Novel N-substituted 4-hydrazino piperidine derivative as a dipeptidyl peptidase IV inhibitor

Article abstract of DOI:10.1016/j.bmcl.2009.07.058

A novel class of N-substituted 4-hydrazino piperidine derivatives were designed, synthesized and evaluated for DPP IV inhibition. The SAR studies on the N-substituted piperidine led to the discovery of compound 22e as a potent DPP IV inhibitor (IC₅₀ 88 nM), which is highly selective over other peptidases. In vivo efficacy indicates that compound 22e stimulates insulin release in response to glucose load and improves glucose tolerance in n5-STZ and Zucker Diabetic Fatty (ZDF) rats.

Full text of DOI:10.1016/j.bmcl.2009.07.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5021–5025
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel N-substituted 4-hydrazino piperidine derivative as a dipeptidyl
peptidase IV inhibitor
a
a
b
Ramesh C. Gupta a, , Laxmikant Chhipa , Appaji B. Mandhare , Shitalkumar P. Zambad ,
*
Vijay Chauthaiwale c, Sunil S. Nadkarni d, , Chaitanya Dutt d,à
a Medicinal Chemistry, Torrent Research Centre, Torrent Pharmaceuticals Ltd, PO Bhat, Gandhinagar 382428, Gujarat, India
b Pharmacology, Torrent Research Centre, Torrent Pharmaceuticals Ltd, PO Bhat, Gandhinagar 382428, Gujarat, India
c Discovery Research, Torrent Research Centre, Torrent Pharmaceuticals Ltd, PO Bhat, Gandhinagar 382428, Gujarat, India
d Torrent Research Centre, Torrent Pharmaceuticals Ltd, PO Bhat, Gandhinagar 382428, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of N-substituted 4-hydrazino piperidine derivatives were designed, synthesized and evalu-
ated for DPP IV inhibition. The SAR studies on the N-substituted piperidine led to the discovery of com-
pound 22e as a potent DPP IV inhibitor (IC50 88 nM), which is highly selective over other peptidases. In
vivo efficacy indicates that compound 22e stimulates insulin release in response to glucose load and
improves glucose tolerance in n5-STZ and Zucker Diabetic Fatty (ZDF) rats.
Received 6 April 2009
Revised 3 July 2009
Accepted 9 July 2009
Available online 12 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
DPP IV inhibitors
GLP-1
N-Substituted 4-hydrazino piperidine
Type-2 diabetes
Type-2 diabetes mellitus (T2DM) is characterized by variable
degrees of insulin resistance and pancreatic beta-cell dysfunction,
which leads to hyperglycemia.1 With the progressive nature of
T2DM, development of drugs that retard or prevent the transition
from the pre-diabetic state of impaired glucose tolerance to overt
diabetes have therapeutic potential in attenuating the rapid rise
in the disease prevalence. Furthermore, with the currently existing
forms of therapy, insulin secretory capacity is ultimately lost in la-
ter stages of diabetes.
Glucagon-like peptide-1 (GLP-1) based therapy is a new para-
digm in the management of T2DM. GLP-1 was shown to retain
insulinotropic action without risk of hypoglycemia when given to
type-2 diabetic patient.2 GLP-1, produced by L-cell in distal small
bowel, stimulates glucose dependent insulin secretion. However,
its effects are short-lived as a result of rapid inactivation by Dipep-
tidyl Peptidase IV (DPP IV). Thus to prolong the beneficial effects of
GLP-1, research has been focused on inhibition of DPP IV enzyme.
DPP IV is an abundantly distributed serine protease located on
endothelial cells of the blood vessels throughout the body and cir-
culates as soluble enzyme. It cleaves peptides containing proline or
alanine at penultimate position from the amino termini of sub-
strate proteins. There are several other DPPs like DPP II, DPP VI,
DPP VII, DPP VIII, and DPP IX, hence the high selectivity of inhibitor
would be an advantage to eliminate side effects associated with
other DPPs.3
For the treatment of T2DM, several DPP IV inhibitors are pro-
gressing through preclinical and clinical trials.4 Some of the se-
lected reversible DPP IV inhibitors have been reported.5 The
crystal structure of DPP IV enzyme is known in the literature.6
These information can be effectively utilized to search for new
classes of DPP IV inhibitors. We have employed these structural
informations to design and synthesize a series of novel substituted
4-hydrazino piperidine derivatives which were evaluated for DPP
IV inhibitory activity. Herein, we report the results of our study
in relation to the synthesis, in vitro DPP IV inhibitory activity,
and SAR of new piperidine analogs. The selectivity and in vivo effi-
cacy profile of one of the potent derivative 22e has also been
discussed.
Novel compounds belonging to N-substituted 4-hydrazino
piperidine series were synthesized by coupling reaction of R(À)3-
(2-chloro-acetyl)-thiazolidine-4-carbonitrile (1), its isomer (2) or
(S)1-(2-chloro-acetyl)-pyrrolidine-2-carbonitrile (3) with com-
pound (8) as described in Scheme 1.
The key intermediate 1 was synthesized starting from R(À)thi-
azolidine-4-carboxylic acid following literature method.7 The syn-
thesis of another key intermediate 8 is described in Scheme 1.
* Corresponding author.
E-mail address: rameshgupta@torrentpharma.com (R.C. Gupta).
Vice President-Product Development.
Director.
à
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.058

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R. C. Gupta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5021–5025
Scheme 1. Synthesis of compounds 10, 25 and 26. Reagents and conditions: (i) Na2CO3, CH2Cl2–water, 0 °C–rt, 3 h; (ii) t-butyl carbazate, CH2Cl2 or IPA, 80–85 °C, 3 h; (iii)
NaBH4, AcOH, CH2Cl2, 0–5 °C, 30 min; (iv) 1 or 2 or 3, K2CO3, KI, EtOAc, 65–70 °C, 18 h; (v) (a) morpholine, CH2Cl2, 0 °C–rt, 30 min; (b) 19v, acetone, 50–60 °C, 8 h; (vi) TFA,
CH2Cl2, 20–25 °C, 30 min; (vii) morpholine, CH2Cl2, 0 °C–rt, 30 min.
The N-protection of 4-piperidone hydrochloride (4) with Fmoc-
succinimide (5) resulted in 6. Reaction of 6 with t-butylcarbazate
in CH2Cl2 or IPA afforded imine 7. Reduction of 7 with NaBH4 in
the presence of AcOH in CH2Cl2 yielded N-protected hydrazino
piperidine (8). The N-alkylation reaction of compound 8 with 1
in the presence of K2CO3 and KI in ethyl acetate afforded 9. The re-
moval of Fmoc -group from 9 was achieved using morpholine in
CH2Cl2 to furnish compound 10.
Deprotection of Boc-group in the presence of TFA yielded 3-[2-
(N-piperidin-4-yl-hydrazino)-acetyl]-thiazolidine-4-carbonitrile
(11) (Scheme 2). Installing different functionalities on nitrogen of
piperidine ring in 10 led to N-acyl, sulfonamides and N-alkylated
compounds (13a–d, 16a–h and 18a–h) as delineated in Scheme
2. Reaction of 10 with various substituted sulfonyl chlorides,
acid chlorides and carbamoyl chlorides in the presence of TEA
in THF afforded compounds (12a–d), (15a–f) and (15g–h),
respectively.
Further N-benzylation or N-alkylation of 10 with substituted
benzyl chlorides or substituted alkyl chlorides in the presence of
KI and K2CO3 yielded their respective Boc protected derivatives
(17a–h). The Boc deprotection of (12a–d, 15a–h and 17a–h) were
carried out in TFA to afford (13a–d, 16a–h and 18a–h) as TFA salts.
The ethyl hydrazine derivative (14) was prepared by the treatment
of compound (13c) with acetaldehyde in MeOH followed by reduc-
tion with NaBH4 (Scheme 2).
chloroacetamide (19a) or various N-substituted chloroacetamides
(19b–v) in the presence of TEA in acetone yielded compounds
20a–v.
The N-substituted chloroacetamide derivatives (19b–v) were
prepared by reacting respective primary amine with chloroacetyl
chloride in the presence of TEA in THF. Deprotection of Boc-group
in compounds 20a–v using TFA afforded respective products 21a–v
as TFA salts. The hydrochloride salt of compounds 21f, 21o, 21t,
21u and 21v were prepared by deprotection of Boc protected com-
pounds 20f, 20o, 20t, 20u and 20v with MeCN–HCl in MeCN, which
resulted to compounds 22a–e. The ethyl hydrazine derivative (23)
was prepared by the treatment of compound 22e with acetalde-
hyde in MeOH followed by reduction with NaBH4, further hydro-
chloride salt was prepared using MeCN–HCl (Scheme 3).
Similarly the compound (24) was prepared from 22e by first,
neutralizing with NH4OH and further reacting with methyl chloro-
formate in DMF in the presence of K2CO3 and then further con-
verted to its hydrochloride salt in MeCN–HCl. Spectroscopic data
for all compounds were in accordance with their established
structures.
All synthesized compounds were tested in vitro for their DPP IV
enzyme inhibitory activity via spectrophotometric method,8 where
Gly-Pro-p-nitroanilide was used as the substrate for the DPP IV en-
zyme from porcine kidney and p-nitroanilide, cleaved from the
substrate and measured at 385 nm. IC50 was determined to com-
pare the DPP IV inhibitory activity of synthesized compounds.
This work is focused on development of a series around com-
pound 11 (IC50 12076 nM). Introduction of aromatic or aliphatic
Synthesis of 21a and various N-substituted acetamide deriva-
tives (21b–v) are shown in Scheme 3. The reaction of 10 with

R. C. Gupta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5021–5025
5023
Scheme 2. Synthesis of compounds 11, 13a–d, 14, 16a–h and 18a–h. Reagents and conditions: (i) RSO2Cl or RCOCl, or substituted carbamoyl chloride, TEA, THF, rt, 2–8 h; (ii)
TFA, CH2Cl2, 20–25 °C, 30 min; (iii) RCH2Cl, K2CO3, KI, THF, 25–80 °C, 8–10 h; (iv) CH3CHO, MeOH, rt, 1 h; (v) NaBH4, MeOH, rt, 1 h; (vi) MeCNÁHCl, MeCN, rt, 1 h.
Scheme 3. Synthesis of compounds 21a–v, 22a–e, 23 and 24. Reagents and conditions: (i) TEA, acetone, 50–60 °C, 8–20 h; (ii) TFA, CH2Cl2, 20–25 °C, 30 min; (iii) MeCNÁHCl,
MeCN, 20–25 °C, 1 h; (iv) CH3CHO, MeOH, rt, 1 h; (v) NaBH4, MeOH, rt, 1 h; (vi) NH4OH, EtOAc; (vii) CH3OCOCl, K2CO3, DMF, rt, 3 h.

5024
R. C. Gupta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5021–5025
Table 2
sulfonyl represented by 13a–d or aromatic or aliphatic carbonyl
group exemplified by 16a–d at nitrogen of piperidine ring of 11
displayed improvement in inhibitory activity (IC50 1449–
6677 nM). The introduction of cyclic urea moiety at nitrogen of
piperidine in compound 11 results in improvement in activity
(16h, IC50 790 nM) as compared to open chain urea derivative
16g. Installing substituted benzyl group in 18a–d or pyridine-3-
yl-methyl in 18e at secondary amino group of piperidine retains
inhibitory activity in sub-micromolar range. The compound with
4-nitrobenzyl group 18b and 4-cyanobenzyl group 18d exhibit bet-
ter activity (IC50 698 nM and 710 nM, respectively) as compared to
unsubstituted benzyl 18c and 4-flourobenzyl derivative 18a. This
indicates that the electron withdrawing substituents on phenyl
ring are more suited for bioactivity (Table 1).
Selectivity of representative compound 22e
IC50 (nM)
Trypsin
DPP IV
88
PEP
DPP II
>104
Elastase
>104
DPP VIII
>105
DPP IX
>105
>104
>104
PEP: prolyl endopeptidase.
500
400
300
8
7
6
5
4
3
2
*
*
*
*
*
200
100
0
Further incorporation of aminocarbonylmethyl group in 11 pro-
duced 21a which showed better activity (IC50 817 nM). The devel-
opment of trends of SAR around 21a and its analogue 21b–v are
shown in Table 1.
0
30
60
90
120
Time (Mins)
Glucose (Vehicle)
Insulin (Vehicle)
Glucose (compound 22e)
Insulin (compound 22e)
Compounds having different substitutions on nitrogen atom of
acetamido group like substituted phenyl 21i–o, heteroaryl such
as pyridine 21t–v, pyrimidine 21s, thaizole 21p–q, benzothiazolyl
21r, and substituted alkyl 21b–c, cycloalkyl 21e–f, tricycloalkyl
21d, benzyl 21h, thiophene-ethyl 21g were prepared to assess
the SAR. The 4-aminosulfonylphenyl 21o group was found to be
the most potent in various substituted phenyl analogs. Among
heteroaryls, compounds with pyridine ring were found to be more
potent as compared to other heteroaryl ring. Various substitutions
on pyridine ring were well tolerated irrespective of their position
on ring as in case of 21t–v.
The substituent N-cyclopropyl-acetamido in 21f showed good
inhibitory activity (IC50 236 nM) whereas introduction of N-cyclo-
propylamino-carbonylmethyl (18g) lowers the activity by about
sixfold, which suggest that an increase in alkyl chain length lead
to the reduction in inhibitory activity.
To investigate the effect of thiazolidine ring, the thaizolidine
derivatives were synthesized. They were found to be more potent
than their corresponding pyrrolidine derivative as observed in case
of 21v (IC50 120 nM) and compound 26 (IC50 3700 nM). Further to
confirm the effect of stereochemistry of cyano group present on
thiazolidine ring, the R(À) isomer 21v was found ꢀ34 times more
potent than its S-isomer (25, IC50 4048 nM).
Among all compounds, 22e was found to be the most potent
compound with IC50 88 nM. However, introduction of small alkyl
group on free nitrogen of hydrazine group in compound 22e like
N-ethyl analog (23, IC50 490 nM) and methoxycarbonyl analog
Figure 1. Effect of 6 weeks treatment of compound 22e (44 mg/kg; n = 14) on
plasma glucose and insulin levels during OGTT in ZDF rats.
(24), leads to the drastic reduction in inhibitory potency (IC50
38450 nM) as compared to compound 22e.
The isozyme selectivity of 22e was investigated and it was
found to be highly selective against other DPPs like PEP, DPP II,
Trypsin, Elastase, DPP VIII, and DPP IX (Table 2).
Compound 22e was further evaluated for its in vivo efficacy in
n5-STZ and ZDF rats. To correlate in vitro DPP IV inhibitory activity
to in vivo scenario, plasma DPP IV inhibition (% of basal) was mon-
itored over 12 h after a single oral administration (22 mg/kg) of
22e in n5-STZ rats. It produces maximum plasma DPP IV inhibition
within 30 min with significant decrease in the activity observed till
4 h (% inhibition with respect to vehicle 39.81 3.71%, p <0.05 vs
vehicle).
In single dose study, compound 22e decreased glucose excur-
sion and the AUC-glucose during oral glucose tolerance test (OGTT)
in n5-STZ rat and was able to stimulate insulin response in a situ-
ation where insulin secretory function is failing, as compared to
that of control. We also evaluated the long term treatment effect
of 22e, after oral administration, in ZDF rats which become insulin
resistant and hyperinsulinemic, when they are five to six weeks
old. Treatment was initiated at the age of 6 weeks. OGTT per-
formed at the end of 6 weeks of treatment showed that compound
22e reduced the glucose excursions and increase insulin release
*
during OGTT in ZDF rats (Fig. 1, p <0.05 vs vehicle).
Thus, compound 22e, has a capacity to augment insulin release
in response to glucose load and thus improve glycemic control
through its inhibitory effect on DPP IV enzyme.
Table 1
DPP IV inhibitory effect of N-substituted piperidine derivatives
Compd
IC50 (nM)
Compd
IC50 (nM)
Compd
IC50 (nM)
In conclusion, we have found that N-(5-chloropyridin-2-yl) sub-
stitution at acetamido group of compound 21a showed better
activity among all compounds. The 4-cyanothiazolidine has poten-
tial to elicit better inhibitory activity (21v IC50 120 nM) as com-
pared to 4-cyanopyrrolidine containing compound 26 with IC50
3700 nM. Free amino of hydrazine moiety is essential for better
activity as it was found that small substitutions on free amino
group of hydrazine leads to reduction in inhibition activity (23–
24). These results suggest that further modification or substitution
on pyridine ring may yield potent compounds with better in vitro
and in vivo efficacy.
11
12,076
6677
1449
1459
2701
3755
866
1534
1793
4149
667
830
1228
790
2228
698
1631
18d
18e
18f
18g
18h
21a
21b
21c
21d
21e
21f
21g
21h
21i
710
4767
638
1760
1022
817
1027
468
523
506
236
415
510
258
359
308
21m
21n
21o
21p
21q
21r
222
200
98
13a
13b
13c
13d
14
16a
16b
16c
16d
16e
16f
16g
16h
18a
18b
18c
226
185
134
143
110
119
120
217
130
124
90
21s
21t
21u
21v
22aa
22ba
22ca
22da
22ea
23a
21j
21k
21l
88
490
38,450
Acknowledgments
240
24a
The authors wish to offer their deep thanks to Dr. Anookh
Mohanan and Dr. Shikha Kumar for their support in preparing
a
Hydrochloride salt.

R. C. Gupta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5021–5025
5025
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Supplementary data
Supplementary data (experimental procedures and results for
in vitro and in vivo DPP IV inhibition assay, experimental proce-
dures of in vivo single dose and long term studies and spectro-
scopic data of compounds 21v, 22e and 26) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.07.058.
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