Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure–activity relationship, in vitro and in vivo evaluation as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2021.127923

The design, synthesis, and biological evaluation of a series novel N1?methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with ¹H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [³H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC₅₀ values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC_50s near ～1 μM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI₅₀ values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.

Full text of DOI:10.1016/j.bmcl.2021.127923

Bioorg. Med. Chem. Lett. 41 (2021) 127923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs):
Synthesis, structure–activity relationship, in vitro and in vivo evaluation as
antitumor agents
a
a
b
c
b
Farhana Islam , Tasdique M. Quadery , Ruoli Bai , Lerin R. Luckett-Chastain , Ernest Hamel ,
c
a,*
Michael A. Ihnat , Aleem Gangjee
a
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, United States
Molecular Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer
b
Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States
c
Department of Pharmaceutical Sciences, The University of Oklahoma College of Pharmacy, Oklahoma City, OK 73117, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo[4,3-d]pyrimidines as in-
Microtubule targeting agents
Structure–activity relationship
Pyrazolo[4,3-d]pyrimidine
Colchicine site
hibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds
involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, charac-
terized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]
colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9
Nuclear Overhauser Effect spectroscopy
and 11–13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42
respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC50s near ~1 M. Compounds 9,
2, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell
μ
M,
μ
1
resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the
standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values
(
≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was
significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumors
in a mouse xenograft model. Collectively, these studies support the further preclinical development of the
pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with
advantages over paclitaxel.
Cancer is the second-largest cause of mortality in the world and
accounted for an estimated 9.6 million deaths in 2018.1 An effective
strategy for cancer therapy is to target the mitotic pathways of rapidly
response to the environment.4 Molecules that bind to tubulin and
interrupt the dynamics of microtubules can be classified as microtubule
targeting agents (MTAs),4 which possess highly diverse chemical
2
5,6
proliferating tumor cells. Microtubules are hollow tube-like assemblies
structures, a few of which are shown in Figure 2.
consisting of heterodimers of two globular protein subunits,
α
- and
Chemotherapy with MTAs has led to improvement in the duration
7
β-tubulin (Fig. 1). Tubulin dimers are longitudinally arrayed to form
protofilaments, and the protofilaments in turn interact laterally to form
the microtubule3 (Fig. 1). Microtubules play essential roles in multiple
eukaryotic cellular processes, such as cell growth and division, motility,
intracellular trafficking, and the ability to adapt cellular shape in
and quality of life for many patients with cancer. Despite their success,
there are several shortcomings associated with MTAs. Treatment with
MTAs can result in drug resistance that often is a major obstacle to
treatment success.7 The clinical utility of the taxanes and the vinca al-
kaloids is severely limited by two major mechanisms of drug resistance:
Abbreviations: CS, colchicine site; CA-1, combretastatin A-1; CA-4, combretastatin A-4; CA-4P, combretastatin A-4 phosphate; DMSO, dimethyl sulfoxide; DIPEA,
N,N-Diisopropylethylamine; (MTAs, microtubule targeting agents; NCI, National Cancer Institute; NOESY, Nuclear Overhauser Effect Spectroscopy; Pgp, P-glyco-
protein; (PDB, Protein Data Bank; SRB, sulforhodamine B; TFA, trifluoracetic acid.
*
Corresponding author at: Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA
1
5282, United States.
E-mail address: gangjee@duq.edu (A. Gangjee).
https://doi.org/10.1016/j.bmcl.2021.127923
Received 4 December 2020; Received in revised form 25 February 2021; Accepted 25 February 2021
Available online 9 March 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Fig. 1. Formation of microtubules: polymerization of dimers of
α
- and β-tubulin, formation of protofilaments and finally cyclization into microtubules. (Modified
2
6
from Kaul et al. ).
Fig. 2. Representative microtubule targeting agents.
the expression of the drug efflux pump P-glycoprotein (Pgp) and over-
success.1
7–19
8
,9
expression of the βIII-isotype of tubulin. Pgp expression is very com-
mon, particularly in patients who have received prior chemotherapy.10
The use of Pgp inhibitors to overcome this resistance has been explored
but is unsuccessful due to side effects, which occur with high doses.11
The overexpression of βIII-tubulin is involved in clinical resistance to
Many reports have shown that CS agents frequently do not display
20
either Pgp or βIII-isotype dependent resistance. Thus, development of
MTAs targeting the CS can circumvent the resistance limitations asso-
ciated with taxanes and vinca alkaloids, and their use should improve
clinical outcomes.2
1,22
Colchicine itself is an approved drug for gout but
1
2,13
14
taxanes and vinca alkaloids in nonsmall cell lung,
breast,
is not employed as an anticancer agent due to its toxic side effects at
higher doses. These include neutropenia, gastrointestinal upset, bone
marrow damage, and anemia.23 Other colchicine binding inhibitors
have demonstrated promise and some are currently in clinical trials as
ovarian,15 and gastric cancers. In addition to the resistance issues, the
complex natural product MTAs have neurotoxicity, low oral bioavail-
ability and narrow therapeutic indexes as major impediments to clinical
16
Table 1
Microtubule depolymerization activity, antiproliferative effects, inhibition of colchicine binding and tubulin assembly.
The increase of activity of lead compounds with a 5‑methyl group
No
Cellular microtubule
depolymerization
EC50 (nM)
Antiproliferative
effects
Inhibition of colchicine binding
Inhibition of tubulin assembly IC50 (µM ± SD)
5
µM inhibitor
0.5 µM inhibitor% inhibition ± SD
MDA-MB-435 IC50
%
inhibition ± SD
(
nM) ± SD
1
>10000
ND
0
ND
ND
2
1200
7.4
–
96 ± 5
60 ± 1
99 ± 2
ND
ND
10 ± 0.6
ND
3
4.3 ± 0.3
79 ± 0.8
ND
4
–
21 ± 1
5
–
–
92 ± 0.2
43 ± 3
72 ± 2
99 ± 0.8
73 ± 3
ND
0.48 ± 0.008
3.3 ± 0.3
0.91 ± 0.03
0.54 ± 0.06
6
–
–
7
–
–
ND
CA-4
9.8
4.4 ± 0.5
84 ± 3
2

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Fig. 3. Lead compounds.
Fig. 4. Compounds 9–17 and the rationale for drug design.
anticancer candidates.24
hydrochloride), BNC105P (sodium 6-methoxy-2-methyl-3-(3,4,5-trime-
thoxybenzoyl)benzofuran-7-yl phosphate), ABT-751 (N-[2-(4-hydrox-
yanilino)pyridin-3-yl]-4-methoxybenzenesulfonamide), CYT-997 (1-
ethyl-3-[2-methoxy-4-[5-methyl-4-[[(1S)-1-pyridin-3-ylbutyl]amino]
pyrimidin-2-yl]phenyl]urea), ZD6126 (phosphoric acid mono-(5-acety-
lamino-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-3-
yl) ester), plinabulin (NPI-2358); ((3Z,6Z)-3-benzylidene-6-[(5-tert-
One of the mostpotent anti-mitotic CSMTAs that gained the attention
of medicinal chemists and cancer biologists is combretastatin A-4 (CA-4,
Fig. 2), a trimethoxyphenyl (TMP) containing stilbenoid.21 CS inhibitors
have been extensively studied, and several have entered clinical trials,
including 2-methoxyestradiol, combretastatin A-4 phosphate (CA-4P)
(
fosbretabulin), the combretastain CA-1P prodrug OXi4503 (3-methoxy-
-phosphonooxy-6-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl]
2
butyl-1H-imidazol-4-yl)methylidene]piperazine-2,5-dione),
MN-029
dihydrogen phosphate), AVE8062 ((2S)-2-Amino-3-hydroxy-N-[2-
methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl]propena-
mide), CKD-516 ((S)-N-(4-(3-(1H-1,2,4-triazol-1-yl)-4-(3,4,5-trimethox-
ybenzoyl)phenyl)thiazol-2-yl)-2-amino-3-methylbutanamide
(methyl N-[6-[4-[[(2S)-2-aminopropanoyl]amino]phenyl]sulfanyl-1H-
benzimidazol-2-yl]carbamate).1
3,14,25
Currently there are no CS agents
approved by the FDA for cancer, and this demonstrates the need to
7
develop additional CS agents for potential clinical use because
3

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Fig. 5. A) Docked pose of lead 3 (pink) with CA-4 (cyan) in the CS. B) Docked pose of 9 (tan) with CA-4 (cyan) in the CS. (Maestro 2020-2. Docked scores of 3 and 9
are ꢀ 9.76 and ꢀ 10.71 kcal/mol, respectively).
overcoming Pgp and βIII-tubulin mediated resistance is required to
improve the overall survival rates observed with MTAs currently in
clinical use.
step of our efforts, we utilized scaffold hopping on the pyrrolo[3,2-d]
pyrimidines as an approach to design novel pyrazolo[4,3-d]pyrimidines.
Scaffold hopping is a drug design strategy to identify structurally diverse
We previously reported27,28 compounds 2–7 (Table 1 and Fig. 3) as
potent CS antiproliferative agents. In the structure–activity relationship
compounds that are similar in activity/property space. An additional
29
aspect incorporated into the pyrazolo[4,3-d]pyrimidine structure was a
2‑chloro substituent. It was observed that a 2‑chloro substituted purine
compound 8 displayed IC50 values of 39.6 ± 2.9, 41.2 ± 3.2 and
58.9 ± 3.1 nM in three human malignant melanoma cell lines A375,
M14 and RPMI7951, respectively.30 Thus, it was of interest to explore
the antitumor activity of these compounds with the novel N1-methyl-
pyrazolo[4,3-d]pyrimidine scaffold with a chlorine substitution at the 5-
position as MTAs. We used various additional structural modifications
for the design of compounds 9–17 (Fig. 4) that include d i) rigidification
or conformational restrictions, ii) homologation and branching on the
alkyl chain and iii) variation in electronic effects on the aromatic ring.
Our optimization effort started by replacing the pyrrole ring in 3
(
SAR) studies of these pyrrolo[3,2-d]pyrimidine based antitubulin
agents, we demonstrated the importance of the 5‑methyl moiety on
compounds 3, 5 and 7 for significant improvement of biological activ-
ities over the 5‑desmethyl compounds 2, 4 and 6. Compound 3 with a
5
‑methyl is 160- and 22-fold more potent in cellular microtubule
depolymerization activity and antiproliferative effects, respectively than
the corresponding 5‑desmethyl 2. Moreover, compounds 5 and 7 are 43-
and 4-fold more potent in inhibiting tubulin assembly respectively, than
compounds 4 and 6. On the basis of our previous studies it was of in-
terest to design a novel series of N1-methylated pyrazolo[4,3-d]pyrim-
idines (Fig. 4) as potential MTAs and as anticancer agents. As the first
Fig. 6. Treatment with compound 9 decreased primary tumor growth in MCF-7 TUBB3 (βIII-tubulin overexpressing) breast cancer cell model. * = P < 0.05; ***
7
P < 0.001 (0.0007 for control vs 9, 0.0003 for PTX vs 9. A) 10 cells/100 uL Matrigel as implanted into fat pad #4 of 8 wk old athymic female mice. The mice were
treated with compound 9 at the maximally tolerated dose (MTD) of 30 mg/kg two times weekly or with paclitaxel at its MTD of 10 mg/kg/week and tumor volumes
were determined. Statistical analysis was performed with two-way ANOVA repeated measures post test. Compound 9 was significantly (P < 0.0001) better than
paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumor volume. B) Animal weights were graphed as percent weight change at day 41 over the
starting weight. Statistical analysis was performed with one-way ANOVA. Only the control and paclitaxel mice lost weight at the end of the study. Sample sizes are 7
for control (an animal was lost toward the end of the experiment) and 8 for PTX and 9.
4

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
3 3
Scheme 1. a) Di-tert-butyl dicarbonate, DCM, rt, 4 h, 72%; b) CH I, DMF, rt, 12 h, 55%; c) CF COOH, THF, rt, 6 h, 69%.
4
Scheme 2. i) Paraformaldehyde, DCM, 12 h; ii) NaBH , DCM, rt, 6-12 h, 56-72% over two steps.
′
with a pyrazolo ring and a 5‑chloro group to afford compound 9. Using
N7-position as a replacement of the monocyclic 4 -methoxyphenyl
group of 9. In compounds 9, 11 and 12, bond rotation restrictions were
anticipated to reduce the energy to adopt the bound conformation, thus
allowing improved inhibitory activity.
3
1
molecular modeling (Maestro 2020–2), we docked several conforma-
tionally restricted analogs in the CS of tubulin. As shown in Fig. 4, the
bioactive conformations of 9 and 10 are determined by three rotatable
′
′
single bonds: 4-position C
–
N bond (bond a), 1 -position C
–
–
N bond
Next, we focused on the homologation and branching of the 4 -OMe
′
32
′
′
(
bond b) and 4 -position C
O bond (bond c). Literature findings
(9) to 4 -OEt (13) and 4 -O-iPr (14) to allow for better penetration into
the hydrophobic pocket of the CS. We also explored the impact of
electron density and hydrogen bonding ability in compounds 15–17.
The distance and the nature of heteroatom substitution affects hydrogen
1
suggest that conformational preference via molecular modeling and H
NMR studies indicate that a methyl group on the aniline nitrogen in 1
and 2 restricts the free rotation of both bond a and bond b (Fig. 3) and
thus restricts the conformation of the anilino ring. Conformational
constraint of a small-molecule inhibitor can enhance the binding affinity
to its intended target protein by reducing conformational entropic costs
bond (HB) strength.35 Thus, it was of interest to isosterically replace the
′
oxygen atom of the 4 -OCH
3
of 9 with a sulfur and synthesize 15.
Compounds 16 and 17 were designed and synthesized to probe the
electron withdrawing effect of fluorine on the inhibition of colchicine
binding and microtubule assembly and on anticancer activity.
upon binding and can also improve the binding selectivity by reducing
accessible, low-energy conformational space.3
3,34
We, therefore,
modeled the conformationally restricted analogs 9, 11, and 12. In
compound 9, the restriction of bond a was accomplished by incorpo-
rating a N7‑methyl group in 10. Compound 11, with the 1,2,3,4-tetrahy-
droquinoline at the N7-position was designed from 9 by further
restricting bond b. Compound 12 restricts both bonds a and b by
incorporating a fused bicyclic 6ʹ-methoxynaphthyl-1ʹ-amino ring at the
Compounds 1–17 were docked in the X-ray crystal structure of the CS
(PDB: 6BS2, 2.65 Å)30 using Maestro, Schr o¨ dinger 2020–2. Multiple
low energy conformations were obtained from docking. Fig. 5 shows the
docked conformation of lead 3 (pink) and compound 9 (tan) superposed
with CA-4 (cyan). The pyrazolo[4,3-d]pyrimidine scaffold of 9–17 form
31
hydrophobic interactions at the
αβ tubulin interface with Alaβ314,
◦
Scheme 3. CH
3
I, K
2
CO
3
, DMF, rt, 12 h (29a: 35%, 29b: 55%) ; b) 10% Pd/C, H
2
, MeOH, 40 psi, 1 h, 72%; c) urea, 180 C, 2 h; d) POCl
3
, pyridine, toluene, 4 h, reflux,
4
2%; e) anilines, acetonitrile, reflux, 4–12 h, 35–49%.
5

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Table 2
Table 3
Inhibition of colchicine binding and tubulin assembly.
Activities of compounds 9, 12, 13 and 16 in a βIII-tubulin overexpressing cell
line.
Compound
No
Inhibition of colchicine binding
Inhibition of tubulin
assembly IC50
Agent
MCF-7
WT
MCF-7 TUBB3
βIII-tubulin
EC50 (nM)
Resistance Ratio
5
µM inhibitor
0.5 µM inhibitor%
inhibition ± SD
(
µM ± SD)
MCF-7 TUBB3/WT
%
EC50 (nM)
inhibition ± SD
9
2.0 ± 0.3
35.2 ± 4.7
7.7 ± 0.7
1.7 ± 0.2
194.2
3.3 ± 0.4
16.8 ± 1.5
9.7 ± 1.0
2.6 ± 0.4
2065.8
1.1
0.5
2
3
4
5
6
7
9
60 ± 1
ND
10 ± 0.6
1
1
1
2
3
6
99 ± 2
79 ± 0.8
ND
ND
1.3
ND
21 ± 1
1.6
92 ± 0.2
43 ± 3
73 ± 3
ND
0.48 ± 0.008
3.3 ± 0.3
0.91 ± 0.03
0.45 ± 0.09
>20
Paclitaxel
10.6
72 ± 2.0
94 ± 3
ND
68 ± 3
′
′
replacement of 4 -OMe with 4 -SMe generated compound 15, which was
equipotent to 9 in the inhibition of colchicine binding assay. Compounds
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
7.1 ± 3
94 ± 0.4
91 ± 1
–
68 ± 2
66 ± 0.7
88 ± 0.007
–
0.42 ± 0.07
0.49 ± 0.01
0.42 ± 0.07
1.1 ± 0.08
0.82 ± 0.02
0.74 ± 0.04
2.9 ± 0.2
0.54 ± 0.06
1
1 and 12, the conformationally restricted analogs of 9, in the colchicine
99 ± 0.4
85 ± 1
binding assay (Table 2), displayed comparable potency to 9 (94 and 91%
at 5
μ
M and 68 and 66% at 0.5
μ
M, respectively). Next, we assessed the
93 ± 0.5
97 ± 0.3
64 ± 5
64 ± 2
77 ± 1
–
84 ± 3
′
effects of homologation and branching at the 4 -O-position of 9. Ho-
′
′
mologation with a single methylene at the 4 -position of 9 (4 -OMe)
CA-4
99 ± 0.8
′
afforded 13 (4 -OEt), which was more potent (99% at 5
μ
M and 88% at
0
.5
μ
M) than both 9 and CA-4 in the colchicine inhibition assay.
Branching with an isopropyl moiety afforded compound 14, which was
Alaβ352 and Ileuβ316 similar to the lead pyrrolo[3,2-d]pyrimidine
scaffold. The pyrazole ring is stacked amid the Metβ257 and Cysβ239.
slightly less potent than 9, with 85% inhibition at 5
μ
M. Introduction of a
′
fluorine atom at the 3 -position of the phenyl ring of 9 yielded com-
The N7-CH
3
moiety of 9 and 11–17 forms hydrophobic interactions with
′
pound 16, which had 97 and 77% inhibition at 5 and 0.5 M, respec-
μ
Alaβ314 and Leuβ253. The 4 -oxygen of compounds 9–14, and 16–17
and the 4 -sulfur of compound 15 are oriented towards the pocket
′
tively, in the inhibition of colchicine binding assay. Compounds 11–16
3
at 5 µM, inhibited the binding of [ H]colchicine by 85–99%, whereas
formed by Thr
α179, Lysβ350, Asnβ256 and Asnβ348. The 5-Cl of 9–17
3
compound 17 showed only a 64% inhibition at 5
μ
M of [ H]colchicine
makes hydrophobic interactions with Leuβ253 and Leuβ240 (only 9 is
shown in Fig. 5B). The N4 of the pyrazolo[4,3-d]pyrimidine ring of all
the compounds makes water mediated hydrogen bond interactions with
binding. Compound 10 was completely inactive in inhibiting colchicine
binding. The lower activity of 17 could be attributed to the strong
′
electron withdrawing effect of the CF
3
moiety on the oxygen of the 4 -
the backbone of Cysβ239 similar to the original X-ray crystallized
ligand.30 The docked score of compound 9 is ꢀ 10.71 kcal/mol and is
position. This oxygen (or sulfur in 16) does interact with the pocket
formed by Thr
179, Lysβ350, Asnβ256 and Asnβ348 at the colchicine
site, and the CF reduces this interaction. Compound 10, lacking the
α
better than the lead compound 3 (ꢀ 9.76 kcal/mol). The docked scores
for 10 is ꢀ 9.32, and for 11–17 are in a range of ꢀ 9.95 to ꢀ 10.65 kcal/
mol. The N7‑desmethyl compound 10 has the lowest docked score of the
designed analogs and was predicted to be the least active.Fig. 6
Boc-protection of 18 afforded 19 in 72% yield. Methylation of 19 to
3
N7‑methyl moiety, did not inhibit tubulin assembly, nor did it have a
significant effect on colchicine binding, emphasizing the critical role of
this methyl moiety at the N7-position in retaining the bioactive
conformation for anti-tubulin activity (described below). The lack of
activity of 10 corroborates the lowest docked score of 10 in the CS ob-
tained from our molecular modeling studies.
3
6
2
0 and subsequent deprotection provided the aniline intermediate 21
Scheme 1). Treatment of 22a-27a with paraformaldehyde, followed by
reduction with NaBH gave the anilino intermediates 22b-27b (Scheme
). In Scheme 3, pyrazole 28 was alkylated with methyl iodide in the
presence of base K CO in DMF, to provide after separation two
(
4
All of the compounds (except 10) were highly active in the tubulin
assembly assay with low to sub micromolar inhibitory IC50 values
2
2
3
(
0.42–1.1
0.91 M, respectively) and CA-4 (0.54
1–13 strongly inhibited the polymerization of tubulin, with IC50 values
0.45, 0.42, 0.49 and 0.42 M, respectively. Compound 10 had no effect
on tubulin polymerization. Compounds 14–16 inhibited the polymeri-
zation of tubulin with an IC50s near ~1 M, 2-fold less potent than CA-4.
μ
M), comparable with lead compounds 5 and 7 (0.48 and
regioisomers, 29a and 29b, in 35 and 55% yields, respectively. In-
1
μ
μM) (Table 2). Compounds 9 and
termediates 29a and 29b were characterized by H NMR and NOESY
1
(
Nuclear Overhauser Enhancement Exchange Spectroscopy). Varying
3
7,38
μ
the base has been used in the literature to alkylate the N1 position.
Our use of K
2 3
CO is the first reported to yield 29a and 29b in 35 and
μ
5
5% yields, respectively. The separation method is discussed in the
1
The potent MTAs 9, 12, 13 and 16 were selected for evaluation of
their inhibitory activities towards the growth of MCF-7 wild-type (WT)
human breast cancer cells and and a MCF-7 cell line overexpressing βIII-
tubulin (data shown in Table 3). Comparing the EC50 values in these cell
lines, compounds 9 and 16 were the most potent of the series (EC50s 2.0
and 3.3 nM for 9 and 1.7 and 2.6 nM for 16 in MCF-7 WT and MCF7-βIII-
tubulin overexpressing cell lines, respectively). Compounds 9 and 16
were 100- and ~600-fold, respectively more active than paclitaxel in the
MCF-7 WT and MCF-7-βIII-tubulin cell lines. On the other hand, com-
pounds 12 and 13 were 5- and 25-fold more potent, respectively, in the
MCF-7 WT cells; and 120- and 220-fold more potent, respectively, in the
MCF-7-βIII-tubulin cell line compared to paclitaxel. Comparison of the
EC50 values in the parental MCF-7 WT and genetically manipulated
MCF-7 βIII cell line allows for the calculation of a relative resistance
value, designated as Rr. This value is calculated by dividing the EC50
value obtained in the βIII-overexpressing MCF-7 cells by the EC50 ob-
tained in the parental MCF-7 WT cells. The isoform βIII is an important
determinant in cellular resistance towards paclitaxel, which is a known
Experimental Section. The H NMRs of 29a and 29b agree with those in
the literature.39 Reduction of the nitro group in 29a, yielded 30, which
was cyclized to 31 with urea. Chlorination of 31 with POCl
3
and pyri-
dine in toluene provided 32. Nucleophilic aromatic substitution of 32
′
′
using anilines 4 -methoxyaniline, 4 -methoxy-N-methylaniline, 21, 22b-
2
7b afforded final compounds 9–17 in 68–84% yields.
Compounds 9–17 and the reference compound CA-4 were evaluated
for inhibitory effects on tubulin polymerization and on the binding of
3
[
H]colchicine to tubulin as an indication of whether their anti-
proliferative effects are the result of an interaction with tubulin
Table 2). CA-4 is a drug candidate in clinical trials,40,41 and it is a highly
(
4
2
potent, competitive inhibitor of the binding of colchicine to tubulin.
3
Except for 10, all the compounds at, 5 µM, inhibited [ H]colchicine
binding to the protein, and the extent of inhibition was similar to that
obtained with CA-4. With equal or more than 94% and 68% inhibition at
5
and 0.5
μ
M, respectively, compounds 9, 11, 13 and 16 showed similar
potency as the lead compounds and CA-4 (Tables 1 and 2). Isosteric
6

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Table 4
breast cancer.
Compound activity in a Pgp overexpressing cell line.
N-Alkylation of the pyrazole scaffold 28 afforded two regioisomers,
29a and 29b. Methylation of unsymmetrically substituted pyrazole de-
a
Compound No
Compound activity in a Pgp overexpressing cell line
Rr
3
9
rivatives usually affords mixtures of both possible alkylated products.
The regiochemistry of both alkylated intermediates 29a and 29b was
Parental OVCAR-8
Pgp overexpressing NCI/ADR-RES
IC50 (nM) ± SD
IC50 (nM) ± SD
1
39
1
identified by H NMR and NOESY spectroscopy (Figs. 7 and 8). In H
9
9.0 ± 0.7
>5000
5.0 ± 0
0.55
–
0.47
0.78
2.0
NMR (only δ values are included, spectra not shown). For the N1-CH
3
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
>5000
protons the chemical shift for 29a and 29b occurred as singlets at 4.02
17 ± 2
8.0 ± 0.7
11 ± 0.7
4.0 ± 1
14 ± 3
and 3.95 ppm, respectively. It is noteworthy that the presence of the
2.0 ± 1
49.0 ± 10
31 ± 2
adjacent C5-carboxylate to the N1-CH
tons of 29a to shift ~0.1 ppm deshielded compared to the N1-CH
9b. The initial studies involved NMR utilizing NOESY spectroscopy,
3
in 29a caused the N1-CH
3
pro-
44 ± 8.0
17 ± 3
0.89
0.54
0.77
0.83
150
1.0
3
of
2
9.0 ± 1
880 ± 40
5.3 ± 2
1.8 ± 0.4
7.0 ± 3
730 ± 100
800 ± 200
1.8 ± 0.4
which is one of the most direct ways to determine spatial proton-proton/
Paclitaxel
CA-4
proton-heteronucleus/heteronucleus-heteronucleus correlations within
39,43
a molecule in the range up to 5 Å or less.
This is also an excellent
method to determine solution conformation. Intermediate 29a was
identified as the methyl 1-methyl-4-nitro-1H-pyrazole-5-carboxylate
regioisomer, as no strong NOE correlation was observed between the H3
Pgp substrate.11,16 The Rr value of paclitaxel was 10.6 (resistance ratio:
βIII-tubulin/WT). Compound 12 had an Rr of 0.5, indicating that it is
able to overcome drug resistance mediated by βIII. Compounds 9, 13,
and 16 also had Rr values ≤1.6, suggesting that they are all poor sub-
strates for βIII-tubulin.
The ability of 9–17 to circumvent Pgp-mediated drug resistance was
evaluated using an ovarian cancer cell line pair (Table 4). The Rr value is
calculated by dividing the IC50 value obtained in the Pgp overexpressing
NCI/ADR-RES cells by the IC50 obtained in the parental OVCAR-8 cells.
The Rr value for paclitaxel was 150 whereas most of the synthesized
compounds had Rr values less than 1. The <1 Rr values indicated that
these compounds were poor substrates for Pgp and hence have minimal
Pgp-mediated transport and consequently should have advantages over
MTAs that are good Pgp substrates.
3
proton and the N1-CH protons (Fig. 7). Intermediate 29b was deter-
mined to be the methyl 1-methyl-4-nitro-1H-pyrazole-3-carboxylate
regioisomer, as a characteristic NOE correlation was observed between
the H5 proton and the N1-CH
and N1-CH cross peak (δ 8.95, 3.95) was dominated by scalar coupling
effects, with evidence of only a NOE proton contributing to it in 29b. For
3
protons (Fig. 8). The observed H5 proton
3
2
9a, the H3 proton occurs at δ 8.36, and, for 29b, the H5 proton is at δ
8
.95. The probable deshielding effects of the H5 proton in 29b is due to
1
the direct electron withdrawing effect of C-5. H NMR and crystal
structure from a previous report, along with the 2D NMR from our
studies, confirm the structure of regioisomers 29a and 29b as
designated.
1
The H NMR spectra of the N7-H analog 10 (Fig. 9) and the N7-CH
3
To determine its antitumor spectrum, compound 9 was tested in the
National Cancer Institute’s panel of 60 human tumor cell lines (NCI-60).
As shown in Table 5, compound 9 exhibited excellent potency with low
GI50 values ≤10 nM against most tumor cell lines, including many
multidrug resistant phenotypes, in the standard NCI testing protocol.
The GI50 values of 9 was single digit nanomolar in 19 tumor cell lines.
Compound 9 was selected for a tubulin III overexpressing, anti-
microtubule drug resistant in vivo xenograft mouse study on the basis of
its nanomolar potency in vitro in the NCI cancer cell line panel and its
potent activities in inhibiting microtubule polymerization and colchi-
cine binding assay. Compound 9 significantly reduced primary tumor
growth vs paclitaxel in the tubulin III overexpressing MCF-7 TUBB3
orthotopic xenograft, with no significant weight loss in the study as
opposed to paclitaxel, which resulted in significant weight loss. Thus
compound 9 is effective in vivo against antimicrotubule drug resistant
analog 9 (Fig. 10) in DMSO‑d6 afforded additional information related
to conformational restriction in 9 as compared to 10. For compound 9,
the “N1-CH ” protons appeared at δ 3.04, whereas for 10 they were
3
significantly deshielded at δ 4.35 ppm. This shielding of the “N1-CH ”
3
protons in 9 was attributed to a diamagnetic anisotropic effect in 9
arising from the proximity of the phenyl ring as shown in Fig. 11 (more
favored anti-conformation for 9 on the basis of the N7-CH
CH groups). The steric bulk of the N7-CH resulting in a steric clash of
the N7-CH and the N1-CH groups in 9 restricts the conformation and
positions the phenyl ring on top of the N1-CH moiety (Fig. 11),
resulting in the observed shielding effect, in the H NMR, on the N1-CH
group in 9 as compared to that in 10. This shielding effect of the phenyl
group on the N1-CH
3
and the N1-
3
3
3
3
3
1
3
3
group (δ ≈ 1.1) was also observed for the N7-CH
3
analogs 11–17. This provides an estimation of the solution conformation
of the active analog 9 compared to the inactive analog 10. In addition,
Table 5
Human cancer cell growth inhibitory activity GI50 (nM) of 9 in NCI 60 cell line panel.
Panel/Cell line
GI50 (nM)
Panel/Cell line
GI50 (nM)
Panel/Cell line
GI50 (nM)
Panel/Cell line
GI50 (nM)
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
Melanoma
LOX IMVI
MALME-3M
M14
Renal Cancer
786-0
9.96
11.23
6.46
9.78
23.56
9.07
8.53
12.07
10.56
3.90
13.23
13.42
8.40
A498
ACHN
MOLT-4
RPMI-8226
SR
19.85
15.29
9.64
5.14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
CAKI-1
RXF 393
SN12C
TK-10
15.72
8.75
HT29
7.60
20.97
17.04
14.58
22.69
15.90
KM12
10.76
6.67
15.76
16.31
10.01
NSCLC
SW-620
CNS Cancer
SF-268
A549/ATCC
EKVX
12.67
15.47
15.41
16.03
9.49
UO-31
12.17
11.43
8.76
HOP-62
SF-295
Ovarian cancer
IGROVI
Breast Cancer
MCF7
HOP-92
SF-539
13.30
6.37
10.81
14.29
11.19
24.12
16.44
9.09
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
SNB-19
13.88
10.81
7.97
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
MDA-MB-231/ATCC
HS 578T
11.87
14.09
4.52
SNB-75
12.93
11.76
14.69
8.24
U251
BT-549
Prostate Cancer
PC-3
T-47D
14.43
10.73
8.89
MDA-MB-468
DU-145
13.93
7

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Fig. 7. NOESY NMR of Intermediate 29a.
8

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
Fig. 8. NOESY NMR of Intermediate 29b.
1
Fig. 9. H NMR of 10.
1
the docked conformation of 9 in Fig. 5B is similar to the H NMR solution
conformation of 9. This indicates a low energy barrier for 9 to adopt the
bound conformation and results in potent activity.
In this study, a series of nine novel N1-methyl-pyrazolo[4,3-d]py-
rimidines were designed and synthesized as anticancer agents that
function as CS binding agents and as inhibitors of tubulin
9

F. Islam et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127923
1
Fig. 10. H NMR of 9.
Fig. 11. Possible explanation of orientation of compound 9.
polymerization. Synthesis of the target compounds involved alkylation
of the pyrazolo scaffold to provide two regioisomers that were sepa-
Therapeutics Program for performing the in vitro evaluation in the NCI
60-cell line panel. This work was supported, in part, by a grant from the
National Institutes of Health, National Cancer Institute (CA142868 (to
A.G), Duquesne University Adrian Van Kaam Chair in Scholarly Excel-
lence (to A.G.); the CTRC Cancer Center Support Grant P30 CA054174
and the Flow Cytometry Shared Resource; and by an NSF equipment
grant for NMR instrumentation (NMR: CHE 0614785). This research was
supported in part by the Developmental Therapeutics Program in the
Division of Cancer Treatment and Diagnosis of the National Cancer
Institute, which includes federal funds under Contract No.
HHSN261200800001E. The content of this publication does not neces-
sarily reflect the views or policies of the Department of Health and
Human Services, nor does mention of trade names, commercial prod-
ucts, or organizations imply endorsement by the U.S. Government.
1
rated, characterized and structurally identified via H NMR and NOESY
spectroscopy. Compounds 9 and 11–13 strongly inhibited the poly-
merization of tubulin with IC50s near ~0.5
μ
M., whereas compounds
1
4–16 inhibited the polymerization of tubulin with IC50s near ~1 μ
M.
Compounds 9, 12, 13 and 16 circumvented βIII-tubulin mediated cancer
cell resistance and compounds 9–17 circumvented Pgp mediated drug
resistance. Compound 9 showed GI50 values ≤10 nM against many
tumor cell lines, including several multidrug resistant phenotypes, in the
standard NCI testing protocol. SAR studies indicated that the N7-CH
3
′
moiety of 9 was crucial for activity and that the 4 -OMeC
6
H
4
was the
best substitution for biological activity. In addition, the lower activity of
1 and 12 compared to 9 and 13 suggested that some flexibility of bond
1
‘
a’ and ‘b’ were necessary for potent activity in this series. Compound 9
was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7
TUBB3 (βIII-tubulin overexpressing) tumor growth in an in vivo study
and is poised for further preclinical development.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127923.
Declaration of Competing Interest
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