Discovery of medium ring thiophosphorus based heterocycles as antiproliferative agents

Article abstract of DOI:10.1016/j.bmcl.2016.12.079

Hydrogen sulfide (H₂S) has been investigated for its potential in therapy. Recently, we reported novel H₂S donor molecules based on a thiophosphorus core, which slowly release H₂S and have improved anti-proliferative activ

Full text of DOI:10.1016/j.bmcl.2016.12.079

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of medium ring thiophosphorus based heterocycles
as antiproliferative agents
Wei Feng a,b, Wisna Novera c, Kaye Peh a, Donavan Neo a, Pondy Murugappan Ramanujulu a,b
,
Philip K. Moore b,d, Lih-Wen Deng c, Brian W. Dymock a,
⇑
a Department of Pharmacy, Block S4A Level 3, 18 Science Drive 4, National University of Singapore, 117543, Singapore
b Life Sciences Institute, Centre for Life Sciences Level 5, 28 Medical Drive, National University of Singapore, 117456, Singapore
c Department of Biochemistry, Block MD7 #04-06, 8 Medical Drive, National University of Singapore, 117596, Singapore
d Department of Pharmacology, UHL Level 5-02R, Lee Kong Chian Wing, 21 Lower Kent Ridge Road, National University of Singapore, 119077, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrogen sulfide (H2S) has been investigated for its potential in therapy. Recently, we reported novel H2S
donor molecules based on a thiophosphorus core, which slowly release H2S and have improved anti-pro-
liferative activity in cancer cell lines compared to the most widely studied H2S donor GYY4137 (1).
Herein, we have prepared new thiophosphorus organic H2S donors with different ring sizes and evaluated
them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17, was
Received 19 August 2016
Revised 21 December 2016
Accepted 29 December 2016
Available online xxxx
found to be the most potent with sub-micromolar IC50s in breast (0.76 lM) and ovarian (0.76 lM) cancer
cell lines. No significant H2S release was detected in aqueous solution for this compound. However, con-
focal imaging showed that H2S was released from 17 inside cells at a similar level to the widely studied
Keywords:
Anti-proliferative agents
Hydrogen sulfide
Thiophosphorus
Organic synthesis
Medium sized rings
H2S donor GYY4137, which was shown to release 10
Comparison of 17 with its non-sulfur oxygen analogue, 26, provided evidence that the sulfur atom is
important for its potency. However, the significant potency observed for 26 (5.94–11.0 M) indicates that
lM H2S after 12 h at a concentration of 400 lM.
l
the high potency of 17 is not entirely due to release of H2S. Additional mechanism(s) appear to be respon-
sible for the observed activity, hence more detailed studies are required to better understand the role of
H2S in cancer with potent thiophosphorus agents.
Ó 2016 Published by Elsevier Ltd.
Hydrogen sulfide is regarded as the third gasotransmitter, after
carbon monoxide and nitrogen monoxide, and has been found to
influence a number of intracellular signaling processes.1 Thus H2S
plays important roles in physiology and pathology, including neu-
romodulation,2 hypertension,3 inflammation,4–6 and pain percep-
tion.7 H2S has also become a hot area of study in cancer.8–10 It
has been reported that attachment of an H2S releasing moiety to
anti-inflammatory drugs such as aspirin and ibuprofen increased
their anticancer activities by 200–1100 fold.12 This indicated the
possibility of using an H2S release approach in cancer drug discov-
ery. Two possible mechanisms underlying the anti-cancer proper-
ties of H2S have been revealed. First, analysis of the cell cycle
showed that treatment of cancer cells (HT-29 colon and MDA
MB-231 breast) with H2S donor compounds, which release H2S
upon coming into contact with water, caused dose-dependent
increases in the percentage of cells in the G0/G1 phases.11,12 Sec-
ond, H2S donors were effective in reducing lipopolysaccharide
(LPS) induced NF-jB activation, thus suppressing the proliferation
of cancer cells.13–15 H2S is released almost instantly from inorganic
salts, such as Na2S and NaHS, however, biologically active organic
donors liberate H2S much more slowly. More importantly, organic
donors appear to deliver H2S into cells more efficiently and have
been the subject of considerable studies in recent years.16–21 How-
ever H2S releasing compounds are reported to require high concen-
trations to achieve meaningful activity in cancer cell lines. Thus
development of more potent organic H2S donors is highly
desirable.
Classes of organic H2S donors based on a range of functional
groups and mechanisms have been developed. For example, Taliani
and Calderone reported the synthesis and vascular effects of
arylthioamides, which release H2S when activated by L
-cysteine.22
S-Arylthiooximes, another class of cysteine-triggered H2S donors,
were found to have structure-dependent H2S releasing kinetics.23
Michael and co-workers reported the development of thiocarba-
mates as H2S donors catalyzed by carbonic anhydrases.24 Another
important class of organic donor is based on the thiophosphrous
core, which releases H2S upon hydrolysis.8 All of these donors
⇑
Corresponding author.
E-mail address: phadbw@nus.edu.sg (B.W. Dymock).
http://dx.doi.org/10.1016/j.bmcl.2016.12.079
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Feng W., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2016.12.079

2
W. Feng et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
realized much slower release of H2S compared to the instanta-
neous release of H2S observed with the inorganic donor NaHS.
GYY4137 (1), the most extensively studied organic H2S donor,
these compounds are still not potent enough to be therapeutically
useful at lower doses desirable for in vivo administration. Hence
we set out to develop new P = S containing H2S donors with a cyclic
structure, based on 3, aiming to discover more potent anti-prolifer-
ative compounds.
has an anti-proliferative IC50 of 368 lM in the MCF-7 breast cancer
cell line (Fig. 1).8 The high dose that is required for 1 to be effective
has hampered its therapeutic application. However, studies on 1
and related compounds has provided evidence that the P = S func-
tionality is an effective H2S releasing functional group. Recently,
we reported the development of new H2S donors based around
the P = S function represented by FW1131 (2) and FW1256 (3)
(Fig. 1).10 In MCF-7 cells, compound 2 blocks proliferation with
Compound 3 was synthesized by reacting phenylthiophospho-
nic dichloride and 2-aminophenol (a ‘dual’ nucleophile).10 In this
work we have expanded the application of new dual nucleophiles
(2-aminoaniline, catechol and ethylenediamine) to synthesize new
5-membered benz-fused different ring sized H2S releasers (Class A
and B compounds 4–13, Table 1, Scheme 1). As observed in our
previously reported work, the H2S releasing ability of thiophospho-
rus based compounds is closely related to their electronic proper-
ties, resulting in different anti-proliferative inhibitory profiles.10
Here, dual nucleophiles with both an aromatic backbone (such as
2-aminoaniline and catechol) and an aliphatic backbone (such as
ethylenediamine) were employed to modify the electronic proper-
ties of the P = S functional group, thus potentially leading to differ-
ent H2S releasing abilities. 2-Aminoaniline, catechol and
ethylenediamine led to the formation of new 5-membered H2S
releasers (Classes A and B, compounds 4–13, Table 1, Scheme 1).
Synthesis with 2-aminobenzyl alcohol and 2-aminobenzylamine
lead to benz-fused 6-membered H2S donors (Class C compounds
14–16, Table 1 and Scheme 1). Appropriate choice of diols led to
the formation of 7- and 8-membered compounds (compounds
17–26) (Table 2 and Schemes 1 and 2). Most of these compounds
are novel, while the synthesis of 4,26 5/17,27 18,28 2429 were previ-
ously reported without cellular activity studies. Besides dual nucle-
ophiles, three sulfur reagents, phenylthiophosphonic dichloride,
Lawesson’s Reagent (LR) and Belleau’s Reagent (BR) were used in
an IC50 of 36.3
lM and the most potent compound, 3, has an IC50
of 5.7 M. Intracellular H2S quantification and confocal imaging
l
showed that 3 produced the highest intracellular H2S concentra-
tions, followed by 2 and then 1. These levels are consistent with
the relative cellular potencies of these compounds and indicates
the importance of measuring H2S levels inside cells. In a parallel
artificial membrane permeability assay (PAMPA),25 2 has a perme-
ability of 19 5 nm/s that is about 10 fold higher than 1, poten-
tially explaining the higher intracellular H2S levels. However,
S
MeO
P
N
S
S
P
HN
S
O
P
PhO
S
N
H
Bn
O
3 (FW1256)
1 (GYY4137)
2 (FW1131)
MCF7 IC50 = 36.3 µM
MCF7 IC50 = 5.7 µM
MCF7 IC50 = 368 µM
Fig. 1. Selected reported organic H2S donors based on the P = S functionality.
Table 1
Structure of H2S donors with 5- and 6-membered rings and their anti-proliferative IC50s
(l
M) in two cancer cell lines and one normal cell line.
7
1
H
N
S
X
X
P
Ph
Ph
6
S
S
R1
2
P
P
N
H
Y
5
N
H
3
R2
4
R2
R2
Class A
Class B
Class C
Compound
Class
R1
R2
X
Y
Yield (%)a
MCF-7 IC50
(
l
M)c
SKOV3 IC50
(l
M)c
WI38 IC50 (l
M)c
1
2
3
4
5
6
7
8
–
–
–
–
H
5-Cl
H
4-OMe
H
5-OMe
5-OMe
H
–
–
H
OMe
OMe
H
OPh
H
OPh
H
–
–
–
–
NH
NH
O
67b
37b
30b
79
22
31
30
39
42
39
11
368 0.7
36.3 1.2
5.7 0.2
>400
281 17
40.7
73.9 0.9
26.2 1.7
32.1 1.9
181 10.3
107 2.4
332 4.6
47.0 1.2
6.12 0.2
>400
193 2.7
29.7 0.05
45.8 2.5
24.1 3.5
27.9 9.3
230 0.1
110 22.4
1977 193
104 0.3
>50
A
A
A
A
A
A
A
A
A
O
NH
O
O
O
O
O
O
O
250
O
40.3 0.05
125 1.2
NA
NA
353 0.9
NA
NH
NH
NH
NTs
9
10
11
H
H
O
O
N
S
Cl
12
13
14
15
16
B
B
C
C
C
–
–
–
–
–
OPh
H
H
OPh
OMe
–
–
NH
O
O
–
–
–
–
–
65
62
54
50
31
>400
165 0.4
>400
29.4 0.3
200
49.0 7.6
164 0.2
>400
48.4 0.4
91.4 4.4
NA
145 0.05
173 0.5
108 0.7
392 2.4
a
b
c
Isolated yield.
10
See Ref.
.
Crystal violet assay.
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W. Feng et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
S
P
Cl
S
X
Y
Et3N
n=1, 2, 3, 4
R
P
Cl
Dual Nucleophile +
Dual Nucleophile +
X, Y = O or N
DCM, 0 o
C
n
S
P
S
S
n=1, 2, 3
X
Y
120 o
C
R
P
X, Y = O or N
S
P
S
R2
R
2 = OMe or OPh
R2
Toluene
n
R2
Scheme 1. General synthetic schemes for all of the H2S donors.
Table 2
Structure of H2S donors with 7- and 8-membered rings and their anti-proliferative IC50s
(l
M) in two cancer cell lines and one normal cell line.
R1
O
O
S
O
S
O
P
O
O
O
O
S
P
S
P
O
P
S
P
O
O
H
H
O
R2
O
R1
OMe
17-21
22
23
24
25
Compound
R1
R2
Yield (%)a
MCF-7 IC50
(l
M)c
SKOV3 IC50
(
l
M)c
WI38 IC50 (l
M)c
17
18
19
20
21
22
23
24
25
26b
H
H
Br
Br
Ph
–
–
–
–
–
OMe
H
OMe
H
H
–
–
–
–
–
28.2
88
16
73
74
15.1
27
29
31
88.7
0.76 0.005
140 0.1
>400
>400
>400
>400
>400
>400
71.1 0.4
11.0 0.05
0.76 0.02
103 0.02
>400
>400
>400
374 0.3
>400
51.2 0.09
51.1 0.3
5.94 0.02
1.38 0.02
ꢀ400
NA
NA
NA
>400
>400
134 2.4
76.2 1.2
18.6 0.3
a
b
c
Isolated yield.
Scheme 2.
Crystal violet assay.
All compounds were tested for their anti-proliferative potency
in MCF-7 (breast) and SKOV3 (ovarian) cancer cells (Table 1). Gen-
erally, compounds derived from 2-aminoaniline (4), catechol (5)
and diaminoethane (12, 13) have higher IC50s than those derived
from 2-aminophenol (3, 7–9) in the MCF-7 cell line. The IC50s var-
ied with changes in substituents. When the nitrogen atom was sul-
O
O
P
Cl
OH
O
Et3N
DCM
P
+
Cl
OMe
O
OH
OMe
26, 88.7%
Scheme 2. Synthesis of non-sulfur compound 26.
fonylated, the IC50s were around 106 to 180
lM (10, 11). The best
activity from Class A compounds, IC50 = 26.2
lM, was obtained
with compound 8, bearing an electron donating methoxy group
at the 5 position. The 6-membered compound 14 was not active
even at its maximum concentration of 400 lM, which is at the
limit of its solubility. Compounds 15 and 16, both with electron
rich phenyl rings directly attached to the phosphorus, had IC50s
the synthesis to deliver different substituents at position R2 aiming
to study the electronic effect of R2 on H2S releasing and anti-prolif-
erative inhibitory ability (Scheme 1). Reactions using phenylthio-
phosphonic dichloride completed in 3 h at 0 °C, in the presence
of triethylamine, leading to 5-, 6-, 7- and 8-membered products
with non-substituted phenyl rings directly attached to the phos-
phorus center. Generally these reactions afforded products in good
yields of up to 88%. Reactions using LR and BR proceeded smoothly
in refluxing toluene but with relatively lower yields due to their
low reactivity. Products containing R2 (OMe or OPh) substituted
phenyls were formed in ring sizes of 5, 6 and 7. All of these prod-
ucts were stable and easily purified by flash column chromatogra-
phy. They were classified based on their ring sizes and the dual
nucleophiles that they were derived from. Efforts to form a 9-
membered ring H2S donor were not successful using this
chemistry.
of 29.4 and 200
against the SKOV3 cell lines. Many of these compounds are weakly
active, with IC50s > 100 M. Compounds 6, 8 and 9 are the three
most potent compounds with IC50s of 29.7, 24.1 and 27.9 M,
lM, respectively. We also tested these compounds
l
l
respectively. These compounds were also tested in a normal
human lung fibroblast cell line, WI38. Therapeutic indices (TI, ratio
of WI38 IC50/cancer cell IC50) were generally positive but in the low
range of 1–3 fold for most compounds, with a few exceptions.
We next studied further ring expansion with 7- and 8-mem-
bered ring compounds synthesized by appropriate choice of dual
nucleophiles (Table 2, Scheme 1). Compounds of class D were
derived from differently substituted 2,20-bisphenols. However,
these compounds have very different IC50s in MCF7 cells.
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4
W. Feng et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
pounds have IC50s ranging from 51.1 to more than 400 lM, signif-
icantly less active than 17.
10
8
1 day
6
Quantification of H2S released in vitro
2 days
3 days
4 days
4
2
From the results above, there is no clear structure activity rela-
tionship. Is H2S playing any role in inhibition of cellular prolifera-
tion? To answer this, we determined the in vitro H2S release
profile10 of all 7-membered ring compounds (Fig. 2) in solution
using an H2S-specific dansyl azide probe (see Supporting Informa-
0
Fig. 2. H2S solution release profile of all 7-membered H2S donors.
tion). However, only 2 lM H2S, which is at the same level as the
blank sample, was detected in vitro for 17 at the very high concen-
tration of 5 mM after incubation at 37 °C for 4 days. Similar low
responses were seen with the other tested compounds with the
exception of 18, 19 and 22 which were slightly higher.
Compound 17 has an IC50 of 0.76
1.38
has a much higher IC50 of 140
The only structural difference between these two compounds is
an OMe group at the R2 position, broadly in line with the SAR
observed in Table 1. Changing the R1 substituent to bromo (19,
20) and phenyl (21) does not improve the activity. Their IC50s
l
M in MCF-7 cells compared to
l
M in WI38 cells giving a TI of nearly 2, while compound 18
l
M and a slightly higher TI of 3.
Considering the potentially complex metabolism process of
these compounds, we believed that compound 17 releases H2S
inside cells. Thus confocal imaging to visualize release of H2S in
cells treated with 17 was carried out,10 in comparison with com-
pounds 1, 2 and 3. (Fig. 3). Although no H2S was detected under
the in vitro conditions, confocal imaging showed similar H2S levels
for 17 inside cells as compound 1. However, the image is much less
brighter than compounds 2 and 3. This result indicates that com-
pound 17 does release H2S inside cells. This experiment was done
over only a 5 h incubation. The effect of H2S release over time
inside cells is not yet fully understood as regards the anti-prolifer-
ative activity of these H2S donors. For the reference compounds,
more H2S leads to a stronger phenotypic response in the assays
increased dramatically to more than 400 lM. Other 7-membered
compounds 22, 23, 24 were derived from tartrate, hydrothal, and
1, 2-benzenedimethanol. Instead of the aromatic structure in com-
pounds of class D, these compounds are aliphatic which signifi-
cantly increases the electron density of the P = S group. However,
their IC50s are much poorer, increasing to more than 400
8-membered compound 25 has an IC50 of 71.1 M. In the SKOV3
cell line, 17 is again the most potent compound with an IC50 of
0.76 M, almost identical to its IC50 in MCF-7 cells. The other com-
lM. The
l
l
Fig. 3. Confocal images showing signals in MCF-7 cells exposed to NaHS, 1, 2, 3 and 17. MCF-7 cells were incubated with 20
incubated with H2S donors for 5 h before imaging. (a, a0) MCF-7 alone (control), (b, b0) probe alone (probe control), (c, c0) 50 M NaHS, (d, d0) 100
f0) 400 M 2. (g, g0) 400 M 17. (h, h0) 400
M 3. (i) Structure of probe 27 used.
l
M probe for 2 h at 37 °C, washed, and then
l
l
M NaHS, (e, e0) 400
l
M 1, (f,
l
l
l
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W. Feng et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Table 3
cannot be solely attributed to the evolution of H2S. Hence more
detailed studies on the intracellular mechanism of action of 17 will
be required to better understand the role of H2S in cancer with
potent thiophosphorus agents.
MTT data with compounds 17 and 26 together with different amounts of Na2S.
Compound
Na2S (
l
M)a
MCF-7 IC50 (l
M)b
–
1000
–
1
10
100
–
1
10
100
20.2% @ 1000
0.82 0.05
1.17 0.39
1.01 0.02
1.44 0.37
69 12.4
lM
17
17
17
17
26
26
26
26
Acknowledgments
This research would not be possible without generous grant
support from the National University of Singapore Faculty of
Science start-up grant (R-148-000-169-133), Drug Development
Unit grant (R-711-000-022-133) and Final Year Project student
support from the Department of Pharmacy, NUS.
59 13.5
52.9 11.9
50.6 4.7
a
Added together with 17/26; forms H2S very rapidly in water.
MTT assay format.
b
A. Supplementary material
used with relatively short timelines. For compound 17, such a sim-
plistic model does not appear to apply. Other parts of the com-
pound structure could also be playing a role perhaps through
other targets, although the data suggests this is likely to be a very
specific interaction due to the poor activity seen with close ana-
logues of 17.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.
079.
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To explore the role of the sulfur atom we then synthesized com-
pound 26, which is the sulfur-negative control of 17 (Scheme 2).
When 1 is hydrolyzed, H2S is released and the sulfur atom is
replaced by oxygen.30 Similarly, when H2S is released from 17,
26 is expected to be formed as the first intermediate. Comparison
of the activities of 17 with 26 will indicate whether the sulfur plays
a role in the activity or not. When tested, 26 was found to have an
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of 0.82
(see Table 2). Compound 26, however, was less potent with an
IC50 of 69 M, 84 fold less than 17. Attempts to add H2S (Na2S @
1, 10 and 100 M) to the media with each compound did not result
lM, comparable to the data obtained with crystal violet
l
l
in any significant changes to the IC50 values. When cells are treated
with a single administration of Na2S on its own, only a weak anti-
proliferative effect is seen at a very high concentration (20.2% @
1 mM). As we have demonstrated in our previous work,10,31 H2S
is most effective when delivered into cells at a slow and continuous
rate. However, it is not clear if this applies to combinations of small
molecules with H2S. Although it is clear that 17 requires the sulfur
atom for optimum potency, further work is required to fully under-
stand any H2S combination effect with compound 26 in order to
characterise the structural requirements and mechanism of action
of 17 (FW1187).
In conclusion, a series of thiophosphorus containing H2S donors
with different ring sizes have been synthesized and tested in MCF-
7 and SKOV3 cancer cell proliferation inhibition assays, as well as
in WI38 fibroblasts. The most potent compound, 17, was effective
in slowing the proliferation of MCF7 and SKOV3 cancer cells at sub-
micromolar concentrations, with a narrow therapeutic window.
Comparing 17 with its negative control, 26, it was clear that the
sulfur significantly contributes to the activity. Combinations of
17 or 26 with Na2S did not significantly change the IC50 values. This
data suggests that the anti-proliferative mechanism of action
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