Synthesis and biological evaluation of SHetA2 (NSC-721689) analogs against the ovarian cancer cell line A2780

Article abstract of DOI:10.1016/j.ejmech.2019.03.010

A series of Flexible Heteroarotinoid (Flex-Het) analogs was synthesized and their biological activities were evaluated against the A2780 ovarian cancer cell line. The objective of this study was to establish structure-activity relationships (SARs) for new Flex-Het derivatives, which were previously inaccessible due to the limited availability of aryl isothiocyanate precursors. The current work developed a synthesis of isothiocyanate 13 and used it to prepare 14 diverse thiourea analogs of the lead compound SHetA2 (1, NSC-721689) from a range of commercial amines. Additionally, five new ureas were prepared along with nine N-benzylthioureas, five derivatives incorporating hydrazine or hydrazide linkers and four desmethyl compounds. Potencies and efficacies were determined for each derivative. Some of the new Flex-Hets displayed high activity with IC₅₀ values ranging from 1.86 to 4.70 μM and 85.6–95.9% efficacies, which are comparable to or better than the lead compound (IC₅₀ 3.17 μM, 84.3% efficacy). Although SHetA2 is scheduled to enter clinical trials in the near future, alternative backup drug candidates have been identified in this work. The new agents possess similar pharmacological properties and retain selective activity against A2780 ovarian cancer cells. Although a mixed SAR was obtained for these analogs, diversified, highly potent molecules were identified for further investigation. In particular, agents 2c-d and 3e-f, which incorporated CF₃ and OCF₃ groups in place of NO₂ on the pendent aryl ring, displayed high activity and excellent differentiation between normal and cancerous cells.

Full text of DOI:10.1016/j.ejmech.2019.03.010

European Journal of Medicinal Chemistry 170 (2019) 16e27
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of SHetA2 (NSC-721689) analogs
against the ovarian cancer cell line A2780
,
*
,
, 2
Baskar Nammalwar a, Richard A. Bunce a 1, K. Darrell Berlin a, Doris M. Benbrook b
,
Coralee Toal b
a Department of Chemistry, Oklahoma State University, Stillwater, OK, 74078-3071, USA
b Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Flexible Heteroarotinoid (Flex-Het) analogs was synthesized and their biological activities
were evaluated against the A2780 ovarian cancer cell line. The objective of this study was to establish
structure-activity relationships (SARs) for new Flex-Het derivatives, which were previously inaccessible
due to the limited availability of aryl isothiocyanate precursors. The current work developed a synthesis
of isothiocyanate 13 and used it to prepare 14 diverse thiourea analogs of the lead compound SHetA2 (1,
NSC-721689) from a range of commercial amines. Additionally, five new ureas were prepared along with
nine N-benzylthioureas, five derivatives incorporating hydrazine or hydrazide linkers and four desmethyl
compounds. Potencies and efficacies were determined for each derivative. Some of the new Flex-Hets
Received 22 November 2018
Received in revised form
3 March 2019
Accepted 4 March 2019
Available online 7 March 2019
Keywords:
SHetA2
NSC-721689
Ovarian cancer
displayed high activity with IC50 values ranging from 1.86 to 4.70
mM and 85.6e95.9% efficacies, which
are comparable to or better than the lead compound (IC50 3.17 M, 84.3% efficacy). Although SHetA2 is
m
scheduled to enter clinical trials in the near future, alternative backup drug candidates have been
identified in this work. The new agents possess similar pharmacological properties and retain selective
activity against A2780 ovarian cancer cells. Although a mixed SAR was obtained for these analogs,
diversified, highly potent molecules were identified for further investigation. In particular, agents 2c-
d and 3e-f, which incorporated CF3 and OCF3 groups in place of NO2 on the pendent aryl ring, displayed
high activity and excellent differentiation between normal and cancerous cells.
6-Isothiocyanatothiochroman
Thiochroman-based anticancer agents
Flexible heteroarotinoids
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer is a lethal gynecological malignancy responsible for more
than 30% of deaths in women in the advanced world. Since this
Cancer has been the leading cause of death world-wide for the
past three decades and has had an enormous impact on economies
and society [1]. It is a complex disease, which creates a tempo-
spatial physiological change within the cell, and ultimately leads
to malignant tumors [2]. Due to the neoplastic nature of tumor cells
and their ability to invade benign tissue, cancer causes malig-
nancies that result in high mortality rates [3]. Although rapid ad-
vances have been made in controlling metastatic cancer, much
work remains to eradicate the disease.
cancer exhibits few symptoms in its early stages, it is often not
diagnosed until it has metastasized to other organs in the body [6].
To date, the details of genetic differences between normal and
cancerous ovarian cells that disrupt apoptosis are not clearly un-
derstood [7]. Although various therapeutic options are available,
the survival and recurrence rates for ovarian cancer are not satis-
factory. Thus, there is an imminent need to develop a drug that can
serve as a chemopreventive agent for ovarian cancer.
Flexible heteroarotinoids (Flex-Hets) are synthetic analogs of
the arotinoid class of retinoids, which function independent of
retinoid receptors and toxicities [8]. Certain retinoids are proven
anticancer agents which regulate cell differentiation during the
apoptosis stage [9e11]. Despite being potent anticancer agents,
arotinoids can exhibit significant toxicity toward normal cells
[10,12]. The introduction of a heteroatom [13] within the arotinoid
framework reduced the toxicity profile of retinoid compounds by
The primary focus of our research involves identifying drug
candidates that can be used to treat ovarian cancer [4,5]. Ovarian
* Corresponding author.
E-mail address: rab@okstate.edu (R.A. Bunce).
Address inquiries on the synthesis work to this individual.
Address inquiries on the biological experiments to this individual.
1
2
https://doi.org/10.1016/j.ejmech.2019.03.010
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
17
1000-fold and resulted in a considerable increase in potency across
various cancer cell lines [14]. Flex-Hets possess excellent differen-
tiation between benign and malignant cells without exhibiting
mutagenicity, carcinogenicity, teratogenicity or toxicity [15e20].
Over the past two decades, considerable changes have been made
within the arotinoid structural framework to identify the lead
molecule SHetA2 (1, NSC-721689) [12]. This agent (Fig. 1) is a Flex-
Het that contains a sulfur heterocycle (Ring A) linked via a thiourea
unit to a 40-nitrophenyl moiety (Ring B) [13,21]. SHetA2 has shown
promising activity not only as a chemotherapeutic drug that in-
the importance of such changes to the binding affinity and potency.
Since a crystal structure of SHetA2 bound to a target protein has not
yet been published, it was not possible to rely on a structure-based
drug design strategy. Thus, to gain information on the active
binding site, we relied on a serendipity approach and guidance
from limited SAR studies of known SHetA2 analogs [13,18,23,25].
After the initial synthesis and screening were completed on
members of 2 and 3, a series of N-benzylthioureas (4a-i) was pre-
pared to identify the importance of analog shape and flexibility
within the binding pocket of mortalin [24,28,29]. Most arotinoids,
including SHetA2, contain a flexible linker which appears to
enhance binding with the receptors. We previously predicted that
increased flexibility within the core structure would facilitate H-
bonding capabilities between the functionalized ligands and the
binding pocket of retinoic acid receptors [5]. The benzyl analogs 4a-
i incorporate a thiourea unit, which should permit further evalua-
tion of linker flexibility as an option to improve binding affinities.
The spatial orientation of the linker may also be a critical parameter
in this particular series of drug candidates. In order to probe this
structural feature, a series of hydrazine and hydrazide derivatives
(5a-e) were also synthesized.
hibits ovarian cancer cells but also as
a chemopreventive
[4,13,22,23]. The differential apoptosis in normal vs cancerous
ovarian cells was evident at the molecular level based on the effect
of SHetA2 on heat shock protein HSPA9 (mortalin), p53 and the Bcl-
2 proteins [24]. Moreover, SHetA2 has a wide therapeutic window
with a No Observed Adverse Effect Level (NOAEL) of >1500 mg/kg/
day (in dogs) and good pharmacokinetics which provides options
for various treatment regimens [12,24e28]. The objective of the
current work was to develop potent analogs of SHetA2 by modu-
lating the pK/pD properties and to evaluate the analogs using
structure-activity relationships (SARs) and diversity oriented syn-
thesis (DOS).
Finally, based on our previous findings with Flex-Hets [29], it
was decided to explore the option of removing the C2 gem-
dimethyl substituents in Ring A, as in targets 6a-d. The removal of
alkyl substituents from this site would reveal the importance of
these groups in the drug scaffold, modulate the log P and log D
properties, and improve the LiPE [e log(IC50) e clogP] of the
compound.
1.1. SAR and DOS study in Flex-Het SHetA2
Although Flex-Het analogs were first reported 15 years ago [13],
a detailed understanding of their SARs has not been established.
However, it has been determined that SHetA2 interacts with the
heat-shock protein mortalin [24,28,29]. To probe the binding
properties of the agents to mortalin, a diverse pool of analogs was
synthesized by incorporating various functional group (FG) sub-
stitutions on Ring B as well as changes in Ring A, and the effects of
these modifications on the biological activity of the drug candidates
were assessed.
Initially, a series of urea (2a-e) and thiourea (3a-n) linked de-
rivatives (Fig. 1) were prepared with various FGs on the phenyl
moiety (Ring B) of the system. By this approach, we altered the
pharmacological activity of the drug scaffold by carefully evaluating
selected groups including chloro, fluoro, methyl, methoxy, hydroxy,
cyano, trifluoromethyl and trifluoromethoxy in Ring B. Functional
group modifications were made at C40 in Ring B to compare the
activity with that of SHetA2. Moreover, a few synthetic analogs
with substitution at C20 and C30 of Ring B were prepared to gauge
2. Results and discussion
2.1. Chemistry
The syntheses of our target analogs are shown in Schemes 1e3.
Previously, derivatizing SHetA2 was limited by the commercial
availability of isothiocyanates as precursors to the thioureas. Thus,
the preparation of an isothiocyanatothiochroman (Scheme 1) was a
crucial goal. Our approach involved the treatment of acet-
amidothiophenol (7) with mesityl oxide (8) in the presence of
triethylamine [30]. Due to the poor reactivity of the thiophenol as a
Michael donor, a two-fold excess of 8 and base were required, along
with a portion-wise addition protocol for the Michael reaction to
afford ketone 9 (87%). The subsequent reaction involved addition of
Fig. 1. Proposed modifications of SHetA2.

18
B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
Scheme 3. Synthesis of desmethyl SHetA2 derivatives 6a-d.
Scheme 1. Synthesis of 6-isothiocyanato-2,2,4,4-tetramethylthiochroman (13).
chlorobenzene readily solubilized the starting material. Refluxing
11 with 6 M HCl in methanol furnished the desired intermediate
aminothiochroman as the hydrochloride salt 12 (95%), which was a
white solid. The conversion of 12 to 13 was achieved by reaction
with CS2, Et3N and (Boc)2O in the presence of 3 mol% DABCO (92%)
[31].
The syntheses of compounds 2a-e, 3a-n, 4a-i and 5a-e are
summarized in Scheme 2. Ureas 2a-e were prepared in high yields
by reacting the free base of 12 [13] with various aryl isocyanates
(14a-e) in THF:DCM (1:1) containing 1.2 equiv. of Et3N at room
temperature (23 ꢁC). Arylthioureas 3a-m were generated by reac-
tion of isothiocyanate 13 with various anilines 15a-m in DCM at
23 ꢁC. Et3N (1.2 equiv.) was added for this procedure as well,
although these reactions seemed to proceed smoothly without
base. A similar reaction of 13 with the free base of 12 [13] was used
to prepare 3n.
N-Benzyl derivatives 4a-i were isolated in high yields from re-
action of 13 with benzylamines 16a-i in DCM containing Et3N at
23 ꢁC. For the synthesis of the hydrazine/hydrazide derivatives 5a-
e, a similar treatment of isothiocyanate 13 with arylhydrazine 17a
or arylhydrazides 18a-d afforded high yields of analogs 5a-e.
Finally, to prepare the C2 desmethyl thiochroman derivatives
6a-d (Scheme 3), acetamidothiophenol (7) and 1-bromo-3-
methylbut-2-ene (19) were allowed to react in the presence of
K2CO3 in acetone to afford 20 (84%) [32]. Thioether 20 then un-
derwent cyclization in the presence of polyphosphoric acid to
furnish thiochroman 21 (72%). A series of other Lewis acid-
mediated cyclization strategies were also explored using AlCl3,
Bi(OTf)3 [33] and OSU-6 [34e36], but none of these catalysts
afforded the desired cyclization product 21. Deprotection of 21 was
achieved by refluxing in 6 M HCl to afford the amine$HCl salt 22
(86%) as an off-white solid. Finally, compound 22 was converted to
thioureas 6a-d by reaction with aryl isothiocyanate derivatives in
the presence of Et3N in THF. In all cases, the final products were
purified by silica gel chromatography and characterized by 1H and
13C NMR as well as elemental analyses.
Scheme 2. Synthesis of analogs 2e5.
methyllithium to 9 at ꢀ50 ꢁC to generate tertiary alcohol 10. An
inverse addition strategy, wherein 9 was added to a methyllithium
solution in THF, resulted in clean product formation in good yield
(61%). The most critical step involved the dehydration-cyclization
of 10 by a Friedel-Crafts reaction, which required chlorobenzene
as the solvent in the presence of AlCl3 to afford 11 (89%). Various
other media were tried, but proved unsuccessful as only
Literature reports have demonstrated that there is a direct cor-
relation between lipophilicity and the ability of a drug to inhibit
cancer cell lines [37]. By increasing the lipophilic nature of a
molecule, it should permeate the cells more effectively, which in

B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
19
turn could enhance the efficacy of the drug. In order to maintain a
good adsorption, distribution, metabolism and excretion (ADME)
profile, the compounds have been designed with log D values be-
tween 3 and 6 to avoid off-target liabilities arising due to human
that of SHetA2. The presence of 40-acetyl (2a) or 20-methoxy-40-
nitro (2e) substitution, resulted in a higher IC50 value for 2a and
lower efficacy values for both. A similar trend was observed in the
thiourea series. Replacing the C40 nitro group with a chlorine (3d)
or a hydroxy (3l) resulted in the former exhibiting improved effi-
cacy relative to SHetA2, while the latter was inferior. Electron
withdrawing groups at C40, such as trifluoromethyl (3e) and tri-
fluoromethoxy (3f), demonstrated comparable potency and greater
efficacy than that of SHetA2. Electron releasing groups, as in 3a-c
and 3i-n, reduced the efficacy considerably. Changing the nitro
position from C40 to C3' (3g) also diminished the potency, sug-
gesting a decreased interaction within the mortalin binding pocket
and a reduced overall log D for the molecule.
ꢀ
Ether-a-go-go Related Gene (HERG), cytochrome P450 (CYP)
interaction, or other transporters. The results show that the log D of
the molecule, along with certain FGs, promote better penetration
and increased efficacy. In most SHetA2 analogs, the FGs present
exerted a greater effect on the efficacy than did the log D of the
molecule.
2.2. Biology
A rather unusual trend was observed for N-benzylthioureas 4a-i.
In these cases, derivatives with electron releasing and electron-
withdrawing groups both exhibited similar IC50 values. All mem-
bers of series 4 exhibited good IC50 values, but the % efficacy varied
from 56.8 3.3 (4i) to 95.3 0.3 (4g), the latter, with a C40 tri-
fluoromethyl, being the best in this series. The orientation of the
benzyl group in the protein binding pocket is likely quite different
from that of the aryl group in some other SHetA2 derivatives.
The hydrazine and hydrazide derivatives 5a-e exhibited variable
potencies with low efficacy. Although these analogs were predicted
The objective of this study was to develop an extensive SAR, to
identify the most active structural components of these new
SHetA2 analogs, and to speculate how the SARs might affect
binding to a putative target protein. The biological effects of the
compounds were assessed using a cytotoxicity assay against the
human A2780 ovarian cancer cell line (Table 1). SHetA2 (1) was
used as a standard for comparison. On initial evaluation of ureas 2a-
e, it was found that replacing the C40 nitro group (Ring B) of SHetA2
with trifluoromethyl (2b), trifluoromethoxy (2c) or cyano (2d)
afforded compounds with activities comparable to or better than
Table 1
Potency and efficacy of SHetA2 analogs at inhibiting ovarian cancer cell line growth.
Cpd
R
L
Ring B
clogD
clogP
IC50
(
m
M)
%Efficacy
1
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
NHC(S)NH
NHC(O)NH
NHC(O)NH
NHC(O)NH
NHC(O)NH
NHC(O)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
C6H4-4-NO2
C6H4-4-C(O)CH3
C6H4-4-CF3
C6H4-4-OCF3
C6H4-4-CN
C6H3-2-OCH3-4-NO2
Ph
C6H3-4-CH3
2-(5,6,7,8-Tetra- hydronaphthyl)
C6H4-4-Cl
5.15
5.36
6.17
6.28
5.26
5.88
5.46
5.79
6.96
6.15
6.32
5.93
4.97
5.93
3.95
5.45
5.45
4.82
4.88
8.05
5.70
6.34
6.29
6.45
5.71
5.75
6.52
5.64
5.15
5.84
4.08
3.85
4.97
5.45
3.36
4.26
4.47
3.86
5.1
5.4
6.2
6.3
5.3
5.9
5.5
5.8
7.0
6.1
6.3
5.9
3.1
5.9
3.9
5.4
5.4
4.8
4.8
8.0
5.7
6.3
6.3
6.4
5.7
5.7
6.5
5.6
5.1
5.8
4.0
3.8
4.9
5.4
3.3
4.2
4.4
3.8
3.17 0.1
13.9 1.5
84.3 0.7
65.1 2.0
93.3 0.1
95.6 0.4
93.3 0.1
73.1 2.1
37.5 2.1
60.0 1.0
73.8 1.3
91.9 0.5
95.9 0.6
95.3 0.4
60.8 2.5
24 2.3
64.2 1.8
59.8 1.2
38.8 0.04
64.0 1.4
56.5 3.3
52.7 1.8
85.6 0.6
88.2 0.3
90.1 0.7
89.4 2.8
64.8 1.4
94.5 0.6
95.3 0.3
89.6 0.9
56.8 3.3
63.6 3.7
67.9 2.6
43.3 2.9
57.4 0.4
61.4 2.9
93.5 1.7
48.7 1.1
64.3 16.2
5.9 0.9
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
4a
4b
4c
4d
4e
4f
3.79 0.05
1.86 0.09
3.93 0.03
3.17 0.21
3.03 0.61
3.11 0.50
2.92 0.43
3.17 0.25
2.86 0.29
3.51 0.07
5.88 0.39
4.62 0.18
3.53 0.12
2.78 0.47
3.04 0.52
5.07 0.32
7.56 0.51
4.04 0.24
3.03 0.47
3.19 0.47
3.31 0.46
2.98 0.44
3.12 0.50
4.70 0.11
3.26 0.43
3.23 0.49
4.82 0.41
7.03 0.31
0.82 0.21
3.15 0.19
3.60 0.18
4.0 0.12
C6H4-4-CF3
C6H4-4-OCF3
C6H4-3-NO2
C6H4-2-CF3
C6H4-4-C(O)NH2
C6H4-4-OCH3
C6H4-3-OCH3
C6H4-4-OH
C6H3-3-OH-4-OCH3
Ring A
CH2Ph
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NH
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHCH2
NHC(S)NHNH
NHC(S)NHNHC(O)
NHC(S)NHNHC(O)
NHC(S)NHNHC(O)
NHC(S)NHNHC(O)
NHC(S)NH
CH2CH2Ph
CH2C6H4-4-CH3
CH2C6H4-4-Cl
CH2C6H4-4-F
CH2C6H4-4-NO2
CH2C6H4-4-CF3
CH2C6H4-4-OCH3
CH2C6H4-4-OH
Ph
4g
4h
4i
5a
5b
5c
5d
5e
6a
6b
6c
6d
Ph
C6H4-4-NO2
C6H4-4-OCF3
C6H3-3,5-CF3
C6H4-4-NO2
C6H4-4-CF3
4.01 0.19
5.81 0.20
5.97 0.24
2.81 0.44
H
H
H
NHC(S)NH
NHC(S)NH
NHC(S)NH
C6H4-4-OCF3
C6H4-4-CO2Et

20
B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
to have improved log P values, the desired effect was not observed.
In the C2 desmethyl series, analog 6a with a C40 nitro offered a
direct comparison with SHetA2, and although it exhibited greater
efficacy, it had a slightly higher IC50 value. The desmethyl tri-
fluoromethyl (6b) and trifluoromethoxy (6c) compounds, which
are close analogs of 2b and 2c, respectively, exhibited reduced ac-
tivity. Placement of an ethoxycarbonyl group at C40 produced a
compound (6d) with minimal efficacy.
Although mixed ovarian cancer cell inhibitory activity was
observed, the data were clear, with the best derivatives having a
trifluoromethyl or trifluoromethoxy in place of the C40 nitro. A
possible rationale for the activities is that the putative target pro-
tein pocket in which SHetA2 binds may be narrow, and even small
positional changes within ligands may not be well tolerated. The
SHetA2 may be binding to lysine [24,29] within the pocket of
mortalin. The differential effect of SHetA2 on cancer cells over
normal cells is also associated with a lack of toxicity [13,22].
Consequently, we evaluated representative compounds 2b and 2c
relative to SHetA2 by comparing the differential effect on A2780
human ovarian cancer cells vs FT-007 normal human fallopian tube
cells [38]. The dose response curve (Fig. 2) demonstrates that
compounds 2b, 2c and SHetA2 exhibited nearly complete inhibition
at C40 promotes the most significant interaction with the protein.
Among N-benzyl derivatives 4a-i, only 4g (40-CF3) showed prom-
ising attributes. Changing the regular 3-atom linkers [NC(O)N]/
[NC(S)N] to 4-atom [C(S)NHC]/[NC(S)NN] and 5-atom [NC(S)
NNC(O)] linkers, as in 5a-e, did not improve the potency, but rather
exhibited the opposite effect. In the case of C2 desmethyl analogs 6,
the 40-NO2 derivative 6a showed promising potency and inhibition
compared to SHetA2, while a similar trend could not be discerned
for 6b-d. As has been observed previously [29], the 40-NO2 group
played a significant role in maximizing the biological activity of all
derivatives, while other electron withdrawing groups exerted var-
iable effects. For ureas 2 and thioureas 3, analogs bearing C40
electron withdrawing groups generally exhibited strong activity.
Two representative compounds, ureas 2b and 2c, demonstrated
similar differential effects on cancer cells over healthy cells, sug-
gesting that the alterations in activity are not due to general toxicity
and may be due to their interaction with the mortalin (HSPA9)
binding protein that is overexpressed in cancer cells compared to
healthy cells [24,28].
4. Experimental section
of cancer cells with IC50 values in the range of 2e4 mM, whereas
4.1. Chemistry
with normal cells these compounds did not show significant inhi-
bition. The compound concentration causing a 50% cell growth
inhibition (IC50) was determined by interpolation from these dose-
response curves. The results reiterate that the SHetA2 family of
compounds does not exhibit any significant toxicity on normal
cells, even at high concentrations, and thereby suggests an effective
therapeutic window for treatment at relatively high doses for
cancer patients.
4.1.1. General methods
Tetrahydrofuran was distilled from lithium aluminum hydride
prior to use. All other commercial reagents and solvents were used
as received. All solutions used in workup procedures were aqueous.
Unless otherwise indicated, all reactions were carried out under dry
N2 in oven-dried glassware. Reactions were monitored by thin layer
chromatography (TLC, Analtech, No 21521) using silica gel GF
plates. Preparative separations were performed by column chro-
matography on silica gel (Davisil®, grade 62, 60e200 mesh) con-
taining UV-active phosphor (Sorbent Technologies, No UV-05)
slurry packed into quartz columns. Band elution for all chromato-
graphic separations was monitored using a hand-held UV lamp.
Melting points were uncorrected. IR spectra were run as CHCl3
solutions on NaCl disks. The 1H and 13C NMR spectra were
measured in the indicated solvents at 400 MHz and 101 MHz,
respectively, using tetramethylsilane as the internal standard with
coupling constants (J) given in Hz.
3. Conclusions
In summary, we have prepared a total of 37 new analogs of
SHetA2 (1, NSC-721689) incorporating broad structural variations,
and the potencies and efficacies are reported. During the course of
our study, a synthetic route to 13 was developed, which allowed us
to prepare and create a SAR for these Flex-Het derivatives. Upon
biological evaluation, many of the new analogs showed comparable
potency to the lead compound by improving the predicted log P
values. The screening of various FGs on the new derivatives allowed
identification of several compounds with greater potency than
SHetA2. In particular, FGs such as CF3 (2b and 3e) and OCF3 (2c and
3f) could be substituted for the 40-NO2 (Ring B) in SHetA2 to
mitigate potential functional group toxicity. In particular, com-
4.1.2. N-(4-(2-methyl-4-oxopentan-2-yl)thio)phenyl)acetamide (9)
To
a stirred solution of acetamidothiophenol (7, 25.0 g,
149.7 mmol) in dry CHCl3 (20 mL) was added triethylamine (15.1 g,
20.8 mL, 149.7 mmol), followed by mesityl oxide (8, 14.7 g, 17.1 mL,
149.7 mmol). The resulting slurry was heated to reflux at 70 ꢁC. Two
additional portions of triethylamine (7.5 g, 10.4 mL, 74.5 mmol) and
8 (7.4 g, 8.6 mL, 74.5 mmol) were added at regular intervals of 4 h,
and the resulting solution was refluxed for 16 h after the final
pound 2c (IC50 1.86
mM, 95.6% efficacy) represents the most potent
analog of SHetA2 (IC50 3.17
mM, 84.3% efficacy) to date against the
A2780 ovarian cancer cell line. The reduced activity for specific
groups at the C20 and C30 positions demonstrates that substitution
Fig. 2. Growth Inhibition Assays of SHetA2, 2b and 2c on A2780 ovarian cancer and FT-007 normal fallopian tube cells.

B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
21
addition. The resulting reaction mixture was cooled, filtered
through Celite® and washed with chloroform (2 ꢂ 50 mL). The
combined organic layers were washed with water (2 ꢂ 100 mL),
saturated NaCl solution, dried (MgSO4), filtered and concentrated
under vacuum to give a yellow oil. The crude reaction mixture was
then purified by silica gel column chromatography eluted with
DCM:EtOAc (1:1) to afford 9 (34.5 g, 87%) as a pale yellow solid, mp
49e51 ꢁC (lit [5] mp 46e49 ꢁC); IR: 3310, 1699, 1676 cmꢀ1; 1H NMR
for 1 h to yield a white solid. The solid was filtered and dried under
vacuum to afford 12 as a white powder (14.0 g, 95%), mp
208e209 ꢁC (lit [5] mp 208e209 ꢁC); IR: 2922, 2853 cmꢀ1; 1H NMR
(DMSO‑d6):
d 10.08 (s, 2H, NH2), 7.48 (s, 1H, Ar-H), 7.22 (d,
J ¼ 8.3 Hz, 1H, Ar-H), 7.09 (d, J ¼ 8.3 Hz, 1H, Ar-H), 1.94 (s, 2H, CH2),
1.42 (s, 6H, 2CH3), 1.40 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d 144.2,
132.0, 130.0, 129.1, 122.0, 121.2, 53.5, 42.6, 35.8, 32.6, 31.6.
(CDCl3):
d
7.90 (br s, 1H, NH), 7.53 (d, 1H, J ¼ 8.8 Hz, 2H, Ar-H), 7.45
4.1.6. 6-Isothiocyanato-2,2,4,4-tetramethylthiochroman (13)
(d, J ¼ 8.8 Hz, 2H, Ar-H), 2.65 (s, 2H, CH2), 2.19 (s, 3H, CH3), 2.15 (s,
To a stirred solution of 12 (10 g, 38.8 mmol) in EtOH (20 mL) was
added CS2 (29.0 g, 22.9 mL, 381 mmol), followed by Et3N (8.03 g,
11.1 mL, 79.5 mmol). The reaction mixture was stirred at 23 ꢁC for a
period of 20 min, and (Boc)2O (8.46 g, 38.8 mmol) dissolved in
ethanol (5 mL) was added. The mixture was then cooled to 0 ꢁC and
DABCO (0.130 g, 1.2 mmol, 3 mol%) in EtOH (2 mL) was added all at
once. With continuous stirring, the reaction mixture was held at
0 ꢁC for 10 min and then was allowed to warm to 23 ꢁC for 20 min
and concentrated under vacuum. The residue was dissolved in Et2O
(300 mL) and the solution was washed with saturated NaCl (50 mL).
The organic layer was dried (MgSO4), filtered and concentrated to
dryness. The compound was further purified on a silica gel column
eluted with increasing concentrations of EtOAc in hexanes to afford
isocyanate 13 (9.40 g, 92%) as a white solid, mp 192e193 ꢁC; IR:
3H, CH3), 1.36 (s, 6H, 2CH3); 13C NMR (CDCl3):
138.3, 126.2, 119.6, 54.3, 47.0, 32.1, 28.0, 24.5.
d 206.9, 168.6, 139.0,
4.1.3. N-{4-[(4-hydroxy-2,4-dimethylpentan-2-yl)thio]phenyl}
acetamide (10)
To
a stirred solution of methyllithium in Et2O (198 mL,
317 mmol, 1.6 M) in THF (300 mL) at ꢀ50 ꢁC was added dropwise a
solution of 9 (28 g, 105.7 mmol) in THF (200 mL) over 30e45 min.
The reaction formed a white precipitate, and the reaction was
allowed to slowly warm to room temperature (23 ꢁC) over a period
of 3 h. Stirring was continued at 23 ꢁC for 1 h. The reaction was then
cooled to 0 ꢁC, and the mixture was quenched by dropwise addition
of ice water (150 mL). After adjusting the solution pH to 6e7 by
addition of 6 M HCl, the solution was extracted with EtOAc
(2 ꢂ 250 mL). The combined organic extracts were washed with
saturated NaCl (1 ꢂ 150 mL), dried (MgSO4), filtered and concen-
trated under vacuum to afford a dark brown liquid. To the crude
product was added CHCl3 (60 mL) with cooling to 0 ꢁC for 1 h which
afforded a yellow solid. The solid was then filtered and dried under
vacuum to afford 10 (18 g, 61%) as a pale yellow solid, mp
141e142 ꢁC (lit [5] mp 138e144 ꢁC); IR: 3400, 3303, 1676 cmꢀ1; 1H
2140 cmꢀ1
J ¼ 8.3 Hz, 1H, Ar-H), 7.24 (d, J ¼ 8.3 Hz, 1H, Ar-H), 1.94 (s, 2H, CH2),
1.40 (s, 6H, 2CH3), 1.46 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
144.0,
; d 7.50 (s, 1H, Ar-H), 7.32 (d,
1H NMR (DMSO‑d6):
d
136.9, 132.2, 130.4, 129.1, 122.0, 121.2, 53.5, 42.6, 35.8, 32.6, 31.6.
4.2. Representative procedure for the formation of the urea
[L ¼ NHC(O)NH] series (2a-e)
NMR (CDCl3): d 7.68 (br s, 1H, NH), 7.52 (m, 4H, Ar-H), 3.50 (br s, 1H,
The free base of 12 (0.113 g, 0.44 mmol) was generated by
shaking a dichloromethane (DCM) solution (10 mL) of 12 with
aqueous sodium bicarbonate, separating the layers, removing the
solvent and reacting the oily product directly with the aryl iso-
cyanates (14a-e) (0.46 mmol) in THF:DCM (1:1, 5 mL) in the pres-
OH), 2.19 (s, 3H, CH3), 1.77 (s, 2H, CH2), 1.34 (s, 6H, 2CH3), 1.33 (s, 6H,
2CH3); 13C NMR (CDCl3):
52.0, 49.2, 32.2, 30.8, 24.6.
d 168.4, 138.8, 138.1, 126.3, 119.6, 72.0,
4.1.4. N-(2,2,4,4-tetramethylthiochroman-6-yl)acetamide (11)
ence of Et3N (56 mg, 77 mL, 0.55 mmol, 1.2 equiv). The reaction
To a stirred solution of 10 (18.0 g, 64.1 mmol) in chlorobenzene
(125 mL) at 60 ꢁC was added portion-wise anhydrous aluminum
chloride (10.2 g, 76.7 mmol) over a period of 45 min (Caution: This
addition was exothermic and typically raised the temperature of
the solvent to its boiling point. External heating was discontinued
before adding the aluminum chloride). Once the addition was
complete, heating was continued at reflux for an additional 90 min.
The reaction mixture was cooled to 23 ꢁC and quenched with ice
cold water (150 mL) to give a thick suspension. The solid was
removed by filtration through Celite® and washed with EtOAc
(2 ꢂ 100 mL). The layers were separated, and the aqueous layer was
extracted with additional EtOAc (2 ꢂ 100 mL). The combined
organic extracts were washed with saturated NaCl, dried (MgSO4),
filtered and concentrated under vacuum to give a yellow oil. The
crude product was purified on a silica gel column using hexanes:
EtOAc (1:1) to afford product 11 (15.0 g, 89%) as a pale yellow solid,
mixture was stirred at 23 ꢁC for 18 h, concentrated, and purified on
a silica gel column eluted with increasing concentrations of EtOAc
in hexanes to afford the desired products.
4.2.1. 1-(4-Acetylphenyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
urea (2a)
Tan solid (0.158 g, 94%), mp 132e133 ꢁC; 1H NMR (DMSO‑d6):
d
9.06 (s, 1H, NH), 8.74 (s, 1H, NH), 7.90 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.58
(superimposed d, J ¼ 8.0 Hz, 2H, Ar-H and s, 1H, Ar-H), 7.20 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 6.99 (d, J ¼ 8.0 Hz, 1H, Ar-H), 2.51 (s, 3H, CH3),
1.91 (s, 2H, CH2), 1.36 (s, 6H, 2CH3), 1.34 (s, 6H, 2CH3); 13C NMR
(DMSO‑d6):
d 195.6, 151.6, 143.8, 142.4, 136.1, 129.7, 129.0, 127.3,
124.4, 116.7, 116.5, 53.1, 41.3, 34.7, 31.6, 30.7, 25.7 (one aromatic C
not observed). Anal. Calcd for C22H26N2O2S: C, 69.08; H, 6.85; N,
7.32. Found: C, 69.24; H, 6.74; N, 7.55.
mp 105e107 ꢁC (lit [5] mp 104e107 ꢁC); IR: 3295, 1662 cmꢀ1
NMR (CDCl3):
;
1H
d
7.60 (br s, 1H, NH), 7.27 (d, J ¼ 2.3 Hz, 1H, Ar-H), 7.20
4.2.2. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(4-
(trifluoromethyl)phenyl)urea (2b)
(dd, J ¼ 8.2, 2.3 Hz, 1H, Ar-H), 7.04 (d, J ¼ 8.2 Hz, 1H, Ar-H), 2.16 (s,
3H, CH3), 1.92 (s, 2H, CH2), 1.39 (s, 6H, 2CH3), 1.35 (s, 6H); 13C NMR
White solid (0.147 g, 82%), mp 141e142 ꢁC; 1H NMR (DMSO‑d6):
(CDCl3):
35.7, 32.4, 31.4, 24.4.
d
168.3, 143.4, 135.1, 128.4, 128.2, 118.7, 118.2, 54.4, 42.0,
d
9.05 (s, 1H, NH), 8.72 (s, 1H, NH), 7.66 (d, J ¼ 8.8 Hz, 2H, Ar-H), 7.62
(d, J ¼ 8.8 Hz, 2H, Ar-H), 7.59 (s, 1H, Ar-H), 7.20 (d, J ¼ 8.2 Hz, 1H, Ar-
H), 6.99 (d, J ¼ 8.2 Hz, 1H, Ar-H), 1.91 (s, 2H, CH2), 1.36 (s, 6H, 2CH3),
4.1.5. 2,2,4,4-Tetramethylthiochroman-6-amine hydrochloride (12)
To a stirred solution of 11 (15.0 g, 57.0 mmol) in MeOH (25 mL)
was added 6 M HCl (100 mL), and the mixture was heated to 90 ꢁC
for 1 h. This mixture was cooled and concentrated to 1/4 of its initial
volume. The resulting crude mixture was cooled to 0 ꢁC (ice bath)
1.34 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d 152.8, 144.0, 143.4, 137.2,
128.4, 126.4 (q, J ¼ 4.2 Hz), 125.4, 125.0 (q, J ¼ 271.0 Hz), 122.1 (q,
J ¼ 32.0 Hz), 118.3, 117.8, 117.6, 54.2, 42.3, 35.8, 32.7, 31.7. Anal. Calcd
for C21H23F3N2OS: C, 61.75; H, 5.68; N, 6.86. Found: C, 61.84; H,
5.90; N, 7.11.

22
B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
4.2.3. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(4-
(trifluoromethoxy)phenyl)urea (2c)
134.5, 133.9, 132.0, 130.1, 128.8, 125.4, 124.0, 123.0, 54.0, 42.3, 35.8,
32.5, 31.6, 21.1. Anal. Calcd for C21H26N2S2: C, 68.07; H, 7.07; N, 7.56.
Found: C, 68.27; H, 6.91; N, 7.74.
Off-white solid (0.145 g, 78%), mp 157e158 ꢁC; 1H NMR
(DMSO‑d6):
d 8.82 (s, 1H, NH), 8.64 (s, 1H, NH), 7.57 (s, 1H, Ar-H),
7.55 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.27 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.19 (d,
4.3.3. 1-(5,6,7,8-Tetrahydronaphthalen-2-yl)-3-(2,2,4,4-
J ¼ 8.5 Hz, 1H, Ar-H), 6.97 (d, J ¼ 8.4 Hz, 1H, Ar-H), 1.90 (s, 2H, CH2),
tetramethylthiochroman-6-yl)thiourea (3c)
1.36 (s, 6H, 2CH3), 1.34 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d
151.9,
Tan solid (0.166 g, 92%), mp 135e136 ꢁC; 1H NMR (CDCl3):
d 7.71
142.3, 141.9, 138.4, 136.3, 127.3, 124.1, 121.1, 119.6 (q, J ¼ 255.3 Hz),
(br s, 2H, 2NH), 7.45 (s, 1H, Ar-H), 7.11 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.09
(s, 2H, Ar-H), 7.05 (dd, J ¼ 8.3, 2.3 Hz, 1H, Ar-H), 7.02 (s, 1H, Ar-H),
2.75 (t, J ¼ 6.2 Hz, 4H, 2CH2), 1.94 (s, 2H, CH2), 1.79 (m, 4H, 2CH2),
118.8, 116.6, 116.4, 53.1, 41.3, 34.7, 31.6, 30.7. Anal. Calcd for
C
21H23F3N2O2S: C, 59.42; H, 5.46; N, 6.60. Found: C, 59.24; H, 5.66;
N, 6.74.
1.42 (s, 6H, 2CH3), 1.38 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 179.9,
144.0, 138.7, 136.5, 134.2, 134.1, 131.8, 130.2, 128.7, 125.8, 124.0,
122.9, 122.7, 54.0, 42.3, 35.8, 32.5, 31.6, 29.4, 29.0, 23.0, 22.8. Anal.
Calcd for C24H30N2S2: C, 70.20; H, 7.36; N, 6.82. Found C, 70.34; H,
7.55; N, 6.70.
4.2.4. 1-(4-Cyanophenyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
urea (2d)
Off-white solid (0.140 g, 87%), mp 114e115 ꢁC; 1H NMR (CDCl3):
d
7.57 (s, 1H, NH), 7.50 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.40 (superimposed
d, J ¼ 8.4 Hz, 2H, Ar-H and s, 1H, Ar-H), 7.21 (s, 1H, NH), 7.06 (d,
J ¼ 8.2 Hz, 1H, Ar-H), 6.91 (d, J ¼ 8.2 Hz, 1H, Ar-H), 1.90 (s, 2H, CH2),
4.3.4. 1-(4-Chlorophenyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (3d)
1.36 (s, 6H, 2CH3), 1.34 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
153.1,
White solid (0.153 g, 89%), mp 137e138 ꢁC. 1H NMR (CDCl3):
144.2, 142.9, 133.9, 133.0, 123.0, 128.9, 120.9, 120.2, 119.2, 119.0,
105.5, 54.1, 42.2, 35.8, 32.4, 31.6. Anal. Calcd for C21H23N3OS: C,
69.01; H, 6.34; N, 11.50. Found: C, 69.36; H, 6.60; N, 12.01.
d 8.00 (br s, 1H, NH), 7.73 (br s, 1H, NH), 7.37 (superimposed d,
J ¼ 8.8 Hz, 2H, Ar-H and s, 1H, Ar-H), 7.35 (d, J ¼ 8.8 Hz, 2H, Ar-H),
7.17 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.03 (dd, J ¼ 8.3, 2.2 Hz, 1H, Ar-H),
1.95 (s, 2H, CH2), 1.43 (s, 6H, 2CH3), 1.38 (s, 6H, 2CH3); 13C NMR
4.2.5. 1-(2-Methoxy-4-nitrophenyl)-3-(2,2,4,4-
tetramethylthiochroman-6-yl)urea (2e)
(CDCl3):
126.3, 124.2, 123.1, 53.9, 42.4, 35.8, 32.5, 31.6. Anal. Calcd for
20H23ClN2S2: C, 61.44; H, 5.93; N, 7.16. Found: C, 61.64; H, 5.90; N,
d 179.8, 144.7, 136.2, 133.03, 133.00, 132.1, 129.3, 129.2,
Yellow solid (0.133 g, 73%), mp 160e161 ꢁC; 1H NMR (CDCl3):
C
d
8.47 (d, J ¼ 8.9 Hz, 1H, Ar-H), 7.93 (d, J ¼ 8.9 Hz, 1H, Ar-H), 7.71 (s,
7.40.
1H, Ar-H), 7.59 (br s, 1H, NH), 7.44 (s, 1H, Ar-H), 7.14 (d, J ¼ 8.2 Hz,
1H, Ar-H), 7.04 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.67 (br s, 1H, NH), 3.90 (s,
4.3.5. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(4-
(trifluoromethyl)phenyl)thiourea (3e)
3H, OCH3), 1.97 (s, 2H, CH2),1.43 (s, 6H, 2CH3),1.38 (s, 6H, 2CH3); 13
C
NMR (CDCl3):
d
152.2, 146.8, 144.3, 142.1, 134.8, 133.9, 130.2, 129.1,
Off-white solid (0.142 g, 76%), mp 147e148 ꢁC; 1H NMR (CDCl3):
121.2, 120.5, 118.1, 117.2, 105.2, 56.3, 54.3, 42.3, 35.8, 32.5, 31.5. Anal.
Calcd for C21H25N3O4S: C, 60.70; H, 6.06; N, 10.11. Found: C, 61.00;
H, 5.94; N, 10.28.
d 8.03 (br s,1H, NH), 7.77 (br s,1H, NH), 7.61 (s, 4H, Ar-H), 7.35 (s,1H,
Ar-H), 7.20 (d, J ¼ 8.2 Hz,1H, Ar-H), 7.04 (d, J ¼ 8.2 Hz,1H, Ar-H),1.97
(s, 2H, CH2), 1.44 (s, 6H, 2CH3), 1.39 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
179.5, 144.9, 141.0, 133.5, 132.6, 129.4, 127.9 (q, J ¼ 33.0 Hz), 126.2
4.3. Representative procedure for the formation of the thiourea
[L ¼ NHC(S)NH] series (3a-n)
(q, J ¼ 3.9 Hz), 124.2, 124.0, 123.9 (q, J ¼ 271.9 Hz), 123.1, 53.8, 42.4,
35.8, 32.5, 31.6. Anal. Calcd for C21H23F3N2S2: C, 59.41; H, 5.46; N,
6.60. Found: C, 59.52; H, 5.62; N, 6.82.
To a stirred solution of 13 (0.115 g, 0.44 mmol) in THF:DCM (1:1,
5 mL) were added substituted anilines (15a-n) (0.46 mmol), fol-
lowed by Et3N (56 mg, 77 mL, 0.55 mmol,1.2 equiv), and the reaction
4.3.6. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(4-
(trifluoromethoxy)phenyl)thiourea (3f)
mixture was stirred at 23 ꢁC for 18 h. The reaction mixture was
concentrated, and the solid was purified on a silica gel column
eluted with increasing concentrations of EtOAc in hexanes to afford
the desired products. Compound 3n was prepared by reacting 13
(0.44 mmol) with the free base of 12 (0.46 mmol). The free base of
12 is known [13].
White solid (0.139 g, 72%), mp 113e114 ꢁC; 1H NMR (CDCl3):
d
8.02 (br s,1H, NH), 7.75 (br s, 1H, NH), 7.47 (d, J ¼ 8.4 Hz, 2H, Ar-H),
7.36 (s, 1H, Ar-H), 7.22e7.16 (m, 3H, Ar-H), 7.03 (d, J ¼ 8.3 Hz, 1H, Ar-
H), 1.96 (s, 2H, CH2), 1.42 (s, 6H, 2CH3), 1.39 (s, 6H, 2CH3); 13C NMR
(CDCl3):
d 179.9, 147.1, 144.8, 136.2, 133.2, 132.9, 129.3, 126.3, 124.3,
123.2, 121.6, 120.4 (q, J ¼ 257.4 Hz), 53.8, 42.4, 35.8, 32.5, 31.6. Anal.
Calcd for C21H23F3N2OS2: C, 57.25; H, 5.26; N, 6.36. Found: C, 57.34;
H, 5.44; N, 6.45.
4.3.1. 1-Phenyl-3-(2,2,4,4-tetramethylthiochroman-6-yl)thiourea
(3a)
White solid (0.148 g, 94%), mp 127e128 ꢁC; 1H NMR (CDCl3):
4.3.7. 1-(3-Nitrophenyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (3g)
d
7.85 (br s, 2H, 2NH), 7.44e7.37 (m, 4H, Ar-H), 7.30e7.23 (m, 2H, Ar-
H), 7.14 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.05 (dd, J ¼ 8.3, 2.3 Hz, 1H, Ar-H),
Yellow solid (0.150 g, 85%), mp 156e157 ꢁC; 1H NMR (DMSO‑d6):
1.95 (s, 2H, CH2), 1.42 (s, 6H, 2CH3), 1.39 (s, 6H, 2CH3); 13C NMR
d 10.11 (s, 1H, NH), 10.08 (s, 1H, NH), 8.53 (s, 1H, Ar-H), 7.95 (d,
(CDCl3):
d
179.8, 144.3, 137.3, 133.7, 132.3, 129.5, 128.9, 126.9, 125.1,
J ¼ 8.2 Hz, 1H, Ar-H), 7.89 (d, J ¼ 8.2 Hz, 1H, Ar-H), 7.60 (t, J ¼ 8.2 Hz,
1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.21 (d, J ¼ 8.4 Hz, 1H, Ar-H), 7.04 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 1.92 (s, 2H, CH2), 1.38 (s, 6H, 2CH3), 1.33 (s, 6H,
124.1, 123.0, 53.9, 42.3, 35.8, 32.5, 31.6. Anal. Calcd for C20H24N2S2:
C, 67.37; H, 6.79; N, 7.86. Found: C, 67.54; H, 6.91; N, 7.73.
2CH3); 13C NMR (DMSO‑d6):
d 178.8, 146.9, 141.9, 140.4, 135.4, 129.0,
4.3.2. 1-(4-Methylphenyl)-3-(2,2,4,4-tetramethylthiochroman-6-
yl)thiourea (3b)
128.9, 127.6, 126.8, 122.0, 121.4, 117.9, 116.9, 52.8, 41.4, 34.7, 31.6,
30.7. Anal. Calcd for C20H23N3O2S2: C, 59.82; H, 5.77; N, 10.46.
Found: C, 59.90; H, 5.82; N, 10.52.
Off-white solid (0.155 g, 95%), mp 162e163 ꢁC; 1H NMR (CDCl3):
d
7.84 (s, 2H, 2NH), 7.43 (s, 1H, Ar-H), 7.23 (d, J ¼ 8.0 Hz, 2H, Ar-H),
7.20 (d, J ¼ 8.0 Hz, 2H, Ar-H), 7.13 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.05 (d,
J ¼ 8.0 Hz,1H, Ar-H), 2.35 (s, 3H, ArCH3),1.94 (s, 2H, CH2),1.42 (s, 6H,
4.3.8. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(2-
(trifluoromethyl)phenyl)thiourea (3h)
2CH3), 1.38 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
179.9, 144.1, 137.1,
Tan solid (0.158 g, 85%), mp 136e137 ꢁC; 1H NMR (DMSO‑d6):

B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
23
d
9.98 (s, 1H, NH), 9.25 (s, 1H, NH), 7.73 (d, J ¼ 7.9 Hz, 1H, Ar-H), 7.68
41.3, 34.6, 31.6, 30.6. Anal. Calcd for C21H26N2O2S2: C, 62.66; H, 6.51;
N, 6.96. Found: C, 62.80; H, 6.44; N, 7.14.
(t, J ¼ 7.8 Hz, 1H, Ar-H), 7.63 (d, J ¼ 2.3 Hz, 1H, Ar-H), 7.58 (d,
J ¼ 7.9 Hz, 1H, Ar-H), 7.47 (t, J ¼ 7.8 Hz, 1H, Ar-H), 7.27 (dd, J ¼ 8.4,
2.3 Hz, 1H, Ar-H), 7.03 (d, J ¼ 8.4 Hz, 1H, Ar-H), 1.92 (s, 2H, CH2), 1.37
4.3.14. 1,3-Bis(2,2,4,4-Tetramethylthiochroman-6-yl)thiourea (3n)
(s, 6H, 2CH3), 1.34 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d
180.3, 141.9,
Tan solid (0.176 g, 83%), mp 142e143 ꢁC; 1H NMR (CDCl3):
d 7.64
136.6, 135.6, 131.9, 131.7, 127.5, 127.1, 126.8, 126.5, 125.5 (q,
J ¼ 4.8 Hz), 123.0 (q, J ¼ 273.3 Hz), 121.7, 121.2, 52.9, 41.4, 34.7, 31.5,
31.7. Anal. Calcd for C21H23F3N2S2: C, 59.41; H, 5.46; N, 6.60. Found:
C, 59.64; H, 5.40; N, 6.93.
(br s, 2H, 2NH), 7.43 (s, 2H, Ar-H), 7.14 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.07
(d, J ¼ 8.3 Hz, 2H, Ar-H), 1.95 (s, 4H, 2CH2), 1.42 (s, 12H, 4CH3), 1.39
(s, 12H, 4CH3); 13C NMR (CDCl3):
d 180.2, 144.0, 132.2, 130.4, 129.1,
122.0, 121.2, 53.5, 42.6, 35.8, 32.6, 31.6. Anal. Calcd for C27H36N2S3:
C, 66.90; H, 7.49; N, 5.78. Found: C, 67.00; H, 7.32; N, 5.90.
4.3.9. 4-(3-(2,2,4,4-Tetramethylthiochroman-6-yl)thioureido)
benzamide (3i)
4.4. Representative procedure for the formation of the
benzylthiourea [L ¼ NHC(S)NHCH2] series (4a-i)
White solid (0.114 g, 65%), mp 172e173 ꢁC; 1H NMR (DMSO‑d6):
d
9.95 (s, 1H, NH), 9.89 (s, 1H, NH), 7.89 (s, 1H, NH), 7.82 (d,
J ¼ 8.3 Hz, 2H, Ar-H), 7.58 (s, 1H, Ar-H), 7.56 (d, J ¼ 8.3 Hz, 2H, Ar-H),
7.27 (s, 1H, NH), 7.23 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.02 (d, J ¼ 8.3 Hz, 1H,
Ar-H), 1.91 (s, 2H, CH2), 1.37 (s, 6H, 2CH3), 1.33 (s, 6H, 2CH3); 13C
To a stirred solution of 13 (0.115 g, 0.44 mmol) in THF:DCM (1:1,
5 mL) were added substituted benzylamines (16a-i) (0.46 mmol),
followed by Et3N (56 mg, 77 mL, 0.55 mmol, 1.2 equiv), and the re-
NMR (DMSO‑d6):
d
178.4, 166.7, 141.8, 141.6, 135.8, 128.9, 127.3,
action was stirred at 23 ꢁC for 18 h. The reaction mixture was
concentrated and purified on a silica gel column eluted with
increasing concentrations of EtOAc in hexanes to afford the desired
products.
127.2, 126.7, 121.8, 121.3, 121.2, 52.8, 41.4, 34.7, 31.6, 30.6. Anal. Calcd
for C21H25N3OS2: C, 63.13; H, 6.31; N, 10.52. Found: C, 63.34; H,
6.50; N, 10.84.
4.3.10. 1-(4-Methoxyphenyl)-3-(2,2,4,4-tetramethylthiochroman-
6-yl)thiourea (3j)
4.4.1. 1-Benzyl-3-(2,2,4,4-tetramethylthiochroman-6-yl)thiourea
(4a)
White solid (0.168 g, 95%), mp 115e116 ꢁC; 1H NMR (CDCl3):
White solid (0.155 g, 95%), mp 149e150 ꢁC; 1H NMR (CDCl3):
d
7.68 (br s, 2H, 2NH), 7.43 (s,1H, Ar-H), 7.28 (d, J ¼ 8.5 Hz, 2H, Ar-H),
d 7.95 (s, 1H, NH), 7.35e7.25 (m, 5H, Ph-H), 7.21 (s, 1H, Ar-H), 7.11 (d,
7.13 (d, J ¼ 8.1 Hz, 1H, Ar-H), 7.05 (d, J ¼ 8.1 Hz, 1H, Ar-H), 6.93 (d,
J ¼ 8.2 Hz, 1H, Ar-H), 6.89 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.18 (br t,
J ¼ 8.5 Hz, 2H, Ar-H), 3.81 (s, 3H, OCH3), 1.94 (s, 2H, CH2), 1.42 (s, 6H,
J ¼ 5.3 Hz, 1H, NHCH2), 4.86 (d, J ¼ 5.3 Hz, 2H, NHCH2Ar), 1.91 (s, 2H,
2CH3), 1.38 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
180.4, 158.7, 144.1,
CH2), 1.40 (s, 6H, 2CH3),1.29 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 180.8,
133.9, 132.0, 129.7, 128.8, 127.5, 124.1, 123.0, 114.8, 55.5, 54.0, 42.3,
35.8, 32.5, 31.6. Anal. Calcd for C21H26N2OS2: C, 65.25; H, 6.78; N,
7.25. Found: C, 65.40; H, 6.83; N, 7.34.
144.8,137.2,132.9,132.5,129.4, 128.8,127.8, 127.8, 124.2, 123.2, 53.8,
49.6, 42.3, 35.7, 32.4, 31.6. Anal. Calcd for C21H26N2S2: C, 68.07; H,
7.07; N, 7.56. Found: C, 68.22; H, 7.14; N, 7.60.
4.3.11. 1-(3-Methoxyphenyl)-3-(2,2,4,4-tetramethylthiochroman-6-
yl)thiourea (3k)
4.4.2. 1-Phenethyl-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (4b)
White solid (0.161 g, 95%), mp 105e106 ꢁC; 1H NMR (CDCl3):
Off-white solid (0.150 g, 89%), mp 122e123 ꢁC; 1H NMR (CDCl3):
d
7.79 (br s, 2H, 2NH), 7.43 (d, J ¼ 2.3 Hz,1H, Ar-H), 7.30 (t, J ¼ 8.1 Hz,
d 7.77 (br s, 1H, NH), 7.26e7.01 (m, 7H, Ph-H, Ar-H), 6.70 (s, 1H, Ar-
1H, Ar-H), 7.14 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.06 (dd, J ¼ 8.3, 2.3 Hz, 1H,
Ar-H), 7.04 (s, 1H, Ar-H), 6.92 (d, J ¼ 8.1 Hz, 1H, Ar-H), 6.81 (dd,
J ¼ 8.1, 2.5 Hz, 1H, Ar-H), 3.81 (s, 3H, OCH3), 1.95 (s, 2H, CH2), 1.42 (s,
H), 5.91 (s, 1H, NH), 3.88 (s, 2H, PhCH2), 2.91 (s, 2H, NHCH2), 1.92 (s,
2H, CH2), 1.42 (s, 6H, 2CH3), 1.30 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
180.7, 144.9, 138.4, 132.8, 132.4, 129.3, 128.73, 128.69, 126.7, 124.3,
123.3, 53.9, 46.3, 42.4, 35.7, 34.9, 32.4, 31.6. Anal. Calcd for
C22H28N2S2: C, 68.71; H, 7.34; N, 7.28. Found: C, 69.00; H, 7.56; N,
6H, 2CH3), 1.39 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 179.6, 160.5, 144.2,
138.2, 133.8, 132.2, 130.3, 128.9, 124.0,123.0, 116.9, 112.6, 110.6, 55.5,
54.0, 42.3, 35.8, 32.5, 31.6. Anal. Calcd for C21H26N2OS2: C, 65.25; H,
6.78; N, 7.25. Found: C, 65.45; H, 6.84; N, 7.38.
7.30.
4.4.3. 1-(4-Methylbenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (4c)
4.3.12. 1-(4-Hydroxyphenyl)-3-(2,2,4,4-tetramethylthiochroman-
6-yl)thiourea (3l)
White solid (0.160 g, 95%), mp 151e152 ꢁC; 1H NMR (CDCl3):
Off-white solid (0.142 g, 87%), mp 93e94 ꢁC; 1H NMR (CDCl3):
d 7.77 (br s, 1H, NH), 7.20e7.15 (m, 3H, Ar-H), 7.13e7.07 (m, 3H, Ar-
d
7.50 (s, 2H, 2NH), 7.42 (s, 1H, Ar-H), 7.24 (d, J ¼ 8.4 Hz, 2H, Ar-H),
H), 6.88 (d, J ¼ 7.5 Hz, 1H, Ar-H), 6.12 (br t, J ¼ 5.2 Hz, 1H, NHCH2),
7.14 (d, J ¼ 8.2 Hz, 1H, Ar-H), 7.05 (dd, J ¼ 8.2, 2.2 Hz, 1H, Ar-H), 6.86
(d, J ¼ 8.4 Hz, 2H, Ar-H), 4.96 (br s, 1H, phenol OH), 1.95 (s, 2H, CH2),
4.81 (d, J ¼ 5.2 Hz, 2H, NHCH2Ar), 2.32 (s, 3H, ArCH3), 1.91 (s, 2H,
CH2), 1.40 (s, 6H, 2CH3), 1.29 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 180.7,
144.8, 137.6, 134.1, 132.8, 132.5, 129.5, 129.4, 127.8, 124.2, 123.1, 53.8,
49.4, 42.3, 35.7, 32.4, 31.6, 21.1. Anal. Calcd for C22H28N2S2: C, 68.71;
H, 7.34; N, 7.28. Found: C, 68.90; H, 7.42; N, 7.52.
1.42 (s, 6H, 2CH3), 1.39 (s, 6H, 2CH3); 13C NMR (CDCl3):
d
180.4,
155.2, 144.2, 133.6, 128.9, 127.8, 124.2, 123.2, 116.9, 116.4, 116.2, 53.9,
42.3, 35.8, 32.5, 31.6. Anal. Calcd for C20H24N2OS2: C, 64.48; H, 6.49;
N, 7.52. Found: C, 64.68; H, 6.67; N, 7.81.
4.4.4. 1-(4-Chlorobenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (4d)
4.3.13. 1-(3-Hydroxy-4-methoxyphenyl)-3-(2,2,4,4-
tetramethylthiochroman-6-yl)thiourea (3m)
Off-white solid (0.164 g, 92%), mp 88e89 ꢁC; 1H NMR (CDCl3):
Tan solid (0.136 g, 77%), mp 152e153 ꢁC; 1H NMR (DMSO‑d6):
d
7.89 (br s, 1H, NH), 7.29 (d, J ¼ 8.2 Hz, 2H, Ar-H), 7.24 (d, J ¼ 8.2 Hz,
d
9.52 (s, 1H, NH), 9.43 (s, 1H, NH), 9.09 (s, 1H, phenol OH), 7.55 (s,
2H, Ar-H), 7.19 (s, 1H, Ar-H), 7.13 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.89 (d,
J ¼ 8.2 Hz, 1H, Ar-H), 6.16 (br t, J ¼ 5.6 Hz, 1H, NHCH2), 4.83 (d,
J ¼ 5.6 Hz, 2H, NHCH2Ar), 1.92 (s, 2H, CH2), 1.41 (s, 6H, 2CH3), 1.30 (s,
1H, Ar-H), 7.22 (d, J ¼ 8.3 Hz, 1H, Ar-H), 6.98 (d, J ¼ 8.3 Hz, 1H, Ar-H),
6.89 (s, 1H, Ar-H), 6.86 (d, J ¼ 8.5 Hz, 1H, Ar-H), 6.75 (d, J ¼ 8.5 Hz,
1H, Ar-H), 3.74 (s, 3H, OCH3), 1.91 (s, 2H, CH2), 1.37 (s, 6H, 2CH3),
6H, 2CH3) 13C NMR (CDCl3):
d 181.1, 144.9, 135.9, 133.6, 133.2, 132.3,
1.32 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d
178.6, 145.7, 144.6, 141.5,
129.5,129.1,128.9,124.4,123.3, 53.7, 48.7, 42.4, 35.7, 32.4, 31.6. Anal.
Calcd for C21H25ClN2S2: C, 62.28; H, 6.22; N, 6.92. Found: C, 62.43;
136.2, 131.6, 126.7, 126.5, 121.9, 121.4, 114.3, 111.7, 111.6, 55.2, 52.9,

24
B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
H, 6.30; N, 7.18.
4.5. Representative procedure for the formation of the hydrazide
[L ¼ NHC(S)NHNH] and benzhydrazide [L ¼ NHC(S)NHNHC(O)]
series (5a-e)
4.4.5. 1-(4-Fluorobenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (4e)
To a stirred solution of 13 (0.115 g, 0.44 mmol) in THF:DCM (1:1,
5 mL) were added arylhydrazine (17a) or arylhydrazides (18a-d)
White solid (0.152 g, 89%), mp 120e121 ꢁC; 1H NMR (CDCl3):
d
7.83 (br s, 1H, NH), 7.26 (m, 2H, Ar-H), 7.19 (s, 1H, Ar-H), 7.12 (d,
(0.46 mmol), followed by Et3N (56 mg, 77 mL, 0.55 mmol, 1.2 equiv),
and the reaction was stirred at 23 ꢁC for 18 h. The reaction mixture
was concentrated and purified on a silica gel column eluted with
increasing concentrations of EtOAc in hexanes to afford the desired
products.
J ¼ 8.0 Hz, 1H, Ar-H), 7.00 (t, J ¼ 8.0 Hz, 2H, Ar-H), 6.88 (d, J ¼ 8.1 Hz,
1H, Ar-H), 6.14 (br t, J ¼ 5.5 Hz, 1H, NHCH2), 4.83 (d, J ¼ 5.5 Hz, 2H,
NHCH2Ar), 1.91 (s, 2H), 1.40 (s, 6H), 1.29 (s, 6H); 13C NMR (CDCl3):
d
180.9, 162.4 (d, J ¼ 246.5 Hz), 144.9, 133.13, 133.10, 132.3, 129.6,
129.5, 124.3, 123.2, 115.7 (d, J ¼ 21.5 Hz), 53.7, 48.7, 42.3, 35.7, 32.4,
31.6. Anal. Calcd for C21H25FN2S2: C, 64.91; H, 6.49; N, 7.21. Found:
C, 65.00; H, 6.34; N, 7.43.
4.5.1. 2-Phenyl-N-(2,2,4,4-tetramethylthiochroman-6-yl)
hydrazine-1-carbothioamide (5a)
Off-white solid (0.113 g, 69%), mp 172e173 ꢁC; 1H NMR (CDCl3):
d
8.92 (br s, 1H, NH), 7.71 (s, 1H, Ar-H), 7.38 (br s, 1H, NH), 7.33 (t,
4.4.6. 1-(4-Nitrobenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-yl)
thiourea (4f)
J ¼ 7.9 Hz, 2H, Ar-H), 7.28 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.09 (d, J ¼ 8.0 Hz,
1H, Ar-H), 7.04 (t, J ¼ 7.9 Hz, 1H, Ar-H), 6.93 (d, J ¼ 7.9 Hz, 2H, Ar-H),
5.90 (br s, 1H, NH), 1.94 (s, 2H, CH2), 1.41 (s, 6H, 2CH3), 1.39 (s, 6H,
Yellow solid (0.142 g, 78%), mp 160e161 ꢁC; 1H NMR (CDCl3):
d
8.18 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.82 (s, 1H, NH), 7.47 (d, J ¼ 8.3 Hz,
2CH3); 13C NMR (CDCl3):
d 181.2, 145.8, 143.2, 134.5, 130.5, 129.7,
2H, Ar-H), 7.23 (s, 1H, Ar-H), 7.16 (d, J ¼ 8.0 Hz, 1H, Ar-H), 6.93 (d,
J ¼ 8.0 Hz, 1H, Ar-H), 6.28 (br t, J ¼ 5.9 Hz, 1H, NHCH2), 5.00 (d,
J ¼ 5.9 Hz, 2H, NHCH2Ar), 1.93 (s, 2H, CH2), 1.42 (s, 6H, 2CH3), 1.33 (s,
128.2, 122.9, 122.7, 121.8, 113.9, 54.2, 42.2, 35.8, 32.5, 31.6. Anal.
Calcd for C20H25N3S2: C, 64.65; H, 6.78; N, 11.31. Found: C, 64.92; H,
7.00; N, 11.56.
6H, 2CH3); 13C NMR (CDCl3):
d 181.8,147.4, 145.2,133.8,132.0,129.5,
128.1, 124.7, 123.9, 123.6, 53.7, 48.3, 42.4, 35.8, 32.4, 31.6 (1 aromatic
C not observed). Anal. Calcd for C21H25N3O2S2: C, 60.70; H, 6.06; N,
10.11. Found: C, 61.02; H, 6.24; N, 10.34.
4.5.2. 2-Benzoyl-N-(2,2,4,4-tetramethylthiochroman-6-yl)
hydrazine-1-carbothioamide (5b)
White solid (0.123 g, 70%), mp 161e162 ꢁC; 1H NMR (CDCl3):
d
10.27 (br s,1H, NH),10.18 (br s,1H, NH), 8.87 (br s,1H, NH), 7.85 (d,
J ¼ 7.6 Hz, 2H, Ar-H), 7.58 (t, J ¼ 7.6 Hz, 1H, Ar-H), 7.53e7.44 (m, 3H,
Ar-H), 7.19 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.09 (d, J ¼ 8.3 Hz, 1H, Ar-H), 1.93
(s, 2H, CH2), 1.41 (s, 6H, 2CH3), 1.34 (s, 6H, 2CH3) 13C NMR (CDCl3):
4.4.7. 1-(2,2,4,4-Tetramethylthiochroman-6-yl)-3-(4-
(trifluoromethyl)benzyl)thiourea (4g)
Tan solid (0.162 g, 84%), mp 116e117 ꢁC; 1H NMR (CDCl3):
d 7.78
d
176.5, 163.2, 143.9, 134.0, 132.8, 131.1, 131.0, 129.1, 128.6, 127.1,
(br s,1H, NH), 7.58 (d, J ¼ 7.9 Hz, 2H, Ar-H), 7.42 (d, J ¼ 7.9 Hz, 2H, Ar-
H), 7.20 (s, 1H, Ar-H), 7.15 (d, J ¼ 7.9 Hz, 1H, Ar-H), 6.92 (d, J ¼ 7.9 Hz,
1H, Ar-H), 6.20 (br t, J ¼ 5.9 Hz, 1H, NHCH2), 4.94 (d, J ¼ 5.9 Hz, 2H,
122.7, 121.9, 54.1, 42.3, 35.8, 32.4, 31.6. Anal. Calcd for C21H25N3OS2:
C, 63.13; H, 6.31; N, 10.52. Found: C, 63.42; H, 6.51; N, 10.82.
NHCH2Ar),1.92 (s, 2H, CH2),1.41 (s, 6H, 2CH3),1.30 (s, 6H, 2CH3); 13
C
4.5.3. 2-(4-Nitrobenzoyl)-N-(2,2,4,4-tetramethylthiochroman-6-yl)
hydrazine-1-carbothioamide (5c)
NMR (CDCl3):
d
181.4, 145.0, 141.6, 133.5, 132.1, 130.8 (q, J ¼ 32.7 Hz),
129.5,127.9, 125.7 (q, J ¼ 3.8 Hz),124.5, 124.0 (q, J ¼ 272.0 Hz),123.4,
53.7, 48.7, 42.4, 35.7, 32.4, 31.6. Anal. Calcd for C22H25F3N2S2: C,
60.25; H, 5.75; N, 6.39. Found: C, 60.35; H, 6.02; N, 6.68.
Yellow solid (0.139 g, 71%), mp 173e174 ꢁC; 1H NMR (DMSO‑d6):
d
10.86 (s, 1H, NH), 9.80 (br s, 2H, 2NH), 8.36 (d, J ¼ 8.4 Hz, 2H, Ar-
H), 8.18 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.20 (d, J ¼ 8.4 Hz,
1H, Ar-H), 7.02 (d, J ¼ 8.4 Hz, 1H, Ar-H), 1.92 (s, 2H, CH2), 1.37 (s, 6H,
2CH3), 1.33 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d 181.0, 163.9, 148.7,
4.4.8. 1-(4-Methoxybenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-
yl)thiourea (4h)
139.0, 137.7, 135.6, 129.8, 127.7, 126.3, 126.0, 124.1, 122.8, 52.8, 41.4,
34.6, 31.6, 30.6. Anal. Calcd for C21H24N4O3S2: C, 56.74; H, 5.44; N,
12.60. Found: C, 57.02; H, 5.66; N, 12.92.
White solid (0.163 g, 93%), mp 154e155 ꢁC; 1H NMR (CDCl3):
d
7.85 (br s, 1H, NH), 7.22 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.19 (s, 1H, Ar-H),
7.10 (d, J ¼ 8.3 Hz, 1H, Ar-H), 6.87 (d, J ¼ 8.3 Hz, 1H, Ar-H), 6.84 (d,
J ¼ 8.3 Hz, 2H, Ar-H), 6.11 (br s, 1H, NH), 4.77 (d, J ¼ 5.2 Hz, 2H,
NHCH2Ar), 3.78 (s, 3H, OCH3), 1.91 (s, 2H, CH2), 1.40 (s, 6H, 2CH3),
4.5.4. N-(2,2,4,4-tetramethylthiochroman-6-yl)-2-(4-
(trifluoromethoxy)benzoyl)hydrazine-1-carbothioamide (5d)
Off-white solid (0.159 g, 75%), mp 158e159 ꢁC; 1H NMR
1.29 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 180.5, 159.3, 144.8, 132.7,
(DMSO‑d6):
d 10.65 (br s, 1H, NH), 9.74 (br s, 2H, 2NH), 8.08 (d,
132.5, 129.4, 129.2, 124.2, 123.1, 114.2, 55.3, 53.8, 49.1, 42.3, 35.7,
32.4, 31.6 (one aromatic carbon missing). Anal. Calcd for
J ¼ 8.3 Hz, 2H, Ar-H), 7.52 (superimposed d, J ¼ 8.3 Hz, 2H, Ar-H and
s, 1H, Ar-H), 7.21 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.01 (d, J ¼ 8.3 Hz, 1H, Ar-
H), 1.92 (s, 2H, CH2), 1.37 (s, 6H, 2CH3) 1.34 (s, 6H, 2CH3); 13C NMR
C
22H28N2OS2: C, 65.96; H, 7.05; N, 6.99. Found: C, 66.24; H, 7.32; N,
7.18.
(DMSO‑d6):
d 181.2, 165.3, 151.2, 142.3, 136.7, 132.2, 130.8, 128.7,
127.3, 124.8 (br), 124.5 (br), 121.1, 120.4 (q, J ¼ 252.3 Hz), 53.9, 42.4,
35.7, 32.7, 31.7. Anal. Calcd for C22H24F3N3O2S2: C, 54.64; H, 5.00; N,
8.69. Found: C, 54.66; H, 4.95; N, 8.82.
4.4.9. 1-(4-Hydroxybenzyl)-3-(2,2,4,4-tetramethylthiochroman-6-
yl)thiourea (4i)
Off-white solid (0.151 g, 89%), mp 149e150 ꢁC; 1H NMR
(DMSO‑d6):
d
9.44 (br s, 1H, NH), 9.31 (s, 1H, NH), 7.92 (br s, 1H, OH),
4.5.5. 2-(3,5-Bis(trifluoromethyl)benzoyl)-N-(2,2,4,4-
7.50 (s,1H, Ar-H), 7.20e7.09 (m, 3H, Ar-H), 6.99 (d, J ¼ 8.4 Hz,1H, Ar-
H), 6.71 (d, J ¼ 8.0 Hz, 2H, Ar-H), 4.58 (s, 2H, NHCH2Ar), 1.90 (s, 2H,
CH2), 1.36 (s, 6H, 2CH3), 1.30 (s, 6H, 2CH3) 13C NMR (DMSO‑d6):
tetramethylthiochroman-6-yl)hydrazine-1-carbothioamide (5e)
White solid (0.153 g, 65%), mp 151e152 ꢁC; 1H NMR (DMSO‑d6):
d
11.05 (s,1H, NH), 9.85 (br s,1H, NH), 9.80 (br s,1H, NH), 8.57 (s, 2H,
d
180.7, 156.9, 143.1, 136.8, 129.4, 127.93, 127.86, 122.6, 122.1, 122.0,
Ar-H), 8.40 (s, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.16 (d, J ¼ 8.3 Hz, 1H, Ar-
115.5, 53.9, 47.4, 42.4, 35.7, 32.6, 31.8. Anal. Calcd for C21H26N2OS2:
C, 65.25; H, 6.78; N, 7.25. Found: C, 65.32; H, 6.84; N, 7.42.
H), 7.03 (d, J ¼ 8.3 Hz, 1H, Ar-H), 1.92 (s, 2H, CH2), 1.37 (s, 6H, 2CH3),
1.33 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d 180.2, 162.7, 141.1, 135.6,

B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
25
134.4, 129.9 (q, J ¼ 33.3 Hz), 128.0 (2C), 126.4, 124.9, 123.9 (br, 2C),
122.5 (q, J ¼ 273.7 Hz), 52.9, 41.4, 34.7, 31.6, 30.6. Anal. Calcd for
23H23F6N3OS2: C, 51.58; H, 4.33; N, 7.85. Found: C, 51.74; H, 4.56; N,
4.7.1. 1-(4,4-Dimethylthiochroman-6-yl)-3-(4-nitrophenyl)
thiourea (6a)
C
Yellow solid (0.134 g, 82%), mp 142e143 ꢁC; 1H NMR (DMSO‑d6):
10.34 (s, 1H, NH), 10.20 (s, 1H, NH), 8.20 (d, J ¼ 8.8 Hz, 2H, Ar-H),
7.94.
d
7.83 (d, J ¼ 8.8 Hz, 2H, Ar-H), 7.54 (s, 1H, Ar-H), 7.20 (d, J ¼ 8.4 Hz,
1H, Ar-H), 7.01 (d, J ¼ 8.5 Hz,1H, Ar-H), 3.02 (d, J ¼ 6.0 Hz, 2H, CH2S),
1.89 (d, J ¼ 6.0 Hz, 2H, CH2), 1.26 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
4.6. Synthesis of desmethyl series (6a-d)
d
179.4, 146.8, 142.7, 142.4, 135.6, 128.3, 126.5, 124.9, 122.8, 122.4,
4.6.1. N-(4-((3-methylbut-2-en-1-yl)thio)phenyl)acetamide (20)
To a stirred solution of acetamidothiphenol (7, 5.0 g, 30.0 mmol)
in acetone (25 mL) was added K2CO3 (8.26 g, 60.0 mmol), followed
by the addition of 1-bromo-3-methylbut-2-ene (19) (5.51 g,
36.5 mmol). The mixture was refluxed for 6 h. The crude reaction
was concentrated to dryness, and the residue was extracted with
EtOAc (3 ꢂ 50 mL). The extract was then washed with saturated
NaCl (50 mL), dried (MgSO4), filtered and concentrated. The crude
product was purified on a silica gel column eluted with increasing
concentrations of EtOAc in hexanes to obtain pure 20 (5.9 g, 84%) as
121.9, 37.4, 33.3, 30.4, 22.8. Anal. Calcd for C18H19N3O2S2: C, 57.89;
H, 5.13; N, 11.25. Found: C, 58.02; H, 5.26; N, 11.32.
4.7.2. 1-(4,4-Dimethylthiochroman-6-yl)-3-(4-(trifluoromethyl)
phenyl)thiourea (6b)
White solid (0.155 g, 89%), mp 173e174 ꢁC; 1H NMR (DMSO‑d6):
d
10.02 (s, 1H, NH), 9.95 (s, 1H, NH), 7.74 (d, J ¼ 8.5 Hz, 2H, Ar-H),
7.67 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.51 (d, J ¼ 1.9 Hz, 1H, Ar-H), 7.19 (dd,
J ¼ 8.4, 1.9 Hz, 1H, Ar-H), 7.01 (d, J ¼ 8.4 Hz, 1H, Ar-H), 3.02 (t,
J ¼ 6.0 Hz, 2H, CH2S), 1.89 (t, J ¼ 6.0 Hz, 2H, CH2), 1.26 (s, 6H, 2CH3);
a colorless oil. 1H NMR (CDCl3):
d
7.43 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.30
13C NMR (DMSO‑d6):
d 178.7, 142.8, 141.3, 134.7, 126.9, 125.4, 125.0
(d, J ¼ 8.3 Hz, 2H, Ar-H), 5.27 (t, J ¼ 7.6 Hz, 1H, ¼CHCH2), 3.49 (d,
(q, J ¼ 3.5 Hz), 123.9 (q, J ¼ 271.5 Hz), 123.2 (q, J ¼ 32.0 Hz), 122.1,
121.8, 121.5, 36.4, 32.2, 29.4, 21.7. Anal. Calcd for C19H19F3N2S2: C,
57.56; H, 4.83; N, 7.07. Found: C, 57.80; H, 5.02; N, 7.14.
J ¼ 7.6 Hz, 2H, ¼CHCH2), 2.17 (s, 3H, CH3CO), 1.54 (s, 3H, CH3), 1.20
(s, 3H, CH3), NH not observed; 13C NMR (CDCl3):
136.3, 131.7, 131.5, 120.2, 119.4, 33.1, 25.7, 24.6, 17.7.
d 168.4, 136.5,
4.7.3. 1-(4,4-Dimethylthiochroman-6-yl)-3-(4-(trifluoromethoxy)
phenyl)thiourea (6c)
4.6.2. N-(4,4-dimethylthiochroman-6-yl)acetamide (21)
Off-white solid (0.132 g, 73%), mp 167e168 ꢁC; 1H NMR (CDCl3):
To stirred polyphosphoric acid (ca. 10 g of 115%) at 70 ꢁC, 20
(4.00 g, 17.0 mmol) was added and heating was continued for 2 h.
The reaction mixture was cooled at 0 ꢁC, poured into ice-cold water
and stirred for 2 h. The compound was extracted with EtOAc
(3 ꢂ 50 mL), washed with saturated NaHCO3 (100 mL), saturated
NaCl (50 mL), dried (MgSO4), filtered and concentrated under vac-
uum. The product was purified on a silica gel column eluted with
increasing concentrations of EtOAc in hexanes to give 21 (2.88 g,
d
7.83 (br s, 1H, NH), 7.62 (br s, 1H, NH), 7.47 (d, J ¼ 8.5 Hz, 2H, Ar-H),
7.33 (s, 1H, Ar-H), 7.22 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.15 (d, J ¼ 8.3 Hz,1H,
Ar-H), 7.01 (d, J ¼ 8.3 Hz,1H, Ar-H), 3.05 (t, J ¼ 6.0 Hz, 2H, CH2S),1.96
(t, J ¼ 6.0 Hz, 2H, CH2), 1.33 (s, 6H, 2CH3); 13C NMR (CDCl3):
d 179.9,
147.1, 144.0, 136.2, 132.6, 132.0, 127.9, 126.2, 124.1, 123.5, 121.7, 120.4
(q, J ¼ 264.0 Hz), 37.0, 33.3, 30.1, 23.1. Anal. Calcd for
C19H19F3N2OS2: C, 55.32; H, 4.64; N, 6.79. Found: C, 55.56; H, 4.80;
N, 6.92.
72%) as a colorless liquid. IR: 3305, 1662 cmꢀ1 1H NMR (CDCl3):
;
d
7.60 (br s, 1H, NH), 7.27 (d, J ¼ 2.3 Hz, 1H, Ar-H), 7.20 (dd, J ¼ 8.2,
4.7.4. Ethyl 4-(3-(4,4-dimethylthiochroman-6-yl)thioureido)
benzoate (6d)
2.3 Hz, 1H, Ar-H), 7.04 (d, J ¼ 8.2 Hz, 1H, Ar-H), 2.16 (s, 3H, CH3CO),
2.72 (t, J ¼ 4.1 Hz, 2H, CH2), 1.90 (t, J ¼ 4.1 Hz, 2H, CH2S), 1.39 (s, 6H,
Tan solid (0.146 g, 83%), mp 159e160 ꢁC; 1H NMR (DMSO‑d6):
2CH3); 13C NMR (CDCl3):
d 168.3, 143.4, 135.1, 128.4, 128.2, 118.7,
d
10.04 (s, 1H, NH), 9.94 (s, 1H, NH), 7.90 (d, J ¼ 8.4 Hz, 2H, Ar-H),
118.2, 54.4, 35.7, 32.4, 24.4. 20.1.
7.68 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.19 (d, J ¼ 8.4 Hz,
1H, Ar-H), 6.99 (d, J ¼ 8.4 Hz, 1H, Ar-H), 4.29 (q, J ¼ 7.1 Hz, 2H,
OCH2CH3), 3.02 (t, J ¼ 6.0 Hz, 2H, CH2S), 1.89 (t, J ¼ 6.0 Hz, 2H, CH2),
1.31 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.26 (s, 6H); 13C NMR (DMSO‑d6):
4.6.3. 4,4-Dimethylthiochroman-6-amine hydrochloride (22)
To a stirred solution of 21 (2.7 g, 11.5 mmol) in methanol (15 mL)
was added 6 M HCl (15 mL). The reaction mixture was heated to
90 ꢁC for 1 h, followed by cooling to 23 ꢁC. The crude reaction
mixture was concentrated to 1/4 of its initial volume and was then
cooled to 0 ꢁC for 1 h to yield a solid. The solid was filtered and dried
under vacuum to afford 22 as an off-white solid (2.27 g, 86%), mp
d
178.4, 164.7, 143.6, 141.2, 134.7, 129.1, 126.8, 125.4, 124.0, 121.7,
121.4, 121.1, 59.8, 37.1, 32.2, 29.4, 21.7, 13.6. Anal. Calcd for
21H24N2O2S2: C, 62.97; H, 6.04; N, 6.99. Found: C, 63.18; H, 6.16; N,
C
7.18.
214e216 ꢁC; IR: 2926, 2794 cmꢀ1 1H NMR (DMSO‑d6):
; d 10.08 (s,
5. Biology
2H, NH2), 7.48 (s, 1H, Ar-H), 7.22 (d, J ¼ 8.3 Hz, 1H, Ar-H), 7.09 (d,
The compounds were dissolved in DMSO at a concentration of
J ¼ 8.3 Hz, 1H, Ar-H), 2.70 (t, J ¼ 4.1 Hz, 2H, CH2S) 1.94 (t, J ¼ 4.1 Hz,
2H, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (DMSO‑d6):
d 144.2, 132.0,
0.01 M. The human ovarian cancer cell line A2780 was plated in 96-
well tissue culture dishes at a concentration of 2500 cells per well
in RPMI medium, supplemented with 10% fetal bovine serum (FBS)
and a mixture of antibiotics and antimitotics. The human fallopian
tube secretory epithelial cell FT-007 primary culture was grown
from a surgical fallopian tube fimbria specimen as previously
described [38]. The FT-007 cells were plated at a concentration of
2500 cells per collagen-coated well in a 96-well tissue culture dish
in DMEM/F-12 medium containing 10% fetal bovine serum (FBS)
and a mixture of antibiotics and antimycotics. The next day, the
cultures were treated in triplicate with compound concentrations
ranging from 0.16 mM to 10 mM. Control cultures were treated with
DMSO solvent only. After 72 h of incubation, the CellTiter 96 Non-
Rad Cell Profileration Assay (Promega) was used to quantify the
130.0, 129.1, 122.0, 121.2, 53.5, 42.6, 35.8, 20.4.
4.7. Representative procedure for the formation of the desmethyl
thiourea series (6a-d)
To a stirred solution of the free base of 22 (0.100 g, 0.44 mmol) in
THF:DCM (1:1, 5 mL) were added substituted aryl isothiocyanates
(0.46 mmol), followed by Et3N (0.53 mmol, 0.54 g, 74 mL, 1.2 equiv),
and the reaction mixture was stirred at 23 ꢁC for 18 h. The reaction
mixture was concentrated, and the solid was purified on a silica gel
column eluted with increasing concentrations of EtOAc in hexanes
to afford the desired products.

26
B. Nammalwar et al. / European Journal of Medicinal Chemistry 170 (2019) 16e27
M.J. Birrer, D.M. Benbrook, Synthesis of flexible sulfur-containing hetero-
arotinoids that induce apoptosis and reactive oxygen species with discrimi-
nation between malignant and benign cells, J. Med. Chem. 47 (2004)
999e1007.
remaining metabolically living cells. After subtracting blank values,
the optical density (OD) readout of the assay for the treated cultures
was divided by the average OD of the control cultures to derive the
Growth Index. GraphPad Prism Version 7.04 software was used to
draw sigmoidal dose-response curves and derive the IC50 values of
two independent experiments. The efficacy represents the maximal
growth inhibition activity exerted by each compound.
[14] A. Dhar, S. Liu, J. Klucik, K.D. Berlin, M.M. Madler, S. Lu, R.T. Ivey, D. Zacheis,
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Conflicts of interest
The authors declare no competing interests.
Acknowledgements
[17] F.A. Mic, A. Molotkov, D.M. Benbrook, G. Duester, Retinoid activation of reti-
noic acid receptor but not retinoid X receptor is sufficient to rescue lethal
defect in retinoic acid synthesis, Proc. Natl. Acad. Sci. Unit. States Am. 100
(2003), 7135-7134.
The authors (RAB and KDB) gratefully acknowledge the support
of this research by the Stephenson Cancer Research Center/Okla-
homa Tobacco Settlement Endowment Trust (TSET). The in-
vestigators are also indebted to the OSU College of Arts and
Sciences for funds to purchase several departmental FT-IR in-
struments and a new A400 MHz NMR for the Statewide NMR fa-
cility. This NMR facility was initially established with support from
NSF (BIR-9512269), the Oklahoma State Regents for Higher Edu-
cation, the W. M. Keck Foundation, and Conoco, Inc. The Oklahoma
Shared Clinical and Translational Resources (OSCTR) Summer
Research Training Program sponsored the efforts of Coralee Toal.
We thank Elangovan Thavathiru for performing some of the ovarian
cancer cell line cytotoxicity assays.
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D.M. Benbrook, Development of flexible-heteroarotinoids for kidney cancer,
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Patent issued July 1, 2003; U. S. patent 6,586,460 B1.
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A. Zakharov, C. Detrisac, M. Pereira, T. Martin-Jimenez, E. Onua, A. Banerjee,
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at
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