Rational design, synthesis, and structure–activity relationships of 5-amino-1H-pyrazole-4-carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.10.012

A series of novel amino-carboxylic based pyrazole as protein tyrosine phosphatase 1B (PTP1B) inhibitors were designed on the basis of structure-based pharmacophore model and molecular docking. Compounds containing different hydrophobic tail (1,2-diphenyl ethanone, oxdiadizole and dibenzyl amines) were synthesized and evaluated in PTP1B enzymatic assay. Structure–activity relationship based optimization resulted in identification of several potent, metabolically stable and cell permeable PTP1B inhibitors.

Full text of DOI:10.1016/j.bmc.2016.10.012

Accepted Manuscript
Rational Design, Synthesis, and Structure–Activity Relationships of 5-Ami-
no-1H-pyrazole-4-carboxylic acid derivatives as protein tyrosine phosphatase
1
B inhibitors
Sujay Basu, Philip Prathipati, Sachin Thorat, Shariq Ansari, Meena Patel,
Vaibhav Jain, Ramana R Jinugu, Sanjay Niranjan, Siddhartha De,
Satyanarayana Reddy
PII:
S0968-0896(16)30927-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.012
BMC 13339
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 August 2016
5 October 2016
8 October 2016
Please cite this article as: Basu, S., Prathipati, P., Thorat, S., Ansari, S., Patel, M., Jain, V., Jinugu, R.R., Niranjan,
S., De, S., Reddy, S., Rational Design, Synthesis, and Structure–Activity Relationships of 5-Amino-1H-pyrazole-4-
carboxylic acid derivatives as proteintyrosine phosphatase 1B inhibitors, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.10.012
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Graphical abstract
.
Rational Design, Synthesis, and Structure–Activity Relationships of 5-Amino-1H-pyrazole-
4
-carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors
1


Sujay Basu , Philip Prathipati, Sachin Thorat, Shariq Ansari, Meena Patel, Vaibhav Jain,
Ramana R Jinugu, Sanjay Niranjan, Siddhartha De, Satyanarayana Reddy.
Drug Discovery Facility, Advinus Therapeutics Ltd, Quantum Towers, Plot-9, Phase-I, MIDC,
Hinjewadi, Pune 411 057, India.
Abstract
A series of novel amino-carboxylic based pyrazole as protein tyrosine phosphatase 1B (PTP1B)
inhibitors were designed on the basis of structure-based pharmacophore model and molecular
docking. Compounds containing different hydrophobic tail (1,2-diphenyl ethanone, oxdiadizole
and dibenzyl amines) were synthesized and evaluated in PTP1B enzymatic assay. Structure–
activity relationship based optimization resulted in identification of several potent, metabolically
stable and cell permeable PTP1B inhibitors.
Keywords: Protein Tyrosine Phosphatase 1B; Pharmacophore; Permeability; Hydrophobic;
Oxadiazole.

Corresponding author. Tel.: +91 20 66539600; fax: +91 20 66539620.
E-mail address: sujay.basu@advinus.com (B, Sujay)
2

1
.
Introduction
Protein tyrosine phosphatase 1B (PTP1B) is an intracellular protein expressed in insulin
1
responsive tissues including the classical insulin targeted tissues such as liver, muscle and fat.
2
PTP1B plays an important role in insulin receptor signaling. PTP1B dephosphorylates the
insulin receptor during its biosynthesis in endoplasmic reticulum as well as after it has been
stimulated by the insulin, and thus play a central role in negative regulation of insulin signaling
3
pathway. Out of several negative regulators, PTP1B has gained importance because of its
specificity and in vivo validation in animal models of diabetes.
The discovery of potent, selective, cell permeable and orally bioavailable PTP1B inhibitors is a
challenging medicinal chemistry objective. PTP1B inhibitors need to have good cellular
penetration, as target is intracellular, and orally available drug is desired. This is formidable task
because of the physical nature of this target. Most of the small PTP1B inhibitors bear highly
4
5
charged phosphonates or multiple acid and peptide functionalities due to which they show poor
cell permeability and oral bioavailability. Out of several small molecule PTP1B inhibitors, only
three small molecule PTP1B inhibitors entered clinical trials (Figure 1) and finally discontinued
due to insufficient efficacy and unwanted side effects. In our earlier communications we have
reported cell permeable and orally bio-available PTP inhibitors. The journey was started with
designing of “heterocyclic based carboxylic acids” that resembles as “head group” and followed
6
by synthesis of the compounds containing “hydrophobic tail”.
3

Ertiprotafib
JTT-551
Trodusquemine
Figure 1. PTP1B inhibitors reached clinical trials.
2
. Inhibitor Design
Multiple chemotypes and their co-crystal structures are known in the literature for PTP1B
inhibitors. We have shortlisted (Figure 2) different head group pharmacophores which have been
7
successfully used to design PTP1B inhibitors.
4

Figure 2. Literature reported PTP1B inhibitors acidic head group pharmacophore.
The active site of PTP1B has evolved to accommodate phosphotyrosine (p-Tyr) residue which
contains two negative charges at physiological pH. Hence the inhibitors developed against
PTP1B were also having negative charges to compete with p-Tyr. But these inhibitors are also
likely to have negative impact on cell permeability. One plausible way to counter this problem is
to increase pKa of terminal –COOH and therefore increase the concentration of the neutral form
8
-10
in physiological environment.
pharmacophore, –NH group next to –COOH group in order to increase pKa of the –COOH
Figure 3). A typical structural design of PTP1B inhibitors is shown in Figure 2, which
We envisaged to design ligands by introduction of new
2
(
comprises of amino pyrazole carboxylic acid based “head group”, an aromatic centre and a
hydrophobic “aryl tail” with a spacer in-between. We have also calculated pKa (using
5

ChemAxon, http://www.chemaxon.com) of designed compounds and a few examples are given
in Table 1. It was found that pKa of –COOH increased after introduction of –NH
2
which may
help to improve cell permeability.
Hydrophobic
Tail
Aromatic
Centre
Linker
Acidic Head Group
Examples of hydrophobic tail
N
O
N
N
O
Figure 3. Amino pyrazole containing carboxylic acid as head group.
6

Table 1. pKa of representative designed ligands.
pKa of –COOH= 3.42
pKa of –COOH= 4.11 (8j)
F
F
F
F
N
N
O
N
O
N
Cl
Cl
H N
2
O
O
HO
HO
pKa of –COOH= 3.41
pKa of –COOH= 4.01 (11f)
O
O
N
N
O
N
N
O
N
N
N
N
H N
2
O
O
HO
HO
Cl
Cl
pKa of –COOH= 3.38
pKa of –COOH= 4.07 (16c)
F
F
N
N
N
N
O
O
N
N
H2N
OH
OH
Cl
Cl
2
.1. Structure-based 3D Pharmacophore model and molecular docking
3
D Pharmacophore model:
The purpose of generating the pharmacophore model was to identify essential features/functional
groups in the PTB1B inhibitors, which are responsible for the inhibitory activity. Moreover, it
7

was also used as a tool to validate the designed ligands. An integrated ligand and structure-based
pharmacophore model was developed for PTP1B inhibitors using PharmaGist and MOE
(
consensus pharmacophore using PCH scheme- Polar, charged, hydrophobic), respectively.
2
QBQ (PTP1B, K
i
: 36 nM)
2ZMM (PTP1B, K : 25 nM)
i
2
ZN7(PTP1B, K
i
: 13 nM)
3CWE (PTP1B, K : 120 nM)
i
Figure 4. Known PTP1B co-crystallized ligands.
The generated pharmacophoric hypothesis was derived using the known PTP1B co-crystallized
ligands (PDB ID: 4B3-2QBQ; 35B-2ZMM; 410-2ZN7; 825-3CWE, Figure 4) as templates. The
structure-based pharmacophore model (Figure 5) consisted of four pharmacophoric features; two
charged features (negative ionizable/hydrogen bond acceptor, Features 1 & 2), two
aromatic/hydrophobic features (Features 3 & 4). The interactions captured by each of the
pharmacophoric features with PTP1B active site is depicted in Figure 5. The pharmacophore
analysis revealed that the difluoromethylene phosphonate (DFMP) of 3CWE or 3-thienyloxy
acetic acid (2QBQ, 2ZMM and 2ZN7) bind in the primary phosphotyrosine binding site and
8

make several hydrogen bonds with the residues of catalytic and WPD loop (Feature 1 in Figure
). In addition, there is a salt bridge between the 2-carboxylic acid of thiophene and Lys120 side
5
chain (Feature 2). Phenyl ring having phosphonate moiety or thiophene ring of the ligands are
located in a hydrophobic pocket and were involved in π–π stacking interactions with Tyr46
(
YRD loop), Phe182 (WPD loop) (Feature 3). The next phenyl ring makes hydrophobic contacts
with aliphatic side chains of Val49 and Ile219 (Feature 4).
Figure 5. Structure-based pharmacophore model of PTP1B inhibitors (2QBQ-Orange; 2ZMM-
Green; 2ZN7-Magenta; 3CWE-Blue color). Abbreviations: Ani (Anionic), Acc (Acceptor), Aro
(
Aromatic), Hyd (Hydrophobic).
The mapping of representative compounds 8j, 11f and 16c on the developed pharmacophore
model revealed their important functional and structural interactions with the PTP1B active site
residues (Figure 6). The carboxylic acid of pyrazole maps to Features 1 and 2 (interacting with
9

catalytic & WPD loop residues), the pyrazole ring and the attached phenyl ring map to Features
3
and 4 respectively (π-π and hydrophobic interactions). The alignment of ligands 8j, 11f and
6c is also shown in pharmacophore model (Figure 6), and the analysis showed that these
1
compounds fit well onto the pharmacophore model in the same manner as potent co-crystallized
PTP1B inhibitors. To complement the above structure-based pharmacophore studies and as an
additional validation of the binding mode predictions of the designed ligands, molecular docking
1
1
was performed using AutoDock Vina. The binding modes of the compounds and details are
depicted in Figure S1 (Supplementary Information).
8
j
11f
1
6c
8j, 11f and 16c (alignment)
Figure 6. Overlay of 8j, 11f and 16c on pharmacophore model and their alignment (8j-blue, 11f-
green, 16c-orange).
10

3
. Chemistry
6
In our previous article we have reported the discovery of isoxazole carboxylic acid-based
potent, selective and orally bio-available PTP1B inhibitors. Herein, our aim was to discover
novel “5-Amino-1H-pyrazole-4-carboxylic acid” as pharmacophore which is connected to an
aromatic ring (phenyl) which in turn connected to a hydrophobic tail group through a methylene
or alkyl amine group. 1,2-diphenyl ethanone, benzylphenyl-1,2,4-oxadiazole and dibenzyl amine
group has been selected as hydrophobic tail for the reason that these hydrophobic tail being used
extensively in PTP1B drug discovery research. With this background, compounds 8a-j, 11a-f,
and 16a-i have been synthesized.
Synthesis of final compounds 8a-j, 11a-f, 16a-i and their analogs are illustrated in Schemes 1-4
below.
i
ii
iii
1
2
3
4
5
o
Scheme 1. Reagents and conditions: (i) Ethanol, 90 C, 48 h, 84%; (ii) BH .THF solution (1M),
3
o
rt; 68%, (iii) PBr
3
, DCM, 10-15 C, 84%.
In scheme 1, compound 3 formation was carried out by reaction of 1 with 2 in ethanol at refluxed
temperature. Compound 3 was then treated with borane-THF in THF at rt to obtain
11

o
corresponding alcohol 4. It was then brominated using PBr
obtain compound 5 in good yield.
3
in DCM at temperature 0-5 C to
i
7
a-j
6
a-j
5
ii
8
a-j
o
Scheme 2. Reagents and conditions: (i) NaH, DMF, 10-15 C, 3 h, 61-98%; (ii) LiOH.H
2
O,
o
dioxane: H
2
O (4:1), 40 C, 16 h, 55-75%.
Synthetic route to compounds 8a–j is outlined in Scheme 2. Key benzyl bromide 5 thus
6
synthesized was treated with ethanone derivatives 6a-j in presence of NaH in DMF at
o
o
temperature10-15 C to obtain 7a-j which on hydrolyzed with LiOH.H
2
O at 40 C provided
target compound 8a-j (Table 2) in overall good yields (55-75%).
12

6
As outlined in scheme 3, oxadiazole derivatives 9a-f were treated with 5 in presence of NaH in
o
DMF at temperature 10-15 C yielded corresponding ester derivatives (10a-f) which on
hydrolyzed with LiOH.H
2
O at rt provided target compounds 11a-f in overall good yields (60-
8
0%).
i
1
0a-f
9
a-f
5
ii
1
1a-f
7
8
9
a; R =H, R =H, R =H
7
8
9
b; R =H, R =OMe, R =F
7
8
9
c; R =H, R =Cl, R =OMe
7
8
9
d; R =F, R =H, R =F
7
8
9
e; R =H, R =Cl, R =F
7
8
9
f; R =F, R =H, R =OMe
o
Scheme 3. Reagents and conditions: (i) NaH, DMF, 10-15 C, 3 h, 55-85%; (ii) LiOH.H
2
O,
o
dioxane: H
2
O (4:1), 40 C, 16 h, 60-80%.
The synthesis of compounds 16a–i is outlined in Scheme 4. Dibenzyl amine derivatives (14a-i)
were prepared by reaction of aldehyde and benzyl amine in dichloroethane in presence of sodium
13

1
2
triacetoxyborohydride and catalytic amount of acetic acid. Dibenzyl amine derivatives (14a-i)
o
were then treated with key benzyl bromide (5) in presence of Cs
2
CO
3
in DMF at 20-25 C to
obtain ester 15a-i which on hydrolyzed with LiOH.H O provided target compounds 16a-i in
2
good overall yields (50-95%).
i
1
4a-i
1
3
1
2
1
0
11
12
a: R =H, R =H, R =H
10
11
12
b: R =F, R =F, R =H
10
11
12
c: R =F, R =F, R =F
10
11
12
d: R =F, R =Cl, R =H
10
11
12
e: R =F, R =OMe, R =H
10
11
12
f: R =F, R =OMe, R =OMe
10
11
12
g: R =Cl, R =F, R =F
10
11
12
h: R =OMe, R =OMe, R =H
10
11
12
i: R =Me, R =OMe, R =H
14

ii
1
4a-i
5
1
5a-i
iii
1
6a-i
o
Scheme 4. Reagents and conditions: (i) Na(OAc)
3
BH, acetic acid, DCE, 20-25 C, 12 h, 43-82%;
o
o
(
ii) Cs
2
CO
3
, DMF, 10-15 C, 16 h, 60-85%; (iii) LiOH.H
2
O, dioxane: H
2
O (4:1), 40 C, 16 h, 50-
9
0%.
4
. Results and discussion
All the derivatives (8a-j, 11a-f and 16a–i) were evaluated in fluorescence based kinetic assay as
6
per the procedure described in our earlier publication.
15

Table 2. Investigation of 1,2-diphenyl ethanone analog as hydrophobic tail.
%
PTP1B Inhibition at
Compd.
R
2
R
3
R
4
R
5
R
6
1
0 μM
100 μM
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
H
H
H
H
H
H
H
F
H
F
H
H
H
H
H
H
H
F
H
H
H
H
9.6 ± 2.1
32.6 ± 1.1
19.0 ± 3.3 43.9 ± 1.5
12.1 ± 4.5 36.8 ± 1.4
H
F
F
0.8 ± 0.7
13.5 ± 0.8 58.5 ± 1.9
4.8 ± 0.9 39.7 ± 4.8
34.4 ± 2.2
F
OMe H
OMe H
OMe H
g
h
i
F
F
F
F
H
H
H
H
Cl
H
H
H
H
H
18.8 ± 3.4 89.5 ± 0.8
10.5 ± 0.3 48.5 ± 1.1
21.6 ± 0.4 82.2 ± 2.3
16.2 ± 3.6 91.5 ± 0.7
F
F
j
F
Cl
Simple unsubstituted compound 8a showed poor activity against PTP1B enzyme assay. To
3
5
explore SAR, small hydrophobic group like –F was incorporated in the phenyl ring (R or R ),
but it also did not improve activity (8b-c). A variety of other di-para-substituents were also
significantly less active (8d-f). Further optimization revealed that incorporation of para-chloro
16

3
2
(
R =Cl) in compound 8c yielded 8g which improves potency. Addition of meta-fluoro (R =F)
substituents in compound 8d and 8g was tolerated and gave 8i and 8j which are equipotent to 8g.
The most potent compounds 8i and 8j were found to be metabolically stable in both mouse
6
(
CLintr: <1 mL/min/g liver) and rat liver CLintr: <2 mL/min/g liver) microsomes. Our initial
SAR was focused on 1,2-diphenyl ethanone containing compounds (Table 2). Then we intended
to synthesize compounds 11a-f by replacing keto group of 1,2-diphenyl ethanone with more rigid
oxadiazole, which further yielded more potent derivatives (Table 3). Unsubstituted compound
8
9
1
1a was found to be inferior to 11b (R =R =F). Attempt was made to improve potency by
introducing different substituents on tail phenyl ring in compound 11b. Small hydrophobic
substitutions like F, Cl and hydrophilic substitution like -OMe (examples 11c-f) yielded
equipotent compounds like 11b. In general para-substituents are more active than meta-
substituents. To understand the metabolic stability of this series compounds 11c-f were tested in
6
rodent liver microsomes and these were found to be metabolically stable as these compounds
have low intrinsic clearance.
17

Table 3. Investigation of oxadiazole analogs as hydrophobic tails.
a
K
i
CLintr
%
PTP1B Inhibition at
(
M)
Compd. R
7
R
8
R
9
(mL/min/g liver)
MLM RLM
1
0 μM
100 μM
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
H
H
H
F
F
12.2 ± 0.5 64.0 ± 2.3
19.0 ± 0.4 94.9 ± 0.3
-
-
-
-
F
H
-
-
H
H
F
Cl
30.8 ± 1.9 97.3 ± 0.2 4.6
25.8 ± 1.8 95.5 ± 0.2 8.1
0.0
1.5
1.3
1.5
3
3
3
2
OMe F
H
OMe 22 ± 1.0
93.3 ± 0.4 8.6
H
Cl
OMe 33.7 ± 0.5 98.3 ± 0.2 4.4
a
Intrinsic clearance < 2 mL/min/g liver or 80% remaining at 30 min is considered as
metabolically stable. MLM: Mouse liver microsomes, RLM: Rat liver microsomes.
We next turned out our attention to the di-benzyl analogs (Table 4), since these compounds
contain tert-nitrogen, it is expected to improve cell permeability. Simple unsubstituted
compound 16a showed better potency than corresponding ethanone and oxadiazole compound.
Attempt was made to improve activity further by introducing para-substituents and small
hydrophobic groups like –F, -Cl, -OMe were found to be tolerated. We found that introduction of
–
Cl improves potency and 16c showed good inhibitory activity in PTP1B enzymatic assay with
18

i
K value of 4.6 μM. Replacing -Cl with –OMe in compound 16c yielded 16d which looses
potency, whereas other disubstituted compounds 16e-f significantly losses potency.
Table 4. Investigation of dibenzylamine analogs as hydrophobic tail.
K
i
CLintr
%
PTP1B Inhibition at
(M)
(mL/min/g
liver)
Compd.
R
10
R
11
R
12
1
0 μM
100 μM
MLM RLM
1
1
1
1
1
1
1
1
1
6a
6b
6c
6d
6e
6f
H
F
F
F
H
H
H
H
H
F
8.9 ± 2.0
40.8 ± 1.2
43.8 ± 2.6
64.0 ± 2.3
98.4 ± 0.7
-
-
-
-
-
-
Cl
97.4 ± 0.9 4.5
2.1
-
3.0
-
OMe 21.6 ± 0.4
Me 5.0 ± 1.0
OMe 9.9 ± 2.9
82.2 ± 2.3
49.7 ± 0.7
-
-
OMe H
OMe H
-
-
76.9 ± 1.3 7.1
95.2 ± 1.1 13
97.2 ± 1.9 11
5.6
2.7
1.2
7.4
2.5
3.0
2.0
4.0
6g
6h
6i
F
F
F
F
F
19.5 ± 1.2
22.5 ± 3.2
Cl
F
OMe OMe 35.07 ± 0.8 94 ± 0.2
4.1
Addition of one more –F, -Cl yielded compound 16g-h, which showed better potency.
Incorporation of -OMe in 16d yielded equipotent compound 16i. This series have shown
moderate to good stability in rodent liver microsomes.
19

Based on our hypothesis to improve the cell permeability by increasing the pka of the
compounds, we have evaluated a few compounds in parallel artificial membrane permeation
assay (PAMPA) at physiological pH (Table 5). Ethanone derivative (8j) showed moderate
intrinsic permeability and oxadiazole derivatives (11c-d) showed good to low intrinsic
permeability. However, dibenzyl derivatives (70c, 70f-i) showed excellent intrinsic permeability
in PAMPA. Furosemide and propanol were used as a positive control in this assay.
Table 5. PAMPA permeability values for a few synthesized compounds.
Permeability
in
Compound
PAMPA at pH 7.4
(
nm/sec)
8
1
1
1
1
1
1
j
15.0
91.0
4.0
1c
1d
6c
6f
6g
6h
106.0
125.0
278.9
498.0
< 1
Furosemide
Propanolol
166
20

5
. Conclusion
In summary, a series of “amino-carboxylic based pyrazole” was discovered as novel
pharmacophore by structure-based pharmacophore model and molecular docking analysis. In
comparison to most of the existing PTP1B inhibitors, our designed molecules contain small
molecular weight, non-phosphonate head groups. Functional groups such as 1,2-diphenyl
ethanone, oxdiadizole and dibenzyl amines were selected as hydrophobic tail. A series of
compounds containing these hydrophobic tails were synthesized and evaluated in PTP1B
enzymatic assay. In general, oxadiazole derivatives (11a-f) and dibenzyl amines (16a-i) were
more potent as compared ethanone (8a-j) derivatives. These compounds were found to be stable
in rodent liver microsomes. Dibenzyl amine derivatives were found to be better cell permeable in
PAMPA than ethanone and oxadiazole derivatives. It is assumed that these PTP1B inhibitors will
act as a lead to identify more potent compounds with good cell permeability and oral
bioavailability for the treatment of Type 2 Diabetes.
6
6
. Experimental section
.1. General synthetic methods
The purity of compounds was determined by HPLC. 1H nuclear magnetic resonance (NMR)
spectra were recorded in the deuterated solvents specified on a Varian 400 spectrometer
operating at 400. The signal of the deuterated solvent was used as internal reference. Chemical
shifts (δ) are given in ppm and are referenced to residual not fully deuterated solvent signal.
Coupling constants (J) are given in Hz. Chemical shifts are reported in parts per million (δ) from
the tetramethylsilane resonance in the indicated solvent (TMS: 0.0 ppm). Data are reported as
follows: chemical shift, multiplicity (br=broad, s=singlet, d=doublet, t=triplet, q=quartet,
21

m=multiplet) and integration. Mass spectra were determined by using Agilent 1200SL-6110
LC/MS (ESI) system using positive-negative switching.
6
.1.1. General procedure for the preparation of 7a-j
To an ice-cold suspension of NaH (1.2 mol equivalent) in DMF (5 fold), a solution of 6a-j (1.0
to 1.4 mol equivalent) in DMF (3 fold) was added drop wise over a period of 10 minutes and
stirred at the same temperature for 30 minutes. A solution of 5 (1.0 mol equivalent) in DMF (2
fold) was added to the reaction mixture at 20-25 °C and stirred at that temperature for 2 h. The
reaction mixture was poured into water and aqueous layer was extracted with ethyl acetate (2 X
1
0 mL). The combined organic layer was successively washed with water and brine, dried over
Na2SO4, filtered and concentrated under vacuum. The crude product was purified by column
chromatography using 5-10% ethyl acetate in hexane as an eluent to obtain title compound (7a-
j).
Ethyl 5-amino-1-[4-[3-(4-chlorophenyl)-2-(3,4-difluorophenyl)-3-oxo-
propyl]phenyl]pyrazole-4-carboxylate (7j)
7
j (0.156 g, 100%) was prepared from 6j (0.097 g, 0.36 mmol) and 5 (0.1 g, 0.30 mmol)
following the general procedure described above as colorless mass.
1
HNMR (DMSO d ): δ 1.26 (t, J = 7.2 Hz, 3H), 3.09 (dd, J = 7.4, 13.8 Hz, 1H), 3.48 (dd, J = 7.4,
6
1
7
2
3.8 Hz, 1H), 4.21 (q, J = 6.8 Hz, 2H), 5.36 (t, J = 7.2 Hz, 1H), 6.25 (br s, 2H), 7.19 (br s, 1H),
.31-7.38 (m, 5H), 7.47-7.49 (m, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.67 (s, 1H), 8.07 (d, J = 8.4 Hz,
+
H); ESI/MS (m/z): 510.1 (M+H)
6
.1.2. General procedure for the preparation of the compounds 8a-j
22

To a solution of 7a-j (1 mol equivalent) in dioxane:H
2
O (4:1) (10 fold), was added LiOH.H O (3
2
o
mol equivalent) and stirred at temperature 40 C for 16 h. The organic volatiles were evaporated
and residue was diluted with water. The aqueous layer was washed with ethyl acetate (2 X 5 mL)
o
and aqueous layer was cooled to 0-5 C. The aqueous layer was acidified with 2(N) HCl and
precipitate was separated, filtered off, washed with water and dried to furnish title product 8a-j.
8
j: 5-Amino-1-[4-[3-(4-chlorophenyl)-2-(3,4-difluorophenyl)-3-oxopropyl]phenyl]pyrazole;
o
1
White solid; mp: 117-118 C; Yield: 49%; Purity: 98.0% by HPLC; HNMR (DMSO d
6
): δ 3.09
(
dd, J = 7.4, 13.8 Hz, 1H), 3.45 (dd, J = 7.4, 13.8 Hz,1H), 5.36 (t, J = 7.4 Hz, 1H), 6.21 (br s,
2
1
H), 7.19 (br s, 1H), 7.34-7.41 (m, 5H), 7.44-7.49 (m, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.64 (s,
+
H), 8.07 (d, J = 8.4 Hz, 2H), 12.07 (br s, 1H); ESIMS m/z: 482.1 (M+H) .
6
.1.3. General procedure for the preparation of the compounds 11a-f
To a solution of 10a-f (1 mol equivalent) in dioxane: H
2
O (4:1) (10 fold), was added LiOH.H O
2
o
(
3 mol equivalent) and stirred at 40 C for 16 h. The organic volatiles were evaporated and
residue was diluted with water. The aqueous layer was washed with ethyl acetate (2 X 5 mL) and
o
aqueous layer was cooled to 0-5 C. The aqueous layer was acidified with 2(N) HCl and
precipitate was separated, filtered off, washed with water and dried to furnish title product 11a-f.
1
1c: 5-Amino-1-[4-[2-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]-2-(4-
fluorophenyl)ethyl]phenyl]pyrazole-4-carboxylic acid, White solid; Yield: 78%; Purity:
1
9
5.0% by HPLC; HNMR (DMSO d
6
): δ 3.45 (dd, J = 8.2, 13.8 Hz, 1H), 3.67 (dd, J = 8.0, 14.0
Hz, 1H), 5.04 (t, J = 8.0 Hz, 1H), 6.23 (br s, 2H), 7.19 (t, J = 8.8 Hz, 2H), 7.41 (br s, 4H), 7.50-
7
5
.53 (m, 2H), 7.64 (br s, 2H), 7.66 (s, 1H), 8.02 (d, J = 8.8 Hz, 2H), 12.08 (br s, 1H).ESIMS m/z:
+
04.2 (M+H) .
23

6
.1.4. General procedure for the preparation of the compounds 16a-i
These compounds were prepared in the same manner as following the general procedure 11a-f
1
6f: 5-Amino-1-[4-[[bis[(4- methoxyphenyl)methyl]amino]methyl
o
]
phenyl]pyrazole-4-carboxylic acid; White solid; mp: 172-174 C; Yield: 52%; Purity: 99.0%
1
by HPLC; HNMR (DMSO d
6
): δ 3.49 (s, 4H), 3.55 (s, 2H), 3.77 (s, 6H), 6.29 (br s, 2H), 6.95
(
d, J = 7.6 Hz, 4H), 7.34 (d, J = 8.0 Hz, 4H), 7.55 (br s, 4H), 7.69 (s, 1H), 12.06 (br s, 1H);
+
ESIMS m/z: 473.2 (M+H) . 16g: 5-Amino-1-[4-[[(3, 4-difluorophenyl)methyl-[(4-
fluorophenyl)methyl]amino]methyl]phenyl]pyrazole-4-carboxylic acid; White solid; mp:
o
1
1
2
4
68-169 C; Yield: 67%; Purity: 95.0% by HPLC; HNMR (DMSO d ): δ 3.53 (s, 4H), 3.56 (s,
6
H), 6.27 (br s, 2H), 7.18 (t, J = 8.8 Hz, 2H), 7.26 (br s, 1H), 7.39-7.45 (m, 4H), 7.50-7.54 (m,
+
H), 7.66 (s, 1H), 12.07 (br s, 1H); ESIMS m/z: 467.2 (M+H) . 16h: 5-Amino-1-[4-[[(4-
chlorophenyl)methyl-[(3,4-difluorophenyl)methyl]amino]methyl]phenyl]pyrazole-4-
o
1
carboxylic acid; White solid; mp: 166-170 C; Yield: 53%; Purity: 98.0% by HPLC; HNMR
DMSO d ): δ 3.53 (s, 4H), 3.56 (s, 2H), 6.26 (br s, 2H), 7.18 (t, J = 8.8 Hz, 2H), 7.20-7.25 (m,
H ), 7.39-7.45 (m, 4H), 7.50-7.54 (m, 4H), 7.66 (s, 1H), 12.05 (br s, 1H); ESIMS m/z: 483.0
(
6
1
Acknowledgements
Authors are grateful to Drs. Kasim A. Mookhtiar and Venkata P. Palle for their support,
management of Advinus Group for encouragement. Analytical departments are being
acknowledged for their help during this work. We thank Dr. Anup Ranade for managing
intellectual property. Advinus publication no. ADV-A-035
24

Supplementary Material
Molecular Docking analysis, Brief of PAMPA screening assay protocol and experimental
procedure.
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