Acyclic triaryl olefins possessing a sulfohydroxamic acid pharmacophore: Synthesis, nitric oxide/nitroxyl release, cyclooxygenase inhibition, and anti-inflammatory studies

Article abstract of DOI:10.1039/c005066k

Nitric oxide (NO) and its reduced form nitroxyl (HNO), effective vasodilation agents that can inhibit platelet aggregation and adhesion, could suppress adverse cardiovascular effects associated with the use of selective COX-2 inhibitors. In this regard, a

Full text of DOI:10.1039/c005066k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Acyclic triaryl olefins possessing a sulfohydroxamic acid pharmacophore:
synthesis, nitric oxide/nitroxyl release, cyclooxygenase inhibition, and
anti-inflammatory studies†
a
a
a
a
a
b
Zhangjian Huang, Carlos Vel a´ zquez, Khaled Abdellatif, Morshed Chowdhury, Sarthak Jain, Julie Reisz,
b
b
a
Jenna DuMond, S. Bruce King and Edward Knaus*
Received 13th April 2010, Accepted 17th June 2010
First published as an Advance Article on the web 22nd July 2010
DOI: 10.1039/c005066k
Nitric oxide (NO) and its reduced form nitroxyl (HNO), effective vasodilation agents that can inhibit
platelet aggregation and adhesion, could suppress adverse cardiovascular effects associated with the use
of selective COX-2 inhibitors. In this regard, a sulfohydroxamic acid (SO
act as a dual NO/HNO donor moiety, was inserted at the para-position of the C2 phenyl ring of acyclic
-alkyl-1,1,2-triaryl olefins previously shown to be potent and highly selective COX-2 inhibitors.
2
NHOH) substituent, that can
2
Although this new group of 1,1-diaryl-2-(4-hydroxyaminosulfonylphenyl)alk-1-enes exhibited weak
inhibition of the constitutive cyclooxygenase-1 (COX-1) and inducible COX-2 isozymes, in vivo studies
showed anti-inflammatory potencies that were generally intermediate between that of the reference
drugs aspirin and ibuprofen. All compounds released NO (5.6–13.5% range) upon incubation with
phosphate buffer which was increased further (8.3–25.6% range) in the presence of the oxidant
K
3
(FeCN ).The low release of HNO in MeOH-buffer (< 2% at 24 h incubation) was much higher at
6
alkaline pH (11–37% range). The concept of designing better anti-inflammatory drugs possessing either
an effective HNO, or dual NO/HNO, donor moiety that are devoid of adverse ulcerogenic and/or
cardiovascular side effects warrants further investigation.
Introduction
Selective cyclooxygenase-2 (COX-2) inhibitors, such as rofecoxib
1
2
3
(
1) celecoxib (2a), valdecoxib (3a) and the acyclic 2-butyl-1,1,2-
4
triaryl (Z)-olefin (4), reduce inflammation and pain by inhibiting
the peripheral production of prostaglandins (see structures in
Fig. 1). Although the risk of gastric irritation and peptic ulceration
associated with the use of selective COX-2 inhibitors is extremely
low, there is an increased incidence of adverse cardiovascular
5
events such as thrombosis and stroke. Highly selective COX-2
inhibitors may decrease the level of the beneficial vasodilator
and platelet aggregation inhibitor prostacyclin (PGI
with a contraindicated increase in the level of the potent platelet
activator and aggregator thromboxane A (TxA ). This biochem-
2
) concurrent
2
2
ical alteration in the arachidonic acid COX biosynthetic pathway
has been attributed to the increased incidence of cardiovascular
6
thrombotic events observed.
The biological activity and biological chemistry of nitrogen ox-
ide species in mammalian systems continues to attract considerable
Fig. 1 Some representative selective cyclooxygenase-2 (COX-2) inhibitors
(1, 2a, 3a), the sulfohydroxamic acid analog of celecoxib (2b), the
sulfohydroxamic acid metabolite of valdecoxib (3b), and an acyclic selective
COX-2 inhibitor (4) with potent anti-inflammatory activity.
7
attention. Nitric oxide (NO) is an effective vasodilation agent, an
8
inhibitor of platelet aggregation and adhesion, and it provides
an attractive method to suppress vascular side effects associated
9
with NSAID use. In recent studies, we showed that a variety
a
Faculty of Pharmacy and Pharmaceutical Sciences, University of Al-
of NO donor ester prodrugs of non-steroidal anti-inflammatory
drugs (NSAIDs) are effectively cleaved by esterases to release
berta, Edmonton, Alberta, Canada T6G 2N8. E-mail: eknaus@pharmacy.
ualberta.ca; Fax: +1 780-492-1217; Tel: +1 780-492-5993
10
b
the parent COX-2 inhibitory compound and NO. Nitroxyl
(HNO), the reduced form of NO, exhibits unique biological
and pharmacological properties compared to other nitrogen
Department of Chemistry, Wake Forest University, Winston-Salem, North
Carolina 27109, USA. E-mail: kingsb@wfu.edu; Fax: +1 336-758-4656;
Tel: +1 336-758-5325
†
Electronic supplementary information (ESI) available: Experimental
11
oxides. In the cardiovascular system, HNO exerts a positive
procedures and spectroscopic data for compounds 6–12, 16–19 and 21–29
1
inotropic cardiac effect that is independent from b-adrenergic sig-
described in the paper are given, and H NMR scans for all compounds
–12, 13a–g, 16–19 and 21–29. See DOI: 10.1039/c005066k
2
+
6
naling by directly enhancing cardiac sarcoplasmic reticulum Ca
4
124 | Org. Biomol. Chem., 2010, 8, 4124–4130
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1
1,12
recycling.
HNO also protects heart tissue against ischemia-
13
reperfusion injury, it effectively inhibits human platelet aggrega-
tion in a rapid and concentration-dependent manner, and it is
resistant to superoxide radical anion. Benzenesulfohydroxamic
acid (PhSO NHOH), well known as Piloty’s acid (PA), can serve as
a HNO and/or NO donor. In this regard, PA can decompose to
14
11
2
15
HNO and sodium phenylsulfinate (C
catalyzed deprotonation mechanism or by the release of NO and
6
H
5
SO Na) via either a base-
2
16
benzenesulfinic acid (C
6
H
5
17a
SO
2
H) upon oxidative decomposition
3
-
17b
by O
2
, H
2
O
2
, [Fe(CN)
3
]
or by metal complexes.
The design of selective COX-2 inhibitors that simultaneously
act as NO/HNO donors, to the best of our knowledge, has not
been reported. In a previous study we showed that 1,1-diphenyl-2-
(
4-methanesulfonylphenyl)hex-1-ene (4) was a highly potent and
selective COX-2 inhibitor that exhibited potent anti-inflammatory
4
activity. It was therefore of interest to determine whether replace-
ment of the MeSO
2
COX-2 pharmacophore present in the (Z)-
olefin 4 by a SO NHOH moiety would provide a hitherto unknown
2
class of compounds that act as dual NO and HNO donors. As
part of this ongoing program, we now describe the synthesis of a
group of acyclic 2-alkyl-1,1,2-triaryl (Z)-olefins possessing a para-
Scheme 2 Reagents and conditions: (a) 4,4¢-difluorobenzophenone,
◦
Zn, TiCl
4
, dry THF, reflux, 4.5 h; (b) ClSO
3
H, CHCl
3
, 25 C, 2 h;
HONHSO
2
, or para-MeONHSO , substituent on the C2 phenyl
2
∑
◦
(
c) HONH
2
HCl, MgO, THF–H
2
O–MeOH = 30 : 2 : 3 (v/v/v), 25 C, 3 h.
ring (13a–g) as illustrated in Schemes 1–3, in vitro NO and HNO
release and COX-1/COX-2 inhibition studies, and evaluation as
anti-inflammatory agents.
◦
Scheme 3 Reagents and conditions: (a) ClSO
3
H, CHCl
3
, 25 C, 4 h;
◦
(
b) isopropanol, DMAP, dry CH
2
Cl
2
, 25 C, 4 h; (c) KMnO
4
, FeCl
3
,
◦
◦
acetone, -78 C for 2 h and then 25 C for 2 h; (d) benzophenone for
Scheme 1 Reagents and conditions: (a) isopropanol, 4-dimethylaminopy-
compound 24, 4,4¢-dimethylbenzophenone for compound 25, Zn, TiCl
4
,
◦
◦
ridine (DMAP), dry CH
2
Cl
2
, 25 C, 4 h; (b) benzophenone for compound
dry THF, reflux, 2.5 h; (e) NaI, acetone, reflux, 16 h; (f) SOCl
2
, DMF, 25 C,
∑
∑
7
, 4,4¢-dimethylbenzophenone for compound 8, Zn, TiCl
4
, dry THF,
1 h; (g) HONH
2
HCl for 13e and 13f; CH
3
ONH
2
HCl aqueous solution
O–MeOH =
◦
reflux, 2.5 h; (c) NaI, acetone, reflux, 16 h; (d) SOCl
2
, DMF, 25 C, 1 h;
O–MeOH = 30 : 2 : 3 (v/v/v), 25 C, 3 h.
for 13g, MgO for 13e and 13f, K
2
CO
3
for 13g, THF–H
2
∑
◦
◦
(
e) HONH
2
HCl, MgO, THF–H
2
30 : 2 : 3 (v/v/v), 25 C, 3 h.
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Results and discussion
designed based on the expectation that: i) a sulfohydroxamic
moiety (SO
similar to a MeSO
of a phenyl ring on a 5-membered heterocyclic ring template
2
NHOH) would act as a COX-2 pharmacophore
Chemistry
2
or H NSO substituent at the para-position
2
2
Reactants that possess a benzenesulfonyl chloride, or ben-
zenesulfohydroxamic acid, moiety are not able to withstand
reductive McMurry olefination reaction conditions. Therefore,
an isopropyl protection strategy involving the reaction of 4-
acetylbenzenesulfonyl chloride (5) with isopropanol was used for
the preparation of isopropyl 4-acetylbenzenesulfonate (6) that was
1–3
that confers potent and selective COX-2 inhibitory activity, ii)
the sulfohydroxamic acids 13a–f will exhibit anti-inflammatory
activity since N-hydroxyvaldecoxib (3b) is an active metabolite
18
25
26
resulting from N-hydroxylation of valdecoxib (3a) in mice and
2
7
humans, and iii) the SO
donor to release NO and/or HNO under different conditions
which could circumvent the adverse vascular side effects of potent
2
NHOH moiety will act as a NO/HNO
15–17
required for the synthesis of the target products 13a and 13b
4
(
Scheme 1). The subsequent McMurry cross-coupling reaction
9,14
COX-2 inhibitors.
of 6 with either benzophenone, or 4,4¢-dimethylbenzophenone,
afforded the respective olefins 7 and 8 which upon reaction with
sodium iodide in acetone at reflux furnished the corresponding
sulfonic acid sodium salts 9 and 10. Treatment of 9 or 10 with
thionyl chloride in DMF, provided the respective sulfonyl chloride
Cyclooxygenase-1 (COX-1) and -2 enzyme inhibition
19
In vitro COX-1/COX-2 enzyme inhibition studies (Table 1) showed
that the N-hydroxysulfonamides 13a–f were weak inhibitors of
COX-1 (IC50 = 6.0 to 53.7 mM range) and COX-2 (IC50 = 44.3 to
2
0
1
1
1 or 12. Subsequent reaction of the sulfonyl chlorides 11 and
2 with hydroxylamine hydrochloride in the presence of the mild
1
1
01 mM range) that resulted in low COX-2 selectivity indexes (SI <
; COX-2 selectivity index = COX-1 IC50/COX-2 IC50). In contrast,
21
base magnesium oxide afforded the respective target products
3a and 13b.
A geometry optimized PM3 calculation for 1,1-di-(4-
1
the N-methoxybenzenesulfonamide analogue 13g did not inhibit
the COX-1 or COX-2 isozymes at the highest test compound
concentration (100 mM). One could interpret this latter structure–
22
fluorophenyl)-2-phenylhex-1-ene (17) showed that the electron
density was highest at the para-position (-0.100) relative to the
ortho (-0.091) and meta (-0.098) position of the C2 phenyl ring.
Accordingly, electrophilic chlorosulfonation of the olefins 16 and
activity observation as an indication that elaboration of the MeSO
2
moiety present in compound 4, or the SO NHOH moiety present
2
in 13f, to a SO
2
NHOMe moiety may not be adaptive to insertion
1
7 provided the expected benzenesulfonyl chlorides 18 and 19 as
illustrated in Scheme 2. A similar PM3 calculation for 1,1-di-
4-methylphenyl)-2-phenylhex-1-ene also indicated that electron
4
into the COX-2 secondary pocket binding site. The COX-2 SI for
1
3a–f remained low (< 1) irrespective of their molecular volumes
(
˚
3
which spanned a range of 315–399 A (see data in Table 1). The
observation that N-hydroxyvaldecoxib (3b, COX-1 IC50 = 96.2 mM;
COX-2 IC50 = 1.1 mM; COX-2 SI = 88) is a much weaker and less
selective COX-2 inhibitor than valdecoxib (3a, COX-1 IC50 = 157.2
mM; COX-2 IC50 = 0.04 mM; COX-2 SI = 3930), in conjunction
with the fact that 3b is a much more potent anti-inflammatory
agent than 3a, indicates that caution must be exercised in the
density was highest at the para-position (-0.062) relative to the
ortho (-0.054) and meta (-0.061) position of the C2 phenyl ring.
However, the chlorosulfonation reaction in this latter instance
occurred on a 4¢-methylphenyl ring rather than at the expected
para-position of the C2 phenyl ring. Reaction of the sulfonyl
chlorides 18 and 19 with hydroxylamine hydrochloride afforded
the respective target product 13c or 13d.
25
interpretation of COX isozyme inhibitory data.
Hex-1-enes 13e–g were prepared using methodologies sim-
ilar to those employed for the preparation of 13a and 13b
(
see Scheme 3). Thus, chlorosulfonation of amylbenzene (20)
Anti-inflammatory activity
furnished the para-chlorosulfonation product 21 in good yield.
The oral anti-inflammatory (AI) activities (% inhibition of inflam-
23
Ferric chloride assisted permanganate benzylic oxidation of
the isopropyl sulfonate 22 afforded the ketone 23 in moderate
yield. After completion of the McMurry cross-coupling reactions,
olefins 24 and 25 were deprotected to give the respective sodium
sulfonate salts 26 and 27. Reaction of the sulfonyl chlorides 28
and 29, prepared from the sodium sulfonate salts 26 and 27,
with hydroxylamine, or methoxyamine, hydrochloride afforded the
respective target products 13e–f or 13g.
-
1
mation for a 100 mg kg oral dose or ED50 value) for the sulfo-
hydroxamic acids 13a–g were determined using a carrageenan-
induced rat foot paw edema model (see data in Table 1). Within
-
1
this group of compounds, 13f (ED50 = 88 mg kg ) and 13e (ED50
=
-
1
1
14 mg kg ), together with 13a, 13c and 13d that showed a 49.0–
5.6% inhibition of inflammation for a 100 mg kg oral dose,
-
1
5
exhibited relatively similar AI activities. Their moderately potent
AI activity is greater than that of the reference drug aspirin, but
less than that of the non-selective COX-2 inhibitor ibuprofen and
The target sulfohydroxamic acids (13a–f), unlike N-
24
hydroxycelecoxib(2b, Fig. 1) which is unstable, are stable prod-
the highly selective COX-2 inhibitors celecoxib and the acyclic
ucts that are readily isolated and purified.
2
(
Z)-olefin 4. The effect of the R -substituent on AI activity was
1
variable. In the prop-1-ene group (13a–c; R = Me), the relative
AI potency order was F ≥ H > Me whereas, in the hex-1-ene
group (13d–f, R = n-Bu) the relative potency order was Me ≥ F >
Drug design rationale
1
A
a
novel group of acyclic triaryl (Z)-olefins possessing
small C2 alkyl substituent, such as 1,1-diphenyl-2-(4-
H. It should be noted that the N-methoxybenzenesulfonamide
methylsulfonylphenyl)hex-1-ene (4) that exhibited optimal COX-
(SO NHOMe) compound 13g, which showed no inhibition of
2
2
7
inhibitory potency (IC50 = 0.014 mM) and selectivity (SI >
124), were recently reported by our group. In this study, the
COX-1 and COX-2 (IC50 > 100 mM), exhibited weak in vivo AI
activity. One plausible explanation for this observation is that the
4
N-hydroxy (methoxy) benzenesulfonamide analogues 13a–g were
SO
2
NHOMe group in 13g undergoes metabolic O-demethylation
4
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Table 1 In vitro COX-1 and COX-2 inhibition, anti-inflammatory (AI), log P and volume data
a
IC50 (mM)
b
AI activity: ED50 (mg kg-1) c
d
˚
3 e
Compound
COX-1
COX-2
COX-2 SI
Log P
Volume/A
f
1
1
1
1
1
1
1
4
3a
3b
3c
3d
3e
3f
40.1
44.0
6.0
45.7
53.7
30.7
100
100
115.9
2.9
44.3
68.8
76.6
76.4
101
85.6
100
<1
<1
<1
<1
<1
<1
—
>7142
1783
2.64
0.13
Moderate
Weak
3.7
4.5
4.0
5.3
5.1
5.9
6.9
5.7
4.3
3.1
1.2
315
349
322
372
365
399
473
362
292
211
153
g
h
Moderate i
Moderate
114 ± 7.9
88 ± 2.3
j
3g
Weak
k
0.014
0.065
2.5
Celecoxib
Ibuprofen
Aspirin
10.8
67.4
128.7
l
l
1.1
2.4
l
l
0.3
a
The in vitro test compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, mM)
is the mean of two determinations acquired using the enzyme immuno assay kit (Catalog No. 560131, Cayman Chemicals Inc., Ann Arbor, MI, USA)
and the deviation from the mean is <10% of the mean value. In vitro COX-2 selectivity index (COX-1 IC50 / COX-2 IC50). Inhibitory activity in a
carrageenan-induced rat paw edema assay. The results are expressed as the ED50 value (mg kg ) at 3 h after oral administration of the test compound. The
b
c
-1
d
e
log P value was calculated using the ChemDraw Ultra program, Version 6.0, CambridgeSoft company. The volume of the molecule, after minimization
f
using the molecular mechanics geometry optimization module, was calculated using the Alchemy 2000 program, Tripos Inc. A 49.0% inhibition of
inflammation was observed at a 100 mg kg- oral dose. A 28.3% inhibition of inflammation was observed at a 100 mg kg oral dose. A 55.6% inhibition
1
g
-1
h
of inflammation was observed at a 100 mg kg- oral dose. A 49.7% inhibition of inflammation was observed at a 100 mg kg oral dose. A 27.0%
1
i
-1
j
-1
k
inhibition of inflammation was observed at a 100 mg kg oral dose. Data taken from a previous report (Bioorg. Med. Chem. 2004, 12, 5929). COX-2
l
inhibition was determined using ovine COX-2. Data taken from a previous report (J. Med. Chem. 2005, 48, 4061). COX-2 inhibition was determined
using ovine COX-2.
to a SO
8
2
NHOH metabolite 13f that exhibits AI activity (ED50
=
strong protein binding to rat serum which results in suppressed
NO release.
8 mg kg- po).
1
Indirect assay of nitroxyl release (HNO) as nitrous oxide (N O)
2
Nitric oxide release
Direct HNO detection continues to be a difficult analytical
challenge. In this regard, gas chromatographic analysis can be used
to detect, and determine the percentage of, HNO release indirectly
The % NO released from the N-hydroxy (methoxy) benzenesul-
fonamide 13a–g and PA upon incubation in i) phosphate-buffered-
saline (PBS at pH 7.4), ii) PBS in the presence of an oxidizing agent,
and iii) PBS containing rat serum were measured by quantitation
of nitrite using the Griess reaction (see data in Table 2). The % NO
released from the sulfohydroxamic acids 13a–f in PBS at pH 7.4
varied over a 4.3–13.5% range which is indicative of slow NO
release. In contrast, the oxidant effect of potassium hexacyano-
by quantitation of nitrous oxide (N
2
O) which arises from HNO
dimerization and dehydration under anaerobic conditions (HNO
2
8
+ HNO → [HONNOH] → N
2
O + H
2
O). Since PA decomposes
to HNO in aqueous alkaline solution, and HNO reacts rapidly
with thiols to form disulfides and hydroxylamine or sulfinamides,
29
HNO release from 13a–g was measured using three MeOH-based
ferrate (K
3
[FeCN
6
]) on the NO release properties of 13a–g was
solvent mixtures (see data in Table 2). In these solvent systems, the
higher (4.4–25.6% range). This latter observation is consistent
with reports that NO release from benzenesulfohydroxamic acid
is facilitated by oxidants. The % NO released from the reference
compound PA in PBS (59.7%), or in PBS containing the oxidant
percentage of N
low (0–2% range). In contrast, in the presence of the base NaOH,
the % N O produced was substantially larger (2–71% range). In
comparison, the % N O produced in the MeOH/NaOH/GSH
2
O arising from 13a–f and PA in MeOH/TBS was
17a
2
2
(
58.4%), was much greater than that observed with 13a–g in similar
NO release experiments. Interestingly, the % NO release from
3a–g and PA was suppressed (0.38–5.72%) in PBS containing
rat serum. One plausible explanation for this observation, since
solvent system was much smaller (0–10% range) since addition of
the thiol glutathione (GSH) reacts with nitroxyl (HNO) thereby
1
resulting in the expected decrease in N O production observed.
2
This latter group of experiments in which the incubation solvent
system contains GSH provides strong evidence for the release of
HNO from 13a–f and the subsequent dimerization of the released
11
NO is not expected to react with serum thiols, could be due to
the likely probability that the highly lipophilic (log P = 3.7–5.9
range; see data in Table 1) sulfohydroxamic acids 13a–f undergo
HNO to N O.
2
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Table 2 Percent (%) nitric oxide and nitrous oxide release from 13a–g and the reference compound Piloty’s acid (PA)
e
%
N
2
O release
a
f
g
h
%
NO release
MeOH/TBS
2h
MeOH/Base
2h
MeOH/Base/GSH
b
c
d
PBS
PBS+Oxidant
PBS+Serum
24h
24h
2h
24h
i
i
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
9.8
11.4
9.4
5.6
7.5
13.5
4.3
59.7
25.3
17.6
25.6
10.7
8.3
13.5
4.4
4.45
3.0
3.2
3.0
5.7
2.2
0.4
3.2
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
36
29
32
40
2
36
0
25
17
11
34
16
37
0
1
1
1 j
10
j
1
5
0
5
0
0
2
4
3
5
0
7
3g
PA
58.4
71
31
a
Percent of nitric oxide released based on a theoretical maximum release of 1 mol of NO/mol of the sulfohydroxamic test compound (13a–g) and the
b
reference agent PA (PhSO
2
NHOH). The result is the mean value of 3 measurements (n = 3) where variation from the mean % value was £ 0.2%. A
-
2
◦
c
solution of the test compound (2.4 mL of a 5.0 x 10 mM) in phosphate buffer at pH 7.4 was incubated at 37 C for 1.5 h. A solution of the test
compound (2.4 mL of a 5.0 x 10- mM) in phosphate buffer at pH 7.4 which contained 5.0 x 10 mM K
2
-2
(FeCN
solution of the test compound (2.4 mL of a 5.0 x 10 mM) in phosphate buffer at pH 7.4, to which 90 mL rat serum had been added, was incubated at 37 C
◦
d
3
6
), was incubated at 37 C for 1.5 h. A
-2
◦
e
for 1.5 h. Percent of nitrous oxide (N
2
O) released, based on the condensation of 2 mol of HNO → 1 mol N
2 2
O + H O. The result is the mean value of
f
3
measurements (n = 3). The HNO donor test compound (13a–g, PA) concentration is 50 mM in each experiment unless otherwise noted. MeOH/TBS
g
solvent is comprised of 0.6 mL MeOH and 0.2 mL of 700 mM Tris buffer solution (TBS) at pH 7.00. MeOH/Base solvent is comprised of 0.6 mL MeOH
h
i
and 0.2 mL of 1M NaOH. MeOH/Base/GSH experiments are 100 mM in glutathione (GSH) unless otherwise noted. Test compound concentration
is 25 mM and GSH concentration is 50 mM. Test compound concentration is 47 mM and GSH concentration is 94 mM.
j
Conclusions
were performed for C, H, N by the Microanalytical Service
Laboratory, Department of Chemistry, University of Alberta.
Silica gel column chromatography was performed using Merck
silica gel 60 ASTM (70–230 mesh). All reagents, purchased from
the Aldrich Chemical Company (Milwaukee, WI), were used
without further purification. The in vivo anti-inflammatory assay
was carried out using a protocol approved by the Health Sciences
Animal Welfare Committee at the University of Alberta.
Experimental chemical procedures and spectroscopic data for,
and biological assays to evaluate, the target compounds 13a–g are
given below—full experimental procedures and spectroscopic data
for the synthesis of compounds 6–12, 16–19 and 21–29 described
in the paper are given in the supporting information.
A group of 2-alkyl-1,1,2-triaryl olefins were synthesized in which
a SO
at the para-position on the C2 phenyl ring (13a–g). Biological
studies showed that the SO NHOH group of compounds 13a–
2
NHOR (R = H, Me) NO/HNO donor moiety was placed
2
f exhibit i) weak in vitro COX-1 and COX-2 inhibitory activity
and show low COX-2 SI’s, ii) moderate in vivo anti-inflammatory
activity that is intermediate between that of the reference drugs
aspirin and ibuprofen, (iii) a pharmacological acceptable stability
in phosphate-buffered saline at pH 7 where NO release is in the
5
.6–13.5% range, that NO release is increased in the presence of the
oxidant potassium hexacyanoferrate (8.3–25.6% range), but NO
release is decreased in the presence of rat serum (2.2–5.7% range),
and iv) a low in vitro release of HNO in MeOH-buffer (< 2% at
2
4 h incubation), and a much higher HNO release at alkaline pH
1
,1 - Diphenyl - 2 - (4 - hydroxyaminosulfonylphenyl)prop - 1 - ene
13a). Magnesium oxide (0.263 g, 6.58 mmol) was added to a
solution of hydroxylamine hydrochloride (0.200 g, 2.87 mmol) in
THF–H O–MeOH (30 mL:2 mL:3 mL, v/v/v) prior to the ad-
dition of 1,1-diphenyl-2-(4-chlorosulfonylphenyl)prop-1-ene (11,
(
11–37% range at 24 h) that is significantly reduced in the presence
(
of GSH. The design of selective COX-2 inhibitors that contain
either an effective HNO, or dual NO/HNO, donor moiety remains
nowadays an important Medicinal Chemistry challenge that offers
a novel and potential drug design concept for the development of
anti-inflammatory drugs that are devoid of adverse ulcerogenic
and/or cardiovascular side effects.
2
0
.300 g, 0.823 mmol). This reaction mixture was vigorously stirred
at 25 C until the sulfonyl chloride had completely disappeared
◦
(
TLC EtOAc–hexane, 1 : 2, v/v) in about 3 h. The reaction mixture
was filtered through a pad of Celite that provided a clear filtrate
which was dried (MgSO ). Removal of the solvent in vacuo
gave a residue that was purification by flash silica gel column
chromatography using n-hexane–EtOAc (2 : 1, v/v) as eluent to
afford the title compound 13a (203 mg, 67.7%) as a white solid,
mp 151–153 C; IR (film): 3411, 3230, 1329, 1168 cm ; H NMR
(DMSO-d ): d 2.07 (s, 3H, CH C), 6.87–6.90 (m, 2H, phenyl
hydrogens), 7.04–7.11 (m, 3H, phenyl hydrogens), 7.22–7.42 (m,
7H, phenyl hydrogens and sulfonylphenyl H-2, H-6), 7.61 (d, J =
7.9 Hz, 2H, sulfonylphenyl H-3, H-5), 9.53 and 9.56 (two d, J =
3.7 and 3.1 Hz, respectively, 1H each, HO–NH). Anal. Calcd for
4
Experimental section
General
◦
-1
1
Melting points were determined on a Thomas–Hoover capil-
lary apparatus and are uncorrected. Infrared (IR) spectra were
recorded as films on NaCl plates using a Nicolet 550 Series II
6
3
C
1
Magna FT-IR spectrometer. H NMR spectra were measured on
a Bruker AM-300 spectrometer with TMS as the internal standard,
where J (coupling constant) values are estimated in Hertz (Hz).
Mass spectra(MS)wererecordedonaWater’s Micromass ZQ4000
mass spectrometer using the ESI ionization mode. Microanalyses
C
21
H
19NO S: C, 69.02; H, 5.24; N, 3.83. Found: C, 69.01; H, 5.34;
3
N, 3.80.
4
128 | Org. Biomol. Chem., 2010, 8, 4124–4130
This journal is © The Royal Society of Chemistry 2010

View Article Online
The physical and spectral data for 13b–g, which were prepared
using similar methodologies, are listed below.
1,1-Di-(4-methylphenyl)-2-(4-methoxyaminosulfonylphenyl)hex-
1-ene (13g). Reaction of the sulfonyl chloride 29 with
methoxylamine hydrochloride that was neutralized with an
1
,1-Di-(4-methylphenyl)-2-(4-hydroxyaminosulfonylphenyl)prop-
aqueous solution of K
white solid (65.1%); mp 138–140 C; IR (film): 3235, 2962, 2921,
2
CO
3
yielded the title compound 13g as a
◦
1
3
3
-ene (13b). Yield, 71.2%; white solid; mp 153–154 C; IR (film):
411, 3233, 1329, 1172 cm ; H NMR (DMSO-d
H, CH C), 2.16 (s, 3H, ArCH ), 2.32 (s, 3H, ArCH
◦
-
1
1
): d 2.08 (s,
), 6.75
-1 1
6
2
3
2
CH
7
868, 1342, 1175 cm ; H NMR (DMSO-d
H, CH CH CH CH ), 1.11–1.17 (m, 4H, CH
.13 (s, 3H, ArCH ), 2.30 (s, 3H, ArCH ), 2.39 (t, J = 6.7 Hz, 2H,
C), 3.60 (s, 3H, OCH ), 6.71 and 6.85 (two d, J = 8.6 and
.9 Hz, respectively, 2H each, tolyl H-3, H-5), 7.07 and 7.18 (two
): d 0.71 (t, J = 6.7 Hz,
6
3
C
3
3
CH CH CH ),
2
2
2
3
2
2
2
3
and 6.90 (two d, J = 7.9 Hz, 2H each, tolyl H-3, H-5), 7.10 and
.20 (two d, J = 7.9 Hz, 2H each, tolyl H-2, H-6), 7.38 (d, J =
.9 Hz, 2H, sulfonylphenyl H-2, H-6), 7.63 (d, J = 7.9 Hz, 2H,
3
3
7
7
2
C
3
sulfonylphenyl H-3, H-5), 9.54 and 9.58 (two d, J = 3.1 and 3.7 Hz,
respectively, 1H each, HO–NH); Anal. Calcd for C23 S: C,
d, J = 7.9 Hz, 2H each, tolyl H-2, H-6), 7.36 (d, J = 7.9 Hz, 2H,
sulfonylphenyl H-2, H-6), 7.63 (d, J = 7.9 Hz, 2H, sulfonylphenyl
H
23NO
3
6
9.84; H, 5.89; N, 3.54. Found: C, 70.00; H, 6.28; N, 3.57.
H-3, H-5), 10.5 (s, 1H, CH
3
O–NH). Anal. Calcd for C27
H
31NO S:
3
C, 72.13; H, 6.95; N, 3.12. Found: C, 72.33; H, 7.03; N, 3.16.
1
,1-Di(4-fluorophenyl)-2-(4-hydroxyaminosulfonylphenyl)prop-
◦
1
(
-ene (13c). Yield, 56.6%; white solid; mp 170–171 C; IR
film): 3410, 3237, 1229, 1173 cm ; H NMR (DMSO-d
In vitro cyclooxygenase inhibition assays
-
1
1
6
): d 2.07
(
s, 3H, CH C), 6.86–6.97 (m, 4H, two 4-fluorophenyl H-3,
3
C
The ability of the test compounds 13a–g listed in Table 1 to
inhibit ovine COX-1 and human recombinant COX-2 (IC50 value,
mM) was determined using an enzyme immuno assay (EIA) kit
H-5), 7.19–7.32 (m, 4H, two 4-fluorophenyl H-2, H-6), 7.37 (d,
J = 7.9 Hz, 2H, sulfonylphenyl H-2, H-6), 7.63 (d, J = 7.9 Hz,
2
1
4
H, sulfonylphenyl H-3, H-5), 9.53 and 9.57 (two d, J = 3.1 Hz,
H each, HO–NH). Anal. Calcd for C21 NO S: C, 62.83; H,
.27; N, 3.49. Found: C, 62.74; H, 4.34; N, 3.50.
(
catalog number 560131, Cayman Chemical, Ann Arbor, MI,
30
H
17
F
2
3
USA) according to a previously reported method.
In vivo anti-inflammatory assay
1
,1-Di-(4-fluorophenyl)-2-(4-hydroxyaminosulfonylphenyl)hex-
◦
1
(
-ene (13d). Yield, 62.3%; white solid; mp 143–145 C; IR
Anti-inflammatory activity was measured using a carrageenan-
induced rat paw edema assay described by Winter et al. Briefly,
-
1
1
31
film): 3418, 3240, 2965, 2929, 2858, 1507, 1226, 1170 cm ; H
NMR (DMSO-d ): d 0.71 (t, J = 7.3 Hz, 3H, CH CH CH CH ),
.13–1.22 (m, 4H, CH CH CH CH ), 2.35 (t, J = 6.7 Hz, 2H,
CH C), 6.80–7.00 (m, 4H, two 4-fluorophenyl H-3, H-5), 7.20–
.32 (m, 4H, two 4-fluorophenyl H-2, H-6), 7.35 (d, J = 8.5 Hz, 2H,
sulfonylphenyl H-2, H-6), 7.64 (d, J = 7.9 Hz, 2H, sulfonylphenyl
H-3, H-5), 9.55 and 9.59 (two d, J = 3.7 and 3.1 Hz, respectively,
6
2
2
2
3
three male Sprague Dawley rats weighing 160–180g were used in
each group. Test compounds suspended in water containing 1%
methyl cellulose, were administered orally at different doses (50–
1
2
2
2
3
2
C
-
1
7
150 mg kg range) 1 h prior to a 0.05 mL subcutaneous injection
of 1% carrageenan in 0.9% NaCl solution under the planter skin of
the right hind paw. Control experiments were identical except that
the vehicle did not contain a test compound. The volume of the
injected paw was measured at 0 and 3 h using a UGO Basile 7141
Plethysmometer (Ser. No 43201) for calculation of % inhibition of
inflammation.
1
5
H each, HO–NH); Anal. Calcd for C24
.23; N, 3.16. Found: C, 65.25; H, 5.33; N, 3.12.
H
23
F
2
NO S: C, 64.99; H,
3
1
,1-Diphenyl-2-(4-hydroxyaminosulfonylphenyl)hex-1-ene (13e).
◦
Yield, 80.3%; white solid; mp 150–152 ; IR (film): 3401, 3243,
-
1
1
2
0
964, 2923, 2857, 1325, 1172 cm ; H NMR (DMSO-d
.67 (t, J = 6.7 Hz, 3H, CH CH CH CH ), 1.12–1.19 (m, 4H,
CH CH CH ), 2.40 (t, J = 7.4 Hz, 2H, CH C), 6.89
d, J = 6.7 Hz, 2H, phenyl hydrogens), 7.00–7.10 (m, 3H,
6
): d
In vitro nitric oxide release assay
2
2
2
3
CH
2
2
2
3
2
C
In vitro nitric oxide release, upon incubation of the test compound
-
2
(
(2.4 mL of 5.0 ¥ 10 mM) with either i) phosphate buffer solution
◦
phenyl hydrogens), 7.23–7.43 (m, 7H, phenyl hydrogens and
sulfonylphenyl H-2, H-6), 7.62 (d, J = 7.9 Hz, 2H, sulfonylphenyl
H-3, H-5), 9.55 and 9.59 (two d, J = 3.7 and 3.1 Hz, respectively,
H each, HO–NH). Anal. Calcd for C24
.18; N, 3.44. Found: C, 70.36; H, 6.22; N, 3.32.
(PBS) at pH 7.4 and 37 C for 1.5 h, ii) PBS containing 50
◦
mM K (FeCN ) at pH 7.4 and 37 C, or iii) PBS at pH 7.4 and
3
6
◦
37 C to which 90 mL rat serum had been added, was determined
by quantification of nitrite produced by the reaction of nitric
oxide with oxygen and water using the Griess reaction. Nitric
oxide release data were acquired for test compounds (13a–g) and
the reference compound Piloty’s acid (PA) using the reported
1
6
H
25NO S: C, 70.73; H,
3
1
,1-Di-(4-methylphenyl)-2-(4-hydroxyaminosulfonylphenyl)hex-
◦
1
-ene (13f). Yield, 78.8%; yellowish solid; mp 153–154 C;
32
procedures.
-
1
1
IR (film): 3407, 3240, 2957, 2921, 2866, 1327, 1170 cm ; H
NMR (DMSO-d ): d 0.72 (t, J = 6.7 Hz, 3H, CH CH CH CH ),
.17–1.18 (m, 4H, CH CH CH CH ), 2.14 (s, 3H, ArCH ), 2.31
s, 3H, ArCH ), 2.40 (t, J = 7.4 Hz, 2H, CH C), 6.73 and 6.87
two d, J = 7.9 Hz, 2H each, tolyl H-3, H-5), 7.08 and 7.19 (two
d, J = 7.9 Hz, 2H each, tolyl H-2, H-6), 7.35 (d, J = 7.9 Hz, 2H,
sulfonylphenyl H-2, H-6), 7.63 (d, J = 7.9 Hz, 2H, sulfonylphenyl
H-3, H-5), 9.55 and 9.59 (two d, J = 3.1 Hz, 1H each, HO–NH).
6
2
2
2
3
Gas chromatographic N O analysis
2
1
(
(
2
2
2
3
3
3
2
C
For headspace analysis, substrate (0.04 mmol) was placed in a
10 mL round bottom flask, which was sealed with a rubber septum
and flushed with inert gas. Solvent (0.8 mL) was added, and
headspace aliquots (0.25 mL) were injected at 2 and 24 h onto a
7890A Agilent Technologies Gas Chromatograph equipped with a
thermal conductivity detector and a 6¢ x 1/8” Porapack Q column.
Anal. Calcd for C26
C, 71.89; H, 6.78; N, 3.23.
H
29NO S: C, 71.69; H, 6.71; N, 3.22. Found:
3
◦
◦
The oven operated at 40 C for 5 min was then ramped to 150 C
This journal is © The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 4124–4130 | 4129

View Article Online
over 4.5 min for a total run time of 9.5 min. The purged packed
inlet with a total flow (He as carrier gas) of 18 mL min and a
D. Das, G. Yu, C. Vel a´ zquez, M. R. Suresh and E. E. Knaus, Bioorg.
Med. Chem., 2009, 17, 5182.
1 J. D. Irvine, D. H. Ritchie, J. L. Favalora, K. L. Andrews, R. E. Widdip
and B. K. Kemp-Harper, Trends Pharmacol. Sci., 2008, 29, 601.
12 N. Paolocci, W. F. Saavedra, K. M. Miranda, C. Martignani, T. Isoda,
J. M. Hare, M. G. Espey, J. M. Fukuto, M. Feelisch, D. A. Wink
and D. A. Kass, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 10463; N.
Paolocci, T. Katori, H. C. Champion, M. E. St. John, K. M. Miranda,
J. M. Fukuto, D. A. Wink and D. A. Kass, Proc. Natl. Acad. Sci.
U. S. A., 2003, 100, 5537.
3 P. Pagliaro, D. Mancardi, R. Rastaldo, C. Penna, D. Gattullo, K. M.
Miranda, M. Feelisch, D. A. Wink, D. A. Kass and N. Paolocci, Free
Radical Biol. Med., 2003, 34, 33.
4 E. Bermejo, D. A. S a´ enz, F. Alberto, R. E. Rosenstein1, S. E. Sara,
E. Bari and M. A. Lazzari, Thromb. Haemost., 2005, 94, 578; M. D.
Hoffman, G. M. Walsh, J. C. Rogalski and N. Kast, Mol. Cell.
Proteomics, 2009, 8, 887.
5 (a) O. Piloty, Ber. Dtsch. Chem. Ges., 1896, 29, 1559; (b) S. B. King and
H. T. Nagasawa, Chemical approaches toward generation of nitroxyl
(HNO), in Methods in Enzymology; ed. L. Packer, Academic Press,
New York, 1998, pp 211-220.
-
1
◦
1
-
1
septum purge flow of 3 mL min was held at 140 C. The back
-
1
detector with a reference flow of 9 mL min and a make-up flow
of 6 mL min- was held at 150 C. The retention time of nitrous
oxide was 2.5 min, and yields were calculated based on a standard
curve for nitrous oxide (Matheson Tri-Gas).
1
◦
1
Acknowledgements
We are grateful to the Canadian Institutes of Health Research
1
(
CIHR) (MOP-14712) for financial support of this research. The
excellent technical assistance of Ying Dong in performing the anti-
inflammatory assays is greatly appreciated.
1
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130 | Org. Biomol. Chem., 2010, 8, 4124–4130
This journal is © The Royal Society of Chemistry 2010