) in DMF
(10 mL/g of compound) was cooled to 0 ꢁC and added NMM

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C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
(0.10 mL, 0.001 mol). To this solution respective substituted phenyl
isocyanates/isothiocyanates (0.0012 mol) was added dropwise
while maintaining the temperature at 0 ꢁC. The reaction mixture
was stirred for 8 h slowly warming to room temperature. DMF was
evaporated under high vacuum and the residue was poured into
about 50 mL ice-cold 90% saturated KHCO3 solution and stirred for
15 min. The precipitated compound was extracted with ethyl
acetate and washed with 5% NaHCO3 solution (2 ꢂ 10 mL), water
(2 ꢂ 10 mL), 0.1 N HCl (2 ꢂ 10 mL) followed by brine solution. The
organic layer was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure, triturated with hexane, filtered
and dried.
3.53 (4H, m, eCH2), 6.62e7.42 (3H, m, Ar-H); Calculated
HRMS ¼ 426.4379; Found HRMS ¼ 449.1775 (Mþ þ Na); Elem.
Anal.: Calcd. for C22H23FN4O4: C: 61.96; H: 5.44; N: 13.14; Found: C:
61.93; H: 5.42; N: 13.10.
4.3.6. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(2 methoxyphenyl)thiourea (4)
Raf ¼ 0.46; Rbf ¼ 0.68; Yield ¼ 89%; M.P. ¼ 100 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1644 (CO), 2032 (CS), 3277 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.82e7.64 (4H, m, Ar-H), 8.00 (1H, s, eNH),
7.06e7.10 (1H, m, eNH), 3.81 (3H, s, eOCH3); Gly ¼ 4.47e4.48 (2H,
d, eaCH2); Heterocycle ¼ 1.88e2.20 (4H, m, eCH2), 2.29e3.04 (1H,
m, eCH), 3.28e3.95 (4H, m, eCH2), 6.82e7.64 (3H, m, Ar-H);
4.3.1. tert-Butyl 2-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-
yl)-2-oxoethylcarbamate (I)
Calculated HRMS
¼
442.5055; Found HRMS
¼
465.1528
(Mþ þ Na); Elem. Anal.: Calcd. for C22H23FN4O3S: C: 59.71; H: 5.24;
Raf ¼ 0.46; Rfb ¼ 0.63; Yield ¼ 85%; M.P. ¼ Gum; IR (Nujol, nmax
,
N: 12.66; S: 7.25; Found: C: 59.75; H: 5.21; N: 12.62; S: 7.21.
cmꢀ1) ¼ 1629 (CO), 3275 (NH); 1H NMR (DMSO-d6,
d ppm):
Boc ¼ 1.43 (9H, s); Gly ¼ 4.27 (2H, d, eaCH2), 8.01e8.05 (1H, t, NH);
Heterocycle ¼ 1.67e2.08 (4H, m, eCH2), 2.08e2.92 (1H, m, eCH),
3.18e3.56 (4H, m, eCH2), 7.25e7.67 (3H, m, Ar-H); Calculated
HRMS ¼ 377.4090; Found HRMS ¼ 400.1532 (Mþ þ Na); Elem.
Anal.: Calcd. for C19H24FN3O4: C: 60.47; H: 6.41; N: 11.13; Found: C:
60.42; H: 6.38; N: 11.16.
4.3.7. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(3-methoxyphenyl)urea (5)
Raf ¼ 0.43; Rbf ¼ 0.66; Yield ¼ 90%; M.P. ¼ 143 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1636 (CO), 3305 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.54e7.46 (4H, m, Ar-H), 7.96 (1H, s, eNH), 7.02e7.08 (1H,
m, eNH), 3.78 (3H, s, eOCH3); Gly ¼ 4.43e4.46 (2H, d, eaCH2);
Heterocycle ¼ 1.67e2.09 (4H, m, eCH2), 2.92 (1H, m, eCH), 3.19e
3.51 (4H, m, eCH2), 6.54e7.46 (3H, m, Ar-H); Calculated
HRMS ¼ 426.4379; Found HRMS ¼ 449.1782 (Mþ þ Na); Elem.
Anal.: Calcd. for C22H23FN4O4: C: 61.96; H: 5.44; N: 13.14; Found: C:
61.91; H: 5.46; N: 13.13.
4.3.2. tert-Butyl 2-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-
carbonyl)pyrrolidine-1-carboxylate (II)
Raf ¼ 0.47; Rbf ¼ 0.65; Yield ¼ 82%; M.P. ¼ 140 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1625 (CO), 3273 (NH); 1H NMR (DMSO-d6,
d ppm):
Boc ¼ 1.43 (9H, s); Pro ¼ 4.07 (1H, t, eaCH), 1.84e1.89 (2H, m, eb
,
gCH2), 3.71e3.75 (2H, m, edCH2); Heterocycle ¼ 1.84e1.89 (4H, m,
eCH2), 2.5 (1H, m, eCH), 3.22e3.61 (4H, m, eCH2), 7.02e7.62 (3H,
m, Ar-H); Calculated HRMS ¼ 417.4727; Found HRMS ¼ 440.2054
(Mþ þ Na); Elem. Anal.: Calcd. for C22H28FN3O4: C: 63.29; H: 6.76;
N: 10.07; Found: C: 63.24; H: 6.79; N: 10.03.
4.3.8. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(3-methoxyphenyl)thiourea (6)
Raf ¼ 0.45; Rbf ¼ 0.67; Yield ¼ 92%; M.P. ¼ 160 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1627 (CO), 2040 (CS), 3272 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.71e7.64 (4H, m, Ar-H), 8.04 (1H, s, eNH),
7.09e7.15 (1H, m, eNH), 3.82 (3H, s, eOCH3); Gly ¼ 4.41e4.43 (2H,
d, eaCH2); Heterocycle ¼ 1.66e2.23 (4H, m, eCH2), 2.91 (1H, m, e
CH), 3.12e3.46 (4H, m, eCH2), 6.71e7.64 (3H, m, Ar-H); Calculated
HRMS ¼ 442.5055; Found HRMS ¼ 465.1536 (Mþ þ Na); Elem.
Anal.: Calcd. for C22H23FN4O3S: C: 59.71; H: 5.24; N: 12.66; S: 7.25;
Found: C: 59.74; H: 5.21; N: 12.61; S: 7.29.
4.3.3. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-phenylurea (1)
Raf ¼ 0.47; Rbf ¼ 0.68; Yield ¼ 89%; M.P. ¼ 160 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1622 (CO), 3283 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.54e7.46 (5H, m, Ar-H), 8.19 (1H, s, eNH), 8.08 (1H, m, e
NH); Gly ¼ 4.45 (2H, d, eaCH2); Heterocycle ¼ 1.64e2.07 (4H, m, e
CH2), 2.93 (1H, m, eCH), 3.21e3.52 (4H, m, eCH2), 6.54e7.46 (3H,
m, Ar-H); Calculated HRMS ¼ 397.4219; Found HRMS ¼ 419.1432
(Mþ þ Na); Elem. Anal.: Calcd. for C21H21FN4O3: C: 63.63; H: 5.34;
N: 14.13; Found: C: 63.58; H: 5.41; N: 14.17.
4.3.9. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(4-methoxyphenyl)urea (7)
Raf ¼ 0.45; Rbf ¼ 0.67; Yield ¼ 89%; M.P. ¼ 147e150 ꢁC; IR (Nujol,
nmax, cmꢀ1) ¼ 1638 (CO), 3304 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.69e7.35 (4H, m, Ar-H), 7.98 (1H, s, eNH), 7.01e7.07 (1H, m,
eNH), 3.81 (3H, s, eOCH3); Gly ¼ 4.39e4.42 (2H, d, eaCH2);
Heterocycle ¼ 1.67e2.09 (4H, m, eCH2), 2.92 (1H, m, eCH), 3.22e
3.53 (4H, m, eCH2), 6.69e7.35 (3H, m, Ar-H); Calculated
HRMS ¼ 426.4379; Found HRMS ¼ 449.1763 (Mþ þ Na); Elem.
Anal.: Calcd. for C22H23FN4O4: C: 61.96; H: 5.44; N: 13.14; Found: C:
61.85; H: 5.40; N: 13.17.
4.3.4. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-phenylthiourea (2)
Raf ¼ 0.46; Rbf ¼ 0.66; Yield ¼ 85%; M.P. ¼ 165 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1628 (CO), 2036 (CS), 3277 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.73e7.68 (5H, m, Ar-H), 9.92 (1H, s, eNH), 9.87
(1H, s, eNH); Gly ¼ 4.44 (2H, d, eaCH2); Heterocycle ¼ 1.69e2.27
(4H, m, eCH2), 2.94 (1H, m, eCH), 3.13e3.47 (4H, m, eCH2),
6.73e7.68 (3H, m, Ar-H); Calculated HRMS ¼ 412.4776; Found
HRMS ¼ 435.1398 (Mþ þ Na); Elem. Anal.: Calcd. for C21H21FN4O2S:
C: 61.15; H: 5.13; N: 13.58; S: 7.77; Found: C: 61.21; H: 5.18; N:
13.51; S: 7.69.
4.3.10. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(4-methoxyphenyl)thiourea (8)
Raf ¼ 0.43; Rbf ¼ 0.66; Yield ¼ 91%; M.P. ¼ 123 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1625 (CO), 2041 (CS), 3275 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.79e7.61 (4H, m, Ar-H), 8.04 (1H, s, eNH),
4.3.5. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(2-methoxy phenyl)urea (3)
7.04e7.12 (1H, m, eNH), 3.83 (3H, s, eOCH3); Gly ¼ 4.41e4.43 (2H,
d, eaCH2); Heterocycle ¼ 1.66e2.23 (4H, m, eCH2), 2.91 (1H, m, e
CH), 3.11e3.48 (4H, m, eCH2), 6.79e7.61 (3H, m, Ar-H); Calculated
HRMS ¼ 442.5055; Found HRMS ¼ 465.1565 (Mþ þ Na); Elem.
Anal.: Calcd. for C22H23FN4O3S: C: 59.71; H: 5.24; N: 12.66; S: 7.25;
Found: C: 59.77; H: 5.20; N: 12.60; S: 7.28.
Raf ¼ 0.47; Rbf ¼ 0.69; Yield ¼ 90%; M.P. ¼ 65 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1617 (CO), 3293 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.62e7.42 (5H, m, Ar-H), 7.98 (1H, s, eNH), 7.03e7.09 (1H,
m, eNH), 3.79 (3H, s, OCH3); Gly ¼ 4.42e4.44 (2H, d, eaCH2);
Heterocycle ¼ 1.67e2.09 (4H, m, eCH2), 2.92 (1H, m, eCH), 3.21e

C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
329
4.3.11. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(2-fluorophenyl)urea (9)
(Mþ þ Na); Elem. Anal.: Calcd. for C21H20ClFN4O3: C: 58.54; H: 4.68;
N: 13.00; Found: C: 58.46; H: 4.61; N: 13.07.
Raf ¼ 0.38; Rbf ¼ 0.53; Yield ¼ 90%; M.P. ¼ 195 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1617 (CO), 3299 (NH); 1H NMR (DMSO-d6,
d
ppm):
4.3.17. 1-(3-Chlorophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)thiourea (15)
Urea ¼ 7.04e7.71 (4H, m, Ar-H), 8.91 (1H, s, eNH), 8.09 (1H, m, e
NH); Gly ¼ 4.41 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m, e
CH2), 2.92 (1H, m, eCH), 3.21e3.47 (4H, m, eCH2), 7.04e7.71 (3H,
m, Ar-H); Calculated HRMS ¼ 414.4045; Found HRMS ¼ 437.1525
(Mþ þ Na); Elem. Anal.: Calcd. for C21H20F2N4O3: C: 60.86; H: 4.86;
N: 13.52; Found: C: 60.81; H: 4.82; N: 13.56.
Raf ¼ 0.46; Rbf ¼ 0.67; Yield ¼ 87%; M.P. ¼ 147 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1615 (CO), 2034 (CS), 3286 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.92e7.95 (4H, m, Ar-H), 10.28 (1H, s, eNH),
9.91 (1H, m, eNH); Gly ¼ 4.41 (2H, d, eaCH2); Heterocycle ¼ 1.66e
2.23 (4H, m, eCH2), 2.91 (1H, m, eCH), 3.09e3.45 (4H, m, eCH2),
6.92e7.95 (3H, m, Ar-H); Calculated HRMS ¼ 446.9247; Found
4.3.12. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
HRMS
¼
469.1021 (Mþ
þ
Na); Elem. Anal.: Calcd. for
oxoethyl)-3-(2-fluorophenyl)thiourea (10)
C21H20ClFN4O2S: C: 56.44; H: 4.51; N: 12.54; S: 7.17; Found: C:
56.38; H: 4.56; N: 12.49; S: 7.23.
Raf ¼ 0.45; Rbf ¼ 0.67; Yield ¼ 88%; M.P. ¼ 158 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1627 (CO), 2028 (CS), 3276 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 7.19e8.21 (4H, m, Ar-H), 10.25 (1H, s, eNH),
4.3.18. 1-(4-Chlorophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)urea (16)
9.91 (1H, m, eNH); Gly ¼ 4.48 (2H, d, eaCH2); Heterocycle ¼ 1.66e
2.23 (4H, m, eCH2), 2.91 (1H, m, eCH), 3.09e3.51 (4H, m, eCH2),
7.19e8.21 (3H, m, Ar-H); Calculated HRMS ¼ 430.4701; Found
Raf ¼ 0.43; Rbf ¼ 0.63; Yield ¼ 88%; M.P. ¼ 88e90 ꢁC; IR (Nujol,
nmax, cmꢀ1) ¼ 1636 (CO), 3294 (NH); 1H NMR (DMSO-d6,
d ppm):
HRMS
¼
453.1365 (Mþ
þ
Na); Elem. Anal.: Calcd. for
Urea ¼ 6.76e7.63 (4H, m, Ar-H), 8.89 (1H, s, eNH), 8.07 (1H, m, e
NH); Gly ¼ 4.47 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m, e
CH2), 2.92 (1H, m, eCH), 3.20e3.50 (4H, m, eCH2), 6.76e7.63 (3H,
m, Ar-H); Calculated HRMS ¼ 430.8591; Found HRMS ¼ 453.1258
(Mþ þ Na); Elem. Anal.: Calcd. for C21H20ClFN4O3: C: 58.54; H: 4.68;
N: 13.00; Found: C: 58.61; H: 4.63; N: 13.09.
C21H20F2N4O2S: C: 58.59; H: 4.68; N: 13.02; S: 7.45; Found: C:
58.53; H: 4.61; N: 13.11; S: 7.49.
4.3.13. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(3-fluorophenyl)urea (11)
Raf ¼ 0.46; Rbf ¼ 0.68; Yield ¼ 89%; M.P. ¼ 105 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1623 (CO), 3272 (NH); 1H NMR (DMSO-d6,
d
ppm):
4.3.19. 1-(4-Chlorophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)thiourea (17)
Urea ¼ 7.04e7.73 (4H, m, Ar-H), 8.85 (1H, s, eNH), 8.15 (1H, m, e
NH); Gly ¼ 4.36 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m, e
CH2), 2.92 (1H, m, eCH), 3.22e3.49 (4H, m, eCH2), 7.04e7.73 (3H,
m, Ar-H); Calculated HRMS ¼ 414.4045; Found HRMS ¼ 437.1536
(Mþ þ Na); Elem. Anal.: Calcd. for C21H20F2N4O3: C: 60.86; H: 4.86;
N: 13.52; Found: C: 60.89; H: 4.80; N: 13.49.
Raf ¼ 0.43; Rbf ¼ 0.62; Yield ¼ 82%; M.P. ¼ 125 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1618 (CO), 2039 (CS), 3280 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.97e7.83 (4H, m, Ar-H), 10.31 (1H, s, eNH),
9.85 (1H, m, eNH); Gly ¼ 4.46 (2H, d, eaCH2); Heterocycle ¼ 1.66e
2.23 (4H, m, eCH2), 2.91 (1H, m, eCH), 3.10e3.45 (4H, m, eCH2),
6.97e7.83 (3H, m, Ar-H); Calculated HRMS ¼ 446.9247; Found
4.3.14. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
HRMS
¼
469.1041 (Mþ
þ
Na); Elem. Anal.: Calcd. for
oxoethyl)-3-(4-fluorophenyl)urea (12)
C21H20ClFN4O2S: C: 56.44; H: 4.51; N: 12.54; S: 7.17; Found: C:
56.49; H: 4.46; N: 12.58; S: 7.27.
Raf ¼ 0.43; Rbf ¼ 0.66; Yield ¼ 91%; M.P. ¼ 110 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1629 (CO), 3279 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 7.04e7.67 (4H, m, Ar-H), 8.95 (1H, s, eNH), 8.05 (1H, m, e
NH); Gly ¼ 4.42 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m, e
CH2), 2.92 (1H, m, eCH), 3.23e3.51 (4H, m, eCH2), 7.04e7.67 (3H,
m, Ar-H); Calculated HRMS ¼ 414.4045; Found HRMS ¼ 437.0951
(Mþ þ Na); Elem. Anal.: Calcd. for C21H20F2N4O3: C: 60.86; H: 4.86;
N: 13.52; Found: C: 60.79; H: 4.82; N: 13.61.
4.3.20. 1-(2-Bromophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)thiourea (18)
Raf ¼ 0.45; Rbf ¼ 0.65; Yield ¼ 86%; M.P. ¼ 145 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1634 (CO), 2043 (CS), 3276 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.92e7.95 (4H, m, Ar-H), 10.28 (1H, s, eNH),
9.91 (1H, m, eNH); Gly ¼ 4.41 (2H, d, eaCH2); Heterocycle ¼ 1.66e
2.23 (4H, m, eCH2), 2.91 (1H, m, eCH), 3.10e3.45 (4H, m, eCH2),
6.92e7.95 (3H, m, Ar-H); Calculated HRMS ¼ 491.3757; Found
HRMS ¼ 513.0498, 515.0611 (Mþ þ Na); Elem. Anal.: Calcd. for
C21H20BrFN4O2S: C: 51.33; H: 4.10; N: 11.40; S: 6.53; Found: C:
51.39; H: 4.17; N: 11.46; S: 6.59.
4.3.15. 1-(2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-
oxoethyl)-3-(4-fluorophenyl)thiourea (13)
Raf ¼ 0.49; Rbf ¼ 0.69; Yield ¼ 85%; M.P. ¼ 145 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1639 (CO), 2032 (CS), 3285 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 7.14e8.05 (4H, s, Ar-H), 10.36 (1H, s, eNH), 9.95
(1H, m, eNH); Gly ¼ 4.41 (2H, d, eaCH2); Heterocycle ¼ 1.66e2.23
4.3.21. 1-(3-Bromophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)urea (19)
(4H, m, eCH2), 2.91 (1H, m, eCH), 3.13e3.49 (4H, m, eCH2), 7.14e
8.05 (3H, m, Ar-H); Calculated HRMS
¼
430.4701; Found
Raf ¼ 0.44; Rbf ¼ 0.63; Yield ¼ 87%; M.P. ¼ 134 ꢁC; IR (Nujol, nmax
,
HRMS Na); Elem. Anal.: Calcd. for
¼
453.0305 (Mþ
þ
cmꢀ1) ¼ 1633 (CO), 3271 (NH); 1H NMR (DMSO-d6,
d ppm):
C21H20F2N4O2S: C: 58.59; H: 4.68; N: 13.02; S: 7.45; Found: C:
58.51; H: 4.59; N: 13.08; S: 7.53.
Urea ¼ 6.74e7.61 (4H, m, Ar-H), 8.45 (1H, s, eNH), 8.07 (1H, m, e
NH); Gly ¼ 4.41 (2H, d, eaCH2); Heterocycle ¼ 1.63e2.23 (4H, m, e
CH2), 2.95 (1H, m, eCH), 3.21e3.54 (4H, m, eCH2), 6.74e7.61 (3H,
m, Ar-H); Calculated HRMS ¼ 475.3101; Found HRMS ¼ 497.0681,
499.0732 (Mþ þ Na); Elem. Anal.: Calcd. for C21H20BrFN4O3: C:
53.07; H: 4.24; N: 11.79; Found: C: 53.01; H: 4.29; N: 11.83.
4.3.16. 1-(3-Chlorophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)urea (14)
Raf ¼ 0.42; Rbf ¼ 0.65; Yield ¼ 82%; M.P. ¼ 93 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1634 (CO), 3291 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.72e7.58 (4H, m, Ar-H), 8.95 (1H, s, eNH), 8.03 (1H, m, e
NH); Gly ¼ 4.42 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m, e
CH2), 2.92 (1H, m, eCH), 3.19e3.50 (4H, m, eCH2), 6.72e7.58 (3H,
m, Ar-H); Calculated HRMS ¼ 430.8591; Found HRMS ¼ 453.1291
4.3.22. 1-(3-Bromophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)thiourea (20)
Raf ¼ 0.46; Rbf ¼ 0.67; Yield ¼ 91%; M.P. ¼ 140 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1625 (CO), 2031 (CS), 3268 (NH); 1H NMR (DMSO-d6,

330
C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
d
ppm): Urea ¼ 6.75e7.54 (4H, m, Ar-H), 8.85 (1H, s, eNH), 8.09 (1H,
HRMS
¼
505.1702 (Mþ
þ
Na); Elem. Anal.: Calcd. for
m, eNH); Gly ¼ 4.39 (2H, d, eaCH2); Heterocycle ¼ 1.64e2.03 (4H,
C25H27FN4O3S: C: 62.22; H: 5.64; N: 11.61; S: 6.64; Found: C: 62.13;
H: 5.69; N: 11.67; S: 6.58.
m, eCH2), 2.96 (1H, m, eCH), 3.24e3.55 (4H, m, eCH2), 6.75e7.54
(3H, m, Ar-H); Calculated HRMS
¼
491.3757; Found
HRMS ¼ 513.0498, 515.0611 (Mþ þ Na); Elem. Anal.: Calcd. for
C21H20BrFN4O2S: C: 51.33; H: 4.10; N: 11.40; S: 6.53; Found: C:
51.39; H: 4.04; N: 11.46; S: 6.58.
4.3.28. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(3-methoxyphenyl)pyrrolidine-1-carboxamide (26)
Raf ¼ 0.43; Rbf ¼ 0.67; Yield ¼ 89%; M.P. ¼ 80 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1631 (CO), 3319 (NH); 1H NMR (DMSO-d6,
d ppm):
4.3.23. 1-(4-Bromophenyl)-3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)-2-oxoethyl)urea (21)
Urea ¼ 6.86e7.46 (4H, m, Ar-H), 7.87 (1H, s, eNH), 3.76 (3H, s, e
,
OCH3); Pro ¼ 4.32e4.44 (1H, t, eaCH), 1.78e2.20 (4H, m, eb gCH2),
Raf ¼ 0.44; Rbf ¼ 0.63; Yield ¼ 88%; M.P. ¼ 118 ꢁC; IR (Nujol,
3.75e3.79 (2H, m, edCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2),
2.55 (1H, m, eCH), 3.16e3.53 (4H, m, eCH2), 6.86e7.46 (3H, m, Ar-
H); Calculated HRMS ¼ 466.5036; Found HRMS ¼ 489.1856
(Mþ þ Na); Elem. Anal.: Calcd. for C25H27FN4O4: C: 64.37; H: 5.83;
N: 12.01; Found: C: 64.29; H: 5.87; N: 12.05.
nmax, cmꢀ1) ¼ 1629 (CO), 3276 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.81e7.78 (5H, m, Ar-H), 8.79 (1H, s, eNH), 8.17 (1H, m, e
NH); Gly ¼ 4.36 (2H, d, eaCH2); Heterocycle ¼ 1.67e2.09 (4H, m,
eCH2), 2.89 (1H, m, eCH), 3.24e3.53 (4H, m, eCH2), 6.81e7.78
(3H, m, Ar-H); Calculated HRMS
¼
475.3101; Found
HRMS ¼ 497.0672, 499.0742 (Mþ þ Na); Elem. Anal.: Calcd. for
C21H20BrFN4O3: C: 53.07; H: 4.24; N: 11.79; Found: C: 53.02; H:
4.29; N: 11.83.
4.3.29. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(3-methoxyphenyl)pyrrolidine-1-carbothioamide (27)
Raf ¼ 0.45; Rbf ¼ 0.65; Yield ¼ 88%; M.P. ¼ 140 ꢁC; IR (Nujol,
nmax, cmꢀ1) ¼ 1626 (CO), 2034 (CS), 3285 (NH); 1H NMR (DMSO-
4.3.24. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
d6,
d
ppm): Thiourea ¼ 6.69e7.61 (4H, m, Ar-H), 7.95 (1H, s, eNH),
,
carbonyl)-N-phenylpyrrolidine-1-carboxamide (22)
3.82 (3H, s, eOCH3); Pro ¼ 4.16 (1H, t, eaCH), 1.95e2.21 (4H, m, eb
gCH2), 3.70e3.73 (2H, m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m,
eCH2), 2.84 (1H, m, eCH), 3.30e3.41 (4H, m, eCH2), 6.69e7.61
Raf ¼ 0.47; Rbf ¼ 0.68; Yield ¼ 83%; M.P. ¼ 126 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1629 (CO), 3284 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.86e7.41 (5H, m, Ar-H), 7.89 (1H, s, eNH); Pro ¼ 4.31e4.44
(3H, m, Ar-H); Calculated HRMS
¼
482.5692; Found
,
(1H, t, eaCH), 1.78e2.21 (4H, m, eb gCH2), 3.75e3.78 (2H, m, e
HRMS Na); Elem. Anal.: Calcd. for
¼
505.1756 (Mþ
þ
dCH2); Heterocycle ¼ 1.98e2.19 (4H, m, eCH2), 2.58 (1H, m, eCH),
3.23e3.57 (4H, m, eCH2), 6.86e7.41 (3H, m, Ar-H); Calculated
HRMS ¼ 436.4777; Found HRMS ¼ 459.1872 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H25FN4O3: C: 66.04; H: 5.77; N: 12.84; Found: C:
66.09; H: 5.71; N: 12.89.
C25H27FN4O3S: C: 62.22; H: 5.64; N: 11.61; S: 6.64; Found: C:
62.14; H: 5.73; N: 11.65; S: 6.56.
4.3.30. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(4-methoxyphenyl)pyrrolidine-1-carboxamide (28)
Raf ¼ 0.45; Rbf ¼ 0.66; Yield ¼ 84%; M.P. ¼ 163 ꢁC; IR (Nujol, nmax
,
4.3.25. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
cmꢀ1) ¼ 1638 (CO), 3304 (NH); 1H NMR (DMSO-d6,
d ppm):
carbonyl)-N-phenylpyrrolidine-1-carbothioamide (23)
Urea ¼ 6.81e7.31 (4H, m, Ar-H), 7.86 (1H, s, eNH), 3.77 (3H, s, e
,
Raf ¼ 0.44; Rbf ¼ 0.65; Yield ¼ 86%; M.P. ¼ 137 ꢁC; IR (Nujol, nmax
,
OCH3); Pro ¼ 4.35e4.47 (1H, t, eaCH), 1.76e2.16 (4H, m, eb gCH2),
cmꢀ1) ¼ 1623 (CO), 2039 (CS), 3281 (NH); 1H NMR (DMSO-d6,
3.71e3.76 (2H, m, edCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2),
2.55 (1H, m, eCH), 3.16e3.54 (4H, m, eCH2), 6.81e7.31 (3H, m, Ar-
H); Calculated HRMS ¼ 466.5036; Found HRMS ¼ 489.1874
(Mþ þ Na); Elem. Anal.: Calcd. for C25H27FN4O4: C: 64.37; H: 5.83;
N: 12.01; Found: C: 64.42; H: 5.91; N: 12.13.
d
ppm): Thiourea ¼ 6.71e7.63 (5H, m, Ar-H), 7.87 (1H, s, eNH);
,
Pro ¼ 4.19 (1H, t, eaCH), 1.91e2.26 (4H, m, eb gCH2), 3.74e3.85 (2H,
m, edCH2); Heterocycle ¼ 2.07e2.26 (4H, m, eCH2), 2.82 (1H, m, e
CH), 3.33e3.47 (4H, m, eCH2), 6.71e7.63 (3H, m, Ar-H); Calculated
HRMS ¼ 452.5433; Found HRMS ¼ 475.1605 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H25FN4O2S: C: 63.70; H: 5.57; N: 12.38; S: 7.09;
Found: C: 63.77; H: 5.63; N: 12.44; S: 7.14.
4.3.31. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(4-methoxyphenyl)pyrrolidine-1-carbothioamide (29)
Raf ¼ 0.45; Rbf ¼ 0.68; Yield ¼ 90%; M.P. ¼ 163 ꢁC; IR (Nujol,
nmax, cmꢀ1) ¼ 1625 (CO), 2041 (CS), 3275 (NH); 1H NMR (DMSO-
4.3.26. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(2-methoxyphenyl)pyrrolidine-1-carboxamide (24)
d6,
d
ppm): Thiourea ¼ 6.73e7.68 (4H, m, Ar-H), 7.89 (1H, s, eNH),
Raf ¼ 0.47; Rbf ¼ 0.68; Yield ¼ 92%; M.P. ¼ 68 ꢁC; IR (Nujol, nmax
,
3.84 (3H, s, eOCH3); Pro ¼ 4.26 (1H, t, eaCH), 1.95e2.21 (4H, m, e
b,
cmꢀ1) ¼ 1634 (CO), 3296 (NH); 1H NMR (DMSO-d6,
d
ppm):
gCH2), 3.77e3.83 (2H, m, edCH2); Heterocycle ¼ 2.04e2.21 (4H,
Urea ¼ 6.94e7.35 (4H, m, Ar-H), 7.86 (1H, s, eNH), 3.77 (3H, s, e
m, eCH2), 2.84 (1H, m, eCH), 3.30e3.41 (4H, m, eCH2), 6.73e7.68
OCH3); Pro ¼ 4.30e4.40 (1H, t, eaCH), 1.74e2.17 (4H, m, eb gCH2),
(3H, m, Ar-H); Calculated HRMS
¼
482.5692; Found
,
3.71e3.72 (2H, m, edCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2),
2.55 (1H, m, eCH), 3.21e3.57 (4H, m, eCH2), 6.94e7.35 (3H, m, Ar-
H); Calculated HRMS ¼ 466.5036; Found HRMS ¼ 489.1947
(Mþ þ Na); Elem. Anal.: Calcd. for C25H27FN4O4: C: 64.37; H: 5.83;
N: 12.01; Found: C: 64.31; H: 5.89; N: 12.08.
HRMS Na); Elem. Anal.: Calcd. for
¼
505.1707 (Mþ
þ
C25H27FN4O3S: C: 62.22; H: 5.64; N: 11.61; S: 6.64; Found: C:
62.24; H: 5.68; N: 11.67; S: 6.54.
4.3.32. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(2-fluorophenyl)pyrrolidine-1-carboxamide (30)
4.3.27. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
Raf ¼ 0.47; Rbf ¼ 0.68; Yield ¼ 89%; M.P. ¼ 138 ꢁC; IR (Nujol, nmax
,
carbonyl)-N-(2-methoxyphenyl)pyrrolidine-1-carbothioamide (25)
Raf ¼ 0.49; Rbf ¼ 0.71; Yield ¼ 86%; M.P. ¼ 104e107 ꢁC; IR (Nujol,
nmax, cmꢀ1) ¼ 1644 (CO), 2031 (CS), 3294 (NH); 1H NMR (DMSO-d6,
cmꢀ1) ¼ 1627 (CO), 3278 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.88e7.35 (4H, m, Ar-H), 7.87 (1H, s, eNH); Pro ¼ 4.28e4.42
,
(1H, t, eaCH), 1.77e2.16 (4H, m, eb gCH2), 3.78e3.82 (2H, m, e
d
ppm): Thiourea ¼ 6.63e7.58 (4H, m, Ar-H), 7.92 (1H, s, eNH),
dCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2), 2.55 (1H, m, eCH),
3.18e3.53 (4H, m, eCH2), 6.88e7.35 (3H, m, Ar-H); Calculated
HRMS ¼ 454.4682; Found HRMS ¼ 477.1738 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24F2N4O3: C: 63.43; H: 5.32; N: 12.33; Found:
C: 63.52; H: 5.26; N: 12.42.
3.83 (3H, s, eOCH3); Pro ¼ 4.16 (1H, t, eaCH), 1.95e2.21 (4H, m, eb
gCH2), 3.77e3.82 (2H, m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m,
eCH2), 2.84 (1H, m, eCH), 3.30e3.41 (4H, m, eCH2), 6.63e7.58
,
(3H, m, Ar-H); Calculated HRMS
¼
482.5692; Found

C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
331
4.3.33. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
carbonyl)-N-(2-fluorophenyl)pyrrolidine-1-carbothioamide (31)
Raf ¼ 0.48; Rbf ¼ 0.66; Yield ¼ 88%; M.P. ¼ 83 ꢁC; IR (Nujol, nmax
(2H, m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m, eCH2), 2.84
(1H, m, eCH), 3.30e3.41 (4H, m, eCH2), 6.65e7.56 (3H, m, Ar-H);
,
Calculated HRMS
¼
486.9884; Found HRMS
¼
509.1287
cmꢀ1) ¼ 1641 (CO), 2027 (CS), 3281 (NH); 1H NMR (DMSO-d6,
(Mþ þ Na); Elem. Anal.: Calcd. For C24H24ClFN4O2S: C: 59.19; H:
d
ppm): Thiourea ¼ 6.79e7.81 (4H, m, Ar-H), 7.85 (1H, s, eNH);
4.97; N: 11.50; S: 6.58; Found: C: 59.11; H: 4.86; N: 11.55; S: 6.51.
Pro ¼ 4.26 (1H, t, eaCH), 1.89e2.21 (4H, m, eb gCH2), 3.60e3.73
(2H, m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m, eCH2), 2.84
(1H, m, eCH), 3.30e3.41 (4H, m, eCH2), 6.79e7.81 (3H, m, Ar-H);
,
4.3.39. N-(4-Chlorophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carboxamide (37)
Calculated HRMS
¼
470.5338; Found HRMS
¼
493.1618
Raf ¼ 0.51; Rbf ¼ 0.70; Yield ¼ 89%; M.P. ¼ 110 ꢁC; IR (Nujol, nmax
,
(Mþ þ Na); Elem. Anal.: Calcd. for C24H24F2N4O2S: C: 61.26; H: 5.14;
cmꢀ1) ¼ 1633 (CO), 3294 (NH); 1H NMR (DMSO-d6,
d ppm):
N: 11.91; S: 6.81; Found: C: 61.21; H: 5.22; N: 11.77; S: 6.74.
Urea ¼ 6.91e7.51 (4H, m, Ar-H), 7.86 (1H, s, eNH); Pro ¼ 4.34e4.43
,
(1H, t, eaCH), 1.66e2.15 (4H, m, eb gCH2), 3.73e3.76 (2H, m, e
4.3.34. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
dCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2), 2.55 (1H, m, eCH),
3.17e3.55 (4H, m, eCH2), 6.91e7.51 (3H, m, Ar-H); Calculated
HRMS ¼ 470.9288; Found HRMS ¼ 493.1457 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24ClFN4O3: C: 61.21; H: 5.14; N: 11.90; Found:
C: 61.26; H: 5.09; N: 11.94.
carbonyl)-N-(3-fluorophenyl)pyrrolidine-1-carboxamide (32)
Raf ¼ 0.48; Rbf ¼ 0.66; Yield ¼ 91%; M.P. ¼ 72 ꢁC; IR (Nujol, nmax
,
cmꢀ1)¼1629(CO),3274(NH);1HNMR(DMSO-d6,
ppm):Urea¼6.88e
d
7.51(4H,m,Ar-H),7.91(1H,s,eNH);Pro¼4.27e4.40(1H,t,eaCH),1.81e
,
2.16 (4H, m, eb gCH2), 3.63e3.71 (2H, m, edCH2); Heterocycle ¼ 1.96e
2.16 (4H, m, eCH2), 2.55 (1H, m, eCH), 3.16e3.57 (4H, m, eCH2),
6.88e7.51 (3H, m, Ar-H); Calculated HRMS ¼ 454.4682; Found
HRMS ¼ 477.1743 (Mþ þ Na); Elem. Anal.: Calcd. for C24H24F2N4O3: C:
63.43; H: 5.32; N: 12.33; Found: C: 63.49; H: 5.22; N: 12.22.
4.3.40. N-(4-Chlorophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carbothioamide (38)
Raf ¼ 0.47; Rbf ¼ 0.68; Yield ¼ 86%; M.P. ¼ 93 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1644 (CO), 2027 (CS), 3282 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.64e7.71 (4H, m, Ar-H), 7.89 (1H, s, eNH);
,
4.3.35. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
Pro ¼ 4.26 (1H, t, eaCH),1.91e2.21 (4H, m, eb gCH2), 3.63e3.71 (2H,
m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m, eCH2), 2.84 (1H, m, e
CH), 3.30e3.41 (4H, m, eCH2), 6.64e7.71 (3H, m, Ar-H); Calculated
HRMS ¼ 486.9884; Found HRMS ¼ 509.1286 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24ClFN4O2S: C: 59.19; H: 4.97; N: 11.50; S: 6.58;
Found: C: 59.07; H: 4.91; N: 11.49; S: 6.69.
carbonyl)-N-(4-fluorophenyl)pyrrolidine-1-carboxamide (33)
Raf ¼ 0.44; Rbf ¼ 0.65; Yield ¼ 92%; M.P. ¼ 63 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1631 (CO), 3271 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.98e7.41 (4H, m, Ar-H), 7.81 (1H, s, eNH); Pro ¼ 4.30e4.40
,
(1H, t, eaCH), 1.76e2.16 (4H, m, eb gCH2), 3.73e3.76 (2H, m, e
dCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2), 2.55 (1H, m, eCH),
3.16e3.53 (4H, m, eCH2), 6.98e7.41 (3H, m, Ar-H); Calculated
HRMS ¼ 454.4682; Found HRMS ¼ 477.2639 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24F2N4O3: C: 63.43; H: 5.32; N: 12.33; Found:
C: 63.49; H: 5.22; N: 12.22.
4.3.41. N-(2-Bromophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carbothioamide (39)
Raf ¼ 0.44; Rbf ¼ 0.67; Yield ¼ 78%; M.P. ¼ 108 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1625 (CO), 2037 (CS), 3281 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.59e7.63 (4H, m, Ar-H), 7.81 (1H, s, eNH);
,
4.3.36. 2-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-
Pro ¼ 4.23 (1H, t, eaCH),1.91e2.29 (4H, m, eb gCH2), 3.71e3.83 (2H,
m, edCH2); Heterocycle ¼ 2.07e2.24 (4H, m, eCH2), 2.74 (1H, m, e
CH), 3.34e3.47 (4H, m, eCH2), 6.55e7.67 (3H, m, Ar-H); Calculated
HRMS ¼ 531.4394; Found HRMS ¼ 553.0762, 555.0698 (Mþ þ Na);
Elem. Anal.: Calcd. for C24H24BrFN4O2S: C: 54.24; H: 4.55; N: 10.54;
S: 6.03; Found: C: 54.29; H: 4.59; N: 10.47; S: 6.07.
carbonyl)-N-(4-fluorophenyl)pyrrolidine-1-carbothioamide (34)
Raf ¼ 0.47; Rbf ¼ 0.69; Yield ¼ 89%; M.P. ¼ 167 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1638 (CO), 2023 (CS), 3275 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.69e7.61 (4H, m, Ar-H), 7.95 (1H, s, eNH);
,
Pro ¼ 4.16 (1H, t, eaCH),1.95e2.21 (4H, m, eb gCH2), 3.70e3.73 (2H,
m, edCH2); Heterocycle ¼ 2.04e2.21 (4H, m, eCH2), 2.84 (1H, m, e
CH), 3.30e3.41 (4H, m, eCH2), 6.69e7.61 (3H, m, Ar-H); Calculated
HRMS ¼ 470.5338; Found HRMS ¼ 493.1546 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24F2N4O2S: C: 61.26; H: 5.14; N: 11.91; S: 6.81;
Found: C: 61.17; H: 5.26; N: 11.83; S: 6.69.
4.3.42. N-(3-Bromophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carboxamide (40)
Raf ¼ 0.45; Rbf ¼ 0.63; Yield ¼ 83%; M.P. ¼ 135 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1629 (CO), 3284 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.83e7.51 (4H, m, Ar-H), 7.74 (1H, s, eNH); Pro ¼ 4.27e4.34
,
4.3.37. N-(3-Chlorophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carboxamide (35)
(1H, t, eaCH), 1.62e2.23 (4H, m, eb gCH2), 3.73e3.81 (2H, m, e
dCH2); Heterocycle ¼ 1.89e2.21 (4H, m, eCH2), 2.59 (1H, m, eCH),
3.19e3.52 (4H, m, eCH2), 6.83e7.51 (3H, m, Ar-H); Calculated
HRMS ¼ 515.3738; Found HRMS ¼ 537.0982, 539.0876 (Mþ þ Na);
Elem. Anal.: Calcd. for C24H24BrFN4O3: C: 55.93; H: 4.69; N: 10.87;
Found: C: 55.99; H: 4.75; N: 10.83.
Raf ¼ 0.47; Rbf ¼ 0.65; Yield ¼ 90%; M.P. ¼ 89 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1633 (CO), 3290 (NH); 1H NMR (DMSO-d6,
d ppm):
Urea ¼ 6.86e7.47 (4H, m, Ar-H), 7.71 (1H, s, eNH); Pro ¼ 4.25e4.38
,
(1H, t, eaCH), 1.67e2.19 (4H, m, eb gCH2), 3.67e3.72 (2H, m, e
dCH2); Heterocycle ¼ 1.96e2.16 (4H, m, eCH2), 2.55 (1H, m, eCH),
3.17e3.59 (4H, m, eCH2), 6.86e7.47 (3H, m, Ar-H); Calculated
HRMS ¼ 470.9228; Found HRMS ¼ 493.8182 (Mþ þ Na); Elem.
Anal.: Calcd. for C24H24ClFN4O3: C: 61.21; H: 5.14; N: 11.90; Found:
C: 61.28; H: 5.23; N: 11.82.
4.3.43. N-(3-Bromophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carbothioamide (41)
Raf ¼ 0.42; Rbf ¼ 0.61; Yield ¼ 91%; M.P. ¼ 113 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1634 (CO), 2041 (CS), 3282 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.68e7.75 (4H, m, Ar-H), 7.86 (1H, s, eNH);
,
4.3.38. N-(3-Chlorophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carbothioamide (36)
Pro ¼ 4.29 (1H, t, eaCH), 1.94e2.26 (4H, m, eb gCH2), 3.67e3.74
(2H, m, edCH2); Heterocycle ¼ 2.09e2.24 (4H, m, eCH2), 2.87
(1H, m, eCH), 3.36e3.453 (4H, m, eCH2), 6.68e7.75 (3H, m, Ar-H);
Calculated HRMS ¼ 531.4394; Found HRMS ¼ 553.0746, 555.0712
(Mþ þ Na); Elem. Anal.: Calcd. for C24H24BrFN4O2S: C: 54.24; H:
4.55; N: 10.54; S: 6.03; Found: C: 54.28; H: 4.61; N: 10.59; S: 6.09.
Raf ¼ 0.46; Rbf ¼ 0.67; Yield ¼ 88%; M.P. ¼ 150 ꢁC; IR (Nujol, nmax
,
cmꢀ1) ¼ 1635 (CO), 2019 (CS), 3279 (NH); 1H NMR (DMSO-d6,
d
ppm): Thiourea ¼ 6.65e7.56 (4H, m, Ar-H), 7.84 (1H, s, eNH);
,
Pro ¼ 4.19 (1H, t, eaCH), 1.89e2.26 (4H, m, eb gCH2), 3.69e3.75

332
C.S. Shantharam et al. / European Journal of Medicinal Chemistry 60 (2013) 325e332
4.3.44. N-(4-Bromophenyl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidine-1-carbonyl)pyrrolidine-1-carboxamide (42)
References
Raf ¼ 0.47; Rbf ¼ 0.65; Yield ¼ 89%; M.P. ¼ 167 ꢁC; IR (Nujol, nmax
,
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,
(1H, t, eaCH), 1.63e2.15 (4H, m, eb gCH2), 3.67e3.72 (2H, m, e
dCH2); Heterocycle ¼ 1.94e2.19 (4H, m, eCH2), 2.61 (1H, m, eCH),
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phosphate buffer saline (PBS) was prepared by mixing NaCl
(137 mM) þ Na2HPO4 (8.1 mM) þ KCl (2.68 mM) þ KH2PO4
(1.47 mM) and pH 10 was adjusted with NaOH (0.25 mM), while
BSA (10 mg/mL) and anhydrous glucose (50 mg/mL) solutions were
prepared in sodium phosphate buffer.
Bovine serum albumin (10 mg/mL) was incubated with glucose
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on the reaction at <2% (v/v). Glycated control contains 20
mL
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L glucose þ 20
m
L sodium phosphate buffer, while blank
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m
L BSA and 40 mL sodium phosphate buffer. The
mixture was incubated at 37 ꢁC for 7 days. After incubation, 6
mL
(100%) of trichloroacetic acid (TCA) was added into each well and
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containing glucose, inhibitor and interfering substance was
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mL phosphate buffer solution.
Evaluation of fluorescence spectrum (excitation 370 nm) and
change in fluorescence intensity (excitation 370 nm to emission
440 nm), based on AGEs were monitored by using spectrofluo-
rimeter (Varioskan, Germany). % Inhibition was calculated using the
formula:
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50 (2002) 2418e2422.
%Inhibition ¼ ½1 ꢀ ðFluorescence of sample=Fluorescence
of glycated sampleÞꢄ ꢂ 100