Synthesis and biological activity of NOSH-naproxen (AVT-219) and NOSH-sulindac (AVT-18A) as potent anti-inflammatory agents with chemotherapeutic potential

Article abstract of DOI:10.1039/c3md00185g

Nitric oxide- (NO) and hydrogen sulfide- (H₂S) releasing naproxen (NOSH-naproxen) and NO and H₂S-releasing sulindac (NOSH-sulindac) were synthesized and their cell growth inhibitory properties were evaluated in four different human cancer cell lines. These cell lines are of adenomatous (colon, pancreas), epithelial (breast), and lymphocytic (leukemia) origin. Using HT-29 human colon cancer cells, NOSH-naproxen and NOSH-sulindac increased apoptosis, and inhibited proliferation. NOSH-naproxen caused a G₀/G₁ block whereas NOSH-sulindac caused a G ₂/M block in the cell cycle. Both compounds exhibited significant anti-inflammatory properties, using the carrageenan rat paw edema model. Reconstitution and structure-activity studies representing a fairly close approximation to the intact molecule showed that NOSH-naproxen was approximately 8000-fold more potent than the sum of its parts in inhibiting cell growth. Our data suggest that these compounds merit further investigation as potential anti-cancer agents.

Full text of DOI:10.1039/c3md00185g

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Medicinal Chemistry Communications
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ARTICLE TYPE
Synthesis and biological activity of NOSH-naproxen (AVT-219) and NOSH-sulindac (AVT-18A)
as potent anti-inflammatory agents with chemotherapeutic potential
Ravinder Kodelaa, Mitali Chattopadhyaya, and Khosrow Kashfia*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
based on the observations that NO and H2S could enhance the
local gastric mucosal defence mechanisms, thereby decreasing
NSAID-induced gastric toxicity.5-8 Here we report on the
synthesis and some of the biological properties of two other NO-
55 and H2S-releasing NSAIDs, NOSH-naproxen and NOSH-
sulindac, Fig. 1.
Nitric oxide- (NO) and hydrogen sulfide- (H2S) releasing
naproxen (NOSH-naproxen) and NO and H2S-releasing
sulindac (NOSH-sulindac) were synthesized and their cell
growth inhibitory properties were evaluated in four different
10 human cancer cell lines. These cell lines are of adenomatous
(colon, pancreas), epithelial (breast), and lymphocytic
(leukemia) origin. Using HT-29 human colon cancer cells,
NOSH-naproxen and NOSH-sulindac increased apoptosis,
and inhibited proliferation. NOSH-naproxen caused a G0/G1
15 whereas NOSH-sulindac caused a G2/M block in the cell
cycle. Both compounds exhibited significant anti-
inflammatory properties, using the carrageenan rat paw
edema model. Reconstitution and structure-activity studies
representing a fairly close approximation to the intact
20 molecule showed that NOSH-naproxen was approximately
8000-fold more potent than the sum of its parts in inhibiting
cell growth. Our data suggest that these compounds merit
further investigation as potential anti-cancer agents.
1. Introduction
25 Non-steroidal anti-inflammatory drugs (NSAIDs) in general and
aspirin in particular are recognized as the prototypical
chemopreventive agents against cancer. However, long-term use
of NSAIDs may lead to significant side effects, mainly
gastrointestinal, ranging from dyspepsia to gastrointestinal
30 bleeding, obstruction, and perforation; renal, and cardiovascular
Figure 1. Structural components of NOSH-naproxen and
NOSH-sulindac. The parent compounds, naproxen and sulindac
60 are shown in the shaded boxes. ADT-OH releases H2S and ONO2
releases NO, both shown in dotted ellipses.
1
(reviewed in ). Amongst patients using NSAIDs, it is estimated
that about 16,500 deaths occur every year in the United States.
This figure is considerably greater than the number of deaths
from multiple myeloma, asthma, cervical cancer, or Hodgkin's
35 disease.2 Unfortunately, many physicians and most patients are
unaware of the magnitude of the problem. The gastric damage is
as a result of direct epithelial damage due to their acidic
properties and also through the breakdown of mucosal defence
mechanisms (leukocyte adherence, decreases in blood flow,
40 bicarbonate and mucus secretions) due to a reduction of mucosal
prostaglandin (PG) synthesis.3 In our search for a “better NSAID”
2. Results and discussion
2.1. Chemistry
NOSH-naproxen (NOSH-NAP, AVT-219) and NOSH-sulindac
65 (NOSH-SUL, AVT-18A) were prepared as described in schemes
1 and 2, respectively. For the synthesis of NOSH-naproxen, the
starting material naproxen (compound 1) was demethylated in the
presence of HBr and AcOH to give compound 2. Further, the
carboxyl group was protected by a tertiary butyl alcohol to obtain
70 compound 3.9 This was esterified with 4-bromobutyric acid in the
presence of DCC/DMAP to compound 4. The bromo moiety in 4
under went nucleophilic displacement with a nitrate group to
we developed NOSH-aspirin,4
a hybrid entity capable of
releasing both, nitric oxide (NO) and hydrogen sulfide (H2S), two
gasotransmitters of physiological significance. Our rational was
45 ____________________________________________________
a
Department of Physiology, Pharmacology and Neuroscience, Sophie
Davis School of Biomedical Education, City University of New York
Medical School, New York, NY 10031, USA. Fax:(212) 650-7692; Tel:
(212) 650-6641; E-mail: kashfi@med.cuny.edu
compound 5. Further, the tertiary butyl group in
5 was
deprotected by TFA to give the acid, which was then coupled
50
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with ADT-OH in the presence of DCC/DMAP to the final
pancreatic epithelial cell line (HPDE, ACBRI 515), and a human
product, NOSH-naproxen, (S)-6-(1-oxo-1-(4-(3-thioxo-3H-1, 2- 60 mammary epithelial cell line (HMEpC), Table 2. An inspection
dithiol-5-yl) phenoxy) propan-2-yl) naphthalen-2-yl 4-(nitrooxy)
butanoate) (Scheme-1).
INSERT Scheme 1 (double column)
of the IC50s at 24h clearly showed a differential response
compared to the various cancer cell lines. For example, the IC50
for NOSH-NAP in HMEpC cells was 13.8 ± 0.9 µM (Table 2)
whereas the IC50 in MCF-7 cells was 0.12 ± 0.01 µM (Table 1).
5
NOSH-sulindac (AVT-18A) (Z)-4-(3-thioxo-3H-1,2-dithiol-5-yl) 65 That is NOSH-NAP was approximately 115-fold more potent in
phenyl
5-(2-(5-fluoro-2-methyl-1-(4-(methylsulfinyl)
acetoxy)-2-((4-(nitrooxy)
the breast cancer cell line. Similarly in the same cell line, NOSH-
SUL was about 150-fold more potent, 15.2 ± 0.7 µM (Table 2)
vs. 0.1 ± 0.01 µM (Table 1). Comparing the IC50s in the
pancreatic normal (HPDE, Table 2) and cancerous (BxPC-3,
benzylidene)-1H-inden-3-yl)
10 butanoyl)oxy) benzoate, was synthesized by using 5-hydroxy
salicylic acid as the starting material, which underwent selective
protection with TBDMSCl to give compound 8.10 This was 70 Table 1) cell lines showed that NOSH-NAP was about 147-fold
esterified with 4-bromobutyric acid in the presence of
DCC/DMAP to compound 9 which underwent nitration by
15 nucleophilic displacement to afford compound 10. This
underwent carboxylation in the presence of NaH2PO4/H2O2,
more potent in the cancer cell line (22.0 ± 1.8 µM vs. 0.15 ± 0.02
µM). For NOSH-SUL the enhanced potency in the same cancer
cell line was >200-fold (>25 µM vs. 0.12 ± 0.01 µM). The IC50s
for NOSH-NAP and NOSH-SUL in the normal human lung
NaClO2 to give compound 11.11 The carboxylic acid in 75 fibroblast cell line (IMP-90) were 20.0 ± 2.5 µM and 21.7 ± 1.7
compound 11 was then coupled with ADT-OH to obtain
compound 12. This underwent TBDMS deprotection in the
20 presence of TBAF/AcOH to give compound 13, which was then
coupled with sulindac in the presence of DCC/DMAP to the final
product, NOSH-sulindac (Scheme-II).
µM, respectively (Table 2). These IC50s are also considerably
higher than the IC50s in the various cancer cell lines (Table 1), the
fold difference being about 70-270-fold. These data indicate that
NOSH-NAP and NOSH-SUL inhibit cell growth preferentially in
80 cancer cell lines compared with normal cell lines.
INSERT Scheme 2 (double column)
INSERT Table 2 (single column)
25 2.2. Biological evaluation
2.2.2. NOSH-naproxen and NOSH-sulindac alter
HT-29 colon cancer cell kinetics.
2.2.1. NOSH-naproxen and NOSH-sulindac are
potent inhibitors of cancer cell growth.
85 Cellular kinetic parameters, that is, proliferation and apoptosis are
two determinants of cellular mass. Therefore, we determined the
effects of NOSH-NAP and NOSH-SUL at their respective IC50s
for cell growth inhibition on these two parameters as a function
of time using HT-29 human colon cancer cells.
The cell growth inhibitory effects of NOSH-NAP, NOSH-SUL
and their respective parent compounds was evaluated using an
30 MTT assay in four different cancer cell lines of different tissue
origin, that being of, colon, breast, pancreas, and leukemia.
NOSH-NAP and NOSH-SUL at 24h strongly inhibited the
growth of these human cancer cell lines in a concentration
dependent manner; the IC50s for cell growth inhibition are shown
35 in Table 1. The data clearly shows that NOSH-NAP and NOSH-
SUL are orders of magnitude more potent than their respective
parent compounds in all the cell lines. For NOSH-NAP, the IC50s
for cell growth inhibition ranged from 80-150 nM, whereas for
the parent compound, naproxen, this range was from 2,350,000-
40 2,775,000 nM. The enhanced potency calculated as the ratio of
IC50 values (NAP/NOSH-NAP) indicated that NOSH-NAP was
16,000-34,000-fold more potent than NAP (Table 1). Similarly,
NOSH-SUL was also significantly more potent than SUL in
inhibiting the growth of these cancer cell lines. The IC50s ranging
45 from 89-270 nM, compared to that of SUL, which ranged from
699,000-980,000 nM. This represents an enhanced potency of
2,500-9,650-fold over SUL (Table 1). These data strongly
suggest that the effect of these modified NSAIDs on cell growth
inhibition is tissue type independent. Further, that NAP which
50 was not as potent as SUL in inhibiting the growth of these cancer
cell lines; became significantly more potent once modified.
However, since the IC50s for cell growth inhibition in each of the
cell line is approximately the same for NOSH-NAP and NOSH-
SUL, one may conclude that the enhanced potency must be due to
55 the released NO and H2S.
90 Cell proliferation: NOSH-NAP and NOSH-SUL reduced PCNA
expression in
a time-dependant manner. In both cases
proliferation was reduced by approximately 50% in 24 hrs, Figs.
2A and 2D respectively.
Cell cycle: Cell cycle progression as measured by DNA content
95 of treated cells using flow cytometry was also affected by NOSH-
NAP and NOSH-SUL. Cells treated with NOSH-NAP at its IC50
accumulated progressively in G0/G1 phase of the cell cycle as a
function of time (Fig. 2B). The cell populations in the different
phases were altered in the following manner compared to control:
100 G0/G1 increased from 42.2 ± 4% to 58.2 ± 7.6%; S phase was
reduced from 29.2 ± 3.3% to 22.3 ± 2.2 %, and G2/M reduced
from 28.7 ± 2.1 to 19.5 ± 2.1%; suggesting a G0/G1 cell cycle
block. This mode of cell cycle arrest has been reported for
NOSH-aspirin in HT-29 colon cancer cells12 and for the parent
105 compound aspirin, in different cancer cell lines.13, 14 A different
pattern emerged for NOSH-SUL, G0/G1 phase of the cell cycle
decreased from 40.9 ± 3.1% to 29.6 ± 2 % as a function of time, S
phase was reduced from 31.7 ± 3.7% to 10.6 ± 2.6 %, while the
G2/M increased from 27.7 ± 4.2% to 59.8 ± 8.1 % (Fig. 2E),
110 suggesting a G2/M block. This mode of cell cycle arrest has been
reported for the parent drug sulindac in SW480 human colon
cancer cells.15
Cell death: NOSH-NAP and NOSH-SUL increased the
proportion of cells undergoing apoptosis in a time-dependant
115 manner as determined by the Annexin V-FITC staining and flow
cytometry. NOSH-NAP treatment of the cells resulted in 9.2 ±
2.6%, 25.3 ± 3.3%, 31.9 ± 4.7%, and 59.2 ± 5.6% of the cells in
INSERT Table 1 (single column)
We also evaluated the effects of NOSH-NAP and NOSH-SUL on
normal human lung fibroblast cells (IMR-90), a human primary
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early apoptotic phase, at 3, 6, 12, and 24 hrs, respectively, 60 Prostaglandins (PGE2) are the main product of cyclooxygenase-
compared to untreated control (Fig. 2C). NOSH-SUL treatment
of the cells also increased the population of cells undergoing
apoptosis but to a lesser extent (Fig. 2F). At 3, 6, 12, and 24 hrs
and percentage of cells undergoing apoptosis were, 2.8 ± 0.4,
mediated arachidonic acid metabolism.1 Comparison of PGE2
content of paw exudates from control, naproxen, sulindac,
NOSH-NAP, and NOSH-SUL-treated animals showed a clear
and significant COX inhibition by all these agents (Fig. 3B).
5
10.1 ± 2.4, 23.2 ± 2.2, and 39.4 ± 4.8, respectively. Therefore, 65 Naproxen and sulindac caused a considerable decrease in PGE2
NOSH-NAP and NOSH-SUL inhibit proliferation of HT-29
colon cancer cells by a combined induction of G0/G1 or G2/M
arrest and apoptosis.
levels from 95 ± 7 pg/mg in the control groups to 15 ± 4 pg/mg
and 12 ± 3 pg/mg, respectively. Treatment with NOSH-NAP and
NOSH-SUL reduced PGE2 levels to 35 ± 3 and 29 ± 2 pg/mg
respectively.
10 INSERT Fig 2 (double column)
70 INSERT Fig 3 (double column)
2.2.3. Effects of NO- and H2S-releasing groups on
cell growth.
2.2.5. NOSH-NSAIDs release both NO and H2S.
NOSH-naproxen and NOSH-sulindac were designed to release
both NO and H2S. In order to show that indeed this was the case
in vivo, blood was collected from vehicle-, NSAID and NOSH-
In order to determine equivalency of NOSH-NAP to the sum of
15 its parts, we carried out a series of reconstitution and structure-
activity studies in HT-29 cells, looking at cell growth inhibition 75 NSAID-treated animals at the end of the carrageenan-induced
using naproxen, the exogenous NO donor SNAP, and ADT-OH
which releases H2S. We examined the combinations of naproxen
plus SNAP, naproxen plus ADT-OH, and naproxen plus SNAP
20 and ADT-OH, and the intact NOSH-NAP. For the combinations,
edema studies. Figure 4 shows that indeed both NO and H2S were
significantly higher in the NOSH-NSAID-treated animals. The
serum concentrations of H2S as determined by the methylene blue
method described in section 4.2.6 are not accurate, as this method
various concentrations of naproxen were combined with different 80 is associated with considerable artifact.17 However, what our data
fixed concentrations of SNAP, ADT-OH, or SNAP and ADT-
OH. Such simulation of intact NOSH-NAP using naproxen plus
SNAP and ADT-OH represents a fairly close approximation to
25 the intact NOSH-NAP. The growth inhibition curves of HT-29
shows are not absolute H2S levels in the serum, but that there
appears to be an increase in some form of sulfide species (H2S +
HS- + and S2- + others) that must be due to the administered
NOSH-NSAIDs.
cells were analyzed with these combinations; the respective IC50s 85 INSERT Fig 4 (single column)
of naproxen in these were evaluated for a possible shift. Table 2
shows that various combinations had a synergistic effect in terms
of cell growth inhibition, but the respective IC50s of naproxen in
30 the combinations were far higher than those of NOSH-NAP. In
3. Conclusions
In the present study, we described the synthesis of NOSH-NAP
particular, the combination of naproxen plus SNAP and ADT-OH
should have given an IC50 for cell growth inhibition comparable
to that of NOSH-NAP. However, the combination gave an IC50 of
780 ± 55 µM, whereas that for NOSH-NAP was 0.09 ± 0.004
35 µM. That is, the intact molecule was approximately 8000-fold
more potent than the combination, or the sum of the parts does
not equal the whole clearly indicative of a strong synergistic
effect. These findings indicate that the combined molecular
components cannot completely account for the biological activity
40 of intact NOSH-NAP and that these constituents may only, in
part, contribute to its activity. Our group has also reported this
phenomenon for NOSH-aspirin.12
and NOSH-SUL designed to release both NO and H2S. These
90 NOSH compounds inhibited the growth of several cancer cell
lines arising from a variety of tissue types such as colon, breast,
pancreas, and T cell leukemia. NOSH-NAP and NOSH-SUL
inhibited the growth of these cancer lines with IC50s ranging
from 80-270 nM at 24hr. These NOSH compounds were more
95 potent than their respective parent counterparts, with enhanced
potency ranging from about 2,500 to greater than 34,000-fold.
Our data indicate that the effect of these NOSH compounds may
be tissue-type independent since both NOSH-NAP and NOSH-
SUL were effective against adenomatous, epithelial, and
100 lymphocytic cancer cell lines. The cell growth inhibitory effects
of the NOSH-NSAIDs were associated with cell cycle arrest,
inhibition of proliferation and induction of apoptosis. NOSH-
NAP and NOSH-SUL also showed strong anti-inflammatory
properties that were comparable to that of their respective parent
105 NSAIDs as demonstrated by measuring the in vivo carrageenan-
induced rat paw oedema, and direct measurement of
cyclooxygenase-dependant production of PGE2. It appears that
these agents have strong antineoplastic potential.
INSERT Table 3 (single column)
2.2.4. NOSH-naproxen and NOSH-sulindac have
45 potent anti-inflammatory properties.
NSAIDs are generally used for treatment of inflammatory
conditions. Therefore, we wanted to compare the COX-dependent
anti-inflammatory activities of NOSH-NAP and NOSH-SUL to
that of their respective parent compound. This was done by using
50 the rat paw edema model, as previously reported.16 After
inducing inflammation in rat’s paw with carrageenan, at 4 hrs
animals receiving vehicle showed an increase in paw volume of
1.1 ± 0.1 mL. In contrast, the increase in paw volume for the
animals receiving naproxen and sulindac was 0.25 ± 0.16 mL and
55 0.22 ± 0.14 mL, respectively. Animals receiving NOSH-NAP and
NOSH-SUL exhibited potent anti-inflammatory properties, the
increase in paw volume being 0.15 ± 0.02 mL and 0.21 ± 0.01
mLin NOSH-NAP and NOSH-SUL treated animals, respectively
(Fig. 3A).
4. Experimental section
110 4.1. Chemistry
All moisture-sensitive reactions were performed under an argon
atmosphere using oven-dried glassware and anhydrous solvents
as previously reported.4 Anhydrous solvents were freshly distilled
from sodium benzophenone ketyl, for THF and DCM was
115 distilled from calcium hydride. Extracts were dried over
anhydrous Na2SO4 and filtered prior to removal of all volatiles
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under reduced pressure. Unless otherwise noted, commercially 60 whole reaction mixture was heated at 70oC for 6h. Reaction
available materials were used without purification. Silica gel
chromatography was performed using 100-200 mesh silica gel
(Natland). Thin layer chromatography was performed using
precoated 250 m plates (Analtech). Nuclear magnetic resonance
mixture was filtered through celite and concentrated, further it
was purified by silica gel column chromatography by using
hexane: ethyl acetate (8:2) as a solvent system to obtain the
compound 5 (575.5 mg, 65%).
5
(NMR) splitting patterns are described as singlet (s), doublet (d), 65 1H NMR (CDCl3,500 MHz): 7.81 (d, J = 8.8 Hz, 1H), 7.77 (d, J =
triplet (t), quartet (q); the value of chemical shifts (δ) are given in
ppm relative to residual solvent (chloroform δ=7.27 for 1HNMR),
and coupling constants (J) are given in hertz (Hz). The mass
10 spectra were recorded on AB SCIEX 4000 QTRAP LC-MS/MS
instrument (EI).
8.3 Hz, 1H), 7.76 (s, 1H), 7.53 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H),
7.20 (dd, J = 8.8, 1.45 Hz,1H), 4.62 (t, J = 6.8 Hz, 2H), 3.76 (q, J
= 7.2 Hz, 1H), 2.78 (t, J = 6.8 Hz, 2H), 2.22 (q, J = 6.8 Hz, 2H),
1.53 (d, J = 7.3 Hz, 3H), 1.38 (s, 9H). ESIMS: m/z 426 (M++Na).
70 4.1.5. Synthesis of compound 6: To the solution of compound 5
(550 mg, 1.36 mmol) in dry DCM (5 mL) was added trifluoro
acetic acid TFA (5 mL) at 0oC then stirred at room temperature
for 30 min. The volatiles were evaporated and the crude product
was washed with water and extracted into DCM. The organic
4.1.1. Synthesis of (7-hydroxynaphthalen-1-yl) acetic acid (2):
To the solution of Naproxen (5g, 23 mmol) in AcOH (50 mL)
was added HBr (47%, 25 mL) and the mixture was refluxed for
15 4h. The whole reaction mixture was then removed under reduced
pressure then washed with water. The obtained precipitate was 75 layers were dried on Na2SO4 and concentrated under reduced
filtered and washed with petroleum ether, then recrystallized
from toluene to give (7-hydroxynaphthalen-1-yl) acetic acid 2
(3.52 g, 75%).
pressure. The solid product was used for further reaction without
further purification (260.0 mg, 55 %).
1H NMR (CDCl3,500 MHz): 7.82 (d, J = 8.8 Hz, 1H), 7.77 (d, J
= 8.3 Hz, 1H), 7.76 (s, 1H), 7.53 (s, 1H), 7.48 (d, J = 8.8 Hz, 1H),
20 1H NMR (CDCl3, 500 MHz): 7.64 (d, J = 8.8 Hz, 1H), 7.62 (s,
1H), 7.58 (d, J = 8.3 Hz, 1H), 7.35 (dd, J = 8.8, 1.46 Hz, 1H), 80 7.21 (dd, J = 8.8, 1.45 Hz, 1H), 4.61 (t, J = 6.8 Hz, 2H), 3.91 (q, J
7.09 (s, 1H), 7.08 (dd, J = 8.8, 1.45 Hz,1H), 3.77 (q, J = 7.3 Hz,
= 7.2 Hz, 1H), 2.78 (t, J = 6.8 Hz, 2H), 2.22 (q, J = 6.8 Hz, 2H),
1H), 1.52 (d, J = 7.3 Hz, 3H). ESIMS: m/z 217 (M++1).
1.60 (d, J = 7.3 Hz, 3H). ESIMS: m/z 370 (M++Na).
4.1.2. Synthesis of compound 3: To the solution of compound 2
4.1.6. Synthesis of NOSH-naproxen (AVT-219): To the
25 (2.39 g, 11.05 mmol) in dry THF (100 mL) at 0oC was added
solution of compound
6
(250.0 mg, 0.72 mmol) in
trifluoro acetic anhydride (13.9 ml. 9.2 g, 66.32 mmol) drop wise 85 dichloromethane was added DCC (148.0 mg, 0.72 mmol), DMAP
and stirred for 4h at same temperature. tert-Butanol (30 mL) was
added drop wise at 0oC and stirred at room temperature for
overnight. At 0oC NH4OH (35 % in water, 6 mL) was added drop
30 wise, and then stirred at room temperature for 30 min. After that
(12.4 mg, 0.07 mmol) at 0oC under argon atmosphere. Then
added ADT-OH ((5-(4-hydroxyphenyl)-3H-1, 2-dithiole-3-
thione) (162.0 mg, 0.72 mmol) and the whole reaction mixture
was stirred at room temperature for 6h. After completion of the
volatiles were evaporated under reduced pressure. The crude 90 reaction as checked by TLC, filtered off and water was added
product was triturated with boiling DCM and the crystalline solid
thus obtained was removed by filtration. The filtrate was washed
with saturated aqueous NaHCO3 and dried over Na2SO4. The
35 organic layer was removed under reduced pressure to give the
tert-butyl ester 3 (2.46 g, 82%) as a white solid.
then extracted into dichloromethane (2x25 ml). Organic solvent
was removed under reduced pressure to get the crude product.
Further it was purified by column chromatography by using
hexane: ethyl acetate (7:3) as a eluent to afford pure NOSH-
95 naproxen, (224.0 mg, 56 % yield).
1H NMR (CDCl3,500 MHz): 7.67 (d, J = 8.8 Hz, 1H), 7.62 (s,
1H), 7.59 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 8.8, 1H), 7.09 (s, 1H),
7.04 (dd, J = 8.8, 1.45 Hz,1H), 3.72 (q, J = 7.3 Hz, 1H), 1.50 (d,
40 J = 7.3 Hz, 3H), 1.38 (s, 9H). ESIMS: m/z 273 (M++1).
1H NMR (CDCl3,500 MHz): 7.86 (d, J = 8.8 Hz, 1H), 7.85 (s,
1H), 7.83 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.57 (d, J
= 1.5 Hz, 1H), 7.55 (dd, J = 8.8 Hz, 1.5 Hz, 1H),7.37 (s, 1H),7.26
(dd, J = 8.6 Hz, 1.4 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 4.52 (t, J =
100 7.2 Hz, 2H), 4.15 (q, J = 7.3 Hz, 1H), 2.56 (t, J = 7.2 Hz, 2H),
2.42 (m, 2H), 1.54 (d, J = 7.3 Hz, 3H). ESIMS: m/z 556 (M++1),
578 (M++Na).
4.1.3. Synthesis of compound 4: To the solution of 4-
bromobutyric acid (614 mg, 3.67 mmol) in dry DCM were added
DCC (757 mg, 3.67 mmol) and DMAP and followed by addition
of compound 3 (1.0 g, 3.67 mmol). The whole reaction mixture
45 was stirred for overnight at room temperature. After completion
4.1.7. Synthesis of compound 9: To the solution of 4-
bromobutyric acid (994 mg, 5.95 mmol) in dry DCM were added
of the reaction DCU was filtered off and the solvent was removed 105 DCC (1.22 mg, 5.95 mmol) and DMAP (102 mg, 0.595 mmol)
under the reduced pressure to obtain the crude product. It was
further purified by column chromatography by using hexane:
ethyl acetate (8:2) as a eluent to obtain the compound 4 (1.05 g,
50 65 %).
and followed by addition of compound 8 (1.5 g, 5.95 mmol). The
whole reaction mixture was stirred for overnight at room
temperature. After completion of the reaction DCU was filtered
off and the solvent was removed under the reduced pressure to
1H NMR (CDCl3,500 MHz): 7.83 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 110 obtain the crude product. It was further purified by column
8.3 Hz, 1H), 7.74 (s, 1H), 7.54 (d, J = 1.45, 1H), 7.47 (dd, J =
8.8, 1.45 Hz, 1H), 7.22 (dd, J = 8.8, 1.45 Hz, 1H), 3.79 (q, J = 7.2
Hz, 1H), 3.58 (t, J = 6.8 Hz, 2H), 2.84 (t, J = 6.8 Hz, 2H), 2.34
55 (q, J = 6.8 Hz, 2H), 1.54 (d, J = 7.3 Hz, 3H), 1.40 (s, 9H).
ESIMS: m/z 421 (M++1).
chromatography by using hexane: ethyl acetate (9:1) as a eluent
to obtain the compound 9 (1.54 g, 65 %).
1H NMR (CDCl3,500 MHz): 9.99 (s, 1H), 7.26 (d, J = 2.4 Hz,
1H), 7.07 (dd, J = 8.2, 2.4 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 3.52
115 (t, J = 6.6 Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H), 2.31 (q, J = 6.6 Hz,
2H), 0.98 (s, 9H), 0.21 (s, 6H). ESIMS: m/z 403 (M++1).
4.1.4. Synthesis of compound 5: To the solution of compound 4
(925 mg, 2.19 mmol) in acetonitrile was added AgNO3 (747 mg,
4.39 mmol) under dark conditions (protecting from light). The
4.1.8. Synthesis of compound 10: To the solution of compound
4 (1.5 g, 3.75 mmol) in acetonitrile was added AgNO3 (1.27 g,
4 | Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C3MD00185G
7.5 mmol) under dark conditions (protecting from light). The 60 (12.4 mg, 0.07 mmol) at 0oC under argon atmosphere. Then
whole reaction mixture was heated at 70oC for 6h. Reaction
mixture was filtered through celite and concentrated, further it
was purified by silica gel column chromatography by using
hexane: ethyl acetate (9:1) as a solvent to obtain the compound
10 (0.79 g, 55%).
added compound 13 (200.0 mg, 0.405 mmol) and the whole
reaction mixture was stirred at room temperature for 6h. After
completion of the reaction as checked by TLC, filtered off and
water was added then extracted into dichloromethane (2x25 ml).
65 Organic solvent was removed under reduced pressure to get the
crude product. Further it was purified by column chromatography
by using 5 % DCM/MeOH as a eluent to afford pure NOSH-
sulindac, (216.0 mg, 63 % yield).
5
1H NMR (CDCl3,500 MHz): 9.97 (s, 1H), 7.27 (d, J = 2.4 Hz,
1H), 7.07 (dd, J = 8.8, 3.2 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 4.62
(t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.5 Hz, 2H), 2.22 (q, J = 6.3 Hz,
10 2H), 1.00 (s, 9H), 0.23 (s, 6H). ESIMS: m/z 384 (M++Na), 406
1H NMR (CDCl3, 500 MHz): 7.95 (d, J = 1.8 Hz, 1H), 7.64-7.74
70 (m, 6H), 7.40 (s, 1H), 7.39 (d, J = 1.42 Hz, 1H), 7.27 (d, J = 8.8
Hz, 2H), 7.20 (m, 3H), 6.97 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 6.58 (t,
J = 7.8 Hz, 1H), 4.50 (t, J = 8.8 Hz, 2H), 3.83 (s, 2H), 2.80 (s,
3H), 2.73 (t, J = 8.8 Hz, 2H), 2.28 (s, 3H), 2.11 (m, 2H). ESIMS:
m/z 832 (M++1), 854 (M++Na).
(M++Na).
4.1.9. Synthesis of compound 11: To the solution of compound
10 (0.75 g, 19.58 mmol) in CH3CN (40 mL) kept 0oC were added
a solution of KH2PO4 (2.0 g) in H2O (15 mL) and 30% H2O2
15 (2.19 mL, 19.58 mmol) and drop wise added a solution of 80%
NaClO2 in H2O (15 mL). After 2h stirring at same temperature
Na2SO3 was added to destroy the excess of H2O2. After
acidification with 6M HCl the mixture was diluted with H2O (100
mL) and extracted twice with DCM (100mL). The organic layer
20 was dried, filtered and concentrated under reduced pressure to
obtain the compound 11 (531.0 mg, 68 %).
75 4.2. Biology
4.2.1. Cell culture: HT-29, human colon adenocarcinoma,
BxPC-3 human pancreatic cancer, MCF-7 (estrogen receptor
positive) breast cancer, and Jurkat T human leukemia cell lines
were obtained from American Type Tissue Collection (Manassas,
80 VA). All cells lines were grown as monolayers except for the
Jurkat T cells which were grown in suspension. The pancreatic
and breast cancer cells were grown in Dulbecco’s modified
Eagle’s medium, the Jurkat T cells were grown in RPMI 1640
medium, and the colon HT-29 cells were grown in McCoy 5A.
85 All media were supplemented with 10% fetal calf serum
(Invitrogen, Carlsbad, CA) penicillin (50 U/ml), and streptomycin
(50 µg/ml) (Invitrogen, Carlsbad, CA). Normal human lung
fibroblast cells IMR-90 (CCL186) were obtained from the
American Type Tissue Collection (Manassas, VA) and
90 maintained in Eagle's Minimum Essential Medium (EMEM)
supplemented with 10% heat-inactivated fetal calf serum (FCS),
1H NMR (CDCl3,500 MHz): 7.54 (d, J = 2.9 Hz, 1H), 7.08 (dd, J
= 8.8, 2.9 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 4.62 (t, J = 6.5 Hz,
2H), 2.77 (t, J = 6.5 Hz, 2H), 2.20 (q, J = 6.3 Hz, 2H), 1.00 (s,
25 9H), 0.24 (s, 6H). ESIMS: m/z 400 (M++Na), 422 (M++Na).
4.1.10. Synthesis of compound 12: To the solution of compound
11 (500.0 mg, 1.25 mmol) in dichloromethane was added DCC
(258.0 mg, 1.25 mmol), DMAP (21.55 mg, 0.125 mmol) at 0oC
under argon atmosphere. Then added ADT-OH ((5-(4-
30 hydroxyphenyl)-3H-1, 2-dithiole-3- thione) (283.0 mg, 1.25
mmol) and the whole reaction mixture was stirred at room
temperature for 6h. After completion of the reaction as checked
by TLC, filtered off and water was added then extracted into
dichloromethane (2 x 25 ml). Organic solvent was removed under
35 reduced pressure to get the crude product. Further it was purified
by column chromatography by using hexane: ethyl acetate (8:2)
as a eluent to afford compound 12 (484.0 mg, 62 % yield).
2
mmol/L L-glutamine and penicillin/streptomycin (100
units/mL). Human primary pancreatic epithelial cells (ACBRI
515) was obtained from Cell Systems Corporation (Kirkland,
95 WA) and maintained in Complete Serum-Free Medium (CSC
Catalog SF-4K0-500D) and with Attachment Factor (CSC
Catalog 4Z0-200) as suggested by manufacturer. Normal human
mammary epithelial (HMEpC) cell line was obtained from Promo
Cell, (Heidelberg, Germany) and was cultured in ready-to-use
100 mammary epithelium cell growth medium as suggested by
manufacturer. Cells were seeded on culture dishes at a density of
25×103 cells/cm2 and incubated at 37°C in 5% CO2 and 90%
relative humidity. Single cell suspensions were obtained by
trypsinization (0.05% trypsin/EDTA), and cells were counted
1H NMR (CDCl3,500 MHz): 7.73 (d, J = 8.8 Hz, 2H), 7.63 (d, J =
2.93 Hz, 1H), 7.42 (s, 1 H), 7.31 (d, J = 8.8 Hz, 2H), 7.12 (dd, J =
40 8.8, 2.99 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 4.52 (t, J = 6.8 Hz,
2H), 2.72 (t, J = 6.8 Hz, 2H), 2.14 (q, J = 6.8 Hz, 2H), 1.00 (s,
9H), 0.24 (s, 6H). ESIMS: m/z 608 (M++1), 631 (M++Na).
4.1.11. Synthesis of compound 13:
A
solution of
tetrabutylammonium fluoride (1 mL, 1.0 mmol) and acetic acid
45 (1 mL) in THF (5 mL) was added to the compound 12 (450.0 mg,
0.722 mmol ) and stirred it for 30 min. After completion of the 105 using a hemocytometer. Viability was determined by the trypan
reaction as checked by TLC, volatiles were removed under
reduced pressure. Furthur thus obtained crude product was
purified by column chromatography by using hexane: ethyl
50 acetate (7:3) as a eluent to get the pure compound 13 (249.0 mg,
72 %).
blue dye exclusion method.
4.2.2. MTT Assay: Cell growth inhibitory effect of NOSH-NAP,
NOSH-SUL and their respective parent NSAID were measured
using a colorimetric MTT assay kit (Roche, Indianapolis, IN).
110 HT-29, BxPC-3, MCF-7, and Jurkat T cells were plated in 96-
well plates at a density of 50,000 cells/well. The cells were
1H NMR (CDCl3,500 MHz): 7.71 (d, J = 8.8 Hz, 2H), 7.70 (d, J =
1.28 Hz, 1H), 7.42 (s, 1 H), 7.27 (d, J = 8.8 Hz, 2H), 7.19 (dd, J =
8.8, 3.0 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 4.54 (t, J = 6.8 Hz,
55 2H), 2.73 (t, J = 6.8 Hz, 2H), 2.15 (q, J = 6.8 Hz, 2H). ESIMS:
m/z 494 (M++1), 516 (M++Na).
incubated for 24
h with different concentrations of test
compounds, after which 10µL of MTT dye (3-[4, 5-
dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), was
115 added to each well, and the plates were incubated for 2 hours at
37ºC. The media was then aspirated, and 100 µL of the
solubilization solution (10% SDS in 0.01 M HCl) was added to
solubilize the formant crystals. The absorbance of the plates was
4.1.12. Synthesis of NOSH-sulindac (AVT-18A): To the
solution of sulindac (acid form, 144.0 mg, 0.405 mmol) in
dichloromethane was added DCC (83.0 mg, 0.405 mmol), DMAP
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Page 6 of 16
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DOI: 10.1039/C3MD00185G
determined on an ELISA reader at 570 nm. Since Jurkat T cells 60 4.2.5. Determination of PGE2 in rat paw exudates: Rats were
are non-adherent, they were added (30,000 cells per well) to
serial dilutions of NOSH-NSAIDs in V-bottom 96-well plates
and incubated for 24 hrs in a humidified 5% CO2 incubator at
37°C. After 24 hrs of incubation, 20 µL of the MTT solution (1.1
euthanized by asphyxiation in a CO2 chamber. After cutting each
hind paw at the level of the calcaneus bone, exudates (oedema
fluid) and some tissue were collected, weighed and placed in a
test tube containing 5 mL of 0.1 M phosphate buffer (pH7.4), 1
5
mg/mL) was added to each well and the plates were incubated for 65 mM EDTA, and 10 µM indomethacin. The mixture was
an additional 4 h. The colored formazan crystals produced from
the MTT was then dissolved by adding 150 µL of dimethyl
sulfoxide (DMSO). The optical density (OD) of the solutions
homogenized and centrifuged for 10 min at 12,000 r.p.m. at 4°C.
PGE2 content in supernatant was determined in duplicate by an
enzyme immunoassay kit following the protocol described by the
manufacturer (Cayman Chemical, Ann Arbor, MI) and previously
70 reported by us.16
10 were measured as indicated above.
4.2.3. Cell kinetics parameters: For proliferation and cell cycle
analysis, HT-29 cells (1×106 cells/mL) were treated for 24 hrs
with various concentrations of NOSH-NAP and NOSH-SUL.
PCNA was determined using an ELISA Kit (Calbiochem, La
15 Jolla, CA), in accordance with the manufacturers protocol as
4.2.6. Determination of plasma NO and H2S levels: The Griess
method was used to estimate plasma NO levels indirectly as the
-
-
concentration of nitrate (NO3 ) and nitrite (NO2 ) using an assay
kit from Cayman Chemical (Ann Arbor, MI) and following the
previously reported.18 For cell cycle analysis, the treated cells 75 manufacturer’s protocol. Plasma was filtered using a 10 KD
(0.5×106) were harvested by trypsinization, centrifugation and
then fixed in 70% ethanol for 10 min at -20°C, pelleted (5000
rpm ×10 min at 4°C), resuspended and incubated in PBS
20 containing 1% FBS/0.5% NP-40 on ice for 5 min. After washing
molecular weight cut-off filter from Millipore (Bedford, MA)
before each analysis, to reduce background absorbance due to the
presence of hemoglobin. After centrifugation for 10 min at 3000
rpm, samples (40 µL/well) were mixed with 10µL nitrate
and addition of propidium iodide (40 µg/mL) to stain for DNA 80 reductase mixture and incubated for 3 h after which Griess
and 200 µg/mL RNase type IIA, cell were analyzed by flow
cytometry. Cell cycle phase distributions of control and treated
cells were obtained using a Coulter Profile XL equipped with a
25 single argon ion laser. For each subset, >10,000 events were
reagents 1 and 2 (50 µL each) were added. Absorbance was read
after 10 min at 540nm using a plate reader. The concentration of
nitrate/nitrite was calculated graphically from a calibration curve
prepared from NaNO2 standard solution, and it is expressed as
analyzed. All parameters were collected in list mode files. Data 85 micromolar nitrate. H2S levels were measured as previously
were analyzed on a Coulter XL Elite Work station using the
Software programs MultigraphTM and MulticycleTM. The
percentage of cells in G0/G1, G2/M, and S phases was determined
30 form DNA content histograms.
described.4, 20, 21Aliquots (100 µL) of rat plasma were mixed with
distilled water (100 µL), Zinc acetate (1% w/v, 250 µL),
trichloroacetic acid (10% w/v, 250 µL), N, N-dimethyl-p-
phenylenediamine sulfate (133 µL, 20 µM) in 7.2M HCl and
To evaluate apoptosis, control and treated cells (0.5×106 90 FeCl3 (133 µl, 30 µM) in 7.2M HCl. The absorbance of the
cells/mL) were washed with and resuspended in 1X Binding
Buffer (Annexin V binding buffer, 0.1 M Hepes/NaOH (pH 7.4),
1.4 M NaCl, 25mM CaCl2) from BD BioSciences (San Diego,
35 CA). Annexin V-FITC and Propidium iodide were then added
resulting mixture (300 µL) was determined after 15 min using a
96-well microplate reader at 670 nm. All samples were assayed in
duplicate and H2S levels were calculated against a calibration
curve of NaHS (1-250 µM). This method overestimates H2S
and the cells incubated at room temperature for 5–15 min in the 95 levels as it measures free H2S, HS- (hydrosulfide anion), and S2-
dark. Cells were then transferred to FACS tubes and analysed by
flow cytometry as described above using Flow Jo software.
(sulfide).22 Therefore, our results presented here indicate the sum
total of these species.
4.2.4. Inflammatory edema model: Our institutional animal care
4.2.7. Statistical analysis: In vitro: data are presented as mean ±
40 and research committee approved the experimental procedure
SEM for 3-7 different sets of plates done in triplicate. In vivo: 3
described here. Male Wistar rats (3 per group) weighing 180–200 100 animals were used per treatment group. Comparison between
g were obtained from Charles River Laboratories International
(Wilmington, MA). The rats were fed standard laboratory chow
and water. On the day of the experiments, rats were given by
45 gavage equimolar doses of naproxen (80 mg/kg), NOSH-
naproxen (188 mg/kg); sulindac (200 mg/kg), NOSH-sulindac
(467 mg/kg); or the vehicle, 0.5% carboxymethylcellulose
solution. One hour post drug administration, freshly prepared,
100 µL of 1% carrageenan, Sigma Chemicals (St. Louis, MO)
50 was subcutaneously injected into the plantar surface of the right
hind paw of the rats following the protocol described by Winter
et al 19. Paw volume was measured using a water displacement
plethysmometer (Model 520, IITC/Life Sciences Instruments,
Woodland Hills, CA) before carrageenan injection and at 4 hr
55 post injection. This time point was chosen because previous
studies had indicted it to be optimum where greatest volume
changes were observed 16. The paw volume measured just prior to
carrageenan injection was used as the control volume. Data are
expressed as the change in paw volume (mL) at 4hr.
treatment groups was performed by one-factor analysis of
variance (ANOVA) followed by Turkey’s test for multiple
comparisons. P < 0.05 was regarded as statistically significant.
5. Acknowledgements
105 Supported in part by NIH grant R24 DA018055. The funding
agency had no role in the study design, collection, analysis and
interpretation of data; in the writing of the manuscript; and in the
decision to submit the manuscript for publication.
6. Notes and references
110 1. K. Kashfi, Adv Pharmacol, 2009, 57, 31-89.
2. M. M. Wolfe, D. R. Lichtenstein and G. Singh, N Engl J Med, 1999,
340, 1888-1899.
3. J. L. Wallace and L. Vong, Curr Opin Investig Drugs, 2008, 9, 1151-
1156.
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DOI: 10.1039/C3MD00185G
4. R. Kodela, M. Chattopadhyay and K. Kashfi, ACS Med Chem Lett,
2012, 3, 257-262.
5. J. L. Wallace, B. Reuter, C. Cicala, W. McKnight, M. Grisham and
G. Cirino, Eur J Pharmacol, 1994, 257, 249-255.
5 6. P. del Soldato, R. Sorrentino and A. Pinto, Trends Pharmacol Sci,
1999, 20, 319-323.
7. S. Fiorucci, E. Antonelli, E. Distrutti, G. Rizzo, A. Mencarelli, S.
Orlandi, R. Zanardo, B. Renga, M. Di Sante, A. Morelli, G.
Cirino and J. L. Wallace, Gastroenterology, 2005, 129, 1210-
10
1224.
8. J. L. Wallace, G. Caliendo, V. Santagada, G. Cirino and S. Fiorucci,
Gastroenterology, 2007, 132, 261-271.
9. V. del Amo, A. P. McGlone, J. M. Soriano and A. P. Davis,
Tetrahedron, 2009, 65, 6370-6381.
15 10. S. Tasler, R. Baumgartner, A. Ammendola, J. Schachtner, T. Wieber,
M. Blisse, S. Rath, M. Zaja, P. Klahn, U. Quotschalla and P.
Ney, Bioorg Med Chem Lett, 2010, 20, 6108-6115.
11. L. Lazzarato, M. Donnola, B. Rolando, E. Marini, C. Cena, G.
Coruzzi, E. Guaita, G. Morini, R. Fruttero, A. Gasco, S.
20
25
30
Biondi and E. Ongini, J Med Chem, 2008, 51, 1894-1903.
12. M. Chattopadhyay, R. Kodela, K. R. Olson and K. Kashfi,
Biochemical and biophysical research communications, 2012,
419, 523-528.
13. E. De Luna-Bertos, J. Ramos-Torrecillas, O. Garcia-Martinez, L.
Diaz-Rodriguez and C. Ruiz, TheScientificWorldJournal,
2012, 2012, 834246.
14. H. Raza, A. John and S. Benedict, Eur J Pharmacol, 2011, 668, 15-
24.
15. D. Xiao, A. Deguchi, G. G. Gundersen, B. Oehlen, L. Arnold and I.
B. Weinstein, Molecular cancer therapeutics, 2006, 5, 60-67.
16. M. Chattopadhyay, C. A. Velazquez, A. Pruski, K. V. Nia, K. R.
Abdellatif, L. K. Keefer and K. Kashfi, J Pharmacol Exp
Ther, 2010, 335, 443-450.
17. K. R. Olson, Biochim Biophys Acta, 2009, 1787, 856-863.
35 18. R. Kodela, M. Chattopadhyay, N. Nath, L. Z. Cieciura, L. Pospishill,
D. Boring, J. A. Crowell and K. Kashfi, Bioorg Med Chem
Lett, 2011, 21, 7146-7150.
19. C. A. Winter, E. A. Risley and G. W. Nuss, Proc. Soc. Exp. Biol.
Med., 1962, 111, 544-547.
40 20. L. Li, G. Rossoni, A. Sparatore, L. C. Lee, P. Del Soldato and P. K.
Moore, Free Radic Biol Med, 2007, 42, 706-719.
21. S. Huang, J. H. Chua, W. S. Yew, J. Sivaraman, P. K. Moore, C. H.
Tan and L. W. Deng, J Mol Biol, 2010, 396, 708-718.
22. Z. W. Lee, J. Zhou, C. S. Chen, Y. Zhao, C. H. Tan, L. Li, P. K.
45
Moore and L. W. Deng, PLoS One, 2011, 6, e21077.
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Medicinal Chemistry Communications
Page 8 of 16
Cell Cycle
A
Proliferation
*
Apoptosis
B
C
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
*
0
3
6 12 24
0
3
6 12 24
0
3
6 12 24
D
E
F
100
100
100
*
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
*
0
3
6 12 24
0
3
6 12 24
0
3
6 12 24
Time, hr
Figure 2. Effect of NOSH-NAP and NOSH-SUL on HT-29 colon cancer cell kinetics. NOSH-NAP and NOSH-
SUL inhibited proliferation by altering cell cycle progression and inducing apoptosis. Cells were treated with vehicle,
or NOSH-NAP (panels A-C) or NOSH-SUL (panels D-F) at their respective IC50 for cell growth inhibition (80 and 90
nM) and analyzed at the indicated times. Panels A and D, proliferation by PCNA antigen expression; Panels B and
E), cell cycle phases by PI staining and flow cytometry; Panels C and F), apoptosis by Annexin V staining and flow
cytometry. Results are mean ± SEM for 3 different experiments performed in duplicate, *P < 0.05, compared to
control.

PageM9eodfic1i6nal Chemistry Communications
A
1.0
*
0.5
0
B
100
80
60
*
40
20
0
Treatment Groups
Figure 3. Anti-inflammatory properties of NOSH-
NAP and NOSH-SUL. Rat paw edema was induced by
carrageenan injection. Panel A, all agents caused a
significant reduction in paw volume at 4hr. Results are
*
mean ± S.E.M. of three rats in each group, P < 0.05
versus vehicle treated rats. Panel B, all agents caused
a significant reduction in PGE
levels in the paw
2
exudate. Results are mean ± S.E.M. for three rats in
each group, *P < 0.01versus vehicle

Medicinal Chemistry CommunPicaagteio1n0sof 16
400
NOx
H2S
†
300
200
100
0
†
Treatment Groups
Figure 4. NO and H 2S levels in vivo after NOSH-
NAP and NOSH-SUL administration. Plasma
concentration of NO
and H S was quantified as
2
x
detailed in Section 4.2.6. Results are mean ± S.E.M. of
three rats in each group. †P < 0.001 versus vehicle
and parent NSAID-treated animals.

Page 11 of 16
Medicinal Chemistry Communications
OH OH
O
O
a
b
O
O
O
O
MeO
HO
HO
1
2
3
4
c
O
d
O
O
Br
O2NO
O
O
O
O
O
5
e
OH
O
f
O
O
O2NO
O2NO
O
O
S
S
6
NOSH-Naproxen
AVT-219
S
a. HBr/AcOH, reflux, 4h; b. Tf2O, t-BuOH, NH4OH; c. 4-bromobutyric acid, DCC/DMAP, DCM, rt, 6h; d. AgNO3/acetonitrile,
70oC, 6h; e. TFA/DCM, 30 min; f. ADT-OH, DCC/DMAP, DCM, rt, 6h.
Scheme-1: Synthetic scheme for the synthesis of NOSH-Naproxen (AVT-219)

Medicinal Chemistry Communications
Page 12 of 16
HO
CHO
OH
TBDMSO
CHO
TBDMSO
CHO
O
a
b
OH
7
8
9
Br
S
O
c
S
S
O
TBDMSO
TBDMSO
e
COOH
O
TBDMSO
CHO
O
O
d
O
12
11
10
ONO2
ONO2
ONO2
S
O
O
O
O
S
f
S
S
F
S
S
O
O
O
O
g
O
HO
O
Me
O
ONO2
O
13
ONO2
Me
O
NOSH-Sulindac
AVT-18A
S
O
a. TBDMSCl/Imidazole, DCM, 18h; b. 4-bromobutyric acid,DCC/DMAP, DCM, rt, 6h; c. AgNO3/acetonitrile, 70°C, 6h; d.
NaH2PO4, H2O2/NaClO2, 0°C, 2h; e. ADT-OH, DCC/DMAP, DCM, rt, 6h; f. TBAF/AcOH, THF, 30 min; g. Sulindac,
DCC/DMAP, DCM, rt, 6h.
Scheme-2: Synthetic scheme for preparation of NOSH-Sulindac (AVT-18A)

Page 13 of 16
Medicinal Chemistry Communications
View Article Online
DOI: 10.1039/C3MD00185G
Table 1: IC50 values at 24 hr for cell growth inhibition in different cancer cell lines.
Origin/Cell line, IC50, µM
Compound
Colon
HT-29
Leukemia
Jurkats
Breast
MCF-7
Pancreas
BxPC-3
NAP
2775 ± 188
0.08 ± 0.01*
~34,000
2550 ± 225
0.10 ± 0.01*
~25,000
2385 ± 145
0.12 ± 0.01*
~20,000
2450 ± 100
0.15 ± 0.02*
~16,000
NOSH-NAP
Potency
enhancement
SUL
800 ± 90
0.089 ± 0.01*
~9,000
699 ± 75
0.27 ± 0.02*
~2,500
965 ± 65
0.10 ± 0.01*
~9,650
980 ± 85
0.12 ± 0.01*
~8,200
NOSH-SUL
Potency
enhancement
Colon, leukemia, breast, and pancreas cancer cell lines were treated with various concentrations
of naproxen (NAP), NOSH-naproxen (NOSH-NAP), sulindac (SUL), and NOSH-sulindac
(NOSH-SUL) as described under Experimental Section/Biology. Cell numbers were determined
at 24 h from which IC50 values were calculated. Results are mean ± S.E.M. of five to seven
different experiments done in triplicate. *P < 0.001 compared to respective parent compound.

Medicinal Chemistry Communications
Page 14 of 16
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DOI: 10.1039/C3MD00185G
Table 2: IC50 values at 24 hr for cell growth inhibition in different human normal cell lines.
Origin/Cell line, IC50, µM
Compound
Lung Fibroblast
IMR-90 (CCL186)
Mammary Epithelial
HMEpC
Pancreatic Epithelial
HPDE (ACBRI 515)
NOSH-NAP
NOSH-SUL
20.0 ± 2.5
21.7 ± 1.7
13.8 ± 0.9
15.2 ± 0.7
22.0 ± 1.8
> 25
Normal human lung fibroblast, mammary epithelial, and pancreatic epithelial cells were treated
with NOSH-naproxen (NOSH-NAP) and NOSH-sulindac (NOSH-SUL) as described under
Experimental Section/Biology. Cell numbers were determined at 24 h from which IC50 values
were calculated. Results are mean ± S.E.M. of three different experiments done in duplicate.

Page 15 of 16
Medicinal Chemistry Communications
View Article Online
DOI: 10.1039/C3MD00185G
Table 3. IC50 values for cell growth inhibition by various components of NOSH-naproxen or
other agents that release NO or H2S alone or in combination.
Compound
IC50 at 24h, µM
2600 ± 150
508 ± 35
Naproxen
SNAP
ADT-OH
35 ± 4
Naproxen + SNAP
Naproxen + ADT-OH
Naproxen + SNAP + ADT-OH
NOSH-naproxen
900 ± 57
300 ± 65
780 ± 55
0.09 ± 0.004†
Cells were treated with various concentrations of the test agents shown above, cell numbers
were determined at 24h from which IC50 values were calculated. Results are mean ± SEM of
†
three different experiments performed in duplicate. P<0.001 compared to all other treatment
groups. The reconstitution experiments at concentrations that provide virtually identical
concentrations of “NOSH-naproxen”, NO and H2S in the media show that the IC50 of the intact
NOSH-naproxen molecule were not obtained, the sum of the parts does not equal the whole.

Graphical Abstract
Medicinal Chemistry CommuPnaicgaeti1o6nosf 16
Anti-inflammatory
NSAID
Cell cycle arrest
NOSH-NSAIDs
NO
Proliferation
Apoptosis
H2S