Synthesis and evaluation of the diarylthiourea analogs as novel anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2015.01.042

Ten p-nitrodiarylthiourea analogs were designed, synthesized and evaluated in breast (MCF-7, T-47D, MDA-MB-453) and prostate (DU-145, PC-3, LNCaP) cancer cell lines for their anticancer activities. The majority of the compounds were able to inhibit the growth of these six cancer cell lines at low micromolar concentrations. Compound 7 was found to be the most potent anticancer agent in this series with GI₅₀ values of 3.16 μM for MCF-7, 2.53 μM for T-47D, 4.77 μM for MDA-MB-453 breast cancer lines and 3.54 μM for LNCaP prostate cancer cell line. These GI₅₀ values were comparable to the parent compound, SHetA2.

Full text of DOI:10.1016/j.bmcl.2015.01.042

Accepted Manuscript
Synthesis and evaluation of the diarylthiourea analogs as novel anti-cancer
agents
Shengquan Liu, Maggie C. Louie, Vanishree Rajagopalan, Guangyan Zhou,
Esmeralda Ponce, Tran Nguyen, Linda Green
PII:
S0960-894X(15)00054-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.01.042
BMCL 22382
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 November 2014
17 January 2015
20 January 2015
Please cite this article as: Liu, S., Louie, M.C., Rajagopalan, V., Zhou, G., Ponce, E., Nguyen, T., Green, L., Synthesis
and evaluation of the diarylthiourea analogs as novel anti-cancer agents, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.01.042
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

FULL TITLE: Synthesis and evaluation of the diarylthiourea analogs as novel anti-cancer agents
AUTHORS:
Shengquan Liua,*, Maggie C. Louiea,c, Vanishree, Rajagopalana , Guangyan Zhoub, Esmeralda Poncec,
Tran Nguyena, and Linda Greenc
aCollege of Pharmacy, Touro University-California, Vallejo, CA 94592, USA
bDepartment of Basic Science, Touro University-California, Vallejo, CA 94592, USA
cDepartment of Natural Science and Mathematics, Dominican University of California, San Rafael,
CA 94901, USA
*Corresponding author
Phone: (707) 638-5983
Fax: (707) 638-5959
Email: shengquan.liu@tu.edu
*Reprint requests should be addressed to the corresponding author.
KEYWORDS: SHetA2; diarylthiourea analogs; anticancer agents; breast cancer; prostate cancer

ABSTRACT:
Ten p-nitrodiarylthiourea analogs were designed, synthesized and evaluated in breast (MCF-7, T-
47D, MDA-MB-453) and prostate (DU-145, PC-3, LNCaP) cancer cell lines for their anticancer
activities. The majority of the compounds were able to inhibit the growth of these six cancer cell
lines at low micromolar concentrations. Compound 7 was found to be the most potent anticancer
agent in this series with GI50 values of 3.16 µM for MCF-7, 2.53 µM for T-47D, 4.77 µM for
MDA-MB-453 breast cancer lines and 3.54 µM for LNCaP prostate cancer cell line. These GI50
values were comparable to the parent compound, SHetA2.
GRAPHICAL ABSTRACT:
ABBREVIATIONS:
SHetA2: N-(2,3-dihydro-2,2,4,4-tetramethyl-6-benzothiopyranyl), N’-(4-nitrophenyl)thiourea;
ATRA: all trans retinoic acid; NOAEL: no observed adverse effect level; MTT: 3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; MTS: MTT coupled with phenazine
methosulfate (soluble form of MTT) ; ER: estrogen receptor; AR: androgen receptor.
Previously, we synthesized N-(2,3-dihydro-2,2,4,4-tetramethyl-6-benzothiopyranyl), N’-(4-
nitrophenyl)thiourea (SHetA2, NSC 726189)1 which has been shown to be a novel potential cancer
prevention agent and is now in preclinical development for cancer prevention through the National
Cancer Institute (NCI) Rapid Access to Intervention Development (RAID) program. SHetA2
evolved from a lead optimization process from all trans-retinoid acid (ATRA, Tretinoin®, mean

GI50 is 51 µM, Figure 1),2-4 a drug used for the treatment of acute promyelocytic leukemia.
However, the exploitation of retinoic acid’s full potential as a chemopreventive and/or a therapeutic
drug, particularly against solid tumors, has been hampered mainly by its local and systemic toxicity
and side effects often associated with its ability to activate nuclear retinoid receptors.2 Thus, efforts
have been made to develop novel compounds that retain retinoid anticancer activity with reduced
retinoid toxicity and side effects. One such compound is SHetA2, a more effective anticancer agent
than ATRA and appears to function without activating retinoid receptors.1 As a result, SHetA2
does not exhibit retinoid toxicities when tested in animal models.3 SHetA2 exerts its selective
anticancer activity through regulating apoptosis, cell growth, differentiation and angiogenesis.5,6
SHetA2 has also been shown to induce proteasomal degradation of cyclin D1,7 generate
mitochondrial swelling and endoplasmic reticulum stress, promote the formation of reactive oxygen
species, and induce apoptosis in cancer cells while sparing normal cells.6 More importantly, it was
reported recently in a dog study, that no toxicity of SHetA2 was observed in any tested dose groups.
The lowest observed adverse effect level (NOAEL) for ShetA2 was not established and was
considered to be above 1,500 mg/kg/day.8
However, SHetA2 may not be an ideal drug candidate due to the following two limitations:
its high lipophilicity and cumbersome six step synthesis. SHetA2’s high lipophilicity (LogP =7.09)
is higher than the upper limit of “Lipinski rule of 5” and the Log P of most marketed drugs,9,10 and
this might contribute to its extremely low (<1%) systemic bioavailability for all doses tested in rat
and its high plasma protein binding (99.3-99.5% at low micromolar concentrations).6,11 This high
lipophilicity may also cause nonselectivity, liver toxicity and drug-drug interactions.12 Furthermore,
the synthesis of SHetA2 involves 6 steps, with low overall yield of 3%, which hinders its large scale
supply and the synthesis of new analogs.13 To address these issues, we have modified the SHetA2
structure based on the previous structure-activity relationship.1,2 We hypothesized that the
nitrophenyl group and the thiourea linker in SHetA2 structure are important for its anticancer
activity. Thus, our modification strategy was to keep these two moieties intact, while replacing the
thiochromane ring with another ring structure. The ring systems were selected so that designed
compounds follow the Lipinski rule, are drug-like, but are not reactive and do not produce reactive
metabolites. In this study, we selected aromatic ring systems so that the designed compounds
resemble SHetA2 but differ in their sizes and shapes. In this way, the structure modification will

reduce the lipophilicity of the designed compounds and simplify the six-step synthesis to one step
using readily available and inexpensive starting materials.
Here, we report the design, synthesis and biological activity of ten diarylthiourea analogs.
These compounds were synthesized according to our previously published procedure.1 The newly
synthesized compounds were evaluated for their ability to inhibit the growth of breast cancer cell
lines-MCF-7, T-47D, and MDA-MB-453 and prostate cancer cell lines-DU-145, LNCaP and PC-3.
The potency of compounds 5 and 7 in this series were shown to be comparable to the parent
compound, SHetA2.
General procedure for the preparation of compounds 1-10 with the overall yield of 19%-87%
from amines and 4-nitrophenyl isothiocyanate is shown in Scheme 1. To a solution of amines in dry
THF at 0oC under nitrogen, 4-nitrophenyl isothiocyanate in dry THF was added dropwise. After the
reaction was completed, the solvent was evaporated; the residue was recrystallized or purified by
flash column chromatography to give the corresponding thiourea product. The chemical structures
of compounds 1-10 were confirmed by 1H, C13 NMR and LC-MS. The Log P, calculated Log P and
the overall yield is summarized in Table 1. Further details of reactions, characterizations, and
bioassays are provided in the Supplementary Data.
The preclinical studies of SHetA2 on cell culture and animal models showed that it is a
promising agent for cancer prevention and treatment without significant toxicity and side effects.6-8
However, SHetA2 is too lipophilic, with a Log P value of 7.09, which can hinder its oral
bioavailability as an anticancer drug, since optimal Log P for most oral drugs is between 1-3. We
have designed the SHetA2 analogs with Log P value between 2.99 and 6.36 (Table 1) so that all our
compounds are less lipophilic than the lead compound SHetA2 with the premise that these
compounds would have better pharmacokinetic profiles. In addition, as previously reported, the six
step synthesis of SHetA2 suffers from a low overall yield of 3% and this procedure involves use of
reagents that are toxic and have unpleasant odor.8 Tallent’s group reported a modified procedure
for the synthesis of key intermediates to the SHetA2 with improved yield, but started with much
more expensive chemicals and used column chromatography after every synthetic step.13 We have
developed a simple, one step synthetic procedure of the target compounds with cheap and
commercially available chemicals with overall yield of 19%-87% (Table 1). Please note that
compounds 1-10 were prepared in one step whereas SHetA2 required 6 steps. By far in the

published literature, only certain diarylurea multi-target kinase inhibitors have chemical structures
related to our compounds.14,15 Sorafenib is among one of these compounds that have been approved
for the treatment of advanced renal cell cancer by the FDA. In addition, the wide diversity of
cellular and molecular targets that can be regulated by judiciously modifying N, N’-diarylureas and
ureas, suggests this part of the molecule may serve as privileged scaffolds for anticancer agents.14,15
The synthesized diarylthiourea analogs 1-10 were tested on three breast cancer cell lines 
MCF7, T-47D and MDA-MB-453 cells (Table 2). While the majority of the compounds showed
some effect on inhibiting growth of all three breast cancer cell lines, diarylthiourea analogs 5 and 7
showed the most significant effects. The GI50 values of compounds 5 and 7 were comparable to the
parent SHetA2 (2.94-6.27 µM and 2.53-4.77 µM vs. 3.27-4.13 µM, respectively), with the T-47D
cells displaying the greatest sensitivity to these compounds when compared to MCF7 and MDA-
MB-453. Since compound 7 appeared to be the most effective in inhibiting the growth of all three
breast cancer cell lines, its ability to inhibit cell growth over a longer period of time was evaluated
(Figure 2 A-C). Results in figure 2 indicate that compound 7 continued to inhibit cell growth 4 days
after treatment and appeared to effectively inhibit the growth of all three breast cancer cell lines.
The diarylthiourea analogs of SHetA2 were also tested on three prostate cancer cell lines—
DU-145, PC-3 and LNCaP. Overall, LNCaP cells were more susceptible to the growth inhibitory
effects of these agents (Table 2). Compound 7 showed the most potent growth inhibitory effect in
LNCaP cells as evidenced by a low GI50 value of 3.54 µM. This value was comparable to the GI50
value (2.25 µM) of SHetA2 in the same cell-line (Table 2). Being the most potent of the
diarylthiourea analogs, compound 7 was tested for its ability to inhibit cell growth over a longer
period of time (Figure 2D). This experiment was carried out in LNCaP cells that were most
vulnerable to the growth inhibitory effects of compound 7. Cell growth was significantly inhibited
with 5 µM compound 7 staring at day 2 and remained so until day 4. A similar trend in growth
inhibition was observed at higher concentrations (10 and 20 µM) as well (Figure 2D). Moreover,
while SHetA2 inhibited the growth of all three prostate cancer cell-lines uniformly, compound 5
and 7 selectively inhibited the growth of just one of the prostate cancer cell line, the LNCaPs.
Here we report a simple, one step synthesis procedure to obtain a variety of diarylthiourea
derivatives of SHetA2 and their biological evaluation in human breast and prostate cancer cells. The
simple syntheses involved a coupling reaction of 4-nitrophenyl isothiocyanate in dry THF with
commercially available amines. Most of these diarylthiourea derivatives of SHetA2 caused growth

inhibition of both human breast and prostate cancer cells. Among them, compounds 5 and 7 showed
potency comparable to the lead compound, SHetA2. The study also validated our previous
hypothesis that the 4-nitrophenyl group and the thiourea linker are important for the anticancer
activity of SHetA2 because all the compounds tested have these moieties. Modification of other
functional groups in SHetA2 resulted in active anticancer agents. Therefore, this study has provided
a new approach to the design and synthesis of the next generation of novel anticancer agents,
perhaps with novel targeted mechanisms of action.
We tested the new diarylthiourea analogs along with SHetA2 on three breast cancer and
three prostate cancer cell lines and our results (Table 2) show that diarylthiourea analog 5 and 7 are
effective in inhibiting cancer cell growth of four cancer cell lines— MCF7, T-47D, MDA-MB-453
(breast cancer cells) and LNCaP (prostate cancer cell). SHetA2 was previously evaluated in the
panel of National Cancer Institute (NCI) human tumor cell lines by the Developmental Therapeutics
Program (DTP) and results showed that SHetA2 inhibited growth of most cancer cells in the
micromolar range. Specifically, reported GI50 values of 4.5 µM for MCF-7, 4.8 µM for T-47D, 5.0
µM for MDA-MB-453, 4.9 µM for DU145 and 5.0 µM for PC-3,6,16 are consistent with our results
shown in Table 2. Our newly synthesized compounds (5 and 7) showed equivalent potency in
comparison to the lead compound SHetA2 (Table 2). Noteworthy are the effects of compounds 5
and 7 on the three prostate cancer cell lines. Both these agents have shown to be more selective in
inhibiting the growth of LNCaP cells in comparison to PC-3 and DU-145 cell-lines. As seen in
Table 2, compound 7 is about six times more potent in inhibiting growth of LNCaP cells than PC-3
and DU145 cells and compound 5 is at least twice as potent in LNCaP cells than in PC-3 and DU-
145 cells. This observation is significant since SHetA2 does not show any such selectivity (Table
2). One explanation for this selectivity could be due to differential androgen receptor signaling
patterns in the three prostate cancer cell lines. Both PC-3 and DU-145 cell-lines do not express
androgen receptors (AR) and are not androgen dependent for their growth, while LNCaP cells
express AR (with a point mutation in the ligand binding domain) and are considered androgen-
dependent cells17. Therefore, we hypothesize that these new analogs, compounds 5 and 7, may
induce growth inhibition in prostate cancer cell lines by acting on specific targets in these cells.
Perhaps the molecular target is androgen receptor, since in addition to the LNCaP cells, all three
breast cancer cell lines that responded to the growth inhibitory effects of compounds 5 and 7

express AR. Whether cell growth inhibition by compounds 5 and 7 involves the regulation of
androgen receptors (AR) requires further investigation.
However, this differential response was not as significant in the breast cancer models.
MCF7 and T-47D cells are both estrogen receptor (ER) positive (presence of ERα) and are
considered hormone-dependent, whereas MDA-MB-453 is ER negative (absence of ERα). While
the GI50 for the ER positive cells were lower in comparison to the ER negative breast cancer cells,
the difference is not statistically significant (Table 2). The differences observed in the breast and
prostate cancer cells might be attributed to different mechanisms of action in the two cancer types.
Although MDA-MD-453 is considered ER negative breast cancer cells, similar to MCF7 and T-
47D, these cells do express ERβ.
In conclusion, this is the first report that demonstrates the growth inhibitory properties of
SHetA2 and a new generation of diarylthiourea analogs on both breast and prostate cancer cells.
While compounds 1-10 are not more potent than the SHetA2, we report for the first time the effects
of p-nitrodiarylthiourea compounds on the inhibition of both breast and prostate cancer cells, along
with a more simplified synthetic scheme. Data from this study indicate that these novel potential
anticancer agents are promising lead compounds for further evaluation. While the mechanism of
action, molecular targets and pharmacokinetic profile of these compounds remain to be elucidated
in future studies, results presented in this study provide a strong foundation for further preclinical
studies of these compounds as potential therapeutic agents for both breast and prostate cancers.
Acknowledgments
This work was support by grant funding from Touro University-CA Intramural Research Award
Program (IRAP) to S.L. and V.R. The authors would like to thank Dr. Michael Ellerby (Touro
University-CA) for his guidance and generous gift of LNCaP, PC-3 and DU-145 prostate cancer
cell lines and Dr. Miriam Gochin Touro University-CA for providing the NMR spectra.
References and notes
1. Liu, S.; Brown, C. W.; Berlin, K. D.; Dhar, A.; Guruswamy, S.; Brown, D.; Gardner, G. J.;
Birrer, M. J.; Benbrook, D. M. J. Med. Chem. 2004, 47, 999.
2. Dawson, M. I.; Eontana, J. A. Mini-Rev. Med. Chem. 2010, 10, 455.

3. Abraham, D. J. (ed): Vitamins. In Burgers’ Medicinal Chemistry and drug discovery; 7th
ed.. Hoboken, NH, John Wiley and Sons, 2010, Vol 6, pp 500 563.
4. Benbrook, D. M. Mini-Rev. Med. Chem. 2002, 2, 277.
5. Myers, T.; Chengedza, S.; Lightfoot, S.; Pan, Y.; Dedmond, D.; Cole, L.; Tang, Y.;
Benbrook, D. M. Invest. New Drugs. 2009, 27, 304.
6. Benbrook, D. M.; Kamelle, S. A.; Guruswamy, S. B.; Lightfoot, A. A.; Rutledge, T. L.;
Could, N. S.; Hannafon, B. N.; Dunn, S. T.; Berlin, D. D. Invest. New Drugs. 2005, 23, 417.
7. Masamha, C. P.; Benbrook, D. M. Cancer Res. 2009, 69(16), 6565.
8. Kabirov, K. K.; Kapetanovic, I. M.; Benbrook, D, M.; Dinger, N.; Mankovskaya, I.;
Zakharov, A.; Detrisac, C.; Pereira, M.; Martin-Jimenez, T.; Onua, E.; Banerjee, A.; van
Breemen, R. B.; Nikolic, D.; Chen, L.; Lyubimov, A, V. Drug Chem. Toxicol. 2013, 36(3),
284.
9. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery. Rev.
1997, 23, 3.
10. Lipinski, C. A. Drug Discov. Today Technol. 2004, 1, 337.
11. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
12. Zhang, Y.; Hua, Y.; Benbrook, D. M.; Covey, J. M.; Dai, G.; Liu, Z.; Chan, K. K. Cancer
Chemother. Pharmacol. 2006, 58, 561.
13. Tallent, C. R.; Twine, C. E.; Risbood, P.; Seltzman, H. H. Org. Prep. Proced. Int. 2007, 39,
96.
14. Denoyelle, D.; Chen, T.; Chen, L.; Wang, Y.; Klosi, E.; Halperin, J.; Aktas, B. H.; Chorev,
M. Bioorg. Med. Chem. Lett. 2012, 22(1), 402.
15. Li, H.; Lv, P.; Yan, T.; Zhu, H. Anticancer Agents in Med. Chem. 2009, 9, 471.
16. Website
of
Developmental
Therapeutics
Program
of
NCI:
http://dtp.nci.nih.gov/dtpstandard/servlet/MeanGraph?searchtype=NSC&searchlist=726189
&outputformat=HTML&outputmedium=page&chemnameboolean=AND&debugswitch=fal
se&assaytype=&testshortname=NCI+Cancer+Screen+Current+Data&dataarraylength=59&
endpt=GI50&button=Mean+Graph&highconc=-4.0. Accessed April 18, 2014.
17. Webber, M. M.; Bello, D.; Quader, S. Prostate. 1996, 29(6), 386.

Supplementary Material: The synthesis procedures, spectrum data of each compound and all
experimental information about bioassay are provided as a separate electronic file. It can be
transformed into PDF format.
FIGURE LEGENDS
Scheme 1: General procedure for the synthesis of the compounds 1-10.
Figure 1: Evolution of SHetA2.
Figure 2: Breast cancer (A) MDA-MB-453, (B) MCF7, (C) T-47D, and prostate cancer (D) LNCaP
cells were plated in 96-plates and treated with varying concentrations of SL-01-18 for 2, 3 and 4
days. Cell growth was analyzed with MTT (A-C) or MTS (D). Means and standard deviations
represent at least two independent experiments done in six replicates.

Table 1. SHetA2 analogs (1-10) produced according to Scheme 1
Compounds
Name
Log Pa
3.83
CLog Pa
3.51
Yield (%)
1
2
SL-01-03
SL-01-05
83
19
ND
5.01
3
SL-01-06
3.84
3.05
23
4
SL-01-08
SL-01-09
SL-01-15
SL-01-18
SL-01-19
SL-01-20
SL-01-21
SHetA2
ND
2.83
4.52
5.39
6.08
4.91
3.54
2.66
5.74
24
87
35
42
70
60
73
3
5
4.11
5.34
6.36
5.24
3.59
2.88
7.09
6
7
8
9
10
Parent
aLog P and Clog P were calculated with ChemDraw.

Table 2. Potency (GI50 values) in µM
Name and Chemical
Structure
Breast cancer cell lines (µM)
Mean SD
Prostate cancer cell lines (µM)
Mean SD
MCF-7
T-47D
DU-145
PC-3
LNCaP
MDA-MB-453
1
16.96 3.351
8.14 1.792
13.48 1.961
35.15 15.42 26.77 13.251
10.58 1.951
5.86 0.412
H
N
H
N
H3C
1
S
H3C
NO2
2
12.81 2.461
9.44 0.121
6.88 0.831
5.87 0.922
19.59 0.681
3
21.62 0.851
ND*
ND*
40.47 28.821
H
H
ND*
ND*
N
N
H C
3
S
N
H
NO
2
SL-1-06
4
5
15.28 3.271
2.94 0.912
ND*
ND*
ND*
23.53 7.111
6.05 0.182
H
N
H
N
ND*
N
S
S
NO2
6.21 1.951
6.27 0.391
13.69 4.151
13.90 1.092
H
N
H
N
H3C
S
NO2
6
7.81 1.192
3.96 0.582
6.20 2.422
17.48 2.201
16.50 1.171
11.32 3.901
7
8
9
3.16 1.792
2.53 0.392
4.77 2.212
21.78 3.981
6.11 1.302
19.96 3.321
19.27 0.411
3.54 0.172
7.29 2.352
13.30 2.731
11.55 3.871
14.49 4.581
13.08 1.741
9.89 1.622
8.68 2.341
27.59 7.221
24.10 5.401
22.56 3.981
12.75 0.601
10
ND*
ND*
ND*
ND*
ND*
SHetA2
4.13 0.572
3.99 0.062
0.82 0.032
3.27 0.332
9.51 0.611
3.47 0.422
4.80 1.532
2.25 1.042
H
N
H
N
S
S
NO
2
All Trans-RA
12.39 0.261
11.64 0.541
12.64 0.381
10.33 0.751
1 Based on linear model
2 Based on exponential model
* ND=Not determined or GI50 >>50µM

Scheme 1

Figure 1

Figure 2
A
0.0 µM
0.5 µM
1.0 µm
0.50
0.40
0.30
0.20
0.10
0.00
MDA-MB 453
5.0 µM
10.0 µM
20.0 µM
0
2
3
4
Days
B
0.80
MCF7
0.60
0.40
0.20
0.00
0
2
3
4
Days
C
D
0.40
0.30
0.20
T-47D
0.10
0.00
0
2
3
4
Days
1.60
1.20
0.80
0.40
LNCaP
0.00
0
2
3
4
Days