Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4- b]pyridines

Article abstract of DOI:10.1021/acs.jmedchem.0c01152

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Full text of DOI:10.1021/acs.jmedchem.0c01152

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Article
Synthesis and structure-activity relationship of
dual-stage antimalarial pyrazolo[3,4-b]pyridines
Scott Eagon, Jared T. Hammill, Martina S. Sigal, Kevin J. Ahn, Julia E. Tryhorn, Grant Koch, Briana Belanger,
Cory A. Chaplan, Lauren Loop, Anna S. Kashtanova, Kenya Yniguez, Horacio Lazaro, Steven P. Wilkinson,
Amy Rice, Mofolusho Falade, Rei Takahashi, Katie Kim, Ashley Cheung, Celine DiBernardo, Joshua
Kimball, Elizabeth A Winzeler, Korina Eribez, nimisha mittal, Francisco-Javier Gamo, Benigno Crespo, Alisje
Churchyard, Irene Garcia-Barbazan, Jake Baum, Marc O. Anderson, Benoît LALEU, and R. Kiplin Guy
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01152 • Publication Date (Web): 18 Sep 2020
Downloaded from pubs.acs.org on September 20, 2020
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Biochemistry
LALEU, Benoît; MMV,
Guy, R. Kiplin; University of Kentucky, College of Pharmacy
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Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial
Pyrazolo[3,4-b]pyridines
a,*
b
c
a
a
d
Scott Eagon , Jared T. Hammill , Martina Sigal , Kevin J. Ahn , Julia E. Tryhorn , Grant Koch , Briana
a
a
a
a
e
f
Belanger , Cory A. Chaplan , Lauren Loop , Anna S. Kashtanova , Kenya Yniguez , Horacio Lazaro , Steven
a
b
b
a
a
a
P. Wilkinson , Amy L. Rice , Mofolusho O. Falade , Rei Takahashi , Katie Kim , Ashley Cheung , Celine
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f
g
g
g
h
DiBernardo , Joshua J. Kimball , Elizabeth A. Winzler , Korina Eribez , Nimisha Mittal , F. Javier Gamo ,
h
i
i
i
j
k
Benigo Crespo , Alisje Churchyard , Irene Gracía-Barbazán , Jake Baum , Marc O. Anderson , Benoît Laleu
and R. Kiplin Guy
b
aDepartment of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, California
93407, USA
bDepartment of Pharmaceutical Sciences, University of Kentucky , Lexington, Kentucky 40508, USA
cDepartment of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, Memphis, Tennessee
8105, USA
3
dDepartment of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California 95064, USA
eSRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, USA
fPromega Biosciences, 277 Granada Drive, San Luis Obispo, California 93401, USA
gSchool of Medicine, University of California San Diego, La Jolla, California 92093, USA
hGlaxoSmithKline, Global Health, DDW, Tres Cantos 28760, Madrid, Spain.
iDepartment of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom
jDepartment of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, USA
kMedicines for Malaria Venture (MMV), P.O. Box 1826, 20, Route de Pré-Bois 1215, Geneva, Switzerland.
ABSTRACT
Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of
deaths each year, primarily in young children and pregnant mothers. Here we report the discovery and
derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest
species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity
against the intraerythrocytic stage of the parasite, as well as little to no toxicity against the human
fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the
liver stage of the parasite, but little activity against the gametocyte stage. Parasitological profile,
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including rate of killing, docking, and molecular dynamics studies suggest that our compounds may
target the Q binding site of cytochrome bc .
o
1
INTRODUCTION
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Malaria continues to remain one of the most deadly infectious diseases on the planet. The
World Health Organization (WHO) reported an estimated 228 million cases in 2018 with nearly 405,000
1
associated deaths, primarily in young children and pregnant women. Despite concerted efforts to
2
combat the spread of Malaria, it continues to have a devastating effect in parts of Asia and Africa. The
latest WHO summary notes that “incidence rate of malaria declined globally between 2010 and 2018.
However, from 2014 to 2018, the rate of change slowed dramatically, remaining at similar levels.”
Malaria in humans is caused by hematoprotozoan parasites of the genus Plasmodium, with P.
3
falciparum accounting for the vast majority of fatalities. The Plasmodium parasite has a complex life
cycle that includes both a liver and intraerythrocytic stage in the human host, significantly increasing the
4
challenge associated with combating infection. While a number of efforts have been made to combat
the disease via vaccines and vector control, small-molecule therapeutics remain a critical component of
5
the fight against malaria. The WHO currently recommends artemisinin-based combination therapies
6
7
(ACTs) to treat complicated forms of infection, but recent reports of emerging resistance against ACTs
8
and a limited number of validated drug targets highlight the need for new treatment options.
We identified a number of promising hit compounds in a high-throughput screen conducted to
identify molecules that stop the growth of P. falciparum in co-cultures with human erythrocytes in
9
vitro. One set of hit compounds was a series of pyrazolo[3,4-b]pyridines detailed in Table 1.
Table 1. Bioactivity and cytotoxicity of pyrazolo[3,4-b]pyridines hits against P. falciparum.
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EC 3D7
EC V1S
EC BJ
(μM)
5
0
50
50
Compound
R1
H
R2
(μM)
strain (μM)
1
0.397 ± 0.135 0.404 ± 0.102
>23
2
3
Et
H
>15
>15
-
0.461 ± 0.126 0.922 ± 0.106
0.555 ± 0.213 0.834 ± 0.137
>23
4
5
H
H
>23
>23
1.19 ± 0.692
0.791 ± 0.148
aEC50 values for the clinically susceptible 3D7 strain are reported as the mean ± SD of five
measurements. EC50 values for the multi-drug resistant V1/S strain (an in vitro culture-adapted clone
of the V1 strain originating in Vietnam that shows resistance to chloroquine, pyrimethamine,
cycloguanil and quinine) are reported as the mean ± SD of four measurements. Human fibroblast cells.
b
c
This is the first report of diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitilres displaying antimalarial
activity, and these initial results suggest that they display comparable activity against drug resistant
strains while simultaneously displaying no cytotoxicity against a human cell line. It should be noted that
the pyrazolopyridine core itself has been reported to have antimalarial activity in a prior publication, but
it was only utilized as a replacement core for chloroquine and only targeting the asexual blood stage of
Plasmodium.10 It also appears that the phenol hydroxyl group is important to activity, as well as
substitution about the phenyl ring at the 2-pyridinyl position. We decided to make a library of
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compounds related to 1 to better understand not only these moieties, but also to probe modifications
about the pyrazole ring as well. To this end, we envisioned three series of compounds investigating the
importance of the phenol hydroxyl group, the effect of varying the p-ethylphenyl system, and the
substitutions about the pyrazole ring, respectively (Figure 1).
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Figure 1. Outline of chemical modifications to probe the structure activity relationship of 1.
RESULTS AND DISCUSSION
Chemistry, Antimalarial Activity and Cytotoxicity
A number of one-pot, multicomponent synthetic procedures for the preparation of
1
1
pyrazolo[3,4-b]pyridines have been reported, including a report detailing a microwave-mediated
procedure in acetic acid.12 We were initially interested in the microwave procedure due to our success
utilizing a microwave-mediated Pictet-Spengler reaction for the preparation of a previous β-carboline
1
3
library, however, we were unable to reproduce the reported results. We next decided to reproduce a
selection of other reported procedures using benzaldehyde, 2-cyanoacetophenone and
3-methyl-1-phenyl-1H-pyrazol-5-amine as our model substrates. We found that heating the reagents
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overnight in DMF at 100 °C overnight provided the highest conversion as observed by H NMR. The
product could then be isolated in modest yields after purification (Scheme 1).
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Scheme 1. Synthesis of pyrazolo[3,4-b]pyridines.
The advantage of this 3-component system was that it allows for easy and selective modification of
functional groups in a single step to prepare the series of modifications outlined in Figure 1 by
selectively varying the appropriate starting reagents, many of which are commercially available.
To establish an initial structure-activity relationship (SAR), we synthesized the first collection of
compounds (Series I) by varying the aldehyde. Due to limited resources, we narrowed our investigation
to aldehydes which were inexpensive and readily available. As a positive control, we also decided to
synthesize hit compound 1. After confirming the identity and purity of the compounds by NMR and
HRMS, we determined the activity of these compounds against the chloroquine sensitive P. falciparum
3
D7 strain (MRA-102) grown in the presence of O-positive erythrocytes, as well as the cytotoxicity
against the human BJ cell line (Table 2).
Table 2. Summary of Series I compounds: antimalarial activity, cytotoxicity, therapeutic index, melting
point and solubility.
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a
b
EC 3D7
EC BJ
(μM)
Solubility
(μM)
5
0
50
Compound
R
T.I.
50
m.p. (°C)
267-269
c
(μM)
1
0.397 ± 0.135
0.692 ± 0.056
0.427 ± 0.096
0.505 ± 0.067
0.502 ± 0.104
0.363 ± 0.048
0.783 ± 0.084
0.508 ± 0.124
0.258 ± 0.047
1.41 ± 0.359
0.704 ± 0.232
0.554 ± 0.050
>20
3.1 ± 0.4
1.2 ± 1
2.4 ± 3
8.5 ± 0.9
1.5 ± 1
1.7 ± 1
1.7 ± 3
6.0 ± 1
2.2 ± 0.9
33 ± 8
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>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
29
47
40
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55
26
39
78
14
28
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225-226
201-203
205-207
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196-198
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0
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12
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11 ± 6
16
7.8 ± 2
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0.278 ± 0.018
>20
>20
72
160-162
198-199
9.9 ± 4
47 ± 20
1
1
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1.79 ± 0.146
2.04 ± 0.343
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>20
>20
172-174
238-240
18 ± 3
2
2
0
1
1.80 ± 0.340
11
5.7 ± 2
0.242 ± 0.026
1.33 ± 0.330
>20
>20
83
15
141-143
148-150
17 ± 2
22
46 ± 20
aEC50 values are reported as the mean ± SD of five measurements. Assay endpoints were normalized from 0%
DMSO only) to 100% inhibition (16 µM Mefloquine). EC50 values are reported as the mean ± SD of six
measurements. Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition (58 µM Idarubicin).
Solubility in PBS buffer, pH 7.4, with DMSO used as the carrier vehicle. The detection limit of the solubility
assay was 100 μM. Albendazole (1.5 ± 1 μM) and carbamazepine (>100 μM) were used as poorly soluble and
highly soluble standards, respectively. Values are reported as the mean ± SD of three measurements.
b
(
c
A few derivatives showed some modest improvement in potency, with a chloro- (10) and
trifluoromethoxy-derivative (17) providing an alternative to the hydroxyl group, which would be
expected to be metabolically vulnerable. Movement or elimination of the hydroxyl group (18, 19 and
2
0) had a strongly deleterious effect, as well as replacement of the o-hydroxy with either a carboxylic
acid (14) or an ethynyl group (22). While steric hindrance is a possible explanation for this loss of
activity, both a methyl sulfone (13) and a methyl ester (21) displayed activity in a similar range to 1.
Most compounds also had fairly high melting points, likely due to extensive pi-pi stacking interactions
and the relatively high molecular weights. Solubility values were also on the low side as expected due to
the lipophilic nature of most of these compounds. While this could possibly be remedied through the
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incorporation of heteroaromatic or smaller alkyl substituents, we were limited in our ability to purchase
these substrates for the purposes of this study and intend to investigate them in future studies once
additional resources can be secured.
1
1
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Next, we prepared our second series of compounds (Series II) by varying the α-cyanoketone
utilized in our reaction. While only a handful of appropriate α-cyanoketones are commercially available,
they are readily prepared from widely available methyl ketone derivatives via a two-step procedure
consisting of an α-bromination by CuBr , followed by a nucleophilic substitution with NaCN (Scheme
2
2).14 These compounds were then tested against both the P. falciparum 3D7 strain and the human BJ
cell line (Table 3).
Scheme 2. Synthesis of α-cyanoketones.
Table 3. Summary of Series II compounds: antimalarial activity, cytotoxicity, therapeutic index, melting
point and solubility.
Solubility
a
b
Compound
R
EC 3D7 (μM) EC BJ (μM)
T.I.
50
m.p. (°C)
267-269
5
0
50
c
(μM)
1
0.397 ± 0.135
>20
3.1 ± 0.4
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0.384 ± 0.078
0.283 ± 0.010
1.32 ± 0.076
22.2 ± 2.23
>20
58
71
16
269-270
261-262
243-244
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0
21.0 ± 2.51
21 ± 10
9.8 ± 1
0.096 ± 0.018 22.6 ± 0.351
235 265-267
308 219-221
27
0.0692 ± 0.016 21.3 ± 2.47
0.113 ± 0.024 22.5 ± 0.859
9.6 ± 7
2
2
8
9
199 240-241
153 241-242
6.5 ± 2
1.8 ± 2
0.131 ± 0.029
>20
3
0
0.254 ± 0.032 22.9 ± 0.841
90
44
81
254-256
234-236
281-282
3.5 ± 2
6.0 ± 2
3.8 ± 2
1.5 ± 0.4
1.7 ± 0.2
6.0 ± 7
11 ± 2
31
0.452 ± 0.167
0.247 ± 0.019
0.127 ± 0.008
0.312 ± 0.036
0.274 ± 0.019
>20
>20
3
3
3
2
3
4
19.0 ± 3.37
>20
150 289-291
64
73
97
165-166
257-258
240-242
35
>20
3
6
0.236 ± 0.050 23.0 ± 0.376
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0.711 ± 0.094
0.214 ± 0.032
0.370 ± 0.058
>20
>20
>20
28
93
54
222-224
236-238
293-294
5 ± 2
13 ± 4
11 ± 2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
39
4
4
0
1
0.115 ± 0.011
0.138 ± 0.034
>20
>20
174
268d
<1
145 178-179
11 ± 6
aEC50 values are reported as the mean ± SD of five measurements. Assay endpoints were normalized from 0%
(DMSO only) to 100% inhibition (16 µM Mefloquine). EC50 values are reported as the mean ± SD of six
measurements. Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition (58 µM Idarubicin).
Solubility in PBS buffer, pH 7.4, with DMSO used as the carrier vehicle. The detection limit of the solubility
assay was 100 μM. Albendazole (1.5 ± 1 μM) and carbamazepine (>100 μM) were used as poorly soluble and
highly soluble standards, respectively. Values are reported as the mean ± SD of three measurements.
Decomposes at this temperature.
b
c
d
Most alkyl-substituted phenyl rings (23-28) were tolerated with little effect on observed potency, with a
t
para- butylphenyl group displaying the most potent activity and highest therapeutic index (27). A
notable exception was observed for 25, where the possible explanation for loss of activity could be the
prevention of a planar configuration by the ortho-methyl group, suggesting that a co-planar orientation
with the pyrazolo[3,4-b]pyridine is preferred in the binding region. A number of other para-substituted
phenyl compounds were investigated (30-35, 37 and 40) and gave no significant change in activity, with
the exception of a methyl sulfone (37). Even a para-morpholino phenyl group (40) displayed
comparable activity to hit compound 1. Indeed, larger aromatic and heteroaromatic ring systems were
also tolerated with negligible change in relative activity (29, 38-39). The wide range of substitutional
flexibility makes this substitution point of the pyrazolo[3,4-b]pyridine core an attractive point to modify
in future pharmacokinetic studies, both in terms of bioavailability and solubility. Indeed, introduction of
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5
6
solubilizing groups and reducing the lipophilicity would be likely next steps in future studies to improve
the physicochemical and pharmacokinetic properties of this chemical series.
Lastly, we decided to make a small group of compounds (Series III) that investigated some
simple substitutions about the pyrazole system, as our original screening set contained no variability
about this area of the core scaffold. Only a few pyrazole-5-amines are readily commercially available,
but our group recently reported a rapid microwave-mediated synthesis of various substituted pyrazole-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-amines that affords access to these substrates in a manner of minutes utilizing a simple aqueous
workup.15 Utilizing this methodology, we were able to prepare a small number of compounds which we
also analyzed as with previous substrates (Table 4).
Table 4. Summary of Series III compounds: antimalarial activity, cytotoxicity, therapeutic index, melting
point and solubility.
a
b
EC 3D7
EC BJ
(μM)
Solubility
(μM)
5
0
50
Compound
R1
R2
T.I.
m.p. (°C)
c
(μM)
1
42
Me
Ph
Ph
Ph
0.397 ± 0.135
0.123 ± 0.025
>20
>20
50
163
267-269
248-249
3.1 ± 0.4
30 ± 4
4
4
3
4
Ph
0.152 ± 0.041
>20
132
19
122-124
21 ± 2
Me
Me
1.04 ± 0.171
>20
311-313
9.6 ± 3
45
0.094 ± 0.009
>20
213
235-237
4.8 ± 2
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Me
0.139 ± 0.019
>20
144
120-122
8.9 ± 7
aEC50 values are reported as the mean ± SD of five measurements. Assay endpoints were normalized from 0%
DMSO only) to 100% inhibition (16 µM Mefloquine). EC50 values are reported as the mean ± SD of six
measurements. Assay endpoints were normalized from 0% (DMSO only) to 100% inhibition (58 µM Idarubicin).
Solubility in PBS buffer, pH 7.4, with DMSO used as the carrier vehicle. The detection limit of the solubility
assay was 100 μM. Albendazole (1.5 ± 1 μM) and carbamazepine (>100 μM) were used as poorly soluble and
highly soluble standards, respectively. Values are reported as the mean ± SD of three measurements.
b
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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0
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
c
Although only small handful of compounds were prepared, a number of alkyl substitutions were
tolerated with the exception of 44, possibly due to either the inability of the phenyl ring to achieve
planarity with the pyrazolo[3,4-b]pyridine core or due to steric repulsion. Solubility remains fairly low
for these compounds, still likely due to the lipophilic nature of the scaffold. Interestingly, incorporation
of a cyclopropyl ring (43) and a para-methyl substitution (46) gave modest potency improvement, along
with a significant reduction in the observed melting point and some improvement in the observed
solubility.
In total, our in vitro results demonstrate that although the ortho-hydroxy group in 1 initially
appeared to be required for good potency (Table 1) some small substitutions to less metabolically
sensitive isosteres in this position are tolerated. In contrast, variation of the 2-pyridinyl aryl system
(
Series II) shows that a large range of substitutions are tolerated without significant changes in observed
potency. A few additions to the N-aryl pyrazole system also showed slight improvement (Series III),
although it is difficult to draw conclusions from such a small set of derivatives. In general, melting points
remain high and solubility low due to the large molecular weight and high lipophilicity of some of these
derivatives.
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3
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1
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1
2
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4
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7
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1
2
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Activity and physiochemical property summary of all tested compounds.
Efficacy of Selected Compounds Versus Blood States of Plasmodium
Three compounds were selected as frontrunner compounds for further efficacy studies.
Compound 27 was selected due to the fact that it had the highest observed potency, while 10 and 17
were selected due to their modest improvement in activity versus our initial hit compound and
presented a chloro- and trifluoromethyl- substitution, respectively, in place of the metabolically
sensitive ortho-hydroxy group.
In order to determine how quickly the selected frontrunner compounds killed blood stage
parasites, a double-colorimetric fluorescence-activated cell sorting (FACS) analysis was used to measure
new invasions of pre-stained erythrocytes by drug treated parasites. Two time points (24h and 48h)
were used to distinguish the rate of killing profile in comparison to chloroquine, pyrimethamine,
atovaquone and artesunate, which were used as standard controls (Table 5 and S1). All three
compounds presented a moderate to slow killing profile with compounds 10 and 17 displaying a slow
killing profile nearly identical to atovaquone, with no inhibition of parasite growth until after 24h (Figure
S1 and S2).
Table 5. Blood stage assays of compounds 10, 17 and 27.
Male
Gamete
Inhibition
Female
Gamete
Inhibition
%
Survival
% Survival
(48h)
Gametocyte
Cmpd.
Structure
a
a
(24h)
EC (μM)
5
0
b
b
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1
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03.63 ±
.51%
52.33 ±
9.07%
1
0
9.61%
2.69%
2.48%
11.55%
-3.94%
-2.29%
>25
>25
>25
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22.21 ±
.02%
32.98 ±
1.55%
17
5
4
0.70 ±
.60%
12.40 ±
2.37%
2
7
5
aValues are reported as the mean ± SD of two measurements. b Data are mean % inhibition at 48h with 1 μM
concentration of compound relative to DMSO carrier control.
The ability of the selected compounds to inhibit the sexual stages of the parasite were also investigated
(
Table 5), since transmission is mediated exclusively by the gametocytes of Plasmodium. Moreover,
sexually mature stage V gametocytes have shown sex-specific sensitivity to most antimalarial
compounds.16 The three frontrunner compounds were tested at a 1 μM concentration to measure the
effect on the growth of both male and female gametocytes, with the results indicating that the
compounds are equally ineffective against both.
Liver Stage Activity of Frontrunner Compounds
Hepatic human transformed cells (HepG2) were pretreated with the frontrunner compounds for
1
8 hours and were then infected with freshly dissected P. berghei Luciferase sporozoites. After 48 hours
of incubation, the viability of P. berghei exoerythrocytic forms was measured by bioluminescence. The
cytotoxicity of the compounds against HepG2 cells was also determined (Table 6). All three compounds
continued to display low cytotoxicity, and compounds 10 and 17 maintained sub-micromolar potency
against the liver stage of the parasite.
Table 6. In vitro liver stage assays of compounds 10, 17 and 27.
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5
5
5
6
P. berghei liver stage
EC HepG2 cells
5
0
Cmpd.
Structure
IC in HepG2 cells
5
0
a
(μM)
a
(μM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
0
7
7
0.928 ± 0.257
0.570 ± 0.020
3.05 ± 0.325
>50
>25
24.4 ± 1.06
aValues are reported as the mean ± SD of two measurements.
Proposed Structural Activity Relationship Based on Molecular Docking & Dynamics Studies
The activity against liver stages, slow killing profile and ineffectiveness against both male and
female gametocytes displayed by the pyrazolo[3,4-b]pyridines mirrors the profile of atovaquone. As
such, we decided to investigate whether or not molecular docking and molecular dynamics techniques
could help explain our observed activity trends, given the assumption that our compounds shared the
same mechanism of action. Atovaquone (marketed in combination with proguanil as Malarone) is a
clinically approved antimalarial targeting the Q binding site of Plasmodium cytochrome bc . While
o
1
cytochrome bc is a validated clinical target, a number of point mutations in the Q binding site have led
1
o
to atovaquone-resistant strains. Fortunately, a crystal structure of atovaquone bound to cytochrome
bc is available (PDB code: 4PD4), allowing for easy comparisons between the known action of
1
atovaquone and the suspected action of pyrazolo[3,4-b]pyridines.
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To begin our docking studies, we first decided to investigate which of the three docking
1
7
18
19
programs available to us (AutoDock Vina , iDock and Smina ) could give the best docking scores
based on known inhibitors. To do this we selected a test set of 12 quinolone compounds believed to
target the Q site of P. falciparum cytochrome bc with EC values that spanned six orders of magnitude
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
50
(
0.066nM to 20000nM).20 We then used the Spearman correlation coefficient (R ) to evaluate the
s
performance of each docking program (Table S2). Our results show that the iDock program gave
superior performance (R = 0.783), so we selected it as the program of choice for docking the
s
pyrazolo[3,4-b]pyridines. We then proceeded to dock our three frontrunner compounds (10, 17 and 27)
to the Q site of cytochrome bc . However, while atovaquone is known to bind the Q site of
o
1
o
cytochrome bc , some 4(1H)-pyridones previously believed to bind the Q site were unexpectedly found
1
o
2
1
to bind to the reductive Q site, most notably compound GW844520. To test this possibility, we also
i
docked our hit compounds to the Q site, and found that the predicted binding energies were less
i
favorable in all cases, suggesting that Q site is preferred (Table S3).
o
The computed docking poses of all three frontrunner compounds along with our original lead
molecule (1) were nearly identical, fitting snugly within the hydrophobic pocket in the Q site, and
o
extending through a hydrophobic barrel formed by nearby alpha helices (Figure 3A). Not surprisingly, all
nearby interacting residues were hydrophobic in nature, and no polar interactions were apparent, in line
with the improved potency observed for the most lipophilic compounds (Figure 3B). The lack of any
apparent hydrogen bonding interaction between the phenol of hit compound 1 (Figure 3C) and the
binding pocket explains why many small substitutions are tolerated at this position with minimal change
of observed activity. The long hydrophobic barrel where the para-ethyl substituted phenyl group of 1
resides explains why most compounds in Series II were measured to have similar potencies despite the
large range of tested substrates, with only 25 having a significant decrease in activity likely due to a
steric interaction between the ortho-methyl substitution and surface of the protein. The methyl group
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1
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2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
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5
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5
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5
5
6
at the 3-position of the pyrazole ring in all docked compounds sticks out away from the protein,
explaining why phenyl and cyclopropyl substitutions in compounds 41 and 42, respectively, had little
change in measured activity despite the drastic change in local size. Additionally, the predicted binding
site of the pyrazolo[3,4-b]pyridines is somewhat different than the known binding site of atovaquone
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 3D). This is significant because the frontrunner compounds are predicted to bind away from
Y279, one of the main residues known to confer resistance to atovaquone upon point mutation.22 Thus,
we would predict that our compounds would be equally effective against atovaquone-resistant strains
of Plasmodium should our proposed binding model prove correct, and we expect to test this claim as a
critical question in future studies.
A.
B.
C.
D.
Figure 3. Proposed binding SAR of pyrazolo[3,4-b]pyridines to the Q site of Plasmodium cytochrome
o
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bc (PDB code: 4PD4). (A) Computing docking pose of 1 (yellow backbone), 10 (magenta backbone), 17
1
(
teal backbone) and 27 (green backbone). (B) Binding residue interactions to 27. (C) 2D SAR of 27 with
nearby residues. Non-polar interactions are shown as forest green lines. (D) Relative position of 27
green backbone) versus atovaquone (peach backbone) to Y279, whose mutation induces resistance to
atovaquone.
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
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5
5
5
5
6
0
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2
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0
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0
1
2
3
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0
1
2
3
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5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
Molecular dynamics (MD) simulations were carried out to examine the ability of inhibitor 27 and
atovaquone to remain bound to the Q site of P. falciparum cytochrome bc1 under equilibrating
0
conditions, and also to provide an estimation of binding energy. The protein-ligand complexes produced
from the docking simulation were embedded into a bi-lipid membrane comprised of 1-palmitoyl-2-
oleoylphosphatidylcholine (POPC) molecules. The lipid-protein-ligand complexes were subjected to 50
ns production runs at 300 K. We observed that the majority of the equilibration was complete within
2
0-30 ns for the complexes, although simulations were continued for a total of 50 ns. Qualitative
examination of the lipid-protein-ligand structures during the trajectories show both 27 and atovaquone
to maintain stable complexes through various stages of production runs, with minimal movement of the
ligands within the putative binding site. For the complex of bc1 with atovaquone, the RMSD increased
slightly from 0 to 25 ns, presumably as the protein accommodated the ligand, and then relaxed into an
apparently stable configuration from 25 ns to 50 ns with the RMSD settling at approximately 3.5 Å, after
a slight decrease in RMSD in the latter part of the experiment (Figure S4). For this complex, the
potential energy also decreased slightly throughout the course of the 50 ns production run. A similar
behavior occurred in the 50 ns simulation of bc1 with inhibitor 27 (Figure S5) and the other inhibitors
evaluated by MD (1, 10, 17; data not presented). The RMSD at equilibrium for this experiment seemed
to stabilize at a slightly lower RMSD of 3.0 Å, compared to the experiment with atovaquone. The
energy profile of cytochrome bc1:27 complex decreased similarly to the complex with atovaquone, the
energy profile decreased similarly to the complex with atovaquone, consistent with a stabilized complex
through the latter ~25 ns of the experiment. Overall, we observed that the majority of the equilibration
was complete within the first ~30 ns for the complexes, making 50 ns production runs suitable for these
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
6
protein-ligand complexes. Binding energies (Gbinding) were estimated using the MM/GBSA and
2
3
MM/PBSA tools and summarized in Table S4. In all cases, our hit compounds were found to have
stronger binding affinities to the Q site than atovaquone. These results support the hypothesis that 1,
o
0
1
2
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4
5
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7
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0
1
2
3
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10, 17, and 27 bind the Q site of P. falciparum cytochrome bc1, and also support the binding poses
0
discovered with docking, due to the lack of significant movement of the compounds through the course
of the experiment.
CONCLUSIONS
We report the synthesis and antimalarial activities of a series of pyrazolo[3,4-b]pyridines
targeting Plasmodium falciparum. Most compounds displayed sub-micromolar potency against the
intraerythrocytic stage of the parasite, with the most potent compound, 27, presenting an EC of
5
0
0
.0692 ± 0.016 μM. Structure-activity analysis of initial hit compounds suggested that the ortho-
hydroxy group in the original hit compound 1 was required for good activity, but subsequent studies
demonstrate that several isosteres with superior metabolic profiles are tolerated well. In contrast, large
variations about the 2-pyridinyl aryl system are accommodated with little impact on activity. Based on
this information, we selected three compounds for future studies. 27 was selected since it had the
highest observed activity, while 10 and 17 were selected as analogues with less metabolically sensitive
substitutions for the ortho-hydroxy group of 27. Additional blood stage assays of these three hit
compounds determined that they had a slow to moderate killing profile and little to no activity against
the gametocyte stage of the parasite, both similar to atovaquone. Liver stage assays against P. berghei
showed that all three hit compounds maintained good potency and all compounds display little to no
toxicity against the human BJ and HepG2 cell lines. Since the Plasmodium lifecycle profile and killing
rate are similar to atovaquone, we used docking and molecular dynamics to see if pyrazolo[3,4-
b]pyridines showed high affinity for known binding regions. Docking results suggest that the hit
compounds bind the hydrophobic pocket of the Q site, while subsequent molecular dynamics
o
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experiments of the docked compounds (and co-crystallized ligand atovaquone) indicate they maintain a
stable binding conformation up to 50ns. Subsequent binding-energy determination studies show that all
hit compounds have more favorable binding energy than atovaquone by the MM/GBSA and MM/PBSA
methods. Moreover, our compounds are predicted to bind away from Y279, one of the main residues
responsible for resistance against atovaquone. Future work will involve studies to verify the mechanism
of action and the synthesis of new derivatives with the aim of further improving potency and solubility.
While a number of antimalarials in current use have very high cLogP (e.g. 10.20 and 6.35 for
lumefantrine and atovaquone respectively), the goal for future studies will be to drive the optimization
based on lipophilic ligand efficiency (LLE) to maximize the chance of obtaining good pharmacokinetic
properties while maintaining dual-action potency against the parasite.
1
1
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0
EXPERIMENTAL SECTION
1
13
General Methods. H and C Nuclear Magnetic Resonance (NMR) spectra were obtained on a
3
00 or 400MHz instrument using CDCl , DMSO-d or acetone-d as the solvent. Chemical shifts were
3
6
6
reported as δ values in parts per million (ppm) relative to the solvent. All reactions were performed
open to the atmosphere unless indicated otherwise. Analytical thin layer chromatography (TLC) was
performed on aluminum-backed 250μm layer silica plates visualized with either 254 or 365nm
wavelengths. Sonication was performed in an Ultrasonic Cleaner GB928. All solvents were used without
purification and no attempts were made to exclude atmospheric moisture. Glassware was dried for at
least 1h in a 90 °C oven prior to use. All reported yields are isolated yields. Frontrunner compounds (10,
1
7 & 27) were assayed to confirm ≥ 95% purity as determined using an HPLC system consisting of a
Shimadzu Prominence UFLC with LC-20ADXR pumps, SIL-20AXR autosampler, CTO-20AC column oven,
and SPD-M20A DAD detector. All HRMS samples were prepared at 1 mg/mL in MeOH, then diluted 1:20
in MeOH for testing with 2 μL injection volume and mass was detected with an ABSciex TripleTOF 5600+
with CDS (Calibrant Delivery System) and DuoSpray Ion Source.
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General Procedure for the Synthesis of α-Cyanoketones. 20mmol of the appropriate acyl
ketone was dissolved in 40mL of ethyl acetate to make a 0.5M solution. 2.6 equivalents of CuBr was
2
added and the mixture refluxed with a condenser for 3-5h until the starting ketone was fully consumed
1
0
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as indicated by TLC or H NMR. Once the reaction was complete, the mixture was cooled, and then the
ethyl acetate was removed under reduced pressure. Hexanes were added to the crude solid and the
mixture was sonicated for 5 min. The hexanes were decanted and a fresh volume was added to the
remaining solids, again sonicating for 5 min. This process was repeated once more, for a total of 3
extractions. The combined hexane layers, which typically appeared as an amber or light yellow solution,
were evaporated under reduced pressure to yield the crude product which typically appeared as yellow
or orange oil. Alternatively, a Soxhlet extraction with hexanes overnight (16h) gives similar isolated
yields.
The crude bromoketone oil was next dissolved in a 5:1 mixture of ethanol/water to give a 0.4M
solution overall. The solution was cooled over ice, and then 3 equivalents of NaCN were added. The
reaction was stirred overnight (16h), allowing the ice to melt. The solution was then diluted with
enough water to roughly double the initial volume. This solution was filtered through a Celite pad to
remove suspended solids. This solution was then acidified by adding concentrated HCl to a stirring
solution. CAUTION! This will cause the evolution of HCN gas! Only perform in a well-ventilated fume
hood! The acidified solution was allowed to stir for 15 minutes. The solution was checked by pH paper
to ensure that it was acidic (pH ≤ 2). If not, additional HCl was added, taking the same precautions
noted above. Once the solution was acidic, it was transferred to a separatory funnel and extracted 3x
with DCM. The organic layers were combined, dried over magnesium sulfate, filtered, and then
1
evaporated under reduced pressure to obtain the final product. Purity was confirmed by H NMR. If
significant impurities are observed, the product can be recrystallized, most typically in isopropanol or
toluene.
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General Procedure for the Synthesis of Pyrazolo[3,4-b]pyridines. The following reaction is for
6
-(4-tert-butylphenyl)-4-(2-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile
(
27) on a 1 mmol scale and is representative. The reagents, 3-(napthalen-2-yl)-3-oxopropanenitrile(195
mg, 1 mmol), 2-hydroxybenzaldehyde (122 mg, 1 mmol), and 3-methyl-1-phenyl-1H-pyrazol-5-amine
173 mg, 1 mmol), were combined in a 50 mL round bottom flask with 5mL DMF (0.2 M overall). The
1
1
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0
1
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(
solution was placed in a 100 C oil bath and left to stir overnight (16h). The solution was then
evaporated using a rotary evaporator with an 80 C water bath to provide the crude as a viscous oil.
Once the flask was cooled to room temperature, deionized water (~25 mL) was added to the round
bottom and the solution was sonicated for 30m, or until the product was finely suspended in solution.
The suspended product was filtered through a Buchner funnel and washed with 2x deionized water. The
isolated crude solid can then be further purified either via trituration, flash chromatography or reverse-
phase HPLC. In the case of 27, the crude product was purified via trituration with toluene and dried to
provide the product as a fine white powder (240 mg, 52%), m.p. 219-221 °C, HRMS (ESI) m/z calculated
+
1
for C H N O [M+H] 459.2185, found 459.2180. H NMR (CDCl , 400MHz):  = 1.42 (s, 9H), 2.19 (s, 3H),
3
0
26
4
3
2
.23 (s, 1H), 7.06 (d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.33-7.39 (m, 2H), 7.49 (m, 1H), 7.52-7.61 (m,
4H), 8.02 (d, J = 8.7 Hz, 2H), 8.33 (d, J = 7.6 Hz, 2H). 13C NMR (CDCl , 101MHz):  = 13.6, 31.2, 34.9,
3
1
1
02.1, 114.1, 116.4, 117.7, 121.1, 121.3, 125.5, 126.2, 129.1, 129.4, 130.4, 131.6, 135.0, 138.9, 144.2,
49.1, 149.1, 150.4, 152.7, 153.5, 160.5.
6
-(4-Ethylphenyl)-4-(2-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbo
nitrile (1). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and salicyaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via flash chromatography on basic alumina using a
3
:1 to 1:1 eluent gradient of hexanes/ethyl acetate to yield the final product as an orange powder (106
+
mg, 25%), m.p. 267-269 °C, HRMS (ESI) m/z calculated for C H N O [M + H] 431.1872, found 431.1881.
2
8
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1
H NMR (CDCl , 400 MHz): δ = 1.33 (t, J = 7.6 Hz, 3H), 2.23 (s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 7.05 (d, J = 8.2
3
Hz, 1H), 7.18 (td, J = 7.5, 0.9 Hz, 1H), 7.32 - 7.41 (m, 4H), 7.47 (td, J = 7.8, 1.7 Hz, 1H), 7.54 (t, J = 8.0 Hz,
H), 7.98 (d, J = 8.3 Hz, 2H), 8.32 (d, J = 7.6 Hz, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.6, 15.2, 28.8, 29.7,
2
3
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9
0
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102.1, 114.2, 116.5, 117.7, 121.0, 121.2, 126.2, 128.1, 129.1, 129.6, 130.4, 131.6, 135.4, 139.0, 144.2,
46.6, 149.2, 150.4, 152.8, 160.7.
-(4-Ethylphenyl)-3-methyl-4-(2-nitrophenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitr
1
6
ile (6). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-nitrobenzaldehyde on a 0.5 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration with absolute ethanol to yield the
final product as a white powder (211 mg, 28%), m.p. 225-256 °C, HRMS (ESI) m/z calculated for
+
1
C H N O [M+H] 460.1773, found 460.1765. H NMR (CDCl , 400 MHz): δ = 1.33 (t, J = 7.6 Hz, 3H),
2
8
21
5
2
3
2.04 (s, 3H), 2.77 (q, J = 7.6 Hz, 2H), 7.37 (m, 3H), 7.55 (m, 3H), 7.84 (m, 1H), 7.92 (m, 1H), 7.99 (d, J = 8.3
Hz, 2H), 8.32 (m, 2H), 8.44 (dd, J = 8.3, 1.0 Hz, 1H). 13C NMR (CDCl , 101 MHz): δ = 13.6, 15.3, 28.8,
3
1
1
00.7, 112.9, 117.1, 121.3, 125.5, 126.5, 128.2, 129.2, 129.6, 129.8, 131.2, 131.4, 134.1, 135.0, 138.8,
43.0, 146.9, 147.5, 149.1, 150.3, 160.3.
4-(2-Cyanophenyl)-6-(4-ethylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonit
rile (7). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-cyanobenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration with methanol to yield the final
+
product as a tan powder (65 mg, 15%), m.p. 201-203 °C, HRMS (ESI) m/z calculated for C H N [M+H]
29 21
5
1
440.1875, found 440.1875. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.14 (s, 3H), 2.79 (q, J =
3
7
7
.6 Hz, 2H), 7.35 - 7.43 (m, 3H), 7.56 (t, J = 8.0 Hz, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.77 (m, 1H), 7.89 (m, 1H),
.98 - 8.01 (m, 3H), 8.32 (d, J = 7.7 Hz, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.9, 15.2, 28.8, 101.4,
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12.9, 113.3, 116.5, 116.9, 121.4, 126.6, 128.2, 129.1, 129.7, 130.0, 130.4, 133.0, 133.3, 135.0, 137.9,
38.7, 143.0, 147.0, 147.8, 150.3, 160.6.
6
-(4-Ethylphenyl)-4-(2-iodophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitri
le (8). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole and
-iodobenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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2
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0
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2
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration with absolute ethanol to yield the
final product as a yellow powder (325 mg, 60%), m.p. 205-207 °C, HRMS (ESI) m/z calculated for
+
1
C H IN [M+H] 541.0889, found 541.0891. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.11
2
8
21
4
3
(
s, 3H), 2.79 (q, J = 7.6 Hz, 2H), 7.49 (m, 8H), 8.06 (m, 3H), 8.38 (d, J = 7.7 Hz, 2H). 13C NMR (CDCl , 101
3
MHz): δ = 13.7, 15.3, 28.8, 96.9, 101.4, 113.4, 116.9, 121.1, 126.3, 128.1, 128.5, 129.1, 129.1, 129.6,
1
31.2, 135.2, 139.0, 139.5, 143.7, 146.8, 150.4, 153.7, 160.5.
4-(2-Bromophenyl)-6-(4-ethylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboni
trile (9). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-bromobenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration with absolute ethanol to yield the
final product as an off-white powder (211 mg, 43%), m.p. 208-209 °C, HRMS (ESI) m/z calculated for
+
1
C H BrN [M+H] 493.1028, found 493.1018. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.13
2
8
21
4
3
s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 7.48 (m, 8H), 7.84 (d, J = 8.1 Hz, 1H), 8.02 (m, 2H), 8.35 (m, 2H). 13C NMR
(
(CDCl , 101 MHz): δ = 13.4, 15.3, 28.8, 101.5, 113.6, 113.7, 117.0, 117.1, 121.2, 122.2, 126.3, 127.2,
3
1
27.8, 128.1, 129.1, 129.6, 130.1, 130.2, 131.3, 131.3, 132.8, 133.2, 133.3, 135.2, 135.4, 138.9, 143.8,
146.8, 149.5, 150.3, 150.9, 160.5.
4
-(2-Chlorophenyl)-6-(4-ethylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboni
trile (10). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-chlorobenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
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pyrazolo[3,4-b]pyridines. Crude product was purified via trituration with absolute ethanol to yield the
final product as an off-white solid (139 mg, 31%), m.p. 198-199 °C, HRMS (ESI) m/z calculated for
+
1
C H ClN [M+H] 449.1533, found 449.1535. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.14
28 21
4
3
(s, 3H), 2.79 (q, J = 7.6 Hz, 2H), 7.5 (m, 9H), 8.01 (d, J = 8.3 Hz, 2H), 8.35 (d, J = 8.4 Hz, 2H). 13C NMR
CDCl , 101 MHz): δ = 13.4, 15.3, 28.8, 101.6, 113.7, 117.1, 121.2, 126.3, 127.2, 128.1, 129.1, 129.6,
0
1
2
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0
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8
9
0
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9
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0
(
3
1
30.1, 130.2, 131.3, 132.8, 133.3, 135.2, 138.9, 143.8, 146.8, 149.5, 150.3, 160.5.
-(4-Ethylphenyl)-4-(2-fluorophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonit
6
rile (11). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-fluorobenzaldehyde on a 0.5 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration of methanol to yield the final
product as a light yellow solid (62 mg, 28%), m.p. 197-199 °C, HRMS (ESI) m/z calculated for C H FN
2
8
21
4
+
1
[M+H] 433.1828, found 433.1826. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.22 (s, 3H),
3
2
8
1
.79 (q, J = 7.6 Hz, 2H), 7.33-7.37 (m,2H), 7.40-7.44 (m, 3H), 7.53 (m, 3H), 7.63 (m, 1H), 8.00 (m, 2H),
.33 (m, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.7, 15.3, 28.8, 102.0, 113.9, 116.3 (d, J = 21 Hz), 117.3,
3
21.3, 121.9 (d, J = 16 Hz), 124.6 (d, J = 3 Hz), 126.3, 128.1, 129.1, 129.6, 130.9 (d, J = 2 Hz), 132.3 (d, J =
8 Hz), 135.2, 138.9, 143.7, 146.3, 146.8, 150.2, 158.0, 160.6 (d, J = 11 Hz).
6-(4-Ethylphenyl)-3-methyl-4-(2-methylphenyl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboni
trile (12). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole
and 2-methylbenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. Crude product was purified via trituration of methanol to yield the final
product as a light beige solid (183 mg, 43%), m.p. 203-204 °C, HRMS (ESI) m/z calculated for C H N
2
9
24
4
+
1
[
M+H] 429.2079, found 429.2081. H NMR (CDCl , 400 MHz): δ = 1.32 (t, J = 8.0 Hz, 3H), 2.04 (s, 3H),
3
2
.21 (s, 3H), 2.77 (q, J = 8.0 Hz, 2H), 7.31 (m, 2H), 7.39-7.49 (m, 4H), 7.51-7.56 (m, 3H), 8.00 (m, 2H), 8.34
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7
8
9
(
m, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.5, 15.3, 19.7, 28.8, 101.6, 113.8, 117.3, 121.1, 126.2, 126.6,
3
1
28.1, 128.4, 129.1, 129.6, 129.9, 130.6, 133.8, 135.3, 135.4, 139.0, 144.1, 146.7, 150.3, 152.7, 160.6.
-(4-Ethylphenyl)-4-(2-methanesulfonylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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7
8
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0
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3
4
5
6
7
8
9
0
1
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3
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7
8
9
0
-5-carbonitrile (13). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-
phenylpyrazole and 2-(methylsulfonyl)benzaldehyde on a 1 mmol scale according to the general
procedure for the synthesis of pyrazolo[3,4-b]pyridines. The crude product was purified via reverse
phase HPLC utilizing a 55%-95% acetonitrile gradient with 0.1% trifluoroacetic acid to isolate the product
as a light brown product (50 mg, 10%), m.p. 196-198 °C, HRMS (ESI) m/z calculated for C H N O S
2
9
24
4
2
+
1
[
M+H] 493.1698, found 493.1703. H NMR (CDCl , 400 MHz): δ = 1.32 (t, 3H, J = 7.6 Hz), 2.02 (s, 3H),
3
2
.77 (m, 2H, J = 7.7 Hz), 3.13 (s, 3H), 7.32 (t, 1H, J = 8.1 Hz), 7.40 (d, 2H, J = 8.1 Hz), 7.53 (m, 3H, J = 8.1
Hz), 7.85 (m, 2H), 7.97 (d, 2H, J = 8.2 Hz), 8.31 (t, 3H, J = 12 Hz). 13C NMR (CDCl , 101 MHz): δ = 14.0,
3
15.6, 45.3, 101.4, 115.0, 118.3, 121.6, 128.5, 129.4, 129.9, 130.6, 131.3, 131.4, 134.2, 135.3, 139.2,
1
39.6, 144.3, 147.2, 150.2, 150.2, 160.1.
-[5-Cyano-6-(4-ethylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl]benzoic acid
14). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole and
2
(
2-carboxybenzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of
pyrazolo[3,4-b]pyridines. The crude product was purified via trituration with methanol to yield the final
product as an off-white solid (110 mg, 39%), m.p. 198-200 °C, HRMS (ESI) m/z calculated for C H N O
2
9
22
4
2
+
1
[
M+H] 459.1821, found 459.1823. H NMR (CDCl , 400 MHz): δ = 1.32 (t, J = 7.6 Hz, 3H), 1.99 (s, 3H),
3
2
.76 (q, J = 7.6 Hz, 2H), 7.31-7.35 (m, 1H), 7.39 (m, 2H), 7.43 (m, 1H), 7.53 (t, J = 7.9 Hz, 2H), 7.69 (m, 1H),
7.79 (m, 1H), 7.97 (d, J = 8.2 Hz, 2H), 8.30-8.35 (m, 3H). 13C NMR (CDCl , 101 MHz): δ = 13.7, 15.3, 28.8,
3
1
1
01.1, 113.5, 117.5, 121.2, 128.1, 128.3, 129.1, 129.6, 130.0, 130.2, 133.3, 135.3, 136.2, 139.0, 143.6,
46.6, 150.2, 152.7, 160.1, 169.2.
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2
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-(4-Ethylphenyl)-3-methyl-1-phenyl-4-[2-(trifluoromethyl)phenyl]-1H-pyrazolo[3,4-b]pyridine
-
5-carbonitrile (15). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-
phenylpyrazole and 2-(triflurormethyl)benzaldehyde on a 0.5 mmol scale according to the general
procedure for the synthesis of pyrazolo[3,4-b]pyridines. The crude product was purified via trituration
with methanol to yield the final product as an off-white (128 mg, 53%), m.p. 193-195 °C, HRMS (ESI) m/z
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
calculated for C H F N [M+H] 483.1796, found 483.1797. H NMR (CDCl , 400 MHz): δ = 1.34 (t, J =
2
9
21
3
4
3
7
.6 Hz, 3H), 2.01 (s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 7.35 (m, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 7.0 Hz,
1H), 7.55 (t, J = 8.0 Hz, 2H), 7.78 (m, 2H), 7.99 (m, 3H), 8.35 (d, J = 7.7 Hz, 2H). 13C NMR (CDCl , 101
3
MHz): δ = 13.3, 15.3, 28.8, 102.1, 114.0, 117.0, 121.1, 123.5 (q, J = 257 Hz), 126.3, 126.8 (q, J = 5.0 Hz),
1
1
28.2, 128.6, 129.0, 129.1, 129.6, 130.1 (d, J = 11 Hz), 132.1, 132.7, 135.0, 138.9, 143.7, 146.8, 149.3,
49.9, 160.0.
6-(4-Ethylphenyl)-4-(2-methoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbo
nitrile (16). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-
phenylpyrazole and 2-methoxybenzaldehyde on a 1 mmol scale according to the general procedure for
the synthesis of pyrazolo[3,4-b]pyridines. The crude product was purified via trituration with absolute
ethanol to yield the final product as an off-white powder (164 mg, 35%), m.p. 196-198 °C, HRMS (ESI)
+
1
m/z calculated for C H N O [M+H] 455.2028, found 445.2038. H NMR (CDCl , 400 MHz): δ = 1.33 (t, J
2
9
24
4
3
=
7
8
7.6 Hz, 3H), 2.16(s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 3.86 (s, 3H), 7.18 (m, 2H), 7.36 (m, 4H), 7.57 (m, 3H),
.99 (d, J = 8.2 Hz, 2H),
.34 (d, J = 7.8 Hz, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.8, 15.3, 28.8, 55.7, 102.2, 111.4, 114.3, 120.8,
3
121.1, 123.0, 126.1, 128.0, 129.1, 129.6, 130.4, 131.6, 135.5, 139.1, 144.2, 146.5, 149.9, 150.4, 156.5,
60.5.
1
6
-(4-Ethylphenyl)-3-methyl-1-phenyl-4-[2-(trifluoromethoxy)phenyl]-1H-pyrazolo[3,4-b]pyridi
ne-5-carbonitrile (17). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-
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phenylpyrazole and 2-trifluoromethoxybenzaldehyde on a 1 mmol scale according to the general
procedure for the synthesis of pyrazolo[3,4-b]pyridines. The crude product was purified via trituration
with absolute ethanol to yield the final product as an off-white flakey solid (131 mg, 26%), m.p. 160-162
+
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
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9
0
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2
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0
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0
°C, HRMS (ESI) m/z calculated for C H F N O [M+H] 499.1745, found 499.1743. H NMR (CDCl , 400
2
9
21
3
4
3
MHz): δ = 1.34 (t, J = 7.6 Hz, 3H), 2.15 (s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 7.35 (m, 1H), 7.42 (d, J = 8.3 Hz,
2
1
H), 7.54 (m, 5H), 7.69 (m, 1H), 8 (m, 2H), 8.35 (d, J = 7.7 Hz, 2H). 13C NMR (CDCl , 101 MHz): δ = 13.5,
3
5.3, 28.8, 102.0, 113.8, 117.0, 119.0, 121.2, 121.2, 126.3, 127.1, 127.6, 128.1, 129.1, 129.6, 131.0,
131.8, 135.1, 138.9, 143.6, 146.3, 146.8, 147.0, 150.2, 160.4.
-(4-Ethylphenyl)-3-methyl-1,4-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (18).
6
Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-phenylpyrazole and
benzaldehyde on a 1 mmol scale according to the general procedure for the synthesis of pyrazolo[3,4-
b]pyridines. The crude product was purified via trituration with methanol to yield the final product as a
+
fine white powder (115 mg, 28%), m.p. 198-199 °C, HRMS (ESI) m/z calculated for C H N [M+H]
2
8
22
4
1
4
7
15.1922, found 415.1923. H NMR (CDCl , 400 MHz): δ = 1.33 (t, J = 7.6 Hz, 3H), 2.16 (s, 3H), 2.78 (q, J =
3
.6 Hz, 2H), 7.33 - 7.36 (m, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.52 - 7.56 (m, 4H), 7.61 - 7.63 (m, 3H), 7.98 (d, J
= 8.2 Hz, 2H), 8.33 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl , 101 MHz): δ = 14.6, 15.2, 28.8, 101.3, 113.6,
3
117.7, 121.2, 126.3, 128.1, 128.6, 128.9, 129.1, 129.6, 129.9, 134.1, 135.4, 139.0, 143.9, 146.7, 150.3,
1
52.7, 160.6.
-(4-Ethylphenyl)-4-(3-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbo
6
nitrile (19). Synthesized from 3-(4-ethylphenyl)-3-oxopropanenitrile, 5-amino-3-methyl-1-
phenylpyrazole and 3-hydroxybenzaldehyde on a 1 mmol scale according to the general procedure for
the synthesis of pyrazolo[3,4-b]pyridines. The crude product was purified via trituration with methanol
to yield the final product as an off-white solid (35 mg, 8%), m.p. 172-174 °C, HRMS (ESI) m/z calculated
+
1
for C H N O [M+H] 431.1872, found 431.1873. H NMR (CDCl , 400 MHz): δ = 1.33 (t, J = 7.6 Hz, 3H),
28 22
4
3
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