4,5,6,7-Tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine as a new scaffold for heat shock protein 90 inhibitors

Article abstract of DOI:10.1016/j.cclet.2015.09.024

Heat shock protein 90 (hsp90) is a promising anticancer drug target. A library of 2,4-dihydroxyphenyl (resorcinol) substituted 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine compounds that target this protein were designed and prepared based on our earlier study. The compounds were tested in five cancer cell lines and seven of them showed notable anticancer activity (IC₅₀ 2-10 μmol/L). The active subset compounds were further subjected to a polarized fluorescent assay and exhibited high binding affinity toward purified hsp90 (IC₅₀ 60-100 nmol/L). These results indicated that the tetrahydro-triazolopyrazine motif of the molecules may represent a novel scaffold for the development of hsp90 inhibitors.

Full text of DOI:10.1016/j.cclet.2015.09.024

Chinese Chemical Letters 27 (2016) 11–15
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
4,5,6,7-Tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine as a new scaffold for
heat shock protein 90 inhibitors
*
Meng-Yi Xu, Ni-Na Xue, Di Liu, Yu-Mei Zhou, Wei Li, Yong-Qiang Li, Xiao-Guang Chen ,
*
Xiao-Ming Yu
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Heat shock protein 90 (hsp90) is a promising anticancer drug target. A library of 2,4-dihydroxyphenyl
(resorcinol) substituted 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine compounds that target this
protein were designed and prepared based on our earlier study. The compounds were tested in five
Received 2 July 2015
Received in revised form 18 August 2015
Accepted 25 September 2015
Available online 12 November 2015
cancer cell lines and seven of them showed notable anticancer activity (IC50 2–10
mmol/L). The active
subset compounds were further subjected to a polarized fluorescent assay and exhibited high binding
affinity toward purified hsp90 (IC50 60–100 nmol/L). These results indicated that the tetrahydro-
triazolopyrazine motif of the molecules may represent a novel scaffold for the development of hsp90
inhibitors.
Keywords:
Hsp90 inhibitor
Anticancer drug
Resorcinol
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Tetrahydrotriazolopyrazine
1. Introduction
study published by Neckers’ team in 1994 [3]. It was demonstrated
that benzoquinone ansamycin geldanamycin (1, Fig. 1) was able to
Heat shock protein 90 (Hsp90) is an ATP-dependent 90 kD
molecular chaperone ubiquitously expressed in eukaryotes [1]. An
array of downstream proteins, which are termed as Hsp90 clients,
are dependent on the chaperon function of Hsp90 during the late
stage of their synthesis and structure maturation. Under stressed
conditions, Hsp90 is also responsible for the structural mainte-
nance and repairing of the clients, to prevent them from
aggregation, dysfunction and degradation.
bind to the ATP-binding cite of Hsp90 located in its N-terminal
domain, and significantly lowered the cellular level of c-Raf, a well-
defined oncogenic Hsp90 client. Since then, research attentions
devoted to this subject has become intense.
A large number of Hsp90 inhibitors based on varied scaffolds,
mostly targeting the N-terminal ATP-binding pocket of Hsp90,
were reported, and nearly twenty of them received clinical studies
for treatment of cancer [4,5]. Among these molecules, resorcinol
containing compounds, e.g. AUY-922 (2) [6], STA-9090 (3) [7] and
AT13387 (4) [8], made a major class. LD-053(5) is a new resorcinol-
type Hsp90 inhibitor identified in our laboratory [9]. It showed
notable anticancer activities in several cellular models and was
proved to be effective in vivo. We report herein an extended study
based on these preliminary results, to demonstrate that the
tetrahydro[1,2,3]triazolopyrazin central segment presented in LD-
053 actually represents a new scaffold for further discovery of
novel Hsp90 inhibitors.
In cancer cells, Hsp90 is 2–10 fold overexpressed and plays
critical role in cell survival, growth and tumor progression [2]. A
number of well-known oncogenic proteins, for example AKT, Eerb-
2, Hif-1a and telomerase, are now known to be Hsp90 clients. As a
consequence, the maintenance of these malignant pathways could
be highly dependent on the chaperone function of Hsp90. It was
hypothesized that inhibition of Hsp90 in cancer cells could result
in the spontaneous depression of its oncogenic clients and thereby
disruption of the related signaling pathways, making the molecu-
lar chaperone a ‘‘one stone two birds’’ anticancer drug target. The
hypothesis, however, was not widely accepted until the seminal
2. Experimental
2.1. Chemicals and instruments
*
Corresponding author.
Starting materials, reagents were purchased from commercial
suppliers (Acros, Alfa Aesar) and used without further purification
E-mail addresses: chxg@imm.ac.cn (X.-G. Chen), mingxyu@imm.ac.cn
(X.-M. Yu).
http://dx.doi.org/10.1016/j.cclet.2015.09.024
1001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

12
M.-Y. Xu et al. / Chinese Chemical Letters 27 (2016) 11–15
18.32, À5.02. IR (cmÀ1): 2228, 1614, 1256, 1219, 1082. HRMS
(ESI+) calcd. for C31H46O6NSi (M + H)+ 556.3089, found 556.3077.
N-(2-Hydroxyethyl)-N-(3-(5-isopropyl-2,4-bis(methoxy-
methoxy)phenyl)prop-2-ynyl)benzamide (9): Tetrabutylammo-
nium fluoride (3.49 g, 8.4 mmol) was added to a solution of
compound 8 (3.33 g, 6.0 mmol) in THF (30 mL). The mixture was
stirred at 40 8C for 6 h and diluted with ethyl acetate (20 mL). The
resulting solution was successively washed with water
(20 mL Â 2) and brine (20 mL). The organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was concentrated and
the residue purified by chromatography on silica gel (petroleum
ether/ethyl acetate, 5/1) to give alcohol 9 (2.12 g, 85%) as light
yellow oil. 1H NMR (400 MHz, CDCl3):
d 7.73–7.36 (m, 5H), 7.21 (s,
1H), 6.88 (s, 1H), 5.22 (s, 2H), 5.20 (s, 2H), 4.29 (m, 2H), 3.98 (m, 2H),
3.91 (m, 2H), 3.51 (s, 3H), 3.48 (s, 3H), 3.23 (m, 1H), 1.20 (d, 6H,
J = 6.9 Hz). 13C NMR (151 MHz, CDCl3):
d 173.14, 157.23, 155.79,
135.19, 131.43, 130.54, 130.37, 128.48, 127.34, 105.24, 101.84,
95.20, 94.48, 86.41, 81.90, 61.72, 56.39, 56.25, 49.44, 41.92, 26.46,
22.71. IR (cmÀ1): 2228, 2101, 1643, 1502, 1262, 1081. HRMS (ESI+)
calcd. for C25H32O6N (M + H)+442.2224, found 442.2205.
Fig. 1. Selected examples of Hsp90 inhibitors.
unless otherwise stated. Pyridine (Py.) was dried over solid
potassium hydroxide and distilled. Tetrahydrofuran (THF) and
toluene were distilled over sodium. Dry dichloromethane (DCM)
was distilled over P2O5 before use. Proton nuclear magnetic
resonance (1H NMR) spectra and carbon-13 (13C NMR) spectra
were recorded on a Varian Mercury-300, Varian Mercury-400,
Varian Avance-500 or Varian System-600 spectrometer. 1H spectra
N-(2-Azidoethyl)-N-(3-(5-isopropyl-2,4-bis(methoxymethox-
y)phenyl)prop-2-ynyl)benzamide (10): CBr4 (1.08 g, 3.2 mmol)
was added portion-wide to a solution of compound 9 (1.20 g,
2.7 mmol) and PPh3 (0.85 g, 3.2 mmol) in dry DMF (15 mL) at 0 8C
under argon atmosphere. Upon completion of addition, the
mixture was stirred for additional 1 h. NaN3 (0.23 g, 3.5 mmol)
was added in one portion and the reaction mixture was stirred for
48 h at room temperature before it was diluted with ethyl acetate
(30 mL). The resulting mixture was successively washed with
water (10 mL Â 3) and brine (10 mL). The organic layer was dried
over anhydrous Na2SO4 and filtered. The filtrate was concentrated
and the residue purified by chromatography on silica gel
(petroleum ether/ethyl acetate, 8/1) to give compound 10
was referenced to the residual solvent (
2.04 ppm forCD3COCD3) or tetramethylsilane (
internal reference. For 13C spectra, chemical shifts are reported
relative to the 77.0 ppm resonance of CDCl3 or the 28.9 ppm
d
7.26 ppm for CDCl3,
d
d
0 ppm, CDCl3) as an
d
d
resonance of CD3COCD3. Coupling constants are reported in Hz.
Infrared (IR) spectra were recorded with Microscope Transmission
on a Nicolet 5700 FT-IR. Optical rotations were measured on a
Perkin-Elmer 240 using a quartz cell with 1 mL capacity and a 10 cm
path length at 20 8C. Mass spectra were recorded on a Thermo
Finnigan LTQ FT mass spectrometry manufacturedby Thermo Fisher
Scientific (San Jose, CA, USA). Column chromatography was
generally performed using HaiyangZCX. II (200–300 mesh) silica
gel. Unless noted otherwise, all compounds isolated by chromatog-
raphy were sufficiently pure by 1H NMR analysis for use in
subsequent reactions.
(0.84 g, 66%) as light yellow oil. 1H NMR (300 MHz, CDCl3):
d
7.68–7.39 (m, 5H), 7.22 (s, 1H), 6.87 (s, 1H), 5.22 (s, 2H), 5.20 (s, 2H),
4.33 (m, 2H), 3.87 (m, 2H), 3.73 (m, 2H), 3.51 (s, 3H), 3.49 (s, 3H),
3.33–3.16 (m, 1H), 1.20 (d, J = 6.9, 6H). 13C NMR (151 MHz, CDCl3):
d
171.60, 157.21, 155.71, 135.41, 131.40, 130.64, 130.08, 128.50,
127.01, 105.38, 102.00, 95.25, 94.46, 86.01, 81.96, 56.30, 56.22,
49.15, 45.27, 42.01, 26.44, 22.68. IR (cmÀ1): 2960.7, 2905.8, 2102.2,
1642.5, 1501.4, 1151.3. HRMS (ESI+) calcd. for C25H31O5N4
(M + H)+467.2289, found 467.2272.
(3-(5-Isopropyl-2,4-bis(methoxymethoxy)phenyl)-6,7-dihy-
dro-[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)(phenyl)methanone
(11): Compound 10 (520 mg, 1.11 mmol) was dissolved in toluene
(10 mL) and heated at reflux for 10 h. The reaction mixture was the
concentrated and the residue was purified by chromatography on
silica gel (petroleum ether/ethyl acetate, 2/1) to give 11 (470 mg,
2.2. Synthesis of compounds 8–13a
N-(2-(tert-Butyldimethylsilyloxy)ethyl)-N-(3-(5-isopropyl-
2,4-bis(methoxymethoxy)phenyl)prop-2-ynyl)benzamide (8): 1-
Iodo-5-isopropyl-2,4-bis(methoxymethoxy)benzene (6) (2.8 g,
7.65 mmol), N-(2-(tert-butyldimethylsilyloxy)ethyl)-N-(prop-2-
ynyl)benzamide (7) (2.67 g, 8.4 mmol) and CuI (0.29 g,
1.53 mmol) were mixed in Et3N (35 mL) (See Supporting
information for preparation of 6 and 7). The mixture was
ultrasonically deoxygenated under an argon atmosphere and
then PdCl2(PPh3)2 (540 mg, 0.77 mmol) was added. The reaction
mixture was heated at 60 8C for 2 h and filtered. The filtrate was
concentrated and the residue was stirred in hexanes (30 mL). The
resulting mixture was filtered again. The filtrate was concentrated
and the residue purified by chromatography on silica gel
91%) as white solid. 1H NMR (500 MHz, DMSO-d6):
d 7.72–7.33 (m,
6H), 7.06–6.71 (m, 1H), 5.24 (s, 2H), 5.04 (s, 2H), 4.52 (s, 2H), 4.34–
3.61 (m, 2H), 3.55–2.91 (m, 9H), 1.36 (m, 6H). 13C NMR (151 MHz,
DMSO-d6):
d 170.31, 155.38, 152.53, 138.96, 138.79, 135.46,
131.01, 130.73, 129.20, 128.47, 127.66, 113.94, 102.10, 95.41,
94.74, 56.34, 46.34, 45.85, 44.86, 44.13, 26.42, 23.23.IR (cmÀ1):
1641.8, 1506.4, 1150.8, 1079.6. HRMS (ESI+) calcd. for C25H31O5N4
(M + H)+467.2289, found 467.2271.
3-(5-Isopropyl-2,4-bis(methoxymethoxy)phenyl)-4,5,6,7-tet-
rahydro-[1,2,3]triazolo[1,5-a]pyrazine (12): Benzamide 11 (4.40 g,
9.43 mmol) was heated at reflux with KOH (1.59 g, 28.4 mmol) in a
mixture of MeOH/H2O (50 mL, v/v, 1/1) for 10 h. After the mixture
was cooled to room temperature, water was added and then
extracted with ethyl acetate. The organic layers were combined,
washed with brine, dried over anhydrous Na2SO4, filtered and
evaporated to dryness to give 12 (3.0 g, 88%) as white solid. This
material is pure enough for the next reaction. 1H NMR (400 MHz,
(petroleum ether/ethyl acetate, 8/1) to give compound
(3.34 g, 95%) as yellow oil. 1H NMR (400 MHz, CDCl3):
7.36 (m, 5H), 7.22 (s, 1H), 6.86 (s, 1H), 5.21 (s, 2H), 5.20 (s, 2H), 4.39
(m, 2H), 4.00–3.58 (m, 4H), 3.51 (s, 3H), 3.48 (s, 3H), 3.32–3.18 (m,
1H), 1.22 (d, 6H, J = 12.3 Hz), 0.90 (s, 9H), 0.08 (s, 6H). 13C NMR
8
d
7.66–
(151 MHz, DMSO-d6):
d 170.91, 157.19, 155.40, 136.41, 131.15,
130.58, 130.00, 128.87, 127.31, 106.10, 102.73, 95.35, 94.59,
87.77, 60.63, 56.41, 56.29, 50.26, 47.57, 41.64, 26.37, 26.20, 23.01,
CDCl3):
d 7.55 (s, 1H), 6.96 (s, 1H), 5.23 (s, 2H), 5.11 (s, 2H),

M.-Y. Xu et al. / Chinese Chemical Letters 27 (2016) 11–15
13
4.42 (t, 2H, J = 5.6 Hz), 4.14 (s, 2H), 3.50 (d, 3H, J = 5.5 Hz), 3.41 (s,
3H), 3.35 (t, 2H, J = 5.6 Hz), 3.33–3.24 (m, 1H), 1.24 (d, 6H,
the reaction mixture through a syringe. The ice bath removed and
the mixture was stirred for 8 h at room temperature before water
was added dropwise at 0 8C to quench the reaction. The mixture
was then extracted with ethyl acetate. The organic layers were
combined, washed with brine and then concentrated. The residue
was purified by chromatography on silica gel (petroleum ether/
ethyl acetate, 1/1) to give 14. 14 was dissolved in HCl-methanol,
and stirred at room temperature for 12 h. Then the reaction
mixture was evaporated under reduced pressure at 35 8C to give
the crude product. The crude product was purified by chromatog-
raphy on silica gel (petroleum ether/ethyl acetate, 1/1) to give 15 as
white solid.
J = 6.9 Hz).13C NMR (151 MHz, CDCl3):
d 155.34, 152.74, 139.27,
131.70, 129.66, 128.14, 114.40, 101.98, 95.54, 94.66, 56.32, 56.22,
46.69, 42.79, 42.52, 26.73, 22.82. IR (cmÀ1): 1658.2, 1112.7,
1053.0. HRMS (ESI+) calcd. for C18H27O4N4 (M + H)+ 363.2027,
found 363.2014.
2-(3-(5-Isopropyl-2,4-bis(methoxymethoxy)phenyl)-6,7-dihy-
dro-[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)ethanol (13): K2CO3
(571 mg, 4.14 mmol) and 2-bromoethanol (1.04 g, 8.29 mmol)
was added to a solution of compound 12 (1.00 g, 2.76 mmol) in dry
acetone (10 mL) in a seal tube. The reaction was kept sealed and
stirred at 50 8C for 48 h. Water was added and the mixture was
extracted with ethyl acetate. The organic layers were combined,
washed with brine, dried over anhydrous Na2SO4 and filtered. The
filtrate was concentrated and residue purified using silica gel
chromatography (petroleum ether/ethyl acetate, 1/1) to give
compound 13 (1.6 g, 95%) as yellow oil. 1H NMR (400 MHz,
Characterization data from compound 15-1 to 15-9 can be
found in Supporting information.
3. Results and discussion
The validated druggability of Hsp90 has provoked numerous
attempts aiming at novel Hsp90 inhibitors, leading to the discovery
of stockpiles of small molecules targeting Hsp90. Despite that the
structures of these agents ranging from semisynthetic natural
product derivatives to target based rational-designed compounds,
chemical space within this scope still remains under-explored.
Novel structures possessing elevated potency, reduced off-target
toxicity and more favorable pharmacokinetic profiles are still of
considerable interest. In this context, randomly discovered LD053
positioned us at a good start point for further investigations, and
hence an extended series of compounds were designed out of this
prototype molecule to tackle a general view of structure activity
relationships.
The molecule of LD053 (5, Fig. 1) virtually comprises three
sections. We intended to retain the framework except that a more
commonly adopted iso-propyl was used in place of the chlorine in
section ‘‘A’’. The major variations, on the other hand, were made to
section ‘‘C’’, into which a series of mono-substituted phenoxyls
were to be introduced with the substituent varying from simple
alkyls, halogens to more polar sulfonylsand the site of substitution
including othro-, meta- and para-.
CDCl3):
d 7.54 (s, 1H), 6.94 (s, 1H), 5.23 (s, 2H), 5.10 (s, 2H), 4.50 (t,
2H, J = 5.6 Hz), 3.84 (s, 2H), 3.70 (s, 2H), 3.51 (s, 3H), 3.41 (s, 3H),
3.30 (m, 1H), 3.11 (t, 2H, J = 5.6 Hz), 2.83–2.75 (m, 2H), 1.24 (d, 6H,
J = 6.9 Hz). 13C NMR (151 MHz, CDCl3):
d 155.43, 152.66, 139.65,
131.84, 129.14, 128.23, 114.22, 102.06, 95.67, 94.63, 58.44, 58.34,
56.37, 56.20, 53.44, 49.44, 45.59, 26.74, 22.81. IR (cmÀ1): 3338.6,
2952.0, 1510.6, 1268.9, 1211.0, 1144.3, 988.1. HRMS (ESI+) calcd.
for C20H31O5N4 (M + H)+407.2289, found 407.2274.
2-(3-(5-Isopropyl-2,4-bis(methoxymethoxy)phenyl)-6,7-dihy-
dro-[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)ethyl
methanesulfo-
nate (13a): Compound 13 (200 mg, 0.49 mmol) and Et3N
(60 mg, 0.59 mmol) were dissolved in dry dichloromethane
(2.5 mL). Methanesulfonyl chloride (62.4 mg, 0.54 mmol) was
added dropwise with stirring at 0 8C. The mixture was kept stirred
for 3 h and quenched by addition of water. The mixture was
extracted with dichloromethane (10 mL Â 3). The organic layers
were combined, washed with brine (20 mL), dried over anhydrous
Na2SO4. After filtration, the filtrate was concentrated under
reduced pressure to yield intermediate 13a. This crude material
was used directly in the next step.
The designed molecules were synthesized as depicted in
Scheme 1. Thus, Sonogashira coupling between 1-iodo-5-
isopropyl-2,4-bis(methoxymethoxy)benzene 6 and N-propargyl
2.3. General method for synthesis of compound 15
Substituted phenol (1.0 equiv) was dissolved in dry DMF. NaH
(3.0 equiv) was added portion-wise at 0 8C under stirring. Upon
completion of addition, stirring was continued for another 0.5 h. A
solution of crude 13a (1 equiv) in dry DMF (1 mL) was injected to
benzamide 7 to give compound 8 [10]. The product was
desilylated (9) and brominated. Upon treatment of the bromo
intermediate with NaN3 in DMF, the resultant azide 10 was
heated at reflux in toluene to effect the intramolecular [3 + 2]
Scheme 1. The synthesis of LD053 analogues. Reagent and conditions: (a) PdCl2(PPh3)2 (0.1 equiv.), CuI (0.2 equiv), Et3N, 60 8C, 95%; (b) TBAF, THF, 40 8C, 85%; (c) CBr4, PPh3,
DMF, 0 8C; (d) NaN3, DMF, r.t., 66% for 2 steps; (e) toluene, reflux, 91%; (f) KOH (3 equiv.), MeOH/H2O (v/v 1/1), 88%; (g) BrCH2CH2OH, K2CO3, dry acetone, 50 8C in seal tube,
95%; (h) MsCl, Et3N, 0 8C; (i) Selected phenols, NaH, DMF, 0 8C to r.t.; (j) HCl–MeOH (1 M), r.t., 65%–88% for 3 steps.

14
M.-Y. Xu et al. / Chinese Chemical Letters 27 (2016) 11–15
Table 1
Cancer cell growth inhibitory effects of compound 15
R
O
HO
N
N
15
OH
N
N
Entry
Compound
R
IC50 (mmol/L)
HCT116
BGC823
A375
MCF7
Mia-paca2
1
15-1a
15-1b
15-1c
15-2a
15-2b
15-2c
15-3a
15-3b
15-3c
15-4a
15-4b
15-4c
15-5a
15-5b
15-5c
15-6a
15-6b
15-6c
15-7a
15-7b
15-7c
15-8a
15-8b
15-8c
15-9a
15-9b
15-9c
AUY-922
o-Me
>100
>100
60.95
>100
15.32
>100
26.53
52.38
>100
>100
>100
>100
>100
3.99
>100
>100
>100
>100
>100
>100
13.99
>100
47.05
>100
4.66
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.66
2
m-Me
p-Me
9.57
91.33
>100
31.85
>100
3
4
o-Et
5
m-Et
72.63
6
p-Et
>100
>100
>100
>100
88.22
>100
>100
>100
>100
>100
>100
6.02
7
o-i-Pr
m-i-Pr
p-i-Pr
o-Cl
>100
>100
>100
>100
>100
>100
>100
8
9
7.37
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>100
>100
>100
>100
m-Cl
p-Cl
>100
>100
12.33
6.90
o-CN
m-CN
p-CN
2.65
2.24
>100
>100
2.29
>100
4.15
>100
4.97
6.02
5.83
>100
>100
5.58
>100
6.60
>100
18.76
6.65
6.15
o-OMe
m-OMe
p-OMe
o-SMe
m-SMe
p-SMe
o-SOMe
m-SOMe
p-SOMe
o-SO2Me
m-SO2Me
p-SO2Me
–
>100
>100
10.22
96.62
7.24
8.63
>100
>100
6.74
>100
4.15
>100
28.71
>100
>100
10.35
18.37
8.33
>100
3.47
>100
11.45
5.18
74.13
7.23
2.78
3.83
4.23
7.41
2.69
8.46
4.56
>100
>100
7.57
13.15
>100
>100
6.22
<0.1
>100
>100
3.21
>100
12.19
0.57
>100
3.80
<0.1
<0.1
<0.1
cycloaddition, giving tetrahydro-[1,2,3]-triazolopyrazin adduct
11 in 91% yield. Removal of the N-benzoyl by using regular basic
hydrolysis condition led to the isolation of 12, which was heated
with 2-bromoethanol and K2CO3 in acetone at 50 8C in a seal
tube to give compound 12. Alcohol 12 was mesylated and
coupled with the selected phenols in the presence of sodium
hydride in DMF to deliver compound 14-1a–14-9c. These
precursors were then stirred in methanolic hydrochloride to
remove the MOM protection, giving the final products (15-1a–
15-9c) mostly in forms of precipitated solids. Structures of these
compounds were summarized in Table 1.
molecules carrying othro-substitution in section C (entries 13, 16,
19, 22 and 25), indicating substitution at this position is
detrimental to the compounds’ anticancer activity. For the active
compounds, on the other hand, a polar substituent, either at meta-
or para-position, seemed necessary; yet more interesting is that
both electron donating and withdrawing groups contributed
positively.
These selected compounds were further subjected to
a
polarized fluorescent assay using the known protocol [9] to
determine their affinities toward hsp90. As it is showed in Table 2,
all compounds appeared highly potent upon binding to purified
hsp90, indicating that the anti-cancer activity of these molecules is
associated with their capability of hsp90 inhibition. It is unfortu-
nate that the high target affinity of our compounds was not fully
reflected in the cellular level examinations, but the results still
evidenced that the 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyra-
zine substructure, or section B of the target molecules, may
represent a new scaffold for the discovery of novel hsp90
inhibitors.
Compounds synthesized as described above were submitted to
MTT assays [9] in five different cancer cell lines to determine their
in vitro effects on cell growth (Table 1), and seven were found
active (entries 14, 15, 18, 20, 23, 24 and 27) with the average IC50
s
lower than 10 mol/L.
m
It is noteworthy that simple aliphatic (chlorine as well)
substituted compounds in this series generally exhibited low
activities (entries 1–12, Table 1). This is also the case for the
Table 2
Hsp90 affinity of the selected compounds.
Compound
R
15-5b
15-5c
15-6c
15-7b
15-8b
15-8c
15-9c
m-CN
p-CN
p-OMe
m-SMe
m-SOMe
p-SOMe
p-SO2Me
IC50
(
mmol/L)
0.090
0.082
0.102
0.060
0.060
0.090
0.064

M.-Y. Xu et al. / Chinese Chemical Letters 27 (2016) 11–15
15
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4. Conclusion
In summary,
a library of analogues was designed and
synthesized based on our randomly discovered prototype hsp90
inhibitor, LD053. In vitro anticancer activities were tested in a
panel of cellular tumor models, and seven of the compounds were
found active (IC50 2–10
mmol/L). The selected active compounds
also exhibited notable binding affinities toward purified hsp90
(IC50 60–100 nmol/L). These results, in combination to our earlier
findings, helped the recognition of the 4,5,6,7-tetrahydro-
[1,2,3]triazolo[1,5-a]pyrazine motif as a new scaffold for hsp90
inhibitors. With the aid of the preliminary structure activity
relationships illustrated in this study, further optimization of the
molecule is ongoing.
(b) F. Zagouri, T.N. Sergentanis, D. Chrysikos, et al., Hsp90 inhibitors in breast
cancer: a systematic review, The Breast 22 (2013) 569–578;
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[5] (a) J.-M. Jia, F. Liu, X.-L. Xu, et al., Synthesis and evaluation of a novel class Hsp90
inhibitors containing 1-phenylpiperazine scaffold, Bioorg. Med. Chem. Lett. 24
(2014) 1557–1561;
Acknowledgment
This project is financially supported by the National Natural
Science Foundation of China (No. 21272279).
(b) J. Ren, J. Li, Y. Wang, et al., Identification of a new series of potent diphenol
HSP90 inhibitors by fragment merging and structure-based optimization, Bioorg.
Med. Chem. Lett. 24 (2014) 2525–2529;
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inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold, J. Med. Chem. 57
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Appendix A. Supplementary data
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fragment based drug design, J. Med. Chem. 53 (2010) 5956–5969.
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