Synthesis and in vitro anti-leukemic activity of structural analogues of JS-K, an anti-cancer lead compound

Article abstract of DOI:10.1016/j.bmcl.2007.12.044

Structural analogues of JS-K, an anti-cancer lead compound, were prepared and their in vitro anti-leukemic activity was determined. The rate of nitric oxide release from the corresponding diazeniumdiolate anions did not appear to affect the anti-leukemic

Full text of DOI:10.1016/j.bmcl.2007.12.044

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 950–953
Synthesis and in vitro anti-leukemic activity of structural
analogues of JS-K, an anti-cancer lead compound
Harinath Chakrapani,a,* Michael M. Goodblatt,a Vidya Udupi,b Swati Malaviya,b
Paul J. Shami,b Larry K. Keefera and Joseph E. Saavedrac,*
aChemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick,
PO Box B, Frederick, MD 21702, USA
bDivision of Oncology, Department of Internal Medicine, University of Utah, Salt Lake City, UT 84112, USA
cBasic Research Program, SAIC-Frederick Inc., NCI-Frederick, Frederick, MD 21702, USA
Received 12 October 2007; accepted 17 December 2007
Available online 4 January 2008
Abstract—Structural analogues of JS-K, an anti-cancer lead compound, were prepared and their in vitro anti-leukemic activity was
determined. The rate of nitric oxide release from the corresponding diazeniumdiolate anions did not appear to affect the anti-leu-
kemic activity of the prodrug forms. Two compounds with potent inhibitory activity and a potentially favorable toxicological profile
were identified.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Glutathione S-transferase (GST), which is frequently
over-expressed in cancer tissue, is a key phase II detox-
ification enzyme that catalyzes the conjugation of elec-
trophilic xenobiotics with glutathione (GSH).1 Our
laboratory has developed several diazeniumdiolate-
based nitric oxide (NO) prodrugs2 that are designed as
substrates for GST, notably O2-(2,4-dinitrophenyl) 1-
[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-dio-
late (JS-K, Scheme 1).3
Scheme 1. The anti-cancer lead compound JS-K, which targets GST-
overexpressing cells, and its mechanism of nitric oxide release.
JS-K is a potent anti-tumor agent against HL-60 human
leukemia xenografts in mice, cutting their growth rate
by about half and inducing significant necrosis in the tu-
mor mass.3a Furthermore, this drug was found to be ac-
tive against: human prostate cancer xenografts in
mice;3a an orthotopic model of liver cancer in rats;3d
and multiple myeloma xenografts in mice.3e In this
study, we synthesized numerous structural analogues
of JS-K and studied their in vitro anti-leukemic activity
by determining the ability of these compounds to inhibit
the growth of HL-60 human leukemia cells.
It is reported that human leukemia cells are sensitive to
cytotoxic effects of nitric oxide.4 While additional path-
ways may be operational, nitric oxide appears to be an
important component of the potent JS-K cytotoxic activ-
ity.3 Nitric oxide release from JS-K is proposed to occur
by nucleophilic aromatic displacement of the spontane-
ously NO-releasing diazeniumdiolate anion2 by glutathi-
one (Scheme 1). In the absence of strong nucleophiles
such as GSH, JS-K is relatively stable in aqueous buffer;
the rate constant for hydrolysis of JS-K in pH 7.4 buffer
at 37 ꢁC was reported as 1 · 10À6 sÀ1, which translates to
a half-life of over a week.3a In the presence of glutathi-
one, however, JS-K nearly completely disappeared with-
in 30 min and a second order rate constant of
1.0 MÀ1 sÀ1 was reported.3a The rate constant for hydro-
lysis of 4-carboethoxy-PIPERAZI/NO in pH 7.4 buffer
Keywords: Nitric oxide; Prodrugs; Glutathione; Glutathione S-trans-
ferase; JS-K; Diazeniumdiolate; Anti-leukemic; Anti-cancer; GST;
Nitric oxide donors; NSAID; Nucleophilic aromatic substitution.
*
Corresponding authors. Tel.: +1 301 846 1601 (H.C.); e-mail:
chakrah@ncifcrf.gov
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.044

H. Chakrapani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 950–953
951
at 37 ꢁC was reported as 1.9 · 10À3 sÀ1, which corre-
sponds to a half-life of 6 min (Scheme 1).3a
Earlier, we prepared a number of JS-K structural ana-
logues and determined their in vitro anti-leukemic activ-
ity.3d However, a systematic study of the effect of
varying the half-life of the diazeniumdiolate ion decom-
position on the anti-proliferative activity of the corre-
sponding O2-aryl prodrug form was not conducted.
Hence, our work was initiated by studying the effect of
varying the rate of dissociation of the diazeniumdiolate
ion to form NO on the cytotoxicity of the corresponding
O2-(2,4-dinitrophenyl) derivative, which was determined
by their ability to inhibit human leukemia HL-60 cell
proliferation.
A number of diazeniumdiolate anions with a range of
half-lives were identified and prepared using reported
methods from the corresponding secondary amine.5 As
listed in Table 1, the reported half-life of decomposition
of PROLI/NO (1) is 2 s while the half-lives of decompo-
sition of 2–5 varied from 78 s to 50 min.5 The O2-(2,4-
dinitrophenyl) derivatives 6–10 of these diazeniumdio-
late anions were prepared using reported methods
(Scheme 2).5c,d
It is noteworthy that the diazeniumdiolate ions PROLI/
NO,5a–c 2,5d and 55d are nitric oxide prodrugs with po-
tential clinical relevance; possible byproducts of NO re-
lease from these compounds, the corresponding
nitrosamines, are reported to have minimal carcinogenic
activity, thus suggesting a favorable toxicological pro-
file.6 For example, N-nitrosoproline, a possible byprod-
uct of PROLI/NO decomposition, was not found to
display any carcinogenic activity in several animal mod-
els (Scheme 3).6e–h
Scheme 2. Synthesis of some O2-(2,4-dinitrophenyl) diazeniumdio-
lates. FDNB, 1-Fluoro-2,4-dinitrobenzene.
Next, we prepared the O2-aryl diazeniumdiolate 11 in
three steps from N-(tert-butoxycarbonyl)piperazine
Scheme 3. Proposed mechanism of nitrosamine formation during
diazeniumdiolate ion decomposition in aqueous buffer.
Table 1. Reported half-lives of some diazeniumdiolate ions in aqueous
buffer and the corresponding O2-arylated prodrug forms
using a reported method (Eq. 1).7 This compound
served as a convenient divergence point in the prepara-
tion of a variety of JS-K analogues by exposing 11 to
a series of electrophiles (Scheme 4).8
The methyl ethers 12a–12d were prepared by treating 11
with the corresponding acyl chloride. The carbamate
portion of JS-K was replaced by a sulfamate functional-
ity in compounds 13a and 13b. Analogues 14a–14c,
where the ester group of the carbamate of JS-K was al-
tered, were prepared in a similar fashion by reaction of
the corresponding acyl chloride with 11.
NO donor
Half-lifea
6.0 min
O2-(2,4-Dinitrophenyl)
derivative
4-Carboethoxy-
PIPERAZI/NO
PROLI/NO, 1
JS-K
2 s
4.3 min
78 s
6
7
2
3
4
5
8
8.3 min
50 min
9
10
a In pH 7.4 buffer at 37 ꢁC.

952
H. Chakrapani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 950–953
Table 2. In vitro anti-leukemic activity of JS-K and its structural
analogues
Entry
Compound
IC50 (lM)
1
2
JS-K
6
0.2–0.5
>50
3.2
1.7
2.5
2.0
5.0
2.3
7.2
4.5
8.8
6.6
6.8
7.4
8.3
6.0
9.8
7.4
9.1
3
7
4
8
5
9
10
6
7
11
8
12a
12b
12c
12d
13a
13b
14a
14b
14c
15a
15b
15c
9
10
11
12
13
14
15
16
17
18
19
derivative 7 and the piperidine analogues 8–10 dis-
played potent but comparable activity, suggesting that
the differences in the rate of NO release from the cor-
responding diazeniumdiolate ions 2–5 did not affect
potency of the diazeniumdiolate prodrug (Table 2,
entries 3–6).
In an earlier study, it was reported that diazeniumdio-
late anions with extended durations of nitric oxide re-
lease were more effective at inhibiting the growth of
this human leukemia cell line than their counterparts
with lesser durations of NO release.4 In this study,
three compounds, DETA/NO, with a half-life of NO
release of 20 h, PAPA/NO, which has a half-life of
30 min, and MAHMA/NO, whose half-life is 2 min,
were tested for their anti-proliferative activity using
the HL-60 human leukemia cell line.4 Among these
compounds, it was reported that the best inhibitor of
cell proliferation was DETA/NO (ID50 = 50 lM), fol-
lowed by PAPA/NO (ID50 = 100 lM), and MAHMA/
NO, which was found to be inactive. In contrast, our
study indicates that there were no observable effects
of altering the rate of NO release on the cytotoxicity
of these prodrugs, suggesting a role for nitric oxide-
independent cytotoxic pathways.3 Furthermore, it is
worth noting that, by releasing NO spontaneously,
DETA/NO, PAPA/NO, and MAHMA/NO act on leu-
kemia cells by a bystander effect while the compounds
described in this study get activated to release NO
intracellularly. Thus, the rate of NO release intracellu-
larly may not be as important for cytotoxicity as when
it is released extracellularly.
Scheme 4. Synthesis of some structural analogues of JS-K from 11.
Diazeniumdiolate-based nitric oxide donors conjugated
with non-steroidal anti-inflammatory drugs (NSAIDs)
are assuming importance as multi-faceted therapeutic
agents.9 Thus, we prepared several JS-K-type NSAID-
conjugates 15a–15c using the precursor 11.
The in vitro anti-leukemic activity of these compounds
was studied using the HL-60 human leukemia cell line
by a reported procedure.3a The IC50 of JS-K was re-
ported as 0.2–0.5 lM (Table 2, entry 1).3a
Although the aqueous solubility of 6 was much higher
than those of the other JS-K analogues prepared in
this study, its cytotoxicity was diminished (Table 2, en-
try 2). The other prodrugs 7–10 were tested in this as-
say and the half-life of the diazeniumdiolate anion in
buffer did not appear to affect the cytotoxicity. The
Earlier, we reported that 2 and 5 decomposed in aque-
ous buffer to release nearly quantitative amounts of
NO, suggesting that nitrosamine formation is a minor
process.5d Due to their potent cytotoxicity and the
potentially favorable toxicological profile of their corre-
sponding parent diazeniumdiolate anions, 7 and 10 are

H. Chakrapani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 950–953
953
3. (a) Shami, P. J.; Saavedra, J. E.; Wang, L. Y.; Bonifant, C.
L.; Diwan, B. A.; Singh, S. V.; Gu, Y.; Fox, S. D.; Buzard,
G. S.; Citro, M. L.; Waterhouse, D. J.; Davies, K. M.; Ji,
X.; Keefer, L. K. Mol. Cancer Ther. 2003, 2, 409; (b) Ren,
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Vallet, S.; Kutok, J. L.; Chauhan, D.; Mitsiades, C. S.;
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noteworthy; further in vitro and in vivo testing of their
anti-cancer activity will be carried out in due course.
The ether derivatives 12a–12d were all found to display
lower potency than JS-K but comparable to that of 11;
the highest cytotoxicity was shown by 12a, whose IC50
was determined as 2.3 lM (Table 2, entries 8–11).
Changing the carbamate functionality to a sulfamate
group (13a and 13b) or varying the ester portion of
the carbamate group (14a–14c) resulted in diminished
anti-proliferative activity (Table 2, entries 12–16). The
IC50 values of all NSAID derivatives were closer to
10 lM (Table 2, entries 17–19). These results are consis-
tent with those of our earlier study, wherein we observed
that minor structural modifications led to substantial
decrease in potency.3d For example, when the ethyl car-
bamate of JS-K was changed to a methyl carbamate, the
potency of the resulting compound diminished from
IC50 0.2–0.5 to 3.5 lM.3d
4. Shami, P. J.; Sauls, D. L.; Weinberg, J. B. Leukemia 1998,
12, 1461.
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K.; Keefer, L. K. J. Pharm. Sci. 2006, 95, 108; (c)
Chakrapani, H.; Showalter, B. M.; Kong, L.; Keefer, L.
K.; Saavedra, J. E. Org. Lett. 2007, 9, 3409; (d) Chakra-
pani, H.; Showalter, B. M.; Citro, M. L.; Keefer, L. K.;
Saavedra, J. E. Org. Lett. 2007, 9, 4551.
While the molecular mechanism of the potent anti-leukemic
activity of JS-K remains to be fully determined, this study
further reinforces the unique features of this compound that
result in a diminution of potency with structural perturba-
tion or conjugation of other potential pharmacophores,
leading us to speculate that more than one cytotoxic path-
way may be involved; the nature of such alternate pathways
is currently being explored in our laboratories.
6. (a) Lijinsky, W. Chemistry and Biology of N-Nitroso
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301; (c) Lijinsky, W. In Chemical Carcinogenesis; Feo, F.,
Pani, P., Columbano, A., Garcea, R., Eds.; Plenum
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Mirvish, S. S.; Bulay, O.; Runge, R. G.; Patil, K. J. Natl.
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Acknowledgments
This research was supported by the Intramural Research
Program of the NIH, National Cancer Institute, Center
for Cancer Research, as well as by National Cancer
Institute Contract N01-CO-12400 to SAIC-Frederick.
Supplementary data
Analytical data for all new compounds. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2007.12.044.
7. Saavedra, J. E.; Booth, M. N.; Hrabie, J. A.; Davies, K. M.;
Keefer, L. K. J. Org. Chem. 1999, 64, 5124.
8. General procedure. A solution of 11 in DCM was reacted
with a solution of one equivalent of the electrophile and
two equivalents of triethylamine. The reaction mixture was
diluted with DCM, washed with dil HCl and aq sodium
bicarbonate. The organic layer was separated, dried
(MgSO4), filtered, and the solvent was removed under
reduced pressure to form a solid. This was then triturated
with ether, filtered, and dried under vacuum.
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