Nitrosothiol esters of diclofenac: Synthesis and pharmacological characterization as gastrointestinal-sparing prodrugs

Article abstract of DOI:10.1021/jm000178w

Despite its widespread use, diclofenac has gastrointestinal liabilities common to nonsteroidal antiinflammatory drugs (NSAIDs) that might be reduced by concomitant administration of a gastrointestinal cytoprotectant such as nitric oxide (NO). A series of novel diclofenac esters containing a nitrosothiol (-S-NO) moiety as a NO donor functionality has been synthesized and evaluated in vivo for bioavailability, pharmacological activity, and gastric irritation. All S-NO-diclofenac derivatives acted as orally bioavailable prodrugs, producing significant levels of diclofenac in plasma within 15 rain after oral administration to mice. At equimolar oral doses, S-NO-diclofenac derivatives (20a-21b) displayed rat antiinfiammatory and analgesic activities comparable to those of diclofenac in the carrageenan-induced paw edema test and the mouse phenylbenzoquinone-induced writhing test, respectively. All tested S-NO-diclofenac derivatives (20a-21b) were gastric-sparing in that they elicited markedly fewer stomach lesions as compared to the stomach lesions caused by a high equimolar dose of diclofenac in the rat. Nitrosothiol esters of diclofenac comprise a novel class of NO-donating compounds having therapeutic potential as nonsteroidal antiinflammatory agents with an enhanced gastric safety profile.

Full text of DOI:10.1021/jm000178w

J . Med. Chem. 2000, 43, 4005-4016
4005
Nitr osoth iol Ester s of Diclofen a c: Syn th esis a n d P h a r m a cologica l
Ch a r a cter iza tion a s Ga str oin testin a l-Sp a r in g P r od r u gs†,‡
Upul K. Bandarage,* Liqing Chen, Xinqin Fang, David S. Garvey, Alicia Glavin, David R. J anero,
L. Gordon Letts, Gregory J . Mercer, J oy K. Saha, J oseph D. Schroeder, Matthew J . Shumway, and
S. William Tam
NitroMed, Inc., 12 Oak Park Drive, Bedford, Massachusetts 01730
Received April 19, 2000
Despite its widespread use, diclofenac has gastrointestinal liabilities common to nonsteroidal
antiinflammatory drugs (NSAIDs) that might be reduced by concomitant administration of a
gastrointestinal cytoprotectant such as nitric oxide (NO). A series of novel diclofenac esters
containing a nitrosothiol (-S-NO) moiety as a NO donor functionality has been synthesized
and evaluated in vivo for bioavailability, pharmacological activity, and gastric irritation. All
S-NO-diclofenac derivatives acted as orally bioavailable prodrugs, producing significant levels
of diclofenac in plasma within 15 min after oral administration to mice. At equimolar oral
doses, S-NO-diclofenac derivatives (20a -21b) displayed rat antiinflammatory and analgesic
activities comparable to those of diclofenac in the carrageenan-induced paw edema test and
the mouse phenylbenzoquinone-induced writhing test, respectively. All tested S-NO-diclofenac
derivatives (20a -21b) were gastric-sparing in that they elicited markedly fewer stomach lesions
as compared to the stomach lesions caused by a high equimolar dose of diclofenac in the rat.
Nitrosothiol esters of diclofenac comprise a novel class of NO-donating compounds having
therapeutic potential as nonsteroidal antiinflammatory agents with an enhanced gastric safety
profile.
Ch a r t 1. Diclofenac and S-NO-Diclofenac
In tr od u ction
Nonsteroidal antiinflammatory drugs (NSAIDs) are
widely used for the treatment of pain, fever, and
inflammation, particularly arthritis.1-3 Among the most
popular NSAIDs, diclofenac (1) (Chart 1) has been
approved in 120 countries since its introduction 25 years
ago4 and is ranked 30th among the top 200 drugs with
respect to new prescriptions and refills in the United
States alone.5
The pharmacological activity of NSAIDs is related to
the suppression of prostaglandin H2 (PGH2) biosynthesis
from arachidonic acid by inhibiting the activity of the
enzyme cyclooxygenases (COXs).6 Recently, it was dis-
covered that COX exists in two isoforms, COX-1 and
COX-2, which are regulated differently.7 COX-1 provides
cytoprotection in the gastrointestinal (GI) tract, whereas
inducible COX-2 mediates inflammation.8 Since most
of the NSAIDs in the market show greater selectivity
for COX-1 than COX-2,9 chronic use of NSAIDs, includ-
ing diclofenac, may elicit appreciable GI irritation,
bleeding, and ulceration2,3 with side effects including
nausea, vomiting, abdominal pain, dyspepsia, and
diarrhea.10-12 The incidence of clinically significant GI
side effects due to NSAIDs is high (over 30%) and causes
some patients to abandon NSAID therapy.5 NSAID-
related GI adverse side effects account for more than
70 000 hospitalizations and 7000 deaths annually in the
United States.13 GI damage from NSAIDs is generally
attributed to two factors: local GI irritation by the
carboxylic acid moiety common to most NSAIDs (“topical
effect”) and decreased tissue prostaglandin production,
which undermines the physiological role of cytoprotec-
tive prostaglandins in maintaining GI health and ho-
meostasis.3,5,14
Synthetic approaches based upon NSAID chemical
modification have been taken with the aim of improving
the NSAID safety profile. One such approach has been
to mask the NSAID carboxylic acid moiety in NSAID
ester prodrugs that hydrolyze in vivo to release the
active parent NSAID.5,14,15 Such NSAID derivatives are
indeed gastroprotective in rodent models of acute NSAID-
induced damage.5,14,15 By itself, though, masking the
carboxylic acid moiety is considered unlikely to amelio-
rate the severe GI complications of chronic NSAID use,
since such prodrugs reduce only the topical irritancy
effect without remediating the NSAID-induced tissue
deficit of beneficial prostaglandins.
† This work was initially presented in part as posters at the 218th
ACS National Meeting, Medicinal Chemistry, New Orleans, LA, Aug.
22-26, 1999 (abstr. # 81), 220th ACS Meeting in WDC, Medicinal
Chemistry, Aug. 20-24, 2000 (abstr. #242), and Digestive Disease
Week Meeting, Orlando, FL, May 16-19, 1999 (abstr. #298).
‡ Authors appear in alphabetical order.
Highly selective COX-2 inhibitors have recently been
developed and marketed as promising gastroprotective
* To whom correspondence should be addressed. Phone: (781) 685
9723. Fax: (781) 275 1127. E-mail: ubandarage@nitromed.com.
10.1021/jm000178w CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/13/2000

4006 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Bandarage et al.
Sch em e 1a
agents.16 Two of the compounds, celecoxib (Celebrex)
and rofecoxib (Vioxx), have been extensively studied and
show exceptional antiinflammatory properties with
reduced GI toxicity.17 Despite the initial enthusiasm
surrounding selective COX-2 inhibitors, questions re-
main regarding their ultimate benefit and safety, since
in certain circumstances COX-2 may be important to
homeostasis.18 Some potential limitations of (long-term)
COX-2 inhibitor therapy include ulcer exacerbation in
high-risk patients, delayed gastroduodenal ulcer heal-
ing, thrombosis due to prostacyclin deficiency, and
kidney toxcity.18,19 Chronic use of COX-2 inhibitors may
affect regulation of female reproduction, bone formation,
and renal functions, since disruption of the gene for
COX-2 impaired those functions in mice.19,20 Thus,
COX-2 inhibitors have not eliminated the need for
improved drugs in the NSAID area.
a
Reagents and conditions: (a) S2Cl2/CCl4, 55 °C; (b) LAH, THF;
(c) t-BuONO, CH2Cl2.
A more recent strategy for devising a gastric-sparing
NSAID, studied by Wallace and colleagues, involves
chemically coupling a nitric oxide (NO)-releasing moiety
to the parent NSAID.21-25 Along with prostaglandins,
NO appears to play an important cytoprotective role in
GI homeostasis and defense by helping to maintain
mucosal blood flow, optimizing mucus gel secretion, and
inhibiting activation of proinflammatory cells.21-25 Thus,
NO may counteract the detrimental effect of COX
inhibition.
of their NO-donating functionality, crystalline tertiary
S-nitrosothiols are particularly stable as compared to
primary and secondary S-nitrosothiols, facilitating their
synthesis and profiling.32,33
We report the synthesis and biological evaluation of
a series of novel diclofenac ester derivatives bearing
tertiary nitrosothiols and demonstrate their effective-
ness as GI-sparing, analgesic, and antiinflammatory
prodrugs.
Syntheses of NO-releasing analogues of several
NSAIDs (aspirin, diclofenac, naproxen, ketoprofen, flur-
biprofen) have been reported. These compounds are
formed by coupling a nitrooxybutyl moiety to the
respective parent NSAID through a short-chain ester
linkage.21-26 Some of these NSAID-derived organic
nitrate esters display comparable antiinflammatory
activity to the respective parent NSAIDs in acute rodent
models, but with reduced GI toxicity.21-26 Furthermore,
in contrast to COX-2 inhibitors and standard NSAID,
a NO-releasing NSAID derivative and a NO donor have
Ch em istr y
Our initial approach to the S-NO-diclofenac target
compounds involved synthesis of nitrosylated tethers
with the aim of coupling them directly to diclofenac
through an ester linkage. To this intent, the three
different classes of NO-containing tethers presented in
Schemes 1-3 were synthesized. Aliphatic aldehydes
with an enolizable proton react with sulfur monochlo-
ride (S2Cl2) to give disulfide-dialdehydes that can be
reduced to tertiary thio alcohols.34,35 We chose this route
to synthesize tertiary thio alcohols 3a -3c starting from
commercially available aldehydes such as 2-methylpro-
panal, 2-ethylbutanal, and cyclohexanecarboxaldehyde
(Scheme 1). We first prepared disulfides 2a -2c in high
yields by reacting the above aldehydes with S2Cl2 in
CCl4 at 55 °C.34 (Upon study of this sulfenation reaction,
we found that tert-butyl methyl ether could substitute
for the CCl4 to form disulfide 2c in 89% yield.) Disulfides
2a -2c were reduced with LiAlH4 to afford thio alcohols
3a -3c in high yields. Subsequently, each thio alcohol
was nitrosylated with tert-butyl nitrite (t-BuONO) in
dichloromethane to give nitrosothiol tethers 4a -4c as
green oils in moderate-to-good yields.
Next, we synthesized a series of nitrosylated tethers
containing a tertiary amine to allow for subsequent salt
formation (Scheme 2). Reductive amination of 2c with
primary amino alcohols of varying chain lengths (n )
2, 3, 4, 5) formed secondary amino disulfides 5a -5d in
good-to-moderate yields. Compound 5a was readily
methylated using aqueous formaldehyde followed by
addition of NaBH4 in methanol to obtain 6a . Reduc-
tion of the disulfide bond in 6a was accomplished with
LiAlH4 at room temperature to afford 7a . Thiol 7a
was nitrosylated with t-BuONO in methanol in the
presence of concentrated HCl to afford nitrosylated
tether 8a . In a similar fashion, nitrosylated tethers 8c
shown existing ulcer-healing properties in rats.23
A
potential limitation of this class of NO donor NSAIDs
arises from their intrinsic nature as indirect sources of
NO. Organic nitrates require metabolic conversion by
as yet ill-defined, likely enzyme-mediated reductive
catabolism in order to produce NO under physiological
conditions.27 Furthermore, tolerance issues may restrict
the therapeutic applicability and efficacy of organic
nitrates.27
A synthetic approach toward NO-tethered diclofenac
prodrugs distinct from organic nitrate esters of NSAID
is depicted in Chart 1. To this intent, we have intro-
duced a nitrosothiol (-S-NO) moiety into diclofenac
through an ester linkage. S-Nitrosothiols are considered
as biological sources of NO and serve as NO sources or
donors without the need for metabolic transforma-
tion.28,29 Although nitrosothiol catabolism is incom-
pletely understood, transition metal-dependent redox
processes and/or enzyme-catalyzed decompositions likely
predominate biological pathways for NO release from
nitrosothiols in vivo. Furthermore, S-nitrosothiols can
directly modulate cell physiology without generating NO
as the effector molecule through S-transnitrosation
reactions, by which the NO group is effectively trans-
ferred from the S-nitrosothiol to the thiol of a target
biomolecule in exchange for a hydrogen.30,31 In terms

Nitrosothiol Esters of Diclofenac
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4007
Sch em e 2a
a
Reagents and conditions: (a) S2Cl2/CCl4; (b) i. NH2(CH2)nOH, CHCl3, reflux, 6 h, ii. NaBH4, MeOH, rt; (c) i. 38% CH2O, MeOH, ii.
NaBH4; (d) LAH, THF; (e) NaBH4, HOAc, rt; (f) t-BuONO, MeOH, HCl.
Sch em e 3a
a
Reagents and conditions: (a) BnBr acetone, K2CO3, rt, 12 h; (b) LAH, THF, reflux, 12 h; (c) t-BuONO, CH2Cl2; (d) Ac2O, pyridine, rt.
and 8d with carbon chain lengths 4 and 5 were
synthesized, starting with 5c and 5d , respectively.
Addition of aqueous formaldehyde to a methanolic
solution of 5b afforded oxazine 9 as a crystalline solid.
Oxazine 9 was opened with NaBH4 in glacial HOAc at
room temperature to give the acyclic disulfide 6b, which
was reduced with LiAlH4 in THF or powdered zinc in
HOAc at room temperature to give the amino thiol 7b
in good yield. The amino thiol 7b was nitrosylated with
t-BuONO to give 8b in high yield (Scheme 2).
Finally, nitrosothiol tethers 12 and 15 were synthe-
sized by replacing the methyl group of 8b with a benzyl
or ethyl group, respectively (Scheme 3). Alkylation of
5b with benzyl bromide in acetone in the presence of
K2CO3 gave the benzylated disulfide 10. Reduction and
nitrosylation were carried out as before to give 12 as
an oil. The N-ethyl derivative was prepared by acety-
lation of 5b with acetic anhydride in pyridine to give
13. Treatment of 13 with excess LiAlH4 cleaved the
O-acetate, reduced the acetamide to the N-ethyl moiety,
and cleaved the disulfide to give 14. Nitrosylation of 14
with acidic t-BuONO gave 15 as an oil.
The nitrosylated tether could be acylated directly by
diclofenac using DCC/DMAP through careful, drop-
wise addition of a solution of DCC in CH2Cl2 to a
mixture of diclofenac, nitrosylated tether, and DMAP
in CH2Cl2 so as to minimize the lactam 18 formation
(Scheme 5). The couplings were usually complete within
30-40 min at 0 °C, and the products 20a -20c,
21a -21d , and 22a and 22b were obtained in moderate-
to-good yield (Scheme 4). On a large scale at room
temperature with longer reaction times, considerable
product decomposition occurred during the coupling
reaction, with evolution of gaseous nitrogen oxides.
Therefore, the reaction temperature was carefully con-
trolled to maximize the yield of desired coupled product.
Compounds 20a -20c were obtained as crystalline solids
after chromatographic purification. Among the series
21a -21d , only 21b was obtained as a crystalline solid,
whereas 21a , 21c, 21d , 22a , and 22b were green oils
readily converted by HCl to amorphous powders. HPLC
analysis showed that crystalline nitrosothiol esters of
diclofenac such as 20b, 20c, and 21b were stable at
room temperature in closed vials for more than 1 year.

4008 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Bandarage et al.
Sch em e 4a
a
Reagents and conditions: (a) 4a -4c, DCC, DMAP, CH2Cl2, 0 °C, 30 min; (b) 8a -8d , DCC, DMAP, CH2Cl2, rt, 30 min; (c) 12, 15,
DCC, DMAP, CH2Cl2, 0 °C, 30 min.
Sch em e 5a
a
Reagents and conditions: (a) DCC, CH2Cl2; (b) diclofenac, DCC, CH2Cl2; (c) Zn, HOAc, rt.
However, most of the amorphous nitrosothiols as HCl
salts decomposed to the corresponding sulfhydryl and
disulfide on standing at room temperature in closed
vials for 2-3 months.
In some cases, the limited stability of the -S-NO
functionality on the tethers or the desire to prepare the
free thiol-containing derivatives required a different
strategy. In such cases, we designed alternative syn-
thetic schemes involving coupling of diclofenac to a
nonnitrosylated, thiol tether followed by nitrosylation
of the free thiol as the final step. Acylation of free thiol-
containing tether 7a with diclofenac using DCC/DMAP
produced varying amounts of O,S-bisacylated 17 and
lactam 18 as byproducts (Scheme 5). However, this
problem was eliminated by acylation of disulfide 6a with
diclofenac and subsequent reduction using powdered
zinc in acetic acid to afford thiol 16 as the major product
in high yield.
P h a r m a cology
Biological characterization of the novel diclofenac
nitrosothiol esters was conducted with the aim of
establishing their bioavailability, analgesic and antiin-
flammatory activities, and gastric tolerance relative to
the sodium salt of the parent NSAID, diclofenac (1). In

Nitrosothiol Esters of Diclofenac
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4009
Ta ble 1. Relative Bioavailability of S-NO-NSAIDs as Plasma
Diclofenac in Mice
mean relative bioavailability slightly, whereas no sig-
nificant difference was noted between propyl (21b) and
butyl (21c). Lowest relative bioavailability (20%) was
observed for 21d , which had the longest (i.e., pentyl)
carbon chain studied. These relative diclofenac bioavail-
ability differences among S-NO-diclofenac derivatives
of varying ester chain length may reflect several factors
operating in vivo: e.g., the ability of the S-NO-diclofenac
esters to interact with the active sites of hydrolases
contributing to their hydrolysis and altered pKa and
lipophilicity of the prodrugs with change in carbon chain
length.5 Since our relative bioavailability data were
obtained in vivo, alteration of spacer chain length in the
S-NO-diclofenac derivatives might also have influenced
compound absorption, tissue uptake/compartmentaliza-
tion, and metabolism which in turn would affect the
appearance of diclofenac from them in the plasma.
Slightly lower relative diclofenac bioavailability was
observed with compounds 22a and 22b which were
synthesized by replacing the N-methyl group of 21b
with benzyl and ethyl substitutions, respectively, as
compared to that observed with 21b (Table 1).
peak diclofenac
(nmol/mL) ( SEM
(n ) 7)
relative plasma
diclofenac AUC0-2 h
(mean %)
compounda
diclofenac sodiumb
23.9 ( 2.9
19.8 ( 2.9
13.4 ( 2.1
15.3 ( 2.3
19.8 ( 2.5
5.5 ( 1.0
7.4 ( 2.3
5.9 ( 1.3
2.4 ( 0.3
6.8 ( 1.8
4.5 ( 2.2
100
80
66
80
100
37
46
42
20
35
35
20a b
20bb
20cb
diclofenac sodiumc
21a c
21bc
21cc
21d c
22a c
22bc
a
All compounds administered to mice at an oral dose of 20 µmol/
kg. Vehicle: 0.5% methocel/5% PEG-400. c Vehicle: 0.5% me-
thocel/50% PEG-400. All values are mean ( SEM (n ) 5-10,
number of animals).
b
all cases, rodent models that are well-established phar-
macological systems were used for in vivo NSAID
profiling.
Rela tive Bioa va ila bility Stu d ies. After adminis-
tration of diclofenac nitrosothiol esters orally to mice,
the level of diclofenac in plasma was measured and
compared to the level produced by oral administration
of diclofenac sodium. This end-point would also indicate
the prodrug nature of these compounds: i.e., whether
the ester bond of the S-NO-diclofenac derivatives is
hydrolyzed in vivo to liberate diclofenac. Diclofenac
levels were determined by HPLC analysis of blood
plasma extracts obtained from 30 min to 2 h postdosing.
The plasma diclofenac area under the curve (AUC) from
0 to 2 h postdosing was determined for each compound.
Because of the range of solubilities encountered among
the diclofenac derivatives studied, vehicles containing
either 5% PEG or 50% PEG (by volume) were used such
that all compounds could be administered orally in the
same dosage state, i.e., as a fine suspension.
Within the S-NO-diclofenac series 20a -20c admin-
istered in aqueous 0.5% methocel-5% PEG, there was
no significant difference in mouse plasma diclofenac
levels when the dimethyl moiety (20a ) was replaced
with a cyclohexyl group (20c) (Table 1). Equimolar doses
of either 20a or 20c resulted in the same level of plasma
diclofenac which was 80% of that from an equimolar
dose of diclofenac sodium (100%). An ethyl moiety (20b)
reduced the apparent relative bioavailability slightly
(80% vs 66% relative to diclofenac sodium).
The extent of in vitro hydrolysis of diclofenac-
morpholine prodrugs incubated in plasma has been
reported to reflect the carbon chain length between the
ester and the tertiary amine function.5 Specifically,
diclofenac-morpholine esters with an even number of
carbons in the chain yielded diclofenac more efficiently
than did prodrugs with an odd number of carbons.5
These data led us to investigate the potential influence
of chain length variations of the alkyl spacer in our
S-NO-diclofenac esters on relative diclofenac bioavail-
ability in the mouse. Varying the chain length of the
alkyl spacer in the series 21a -21d altered relative
diclofenac bioavailability over an approximately 2-fold
range when the compounds were administered in 0.5%
methocel-50% PEG (Table 1). Increasing the chain
length from ethyl (21a ) to propyl (21b) increased the
In summary, oral administration of all the novel
S-NO-diclofenac esters delivered diclofenac in the plasma
to varying degrees, likely depending upon some salient
structural features identified above, particularly chain
length of the alkyl spacer and substituent at the tertiary
amine group. In all cases, however, plasma diclofenac
peaked within 30 min following oral adminstration, as
would be expected if the S-NO-diclofenac esters were
NSAID prodrugs in vivo.
An a lgesic Activity. The phenylbenzoquinone (PBQ)-
induced writhing model in the mouse36 was used to
assess the analgesic activity of diclofenac and selected
S-NO-diclofenac derivatives (20a -20c, 21a , 21b) (Table
2). At an oral dose of 100 µmol/kg, all of the S-NO-
diclofenac compounds evidenced analgesic activity, and
diclofenac sodium itself at this dose inhibited writhing
by 84%. The order of potency is 20b g diclofenac sodium
> 21a > 20c ) 21b > 20a . These data indicate that
our synthetic diclofenac analogues are analgesics.
An tiin fla m m a tor y Activity. Injection of carrag-
eenan into the rat foot pad causes local inflammation
and edema, evident by an increase in paw volume of
1.2 mL at 3 h postinjection.28 An NSAID administered
orally just prior to the carrageenan injection acts as an
antiinflammatory agent and attenuates the increase in
paw volume.37 A relatively high equimolar oral dose (100
µmol/kg) of either diclofenac sodium or S-NO-diclofenac
derivatives (21a , 21b) elicited a comparable (≈58%)
suppression of carrageenan-induced paw inflammation
(Table 2). These results indicate that after oral admin-
istration, esters 21a and 21b are antiinflammatory
agents with similar efficacy to the parent NSAID,
diclofenac.
Ga str ic Lesion Test. To characterize the GI safety
profiles of our S-NO-diclofenac compounds, experiments
were performed with fasted male rats to measure the
extent of NSAID-induced stomach lesions.38,39 Diclofen-
ac sodium at a high oral dose (100 µmol/kg) caused
significant stomach lesions with a lesion score of >40
mm, whereas equimolar oral doses of 20a -20c, 21a ,
and 21b caused negligible lesions (Table 3, entries 3-5,
9, 10). Likewise, at lower doses (5, 25, or 50 µmol/kg),

4010 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Ta ble 2. Analgesic and Antiinflammatory Effects of S-NO-NSAIDs
Bandarage et al.
analgesic effect in PBQ-induced
writhing test (mice)
antiinflammatory effect in carrageenan-induced
paw edema test (rat)
compounda
% inhib of writhing
activity ratio vs diclofenacb
% inhib of paw volume increase
activity ratio vs diclofenacb
vehiclec
diclofenacc
20a c
0 (27)
84e (18)
32e (9)
95e (9)
58e (9)
0 (27)
1.0
0.4
1.1
0.7
NDf
ND
ND
0 (20)
53e (10)
55e (15)
58e (5)
20bc
20cc
vehicled
diclofenacd
21a d
84e (26)
70e (27)
53e (9)
1.0
0.8
0.6
1.0
1.0
1.1
21bd
a
b
All compounds administered at an oral dose of 100 µmol/kg. Activity ratio ) (% inhibition with the compound at 100 µmol/kg, po)/(%
inhibition with diclofenac at 100 µmol/kg, po). c Vehicle: 0.5% methocel/5% PEG-400. Vehicle: 0.5% methocel/50% PEG-400. e Difference
d
statistically significant from respective vehicle (p < 0.05). All values are mean ( SEM (the number of animals in parentheses). f ND, not
determined.
Ta ble 3. Gastric Toxicity Studies in Rats
Ch a r t 2. Sulfhydryls 23 and 24 and Flurbiprofen
Nitrooxybutyl Ester 25
entry
compounda
vehicleb
gastric lesion score (mm)
1
2
3
4
5
6
7
8
9
10
11
12
13
0
diclofenac sodiumb
20a b (SNO)
20bb (SNO)
20cb (SNO)
vehiclec
41.3 ( 5.4
0.7 ( 0.4
0.4 ( 0.2
2.0 ( 0.8
0
54.3 ( 3.9
0.8 ( 0.5
1.1 ( 0.9
0.7 ( 0.4
6.7 ( 1.1
10.2 ( 2.8
28.3 ( 4.2
diclofenac sodiumc
20cc (SNO)
21a c (SNO)
21bc (SNO)
23c (SH)
16c (SH)
24c (SH)
a
All compounds administered at an oral dose of 100 µmol/kg
to 5-10 rats/group. Vehicle: 0.5% methocel/5% PEG-400. c Ve-
hicle: 0.5% methocel/50% PEG-400.
b
diclofenac still produced appreciable stomach lesions,
whereas our S-NO-diclofenac esters were devoid of
apparent GI toxicity (data not shown).
tives. To this intent, GI lesion scores were compared in
rats 18 h after a single oral dose (100 µmol/kg) of either
diclofenac (Table 3, entry 7), the S-NO derivatives 20c,
21a , and 21b (Table 3, entries 8-10), or their respective
des-NO analogues 23, 16, and 24 (Chart 2) (Table 3,
entries 11-13). All of these sulfhydryl (SH) derivatives
induced fewer gastric lesions than did diclofenac but
more gastric lesions than each respective nitrosothiol
derivative. The limited, NO-independent gastroprotec-
tion afforded by the sufhydryl derivatives may reflect a
“prodrug effect” (i.e., intact absorption) and/or GI cyto-
protection by the free nucleophilic thiol group. Sulfhy-
dryls with gastric-sparing properties have been identi-
fied.40
NO itself may support or promote several endogenous
GI defense mechanisms involving increased mucus and
bicarbonate secretion, increased mucosal blood flow, and
prevention of neutrophil adherence to the vascular
endothelium, all of which are believed capable of
minimizing chemically induced gastric injury.21-26 Very
recently, it has been suggested that prevention of gastric
damage by a nitrooxybutyl ester of aspirin may reflect,
at least in part, an NO-mediated suppression of the
NSAID-induced upregulation of gastric cysteine pro-
teases in the caspase cascade, which modulates pro-
grammed cell death (apoptosis).41
The mechanism of gastroprotection by NSAIDs func-
tionalized with NO-donating groups is poorly defined
but likely complex and multifactorial, given that several
aspects of GI physiology are influenced or regulated by
NO.21-25 The limited published data in this regard come
mainly from studies on NSAID nitrooxybutyl esters.
Masking the carboxylic acid group of diclofenac as an
ester prodrug may itself attenuate the topical irritation
component of NSAID-induced gastropathy and elicit an
acute GI-sparing effect by reducing the local intracel-
lular accumulation of irritant NSAID in stomach tissue
as the free acid.5,14,15 However, a flurbiprofen nitrooxy-
butyl ester 25 (Chart 2) was gastroprotective even after
systemic administration as compared to the parent
NSAID itself, suggesting that the GI-sparing property
of the NO-releasing derivative was not completely
dependent upon a tissue-contact-related mechanism.21
This observation further suggests that the flurbiprofen
nitrooxybutyl ester containing organic nitrate was not
acting solely as a “prodrug.” Given evidence that NSAIDs,
whether derivatized to NO-donating nitrooxybutyl es-
ters or not, comparably inhibit PG synthesis in the
gastric mucosa,21 current focus is on the potential for
bioactive NO (or NO-derived metabolites) to account for
the acute GI-sparing effects of NO-releasing NSAIDs.
Con clu sion s
An attempt was made to gain some pharmacological
insight into the role of the NO moiety in the gastropro-
tection afforded by the nitrosothiol diclofenac deriva-
A series of novel diclofenac ester derivatives bearing
tertiary nitrosothiols has been synthesized. These com-
pounds are NO-releasing diclofenac prodrugs that yield

Nitrosothiol Esters of Diclofenac
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4011
H); 13C NMR (CDCl3) δ 28.3, 46.3, 73.29; HRMS m/z calcd for
C4H11OS (M + 1) 107.0530, found 107.0525. Compounds 3b
and 3c were synthesized in a manner similar to the synthesis
of 3a . Spectroscopic data are reported elsewhere.34,35
2-Meth yl-1-h yd r oxyp r op a n e-2-n itr osoth iol (4a ). A solu-
tion of 3a (4.40 g, 41.5 mmol) in CH2Cl2 (50 mL) was added to
t-BuONO (90% tech, 5.50 mL, 41.5 mmol). The resulting green
reaction mixture was stirred for 10 min at room temperature
and then evaporated under reduced pressure at 40 °C to
furnish 4a (4.60 g, 82%) as a dark green oil: 1H NMR (CDCl3)
δ 1.90 (s, 6 H), 1.95 (brs, 1H), 4.17 (s, 2 H); 13C NMR (CDCl3)
δ 25.1, 57.7, 70.5; HRMS m/z calcd for C4H10NO2S (M + 1)
136.0432, found 136.0435.
2-E t h yl-2-(n it r osot h io)b u t a n -1-ol (4b ). Compound 4b
was synthesized in a manner similar to the synthesis of 4a
from 3b.34 4b was obtained as a green oil: 1H NMR (CDCl3) δ
1.90 (t, J ) 7.4 Hz, 6 H), 2.29 (m, 4 H), 2.16 (brs, 1H), 4.34 (s,
4 H); 13C NMR δ 7.9, 26.8, 66.5, 67.0.
[(Nitr osoth io)cycloh exyl]m eth a n -1-ol (4c). Compound
4c was synthesized in a manner similar to the synthesis of
4a from 3c.35 4c was obtained as a green oil: 1H NMR (CDCl3)
δ 1.45-1.85 (m, 5 H), 2.05-2.13 (m, 3 H), 2.45-2.55 (m, 2 H),
4.26 (s, 2 H); 13C NMR δ 21.9 (2C), 25.6, 33.2 (2 C), 63.6, 70.8.
2-{([({[(2-H yd r oxyet h yl)a m in o]m et h yl}cycloh exyl)-
d isu lfa n yl]m eth yl)a m in o}eth a n -1-ol (5a ). A mixture of 2c
(10 g, 35 mmol), ethanolamine (4.26 g, 69.8 mmol) and MgSO4
(10 g) in dry CHCl3 (100 mL) was heated under reflux for 8 h.
The solid was removed by filtration, and the solvent was
evaporated under reduced pressure to obtain a viscous yellow
liquid. The crude product was dissolved in MeOH (125 mL),
and NaBH4 (3.3 g, 87.3 mmol) was added in small portions
over 10 min. The resulting solution was stirred at room
temperature for 1 h. The solvent was evaporated, and the
crude material was dissolved in a mixture of water (200 mL)
and EtOAc (100 mL). The organic layer was recovered, and
the aqueous layer was extracted with EtOAc (100 mL). The
combined organic layers were dried over Na2SO4 and evapo-
rated under reduced pressure to give a colorless viscous liquid.
This product was further purified as follows: The liquid 5a
was dissolved in Et2O (50 mL), and HCl in Et2O was added
dropwise to form a white salt. The salt was washed with Et2O
(2 × 50 mL), and the solid was dissolved in water (100 mL).
The aqueous solution was washed with Et2O (100 mL), and
the Et2O layer was discarded. The aqueous layer was made
basic with 15% NH4OH (10 mL) to form a white suspension
which was extracted with EtOAc (2 × 50 mL). The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure to give 5a (12.2 g, 93%) as a viscous liquid: 1H NMR
(CDCl3) δ 1.20-1.95 (m, 20 H), 2.66 (s, 4 H), 2.78 (t, J ) 5.2
Hz, 4 H), 3.61 (t, J ) 5.2 Hz, 4 H); 13C NMR δ 22.7, 25.8, 34.3,
51.5, 54.7, 60.6, 68.2. Anal. (HCl salt) (C18H38 N2O2S2Cl2) C,
H, N, S, Cl.
3-{([({[(3-Hyd r oxyp r op yl)a m in o]m et h yl}cycloh exyl)-
d isu lfa n yl]m eth yl)a m in o}p r op a n -1-ol (5b). Compound 5b
was synthesized in a manner similar to the synthesis of 5a ,
using propanolamine (10.5 g, 140 mmol), 2c (20 g, 70 mmol)
and NaBH4 (5.3 g, 139.6 mmol) to give 5b (27.5 g, 97%) as an
oil: 1H NMR (CDCl3) δ 1.25-1.80 (m, 24 H), 2.67 (s, 4 H), 2.91
(t, J ) 5.5 Hz, 4 H), 3.84 (t, J ) 5.3 Hz, 4 H); 13C NMR δ 22.2,
25.8, 30.4, 34.3, 50.6, 54.4, 57.1, 64.7; MS m/z 405 (M + 1).
2-{([({[(2-H y d r o x y e t h y l)m e t h y la m in o ]m e t h y l}c y -
cloh exyl)d isu lfa n yl]m eth yl)a m in o}eth a n -1-ol (6a ). A mix-
ture of 5a (12.2 g, 32.4 mmol), 38% formaldehyde (35 mL) and
methanol (70 mL) was stirred at room temperature under N2
for 12 h. The solution was diluted with water (100 mL) and
extracted with EtOAC (3 × 100 mL). The organic layer was
dried over Na2SO4 and evaporated under reduced pressure to
give an oil. The crude product was dissolved in MeOH (120
mL), and NaBH4 (3.05 g, 80.6 mmol) was added in small
portions over 10 min. The resulting solution was stirred at
room temperature for 1 h. Solvent was evaporated, and the
crude material was dissolved in a mixture of water (200 mL)
and EtOAc (100 mL). The organic layer was separated, and
the aqueous layer was extracted with EtOAc (2 × 100 mL).
the parent NSAID in vivo and have good antiinflam-
matory and analgesic activities in rodent models. At
equimolar concentrations, the S-NO-diclofenac deriva-
tives were gastric-sparing relative to diclofenac sodium
in rats. The relative diclofenac bioavailability of the
S-NO-diclofenac esters in vivo varied among the struc-
tures examined, with the carbon chain length of the
alkyl tether a particular influence. Our data suggest
that S-NO prodrugs may represent a safer chemical
alternative to diclofenac for the treatment of inflam-
matory diseases and pain. The molecular mechanism
of gastroprotection by the S-NO-diclofenac derivatives
likely involves the action of NO and is multifactorial
and thus awaits elucidation. Ultimately, any potential
advantage of S-NO-NSAIDs over standard NSAIDs or
COX-2 inhibitors would need to be demonstrated in the
clinic.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents and dry solvents were
obtained from commercial sources and used without further
purification. Flash chromatography42 was performed on silica
1
gel (Merck, 230-400 mesh). H and 13C NMR were recorded
on a Bruker AMX-300 instrument. HPLC was performed with
a Beckman liquid chromatograph equipped with a variable-
wavelength spectrophotometric detector and System Gold
software. The purity of some testing compounds was checked
by HPLC with two diverse solvent systems: system A - linear
gradient solvent system of 0.1% TFA in water/CH3CN from
50/50 to 5/95 in 20 min and flow rate 1 mL/min; system B -
0.1% TFA in water/MeOH from 50/50 to 5/95 in 20 min and
flow rate 1 mL/min (λ ) 254 nm on a µ-Bondapak C18 (3.9 ×
150 mm) column). Low-resolution mass spectra were recorded
on a Perkin-Elmer API-150EX spectrometer with atmospheric
pressure turbo ion spray. High-resolution mass spectra (HRMS)
were obtained from the Auburn Mass Spectra Center, Auburn,
AL. Elemental analyses were obtained from Robertson Microlit
Laboratories, Madison, NJ .
1-[(F or m ylcycloh e xyl)d isu lfa n yl]cycloh e xa n e ca r -
ba ld eh yd e (2c). (a ) To a stirred solution of cyclohexanecar-
boxaldehyde (100 g, 89 mmol) in CCl4 (100 mL) was added
sulfur monochloride (36.4 mL, 91 mmol) dropwise at 50 °C.
After a short lag (15 min), evolution of HCl gas began. After
HCl evolution ceased, the mixture was stirred at 55 °C for 1
h, then cooled to room temperature. The CCl4 was evaporated
to produce a yellow solid, and the solid was placed in a sintered
glass funnel and washed with hexane (3 × 100 mL) to give 2c
(114 g, 89%) as a white solid: mp 85-88 °C.
(b) Sulfur monochloride (2 mL, 25 mmol) and cyclohexan-
ecarboxaldehyde (6 mL, 49.6 mmol) were dissolved in tert-butyl
methyl ether (30 mL). The flask was placed in an oil bath at
55 °C, and the solution was stirred for 2 h. The flask was
removed from the oil bath and set at room temperature for 3
h, until crystals formed. The flask, containing a layer of
crystals, was placed in the freezer overnight, and the mother
liquor was then decanted off. The crystals were washed with
several small portions of hexane (15 mL) until the hexane wash
did not turn yellow. The crystals were then dried in vacuo to
1
give 2c (6.3 g, 89%) as a white solid: mp 85-88 °C; H NMR
(CDCl3) δ 1.24-1.33 (m, 6 H), 1.42-1.46 (m, 6 H), 1.62-1.69
(m, 4 H), 1.94-1.99 (m, 4 H), 8.94 (s, 2 H); 13C NMR δ 23.0,
25.1, 30.3, 60.8, 194.3. Anal. (C14H22O2S2) C, H, N, S.
2-Meth yl-1-h yd r oxyp r op a n e-2-th iol (3a ). To a stirred
solution of 2a (15.0 g, 85.7 mmol) in THF (100 mL) was added
dropwise 1 M LiAlH4 in THF (85.7 mL, 85.7 mmol), and the
reaction mixture was stirred at room temperature under N2
for 1 h. The mixture was poured onto ice, treated with 3 N
HCl (150 mL), and extracted with EtOAc (2 × 200 mL). The
combined organic layers were dried over Na2SO4, filtered, and
evaporated to give 3a (12.8 g, 71%) as a colorless oil: 1H NMR
(CDCl3) δ 1.36 (s, 6 H), 1.63 (s, 1 H), 2.25 (brs, 1 H), 3.44 (s, 2

4012 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Bandarage et al.
The combined organic layers were dried over Na2SO4 and
evaporated under reduced pressure to give 6a (11.6 g, 88.5%)
as a colorless viscous liquid. The product solidified on stand-
SO4‚10H2O (5 g). A solution of 10% MeOH in CH2Cl2 (50 mL)
was added, and the precipitated solid was removed by filtra-
tion. The solid was washed with 10% MeOH in CH2Cl2 (2 ×
50 mL). The combined filtrate was concentrated under reduced
pressure and dried in vacuo for 3 h to give 7b (4.9 g, 97%) as
a viscous liquid: 1H NMR (CDCl3) δ 1.11-1.78 (m, 12 H), 2.33
(s, 3 H), 2.46 (s, 2 H), 2.69 (t, J ) 6.3 Hz, 2 H), 3.75 (t, J ) 5.1
Hz, 2 H); 13C NMR δ 22.1, 25.8, 37.8, 44.5, 51.5, 60.4, 63.5,
73.34; MS m/z 218 (M + 1).
(b) Powdered zinc (10 g, 153 mmol) was suspended in a
solution of 6b (5.3 g, 12.3 mmol) in glacial HOAc (40 mL). The
resulting slurry was stirred at room temperature under N2
for 80 min. The inorganic solid was removed by filtration and
washed with HOAc (30 mL). The filtrate was poured onto
crushed ice (∼200 g) and made basic with concentrated NH4-
OH (90 mL). The white precipitate was extracted with EtOAc
(3 × 150 mL) and washed with brine (2 × 100 mL), then water
(2 × 100 mL). The organic layer was dried over Na2SO4 and
evaporated under reduced pressure to give 7b (4.5 g, 84.3%)
as an oil.
7b‚HCl. A saturated solution of HCl in dry Et2O was added
dropwise to a solution of 7b (4.9 g, free base) in dry Et2O (50
mL) to form an insoluble gummy precipitate. The solvent was
decanted, and the residue was washed with Et2O (2 × 50 mL)
and dried in vacuo for 12 h to give 7b‚HCl (4.8 g) as a gummy
semisolid.
2-{Met h yl[((n it r osot h io)cycloh exyl)m et h yl]a m in o}-
eth a n -1-ol (8a ). Nitrosylated tether 8a was synthesized as
similar to 8b using 7a (1.05 g, 5.2 mmol) MeOH (7 mL),
concentrated HCl (0.5 mL) and t-BuONO (0.76 g, 6.5 mL). The
crude product was flash chromatographed on silica gel eluting
with EtOAc/hexane (5:1) afforded 8a (0.2 g, 17%): 1H NMR
(CDCl3) δ 1.42-1.75 (m, 6 H), 2.02-2.11 (m, 2 H), 2.33 (s, 3
H), 2.48-2.56 (m, 2 H), 2.7 (t, J ) 6 Hz, 2 H), 3.23 (s, 2 H),
3.53 (t, J ) 6 Hz, 2 H); 13C NMR δ 22.14, 25.6, 34.7, 44.1,
58.9, 61.7, 64.2, 69.3; MS m/z 233 (M + 1), 202 (M - 30, -NO).
3-{Met h yl[((n it r osot h io)cycloh exyl)m et h yl]a m in o}-
p r op a n -1-ol (8b). t-BuONO (3.03 mL, 25.9 mmol) was added
to a stirred solution of 7b (4.5 g, 20.7 mmol) and concentrated
HCl (1.5 mL) in MeOH (30 mL) under N2 at room temperature.
The green solution was stirred for 18 min and poured onto
crushed ice (∼200 g). The mixture was made basic with 10%
Na2CO3 (35 mL) and extracted with EtOAc (2 × 120 mL). The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure to give 8b as a green viscous oil (4.8 g,
94%): TLC Rf ) 0.23 (hexane/EtOAc, 1:1; green); 1H NMR
(CDCl3) δ 1.45-1.80 (m, 8 H), 2.09 (m, 2 H), 2.38 (s, 3 H), 2.53
(d, J ) 14 Hz, 2 H), 2.74 (t, J ) 5.8 Hz, 2 H), 3.24 (s, 2 H),
3.77 (t, J ) 5.2 Hz, 2 H); 13C NMR δ 21.9, 25.3, 28.7, 34.3,
44.2, 60.1, 63.1, 63.7, 69.7.
4-{Met h yl[((n it r osot h io)cycloh exyl)m et h yl]a m in o}-
bu ta n -1-ol (8c). t-BuONO (3 mL, 25.97 mmol) was added to
a stirred solution of 7c (5.0 g, 21.6 mmol) and concentrated
HCl (2 mL) in EtOH (25 mL) under N2 at room temperature.
The resulting green solution was stirred for 15 min and poured
onto crushed ice (∼100 g). The mixture was made basic with
10% Na2CO3 (20 mL) and extracted with EtOAc (2 × 50 mL).
The organic layer was dried over Na2SO4 and concentrated.
(During the workup, some 8c decomposed.) Flash chromatog-
raphy of the crude material on silica gel eluting with EtOAc/
hexane (1:1) afforded 8c (0.89 g, 16%) as a green viscous oil:
1H NMR (CDCl3) δ 1.33-1.70 (m, 10 H), 2.01-2.12 (m, 2 H),
2.26 (s, 3 H), 2.26-2.50 (m, 4 H), 3.12 (s, 2 H), 3.53 (t, J ) 5.7,
2 H); 13C NMR δ 22.2, 25.1, 25.5, 31.3, 34.5, 44.7, 60.9, 62.9,
64.1, 69.5.
1
ing: mp 65-70 °C; H NMR (CDCl3) δ 1.23-1.65 (m, 20 H),
2.34 (s, 6 H), 2.59 (s, 4 H), 2.65 (t, J ) 5.3 Hz, 4 H), 3.60 (t, J
) 5.3 Hz, 4 H); 13C NMR δ 22.3, 25.6, 33.4, 44.2, 55.9, 59.2,
61.7, 67.4. Anal. (C20H40N2O2S2) C, H, N, S.
2-{Meth yl[(su lfa n ylcycloh exyl)m eth yl]a m in o}eth a n -1-
ol (7a ). To a stirred solution of 6a (11.6 g, 28.7 mmol) in dry
THF (100 mL) was added 1 M LiAlH4 in THF (43 mL, 43
mmol) dropwise at room temperature under N2. The resulting
clear solution was stirred at room temperature for 1 h. The
excess LiAlH4 was destroyed by careful addition of solid Na2-
SO4‚10H2O (2 g). EtOAc (100 mL) was added, and the
precipitate was removed by filtration. The precipitate was
washed with 10% MeOH in CH2Cl2 (2 × 50 mL), and the
combined filtrate was evaporated under reduced pressure to
give 7a (9.2 g, 79%) as a viscous liquid: 1H NMR (CDCl3) δ
1.05-1.25 (m, 2 H), 1.45-1.85 (m, 8 H), 2.34 (s, 3 H), 2.50 (s,
2 H), 2.64 (t, J ) 5.4 Hz, 2 H), 3.56 (t, J ) 5.4 Hz, 2 H); 13C
NMR δ 22.2, 25.9, 38.2, 44.8, 52.2, 59.4, 62.1, 72.2; HRMS m/z
calcd for C10H20NOS (M+ - 1) 202.1265, found 202.1261.
5-{Meth yl[(su lfa n ylcycloh exyl)m eth yl]a m in o}p en ta n -
1-ol (7d ). Compound 7d was synthesized in a manner similar
to that of 7a using disulfide 6d (7.4 g, 15.14 mmol), 1 M LiAlH4
(30 mL, 30 mmol) and THF (50 mL). Workup similar to that
of 7a gave 7d (6.1 g, 82%) as a viscous liquid: 1H NMR (CDCl3)
δ 0.95-1.60 (m, 16 H), 2.16 (s, 3 H), 2.26 (s, 2 H), 2.32 (t, J )
7.4 Hz, 2 H), 3.45 (t, J ) 6.6 Hz, 2 H); 13C NMR (CDCl3) δ
22.4, 23.4, 25.9, 27.4, 32.7, 37.9, 44.9, 52.3, 60.6, 62.7, 72.1;
MS m/z 246 (M + 1).
Di(1,3-oxa za p er h yd r oin -3-ylm eth yl)cycloh exyl Disu l-
fid e (9). Propanolamine (15.7 g, 209.5 mmol) in MeOH (50
mL) was added to a stirred suspension of disulfide 2c (30 g,
104.7 mmol) in MeOH (150 mL) at room temperature. The
disulfide was gradually dissolved, forming a light brown
solution which was stirred at room temperature for 3 h. NaBH4
(4 g, 104.73 mmol) was added in small portions over 10 min,
and the reaction mixture was stirred at room temperature for
1 h. Formaldehyde solution (38%, 120 mL) was added, and
the resulting cloudy solution was stirred at room temperature
for 1 h. The flask was placed in a freezer (-20 °C) for 12 h
after which time the clear supernatant was decanted. The
gummy mass remaining in the flask was shaken well with
MeOH (50 mL) to produce a white solid which was filtered
and dried in vacuo (6 h) to give 9 (34 g, 75.7%) as a white
powder. Compound 9 (24 g, 56 mmol) was dissolved in boiling
MeOH (150 mL), and the resulting clear solution was left in a
freezer for 12 h. The solid was filtered and dried in vacuo to
give 9 (21 g) as a white crystalline solid: mp 65-66 °C; 1H
NMR (CDCl3) δ 1.20-1.80 (m, 24 H), 2.92 (s, 4 H), 3.06 (t, J )
5.3 Hz, 4 H), 3.93 (t, J ) 5.3 Hz, 4 H), 4.39 (s, 4 H); 13C δ 22.1,
22.7, 25.6, 32.3, 52.6, 55.8, 60.9, 67.7, 86. Anal. (C22H40N2O2S2)
C, H, N, S.
3-{([({[(3-H y d r o x y p r o p y l)m e t h y la m in o ]m e t h y l}-
cycloh exyl)d isu lfa n yl]m eth yl)a m in o}eth a n -1-ol (6b). To
a stirred solution of 9 (4.6 g, 10.7 mmol) in glacial HOAc (30
mL) was added NaBH4 (1 g, 26.3 mmol) in small portions over
5 min at room temperature, and the resulting solution was
stirred for 4 h. The solution was poured onto crushed ice (10
g), made basic with concentrated NH4OH, and extracted with
EtOAc (3 × 50 mL). The combined organic layers were dried
over Na2SO4 and evaporated to give 6b (4.6 g, 99%) as a pale
yellow oil: 1H NMR (CDCl3) δ 1.20-1.65 (m, 24 H), 2.29 (s, 6
H), 2.48 (s, 4 H), 2.66 (t, J ) 5.8 Hz, 4 H), 3.76 (t, J ) 5.2 Hz,
4 H); 13C NMR δ 22.4, 25.6, 28.7, 33.4, 44.4, 55.7, 60.8, 64.0,
68.2; MS m/z 433 (M + 1), 216; HRMS m/z calcd for C22H44N2-
O2S2 (M + 1) 433.2922, found 433.2910.
5-{Met h yl[((n it r osot h io)cycloh exyl)m et h yl]a m in o}-
p en ta n -1-ol (8d ). t-BuONO (3 mL, 25.97 mmol) was added
to a stirred solution of 7d (4.00 g, 14.93 mmol) and concen-
trated HCl (2 mL) in CH2Cl2 (20 mL) under N2 at room
temperature. The resulting green solution was stirred for 15
min and poured onto crushed ice (∼100 g). The mixture was
made basic with 10% Na2CO3 (20 mL) and extracted with
EtOAC (2 × 50 mL). The organic layer was dried over Na2-
SO4 and evaporated under reduced pressure. (During the
3-{Meth yl[(su lfa n ylcycloh exyl)m eth yl]a m in o}p r op a n -
1-ol (7b). (a ) To a stirred solution of 1 M LiAlH4 in THF (18
mL, 18 mmol) was added 9 (5.00 g, 11.66 mmol) in THF (25
mL) dropwise at room temperature under N2. The resulting
clear solution was stirred at room temperature for 3 h, and
the excess LiAlH4 was destroyed by careful addition of Na2-

Nitrosothiol Esters of Diclofenac
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4013
workup, some of the desired product decomposed.) Flash
chromatography of the crude material on silica gel eluting with
EtOAc/hexane (1:1) afforded 8d (0.8 g, 14%) as a green oil:
1H NMR (CDCl3) δ 1.1-1.8 (m, 12 H), 2.01-2.19 (m, 2 H), 2.25
(s, 3 H), 2.25-2.45 (m, 4 H), 3.05 (s, 2 H), 3.54 (t, J ) 6.6, 2
H); 13C NMR δ 22.3, 23.3, 25.5, 27.2, 32.6, 34.3, 44.9, 60.4,
62.8, 64.5, 68.7; MS m/z 275 (M + 1).
onto crushed ice (100 g). The mixture was made basic with
10% Na2CO3 (20 mL) and extracted with EtOAc (2 × 50 mL).
The organic layer was dried over Na2SO4 and concentrated.
Flash chromatography of the crude material on silica gel
eluting with EtOAc/hexane (1:2) afforded 15 (1.3 g, 32.6%) as
a green viscous oil: 1H NMR (CDCl3) δ 0.91 (t, J ) 7.1, 3 H),
1.30-1.66 (m, 8 H), 1.98-2.07 (m, 2 H), 2.42-2.53 (m, 2 H),
2.61 (q, J ) 7.1 Hz, 2 H), 2.71 (t, J ) 6.1 Hz, 2 H), 3.13 (s, 2
H), 3.68 (t, J ) 5.5 Hz, 2 H); 13C NMR δ 10.4, 22.2, 25.4, 28.4,
34.5, 48.2, 55.5, 63.4, 63.8, 66.3.
3-{Ben zyl[(su lfa n ylcycloh exyl)m eth yl]a m in o}p r op a n -
1-ol (11). Solid K2CO3 (10 g) was added to a stirred solution
of disulfide 5b (6.5 g, 16.06 mmol) and benzyl bromide (3.8
mL, 32.12 mmol) in acetone (75 mL). The solution was heated
at 60 °C for 12 h. The solid was filtered, and the filtrate was
concentrated. The crude product was flash chromatographed
on silica gel eluting with EtOAc/Hex (1:1) to afford disulfide
10 (8.6 g, 91.6%) as a viscous oil. Compound 10 (8.00 g, 13.67
mmol) was dissolved in THF (50 mL), and 1 M LiAlH4 in THF
(26 mL, 26 mmol) was added dropwise at 0 °C. The mixture
was then heated at 60 °C for 18 h and then the reaction
mixture was cooled to room temperature. Solid Na2SO4‚10H2O
(∼10 g) was added in small portions over 5 min to form a white
precipitate. A solution of MeOH/CH2Cl2 (1:10, 100 mL) was
added, and the solid was removed by filtration. The solid was
washed with MeOH/CH2Cl2 (1:10, 100 mL). The filtrate was
concentrated and dried in vacuo to give 11 (5.6 g, 70%) as an
oil: 1H NMR (CDCl3) δ 1.00-1.80 (m, 8 H), 2.57 (s, 2 H), 2. 70
(t, J ) 6.5 Hz, 2H), 3.68 (s, 2 H), 3.41-3.71 (m, 2 H), 7.15-
7.36 (m, 5 H); 13C NMR δ 22.2, 25.8, 29.1, 36.0, 38.2, 54.9,
60.1, 62.5, 68.9, 127.2, 128.3, 129.3, 138.6; MS m/z 294 (M +
1); HRMS m/z calcd for C17H26NOS (M - 1) 292.1734, found
292.1730.
2-Meth yl-2-(n itr osoth io)pr opyl 2-{2-[(2,6-Dich lor oph en -
yl)a m in o]p h en yl}a ceta te (20a ). A solution of diclofenac
(8.30 g, 28.0 mmol), DCC (5.80 g, 28.0 mmol), and 4a (3.80 g,
28.0 mmol) in CH2Cl2 (100 mL) was stirred at room temper-
ature for 40 min. The precipitate was removed by filtration,
and the filtrate was concentrated. The crude material was
purified by flash chromatography on silica gel eluting with
EtOAc/hexane (1:9) to give a green oil which was dissolved in
EtOAc/pentane (1:5) and left in a freezer (-20 °C) for 12 h to
produce 20a (8.2 g, 71%) as red rods: TLC Rf ) 0.62 (EtOAc/
1
hexane, 1:9); mp 48 °C; H NMR (CDCl3) δ 1.89 (s, 6 H), 3.84
(s, 2 H), 4.72 (s, 2 H), 6.54-7.36 (m, 8 H); 13C NMR δ 25.7,
38.2, 54.7, 71.4, 118.3, 122.1, 123.9, 124.0, 128.0, 129.3, 137.7,
142.6, 171.9. Anal. (C18H18N2O3SCl2) C, H, N, S, Cl.
2-Eth yl-2-(n itr osoth io)bu tyl 2-{2-[(2,6-Dich lor oph en yl)-
a m in o]p h en yl}a ceta te (20b). DCC (2.43 g, 21.46 mmol) in
CH2Cl2 (25 mL) was added dropwise to a stirred solution of
4b (1.75 g, 10.72 mmol), diclofenac (3.17 g, 10.72 mmol), and
DMAP (0.10 g) in dry CH2Cl2 (25 mL) at 0 °C over 15 min.
The suspension was then stirred at 0 °C for 30 min. The solid
was removed by filtration and washed with CH2Cl2 (25 mL).
The filtrate was concentrated under reduced pressure at 40
°C and chromatographed on silica gel eluting with EtOAc/
hexane (1:10) to afford 20 b (4.3 g, 91%) as a green oil which
crystallized on standing in a freezer (-20 °C) for 12 h: mp
50-51 °C; TLC Rf ) 0.80 (hexane/EtOAc, 9:1); 1H NMR
(CDCl3) δ 0.88 (t, J ) 7.5, 6 H), 2.04-2.24 (m, 4 H), 3.68 (s, 2
H), 4.72 (m, 2 H), 6.41 (d, J ) 7.9 Hz, 1 H), 6.66 (s, 1 H), 6.79-
7.12 (d, J ) 7.4 Hz, 1 H), 7.20 (d, J ) 8.1 Hz, 2 H); 13C NMR
δ 8.1, 27.7, 38.3, 63.0, 68.1, 118.4, 122.1, 124.0, 124.1, 128.1,
128.8, 129.4, 130.8, 137.7, 142.6,172.1; MS m/z 441 (M + 1).
Anal. (C20H22N2O3SCl2) C, H, N, S, Cl.
3-({[(Nit r osot h io)cycloh exyl]m et h yl}b en zyla m in o)-
p r op a n -1-ol (12). t-BuONO (2.8 mL, 21.3 mmol) was added
to a stirred solution of 11 (5.0 g, 20.7 mmol) and concentrated
HCl (2 mL) in MeOH (30 mL) under N2 at room temperature.
The workup similar to 8b gave crude material which was
chromatographed on silica gel eluting with EtOAc/hexane (1:
2) to afford 12 (4.24 g, 76%): TLC Rf ) 0.53 (hexane/EtOAc,
1
2:1, green); H NMR (CDCl3) δ 1.40-1.80 (m, 6 H), 1.76 (t, J
) 6.1 Hz, 2 H), 2.01-2.09 (m, 2 H), 2.43-2.48 (m, 2 H), 2.74
(t, J ) 6.5 Hz, 2 H), 3.34 (s, 2 H), 3.69 (t, J ) 5.8 Hz, 2 H),
3.74 (s, 2 H), 7.15-7.40 (m, 5 H); 13C NMR 22.1, 25.5, 28.9,
34.6,, 55.1, 59.9, 62.6, 63.8, 65.8, 127.3, 128.3, 129.3, 138.2;
MS m/z 323 (M+ + 1).
[(Nitrosothio)cyclohexyl]methyl 2-{2-[(2,6-Dichlorophen-
yl)a m in o]p h en yl}a ceta te (20c). Compound 20c was syn-
thesized in a manner similar to the synthesis of 20b using 4c
(2 g, 11.4 mmol), DCC (2.8 g, 13.5 mmol), diclofenac (4.0 g,
13.5 mmol) and DMAP (0.3 g, 2.4 mmol) in CH2Cl2 (100 mL).
Chromatography of the crude product on silica gel eluting with
EtOAc/hexane (1:40) and followed by recrystallization from
EtOAc/MeOH (1:4) afforded 20c (4.0 g, 77%) as a green solid:
Aceta te 13. Acetic anhydride (7.8 g, 77.1 mmol) was added
to a stirred solution of disulfide 5b (5.2 g, 12.8 mmol), DMAP
(0.1 g) and Et3N (2 mL) in CH2Cl2 (50 mL). The solution was
heated at 60 °C for 4 h, stirred at room temperature for 12 h
and concentrated. The crude product was flash chromato-
graphed on silica gel eluting with MeOH/CH2Cl2 (1:19) to
afford disulfide 13 (5.3 g, 72%) as a viscous oil: 1H NMR
(CDCl3) δ 1.00-1.95 (m, 24 H), 2.30 (s, 6 H), 2.38 (s, 6 H),
3.42-3.52 (m, 8 H), 4.03 (t, J ) 5.9 Hz, 4 H); 13C NMR δ 20.8,
21.6, 22.1, 25.5, 26.4, 27.9, 33.8, 47.8, 52.7, 56.3, 61.7, 170.8,
171.8; MS m/z 573 (M + 1).
3-{Eth yl[(su lfa n ylcycloh exyl)m eth yl]a m in o}p r op a n -1-
ol (14). Compound 13 (5.2 g, 9.1 mmol) was dissolved in THF
(50 mL), and 1 M LiAlH4 in THF (18 mL, 18 mmol) was added
dropwise at 0 °C. The mixture was then heated at 60 °C for
18 h and then the reaction mixture was cooled to room
temperature. Solid Na2SO4‚10H2O (∼10 g) was added in small
portions over 5 min to form a white precipitate. A solution of
MeOH/CH2Cl2 (1:10, 100 mL) was added, and the solid was
removed by filtration. The solid was washed with MeOH/CH2-
Cl2 (1:10, 100 mL). The filtrate was concentrated and dried in
vacuo to give 11 (3.3 g, 78%) as an oil: 1H NMR (CDCl3) δ
0.96 (t, J ) 5.2 Hz, 2H), 1.19-1.80 (m, 13 H), 2.52 (s, 2 H), 2.
72 (t, J ) 5.8 Hz, 2 H), 2.62 (q, J ) 7.1 Hz, 2 H), 3.76 (t, J )
5.2 Hz, 2 H); 13C NMR δ 10.3, 22.4, 25.5, 28.4, 33.3, 48.2, 55.6,
55.7, 63.4, 64.9; MS m/z 232 (M + 1).
1
mp 61-63 °C; H NMR (CDCl3) δ 1.33-1.78 (m, 6 H), 2.02-
2.12 (m, 2 H), 2.40-2.51 (m, 2 H), 3.82 (s, 2 H), 4.80 (s, 2 H),
6.55 (d, J ) 8 Hz, 1 H), 6.79 (s, 1 H), 6.92-7.00 (m, 2 H), 7.10-
7.20 (m, 2 H), 7.34 (d, J ) 8 Hz, 2 H); 13C NMR δ 21.7, 25.3,
33.2, 38.4, 60.2, 71.3, 118.4, 122.1, 124.0, 124.1, 128.1, 128.8,
129.4, 130.8, 137.7, 142.7, 172.2. Anal. (C21H22N2O3SCl2) C,
H, N, S, Cl.
2-(Me t h yl[(n it r osot h ioc yc loh e xyl)m e t h yl]a m in o)-
et h yl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a cet a t e
(21a ). (a ) t-BuONO (0.46 g, 4.42 mmol) was added to a stirred
solution of 16.HCl (2.28 g, 4.42 mmol) in CH2Cl2 (50 mL) at
-78 °C. The cooling bath was removed, and the green solution
was stirred for 10 min and then concentrated under reduced
pressure to give a green foam. The green foam was dissolved
in EtOAc (25 mL), ice (∼5 g) was added, and the mixture was
first washed with 10% Na2CO3 (10 mL) and then with water
(25 mL). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to give an oil. Flash
chromatography of the crude product on silica gel eluting with
EtOAc/hexane (1:19) afforded 21a (1.92 g, 85.2%) as a green
viscous oil.
3-(E t h yl{[(n it r osot h io)cycloh e xyl]m e t h yl}a m in o)-
p r op a n -1-ol (15). t-BuONO (1.9 mL, 18.7 mmol) was added
to a stirred solution of the HCl salt of crude 14 (4.0 g, 14.9
mmol) in CH2Cl2 (25 mL) under N2 at room temperature. The
resulting green solution was stirred for 15 min and poured
(b) Alternatively, compound 21a was synthesized in a
manner similar to the synthesis of 21b using DCC (0.49 g,
2.38 mmol), 8a (0.46 g, 1.98 mmol), diclofenac (0.59 g, 2.00

4014 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Bandarage et al.
mmol), DMAP (0.025 g) and CH2Cl2 (15 mL). Chromatography
of the crude coupling product on silica gel eluting with EtOAc/
hexane (1:9) afforded 21a (1.0 g, 98%) as a green oil: TLC Rf
) 0.47 (Hex/EtOAc, 9:1, green); 1H NMR (CDCl3) δ 1.25-1.70
(m, 6 H), 2.05-2.14 (m, 2 H), 2.35-2.44 (m, 2 H), 2.37 (s, 3
H), 2.81 (t, J ) 5.9 Hz, 2 H), 3.19 (s, 2 H), 3.80 (s, 2 H), 4.21
(t, J ) 5.9 Hz, 2 H), 6.54 (d, J ) 8.0 Hz, 1 H), 6.90-6.99 (m,
3 H), 7.08-7.13 (m, 1 H), 7.20 (d, J ) 7.4 Hz, 1 H), 7.32 (d, J
) 8.0 Hz, 2 H); 13C NMR δ 22.2, 25.6, 34.0, 38.6, 45.0, 58.4,
63.0, 64.4, 69.0, 118.2, 122.0, 124.0, 124.2, 128.0, 128.8, 129.4,
130.9, 137.8, 142.7, 172.2; HPLC tR ) 4.6 min (system A, 98%).
J ) 6.7 Hz, 2 H), 6.49 (d, J ) 7.9 Hz, 1 H), 6.87-6.95 (m, 3
H), 7.04-7.09 (m, 1H), 7.18 (dd, J ) 1.1 and 7.4, 1 H), 7.28 (d,
J ) 8.0 Hz, 2 H); 13C NMR δ 22.3, 23.4, 25.6, 27.1, 28.5, 34.3,
38.7, 44.9, 60.3, 64.5, 65.3, 68.8, 118.2, 121.9, 123.9, 124.4,
127.9, 128.8, 129.4, 130.8, 137.8, 142.7, 172.4; MS m/z 552 (M
+ 1); HPLC tR ) 5.4 min (system A, 98.4%).
3-([(Nit r o sot h io c yc loh e xy l)m e t h yl]b e n zy la m in o )-
p r op yl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a ceta te
(22a ). Compound 22a was synthesized in a manner similar
to the synthesis of 21b using DCC (0.87 g, 4.23 mmol), 12 (0.82
g, 2.54 mmol), diclofenac (1.04 g, 3.53 mmol), DMAP (0.025 g)
and CH2Cl2 (50 mL). Chromatography of the crude product
on silica gel eluting with EtOAc/hexane (1:19) afforded 22a
(1.20 g, 80%) as a green oil: TLC Rf ) 0.58 (hexane/EtOAc
21a ‚HCl. A saturated solution of HCl gas in Et2O was added
dropwise to a solution of 21a (1.8 g) in dry Et2O (30 mL) to
form an insoluble green gum. The solvent was evaporated
under reduced pressure to give a green foam, which was
triturated with hexane (25 mL) to afford a green suspension.
The solvent was evaporated under reduced pressure, and the
product was dried in vacuo for 12 h to give 21a ‚HCl (1.86 g)
1
9:1); H NMR (CDCl3) δ 1.30-1.80 (m, 8 H), 2.01-2.12 (m, 2
H), 2.30-2.68 (m, 4 H), 3.26 (s, 2 H), 3.67 (s, 2 H), 3.69 (s, 2
H), 4.03 (t, J ) 6.3 Hz, 2 H), 6.50 (d, J ) 7.9 Hz, 1 H), 6.78-
6.96 (m, 3 H), 7.01-7.14 (m, 2 H), 7.16-731(m, 7H); 13C NMR
δ 22.3, 25.6, 25.9, 34.5, 38.6, 52.1, 60.7, 63.4, 64.5, 65.9, 118.2,
122.0, 124.1, 124.4, 127.2, 127.9, 128.3, 128.7, 128.9, 129.5,
130.9, 137.8, 139.2, 142.7, 172.4; MS m/z 601 (M + 1); HPLC
tR ) 6.5 min (system A, 99%).
1
as a green powder: mp 105-107 °C dec; H NMR (CDCl3) δ
1.50-1.90 (m, 6 H), 2.52 (bs, 2 H), 2.88 (s, 3 H), 3.35-3.52 (m,
2 H), 3.80-4.20 (m, 2 H), 3.82 (s, 2 H), 4.72 (brs, 2 H), 6.52 (d,
J ) 7.6 Hz, 2 H), 6.92 (t, J ) 7.3 Hz, 1 H), 7.01 (t, J ) 7.7 Hz,
1 H), 7.09-7.22 (m, 2 H), 7.34 (d, J ) 8.0 Hz, 2 H), 12.61 (brs,
1 H).
3-(E t h y l[(n it r o s o t h io c y c lo h e x y l)m e t h y l]a m in o )-
p r op yl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a ceta te
(22b). Compound 22b was synthesized in a manner similar
to the synthesis of 21b using DCC (4.4 g, 21.46 mmol), 15 (1.1
g, 4.22 mmol), diclofenac (1.37 g, 4.64 mmol), DMAP (0.025 g)
and CH2Cl2 (50 mL). Chromatography of the crude product
on silica gel eluting with EtOAc/hexane (1:9) afforded 22b (1.65
g, 73%) as a green oil: TLC Rf ) 0.33 (hexane/EtOAc, 9:1); 1H
NMR (CDCl3) δ 0.84 (t, J ) 7.0, 3 H), 1.22-1.70 (m, 8 H), 1.96-
2.04 (m, 2 H), 2.33-2.55 (m, 6 H), 3.06 (s, 2 H), 3.72 (s, 2 H),
4.08 (t, J ) 6.4 Hz, 2 H), 6.46 (d, J ) 7.9 Hz, 1 H), 6.85-6.92
(m, 3 H), 7.01-7.06 (m, 1H), 7.14 (d, J ) 7.4 Hz, 1H), 7.25 (d,
J ) 8.0 Hz, 2 H); 13C NMR δ 11.1, 22.3, 25.6, 26.4, 34.2, 38.7,
48.9, 51.5, 63.5, 64.6, 66.0, 118.2, 122.0, 124.0, 124.4, 127.9,
128.8, 129.5, 130.8, 137.8, 142.7, 172.4; MS m/z 538 (M + 1);
HPLC tR ) 8.4 min (system A, 95%). Anal. (C26H33N3O3SCl2)
C, H, N, S, Cl.
2-{[({[([{2-(2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}-
a c e t y lo x y )e t h y l}m e t h y la m in o ]m e t h y l)c y c lo h e x y l]-
d isu lfa n yl}cycloh exyl)m et h yl]a m in o}et h yl 2-{2-[(2,6-
Dich lor op h en yl)a m in o]p h en yl}a ceta te (19). DCC (6.1 g,
29.6 mmol) in CH2Cl2 (160 mL) was added dropwise to a stirred
solution of 6a (12.0 g, 29.8 mmol) and diclofenac (24.2 g, 81.7
mmol) in CH2Cl2 (160 mL) over 1 h at room temperature. The
mixture was then stirred for an additional 24 h at room
temperature. The solid was removed by filtration and washed
with CH2Cl2 (100 mL). The filtrate was concentrated under
reduced pressure, and the residue was dissolved in Et2O. A
saturated solution of HCl gas in Et2O was added to the solution
until it became acidic. The precipitated yellow powder was
filtered onto a Buchner funnel and washed with Et2O (600
mL). The resulting white powder was dried in vacuo to afford
19‚HCl (26.0 g, 84%): mp 162-165 °C; 1H NMR (CDCl3) δ
1.20-1.90 (m, 10 H), 2.36 (s, 3 H), 2.55 (s, 2 H), 2.78 (t, J ) 6
Hz, 2 H), 3.82 (s, 2 H), 4.23 (t, J ) 6 Hz, 2 H), 6.54 (d, J ) 8
Hz, 1 H), 6.92-7.0 (m, 3 H), 7.09-7.25 (m, 2 H), 7.33 (d, J )
8 Hz, 2 H); 13C NMR δ 22.3, 32.6, 38.7, 45.3, 55.9, 58.5, 63.2,
118.2, 121.9, 123.9, 124.3, 127.9, 128.8, 129.4, 130.8, 137.8,
142.7, 172.3.
3-(Me t h yl[(n it r osot h ioc yc loh e xyl)m e t h yl]a m in o)-
p r op yl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a ceta te
(21b). DCC (4.4 g, 21.46 mmol) in CH2Cl2 (30 mL) was added
dropwise to a stirred solution of 8b (4.2 g, 17 mmol), diclofenac
(5.3 g, 17.9 mmol), and DMAP (0.15 g) in dry CH2Cl2 (30 mL)
at 0 °C over 15 min. The suspension was then stirred at 0 °C
for 30 min. The precipitate was removed by filtration and
washed with CH2Cl2 (25 mL). The filtrate was concentrated
at 40 °C. Hexane (100 mL) was added, and the precipitated
solid was removed by filtration. The filtrate was concentrated
under reduced pressure to give an oil which was dissolved in
EtOAc (10 mL) and MeOH (40 mL). The solution was filtered,
and the filtrate was heated gently at 40 °C for 2 min and then
left in the freezer (- 20 °C) overnight (12 h). The green crystals
which formed were collected by filtration and dried in vacuo 6
h to give 21b (8.4 g, 94%): mp 58-60 °C; TLC Rf ) 0.46
1
(hexane/EtOAc, 9:1); H NMR (CDCl3) δ 1.40-1.77 (m, 6 H),
1.84 (p, J ) 6.8 Hz, 2 H), 2.08-2.18 (m, 2 H), 2.35 (s, 3 H),
2.47 (d, J ) 13.9 Hz, 2 H), 2.58 (t, J ) 7.1 Hz, 2 H), 3.16 (s, 2
H), 3.85 (s, 2 H), 4.22 (t, J ) 6.4 Hz, 2 H), 6.61 (d, J ) 7.9 Hz,
1 H), 6.97-7.05 (m, 3H), 7.16 (t, J ) 5 Hz, 1H), 7.28 (d, J )
7.4 Hz, 1H), 7.38 (d, J ) 8.0 Hz, 2 H); 13C NMR δ 22.2, 25.5,
26.7, 34.2, 38.6, 44.5, 56.5, 63.2, 64.4, 68.9, 118.2, 121.9, 123.9,
124.3, 127.8, 128.8, 129.4, 130.8, 137.8, 142.7, 172.3. Anal.
(C25H31N3O3SCl2) C, H, N, S, Cl..
4-(Me t h yl[(n it r osot h ioc yc loh e xyl)m e t h yl]a m in o)-
b u t yl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a cet a t e
(21c). Compound 21c was synthesized in a manner similar to
the synthesis of 21b using DCC (0.77 g, 3.7 mmol), 8c (0.65 g,
2.49 mmol), diclofenac (0.927 g, 3.13 mmol), DMAP (0.025 g)
and CH2Cl2 (40 mL). Chromatography of the crude product
on silica gel eluting with EtOAc/hexane (1:9) afforded 21c (1.1
g, 82%) as a green oil: 1H NMR (CDCl3) δ 1.20-1.70 (m, 10
H), 2.00-2.15 (m, 2 H), 2.21 (s, 3 H), 2.28-2.42 (m, 4 H), 3.04
(s, 2 H), 3.75 (s, 2 H), 4.08 (t, J ) 6.6 Hz, 2 H), 6.48 (d, J ) 7.9
Hz, 1 H), 6.86-6.94 (m, 3 H), 7.03-7.08 (m, 1 H),7 0.16-7.19
(m, 1H), 7.27 (d, J ) 8.1 Hz, 2 H); 13C NMR δ 22.3, 23.9, 25.6,
26.3, 34.3, 38.7, 44.7, 59.9, 64.6, 65.3, 68.8, 118.2, 122.0, 124.0,
124.4, 127.9, 128.8, 129.5, 130.8, 137.8, 142.7, 172.4; MS m/z
538 (M + 1); HPLC tR ) 4.9 min (system A, 100%).
2-{Meth yl[(su lfan ylcycloh exyl)m eth yl]am in o}eth yl 2-{2-
[(2,6-Dich lor op h en yl)a m in o]p h en yl}a ceta te (16). (a ) DCC
(1.21 g, 5.89 mmol) in CH2Cl2 (40 mL) was added dropwise to
a stirred solution of 7a (1.0 g, 4.91 mmol) and diclofenac (1.45
g, 4.91 mmol) in dry CH2Cl2 (50 mL) over 1 h. The suspension
was then stirred at room temperature for 2 h. The solid was
removed by filtration, and then was washed with CH2Cl2 (2 ×
20 mL). The filtrate was concentrated under reduced pressure,
and the crude material was triturated with hexane (2 × 25
mL) and filtered to remove DCU and the lactam byproduct
(18). The solvent was evaporated to give a viscous oil which
was chromatographed on silica gel eluting with EtOAc/hexane
(1:19) afforded 16 (1.35 g, 57%): TLC Rf ) 0.35 (hexane/EtOAc,
5-(Me t h yl[(n it r osot h ioc yc loh e xyl)m e t h yl]a m in o)-
p en tyl 2-{2-[(2,6-Dich lor op h en yl)a m in o]p h en yl}a ceta te
(21d ). Compound 21d was synthesized in a manner similar
to the synthesis of 21b using DCC (0.75 g, 3.66 mmol), 8d (0.80
g, 2.91 mmol), diclofenac (0.93 g, 3.05 mmol), DMAP (0.1 g)
and CH2Cl2 (40 mL). Chromatography of the crude product
on silica gel eluting with EtOAc/Hex (1:9) afforded 21d (1.1 g,
1
69%) as a green oil: TLC Rf ) 0.40 (hexane/EtOAc, 9:1); H
NMR (CDCl3) δ 1.18-1.66 (m, 12 H), 2.03-2.12 (m, 2 H), 2.25
(s, 3 H), 2.31-2.47 (m, 4 H), 3.05 (s, 2 H), 3.76 (s, 2 H), 4.08 (t,

Nitrosothiol Esters of Diclofenac
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 4015
1
9:1; KMNO4 brown); H NMR (CDCl3) δ 1.15-1.18 (m, 1 H),
pound bioavailability was indexed as the amount of circulating
diclofenac present in mouse blood after oral dosing. Male CD-1
mice (Charles River Laboratories, Wilmington, MA) were
fasted overnight (≈17 h) with water ad libitum. Mice were
administered by oral gavage either a fixed dose (20 µmol/kg)
of diclofenac sodium or an equimolar dose of a S-NO-diclofenac
derivative as the appropriate suspension. The mice were
anesthetized by ketamine (90 mg/kg)/xylazine (10 mg/kg) at
30, 60 and 120 min postdosing. A different group of mice was
used for each time point, and blood samples were collected
through cardiac puncture into a heparinized syringe. The
heparinized blood was then centrifuged at 4 °C for 7 min at
850g, and the plasma was collected and stored at -20 °C until
analysis. Plasma diclofenac was quantified in organic extracts
by HPLC based upon the response of the spectrophotometric
detector to known amounts of authentic diclofenac with
meclofenamic acid as the internal standard.43 Relative bio-
availability over the 2-h period was then calculated and
expressed relative to that of an equimolar dose (20 µmol/kg)
of diclofenac in the same vehicle as the integrated area under
the pharmacokinetic plot of blood diclofenac level vs time after
dosing. The peak diclofenac plasma concentration within the
2-h sampling period was also quantified. Mice administered
vehicle only evidenced no circulating diclofenac (data not
shown). Data are expressed as mean plasma levels of di-
clofenac ( SEM.
An a lgesic Activity (Wr ith in g Test). The phenylbenzo-
quinone (PBQ)-induced writhing test was used to determine
analgesic activity.36 Male CD-1 mice weighing 20-25 g were
fasted overnight (≈17 h) with water ad libitum. Vehicle or test
compound (100 µmol/kg) was administered by oral gavage as
a suspension with a constant dose volume of 2.5 mL/kg body
weight at least 9 mice/group. One hour after dosing, each
mouse received an intraperitoneal injection (2 mg/kg body
weight) of PBQ solution. Five minutes after the PBQ injection,
the number of writhes by each mouse over the next 5-min
period was scored. Analgesic activity is expressed as the
inhibition of the PBQ-induced writhing due to test compound.
An tiin fla m m a tor y Activity (P a w Ed em a Test). Antiin-
flammatory activity was assessed in the carrageenan-induced
paw edema assay.37 Male Sprague-Dawley rats (200-250 g)
(Charles River Laboratories, Wilmington, MA) were fasted
overnight (≈17 h) with water ad libitum. A test compound (100
µmol/kg) or appropriate vehicle was administered by oral
gavage at a constant dose volume of 5 mL/kg body weight, with
at least 5 rats/group. One hour after dosing, 50 µL of 1%
carrageenan solution was injected into the subplantar region
of the right front paw. Paw volume was measured immediately
and 3 h after carrageenan injection by a water-displacement
method. Antiinflammatory activity was quantified as the
incremental reduction of the carrageenan-induced increase in
paw volume due to test compound.
1.29-1.80 (m, 9 H), 2.12 (bs, 1 H), 2.42 (s, 3 H), 2.52 (s, 2 H),
2.86 (t, J ) 5.9 Hz, 2 H), 3.84 (s, 2 H), 4.28 (t, J ) 5.9 Hz, 2
H), 6.56 (d, J ) 7.9 Hz, 1 H), 6.93-7.00 (m, 3 H), 7.14 (t, J )
6.7 Hz, 1 H), 7.23 (d, J ) 6.7 Hz, 1 H), 7.34 (d, J ) 8.0 Hz, 2
H), 13C NMR δ 22.3, 25.9, 37.6, 38.6, 45.2, 52.2, 58.5, 60.2,
63.1, 72.1, 118.1, 121.9, 123.9, 124.1, 127.8, 128.7, 129.3, 130.8,
137.7, 142.6, 172.2; MS m/z 510 (M + 1). Anal. (C24H30N2O2-
Cl2S2) C, H, N, S, Cl.
(b) 19 (18 g, 18.1 mmol) was dissolved in glacial HOAc (300
mL), and powdered zinc (36 g, 550 mmol) was added to this
solution. The resulting slurry was stirred under N2 for 6 h at
room temperature, and the inorganic solid was removed by
filtration. The solid was washed with HOAc (50 mL), and the
residue was made basic with aqueous 15% NH4OH. The
product was then extracted with EtOAc (2 × 150 mL). The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure to give an oil, which was dissolved in Et2O
(100 mL). A saturated solution of HCl gas in Et2O was added
to acidify the Et2O solution. The precipitate was filtered and
washed with Et2O (2 × 100 mL). The resulting white solid was
dried in vacuo for 12 h to afford 16‚HCl (16 g, 86%): mp 113
1
°C; H NMR (CDCl3) δ 1.14-1.30 (m, 1 H), 1.34-2.00 (m, 9
H), 2.95-3.01 (m, 1 H), 2.96 (d, J ) 4.2 Hz, 3 H), 3.30 (d, J )
13.5 Hz, 1 H), 3.43-3.60 (m, 3 H), 3.86 (s, 2 H), 4.66-4.82 (m,
2 H), 6.51-6.54 (m, 2 H), 6.94 (t, J ) 7.4 Hz, 1 H), 7.02 (t, J
) 8.0 Hz, 1 H), 7.06-7.16 (m, 1 H), 7.20 (d, J ) 7.4 Hz, 1 H),
7.35 (d, J ) 8.0 Hz, 2 H), 11.66 (bs, 1 H).
(Su lfan ylcycloh exyl)m eth yl 2-{2-[(2,6-Dich lor oph en yl)-
a m in o]p h en yl}a ceta te (23). DCC (2.25 g, 10.94 mmol) in
CH2Cl2 (25 mL) was added dropwise to a stirred solution of 3c
(1.60 g, 10.95 mmol) and diclofenac (2.72 g, 9.12 mmol) in dry
CH2Cl2 (25 mL) over 30 min at 0 °C. The suspension was then
stirred at 0 °C for 1 h. Workup and purification (EtOAc/hexane,
1:19) similar to 16 afforded 23 (2.45 g, 63%): 1H NMR (CDCl3)
δ 1.17-1.80 (m, 10 H), 3.88 (s, 2 H), 4.18 (s, 2 H), 6.57 (d, J )
7.9 Hz, 1 H), 6.84 (bs, 1 H), 6.96-7.02 (m, 2 H), 7.12-7.17 (m,
1 H), 7.26 (d, J ) 6.7 Hz, 1 H), 7.35 (d, J ) 8.0 Hz, 2 H); 13C
NMR δ 21.8, 25.7, 36.0, 38.5, 48.4, 74.2, 118.4, 122.1, 124.0,
124.4, 128.0, 128.8, 129.5, 130.8, 137.8, 142.7, 172.1; MS m/z
425 (M + 1); HPLC tR ) 9.0 min (system A, 100%). Anal.
(C21H23NO2Cl2S) C, H, N, S, Cl.
3-{Met h yl[(su lfa n ylcycloh exyl)m et h yl]a m in o}p r op yl
2-{2-[(2,6-Dich lor oph en yl)am in o]ph en yl}acetate (24). DCC
(1.45 g, 7.03 mmol) in CH2Cl2 (25 mL) was added dropwise to
a stirred solution of 7b (1.53 g, 7.03 mmol) and diclofenac (2.08
g, 4.91 mmol) in dry CH2Cl2 (25 mL) over 1 h at room
temperature. The suspension was then stirred at room tem-
perature for 1 h. Workup and purification (EtOAc/hexane, 1:9)
similar to 16 afforded 24 (0.65 g, 20%): TLC Rf ) 0.58 (hexane/
EtOAc, 4:1; KMNO4 brown); 1H NMR (CDCl3) δ 0.99-1.80 (m,
12 H), 1.94 (bs, 1 H), 2.23 (s, 3 H), 2.34 (s, 2 H), 2.47 (t, J )
7.2 Hz, 2 H), 3.70 (s, 2 H), 4.13 (t, J ) 6.5 Hz, 2 H), 6.45 (d, J
) 7.9 Hz, 1 H), 6.82-6.90 (m, 3 H), 6.99-7.04 (m, 1 H), 7.12
(d, J ) 7.4 Hz, 1 H), 7.23 (d, J ) 8.0 Hz, 2 H); 13C NMR δ
22.4, 25.9, 26.9, 37.8, 38.6, 44.7, 52.1, 56.7, 63.4, 72.2, 118.2,
121.9, 123.9, 124.3, 127.8, 128.8, 129.4, 130.7, 137.7, 142.6,
172.2; MS m/z 495 (M + 1); HPLC tR ) 4.3 min (system A,
100%). Anal. (C25H32N2O2Cl2S1) C, H, N, S, Cl.
Biologica l Assa ys Com p ou n d F or m u la tion . All test
compounds, including diclofenac sodium (Sigma Chemical Co.,
St. Louis, MO), were formulated just prior to administration
in either 5% (by volume) or 50% (by volume) aqueous propylene
glycol (PEG) containing 0.5% methocel in order to obtain a
homogeneous suspension for oral animal dosing. The more
lipophilic S-NO-diclofenac derivatives (i.e., 21a -21d , 22a , 22b)
required the greater PEG concentration. Procedurally, the
desired amount of test compound was weighed and dissolved
in aqueous PEG and then diluted with 0.5% methocel until
the 5% or 50% concentration was attained. In all experiments,
comparison between diclofenac sodium and a given S-NO-
diclofenac derivative was made with the two compounds
administered in the same vehicle.
Ga str ic Lesion Assessm en t. Male Sprague-Dawley rats
(180-200 g) were fasted for 24 h with water ad libitum and
then dosed by oral gavage with diclofenac sodium or a S-NO-
diclofenac derivative administered in a constant volume of 5
mL/kg at a final dose of 100 µmol/kg. Fasting was continued
for an additional 18 h, at which time the animals were
euthanized with CO2. The stomachs were dissected along the
greater curvature, washed with a directed stream of 0.9%
saline, pinned open on a sylgard-lined Petri dish, and exam-
ined by light microscopy to assess the presence of gastric
lesions.39 Gastric lesion score is expressed in mm of lesion and
calculated by summing the length of all lesions in a given
stomach.
Sta tistics. All data are expressed as the mean ( SEM and
were analyzed by performing analysis of variance followed by
a Student-Neuman-Keuls post hoc test to determine the
significance level with a Super Anova computer program.
Ack n ow led gm en t. The authors thank Dr. George
W. Goodloe at Auburn Mass Spectroscopy Center for
high-resolution mass spectra, Dr. Radha Iyengar at
NitroMed, Inc. for helpful comments, and Dr. Michael
Rela tive Bioa va ila bility Deter m in a tion . Relative com-

4016 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Bandarage et al.
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J . Ernest (VP, Exploratory Development) at R. W.
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