Synthesis, structure-activity relationships, and in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and orally active cyclooxygenase-2 inhibitors. 1. Thiazolone and oxazolone series

Article abstract of DOI:10.1021/jm9805081

Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). Initial mass screening and subsequent structure-activity relat

Full text of DOI:10.1021/jm9805081

J . Med. Chem. 1999, 42, 1151-1160
1151
Syn th esis, Str u ctu r e-Activity Rela tion sh ip s, a n d in Vivo Eva lu a tion s of
Su bstitu ted Di-ter t-bu tylp h en ols a s a Novel Cla ss of P oten t, Selective, a n d
Or a lly Active Cyclooxygen a se-2 In h ibitor s. 1. Th ia zolon e a n d Oxa zolon e Ser ies1
Yuntao Song,*,† David T. Connor,† Robert Doubleday,† Roderick J . Sorenson,† Anthony D. Sercel,†
Paul C. Unangst,† Bruce D. Roth,† Richard B. Gilbertsen,‡ Kam Chan,‡ Denis J . Schrier,‡ Antonio Guglietta,‡
Dirk A. Bornemeier,§ and Richard D. Dyer§
Departments of Chemistry, Biochemistry, and Immunopathology, Parke-Davis Pharmaceutical Research, Division of
Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received September 4, 1998
Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory
agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs
(NSAIDs). Initial mass screening and subsequent structure-activity relationship (SAR) studies
have identified 4b (PD138387) as the most potent and selective COX-2 inhibitor within the
thiazolone and oxazolone series of di-tert-butylphenols. Compound 4b has an IC50 of 1.7 µM
against recombinant human COX-2 and inhibited COX-2 activity in the J 774A.1 cell line with
an IC50 of 0.17 µM. It was inactive against purified ovine COX-1 at 100 µM and did not inhibit
COX-1 activity in platelets at 20 µM. Compound 4b was also orally active in vivo with an ED40
of 16 mg/kg in the carrageenan footpad edema (CFE) assay and caused no gastrointestinal
(GI) damage in rats at the dose of 100 mg/kg but inhibited gastric prostaglandin E2 (PGE2)
production in rats’ gastric mucosa by 33% following a dose of 100 mg/kg. The SAR studies of
this chemical series revealed that the potency and selectivity are very sensitive to minor
structural changes. A simple isosteric replacement led to the reversal of selectivity.
Ch a r t 1
In tr od u ction
Prostaglandins are important mediators of inflam-
mation2 and also provide cytoprotection in the stomach
and intestine.3 Consequently, common nonsteroidal
antiinflammatory drugs (NSAIDs) which inhibit cy-
clooxygenase (COX), a key enzyme involved in prosta-
glandin production,2 have had major side effects, such
as gastrointestinal (GI) hemorrhage and ulceration.4
Separation of toxicity from efficacy has been a major
focus in the search for new NSAIDs. Recently, it was
discovered that COX exists in two isoforms, COX-1 and
COX-2,5 and that the two isoforms are regulated dif-
ferently: COX-1 is expressed constitutively, whereas
COX-2 is transiently upregulated by proinflammatory
mediators and downregulated by corticosteroids.6 These
discoveries suggest the possibility that constitutive
COX-1 protects the GI tract, whereas inducible COX-2
mediates inflammation. This difference in function
provides an opportunity to separate toxicity from ef-
ficacy of NSAIDs by selectively inhibiting COX-2. In-
deed, in vivo studies with several known selective
COX-2 inhibitors, 1 (SC-58125),7 2 (NS-398),8 and 3 (L-
745,337)9 (Chart 1), have shown that they have potent
antiinflammatory activity with little GI toxicity.
tor strategy. The possibility that these compounds would
be selective COX-2 inhibitors was investigated, and a
structure-activity relationship (SAR) for selective in-
hibitors of COX-2 was developed in several series. In
this paper, we report the SAR studies on thiazolone and
oxazolone series, as well as pharmacological evaluations
of selective inhibitors in carrageenan footpad edema
(CFE) assay and in a hyperalgesia model. The GI safety
profiles of selected compounds are also described.
Ma ss Scr een in g
The Parke-Davis chemical library was screened for
compounds that would inhibit the conversion of [14C]-
arachidonic acid by sheep placental COX-2 (oCOX-2)
and ram seminal vesicle COX-1 (oCOX-1). Substituted
2,6-di-tert-butylphenols with a generic structure of I
(Chart 2) were identified as potent and selective inhibi-
tors. This chemical class was attractive to us for several
reasons: first, substituted di-tert-butylphenols are struc-
turally different from the reported selective COX-2
inhibitors (1-3).7-9 Second, BF-389,11 a di-tert-bu-
tylphenol derivative, has been shown to have potent
inhibitory effects toward COX-2 activity in intact cells
Prior to the discovery of two isoforms of the cyclooxy-
genase, our efforts on a dual cyclooxygenase and 5-li-
poxygenase inhibitor program10 had identified a number
of potent COX inhibitors. However, some potent COX
inhibitors, lacking inhibitory activity toward 5-lipoxy-
genase, were not fully evaluated under the dual inhibi-
† Department of Chemistry.
‡ Department of Immunopathology.
§ Department of Biochemistry.
10.1021/jm9805081 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/17/1999

1152 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
Ta ble 1. Compounds 5-7 Shown in Scheme 1
Ch a r t 2
no.
6a , 7a
R1
R2
H
H
5b, 6b, 7b
5c, 6c, 7c
5e, 6e, 7e
5f, 6f, 7f
5 g, 6g, 7g
5h , 6h , 7h
OH
OH
OH
OMe
OMe
H
Br
I
i-Pr
i-Pr
t-Bu
t-Bu
(IC50 ) 0.09 µM) with a 5-fold selectivity favoring COX-
2. However BF-389 was a nonselective and weak COX
inhibitor based on purified enzyme assays with IC50
values of 24 µM against COX-2 and 12 µM against COX-
1. Di-tert-butylphenol derivatives 4a ,b (PD138387) are
more potent and selective for COX-2 versus COX-1
based on enzyme assays (Table 2). Third, several
compounds in this class are known antiinflammatory
agents, and some of them have a favorable GI safety
profile. Compounds 4a ,b are congeners of 4f (CI-1004)
which is currently under clinical development as a dual
inhibitor of cellular leukotriene and prostaglandin
production. Consequently, a great deal of knowledge
about the pharmacokinetic and toxicological properties
of this series had been accumulated. Therefore, 4a ,b
were chosen as chemical leads for further SAR studies.
Sch em e 1. Synthesis of Thiazolone Derivatives
Com p ou n d Eva lu a tion s
The primary assays for the compounds described in
this SAR study were two purified enzyme assays.
Recombinant human COX-2 (rhCOX-2) and ram semi-
nal vesicle COX-1 (oCOX-1) were used to measure the
inhibitory activity against isolated enzymes in a cell-
free environment. Compounds of interest were also
tested for their inhibitory effects toward the activity of
both enzymes in a cellular environment. Human platelet-
rich plasma was used for the evaluation of COX-1
activity and a murine macrophage cell line (J 774A.1)
for COX-2. Selected compounds were also evaluated in
the CFE, in a hyperalgesia model in mice, and in GI
safety models for gastric toxicity and inhibition of PGE2
synthesis in rats. SAR development was primarily
guided by biological evaluations in the two purified
enzyme assays.
Ch em istr y
In the case of the oxazolones, reaction of 10 (Scheme
3) with O-methylhydroxylamine or hydroxylamine in
ethanol at reflux failed to give the desired products.
Instead, a ring-opened product 2012 was isolated in the
reaction of 10 with O-methylhydroxylamine. However,
reactions in ethanol at room temperature, using 0.8
equiv of amine, did furnish the desired products 11a -d
(Table 2), albeit in low yields.
Thiazolones 8a ,ea ,eb,f-h were prepared according
to a procedure reported earlier by Unangst et al.10a
(Scheme 1). Knovevenagel condensation of aldehydes
5b,c,e-h with rhodanine gave 6b,c,e-h in good to
excellent yields. Methylation with methyl iodide pro-
vided thiazolones 7a -c,e-h in excellent yields. Reac-
tion of 7a ,e-h with appropriate amines afforded thi-
azolones 8a ,ea ,eb,f-h . Compounds 5-7 are listed in
Table 1.
Thiazolones 4c,d ,g,h -n (Tables 2 and 4; Table 4 is
provided as Supporting Information) were synthesized
by reaction of thiazolones 9 with appropriate amines.
Compounds 4o,p were prepared by acylation and sul-
fonylation of 4f, respectively (Scheme 2).
The preparation of 4a ,b,e,f,q-s, 9, 10, 11e, 12a -c,
13-15, 16a -d , 17a ,b, 18a -e, and 19a ,b has been
reported previously.13-16
Resu lts a n d Discu ssion
Str u ctu r e-Activity Rela tion sh ip s a n d Discu s-
sion of th e En zym e Da ta . COX enzyme catalysis is

COX-2 Inhibitors. 1. Thiazolones and Oxazolones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1153
Sch em e 2. Preparation of Di-tert-butylphenol-Derived
Thiazolone Analogues
or COX-1. COX-2 and COX-1 are highly homologous
enzymes that use similar catalytic mechanisms, yet 4b
showed good selectivity for COX-2 over COX-1, which
suggests that the two tert-butyl groups or the thiazolone
moiety or the combination of both must be the structural
elements which are responsible for the selectivity
toward the rhCOX-2 observed with 4b.
To investigate the substitution effects of R2, we
focused on three selective COX-2 inhibitors (4b, 9, and
12b) and one selective COX-1 inhibitor (4e) and changed
the tert-butyl groups to other smaller lipophilic groups.
The SAR studies reveal that the potency and selectivity
requirements of R2 are dependent upon the nature of
R4. When R4 is NHOMe or NHC(dNH)NH2, compounds
with both tert-butyl (4b ,e) and isopropyl (8ea ,eb )
substitutions as R2 gave potent and selective com-
pounds. When R4 is SMe or SH, the change of tert-butyl
groups to any of the following groups: isopropyl (7e and
13), I (7c), Br (7b and 14), or OMe (15), abolished the
activity against both enzymes. Selective inhibition,
toward COX-2 or COX-1, favors tert-butyl groups as R2.
In the case of 4b and 8ea , the potency and selectivity
of the two compounds were similar. The tert-butyl group
still is optimal since 8ea failed to inhibit COX-2 in the
cellular assay. Another reason for favoring tert-butyl
groups is the prevention of potential O-glucuronidation
of the phenolic hydroxy group in vivo. It has been
observed in series of antiinflammatory dialkylphenols
that two tert-butyl groups flanking the OH group are
required to retain in vivo antiinflammatory potency.19
Sch em e 3. Preparation of Oxazolone Derivatives
Having established the di-tert-butylphenol as optimal,
we focused on the SAR around the thiazolone portion
of the molecule in this investigation. Isosteric replace-
ment of the sulfur atom with an oxygen atom resulted
in the reversal of selectivity. Several thiazolones were
selective COX-2 inhibitors; in contrast, the correspond-
ing oxazolones were selective COX-1 inhibitors. This
phenomenon was observed when R4 is NHOMe (4b vs
11b), NHOH (4a vs 11a ), NHOEt (4c vs 11c), and NHO-
allyl (4d vs 11d ), as well as when R4 is OH (17a vs 17b),
SH (19a vs 19b), or SMe (9 vs 10). The only exception
is when R4 is NHC(dNH)NH2, where the trend is
reversed: the thiazolone 4e was much more selective
for COX-1 than the corresponding oxazolone 11e. The
reason for the observed differences in activity between
thiazolones and oxazolones is not clear.
thought to involve radical intermediates,17 especially the
phenoxy radical formed on a tyrosine residue.18 Our
chemical leads are substituted phenols which are ca-
pable of forming phenoxy radicals. Therefore, it is
possible that the substituted phenol inhibitors could
simply function as radical scavengers. To address this
issue, compounds 8g,f, replacing the exchangeable
proton with a methyl group, 8h , deleting the phenolic
hydroxy group, and 8a , removing both hydroxy and tert-
butyl groups, were synthesized and evaluated in puri-
fied enzyme assays and cellular assays. As shown in
Tables 2 and 4, against COX-2, 8g was about 40-fold
less active than 4b and 8f was essentially inactive. The
deshydroxy compounds 8h ,a were also inactive against
both enzymes. The data demonstrate the importance of
the hydroxyl group for the inhibitory activity against
COX-2.
Isosteric replacement of the sulfur atom with a NMe
group abolished the activity or decreased the potency
against COX-2 independent of R4 (19a vs 16a , 9 vs 16b,
4r vs 16c, 4e vs 16d ). The isosteric replacement of the
sulfur atom with a NH group, however, affected the
activity and selectivity differently, depending on the
nature of R4. For example, when R4 is SMe or OH, The
change of a sulfur atom to NH (12a ,c) gave inactive
compounds against both enzymes (9 vs 12c, 17a vs 12a ).
When R4 is SH, the change of the sulfur atom to a NH
group resulted in compound 12b with increased potency
against both enzymes, but the selectivity was decreased
(19a vs 12b). The change of R4 from NHOMe to
NHCNHCNH2 also resulted in reversal of selectivity
leading to a potent and selective COX-1 inhibitor, 4e.
Compounds with a guanadine group as R4 are selective
COX-1 inhibitors in all cases studied: when X is S, 4e;
O, 11e; or NMe, 16d . Here again, the thiazolone and
It was clear at the beginning that the inhibitory effect
of these substituted di-tert-butylphenols, whether by
radical scavenging or not, is selective for either COX-2

1154 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
Ta ble 2. Enzyme and Cellular Activity of Benzylidene Derivatives 4, 8-12, and 16-19 and 1, 2, and Indomethacina,b,e
IC50 (µM) ( SEM
IC50 (µM)
rhCOX-2
1.5
COX-2
COX-1
compd
X
R1
R2
R3
R4
oCOX-1
J 774A.1
hplatelet
4a
4b
4c
4d
4e
4f
4q
4r
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
i-Pr
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
NHOH
NHOMe
NHOEt
NHO-allyl
NHC(dNH)NH2
NH2
NMeOMe
NHCN
NHOMe
NHC(dNH)NH2
SMe
SMe
NHOMe
NHOEt
52
0.17 ( 0.10
0.17 ( 0.070
0.27 ( 0.14
1.6 ( 1.0
0.43 ( 0.32
0.069 ( 0.027
1.6 ( 0.22
0.23 ( 0.15
NA
9.2 ( 2.7
1.7
3.2
2.7
21
NA
NA
NA
0.0061
NA
51
NA
NA
0.22
63
1.6
7.3
3.5
4
1.2
1.3
0.027
42
0.095
0.46
24
NA
NA
0.0122
NA
NA
NA
0.25 ( 0.50
0.60 ( 0.19
5.8 ( 0.056
NA
41%@10 µMc
18
55
0.57
1.8
8ea
8eb
9
NA
i-Pr
0.19 ( 0.96
2.7 ( 0.80
14.4 ( 5.7
6.4 ( 2.0
NA
NA
NA
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
1.8
1.6 ( 0.19
10
NAd
NA
79
0.54 ( 0.29
0.79 ( 0.33
0.65 ( 0.081
1.9 ( 0.18
11b
11c
11d
11e
12b
16d
17a
17b
18e
19a
1
NHO-allyl
NHC(dNH)NH2
SH
NHC(dNH)NH2
OH
26
5.1
0.26
81
4.7
34
12
1.5
0.44 ( 0.17
0.083 ( 0.049
0.75 ( 0.48
0.06 ( 0.030
0.071 ( 0.051
0.18 ( 0.061
0.39 ( 0.23
0.070 ( 0.047
0.022 ( 0.008
0.0064 ( 0.001
4.4 ( 1.5
0.99 ( 0.26
1.5 ( 0.45
9.3 ( 4.4
1.2 ( 0.33
0.59 ( 0.23
NA
NH
NMe
S
O
S
OH
NHC(dNH)NH2
SH
S
0.31
0.19
5.8
NA
2
3.3 ( 0.87
0.0082 ( 0.001
indomethacin
a
NA, not active (in purified enzyme assays, NA is defined as less than 20% inhibition at 10 µM; in cellular assays, NA is defined as
b
IC50 value greater than 20 µM). Standard errors for the IC50 determinations in purified enzyme assays averaged 25% for rhCOX-2 and
14% for oCOX-1. c Percent inhibition at 10 µM. Found to be inactive against oCOX-2, not further tested. e Enzyme and cellular activity
d
of the following compounds is summarized in Table 4 which is provided as Supporting Information: 4g-p , 4s, 7b,c,e, 8a ,f-h , 11a ,
12a ,c, 13-15, 16a -c, 18a -d , and 19b.
oxazolone showed marked differences in selectivity:
thiazolone 4e was more than 3400-fold selective, whereas
the oxazolone 11e is only 4-fold selective for COX-1
inhibition.
with bulkier substituents on the oxygen atom (4l-n )
are not active against either enzyme.
The effect of R3 on potency and selectivity was also
examined. In most cases, the methyl substitution abol-
ished activity against both enzymes (18a -d ). When R4
is NHC(dNH)NH2, the change of H to a methyl group
decreased the potency against COX-1 (4e vs 18e).
In summary, potency and selectivity are extremely
sensitive to minor changes in chemical structure within
this chemical series. The degree of sensitivity might be
best illustrated by the results of isosteric replacement
studies where the selectivity was completely reversed
when a sulfur atom was replaced by an oxygen atom in
all the cases studied. The phenolic OH group is essential
for potency, and the change of the tert-butyl group to
other groups mainly affected potency, whereas changes
around the thiazolone moiety affected both potency and
selectivity.
En zym e P oten cy ver su s Cellu la r Activity. Evalu-
ation of the cellular and isolated enzyme data revealed
no apparent correlation between the two for either
potency or selectivity. However, in general, compounds
that were selective for COX-2 in the enzyme assays
tended to retain their selectivity in the cellular assays
(4a -d and 17a as examples). In the case of compounds
showing selectivity for COX-1 in the enzyme assays,
selectivity observed in the isolated enzyme assays was
not predictive of selectivity in the cellular assays. For
Within the thiazolone series, we investigated the
effect of R4 in detail. When the proton on the nitrogen
was replaced with a methyl group, 4q, both the potency
and selectivity were decreased. We kept the proton and
changed the other group on the nitrogen to an array of
substituents ranging from electron-donating to electron-
withdrawing groups (4g-j,b,s,f,r ,o,p ), so that the pro-
ton on the nitrogen could have a range of acidity.
However, we did not observe any correlation between
the potency and electronic nature of the substituents.
It seems that the nature of the chemical bond between
the nitrogen atom and the substituents attached to it
is critical for determining potency and selectivity. An
N-O bond gives compounds (4a -d ) which are potent
and selective COX-2 inhibitors. N-H bond (4f) leads to
weak COX-2 inhibition. An N-C (4r ,s,o) and N-N (4g-
j) bonds lead to loss of potency against both enzymes.
In all cases studied, it is quite clear that the N-O bond
is exclusively preferred for potency.
We also studied the effect of bulk on the potency and
selectivity within a compound series where X ) S, R1
) OH, R2 ) t-Bu, R3 ) H, and R4 contains an N-O bond.
Compounds are potent and selective when R4 is NHOH
or NHOR, where R is lower alkyl (4a -d ). Compounds

COX-2 Inhibitors. 1. Thiazolones and Oxazolones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1155
Ta ble 3. In Vivo Activity of Thiazolones 4b‚choline, 4q, and Standards 1, 2, and Indomethacin
ED40 (mg/kg)
lesion frequency
(%)
damaged
area (%)
ulcer
index
PGE2 synthesis inhibition
compd
CFEa
hyperalgesiab
(% ( SEM)
4b‚cholinec
15.7
8.1
0.61
>30
1.6
0.1
0.06
7.4
0.3
0.6
0.00
25.00
0.00
0.00
75
0.00
0.04
0.00
0.00
0.18
0.00
0.91
0.00
0.00
13.4
32.8 ( 4.43
52.2 ( 14.5
30.7 ( 12.1
-1.58 ( 13.5
96.5 ( 0.47
4q
2
1
indomethacin
a
b
Compounds dosed orally, 10 rats/test group. Compounds dosed orally, 8 mice/test group. c Compound 4b‚choline is the choline salt
of 4b.
example, the most selective COX-1 inhibitor, 4e, was
potency and selectivity profiles observed with 4b,q in
vitro: 4b is the most selective COX-2 inhibitor in this
chemical series and did not show GI toxicity when tested
as a choline salt, whereas 4q is a less selective COX-2
inhibitor and did show some ulceration in rats.
almost equally potent in inhibiting the function of both
enzymes in cellular assays, and several compounds
(11b-d a n d 17b) that are selective COX-1 inhibitors
in the enzyme assays have the opposite selectivity in
cellular assays. Illustrative of the lack of correlation
between the purified enzyme and cellular assays, in
several instances, compounds (4f,s) inactive in both
enzyme assays could be very potent in the corresponding
cellular assays leading to the speculation that these
compounds may possess other pharmacological activity
(vide infra).
Within this chemical series, compounds 4b,c were
identified as the most potent and selective COX-2
inhibitors based on the data from both isolated enzyme
and cellular assays. The most potent and selective
COX-1 inhibitors, based on the enzyme assay, are 4e,
17b, and 16d . Thus compound 4b was selected for
evaluation in vivo.
In Vivo Eva lu a tion s. Selective COX-2 inhibitors,
based on both enzyme and cellular assays, were tested
in the CFE and hyperalgesia assays. The results are
shown in Table 3. Compound 4b, the most potent and
selective inhibitor in vitro, was tested in vivo as its
choline salt. The choline salt of 4b (4b‚choline) was
active in CFE (ID40 ) 16 mg/kg) and also had potent
analgesic activity in the acetic acid writhing test (ED40
) 0.1 mg/kg). However, compound 4q, a weak and less
selective COX-2 inhibitor, was more potent than 4b‚
choline in CFE (ID40 ) 8.1 mg/kg) and very potent in
the analgesic assay (ED40 ) 0.06 mg/kg), suggesting that
additional pharmacological properties of the substituted
di-tert-butylphenols may contribute to efficacy in vivo.
Compounds 4b‚choline and 4q, together with refer-
ence standards indomethacin, 1, and 2, were further
evaluated for their gastric toxicity and inhibition of
gastric PGE2 synthesis in rats (Table 3). Compound 1
was evaluated here even though it did not show activity
in CFE in our hands. As expected for selective COX-2
inhibition, compounds 1 and 2 did not cause ulceration
at the dose of 100 mg/kg. Compound 2 inhibited PGE2
production in the rats’ gastric mucosa by 31%. Slight
enhancement of PGE2 production was observed with 1.
Indomethacin, on the other hand, caused ulcers in rats
with an ulcer index (ulcer index ) % frequency × % of
gastric area damaged) of 13. No GI damage was detected
with 4b‚choline at a dose of 100 mg/kg, although 33%
inhibition of PGE2 production was observed, which is
about the same as that observed with 2. Minimal GI
damage was detected with 4q at a dose of 100 mg/kg,
and 52% inhibition of PGE2 production was observed,
slightly higher than observed with 4b‚choline. The
gastric toxicity and inhibition of PGE2 synthesis in vivo
observed with 4b‚choline and 4q are consistent with the
Con clu sion
SAR studies on these thiazolone- and oxazolone-
derived 2,6-di-tert-butylphenol series have shown that
potency and selectivity are extremely sensitive to minor
changes in chemical structure. The isosteric replace-
ment of a sulfur atom with an oxygen resulted in
reversal of selectivity in all cases studied. Several
compounds showed good potency and selectivity against
COX-2 in both its purified form and a cellular environ-
ment. Compound 4b, the most potent and selective
compound identified in this study, was an orally active
antiinflammatory agent and lacked GI side effects when
tested as a choline salt, further substantiating the
hypothesis that the antiinflammatory activity of NSAIDs
is derived from inhibition of COX-2 and their GI toxicity
from inhibition of COX-1. Compounds reported here
represent a new class of selective COX-2 inhibitors that
are structurally different from those previously re-
ported. Further improvements on the potency of these
compounds could lead to novel and safe antiinflamma-
tory agents with potential therapeutic utilities.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Thomas-
Hoover capillary apparatus and are uncorrected. Infrared
spectra were recorded using KBr disks on a Nicolet MX-1 FTIR
spectrometer. Elemental analyses were performed by Robert-
son Microlit, Inc. (Madison, NJ ) and were within (0.4% of the
theoretical values, unless indicated otherwise. Proton NMR
spectra were recorded on a Bruker AM 400-MHz spectrometer,
with chemical shifts reported in δ units relative to internal
TMS. Mass spectra were obtained by using a Micromass Trio-
2000 (APCI), PlatformLC (APCI), or Micromass Trio-2A (EI
and CI) mass spectrometer. Reactions were generally run
under a nitrogen atmosphere. Organic solutions were concen-
trated at house vacuum on a rotary evaporator. Flash chro-
matography was performed with ICN Biomedicals silica gel
60, 63-200 µm, according to the method of Still.20
P u r ified En zym e Assa ys. Mass screening of the Parke-
Davis compound library was carried out using commercially
obtained sheep placental COX-2 (oCOX-2; Cayman Chemical,
Ann Arbor, MI). For all the compounds included in the SAR
studies, IC50 values against purified enzymes were determined
using recombinant human COX-2 (rhCOX-2, purified from
baculovirus-infected SF-9 cells21) and commercially obtained
ram seminal vesicle COX-1 (oCOX-1; Cayman Chemical, Ann
Arbor, MI). Inhibition assays were conducted in 50 mM
phosphate buffer, pH 7.5, containing 2 mM epinephrine as
cofactor for the COX-catalyzed peroxidase reaction and 20 µM
[14C]arachidonic acid as substrate. rhCOX-2 or oCOX-1 in
Tween-20-containing buffer was added in sufficient amount

1156 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
to convert 20-30% of added substrate to products during 1-min
incubation. Reaction products were separated and measured
by radiometric HPLC, and percent inhibition was computed
by comparison of compound-treated to vehicle control incuba-
tions. The concentration of compound causing 50% inhibition
(IC50) was estimated using the software package Kaleida-
Graph, version 3.0.1, running on a Macintosh Centris 650
using operating system 7.1. Percent inhibition versus inhibitor
concentration data were fit to the two-parameter equation: %
the CUE3 imaging software. To take into account both the
frequency and extent of gastric damage, data were expressed
as ulcer index (ulcer index ) % frequency × % of gastric area
damaged). Indomethacin was also tested and used as a positive
control.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h ydr oxyben zyliden e)-2-(m eth -
oxya m in o)t h ia zol-4-on e Ch olin e Sa lt (4b‚ch olin e). A
solution of 80% choline in water (0.84 g, 4.1 mmol) was added
to a stirred suspension of (Z)-5-(3,5-di-tert-butyl-4-hydroxy-
benzylidene)-2-(methoxyamino)thiazol-4-one (4b) (1.50 g, 4.1
mmol) in 40 mL of ethanol under an inert atmosphere and
heated to reflux. After 1 h the resulting solution was stripped
of solvent under reduced pressure to afford a glass, which was
crystallized from 10-15 mL of ethyl acetate. The suspension
was cooled and the solid filtered off, washed twice with ethyl
acetate and then twice with ether, and dried to afford the pure
product in two crops (1.7 g, 89% yield): mp 170 °C; IR (KBr)
inhibition ) 100/(1 + (inhibitor concentration/IC50)
slope), and
best fits for IC50 and slope coefficient were determined by least-
squares analysis. Standard errors for replicate determinations
averaged 25% for rhCOX-2 and 14% for oCOX-1.
Cellu lar Assays. COX-1 assays utilized platelet-rich plasma
(PrP) from NSAID-free normal human volunteers. J 774A.1
(J 7), a murine macrophage cell line, was used to evaluate COX-
2, expression of which was induced by overnight incubation
with LPS (1 µg/mL) prior to assay. All tests were performed
in serum- and plasma-free medium except PrP, which had a
final plasma concentration of 3.1% (autologous). Assays were
performed in flat bottom 96-well plates (100 µL of cell
preparation, 100 µL of drug dilution). After a 60-min prein-
cubation of cells and test compounds, samples were spiked
with 3 µg of arachidonic acid and incubated from from 30 min
and the reaction stopped by the addition of 25 µL of 80 µM
indomethacin. TxB2 and PGE2 concentrations in clarified cell
supernatants were determined by EIA from Cayman Chemical
Co. (TxB2) and Assay Designs (PGE2), both in Ann Arbor, MI.
Percent inhibition was determined using the formula: {1 -
[(drug - medium)/(maximum - medium)]} × 100, where
“drug” is the pg/mL of PGE2 or TxB2 (prostanoid) in drug-
treated samples, “medium” is the average prostanoid in control
samples not treated with exogenous arachidonic acid, and
“maximum” is the average prostanoid in samples treated with
exogenous arachidonic acid in the absence of any drug sample.
IC50 values were determined by regression analysis of a plot
of percent inhibition (ordinate) versus drug concentration
(abscissa) using TableCurve2D (J andel, San Rafael, CA).
Ca r r a geen a n F ootp a d Ed em a . Male rats were injected
in the right hind paw with 0.1 mL of a 1% solution of
carrageenan in saline. Compounds were suspended in vehicle
(0.5% hydroxypropyl-methylcellulose (HPMC) containing 0.2%
Tween 80) and administered orally (10 mL/kg) 1 h before
carrageenan injection. The dose volume was adjusted to 5.0
mL with water. Paw volume was measured by mercury
plethysmography 5 h after carrageenan injection. Swelling was
compared in the compound- and vehicle-treated groups to
obtain percent inhibitions. ID40 values were determined by
linear regression analysis (see ref 22 for the details). A
Student’s t-test was done on each experimental group (com-
pared to vehicle) to evaluate statistical significance.
Hyp er a lgesia . Analgesic activity was measured by the
acetic acid-induced writhing test as previously described.23
Male Swiss-Webster mice were fasted for 16 h prior to oral
administration of compounds or vehicle (HPMC with Tween
80). Sixty minutes later, each animal was injected intraperi-
toneally with 0.6% acetic acid in saline (10 mL/kg). Writhing
was observed and tallied for 5 min, beginning 7 min after the
acetic acid injection. ID40 values were determined by linear
regression analysis. A two-way ANOVA was used to determine
the level of statistical significance for each experimental group.
Ga str ic Toxicity a n d In h ibition of P GE 2 Syn th esis in
Ra ts. Sprague-Dawley male rats were administered 100 mg/
kg of the compounds under study in 1 mL of 1% CMC solution.
Four hours later the animals were sacrificed. The stomachs
were removed and opened along the greater curvature. Their
images were digitized and stored on an optical disk using a
486-based computer equipped with CUE3 system imaging
analysis software. Two 6-mm biopsies were taken from a
constant region located in each side of the glandular portion
of the stomach, and their PGE2 content was measured using
a commercially available ELISA kit (Assay Designs, Inc., Ann
Arbor, MI). On the retrieved electronic image, the presence of
gastric damage was determined and its extent measured using
1
3420, 2955, 1549 cm-1; H NMR (DMSO-d6) δ 1.39 (s, 18H, 2
× t-Bu), 3.10 (s, 9H, HOCH2CH2N(CH3)3), 3.40 (m, 2H, HOCH2-
CH2N(CH3)3), 3.62 (s, 3H, OCH3), 3.83 (m, 2H, HOCH2CH2N-
(CH3)3), 7.04 (s, 1H, Hvinyl), 7.24 (s, 2H, 2 × Harom); MS (+APCI)
m/z 362 (parent MH+). Anal. (C24H39N3O4S) C, H, N.
Gen er a l P r oced u r e A for th e P r ep a r a tion of 4c,d ,g,-
l,m ,n . To a mixture of 910a (1 equiv), amine hydrochloride (1.1
equiv), and ethanol (25 mL/1 g of 9) was added potassium tert-
butoxide (1.1 equiv). The resultant reaction mixture was
refluxed for 2 h, then cooled to ambient temperature, and
poured onto ice-water. The precipitate formed was filtered
and recrystallized from the appropriate solvent to give the
desired product.
Gen er a l P r oced u r e B for th e P r ep a r a tion of 4h ,i,j,k .
To a mixture of 910a (1 equiv), amine (1.5 equiv), and ethanol
(25 mL/1 g of 9) was added potassium tert-butoxide (1.5 equiv).
The resultant reaction mixture was refluxed for 2 h, then
cooled to ambient temperature, and poured onto ice-water.
A cloudy red solution was obtained. Upon acidification with
concentrated aqueous HCl to pH ) 1, a precipitate formed.
Filtration and further purification gave the desired product.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h yd r oxyben zylid en e)-2-(eth -
oxya m in o)th ia zol-4-on e (4c). Compound 4c was prepared
by subjecting O-ethylhydroxylamine hydrochloride (0.443 g,
4.54 mmol) to general procedure A; 0.69 g (44%) of pure 4c
was obtained as a beige solid after two recrystallizations
successively from CH3OH/CH3CN and EtOAc/hexanes: IR
3624, 3606, 1689, 1637, 1606, 1593, 1438, 1424, 1241, 1206,
1050 cm-1 1H NMR (DMSO-d6) δ 1.22 (t, 3H, J ) 6.99 Hz,
;
CH3), 1.41 (s, 18H, 2 × t-Bu), 4.05 (q, 2H, J ) 6.99 Hz, CH2),
7.35 (s, 2H, 2 × Harom), 7.51 (s, 1H, Hvinyl), 7.66 (bs, 1H, OH),
12.05 (bs, 1H, NH); mp 252-254 °C; MS (+APCI) m/z 377
(MH+). Anal. (C20H28N2O3S) C, H, N.
(Z)-2-(Allyloxya m in o)-5-(3,5-d i-ter t-b u t yl-4-h yd r oxy-
ben zylid en e)th ia zol-4-on e (4d ). Compound 4d was pre-
pared by subjecting O-allylhydroxylamine hydrochloride to
general procedure A. Crude product, a yellow solid, was
triturated with methanol to give 1.2 g (56%) of pure 4d as a
light-yellow solid: 1H NMR (DMSO-d6) δ 1.41 (s, 18H, 2 ×
t-Bu), 4.54 (dt, 2H, J ) 5.55, 1.21 Hz, CH2), 5.21-5.33 (m,
2H, 2 × Hvinyl), 5.95-6.05 (m, 1H, 2 × Hvinyl), 7.34 (s, 2H, 2 ×
H
arom), 7.53 (s, 1H, Hvinyl), 7.67 (bs, 1H, OH), 12.09 (bs, 1H,
NH); mp 222-224 °C; MS (+APCI) m/z 389 (MH+). Anal.
(C21H28N2O3S) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h yd r oxyben zylid en e)-2-(p yr -
r olid in -1-yla m in o)th ia zol-4-on e (4g). Compound 4g was
prepared by subjecting 1-aminopyrrolidine hydrochloride to
general procedure A; 1.30 g (78%) of pure 4g was obtained as
a yellow solid after trituration with CH3CN: IR 3628, 3353,
1703, 1671, 1642, 1618, 1598, 1436, 1422, 1194 cm-1; 1H NMR
(DMSO-d6) δ 1.41 (s, 18H, 2 × t-Bu), 1.78 (m, 4H, 2 × CH2),
2.86 (m, 4H, 2 × CH2), 7.36 (s, 2H, 2 × Harom), 7.50 (s, 1H,
H
vinyl), 7.61 (bs, 1H, OH), 11.75 (bs, 1H, NH); mp 265-267 °C;
MS (+APCI) m/z 402 (MH+). Anal. (C22H31N3O2S) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h yd r oxyben zylid en e)-2-(m or -
p h olin -4-yla m in o)th ia zol-4-on e (4h ). Compound 4h was
prepared by subjecting N-aminomorpholine to general proce-

COX-2 Inhibitors. 1. Thiazolones and Oxazolones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1157
dure B. Crude product, a red solid, was triturated successively
with methanol/water and Et2O/hexanes to give 0.80 g (34%)
of pure 4h as a yellow solid: 1H NMR (DMSO-d6) δ 1.42 (s,
18H, 2 × t-Bu), 2.75 (m, 4H, 2 × CH2), 3.68 (m, 4H, 2 × CH2),
7.39 (s, 2H, 2 × Harom), 7.51 (s, 1H, Hvinyl), 7.63 (bs, 1H, OH);
mp >270 °C; MS (+APCI) m/z 418 (MH+). Anal. (C22H31N3O3S)
C, H, N.
Filtration and the subsequent drying under vacuum gave 0.11
g (27%) of 4o as a yellow solid: IR 3619, 1723, 1695, 1587,
1
1559, 1438, 1424, 1165 cm-1; H NMR (DMSO-d6) δ 1.42 (s,
18H, 2 × t-Bu), 2.23 (s, 3H, CH3), 7.48 (s, 2H, 2 × Harom), 7.78
(s, 1H, Hvinyl), 7.82 (s, 1H, OH), 12.82 (bs, 1H, NH); mp >270
°C; MS (+APCI) m/z 375 (MH+). Anal. (C20H26N2O3S‚0.20H2O)
C, H, N.
(Z)-5-(3,5-Di-ter t-b u t yl-4-h yd r oxyb en zylid en e)-2-h y-
d r a zin oth ia zol-4-on e (4i). Compound 4i was prepared by
subjecting hydrozane to general procedure B. Crude product,
a yellow solid, was triturated with acetonitrile and filtered,
and the filtrate was mixed with water forming a yellow
precipitate. Filtration and subsequent drying at 70 °C under
vacuum gave 0.51 g (18%) of pure 4i as a yellow solid: 1H NMR
(DMSO-d6) δ 1.42 (s, 18H, 2 × t-Bu), 5.45 (bs, 2H, NH2), 7.38
(s, 2H, 2 × Harom), 7.47 (s, 1H, Hvinyl), 7.57 (bs, 1H, OH); mp
245-246 °C; MS (+APCI) m/z 348 (MH+). Anal. (C18H25N3O2S)
C, H, N.
(Z)-N-[5-(3,5-Di-ter t-b u t yl-4-h yd r oxyb en zylid en e)-4-
oxo-4,5-d ih yd r ot h ia zol-2-yl]m et h a n esu lfon a m id e (4p ).
To a mixture of compound 4f10a (1.77 g, 4.79 mmol), Et3N (2.40
mL, 17.2 mmol), and dichloromethane (60 mL) was added
methanesulfonyl chloride (0.450 mL, 5.75 mmol). The solid
dissolved rapidly upon addition of methanesulfonyl chloride.
The resultant reaction mixture was stirred for 30 min at
ambient temperature. TLC indicated that there was still some
4f left; 0.4 mL of methanesulfonyl chloride was added; 10 min
after addition of the additional methanesulfonyl chloride, the
reaction solution was diluted with 40 mL of dichloromethane,
washed successively with 2% aqueous HCl (2 × 60 mL), brine
(2 × 60 mL), and water (2 × 60 mL), and then dried over Na2-
SO4. The dry solution was filtered through a pad of silica gel,
and the silica gel was washed with 20-40% of methanol in
dichloromethane (2 × 60 mL); the filtrate was concentrated
in vacuo to afford a solid. Recrystallization from EtOAC/
hexanes followed by trituration with CH3CN gave 0.3 g (15%)
of 4p as a bright-yellow solid: IR 3580, 1696, 1582, 1440, 1423,
(Z)-5-(3,5-Di-ter t-bu tyl-4-h ydr oxyben zyliden e)-2-([1,2,4]-
tr ia zol-4-yla m in o)th ia zol-4-on e (4j). Compound 4j was
prepared by subjecting 4-amino-1,2,4-triazole to general pro-
cedure B. Crude product, a yellow solid, was triturated
successively with methanol and CH3CN/EtOAc to give 0.93 g
(42%) of pure 4j as a beige solid: 1H NMR (DMSO-d6) δ 1.35
(s, 18H, 2 × t-Bu), 7.30 (s, 2H, 2 × Hphenol), 7.75 (s, 1H, Hvinyl),
7.82 (bs, 1H, OH), 8.76 (s, 2H, 2 × Htriazole); mp >270 °C; MS
(+APCI) m/z 400 (MH+). Anal. (C20H25N5O2S) C, H, N.
(Z)-{N-[5-(3,5-Di-ter t-b u t yl-4-h yd r oxyb en zylid en e)-4-
oxo-4,5-d ih yd r oth ia zol-2-yl]a m in ooxy}a cetic Acid (4k ).
Compound 4k was prepared by subjecting (aminooxy)acetic
acid hemihydrochloride to general procedure B. Crude product,
a yellow solid, was recrystallized from CH3CN/H2O, followed
by trituration with dichloromethane to give 2.2 g (66%) of pure
4k as a yellow solid: 1H NMR (DMSO-d6) δ 1.44 (s, 18H, 2 ×
t-Bu), 4.57 (s, 2H, CH2), 7.36 (s, 2H, 2 × Harom), 7.55 (s, 1H,
1346, 1196, 965 cm-1
;
1H NMR (CDCl3) δ 1.48 (s, 18H, 2 ×
t-Bu), 3.14 (s, 3H, CH3), 5.77 (s, 1H, OH), 7.40 (bs, 2H, 2 ×
Harom), 7.83 (s, 1H, Hvinyl), 8.79 (bs, 0.54H, NH), 10.25 (bs,
0.46H, NH); mp 235-237 °C; MS (+APCI) m/z 411 (MH+).
Anal. (C19H26N2O4S2) C, H, N.
3,5-Diisop r op yl-4-m eth oxyben za ld eh yd e (5f). Dimethyl
sulfate (3.3 g, 26 mmol) was added to a stirred mixture of 3,5-
diisopropyl-4-hydroxybenzaldehyde (4.5 g, 22 mmol) and po-
tassium carbonate (3.9 g, 28 mmol) in 100 mL of acetone under
an inert atmosphere and heated to reflux. After 2.5 h the
mixture was allowed to cool, and most of the acetone was
removed under reduced pressure. The residue was partitioned
between 200 mL of water and 200 mL of dichloromethane, and
the layers were separated. The aqueous phase was extracted
with 100 mL of dichloromethane, and the organic extracts were
combined, washed with 0.5 M K2CO3, water, and saturated
brine, and then dried over MgSO4. The solvent was removed
under reduced pressure to leave the product as a clear oil (4.9
g) of sufficient purity for the next reaction: 1H NMR (DMSO-
d6) δ 1.23 (d, 12H, 4 × CH3), 3.31 (septet, 2H, HCMe2), 3.75
(s, 3H, OCH3), 7.61 (s, 2H, ArH), 9.89 (s, 1H, CHO).
H
vinyl), 7.68 (bs, 1H, OH); mp 229-231 °C dec; MS (+APCI)
m/z 332 (M - OCH2CO2H + H+). Anal. (C20H26N2O5S) C, H,
N.
(Z)-2-(ter t-Bu toxyam in o)-5-(3,5-di-ter t-bu tyl-4-h ydr oxy-
ben zylid en e)th ia zol-4-on e (4l). Compound 4l was prepared
by subjecting O-tert-butylhydroxylamine to general procedure
A. Crude product, a yellow solid, was recrystallized twice from
hexanes to give 2.4 g (63%) of pure 4l as a light-yellow solid:
1H NMR (DMSO-d6) δ 1.26 (s, 9H, O-t-Bu), 1.41 (s, 18H, 2 ×
t-Bu), 7.35 (s, 2H, 2 × Harom), 7.49 (s, 1H, Hvinyl), 7.64 (bs, 1H,
OH), 12.02 (bs, 1H, NH); mp 177-179 °C; MS (+APCI) m/z
405 (MH+). Anal. (C22H32N2O3S) C, H, N.
(Z)-2-(Ben zyloxya m in o)-5-(3,5-d i-ter t-bu tyl-4-h yd r oxy-
ben zylid en e)th ia zol-4-on e (4m ). Compound 4m was pre-
pared by subjecting O-benzylhydroxylamine hydrochloride to
general procedure A. Crude product, a yellow solid, was
recrystallized successively from EtOAc/hexane, CH3CN/H2O,
CH3OH/CH3CN/H2O, and CH3CN to give 0.34 g (8%) of pure
4m as a light-yellow solid: 1H NMR (DMSO-d6) δ 1.42 (s, 18H,
2 × t-Bu), 5.07 (s, 2H, CH2Ph), 7.30-7.40 (m, 7H, 7 × Harom),
7.52 (s, 1H, Hvinyl), 7.68 (bs, 1H, OH), 12.07 (bs, 1H, NH); mp
193-194 °C; MS (+APCI) m/z 439 (MH+). Anal. (C25H30N2O3S)
C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h yd r oxyben zylid en e)-2-(p h e-
n oxya m in o)th ia zol-4-on e (4n ). Compound 4n was prepared
by subjecting O-phenylhydroxylamine hydrochloride to general
procedure A. Crude product, a yellow solid, was recrystallized
twice from acetonitrile/water to give 1.3 g (57%) of pure 4n as
golden plates: 1H NMR (DMSO-d6) δ 1.42 (s, 18H, 2 × t-Bu),
7.05-7.38 (m, 5H, 5 × Harom), 7.40 (s, 2H, 2 × Harom), 7.62 (s,
1H, Hvinyl), 7.72 (bs, 1H, OH), 12.45 (bs, 1H, NH); mp 194 °C
dec; MS (+APCI) m/z 425.3 (MH+). Anal. (C24H28N2O3S) C, H,
N.
(Z)-5-(3,5-Diisopr opyl-4-h ydr oxyben zyliden e)-2-th ioxo-
th ia zolid in -4-on e (6e). A mixture of 3,5-diisopropyl-4-hy-
droxybenzaldehyde (5e)24 (5.7 g, 28 mmol), rhodanine (3.5 g,
25 mmol), sodium acetate (7.5 g), and acetic acid (40 mL) was
stirred and heated to reflux under an inert atmosphere. After
16 h the mixture was allowed to cool, then poured into water
(500 mL), and stirred vigorously for 1.5-2 h. The precipitate
was filtered off, washed three times with water, and then
dried. Recrystallization from acetonitrile afforded 6e (6.8 g,
85%): mp 200-200 °C; IR (KBr) 2962, 1706, 1575, 1431 cm-1
;
1H NMR (DMSO-d6) δ 1.20 (d, 12H, CH3), 3.34 (m, 2H,
HCMe2), 7.27 (s, 2H, ArH), 7.61 (s, 1H, olefin), 9.15 (s, 1H,
OH), 13.69 (br s, 1H, NH). Anal. (C16H19NO2S2) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tylben zyliden e)-2-th ioxoth iazolidin -
4-on e (6h ). Prepared from 5h 25 and rhodanine by the proce-
dure described in the preparation of 6e. Recrystallization of
the purified product from ethyl acetate gave 11a in 63%
yield: mp 186-188 °C; IR (KBr) 2958, 1696, 1583, 1441 cm-1
;
1H NMR (DMSO-d6) δ 1.32 (s, 18H, CH3), 7.42 (s, 2H, ArH),
7.55 (s, 1H, olefin), 7.70 (s, 1H, ArH), 13.8 (br s, 1H, NH). Anal.
(C18H23NOS2) C, H, N.
(Z)-N-[5-(3,5-Di-ter t-b u t yl-4-h yd r oxyb en zylid en e)-4-
oxo-4,5-d ih yd r oth ia zol-2-yl]a ceta m id e (4o). To a mixture
of 4f10a (0.42 g, 1.1 mmol) and acetic anhydride (10 mL) was
added pyridine (5 mL). The resultant reaction solution was
stirred at ambient temperature for 90 min forming a precipi-
tate. The reaction solution was diluted with ether (20 mL).
(Z)-5-(3,5-Diiod o-4-h yd r oxyb en zylid en e)-2-t h ioxot h i-
a zolid in -4-on e (6c). Prepared from 5c26 and rhodanine by
the procedure described in the preparation of 6e. The crude
product was triturated in ethanol twice and then diethyl ether
twice to give 6c in 99% yield: mp >285 °C; IR (KBr) 3279,

1158 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
1
1720, 1592, 1569, 1455 cm-1; H NMR (DMSO-d6) δ 7.51 (s,
(septet, 2H, HCMe2), 3.72 (s, 3H, OCH3), 7.45 (s, 2H, ArH),
7.85 (s, 1H, olefin). Anal. (C18H23NO2S2) C, H, N.
1H, olefin), 7.96 (s, 2H, ArH). Anal. (C10H5I2NO2S2) C, H, N.
(Z)-5-(3,5-Diisopr opyl-4-m eth oxyben zyliden e)-2-th ioxo-
th ia zolid in -4-on e (6f). A mixture of 3,5-diisopropyl-4-meth-
oxybenzaldehyde (5f) (4.9 g, 22 mmol), rhodanine (3.0 g, 22
mmol), and â-alanine (1.6 g, 18 mmol) in acetic acid (50 mL)
was stirred and heated to reflux under an inert atmosphere.
After 2 h the mixture was cooled, poured into water (300 mL),
and stirred vigorously for 0.5 h. The precipitate was filtered
off, washed three times with water, and then dried to afford
6f (6.8 g, 92%): mp 169-170 °C. Recrystallization of a sample
from acetonitrile afforded the analytically pure product: mp
(Z)-5-(3,5-Di-ter t-bu tyl-4-m eth oxyben zylid en e)-2-(m eth -
ylsu lfa n yl)th ia zol-4-on e (7g). Prepared from 6g and iodo-
methane by the procedure described in the preparation of 7a .
Recrystallization from acetonitrile gave 7g in 46% yield: mp
132-134 °C dec; IR (KBr) 2965, 1700, 1599, 1461 cm-1 1H
;
NMR (DMSO-d6) δ 1.42 (s, 18H, CH3), 2.83 (s, 3H, SCH3), 3.68
(s, 3H, OCH3), 7.57 (s, 2H, ArH), 7.87 (s, 1H, olefin). Anal.
(C20H27NO2S2) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tylben zyliden e)-2-(m eth ylsu lfan yl)-
th ia zol-4-on e (7h ). Prepared from 6h and iodomethane by
the procedure described in the preparation of 7a . Recrystal-
lization from ethanol gave 7h in 45% yield: mp 123-125 °C;
IR (KBr) 2963, 1705, 1600, 1474 cm-1; 1H NMR (DMSO-d6) δ
1.31 (s, 18H, CH3), 2.82 (s, 3H, SCH3), 7.48 (s, 2H, ArH), 7.53
(s, 1H, ArH), 7.89 (s, 1H, olefin). Anal. (C19H25NOS2) C, H, N.
(Z)-5-Ben zyliden e-2-(m eth oxyam in o)th iazol-4-on e (8a).
Potassium tert-butoxide (0.37 g, 3.2 mmol) was added to a
stirred mixture of 7a (0.7 g, 3.0 mmol) and methoxylamine
hydrochloride (0.27 g, 3.2 mmol) in 20 mL of ethanol under
an inert atmosphere. The mixture was heated to reflux for 8
h, then, after being allowed to cool, was poured into 200 mL
of water, and was stirred. After approximately 2 h the
precipitate was filtered off, washed three times with water,
once with ethanol, and once with ether and then dried. The
crude product was recrystallized from ethanol and dried to
afford the product 8a (0.3 g, 43%) as fluffy yellow crystals:
172-174 °C; IR (KBr) 3150, 2963, 1698, 1589, 1435 cm-1; H
NMR (DMSO-d6) δ 1.22 (d, 12H, CH3), 3.30 (septet, 2H,
HCMe2), 3.72 (s, 3H, SCH3), 7.37, 2H, ArH), 7.65 (s, 1H, olefin),
13.83 (br s, 1H, NH). Anal. (C17H21NO2S2) C, H, N.
1
(Z)-5-(3,5-Dibr om o-4-h ydr oxyben zyliden e)-2-th ioxoth i-
a zolid in -4-on e (6b). Prepared from 5b26 and rhodanine by
the procedure described in the preparation of 6f. The crude
product was triturated in ethanol twice and then diethyl ether
twice to give 6b in 89% yield: mp >300 °C; IR (KBr) 1726,
1580, 1473, 1144 cm-1 1H NMR (DMSO-d6) δ 7.56 (s, 1H,
;
olefin), 7.78 (s, 2H, ArH). Anal. (C10H5Br2NO2S2) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-m eth oxyben zyliden e)-2-th ioxo-
th ia zolid in -4-on e (6g). Prepared from 5g27 and rhodanine
by the procedure described in the preparation of 6f. Recrys-
tallization of the purified product from ethanol gave 6g in 50%
yield: mp 229-232 °C; IR (KBr) 3457, 2961, 1692, 1596, 1409
cm-1; H NMR (DMSO-d6) δ 1.41 (s, 18H, CH3), 3.68 (s, 3H,
1
mp 183-184 °C; IR (KBr) 3170, 3006, 1714, 1643, 1333 cm-1
;
1H NMR (DMSO-d6) δ 3.82 (s, 3H, OCH3), 7.47 (tr, 1H, ArH),
7.53 (tr, 2H, ArH), 7.61 (d, 1H, ArH), 7.63 (s, 1H, olefin) 12.26
(s, 1H, NH); MS (+APCI) m/z 235 (MH+). Anal. (C11H10N2O2S)
C, H, N.
SCH3), 7.50 (s, 2H, ArH), 7.68 (s, 1H, olefin), 13.8 (br s, 1H,
NH). Anal. (C19H25NO2S2) C, H, N.
(Z)-5-Ben zyliden e-2-(m eth ylsu lfan yl)th iazol-4-on e (7a).
Iodomethane (1.8 g, 12.9 mmol) was added to a stirred mixture
of 6a 28 (2.0 g, 9.0 mmol) and diisopropylethylamine (1.4 g, 10.6
mmol) in ethanol (50 mL) at room temperature under an inert
atmosphere. After 1.5 h the mixture was poured into water
(200 mL) and stirred for an additional 2 h. The precipitate
was filtered off, washed three times with water, dried, and
then chromatographed on a column of silica gel in chloroform
to afford the pure 7a (1.1 g, 52%): mp 147-148 °C; IR (KBr)
(Z)-5-(3,5-Diisop r op yl-4-h yd r oxyben zylid en e)-2-(m eth -
oxya m in o)th ia zol-4-on e (8ea ). Prepared from 7e and meth-
oxylamine hydrochloride by the procedure described in the
preparation of 8a . Recrystallization from dimethylformamide/
methanol gave 8ea in 70% yield: mp 282 °C dec; IR (KBr)
3476, 2951, 1698, 1639, 1591 cm-1; 1H NMR (DMSO-d6) δ 1.18
(d, 12H, CH3), 3.34 (m, obscured by water peak, HCMe2), 3.82
(s, 3H, OCH3), 7.24 (s, 2H, ArH), 7.57 (s, 1H, olefin), 8.90 (s,
1H, OH), 12.06 (s, 1H, NH); MS (+APCI) m/z 335 (MH+). Anal.
(C17H22N2O3S) C, H, N.
1
3006, 1698, 1601, 1464 cm-1; H NMR (DMSO-d6) δ 2.84 (s,
3H, SMe), 7.50-7.58 (m, 3H, ArH), 7.68 (d, 2H, ArH), 7.86 (s,
1H, olefin). Anal. (C11H9NOS2) C, H, N.
(Z)-5-(3,5-Dibr om o-4-h yd r oxyben zylid en e)-2-(m eth yl-
su lfa n yl)th ia zol-4-on e (7b). Prepared from 6b and iodo-
methane by the procedure described in the preparation of 7a .
Recrystallization from dimethylformamide gave 7b in 86%
N-[5-(4-H yd r oxy-3,5-d iisop r op ylb en zylid en e)-4-oxo-
4,5-d ih yd r oth ia z-2-yl]gu a n id in e (8eb). Prepared from 7e
and guanidine hydrochloride by the procedure described in the
preparation of 8a . Trituration in boiling ethyl acetate gave 8eb
in 69% yield: mp 240-241 °C dec; IR (KBr) 3392, 2963, 1653,
yield: mp 277 °C dec; IR (KBr) 3282, 1685, 1574, 1463 cm-1
;
1H NMR (DMSO-d6) δ 2.84 (s, 3H, SCH3), 7.75 (s, 1H, olefin),
7.85 (s, 2H, ArH); MS (+APCI) m/z 410 (MH+). Anal. (C11H7-
NO2S2Br2) C, H, N.
1582, 1466 cm-1 1H NMR (DMSO-d6) δ 1.17 (d, 12H, CH3),
;
3.32 (m, obscured by water peak, HCMe2), 7.24 (s, 2H, ArH),
7.35 (br s, 1H, OH), 7.51 (s, 1H, olefin), 8.25 (br s, ∼2H, NH);
MS (+APCI) m/z 347 (MH+). Anal. (C17H22N4O2S) C, H, N.
(Z)-5-(3,5-Diisop r op yl-4-m eth oxyben zylid en e)-2-(m eth -
oxya m in o)th ia zol-4-on e (8f). Prepared from 7f and meth-
oxylamine hydrochloride by the procedure described in the
preparation of 8a . The crude product was first purified by
chromatography on silica gel (chloroform/ethyl acetate, 95:5)
and then recrystallized from acetonitrile to give 8f in 16%
yield: mp 165-167 °C dec; IR (KBr) 2965, 1692, 1634, 1610
(Z)-5-(3,5-Diiod o-4-h yd r oxyben zylid en e)-2-(m eth ylsu l-
fa n yl)th ia zol-4-on e (7c). Prepared from 6c and iodomethane
by the procedure described in the preparation of 7a . Recrys-
tallization from dimethylformamide gave 7c in 47% yield: mp
1
240 °C dec; IR (KBr) 3420, 1684, 1595, 1485 cm-1; H NMR
(DMSO-d6 + TFA) δ 2.81 (s, 3H, SCH3), 7.69 (s, 1H, olefin),
8.01 (s, 2H, ArH); MS (+APCI) m/z 504 (MH+). Anal. (C11H7I2-
NO2S2) C, H, N.
1
cm-1; H NMR (DMSO-d6) δ 1.22 (d, 12H, CH3), 3.28 (septet,
(Z)-5-(3,5-Diisop r op yl-4-h yd r oxyben zylid en e)-2-(m eth -
ylsu lfa n yl)th ia zol-4-on e (7e). Prepared from 6e and iodo-
methane by the procedure described in the preparation of 7a .
Recrystallization from acetonitrile gave 7e in 78% yield: mp
2H, HCMe2), 3.71 (s, 3H, ArOCH3), 3.83 (s, 3H, NOCH3), 7.36
(s, 2H, ArH), 7.57 (s, 1H, olefin), 12.19 (s, 1H, NH); MS
(+APCI) m/z 349 (MH+). Anal. (C18H24N2O3S) C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-m eth oxyben zyliden e)-2-(m eth -
oxya m in o)th ia zol-4-on e (8g). Prepared from 7g and meth-
oxylamine hydrochloride by the procedure described in the
preparation of 8a . The crude product was first purified by
chromatography on silica gel (chloroform/ethyl acetate, 98:2)
and then recrystallized from ethanol to give 8g in 43% yield:
1
195-197 °C; IR (KBr) 3429, 2957, 1677, 1577, 1453 cm-1; H
NMR (DMSO-d6) δ 1.19 (d, 12H, CH3), 2.82 (s, 3H, SCH3),
3.28-3.38 (m, 2H, HCMe2), 7.34 (s, 2H, ArH), 7.80 (s, 1H,
olefin), 9.17 (br s, 1H, OH); MS (+APCI) m/z 336 (MH+). Anal.
(C17H21NO2S2) C, H, N.
(Z)-5-(3,5-Diisop r op yl-4-m eth oxyben zylid en e)-2-(m eth -
ylsu lfa n yl)t h ia zol-4-on e (7f). Prepared from 6f and iodo-
methane by the procedure described in the preparation of 7a .
Recrystallization from acetonitrile gave 7f in 46% yield: mp
mp 224-225 °C dec; IR (KBr) 2961, 1692, 1638, 1607 cm-1
;
1H NMR (DMSO-d6) δ 1.41 (s, 18H, CH3), 3.67 (s, 3H, ArOCH3),
3.82 (s, 3H, NOCH3), 7.48 (s, 2H, ArH), 7.58 (s, 1H, olefin),
12.18 (br s, 1H, NH); MS (+APCI) m/z 377 (MH+). Anal.
(C20H28N2O3S) C, H, N.
1
114-116 °C; IR (KBr) 2961, 1700, 1591, 1470 cm-1; H NMR
(DMSO-d6) δ 1.22 (d, 12H, CH3), 2.83 (s, 3H, SCH3), 3.28

COX-2 Inhibitors. 1. Thiazolones and Oxazolones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1159
(Z)-5-(3,5-Di-ter t-bu tylben zylid en e)-2-(m eth oxya m in o)-
th ia zol-4-on e (8h ). Prepared from 7h and methoxylamine
hydrochloride by the procedure described in the preparation
of 8a . Recrystallization from ethanol gave 8h in 30% yield:
mmol). The mixture was stirred for 15 min and then filtered
into a solution of 1010a (3.1 g, 8.9 mmol) in 60 mL of ethanol.
The reaction mixture was stirred at reflux for 15 h, then cooled,
and filtered. The filtrate was condensed about 50% and added
to 450 g of ice and water. The precipitated solid was filtered,
washed with water, and dissolved in ethyl acetate. The organic
solution was washed with brine, dried over Na2SO4, and
evaporated. The residue was recrystallized from aqueous
ethanol to yield 1.8 g (48%) of 20: mp 123-125 °C; IR 3613,
3363, 1706, 1579, 1054 cm-1; 1H NMR (CDCl3) δ 1.37 (s, 18H,
2 × t-Bu), 2.26 (s, 3H, SMe), 3.76 (s, 2H, CH2), 3.83 (s, 3H,
NOMe), 4.04 (s, 3H, NOMe), 5.04 (s, 1H, ArOH), 7.07 (s, 1H,
Harom), 7.22 (s, 1H, Harom), 9.76 (bs, 1H, enolic OH); MS (EIMS)
m/z 424 (M+). Anal. (C21H33N3O4S) C, H, N.
mp 210-211 °C dec; IR (KBr) 2963, 1697, 1638, 1607 cm-1
;
1H NMR (DMSO-d6) δ 1.32 (s, 18H, CH3), 3.83 (s, 3H, OCH3),
7.42 (s, 2H, ArH), 7.49 (s, 1H, ArH), 7.62 (s, 1H, olefin), 12.21
(s, 1H, NH); MS (+APCI) m/z 347 (MH+). Anal. (C19H26N2O2S)
C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h ydr oxyben zyliden e)-2-(m eth -
oxya m in o)oxa zol-4-on e (11b). Methoxylamine hydrochloride
(0.19 g, 2.3 mmol) in 75 mL of ethanol was cooled in an ice
bath and treated with potassium tert-butoxide (0.25 g, 2.2
mmol). The mixture was stirred for 15 min and then filtered
into a solution of 1010a (1.0 g, 2.9 mmol) in 60 mL of ethanol.
The reaction mixture was stirred in a ice bath for 2 h and then
for 1 h at room temperature. The reaction mixture was filtered;
the filtrate was concentrated to one-half of the original volume
and poured onto 450 mL of ice-water. The mixture was
extracted with ethyl acetate, and the combined organic layers
were washed with brine, dried over sodium sulfate, and then
evaporated to an oil. The oil was purified by flash chromatog-
raphy (elution with 2.5-5% methanol in dichloromethane).
Recrystallization from aqueous ethanol gave 0.22 g (29%) of
Ack n ow led gm en t. We thank Dr. Gary McClusky
and staff for microanalytical and spectral data, Brian
Tobias for special NMR studies, J . Ron Rubin for
crystallographic studies, and Godwin Okonkwo, Keith
Laemont, and Mark Lesch for animal testing.
Su p p or tin g In for m a tion Ava ila ble: Enzyme and cel-
lular activity of compounds 4g-p ,s, 7b,c,e, 8a ,f-h , 11a , 12a ,c,
13-15, 16a -c, 18a -d , and 19b (Table 4). This information
is available free of charge via the Internet at http://pubs.
acs.org.
11b: mp 204-207 °C; IR 3628, 3608, 1740, 1706, 1328 cm-1
;
1H NMR (DMSO-d6) δ 1.393 and 1.397 (s, 18H, 2 × t-Bu), 3.70
and 3.72 (s, 3H, OCH3), 6.51 and 6.54 (s, 1H, Hvinyl), 7.48 and
7.49 (s, 1H, OH), 7.56 (s, 1H, Harom), 7.67 (s, 1H, Harom); MS
(EIMS) m/z 346 (M+). Anal. (C19H26N2O4) C, H, N.
Refer en ces
(1) Presented in part: (a) Song, Y.; Connor, D. T.; Sorenson, R. J .;
Doubleday, R.; Sercel, A. D.; Unangst, P. C.; Gilbertsen, R. B.;
Chan, K.; Bornemeier, D. A.; Dyer, R. D. 2,6-Di-tert-butylphe-
nols: A New Class of Potent and Selective COX-2 Inhibitor.
Poster presentation at the 8th International Conference of the
Inflammation Research Association, Hershey, PA, October 1996;
Abstract P86. (b) Song, Y.; Connor, D. T.; Sorenson, R. J .;
Doubleday, R.; Sercel, A. D.; Unangst, P. C.; Gilbertsen, R. B.;
Chan, K.; Bornemeier, D. A.; Dyer, R. D. 2,6-Di-tert-butylphe-
nols: A New Class of Potent and Selective COX-2 Inhibitor.
Inflamm. Res. Suppl. 2 1997, 46, S141-S142. (c) Song, Y.;
Connor, D. T.; Doubleday, R.; Sorenson, R. J .; Sercel, A. D.;
Unangst, P. C.; Cornell, W.; Gilbertsen, R. B.; Chan, K.; Schrier,
D. J .; Laemont, K.; Okonkwo, G. C.; Guglietta, A.; Bornemeier,
D. A.; Dyer, R. D. Novel Di-tert-butylphenols as Selective COX-2
Inhibitors and Orally Active and Safe Antiinflammatory Agents.
Poster presentation at the 213th ACS National Meeting, San
Francisco, CA, Apr 13-17, 1997; Abstract 073.
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Selective Inhibition of Inducible Cyclooxygenase 2 in vivo Is
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(Z)-5-(3,5-Di-ter t-b u t yl-4-h yd r oxyb en zylid en e)-2-(h y-
d r oxya m in o)oxa zol-4-on e (11a ). Prepared from hydroxyl-
amine hydrochloride (0.38 g, 5.5 mmol), potassium tert-
butoxide (0.55 g, 4.9 mmol), and 1010a (2.3 g, 6.6 mmol) by the
procedure described in the preparation of 11b, except that the
reaction was stirred at room temperature for 5 h. Recrystal-
lization of the purified product from aqueous ethanol gave 0.20
g (13%) of 11a : mp 220-224 °C; IR 3631, 1752, 1711, 1331,
1
1239 cm-1; H NMR (DMSO-d6) δ 1.393 and 1.398 (s, 18H, 2
× t-Bu), 6.44 and 6.47 (s, 1H, Hvinyl), 7.43 and 7.44 (s, 1H,
phenol OH), 7.56 (s, 1H, Harom), 7.67 (s, 1H, Harom); 9.61 and
9.69 (s, 1H, NHOH), 11.90 and 12.25 (br s, 1H, NH); MS
(EIMS) m/z 333 (MH+). Anal. (C18H24N2O4) C, H, N.
(Z)-2-(Allyloxya m in o)-5-(3,5-d i-ter t-b u t yl-4-h yd r oxy-
ben zylid en e)oxa zol-4-on e (11d ). Prepared from O-allylhy-
droxylamine hydrochloride (0.50 g, 4.6 mmol), potassium tert-
butoxide (0.49 g, 4.4 mmol), and 1010a (2.0 g, 5.8 mmol) by the
procedure described in the preparation of 11b, except that the
reaction mixture was stirred at -40 °C for 15 h. Several
recrystallizations of the purified product from aqueous ethanol
gave 0.29 g (18%) of 11d : mp 187-189 °C; IR 3617, 1739, 1705,
1332, 1044 cm-1; 1H NMR (DMSO-d6) δ 1.39 (s, 18H, 2 × t-Bu),
4.40-4.45 (m, 2H, CH2CHdCH2), 5.19-5.38 (m, 2H, CH2CHd
CH2), 5.93-6.01 (m, 1H, CH2CHdCH2), 6.51 and 6.54 (s, 1H,
ArCHd), 7.48 and 7.49 (s, 1H, OH), 7.55 (s, 1H, Harom), 7.67
(s, 1H, Harom); MS (EIMS) m/z 372 (M+). Anal. (C21H28N2O4)
C, H, N.
(Z)-5-(3,5-Di-ter t-bu tyl-4-h yd r oxyben zylid en e)-2-(eth -
oxya m in o)oxa zol-4-on e (11c). Prepared from ethoxylamine
hydrochloride (0.45 g, 4.6 mmol), potassium tert-butoxide (0.49
g, 4.4 mmol), and 1010a (2.0 g, 5.8 mmol) by the procedure
described in the preparation of 11b, except that the reaction
was stirred at -30 °C for 4 h. Several recrystallizations of the
purified product from aqueous ethanol gave 0.11 g (7%) of
11c: mp 214-217 °C; IR 3627, 3614, 1735, 1708, 1328 cm-1
;
1H NMR (DMSO-d6) δ 1.18-1.23 (m, 3H, OCH2CH3), 1.39 and
1.40 (s, 18H, 2 × t-Bu), 3.90-3.97 (m, 2H, OCH2CH3), 6.49
and 6.52 (s, 1H, Hvinyl), 7.47 and 7.48 (s, 1H, OH), 7.55 (s, 1H,
H
arom), 7.68 (s, 1H, Harom); MS (EIMS) m/z 360 (M+). Anal.
(C20H28N2O4) C, H, N.
N-[3-[3,5-Bis(1,1-d im et h ylet h yl)-4-h yd r oxyp h en yl]-1-
h yd r oxy-2-(m eth oxyim in o)p r op ylid en e]-N′-m eth oxyca r -
ba m id oth ioic Acid Meth yl Ester (20). Methoxylamine
hydrochloride (1.6 g, 19.2 mmol) in 75 mL of ethanol was cooled
in ice and treated with potassium tert-butoxide (2.1 g, 18.7

1160 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
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