Synthesis and biological evaluation of a novel human stem/progenitor cells proliferation activator: 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl) thiosemicarbazide (Stemazole)

Article abstract of DOI:10.1016/j.ejmech.2011.04.017

Stem/progenitor cells are crucial for cell-based therapy and regenerative medicine, and their application in clinical and basic research requires a large supply of cells. To identify effective stem/progenitor cell proliferation activators, we synthesised a series of new 4-(4-(5-mercapto-1,3,4-oxadiazol-2- yl)phenyl) thiosemicarbazide (named Stemazole) derivatives. Preliminary evaluation of the structure-activity relationship (SAR) and the biological activities of the compounds were determined with a luminescent cell viability assay. The identified leading compound, Stemazole, exhibited remarkable proliferation-promoting activity in human hippocampal stem/progenitor cells (HSCs) in a time-dependent and concentration-dependent manner. The proliferation-promoting activity of Stemazole was further confirmed against a panel of human stem/progenitor cells derived from each of the three blastoderm layers. In conclusion, Stemazole is a novel activator of stem cells proliferation.

Full text of DOI:10.1016/j.ejmech.2011.04.017

European Journal of Medicinal Chemistry 46 (2011) 2930e2936
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a novel human stem/progenitor
cells proliferation activator: 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)
thiosemicarbazide (Stemazole)
*
Ying Sun, Wei Wang, Yuanyuan Sun, Mei Han
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Stem/progenitor cells are crucial for cell-based therapy and regenerative medicine, and their application
in clinical and basic research requires a large supply of cells. To identify effective stem/progenitor cell
proliferation activators, we synthesised a series of new 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)
thiosemicarbazide (named Stemazole) derivatives. Preliminary evaluation of the structureeactivity
relationship (SAR) and the biological activities of the compounds were determined with a luminescent
cell viability assay. The identified leading compound, Stemazole, exhibited remarkable proliferation-
promoting activity in human hippocampal stem/progenitor cells (HSCs) in a time-dependent and
concentration-dependent manner. The proliferation-promoting activity of Stemazole was further
confirmed against a panel of human stem/progenitor cells derived from each of the three blastoderm
layers. In conclusion, Stemazole is a novel activator of stem cells proliferation.
Received 31 January 2011
Received in revised form
21 March 2011
Accepted 4 April 2011
Available online 12 April 2011
Keywords:
Human stem/progenitor cell
4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)
phenyl) thiosemicarbazide
Stemazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cell viability assay
Proliferation activator
1. Introduction
widespread use in cell culture, these activators have obvious
shortcomings. For example, the composition of serum is complex
Stem cell research is an active and highly attractive field of
research [1e3]. Diseases due to the loss or damage of functional
cells, such as Alzheimer’s disease and Parkinson’s disease, can be
treated by mobilising endogenous stem cells to proliferate and/or
differentiate or by regenerative medicine through exogenous stem
cell-based replacement therapies. The application of stem cells in
clinical and basic research purposes currently requires a large
quantity of stem/progenitor cells. However, the number of stem
cells available in vivo is insufficient for research and clinical needs,
and the propagation of stem/progenitor primary cultures in vitro is
challenging. Effective activators of stem cell proliferation that can
mobilise and expand stem cells are urgently needed. Consequently,
their development and application is one of the most fundamental
and important priorities in stem cell research [4,5].
and incompletely defined. Stem cells that are cultured long-term in
the presence of serum tend to differentiate [8,9]. Irradiated feeder
cells presumably secrete indistinct cell growth factors that
encourage stem cell expansion but add undesirable complexity to
the stem cell microenvironment. Exogenous growth factors, such as
basic fibroblast growth factor (bFGF) or leukaemia inhibitory factor
(LIF), can support adult neural stem cell expansion under serum-
free conditions, but their ability to independently support long-
term maintenance proliferation of other types of adult stem cells
in the absence of serum or feeder cells is unclear [6,10].
Chemical approaches, including small molecules, have been the
focus of increasing interest as an alternative to traditional stem cell
proliferation activators. Chemical genetics has been used to identify
several small molecules capable of promoting stem cell prolifera-
The most frequently used stem cell proliferation activators are
serum, feeder cells and exogenous growth factors [6,7]. Despite
tion [11e13]. For example,
a
high-throughput screen of
50,000 compounds identified a heterocyclic compound, SC1, that
propagated murine embryonic stem cells (ES) in an undifferenti-
ated, pluripotent state under chemically defined conditions in the
absence of feeder cells, serum and LIF [14]. The same author,
S. Ding, later reported two small molecules, Tzv and Ptn, which
promoted human ES cells survival and self-renewal, respectively
[15]. Other significant small molecules have also been identified. A
high-throughput screen of 1,400,000 compounds identified eight
Abbreviations: Stemazole, 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl) thiose-
micarbazide; Br-Stemazole, 4-(2-bromo-4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)
thiosemicarbazide; HSCs, human hippocampal stem/progenitor cells; PSCs, human
pancreatic stem/progenitor cells; CSCs, human cardiac stem/progenitor cells.
* Corresponding author. Tel.: þ86 10 62207786; fax: þ86 10 58802075.
E-mail address: hanmei@bnu.edu.cn (M. Han).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.017

Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
2931
compounds with unpublished structures that stimulated mouse
adult SVZ progenitor cell proliferation [16]. The natural product
6-bromoindirubin-30-oxime (BIO) maintains human or murine ES
cells in an undifferentiated pluripotent state in conditioned
medium via activation of the Wnt signalling pathway and promotes
adult myocardial cell proliferation [17,18].
Although the number of known small molecule proliferation
activators is small, natural or synthesised small molecule prolifera-
tion activators have distinct advantages [14,19e23]. First, small
molecule compounds allow the proliferative culture conditions to be
chemically defined, precisely controlling stem cell fate and avoiding
the influence of indistinct exogenous factors in the stem cell culture
environment [24]. Second, small molecules might simultaneously
modulate multiple specific targets in a synergistic manner, poten-
tially increasing the homogeneity of the harvested stem cells. Third,
small molecules act in a temporal and adjustable way, making them
useful tools for manipulating stem cells and investigating the
complex molecular mechanisms that modulate stem cell fate. Fourth,
small molecules have the greatest potential for development into
new drugs targeted at stem cells [25]. Future stem cell drugs could
treat or prevent diseases due to the loss or damage of functional cells
by regulating the proliferation and/or differentiation of internal stem
cells and rebuilding lost or damaged functional cells. Small molecule
stem cell drugs not only resolve the limitations of adult stem cell
resources and the ethical concerns associated with ES cells but also
avoid immune rejection and operational sequelae resulting from
stem cell transplantation. In light of these advantages, small mole-
cules have become the subject of intense interest and research.
We performed a high-throughput chemical screen of approxi-
mately25,000compoundstoidentifysmallmoleculesthatpromoted
stem cell proliferation. As a result of the screen, one compound,
a 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl) thiosemicarbazide
(Stemazole), wasused forsubsequentresearch.Basedontheskeleton
structure of Stemazole (Scheme 1), we designed several novel
heterocyclic small molecules and synthesised them in this study. The
novel compounds were characterised by 1H NMR,13C NMR and mass
spectrometry. Preliminary evaluations of the structureeactivity
relationship (SAR) were performed to gain insight into the substi-
tution pattern required for the greatest biological potency in human
foetal-derived hippocampal stem/progenitor cells (HSCs) under
serum-free culture conditions. From the SAR analysis, we identified
thestrongestproliferation-promotingcompound1, Stemazole, asthe
lead compound, which was used in the subsequent time-response
and dose-response studies. To further confirm the proliferation-
promoting activity of Stemazole, we assessed its effect on a panel of
human stem/progenitor cells derived from each of the three blasto-
derm layers (HSCs, ectoderm; PSCs, human pancreatic stem/
progenitor cells, endoderm; and CSCs, human cardiac stem/progen-
itor cells, mesoderm).
Scheme 2. The synthetic route to Stemazole (1) and its derivatives (2e7).
Stemazole (1) was prepared in four steps. Compound I (methyl
4-aminobenzoate) was used as the starting material. Compound III
(4-aminobenzohydrazide) was first synthesised from starting
material I and hydrazine hydrate by hydrazinolysis. Compound IV
was obtained by cyclisation of compound III and carbon disulphide.
Compound IV plus thiophosgene afforded product V (5-(4-iso-
thiocyanatophenyl)-1,3,4-oxadiazole-2-thiol). Lastly, Stemazole (1)
was obtained from V by a thiosemicarbazone reaction with
hydrazine hydrate. The series of Stemazole derivatives was syn-
thesised by a similar procedure. All compounds were stable in the
solid state and were soluble in dimethyl sulfoxide (DMSO).
2. Results and discussion
2.1. Chemistry
The synthesis of the series of new Stemazole derivatives is
illustrated in Scheme 2.
2.2. Biological activities
2.2.1. Preliminary structureeactivity relationship evaluation
To evaluate the SAR of Stemazole and its derivatives, we used
HSCs as our stem cell scanning model. DMSO was used as the
solvent for the tested compounds and as the negative control. The
Scheme 1. Chemical structures of the 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)
thiosemicarbazide scaffold (A) and Stemazole (B).

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Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
Table 1
effects of the small molecules were represented by cell viability (%).
The cell viability (%) was defined as the percentage change in cell
viability relative to the DMSO negative control: cell viability
(%) ¼ cell viabilitysample/cell viabilityDMSO ꢀ 100%. The screening
experiment identified obvious differences in the biological activi-
ties of the compounds (Fig.1). The EC50 and IC50 values of the tested
compounds promoting/inhibiting HSCs proliferation are shown in
Table 1. Compounds 1 (Stemazole) and 7 (bromine-substituted
Stemazole) promoted stem cell proliferation in vitro. Compound 3
(benzyl-substituted Stemazole) had no activity. Compounds 4
(phenyl-substituted Stemazole), 5 (para-chlorophenyl-substituted
Stemazole) and 6 (para-anisyl-substituted Stemazole) were signif-
icantly cytotoxic. Compound 2 (methyl-substituted Stemazole) was
weakly cytotoxic at high concentrations. The R1 position can likely
tolerate a substituent; compound 7 (Br-Stemazole), which contains
a bromine substituent at the R1 position, was similar to Stemazole
with respect to its proliferation-promoting activity.
The EC50 and IC50 values of active compounds promoting/inhibiting HSCs
proliferation.
No. of compounds
EC50
(
mM)
IC50(mM)
1
2
3
4
5
6
7
7.5
ND
ND
ND
ND
ND
10.0
ND
ND
ND
8.3
3.2
42.7
ND
“ND’’ means “Not Detected’’.
The EC50 (median effective concentration) represents the concentration of
a compound required to induce a 50% promoting-proliferation effect. The IC50 (half-
inhibitory concentration) represents the concentration of a compound required to
induce a 50% against-proliferation effect.
observation, cell number counting and a cell viability assay. As
expected, cell proliferation increased with time after the addition of
Stemazole, which continued until the end of the experiment. The
cell number increased approximately three-fold after six days,
which was similar to the bFGF positive control (Fig. 2).
To identify a lead compound for subsequent research, we further
compared the biological activity of Stemazole and Br-Stemazole in
HSCs. Although the proliferation-promoting activity of Br-Stemazole
was similar to that of Stemazole, its effective toxicity window was
more narrow than Stemazole; at approximately 100
mM, Br-
2.2.3. Stemazole showed similar promoting-proliferation activity
with bFGF and no observed differentiation potential on HSCs
To be useful, new activators should generally be equivalent or
surpass existing activators. Therefore, we compared the effects of
Stemazole and the frequently used stem cell growth factor bFGF
(positive control). The experimental results revealed that both
Stemazole exhibited weak cytotoxicity, which was not observed
with Stemazole at this concentration. The maximum proliferation
effect of Br-Stemazole was also lower than that of Stemazole (Fig. 1).
Based on its proliferation-promoting activity and relatively low
cellular toxicity, compound 1, Stemazole, was selected from this set
of new compounds as the lead compound for subsequent research.
Stemazole (population doubling timeStemazole
¼
48.5 h,
concentrationStemazole ¼ 41.6 M) and bFGF (population doubling
m
2.2.2. The promoting-proliferation effects of Stemazole on HSCs are
time- and dose-dependent
Stemazole was evaluated by dose-response assays and time-
response assays in HSCs under serum-free conditions without the
growth factor bFGF. Stemazole had a very clear dose-response
timebFGF ¼ 47.0 h, concentrationbFGF ¼ 20 ng/mL) significantly
promoted HSCs proliferation relative to DMSO (negative control,
population doubling timeDMSO ¼ 73.6 h, concentrationDMSO ¼ 0.08%).
The proliferation-promoting effects of both molecules were similar
whether cells were treated for four or six days. No significant differ-
ences were observed between Stemazole and bFGF (Fig. 3).
relationship between approximately 5 and 40 mM. The maximum
effect was approximately three-fold higher relative to the DMSO
negative control.
The proliferation-promoting activity of Stemazole was also
distinctly time-dependent in HSCs. We performed a six-day
proliferation experiment. The cells were measured every two days
by three methods, including direct phase contrast microscope
Additionally, the morphology of the cells treated with Ste-
mazole and bFGF were evaluated. Again, the effects of Stemazole
and bFGF were very similar. HSCs treated with the DMSO nega-
tive control appeared somewhat attached and inactive, whereas
the cells treated with Stemazole or bFGF appeared clear, round
Fig. 1. The structureeactivity relationship of Stemazole derivatives and identification of the lead compound. (A) Response of HSCs to Stemazole derivatives. HSCs were treated with
Stemazole (1) and its derivatives (2e7) for four days, and their effects were analysed with a CellTiter-Glo luminescent cell viability assay. Compounds 1 and 7 promoted stem cell
proliferation, while compounds 4, 5 and 6 exhibited significant cytotoxicity. DMSO-treated cells were used as the control (100%). (B) Comparison of the biological activities of
Stemazole (1) and Br-Stemazole (7) on HSCs. HSCs were treated with 1 or 7 for four days. Compounds 1 and 7 exhibited similar biological activities, but the effective toxicity window
of 1 was wider than that of 7, and the maximum proliferation effect of 1 was higher than that of 7. The values presented are the mean ꢁ SD.

Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
2933
Fig. 2. The proliferation of HSCs treated with Stemazole. (A) Phase contrast images of HSCs treated with various concentrations of Stemazole, DMSO (negative control) and bFGF
(positive control) at the indicated times. Bar: 50 m. (B) The proliferation of HSCs treated with various concentrations of Stemazole as assessed by cell number counting at the indicated
m
times. *P < 0.05 compared to DMSO. **P < 0.001 compared to DMSO. The values presented are the mean ꢁ SD. (C) The proliferation of HSCs treated with various concentrations of
Stemazole as assessed by cell viability assays at the indicated times. *P < 0.05 compared to DMSO. **P < 0.001 compared to DMSO. The values presented are the mean ꢁ SD.
and compact in shape, indicating good cell viability and possible
proliferation in progress. No morphological differentiation such
as axonal growth or adipose drop appearance was visible
(Fig. 2A).
2.2.4. Stemazole has similar proliferation-promoting effects on
different stem/progenitor cells
In addition to HSCs, we also examined PSCs and CSCs. Humans
develop from fertilised eggs through three embryonic germ layers,

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Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
Fig. 3. Stemazole and bFGF have similar proliferation-promoting properties. (A) Comparison of the proliferation-promoting activities of Stemazole and bFGF in HSC cells as assessed
by a cell number counting assay. Both Stemazole and bFGF significantly promoted proliferation relative to the DMSO negative control. There was no significant difference between
Stemazole and bFGF. DMSO: 0.8%; Stemazole: 41.33
proliferation-promoting activities of Stemazole and bFGF in HSC cells as assessed by the CellTiter R-Pro luminescent cell viability assay. Both Stemazole and bFGF significantly
m
M; bFGF: 20 ng/mL. The values presented are the mean ꢁ SD. **P < 0.001 compared to DMSO. (B) Comparison of the
promoted proliferation relative to the DMSO negative control. There was no significant difference between Stemazole and bFGF. DMSO: 0.8%; Stemazole: 41.33
The values presented are the mean ꢁ SD. **P < 0.001 compared to DMSO.
mM; bFGF: 20 ng/mL.
the ectoderm, endoderm and mesoderm. When testing the
promoting-proliferation activity of a compound on stem cells, the
best and most comprehensive technique is to examine stem cells
derived from the above three embryonic germ layers. In the pre-
sented study, we selected three representative stem cells, including
hippocampal stem/progenitor cells (HSCs, representing ectoderm
stem cells), human pancreatic stem/progenitor cells (PSCs, repre-
senting endoderm stem cells), and human cardiac stem/progenitor
cells (CSCs, representing mesoderm stem cells), to evaluate the
promoting-proliferation activity of Stemazole. These stem/
progenitor cells were treated with Stemazole for four days. Cell
viability assays showed that all the tested stem/progenitor cells had
different but significant levels of proliferation. Among the
progenitor cells, the ectoderm-derived HSCs appeared to be the
most sensitive to Stemazole. The maximum proliferation effect was
approximately three-fold higher relative to DMSO. The endoderm-
derived PSCs responded to Stemazole in a similar fashion as the
HSCs. The mesoderm-derived CSCs also responded to Stemazole,
but the response was slightly weaker than in the other two cell
lines (Fig. 4). The difference is likely because the different types and
developing stages of the stem cells expand at different speeds, and
their responses to the same regulator are often not identical. In
summary, Stemazole exerted significant proliferative effects on
stem/progenitor cells derived from the different germ layers.
3. Conclusion
Seven Stemazole derivatives of previously unpublished struc-
ture were synthesised and characterised by 1H NMR, 13C NMR and
mass spectrometry. Preliminary SAR studies revealed that Stema-
zole (1) and Br-Stemazole (7) possessed significant stem/progenitor
cell proliferation-promoting activity. In contrast, compounds 4, 5
and 6 were noticeably cytotoxic. Compound 2 was weakly cyto-
toxic, and compound 3 had no influence on the human HSCs tested.
Stemazole was the strongest stem/progenitor cell proliferation
activator among the set of tested compounds, and it stimulated
human HSCs proliferation in
a time-dependent and dose-
dependent manner. The observed proliferation-promoting effect
was reproduced in HSCs, PSCs and CSCs, which represent stem/
progenitor cells from all three human blastoderm layers. The
results suggest that Stemazole is a novel and potent general acti-
vator of stem/progenitor cell expansion and has the ability to play
a critical role in the survival and expansion of multiple types of
stem/progenitor cells in vitro. Thus, Stemazole has significant
potential as a prospective stem cell drug. Identification of the effect
of Stemazole on stem/progenitor cells in vivo and elucidation of the
molecular mechanism underlying the proliferative phenomenon
are in progress.
Fig. 4. The proliferation of multiple types of stem/progenitor cells derived from each of
the three blastoderm layers treated with Stemazole for four days. HSCs: human
hippocampal stem/progenitor cells, ectoderm; PSCs: Human pancreatic stem/progen-
itor cells, endoderm; CSCs: Human cardiac stem/progenitor cells, mesoderm. All three
human blastoderm-derived stem/progenitor cells responded to Stemazole. HSCs were
most sensitive to Stemazole, PSCs responded to Stemazole in a similar fashion. CSCs
also responded to Stemazole, but the response was slightly weaker than in the other
two cell lines. *P < 0.05 compared to DMSO. **P < 0.001 compared to DMSO. The
values presented are the mean ꢁ SD.

Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
2935
4. Experimental protocols
(2C), 124.73, 123.14, 121.01; HRMS (ESI) found: 268.0331,
[C9H9N5OS2 þ H]þ calcd: 268.0327.
4.1. Materials and general methods
4.2.2. Synthesis of 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)-
1-methythiosemicarbazide (2)
Compound 2 was obtained from compound V (235 mg) and 40%
methyl hydrazine aqueous solution (233.5 mL) following the
general procedure as a white solid (76 mg). Total yield 10.1%, 1H
Chemical reagents and organic solvents were purchased from
SigmaeAldrich (MO, USA) unless otherwise mentioned. All com-
mercial reagents and solvents for the reactions were of analytical
grade. The structures of the novel synthetic compounds were
identified by 1H NMR,13C NMR and mass spectrometry. NMR spectra
were obtained on a Bruker Avance III 400 in DMSO-d6 (1H: 400 MHz,
13C: 100 MHz). Chemical shifts are reported in parts per million
(ppm) downfield from tetramethylsilane (TMS). Spin multiplicities
are described as s (singlet), d (doublet), t (triplet), q (quartet) and m
(multiplet). Coupling constants are reported in Hertz (Hz). Mass
spectra (ES) were determined on a high-resolution Micromass LCT
Premier XE (Waters, UK). Thin-layer chromatography was per-
formed on pre-coated Kieselgel 60 F254 plates (Merck, GER).
Dulbecco’s Modified Eagle Media, Nutrient Mixture F-12
(DMEM/F12), Dulbecco’s Modified Eagle Media (DMEM), low-
glucose Dulbecco’s Modified Eagle Media, Iscove’s modified Dul-
becco’s medium (IMDM) and foetal bovine serum (FBS) were
purchased from Gibco (CA, USA). Basic fibroblast growth factor
(bFGF), B27 and epidermal growth factor (EGF) were purchased
from Invitrogen (CA, USA). DMSO was purchased from Sigma-
eAldrich (MO, USA). The CellTiter-GloÔ luminescent cell viability
assay kit was purchased from Promega (WI, USA).
NMR (400 MHz, DMSO-d6):
d
3.541 (s, 3H), 7.710 (d, J ¼ 11.6 Hz, 2H),
7.787 (d, J ¼ 11.2 Hz, 2H); 13C NMR (100 MHz, DMSO-d6):
d 178.33,
177.64, 160.51 (2C), 142.98 (2C), 125.73, 123.14, 117.92, 42.22; HRMS
(ESI) found: 282.0483, [C10H11N5OS2 þ H]þ calcd: 282.0483.
4.2.3. Synthesis of 1-benzyl-4-(4-(5-mercapto-1,3,4-oxadiazol-2-
yl)phenyl)thiosemicarbazide (3)
Compound 3 was obtained from compound V (235 mg) and
benzyl-hydrazine hydrochloride (319.2 mg) following the general
procedure as a white solid (60 mg). Total yield 2.1%, 1H NMR
(400 MHz, DMSO-d6):
d
5.327 (s, 2H), 7.333(m, 5H), 7.792 (d, 2H),
179.08, 177.25,
7.889 (d, 2H); 13C NMR (100 MHz, DMSO-d6):
d
160.45 (2C), 143.42 (2C), 136.36, 128.45, 127.79, 127.31, 126.06 (2C),
123.55 (2C), 117.47, 56.20; HRMS (ESI) found: 358.0795,
[C16H15N5OS2þH]þ calcd: 358.0796.
4.2.4. Synthesis of 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)-
1-phenylthiosemicarbazide (4)
Compound 4 was obtained from compound V (235 mg) and
phenylhydrazine (216.28 mg) following the general procedure as
a brownish yellow solid (122 mg). Total yield 13.2%, 1H NMR
4.2. General procedures for the preparation of Stemazole 1 and
derivatives 2 through 7
(400 MHz, DMSO-d6):
d 6.817e6.733 (m, 3H), 7.222e7.184 (m, 2H),
Compounds 1e7 were prepared according to Scheme 2. The
details of the experimental protocol are provided below.
7.774 (d, J ¼ 8.40 Hz, 2H), 7.866 (d, J ¼ 8.80 Hz, 2H), 8.093 (s, 1H),
9.898 (s, 1H), 10.098 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d
180.84, 177.25, 160.40 (2C), 147.69 (2C), 142.77, 128.86, 125.89,
4.2.1. Synthesis of 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)
thiosemicarbazide (1)
124.77,120.01,117.99 (2C),113.11 (2C); HRMS (ESI) found: 344.0646,
[C15H13N5OS2 þ H]þ calcd: 344.0640.
Compound I (45 g) was added in batches to a solution of 450 mL
of hydrazine hydrate. The mixture was heated to reflux for 1 h and
was monitored by TLC. After cooling to r.t., the excess hydrazine
hydrate was evaporated under reduced pressure. The residue was
poured into 50 mL of methanol, and the white solid compound III
was filtered and dried to afford 45 g.
4.2.5. Synthesis of 1-(4-chlorophenyl)-4-(4-(5-mercapto-1,3,4-
oxadiazol-2-yl)phenyl)thiosemicarbazide (5)
Compound 5 was obtained from compound V (235 mg) and 4-
Chloro-phenylhydrazine hydrochloride (358.1 mg) following the
general procedure as a brownish yellow solid (78 mg). Total yield
KOH (22 g) was added at 0 ꢂC to a stirred solution of compound
III (30 g) in anhydrous ethanol (400 mL) and DMF (50 mL). The
reaction mixture was stirred for 10 min under N2. To this mixture,
CS2 (45 g) was added dropwise at 0 ꢂC. The mixture was warmed to
ambient temperature for 0.5 h and heated to reflux overnight until
no starting material remained. The pH was adjusted to pH 4 with
2 N HCl, and the solid was filtered and dried to afford 19 g of yellow
solid compound IV.
7.7%, 1H NMR (400 MHz, DMSO-d6):
d
6.745 (d, J ¼ 8.80 Hz, 2H),
7.260 (d, J ¼ 8.80 Hz, 2H), 7.780 (d, J ¼ 8.80 Hz, 2H), 7.845 (d,
J ¼ 8.80 Hz, 2H), 8.251 (s, 1H), 9.933 (s, 1H), 10.111 (s, 1H); 13C NMR
(100 MHz, DMSO-d6):
d 180.89, 179.26, 170.26 (2C), 160.59 (2C),
146.92 (2C), 140.61, 128.62, 124.96, 124.73, 124.16, 114.54 (2C); MS
(EMS): m/z [M þ H]þ 378.3 (100); HRMS (ESI) found: 378.0248,
[C15H12N5OS2Cl þ H]þ calcd: 378.0250.
To a stirred solution of compound IV (6.1 g) in acetone (40 mL),
sulphur phosgene (2.9 mL) in acetone (11 mL) and saturated
NaHCO3 were added dropwise simultaneously at 0 ꢂC. The mixture
was then warmed to ambient temperature for 0.5 h. The mixture
was poured into water to quench the reaction. The reaction was
extracted with EtOAc, and the combined solution was dried with
Na2SO4, filtered and evaporated under reduced pressure to give
5.7 g of yellow solid compound V.
4.2.6. Synthesis of 4-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)-
1-(4-methoxyphenyl)thiosemicarbazide (6)
Compound 6 was obtained from compound V (235 mg) and 4-
methoxy-phenylhydrazine hydrochloride (349.26 mg) following
the general procedure as a light brownish yellow solid (184 mg).
Total yield 18.3%, 1H NMR (400 MHz, DMSO-d6):
d 3.741 (s, 3H),
6.927(d, J ¼ 8.80 Hz, 2H), 7.319 (d, J ¼ 8.80 Hz, 2H), 7.792 (d,
J ¼ 9.20 Hz, 2H), 7.966 (d, J ¼ 8.80 Hz, 2H); 13C NMR (100 MHz,
Compound V (100 g) was suspended in toluene (1000 mL), and
hydrazine hydrate (42.5 mL) was slowly added. The mixture was
stirred at r.t. under N2 until no starting material remained. The
organic layer was separated, and the solid was filtered and washed
with ethanol to afford 60 g of white solid compound 1, Stemazole.
DMSO-d6): d 178.92, 177.26, 160.45 (2C), 157.77 (2C), 143.21(2C),
138.71,128.63,126.04,123.43,117.49,113.70 (2C), 55.32; HRMS (ESI)
found: 374.0748, [C16H15N5O2S2þH]þ calcd: 374.0745.
4.2.7. Synthesis of 4-(2-bromo-4-(5-mercapto-1,3,4-oxadiazol-2-
yl)phenyl)thiosemicarbazide (7)
Total yield 14.6%, 1H NMR (400 MHz, DMSO-d6):
d
5.002 (s, 1H),
7.056 (s, 3H), 7.727 (d, J ¼ 8.40 Hz, 2H), 7.871(d, 2H), 9.285 (S, 1H);
13C NMR (100 MHz, DMSO-d6):
179.44, 179.05, 160.68 (2C), 140.34
To a stirred solution of compound I (1.51 g) in acetic acid Br2
d
(1.59 g) was added at 0 ꢂC. A large quantity of white solid was

2936
Y. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 2930e2936
obtained. TCL result showed that no starting material remained. The
solid was purified by column chromatography on silica gel
(DCM:CH3OH ¼ 20:1) to afford a white solid compound II (1.4g). The
subsequent reaction followed the procedure outlined in Section 4.2.1.
Total yield 0.7%, brownish yellow, 1H NMR (400 MHz, DMSO-d6):
the luciferase/luciferin reaction. In the presence of Mg2þ and ATP,
the luminescence produced was proportional to the amount of ATP
present, which is an indicator of cellular metabolic activity. The
luminescent signal was directly proportional to the number of
viable cells. Briefly, the assay was performed by adding CellTiter-
GloÔ reagent directly to cells in culture. After mixing and incu-
bating for 10 min at ambient temperature, luminescence was
measured with a Multilabel Counter (PE, 1420, VICTOR3TM).
d
7.034 (s, 3H), 7.731 (dd, J1 ¼ 11.4 Hz, J2 ¼ 1.60 Hz, 1H), 7.955 (d,
J ¼ 1.60 Hz, 1H), 8.647 (d, J ¼ 8.80 Hz, 1H), 9.556 (s, 1H); 13C NMR
(100 MHz, DMSO-d6):
d 179.38, 178.73, 159.26, 138.54, 128.52,
124.86, 123.93, 121.62, 116.51; HRMS (ESI) found: 345.9439,
[C9H8N5S2BrO þ H]þ calcd: 345.9432.
4.3.5. Statistical analysis
All experiments were performed at least in triplicate at each
concentration level and repeated three times. Data are presented as
the mean ꢁ SD. Statistical comparisons were performed using
a Student’s t-test. A value of P < 0.05 was considered statistically
significant. A value of P < 0.001 was considered markedly signifi-
cant. The EC50 and IC50 values of the compounds were calculated
based on the relationship of cell viability vs. the corresponding
concentration by the SPSS software (version 13.0).
4.3. Biology
4.3.1. Preparation of stem/progenitor cells
Human stem/progenitor cells were kindly supplied by Prof.
Lingsong Li (Peking University Stem Cell Research Center, Beijing,
China). The stem/progenitor cells were maintained according to
documented methods [26e29].
4.3.1.1. Maintenance of HSCs. The HSCs were maintained in DMEM/
F12 supplemented with 20 ng/mL bFGF, 20 ng/mL EGF, 2% B27,
100 IU/mL penicillin and 100 IU/mL streptomycin at 37 ꢂC in a 5%
CO2-humidified atmosphere. The medium was refreshed every
2e3 days. The cells were passaged by gently triturating the
resulting neurospheres into a quasi-single cell suspension once per
week. The sixth passage HSCs were used to evaluate the biological
characteristics of Stemazole.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (grant number 30672491) and the National
Key Technology Research and Development Program (grant
number 2008BAI49B04). The authors would like to thank Prof.
Lingsong Li for the stem/progenitor cells supply and Dr. Jun Wen for
synthesis guidance.
4.3.1.2. Maintenance of PSCs. PSCs were expanded in DMEM/F12
(7.33 mM glucose) with 10% FBS, 20 ng/mL bFGF, 100 IU/mL peni-
cillin and 100 IU/mL streptomycin at 37 ꢂC in a 5% CO2-humidified
atmosphere. The cells proliferated rapidly in the expansion
medium. The sixth passage PSCs were used to evaluate the bio-
logical characteristics of Stemazole.
References
[1] J.C. Gerlach, K. Zeilinger, Int. J. Artif. Organs 25 (2002) 83e90.
[2] S. Kume, Dev. Growth Differ. 47 (2005) 393e402.
[3] S.A. Moody, Biol. Cell 97 (2) (2005) 111.
[4] R. Lanza, N. Rosenthal, Sci. Am. 290 (2004) 92e99.
[5] G.V. Glinsky, Cell Cycle 7 (2008) 3292e3293.
[6] A. Gritti, E.A. Parati, L. Cova, P. Frolichsthal, R. Galii, E. Wanke, L. Faravelli,
D.J. Morassutti, F. Roisen, D.D. Nickel, A.L. Vescovi, J. Neurosci. Res. 16 (1996)
1091e1100.
[7] A.L. Vescovi, B.A. Reynolds, D.D. Fraser, S. Weiss, Neuron 11 (1993)
951e966.
[8] H.F. Chen, H.C. Kuo, C.L. Chien, C.T. Shun, Y.L. Yao, P.L. Ip, C.Y. Chuang,
C.C. Wang, Y.S. Yang, H.N. Ho, Hum. Reprod. 22 (2007) 567e577.
[9] A. Shahdadfar, K. Fronsdal, T. Haug, F.P. Reinholt, J.E. Brinchmann, Stem Cells
23 (2005) 1357e1366.
[10] B.A. Reynolds, W. Tetzlaff, S. Weiss, J. Neurosci. 12 (1992) 4565e4574.
[11] Y. Xu, Y. Shi, S. Ding, Nature 453 (2008) 338e344.
[12] Y. Shi, J.T. Do, C. Desponts, H.S. Hahm, H.R. Scholer, S. Ding, Cell Stem Cell 2
(2008) 525e528.
4.3.1.3. Maintenance of CSCs. CSCs were maintained in IMDM
supplemented with 10% FBS, 2-mercaptoethanol, 100 IU/mL peni-
cillin and 100 IU/mL streptomycin at 37 ꢂC in a 5% CO2-humidified
atmosphere. The cells were passaged to the next generation upon
reaching greater than 90% confluence. The third passage CSCs were
used to evaluate the biological characteristics of Stemazole.
4.3.2. Measurement of proliferation-promoting activity
Exponentially growing cells were seeded on 384-well black wall
clear bottom plates (Corning, USA) with a Multidrop Combi
(Thermal, USA) at a density of 1 ꢀ 103 cells/well in basic culture
medium and incubated for 24 h. Serial dilutions of the small
molecules under investigation were added to the planted cells.
DMSO was used as the negative control, and bFGF was used as the
positive control. Cells were exposed to the synthetic compounds for
4 days (in the time-response assay, the exposure times were 2 days,
4 days and 6 days). The proliferation-promoting activities of the
tested compounds were assessed by cell counting and a CellTiter-
GloÔ luminescent cell viability assay.
[13] E. Theodorou, G. Dalembert, C. Heffelfinger, E. White, S. Weissman,
L. Corcoran, M. Snyder, Genes Dev. 23 (2009) 575e588.
[14] S. Yao, S. Chen, J. Clark, E. Hao, G.M. Beattie, A. Hayek, S. Ding, Proc. Natl. Acad.
Sci. U.S.A. 103 (2006) 6907e6912.
[15] Y. Xu, X.W. Zhu, H.S. Hahm, W.G. Wei, E.G. Hao, A. Hayek, S. Ding, Proc. Natl.
Acad. Sci. U.S.A. 107 (2010) 8129e8134.
[16] Y.P. Liu, R. Lacson, J. Cassaday, D.A. Ross, A. Kreamer, E. Hudak, R. Peltier,
D. McLaren, I. Munoz-Sanjuan, F. Santini, B. Strulovici, M. Ferrer, J. Biomol.
Screen. 14 (2009) 319e329.
[17] N. Sato, L. Meijer, L. Skaltsounis, P. Greengard, A.H. Brivanlou, Nat. Med. 10
(2004) 55e63.
[18] A.S. Tseng, F.B. Engel, M.T. Keating, Chem. Biol. 13 (2006) 957e963.
[19] S. Ding, P.G. Schultz, Nat. Biotechnol. 22 (2004) 833e840.
[20] S. Ding, T.Y.H. Wu, A. Brinker, E.C. Peters, W. Hur, N.S. Gray, P.G. Schultz, Proc.
Natl. Acad. Sci. U.S.A. 100 (2003) 7632e7637.
[21] S.B. Chen, S. Hilcove, S. Ding, Mol. Biosyst. 2 (2006) 18e24.
[22] S. Ding, P.G. Schultz, Curr. Top. Med. Chem. 5 (2005) 383e396.
[23] N. Emre, R. Coleman, S. Ding, Curr. Opin. Chem. Biol. 11 (2007) 252e258.
[24] H. Wu, J. Xu, Z.P. Pang, W. Ge, K.J. Kim, B. Blanchi, C. Chen, T.C. Sudhof,
Y.E. Sun, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 13821e13826.
[25] M. Han, W. Zhang, J. Li, Y. Sun, Y. Zhu, Y. Wang, Zhongguo Yaolixue Tongbao
24 (2008) 841e844.
[26] B.A. Reynolds, S. Weiss, Science 255 (1992) 1707e1710.
[27] O. Brustle, A.C. Spiro, K. Karram, K. Choudhary, S. Okabe, R.D. McKay, Proc.
Natl. Acad. Sci. U.S.A. 94 (1997) 14809e14814.
4.3.3. Cell counting
Cell images were captured at the tested time points with
a digital DP70 colour camera (Olympus, Japan) connected to a IX-
51 microscope (Olympus, Japan). Three cell pictures were counted
for each group. The total cell numbers in the pictures were counted
with a haemocytometer.
4.3.4. Cell viability assay
The viability of the cells was quantitatively analysed by the
CellTiter-GloÔ luminescent cell viability assay, which is based on
[28] H. Huang, X. Tang, Lab. Invest. 83 (2003) 539e547.
[29] A.P. Beltrami, L. Barlucchi, D. Torella, M. Baker, F. Limana, S. Chimenti,
H. Kasahara, M. Rota, E. Musso, Cell 114 (2003) 763e776.