Design, synthesis, and bioevaluation of pyrazolo[1,5-a]pyrimidine derivatives as tubulin polymerization inhibitors targeting the colchicine binding site with potent anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2020.112519

A series of Pyrazolo[1,5-a]Pyrimidine analogs were designed and synthesized as novel tubulin inhibitors. Among them, compounds 1a and 1b showed the highest antiproliferative activity against a panel of cancer cell lines with average IC₅₀ values

Full text of DOI:10.1016/j.ejmech.2020.112519

Journal Pre-proof
Design, Synthesis, and Bioevaluation of Pyrazolo[1,5-a]Pyrimidine Derivatives as
Tubulin Polymerization Inhibitors Targeting the Colchicine Binding Site with Potent
Anticancer Activities
Gang Li, Yuxi Wang, Ling Li, Yichan Ren, Xin Deng, Jin Liu, Wei Wang, Meihua Luo,
Shuwen Liu, Jianjun Chen
PII:
S0223-5234(20)30491-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112519
EJMECH 112519
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 March 2020
Revised Date: 21 May 2020
Accepted Date: 29 May 2020
Please cite this article as: G. Li, Y. Wang, L. Li, Y. Ren, X. Deng, J. Liu, W. Wang, M. Luo, S. Liu,
J. Chen, Design, Synthesis, and Bioevaluation of Pyrazolo[1,5-a]Pyrimidine Derivatives as Tubulin
Polymerization Inhibitors Targeting the Colchicine Binding Site with Potent Anticancer Activities,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112519.
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Design, Synthesis, and Bioevaluation of Pyrazolo[1,5-a]Pyrimidine Derivatives as
Tubulin Polymerization Inhibitors Targeting the Colchicine Binding Site with Potent
Anticancer Activities
Gang Li1, Yuxi Wang2, Ling Li3, Yichan Ren3, Xin Deng3, Jin Liu3, Wei Wang3, Meihua
Luo1, Shuwen Liu3, Jianjun Chen3*
1Department of oncology, Shunde Hospital, Southern Medical University (The First
People’s Hospital of Shunde), Foshan, 528300, China
2State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University, and Collaborative Innovation, Center of Biotherapy, Chengdu, Sichuan
610041, China
3School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New
Drug Screening, Southern Medical University, Guangzhou 510515, China.
*Corresponding Author: Jianjun Chen. School of Pharmaceutical Sciences, Guangdong
Provincial Key Laboratory of New Drug Screening, Southern Medical University,
Guangzhou 510515, China. Email: jchen21@smu.edu.cn.
ABSTRACT
A series of Pyrazolo[1,5-a]Pyrimidine analogues were designed and synthesized as novel
tubulin inhibitors. Among them, compounds 1a and 1b showed the highest
antiproliferative activity against a panel of cancer cell lines with average IC50 values of
24.8 nM and 28 nM, respectively. We determined the crystal structures of 1a and 1b in

complex with tubulin and confirmed their direct binding to the colchicine site.
Compounds 1a and 1b also effectively inhibited tubulin polymerization in vitro, induced
cell cycle arrest in G2/M phase, and inhibited cancer cell migration. In addition,
compound 1b exhibited high metabolic stability in human liver microsomes. Finally, 1b
was highly effective in suppressing tumor growth in a B16-F10 mouse melanoma model
without apparent toxicity. In summary, these results suggest that 1b represents a
promising tubulin inhibitor worthy of further investigation.
Keywords: Tubulin inhibitor, Colchicine binding site, Pyrazolo[1,5-a]pyrimidine,
Antitumor
1. Introduction
Microtubules are composed of α-tubulin and β-tubulin, which are the key
components of cytoskeleton[1-3]. Due to the critical role of microtubules in mitosis,
maintenance of cellular shape, and intracellular transport, microtubules are regarded as an
excellent target for anticancer therapeutic agents such as microtubule targeting agents
(MTAs). MTAs (e.g. paclitaxel, vinblastine and colchicine) interfere with the dynamic
equilibrium of tubulin/microtubule through the well-known six binding sites[4] (taxane,
vinca, laulimalide, maytansine, pironetin and colchicine sites), which could lead to cell
cycle arrest and limit cell proliferation thus resulting in cell death[5-7]. MTAs can be
divided into two classes: microtubule stabilizers (e.g. taxanes) and microtubule
destabilizers (vinca alkaloids and colchicine). Current available MTAs such as paclitaxel,

vincristine, and vinblastine bind to the taxanes or vinca alkaloids sites in tubulin and have
enjoyed considerable clinical success, however, the further application of these MTAs is
often hampered by several drawbacks including low water solubility and the development
of multidrug resistance[8-10]. During the last 10 years, the search for MTAs that can
potentially overcome the above disadvantages has been intensified, and extensive
research suggest that colchicine binding site inhibitors (CBSIs) may provide better
clinical outcomes compare to other MTAs such as taxanes[11-13].
We have previously developed several series of CBSIs including SMART
(4-substituted methoxybenzoyl-aryl-thiazoles)[14], ABI (2-aryl-4-benzoyl-imidazoles)[15,
16] and SAI (substituted-2-aryl imidazoles) analogues[17], which showed potent in vitro
antiproliferative activities with IC50 in the nanomolar range and high in vivo antitumor
efficacy, more importantly, they could effectively overcome P-gp mediated MDR
(multi-drug resistance). Despite the favorable pharmacodynamic profiles, the ABI and
SMART analogs contain a metabolically labile carbonyl (ketone) group between the
B-ring and C-ring (Figure 1), which is prone to metabolic reductions by human liver
microsomes[18, 19]. In an effort to improve the metabolic stability, we designed a series
of nonketones, namely, the ring-fused pyrazolo[1,5-a]pyrimidine analogues, by
incorporating the carbonyl moiety into a six-membered pyrimidine ring, as the
ring-fusion strategy is a common approach frequently applied in medicinal chemistry to
improve the stability of a compound[20, 21]. We chose the pyrazolo[1,5-a]pyrimidine
scaffold as the bioisostere of the B-ring and ketone of the SMART and ABI analogs,

because the pyrazole and pyrimidine groups are widely used templates in many drugs[22,
23]. While for the A-ring, we explored the indole group, substituted phenyl and other
aromatic groups. The 3,4,5-trimethoxy phenyl (TMP) group was maintained as the C-ring
of the target compounds because the TMP moiety is an important pharmacophoric feature
for antitumor activity and has to be kept intact[24]. We herein describe the synthesis and
biological evaluation of the novel nonketone tubulin inhibitors that contain the
pyrazolo[1,5-a]pyrimidine moiety as potential anticancer agents.
Figure 1. Design of target compounds by ring-fusion strategy
2. RESULTS AND DISCUSSION
2.1. Chemistry
Compounds 1a-y and 1aa-af were synthesized according to the synthetic route
illustrated in Scheme 1. Firstly, 3,4,5-Trimethoxyacetophenone was condensed with N,
N-dimethylformamide dimethyl acetal in DMF to afford intermediate 2, followed by a
ring closure reaction with 3-bromo-1H-pyrazol-5-amine to give the key intermediate 3,
then compound 3 was coupled with appropriate aryl boronic acids to generate compounds
1a-y. The phenylsulfonyl group of compound 1y was removed to produce compound 1aa.
Introduction of a methyl and a hydroxymethyl group to the N-1 position on the indole
moiety of 1aa provided compounds 1ab and 1ac, respectively. The conversion of

compound 1q to 1ad was achieved by reducing the 4-ethoxycarbonyl moiety in the
presence of LiAlH4. Compound 1ae was prepared by methylating the indole N-1 position
of 1c. Compound 1af was obtained by reducing the nitro group of compound 1h with
Pd/C under H2 atmosphere.
Scheme 1. Synthesis of compounds 1a-y, 1aa-af, 4a-b and 7
Reagents and conditions: (a) N, N-dimethylformamide dimethyl acetal, DMF, 120 oC, 6h,
88.7%; (b) 3-bromo-1H-pyrazol-5-amine, AcOH, 80 oC, 8h, 93.3%; (c)
4-bromo-1H-pyrazol-5-amine, AcOH, 80 oC, 8h, 71.2%; (d) appropriate aryl boronic acid,
Pd(dppf)Cl2, Na2CO3 aq., DMF, 95 oC, 12h; (e) NaOH aq., EtOH, 50 oC, 8h; (f) NaH, dry
o
THF, CH3I, rt, 4h; (g) 37 % HCHO aq., 10 % NaOH, EtOH, 30 C, 4h;(h) LiAlH4, dry
THF, rt, 4h; (i) Pd/C, H2, CH3OH/THF, rt, overnight.

The second series of compounds 7 and 4a-b (A-ring as the 4-fluorophenyl group
instead of 3,4,5-trimethoxyphenyl moiety) were obtained by a similar method shown in
Scheme 1. Compound 11 (the positions of the indole group and 3,4,5-trimethoxy phenyl
group were switched) was synthesized following the synthetic route outlined in Scheme 2.
The 3-acetylindole was protected by the phenylsulfonyl group followed by a
condensation reaction with N, N-dimethylformamide dimethyl acetal to give intermediate
9. Compound 9 was cyclized with 3-bromo-1H-pyrazol-5-amine to provide the indazole
intermediate 10. Compound 10 was subject to a Suzuki coupling reaction with
3,4,5-trimethoxyphenylboronic acid followed by the deprotection of the phenylsulfonyl
group to produce compound 11.
Scheme 2. Synthesis of compound 11
Reagents and conditions: (a) NaH, dry THF, benzenesulfonyl chloride, 25 oC, 6h, 87.2%;
(b) N, N-dimethylformamide dimethyl acetal, DMF, 120 oC, 6h, 89.5%; (c)
3-bromo-1H-pyrazol-5-amine,
CH3COOH,
80
oC,
8h,
93.8
%;
(d)
3,4,5-trimethoxyphenylboronic acid, Pd(dppf)Cl2, Na2CO3 aq., DMF, 95 oC, 12 h, 76.8 %;
(e) NaOH aq., EtOH, 50 oC, 8h, 70.2 %.
2.2. Biological evaluation
2.2.1 In vitro Antiproliferative Activity

The antiproliferative activities of the newly synthesized compounds were evaluated
against five cancer cell lines (Hela, MCF-7, A549, HCT-116, B16-F10) using the CCK-8
assay with colchicine and paclitaxel as positive controls. In general, most of the
pyrazolo[1,5-a]pyrimidine-based compounds (e.g. 1a-1g, 1i-1l) showed high
antiproliferative activities with IC50 values in the nanomolar range (e.g. from 3 nM to 1
µM, Table 1). Compounds with electron-donating groups on the phenyl A-ring [e.g. 1b
(4-methylphenyl, average IC50 = 28 nM), 1d (4-vinyl, IC50 = 789 nM), 1e (3,4-dimethyl,
IC50 = 238 nM), 1f (4-N,N-dimethyl, IC50 = 855 nM), 1j (4-methoxy, IC50 = 536 nM),
1ad (4-methylol, IC50 = 914 nM) and 1af (3-amino-4-methoxy, IC50 = 157 nM)],
displayed higher activities than the ones with electron-withdrawing groups [e.g. 1h
(3-nitro-4-methoxy, IC50 > 10 µM), 1i (3-fluoro-4-methoxy, IC50 = 1.5 µM), 1q
(4-ethoxycarbonyl, IC50 > 10 µM), 1s (4-fluoro, IC50 = 4.1 µM), 1t (3-cyano, IC50 > 10
µM), 1w (4-chloro, IC50 = 2.8 µM)], or the unsubstituted counterpart (1g, IC50 = 1.9 µM).
Among them, compound 1b (4-methylphenyl A-ring) exhibited the highest activity with
an average IC50 value of 28 nM, which is comparable to that of paclitaxel (IC50 = 19.6
nM), and is slightly better than colchicine (IC50 = 67 nM). Replacing the 4-methyl group
of 1b with larger groups such as morpholinyl (1o, IC50 > 10 µM), acetyl (1u, IC50 = 3.9
µM), trifluoromethyl (1v, IC50 = 1.9 µM), and t-butyl (1x, IC50 > 10 µM), led to
diminished activity. Compound 1n with a 3,4,5-trimethoxypheny A-ring showed
essentially a total loss of activity (IC50 > 10 µM). In addition to the phenyl A-ring, we
also introduced an indole moiety as the A-ring, and explored the influence of the

positions of the indole moiety connecting to the central pyrazolo[1,5-a]pyrimidine ring
on antiproliferative activities. For the N-methylated indole analogs (1a, 1ab, 1ae), the
5-indolyl compound 1a showed better activity (IC50 = 24.8 nM) than the 3-indolyl analog
1ab (IC50 = 556.6 nM) and the 6-indolyl analog 1ae (IC50 value of 556.8 nM). For the
3-indolyl analogs, Compound 1aa with no substitution on the indole NH (IC50 = 76.8 nM)
and 1ac with the N-methylol group (IC50 = 102 nM) displayed higher activity than 1ab
with the N-methyl group (IC50 = 556.6 nM).
Table 1 Anti-proliferative activities of compounds against five cancer cell linesa.
IC50 (μM) b
Compound
Hela
MCF-7
A549
HCT-116
B16-F10
1a
1b
1c
1d
1e
1f
0.019 ± 0.001
0.021 ± 0.001
0.125 ± 0.011
0.882 ± 0.075
0.241 ± 0.021
0.378 ± 0.032
0.458 ± 0.042
>10
0.003 ± 0.001
0.047 ± 0.004
0.023 ± 0.003
0.193 ± 0.027
0.026 ± 0.003
0.044 ± 0.003
0.254 ± 0.021
>10
0.015 ± 0.001
0.003 ± 0.001
0.170 ± 0.015
1.029 ± 0.312
0.272 ± 0.021
1.103 ± 0.103
0.208 ± 0.014
>10
0.039 ± 0.003
0.048 ± 0.005
0.187 ± 0.016
1.055 ± 0.389
0.261 ± 0.019
2.051 ± 0.197
3.876 ± 0.331
>10
0.048 ± 0.004
0.021 ± 0.002
0.055 ± 0.008
0.788 ± 0.065
0.391 ± 0.034
0.702 ± 0.084
4.734 ± 0.420
>10
1g
1h
1i
0.236 ± 0.022
0.373 ± 0.030
0.183 ± 0.010
0.125 ± 0.011
0.545 ± 0.048
0.376 ± 0.031
2.840 ± 0.232
0.404 ± 0.038
3.952 ± 0.331
1.405 ± 0.102
1j

0.160 ± 0.012
0.097 ± 0.007
4.226 ± 0.390
>10
0.242 ± 0.021
0.279 ± 0.023
1.076 ± 0.092
>10
0.643 ± 0.062
0.233 ± 0.016
5.731 ± 0.502
>10
1.53 ± 0.096
2.436 ± 0.212
6.152 ± 0.562
>10
1.805 ± 0.324
1.473 ± 0.130
2.220 ± 0.182
>10
1k
1l
1m
1n
>10
>10
>10
>10
>10
1o
1.831 ± 0.227
>10
0.274 ± 0.026
>10
1.147 ± 0.317
>10
2.106 ± 0.282
>10
1.238 ± 0.268
>10
1p
1q
3.768 ± 0.436
5.836 ± 0.475
>10
0.268 ± 0.021
2.065 ± 0.178
5.716 ± 0.530
5.033 ± 0.761
>10
3.070 ± 0.220
4.544 ± 0.402
>10
1r
1.280 ± 0.094 3.797 ± 0. 352
1s
>10
>10
1t
1u
1.810 ± 0.105
1.840 ± 0.179
1.488 ± 0.125
>10
5.229 ± 0.497
2.444 ± 0.221
2.321 ± 0.210
>10
4.098 ± 0.386
2.069 ± 0.184
3.065 ± 0.285
>10
4.394 ± 0.326
1.862 ± 0.138
3.335 ± 0.296
>10
3.933 ± 0.321
1.772 ± 0.158
4.091 ± 0.385
>10
1v
1w
1x
0.195 ± 0.012
0.399 ± 0.035
0.103 ± 0.009
0.456 ± 0.038
0.187 ± 0.011
0.283 ± 0.015
0.085 ± 0.007
0.095 ± 0.008
0.019 ± 0.003
1.067 ± 0.088
0.074 ± 0.006
0.027 ± 0.003
0.029 ± 0.003
1.342 ± 0.106
0.167 ± 0.013
1.100 ± 0.101
0.829 ± 0.075
0.138 ± 0.010
0.035 ± 0.004
0.762 ± 0.065
0.128 ± 0.010
0.861 ± 0.074
1.037 ± 0.097
0.251 ± 0.015
0.040 ± 0.005
0.185 ± 0.013
0.093 ± 0.007
1.086 ± 0.119
0.657 ± 0.058
0.088 ± 0.006
1aa
1ab
1ac
1ad
1ae
1af

5.211 ± 0.429
4.579 ± 0.338
>10
3.518 ± 0.442
>10
2.157 ± 0.184
>10
4.784 ± 0.569
>10
4a
4b
7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
11
Colchicine 0.081 ± 0.006
0.018 ± 0.001
0.004 ± 0.001
0.107 ± 0.009
0.012 ± 0.001
0.042 ± 0.004
0.020 ± 0.001
0.087 ± 0.006
0.021 ± 0.002
PTX
0.041 ± 0.005
a
Cells were treated with different concentrations of the compounds for 48 h. Cell
viability was measured by the CCK8 assay as described in the Experimental Section.
b
IC50: the half maximal inhibitory concentration. IC50 values are indicated as the mean ±
SD (standard error) of at least three independent experiments.
Compound 7 (Table 1) with a 3-indolyl moiety is essentially inactive (IC50 >10 μM)
probably due to their “straight” conformation which is not preferred for binding to the
colchicine binding site in tubulin as compared to the “bent” conformation of compound
1aa (IC50 = 76.8 nM, Figure 2). Similar results were also observed by Zhang’s
Group[25].
Figure 2. “Bent” and “Straight” conformations of target compounds.

In addition to the phenyl and indole A-ring, we also investigated other A-ring
moieties, for example, 1-ethyl-carbazole (1k, IC50 = 876 nM), 1-methyl-indazole (1l, IC50
= 903 nM), and 1-methyl-pyrazole (1p, IC50 = 1.3 μM), these compounds displayed
similar activity to that of 1f (IC50
=
855 nM). The detailed
Structure-Activity-Relationships (SARs) of this series of compounds are summarized in
Figure 3.
Figure 3. SARs of target compounds.
As a comparison, the compounds with a 4-fluorophenyl C-ring instead of a
3,4,5-trimethoxy phenyl C-ring displayed poor activity (IC50 of 4.0 μM and >10 μM for
4a and 4b, respectively) than the corresponding 3,4,5-trimethoxy phenyl C-ring analogs
1a and 1b (IC50 of 24.8 nM and 28 nM for 1a and 1b, respectively, Table 1). These
results suggest that the 3,4,5-trimethoxy C-ring is optimal for activity which is consistent
with our previous findings[17, 24]. Compound 11 showed little or no activity (IC50 >10
μM) due to the switching of the positions of indole A-ring and 3,4,5- trimethoxy phenyl
C-ring.
2.2.2. Inhibitory effects of 1a and 1b on tubulin polymerization

To understand the mechanism of action, we conducted a tubulin polymerization
o
assay (at 37 C without preincubation of tubulin with compounds) using the two most
active compounds, 1a and 1b, along with colchicine and CA-4 as positive controls. As
shown in Figure 4, the positive control compounds, colchicine and CA-4 inhibited
tubulin polymerization with an IC50 of 7.5 μM and 1.1 μM (Figure 4A, 4B), respectively;
while compounds 1a and 1b inhibited tubulin polymerization with IC50 values of 6.7 μM
(Figure 4C) and 2.1 μM (Figure 4D), respectively. It is well-known that the binding of
colchicine to tubulin is slow, temperature-dependent and slowly reversible, therefore, we
studied the effects of preincubation of tubulin with test compounds at different
o
temperatures (e.g. 30, 37 C) prior to the addition of GTP. As shown in Figure S1
(supporting information), temperature has little effect on the binding of test compounds
o
to tubulin [IC50 of 7.0, 6.4, 2.1
µ
M at 30 C without preincubation (Figure S1A, S1B,
o
S1C) vs 7.5, 6.7, 2.1
µ
M at 37 C without preincubation (Figure S1D, S1E, S1F) for
colchicine, 1a, and 1b, respectively]. While the preincubation of tubulin with compounds
moderately increased the binding affinity of test compounds to tubulin [IC50 of 5.4, 5.7,
1.6
µ
M at 37 oC with 5 min preincubation (Figure S1G, S1H, S1I) vs 7.5, 6.7, 2.1
µM at
o
37 C without preincubation (Figure S1D, S1E, S1F) for colchicine, 1a, and 1b,
respectively].

Figure 4. The inhibition of tubulin polymerization in vitro (at 37 oC without preincubation
of tubulin with compounds) by colchicine (A), CA-4 (B), 1a (C) and 1b (D).
2.2.3. Cell Cycle Study by flow cytometry
Tubulin polymerization inhibitors are known to impact cell division, therefore, we
evaluated the effects of the most potent compounds 1a and 1b on the cell cycle of MCF-7
cancer cells using flow cytometry. As shown in Figure 5, both 1a and 1b arrested cell
cycle at G2/M phase in a dose-dependent manner. Compared with the control cells
incubated with DMSO (Figure 5A), the incubation with colchicine (Figure 5B), 1a
(Figure 5C, 5D) and 1b (Figure 5E, 5F) drastically increased the percentage of cells
arrested in G2/M phase.

Figure 5. The cell cycle of MCF-7 cancer cells treated with 1a and 1b. (A) Vehicle
control (DMSO); (B) colchicine, 10 nM; (C) 1a, 5 nM; (D) 1a, 10 nM; (E) 1b, 5 nM; (F)
1b, 10 nM.
2.2.4. Inhibition of Cancer Cell Migration
One of the important features of tumor cells is their ability to migrate to distant
organs, leading to the formation of metastasized tumors. Our previous studies have
shown that microtubule polymerization inhibitors are able to inhibit tumor metastasis[26],
and similar results were also observed by other research groups [27, 28]. Therefore, we
evaluated the effects of compound 1b on cancer cell migration utilizing a wound healing
assay. As shown in Figure 6, the wound closure was potently suppressed by 1b in a
dose-dependent manner, as compared to the control.

Figure 6. Effects of 1b on MCF-7 cancer cell migration
2.2.5. Analysis of Immunofluorescence Staining
Microtuble dynamics play an essential role in cancer cell growth. To study whether
compound 1b is able to disrupt microtuble dynamics, we conducted an
immunofluorescent assay in B16-F10 cells. As shown in Figure 7, the microtubule
network in the control cells (DMSO, Figure 7A) exhibits normal arrangement and
organization with the hair like, slim, and fibrous microtubules (green) wrapped around
the cell nucleus (blue). Whereas in the PTX-treated cells (Figure 7B), the microtubules
were stabilized as seen from the increased density of microtubules around the nuclei,
which is consistent with PTX’s mechanism of action as a microtubule stabilizer. For the
cells treated with colchicine (160 nM, Figure 7C) or 1b (40 nM, Figure 7D), the
microtubules became short and wrapped around the nucleus in comparison with the
control, indicating the disruption of microtubule network. These results suggest that 1b
could destabilize microtubule by inhibiting tubulin polymerization and disturbing
microtubule networks, similar to colchicine and CA-4.

Figure 7. Effects of tested compounds on the cellular microtubule networks visualized by
immunofluorescence microscopy. B16-F10 cells were treated with vehicle control 0.1%
DMSO (A), PTX (40 nM) (B), Colchicine (160 nM) (C) and 1b (40 nM) (D) for 6 h.
Then, the cells were fixed and stained with anti-β-tubulin-FITC antibody (green), Alexa
Fluor 488 dye and counterstained with DAPI (blue). The detection of the fixed and
stained cells was performed by Carl Zeiss inverted microscope with plan-apochromat

63x/1.40 objective.
2.3 In vitro Metabolic Stability Studies
To test our hypothesis that the incorporation of the metabolically labile carbonyl
linker into a cyclic structure could effectively block the metabolic reduction of the
carbonyl group, thus prolonging the half-life of the compounds, we measured the
metabolic stability for 1a and 1b (two of the most potent compounds) in human liver
microsomes (HLM) and mouse liver microsomes (MLM). As shown in Table 2,
compound 1b exhibited significantly better metabolic stability in both HLM and MLM
(T1/2 = 125 and 120 min) than that of compound 1a (T1/2 = 20 min and 7 min) and the
SMART compound (T1/2 = 17 min and < 5min in HLM and MLM, respectively).
Table 2. Half-Lives of 1b in liver microsomes of different species
T1/2 (min)
Compounds
Human
20 ± 1.2
125 ± 9.2
17
mouse
7 ± 0.5
120 ± 8.0
<5
1a
1b
SMART[20]
2.4. X-ray Structures of 1a and 1b in Complex with Tubulin
To investigate the binding interactions of compounds with tubulin, we solved the
crystal structures of compounds 1a and 1b in complex with T2R-TTL (composed of two
α/β tubulin dimers, the stathmin-like protein RB3, and tubulin tyrosine ligase) (PDB code:

6LSN for 1a, 6LSM for 1b). The X-Ray/crystallographic data and structure refinement
statistics are presented in Table 3.
Table 3. X-Ray data collection and refinement statistics.
T2R-TTL-1a
T2R-TTL-1b
(PDB code: 6LSN)
(PDB code: 6LSM)
Data collection
Space Group
P212121
P212121
Cell dimensions
a, b, c (Å)
105.296, 158.458,181.959
105.553, 158.081,182.23
90, 90, 90
α,
β,
γ
(°)
90, 90, 90
Resolution (Å)
50-2.45(2.49-2.45) a
30-2.75 (2.8-2.75) a
0.129 (0.726)
0.997 (0.601)
12.8 (1.83)
b
Rmerge
CC1/2
13.1(100.3)
0.992(0.620)
9.12(1.0)
c
I/δ(I)
Completeness (%)
Redundancy
100(99.8)
6.6(5.9)
100 (100)
11.4 (10.1)
Structure refinement
No. reflections
739524 (112697)
0.1789/0.2227
909059 (79742)
0.1813/0.2271
17766
17446
214
R
work / Rfree
No. atoms
Protein
Ligand/ion
water
18041
17448
217
376
106
Average B factor
Protein
61.26
61.47
61.64
61.73
Ligand/ion
water
59
52.7
59
53.5
R.m.s. deviations
Bond lengths (Å)
Bond angle (°)
0.01
1.17
0.01
1.18
Ramachandran plot
Favored (%)
Allowed (%)
Outliers (%)
96.88
3.12
0
96.55
3.45
0
a
b
Values in parentheses are for highest-resolution shell.
Rmerge as outlined by
c
Diederichs and Karplus[29]. CC1/2 is the Pearson correlation coefficient of two-half

data sets as described by Karplus and Diederichs[30].
As anticipated, compounds 1a and 1b are bound to the colchicine binding site of
tubulin. The binding pocket is located at the interface of α- and β-tubulin, as opposed to
the GTP molecule which occupies a pocket in the α-tubulin (Figure 8A, 8D). There are
two water-bridged hydrogen bonds formed between 1a and β-tubulin: the oxygen atom of
the middle methoxy from the TMP (3,4,5-trimethoxyphenyl) moiety of 1a with the -NH
in β-Cys241 and -C=O in β-Val238 (Figure 8B and 8C). This water mediated hydrogen
bonding was also observed in several other crystal structures of tubulin in complex with
colchicine binding site inhibitors [31, 32]. As for 1b, one hydrogen bond was formed
between it and β-tubulin: the middle methoxy group of the TMP (3,4,5-trimethoxyphenyl)
with the -C=O of Val238 (Figure 8E and 8F). In addition, the pyrazolo[1,5-a]pyrimidine
moiety of compounds 1a and 1b is firmly wrapped in the colchicine-binding site with the
formation of a tight hydrophobic “sandwich” by the side-chains of β-Lys254, β-Leu255
and β-Asn258 from one side and that of β-Leu248 from the other side[32, 33].

Figure 8. Complex structures of 1a (6LSN) and 1b (6LSM) with tubulin protein. (A, D)
The overall structures of two compounds (1a and 1b) in complex with tubulin. Colored in
light green is the α units of T2R-TTL complex while colored in cyan is the β units; GTP
and compounds 1a and 1b are shown in yellow and pink spheres, respectively. (B, E)
Detailed interactions between 1a/1b and tubulin at the colchicine binding domain. (C, F)
90o clockwise rotation of corresponding structures along x axis. Compound-interacting
residues are presented as grey sticks in β-subunit of tubulin, and are labeled in black.
Oxygen and nitrogen atoms are colored read and blue, respectively. Hydrogen bonds are
indicated with blue dashed lines.
2.5. In vivo Antitumor Activity
Based on the high in vitro antiproliferative activity and excellent metabolic stability,
compound 1b was selected to evaluate the in vivo antitumor efficacy using a melanoma
B16-F10 tumor model in C57/BL mice. Each mouse was injected subcutaneously with
0.2 million B16-F10 melanoma cells. When tumors reached ~100 mm3 in volume and

were palpable, mice were randomized into control or treatment (1b and paclitaxel) groups,
and treated by intraperitoneal injection of compound 1b (10, 20 m/kg), paclitaxel (PTX,
10 m/kg), or a vehicle solution for 16 days. The tumor volume/size and body weights of
the mice were monitored and recorded every other day. As shown in Figure 9, compound
1b dose-dependently decreased tumor volume and tumor weight (Figure 9A, 9B). At the
dose of 10 mg/kg, 1b decreased the tumor volume and tumor weight by 40.1 % and
38.6 %, respectively; At the dose of 20 mg/kg, 1b significantly decreased the tumor
volume and tumor weight by 77.4 % and 74.5 %, respectively, which is better than that of
PTX (63.8 % and 60.4 % for decreasing the tumor volume and tumor weight, respectively,
at a dose of 10 mg/kg, Figure 9C). Moreover, no significant body weight loss was
observed in any of the treatment groups during the experiment (Figure 9D).

(b)
(a)
1
2
3
4
(c)
(d)
***
38.6 %
**
60.4 %
***
74.5 %
Figure 9. 1b inhibits melanoma tumor growth in vivo. After administering vehicle (group
1), PTX (10 m/kg, group 2), 1b (10 m/kg, group 3) and 1b (20 m/kg, group 4) for 16 days,
the mice were sacrificed, and tumors were excised and weighed. (a) The images of
excised tumors in each group. (b) Changes of tumor volume during treatment. (c) Weight
of the excised tumors of each group. (d) Changes of mice body weight during treatment.
The data were presented as the mean ± SEM ** P < 0.01, ***P < 0.001, significantly
different compared with the control by t test, n = 6.
In addition, pathohistological analysis utilizing H
＆E staining of the major organs
(e.g. liver and kidney) collected at end of the in vivo study showed that there is no
apparent organ-related toxicities (Figure 10). Overall, these data suggested that
compound 1b was highly efficacious in inhibiting melanoma tumor growth in vivo with

no obvious toxicity.
Figure 10. HE staining of major organs (liver and kidney) of mice. No abnormality or
apparent toxicities were observed.
3. Conclusion
In summary, a series of pyrazolo[1,5-a]pyrimidine derivatives were designed and
synthesized as tubulin polymerization inhibitors. Among them, compounds 1a and 1b
exhibited the highest antiproliferative activities against five cancer cell lines in vitro.
Further, compounds 1a and 1b were able to inhibit tubulin polymerization in vitro in a
dose dependent manner. We also solved the high-resolution X-Ray crystal structure of
compounds 1a and 1b in complex with tubulin, which confirmed the direct binding of 1a
and 1b to the colchicine binding site in tubulin, validating the mode of action of 1a and
1b as tubulin inhibitors. Further mechanism studies indicated that 1a and 1b induced cell
cycle arrest in G2/M phase, and inhibited cancer cell motility and migration. Importantly,
1b exhibited excellent in vitro metabolic stability on human and mouse liver microsomes.
Finally, 1b was highly effective in suppressing tumor growth in a B16-F10 mouse

melanoma model without apparent toxicity. Taken together, these results suggest that
compound 1b represents a promising tubulin inhibitor deserving further investigation.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all chemicals/reagents were purchased from commercial
suppliers and were used without further purification. The reference compounds
colchicine and paclitaxel were purchased from InvivoChem (Libertyville, IL 60048,
USA). Thin layer chromatography (TLC) analysis of reaction mixtures was performed on
Dynamic adsorbents silica gel F-254 TLC plates with fluorescent indicator visualized at
254 nm and 365 nm. Column chromatography for purification of compounds was
1
13
performed on silica gel (200-300 mesh). H NMR (400 MHz) and C NMR (100 MHz)
spectra were recorded with a Bruker AVANCE 400MHz spectrometer. Chemical shifts
were reported in ppm units (parts per million) and all coupling constants (J values) were
reported in Hertz (Hz). High resolution mass spectra were acquired using Bruker
micrOTOF-Q instrument with an ESI ion source. The purity of the final compounds
(>95%) was tested via HPLC on a Shimadzu LC-20A HPLC system installed with a UV
detector. The HPLC methods was conducted using a 5 μm C-18 column (4.6 × 150 mm)
o
at 40 C and a flow rate of 1 mL/min. HPLC gradient, solvent A (water) and solvent B
(methanol), 0-1 min 75 % B, 1-15 min 75-90 % B (linear gradient), 15-20 min 90% B.
UV detection at 254 nm. Purities of the compounds were established by integrating the
areas for all peaks detected and reported for each compound in the following section.

4.1.1. General synthetic procedure for the key intermediate
3
To a solution of 3,4,5-trimethoxyacetophenone (10 g, 47.6 mmol) in 40 mL DMF
was added N, N-dimethylformamide dimethyl acetal (6.8 g, 57.1 mmol). The mixture was
o
stirred at 120 C for 6 h, then the reaction was quenched by H2O, and extracted with
CH2Cl2. concentrated in vacuo to provide the crude product, which was purified by
column chromatography to give intermediate 2. To a solution of 2 (2.4 g, 9.0 mmol) in 10
mL AcOH was added 3-bromo-1H-pyrazol-5-amine (1.5 g, 9.2 mmol). The mixture was
o
stirred at 80 C for 8h, after the reaction completed, the cooled mixture was deposited
dropwise in H2O, and the precipitate solid was filtered, dried to obtain intermediate 3.
4.1.2. General synthetic procedure for 1a-y
To a solution of 3 (100 mg, 0.275 mmol) and corresponding boronic acid (0.360
mmol) in 5 mL DMF and 1 mL H2O, Na2CO3 (73 mg, 0.688 mmol), Pd(dppf)Cl2 (10 mg,
o
0.014 mmol) was added. The mixture was stirred at 95 C for 12h, then the reaction was
quenched by H2O, and extracted with CH2Cl2. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to provide the crude
product, which was purified by column chromatography with DCM/MeOH (100:1) to
give pure compounds 1a-y.
4.1.3. General synthetic procedure for 4a-b
The compounds 4a-b were prepared from 4-fluoroacetophenone following the same
procedure as described in preparation of compounds 1a-y.
4.1.4. General synthetic procedure for 1aa

To a solution of 1y (100 mg, 0.185 mmol) in 10 mL EtOH, 1mL H2O was added
o
NaOH (72 mg, 1.8 mmol). The mixture was stirred at 70 C for 8h, then the reaction
mixture was poured into water and extracted with CH2Cl2. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
provide the crude product. Which was purified by column chromatography with
DCM/MeOH (90:1) to give the pure compound 1aa.
4.1.5. General synthetic procedure for 1ab, 1ae
To a solution of 1aa (100 mg, 0.250 mmol) in 10 mL dry THF was added NaH (60
o
mg, 2.5 mmol). after stirring for 2h at 0 C, then 0.6 mL CH3I was added, the mixture
was stirred at room temperature for 4h. the reaction was quenched by H2O, and extracted
with CH2Cl2. The combined organic layers were washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to provide the crude product, which was purified by
column chromatography with DCM/MeOH (85:1) to give pure compound 1ab. The
synthesis of compound 1ae is similar to compound 1ab.
4.1.6. General synthetic procedure for 1ac
To a solution of 1aa (50 mg, 0.125 mmol) in 5 mL EtOH was added 1 mL 10%
NaOH aqueous, 1 mL 37% HCHO aqueous. after stirring for 4h at room temperature.
then the reaction mixture was poured into water and extracted with CH2Cl2. The
combined organic layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo to provide the crude product, which was purified by column
chromatography with DCM/MeOH (80:1) to give pure compound 1ac.

4.1.7. General synthetic procedure for 1ad
To a solution of 1q (100 mg, 0.231 mmol) in 10 mL dry THF was added LiAlH4 (87
mg, 2.3 mmol, the mixture was stirred at room temperature for 4h. The reaction was
quenched by H2O, and extracted with CH2Cl2. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to provide the crude
product, which was purified by column chromatography with DCM/MeOH (85:1) to give
pure compound 1ad.
4.1.8. General synthetic procedure for 1af
To a solution of 1h (50 mg, 0.115 mmol) in 2 mL THF, 3mL MeOH was added 10
mg 10% Pd/C. The reaction system was replaced by nitrogen three times, hydrogenated
with H2 at room temperature and monitored by TLC. The catalyst was filtered and the
solvent was removed in vacuum to afford the compound 1af.
4.1.9. General synthetic procedure for
7
To a solution of 2 (2.4 g, 9.0 mmol) in 10 mL AcOH was added
4-bromo-1H-pyrazol-5-amine (1.5 g, 9.2 mmol). The mixture was stirred at 80 oC for 8h,
after the reaction completed, the cooled mixture was deposited dropwise in H2O, and the
precipitate solid was filtered, dried to obtain the intermediate 5. Then compound 7 was
synthesized according to the synthesis procedure of 1aa.
4.1.10. General synthetic procedure for 11
To a solution of 3-acetylindole (5 g, 31.4 mmol) in 30 mL dry THF was added NaH,
after 6 hours, benzenesulfonyl chloride (6.6 g, 37.7 mmol) was added. The mixture was

o
stirred at 25 C for 6h, then the reaction was quenched by H2O, and extracted with
CH2Cl2, and concentrated in vacuo to provide the intermediate 8. Then compound 11 was
synthesized according to the synthesis procedure of 1n.
4.1.11. 2-(1-methyl-1H-indol-5-yl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine
(1a). Yield: 81.5 %; mp 177-179 oC; 1H NMR (400 MHz, CDCl3)
δ 8.43 (s, 1H), 8.31 (s,
1H), 7.94 (d, J = 8.5 Hz, 1H), 7.59 (s, 2H), 7.36 (d, J = 11.5 Hz, 1H), 7.11 (s, 1H), 7.06 (s,
13
1H), 6.85 (s, 1H), 6.56 (s, 1H), 4.01 (s, 3H), 3.99 (s, 3H), 3.77 (s, 3H). C NMR (101
MHz, CDCl3) δ 157.19, 153.00, 151.33, 148.53, 145.53, 140.37, 137.23, 129.63, 128.68,
126.15, 124.19, 120.32, 119.23, 109.43, 107.12, 106.22, 101.60, 92.92, 60.95, 56.32,
32.83. HRMS m/z: calcd for C24H22N4O3 [M+H]+ 415.1765, found 415.1783. Purity:
98.1 % by HPLC (tR = 7.93 min).
4.1.12. 2-(p-tolyl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine (1b). Yield:
88.0 %; mp 155-157 oC; 1H NMR (400 MHz, CDCl3)
δ 8.48 (d, J = 4.4 Hz, 1H), 7.92 (d,
J = 8.1 Hz, 2H), 7.54 (s, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.06 (s, 1H), 6.91 (d, J = 4.4 Hz,
1H), 4.00 (s, 3H), 3.98 (s, 6H), 2.41 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 155.83,
153.03, 151.17, 148.71, 145.85, 140.43, 138.97, 129.97, 129.42, 126.31, 125.92, 115.19,
107.04, 106.57, 93.24, 60.96, 56.32, 21.34. HRMS m/z: calcd for C22H21N3O3 [M+H]+
376.1656, found 376.1676. Purity: 98.5 % by HPLC (tR = 9.56 min).
4.1.13. 2-(1H-indol-6-yl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine (1c).
Yield: 80.2 %; 1H NMR (400 MHz, CDCl3)
δ 8.49 (d, J = 4.4 Hz, 1H), 8.40 (s, 1H), 8.09
(s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.56 (s, 2H), 7.13 (s, 1H), 6.91