Design, synthesis and biological evaluation of novel pyrazolo-pyrimidinones as DPP-IV inhibitors in diabetes

Article abstract of DOI:10.1016/j.bmcl.2015.09.015

We report the design, synthesis, biological activity and docking studies of series of novel pyrazolo[3,4-d]pyrimidinones as DPP-IV inhibitors in diabetes. Molecules were synthesized and evaluated for their DPP-IV inhibition activity. Compounds 5e, 5k, 5o and 6a were found to be potent inhibitors of DPP-IV enzyme. Amongst all the synthesized compounds, 6-methyl-5-(4-methylpyridin-2-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (5k) was found to be the most active based on in vitro DPP-IV studies and also exhibited promising in vivo blood glucose lowering activity in male Wistar rats.

Full text of DOI:10.1016/j.bmcl.2015.09.015

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel pyrazolo-
pyrimidinones as DPP-IV inhibitors in diabetes q
Sneha R. Sagar a,c,y, Jessica K. Agarwal a, Dhaivat H. Pandya c, Ranjeet Prasad Dash b, Manish Nivsarkar b,
Kamala K. Vasu c,
⇑
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research—Ahmedabad, S. G. Highway, Thaltej, Ahmedabad 380054, Gujarat, India
b Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad 380054,
Gujarat, India
c Department of Medicinal Chemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad 380054, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis, biological activity and docking studies of series of novel pyrazolo[3,4-d]
pyrimidinones as DPP-IV inhibitors in diabetes. Molecules were synthesized and evaluated for their DPP-
IV inhibition activity. Compounds 5e, 5k, 5o and 6a were found to be potent inhibitors of DPP-IV enzyme.
Amongst all the synthesized compounds, 6-methyl-5-(4-methylpyridin-2-yl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5k) was found to be the most active based on in vitro DPP-IV studies and also
exhibited promising in vivo blood glucose lowering activity in male Wistar rats.
Received 7 July 2015
Revised 19 August 2015
Accepted 7 September 2015
Available online xxxx
Keywords:
Ó 2015 Published by Elsevier Ltd.
DPP-IV enzyme
Pyrazolo-pyrimidinones
Molecular docking
Diabetes mellitus is recognized as world’s major health problem
affecting millions of people. It is characterized by abnormally high
levels of plasma glucose that occurs due to the variable degrees of
insulin resistance produced by the pancreatic b cells and dysfunc-
tion of b cells.1
Insulin secretion enhancement by pancreatic islet b-cells has
been a major goal for the treatment of Type 2 Diabetes2 but these
treatments have side effects viz. severe hypoglycemia, weight gain,
edema and nausea. In the area of diabetes, researchers have found
various novel targets. One such target that has been found very
effective in modulating the disease physiology is Dipeptidyl Pepti-
dase-IV (DPP-IV) enzyme. DPP-IV is the major enzyme involved in
the regulation of incretin hormone. It is a serine protease that
DPP IV belongs to a family of enzyme that contains several clo-
sely related members like DPP-II, DPP-VIII, DPP-IX and FAP-a 5
In
.
order to inhibit DPP-IV enzyme, selectivity is of prime importance.
Currently available ‘Gliptins’ namely, Sitagliptin, Vildagliptin, Sax-
agliptin, Alogliptin, Linagliptin, and Gemigliptin5–11 are associated
with several side effects including nasopharyngitis, headache, nau-
sea, hypersensitivity, skin reactions and pancreatitis. Pancreatitis is
the most severe side effect of DPP-IV inhibitors.5 The side-effects
observed with the ‘Gliptins’ are specific to the molecules which
inhibit other substrates rather than DPP-IV per se. To avoid the side
effects associated with the inhibition of other members of DPP
family like DPP-II, DPP-VIII, and DPP-IX it becomes necessary to
design selective inhibitors of DPP-IV enzyme.
cleaves the N-terminal dipeptide with a preference for
L
-proline
Large numbers of structurally diverse molecules were designed
against DPP-IV enzyme inhibition. We report here new chemical
classes of DPP-IV inhibitors containing fused pyrazolo-pyrimidi-
nones. As mentioned earlier, DPP-IV cleaves at proline residue of
N-terminal site. Incorporation of a small electron rich heterocycle
at the proline site has ability to provide reactive center to accept
DPP-IV’s catalytic activity for its inhibition, thus it can impart the
potency of molecule.12 Pyrazole scaffold has electron rich ‘N’ con-
taining center in its structure. Thus it has ability to serve as proline
mimics and expected to cleave DPP-IV enzyme.13 In addition, sev-
eral reported molecules containing pyrazole moiety showed potent
inhibition of DPP-IV action.5,14,15 Whereas pyrimidine is a privi-
leged structure and important for its biological activity.16 Ring ‘N’
or
L
-alanine at the penultimate position.3 DPP-IV inhibitors stabi-
lize endogenous GLP-1 and induce insulin secretion in a glucose-
dependent manner in contrast to insulin tropic agents which
release insulin in glucose independent manner and manifest hypo-
glycemia as a side effect.2 The inhibition of DPP-IV results in higher
levels of circulating active GLP-1 and improves glucose tolerance.4
q
Communication Ref. No.: NIPERA/55/6/2015.
⇑
Corresponding author. Tel.: +91 79 27439375; fax: +91 79 27450449.
E-mail address: kamalav@perdcentre.com (K.K. Vasu).
Sneha R. Sagar is registered research scholar in Institute of Pharmacy, Nirma
y
University, India.
http://dx.doi.org/10.1016/j.bmcl.2015.09.015
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Sagar, S. R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.015

2
S. R. Sagar et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
of pyrimidinone has ability to form H-boding interaction with
Arg125 present in S2 pocket of DPP-IV enzyme.17 Pyrimidinone
has 10,000 fold selectivity for DPP-IV over DPP-VIII and DPP-IX
as ‘AC@O’ forms H-bonding interaction with Tyr631.2,12,18 This
special chemical feature imparts its selectivity towards other
DPP’s. Two pharmacophores, pyrazoles13 and pyrimidinones17
both independently act on DPP-IV enzyme so combining these
two pharmacophores in a single molecule may lead to a potent
and selective inhibitor of DPP-IV enzyme (see Fig. 1).
As depicted in Scheme 1, synthesis of pyrazolo[3,4-d]pyrimidi-
none based DPP-IV inhibitors (5a–5t) and (6a–6e) were synthe-
sized. Pyrazole synthesis was carried out by reaction of
Figure 1. Designed pyrazolo-pyrimidinones. (Refer Table 1 for specific substitutions R1, R2 and R3).
Scheme 1. Synthesis of pyrazolo-pyrimidinones. R1 = -phenyl, -4-methoxyphenyl. R2 = -methyl, -trifluoromethyl. R3 = -phenyl, -4-fluoromethyl, -4-ethylphenyl, -3-
chlorophenyl, -4-methyl-pyridine-2-yl, -5-bromo-pyridine-2-yl-5-nitro-pyridine-2-yl, -4-(4-chlorophenyl)thiazol-2-yl, -3-methyl-thiophene-2,4-dicarboxylate-5-yl, etc.
Reagents and conditions: (a0) Hydrazines (1.0 equiv), ethanol, 80 °C, 6 h, (b0) NaOH (3.0 equiv), ethanol, 80 °C, 7 h, (c0) anhydrides (6.0 equiv), tetrahydrofuran, 70 °C, 10 h, (d0)
amine derivatives (2.0 equiv), dimethylformamide, 150 °C, 15 h.
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S. R. Sagar et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
List of synthesized compounds (compounds 5a to 5t and 6a to 6e) and its DPP-IV inhibitory activity
R2
NH
R1
N
O
N
O
R3
R2
R3
O
N
N
H
N
N
N
R1
Compounds 5a to 5t
Compounds 6a to 6e
R1
R2
R3
% yielda
IC50
(lM)
b
Code
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
6a
6b
6c
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4-Methoxyphenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
Phenyl
4-Methoxyphenyl
Phenyl
Phenyl
Phenyl
4-Fluorophenyl
4-Ethylphenyl
3-Chlorophenyl
2,4,6-Trimethylphenyl
4-Fluorophenyl
Benzyl
2,4-Difluorophenyl
Pyridine-2-yl
Pyrimidine-2-yl
4-Methylpyridine-2-yl
5-Bromopyridine-2-yl
5-Nitropyridine-2-yl
Pyridine-2-yl
4-Methylpyridine-2-yl
5-Bromopyridine-2-yl
5-Nitropyridine-2-yl
4-(4-Chlorophenyl)thiazol-2-yl
3-Methylthiophene-2,4-dicarboxylate-5-yl
3-Methylthiophene-2,4-dicarboxylate-5-yl
4-Fluorophenyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
70
60
70
85
60
60
55
50
54
60
49
48
41
46
52
38
55
51
58
42
70
75
35
45
25
ND
ND
ND
ND
1.32 0.09
ND
ND
ND
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Trifluoromethyl
Trifluoromethyl
Methyl
Methyl
Methyl
4.0 0.02
24 0.13
1.06 0.09
11 0.1
ND
ND
2.23 0.02
ND
ND
100 0.03
ND
ND
1.25 0.04
ND
4-Chlorophenyl
Phenyl
Phenyl
Phenyl
5-Bromopyridine-2-yl
4-(4-Chlorophenyl)thiazol-2-yl
Pyridine-2-yl
ND
ND
ND
6d
6e
Sitagliptin
0.018 0.00120
ND not determined because of their low inhibitory activities.
Potent DPP-IV inhibitors are given in bold.
a
Isolated yield.
b
IC50 value represents the concentration of each compound resulting in 50% inhibition.
hydrazine derivatives with ethylethoxymethylene cyanoacetate
these compounds were found to be 1.06
lM for 5k, 2.23 lM for
(1) to obtain substituted ethyl 5-amino-1H-pyrazole-4-carboxylate
(2).19 Then compounds (2) were hydrolyzed to get substituted 5-
amino-1H-pyrazole-4-carboxylic acids (3). Acids then reacted with
anhydride derivatives to obtain substituted pyrazolo[3,4-d][1,3]
oxazin-4(1H)-ones (4) which on condensation with different ami-
nes produced a series of various 1H-pyrazolo[3,4-d]pyrimidin-4
(5H)-one derivatives (5). Uncyclized product of this reaction was
isolated (6) and also checked for its DPP-IV inhibition. All the com-
pounds were purified by column chromatography and character-
ized by various spectroscopic techniques (1H NMR and 13C NMR).
Purity of the compounds was checked by HPLC chromatogram
(see Supplementary data for spectral data).
The in vitro DPP-IV inhibition was determined in compounds
(5a to 5t) and (6a to 6e). All the molecules were screened for their
DPP-IV enzyme inhibition activity using colorimetric assay. Com-
pounds were incubated with human recombinant DPP-IV enzyme
and Gly-Pro-p-nitroanilide substrate.20 Cleavage of Gly-Pro-p-ni-
troanilide and formation of p-nitroaniline was measured by using
micro-plate reader (Biotek, India). Compounds 5e, 5i, 5j, 5k, 5l
and 5o and 6a showed DPP-IV inhibition activity in micro molar
range. DPP-IV inhibition varied across all the tested compounds
depending on their substitution. Trimethyl phenyl group in com-
pound 5e, 4-methylpyridine group in compound 5k and compound
5o showed potent inhibition of DPP-IV enzyme (Table 1). The activ-
ity was quantified in terms of percentage inhibition of DPP-IV
enzyme and was found to be 82%, 81%, 75% and 72% for compounds
5o and 1.32 M for 5e and 1.25 M for 6a.
l
l
Molecules which showed promising activity from in vitro stud-
ies were tested for their cytotoxicity on normal cells using isolated
rat macrophages.21 None of them showed cytotoxicity towards
normal cells. Further compounds were evaluated for their in vivo
efficacy in male Wistar rats. All experimental protocols were
reviewed and approved by the Institutional Animal Ethics Commit-
tee prior to initiation of the experiment (Registration No. PERD/
IAEC/2013/0011).
A combination of high-fat-diet and low dose of streptozotocin
(dose: 40 mg/kg) was administered to 6–8 weeks old male Wistar
rats (200–250 g body weight) for induction of hyperglycemia.22
The animals were divided in 6 groups (n = 6), normal control group,
disease control group, positive control group (Sitagliptin), com-
pound 5e test group, compound 5k test group and compound 6a
test group. The dose of sitagliptin was 10 mg/kg whereas the test
compounds were dosed at 50 mg/kg bodyweight. All the com-
pounds were administered orally, as a suspension in 0.2% agar.
Blood glucose level (as shown in Fig. 2) was measured by using
automated biochemical analyser (Em360, Transasia, Germany).
Blood glucose lowering was observed in all animals treated with
the test molecules compared to disease control animals. Blood glu-
cose level decreased in disease control group from 515.0 mg/dl to
136.4 mg/dl for sitagliptin, 162.6 mg/dl for 5e, 198.5 mg/dl for 5k
and 193.2 mg/dl for 6a treated animals after 15 days of treatment.
To see the possible interactions of the newly synthesized
ligands with the DPP-IV enzyme, docking study was carried out
5k, 5o, 5e and 6a, respectively, at 100 lM concentration. IC50 of
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S. R. Sagar et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 2. In vivo results of blood glucose level. Statistical analysis was done by One-way ANOVA. Data shown are mean SEM (n = 6) *p <0.05. Normal control: vehicle.
disease control: streptozotocin (40 mg/kg) + high-fat-diet. Positive control: sitagliptin (10 mg/kg). Compound 5e: dose (50 mg/kg), compound 5k: dose (50 mg/kg),
compound 6a: dose (50 mg/kg).
using surflex dock module of SYBYL X-2.0. The X-ray structure of
the DPP-IV enzyme (PDB ID: 2RGU) was obtained from the protein
databank and the protein structure was prepared. For docking
study, the ligands were geometrically optimized and Gasteiger–
Huckel charges were assigned to the structure. The pyrazolo-
pyrimidinone group of the derivatives docked into the binding site
pocket and compared with the standard drug sitagliptin (Fig. 3).
Figure 3A illustrates an overlay of the docking stimulated 3D
structural model of compound 5k and sitagliptin with DPP-IV
enzyme. H-bonding interactions were shown in yellow dotted line.
Compound 5k shows important interactions with DPP-IV enzyme
similar to that of sitagliptin. ‘N’ of pyrimidinone moiety shows
Figure 3B illustrates an overlay of the docking simulated 3D
structural model of low potent compound 5b and sitagliptin bind-
ing to DPP-IV enzyme. From the docking result of this compound it
is clear that overlay of compound 5b did not match with sitaglip-
tin. Compound 5b did not show interaction with important resi-
dues. Compound 5b shows H-bonding interaction with Ser630
and Tyr631 in S1 pocket but not showing any interaction with
Arg125 in S2 pocket. Additional H-bonding interactions observed
with residues like Gly632, Gly633, Trp629, and Lys554. Total Score
and CScore of compound 5b was 3.6 and 2, respectively (see
Table 2).
Docking results of some of the representative molecules were
depicted in Table 2. Total Score value of docked compound implies
binding capacity of ligand. CScore represents ranking of the affinity
of ligands bound to the active site of a receptor. CScore is generated
by combination of G_Score, D_Score, PMF_Score and ChemScore.23
Compound 5e does not have ‘N’ containing heterocycle in its cat-
alytic region so, H-bonding with Asn710 is not present. Though
compound 5e has other important residues for interaction like
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554, His740 and
Arg125. Total Score of compound 5e was 6.5 which is less com-
pared to compound 5k but it is showing promising activity in
in vitro studies. Compounds 5i, 5j and 5l incorporate well into bid-
ing site having pyridine, pyrimidine and 5-bromo-pyridine moiety.
Compound 5o shows additional binding interactions with Gly632,
H-bonding with Arg125. This site is also responsible for p–p inter-
action in its S2 pocket. ‘AC@O’ group fits well into the S1 pocket of
enzyme having important residues Ser630 and Tyr631. Free ‘N’ of
pyrazole moiety interacted with His740. Whereas phenyl moiety
of pyrazole accommodate well in hydrophobic S1 pocket of
enzyme. 4-Methylpyridine moiety of compound 5k is responsible
for H-bonding interaction with Asn710 in catalytic region of
enzyme. The inhibitory potency of compound 5k is higher since
the docking results show that compound 5k fits very well into
the binding pocket of DPP-IV enzyme. Total Score and Consensus
Score (CScore) of compound 5k was 7.69 and 5, respectively. Sita-
gliptin shows additional binding with Glu205 and Ser209 in S2
pocket and Val207 in S1 pocket (see Table 2).
A
B
Figure 3. Docking study results (PDB ID: 2RGU). (H-bonding interactions were shown in yellow dotted line and amino acid residues presented in green color). (A) Overlay of
compound 5k (blue sticks) with Sitagliptin (red sticks): H-bonding interactions of compound 5k: Ser630, Tyr631, His740, Tyr662, Asn710, and Arg125. H-bonding
interactions of Sitagliptin: Ser630, Tyr631, Tyr662, Glu205, Val207, Asn710, Ser209. (B) Overlay of compound 5b (pink sticks) with Sitagliptin (red sticks): H-bonding
interactions of compound 5b: Ser630, Tyr631, Gly632, Gly633, Trp629, and Lys554. H-bonding interactions of Sitagliptin (red sticks): Ser630, Tyr631, Tyr662, Glu205, Val207,
Asn710, Ser209.
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S. R. Sagar et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 2
Docking results of compounds with interacting binding residues
Ligand
Docking score
Total score
Interacting residues with docked ligand
CScore
Compound 5e
Compound 5i
Compound 5j
Compound 5k
Compound 5l
Compound 5o
Compound 6a
Compound 5b
Compound 5n
Compound 5t
Compound 6e
Sitagliptin
6.5
5
5
4
5
4
4
4
2
3
4
3
5
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554, His740, Arg125
Ser630, Tyr631, Tyr662, Asn710, His740, Arg125
Ser630, Tyr631, Tyr662, Asn710, Gly632, His740, Arg125
Ser630, Tyr631, Tyr662, Asn710, His740, Arg125
Ser630, Tyr631, Gly632, His 740, Arg125
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554, Asn710, Arg125
Ser630, Tyr631, Arg358, Asn710, Glu205, Tyr547, Arg125
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554
Ser630, Tyr631, Tyr662, Asn710, Arg125
7.56
7.41
7.69
7.43
7.57
7.0
3.6
4.0
6.0
3.9
Ser630, Tyr631, Gly632, Gly633, Trp629, Lys554
Ser630, Tyr631, Tyr662, Glu205, Val207, Asn710, Ser209
9.01
Total score: Binding capacity of ligand with receptor.
CScore: ranges from 0 to 5 (higher the score higher is the affinity of ligand with receptor).
which has shown good binding affinity but they did not show good
in vitro inhibitory activity as observed in compounds 5s and 5t.
Based on in vitro and docking studies, we could derive a struc-
ture activity relationship (SAR) of synthesized molecules but some
of the molecules did not fit into this SAR. Compound 5e does not
have ‘N’ containing heterocycle but shows potent inhibition of
DPP-IV action. Whereas in some molecules like 5m, 5n, 5p and
5q ‘N’ containing heterocycle did not show beneficial effect on
inhibition of enzyme. Based on SAR we could come to a conclusion
that in order to enhance potency of compound we should modify
substitutions on S2 pocket of enzyme. ‘ANH’ and ‘ANH2’ has ability
to form salt bridge with Glu205 and Glu206 and H-bonding inter-
action with Arg356 and Phe357. This study will be helpful for fur-
ther developing of these molecules with better activity (see Fig. 4).
In summary, a novel scaffold of pyrazolo[3,4-d]pyrimidinones
was designed using a dual pharmacophore approach and a number
of structurally diverse molecules containing the aforementioned
scaffold were synthesized. Most of the synthesized compounds
were found active from in vitro studies and a few from in vivo
experiments. The most potent molecule of this series will be taken
as a lead to optimize it further as an anti-diabetic drug acting
through DPP-IV inhibition.
A
Acknowledgements
The authors would like to acknowledge NIPER Ahmedabad and
B.V. Patel PERD Centre for providing research facilities. One of the
authors would like to acknowledge NIRMA University for registra-
tion as research scholar.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.09.
015.
B
Figure 4. Structure activity relationship (SAR) study of synthesized molecules. (A)
Structure activity relationship (SAR) study of compounds 5a–5t. (B) Structure
activity relationship (SAR) study of compounds 6a–6e.
References and notes
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Please cite this article in press as: Sagar, S. R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.015