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. 2. 1,3,4- and 1,2,4-thiadiazole series

Article abstract of DOI:10.1021/jm980570y

Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lac

Full text of DOI:10.1021/jm980570y

J . Med. Chem. 1999, 42, 1161-1169
1161
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. 2. 1,3,4- a n d 1,2,4-Th ia d ia zole Ser ies1
Yuntao Song,*,† David T. Connor,† Anthony D. Sercel,† Roderick J . Sorenson,† Robert Doubleday,†
Paul C. Unangst,† Bruce D. Roth,† Vlad G. Beylin,§ Richard B. Gilbertsen,‡ Kam Chan,‡ Denis J . Schrier,‡
Antonio Guglietta,‡ Dirk A. Bornemeier,| and Richard D. Dyer|
Departments of Chemistry, Chemical Development, Biochemistry, and Immunopathology, Parke-Davis Pharmaceutical
Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received October 7, 1998
Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is
expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been
shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side
effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies
have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had
IC50 values of 0.14 and 100 µM against recombinant human COX-2 and purified ovine COX-1,
respectively. It inhibited COX-2 activity in the J 774A.1 cell line with an IC50 of 0.18 µM and
inhibited COX-1 activity in platelets with an IC50 of 3.1 µM. The choline salt of compound 6b
was also orally active in vivo with an ED40 of 7.1 mg/kg in the carrageenan footpad edema
(CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited
gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI
damage. SAR studies of this chemical series revealed that the potency and selectivity are very
sensitive to minor structural changes.
In tr od u ction
tive COX-2 inhibitors: 1 (SC-58125),7 2 (NS-398),8 3 (L-
745,337),9 and 4 (PD 138387)10 (Chart 1).
Nonsteroidal antiinflammatory drugs (NSAIDs) are
important therapeutic agents for the treatment of pain
and inflammation. However, their therapeutic use is
often limited by common side effects, such as gas-
trointestinal (GI) hemorrhage and ulceration.2 There-
fore, a major challenge of the pharmaceutical industry
is to develop drugs that have antiinflammatory activi-
ties but lack the toxic side effects associated with
currently used NSAIDs. A major mechanism of action
of NSAIDs is lowering prostaglandin production through
inhibition of cyclooxygenase (COX), a key enzyme in
prostaglandin biosynthesis.3 Because prostaglandins
have dual functions, mediation of inflammation3 and
cytoprotection in the stomach and intestine,4 long-term
usage of NSAIDs to relieve the symptoms of inflamma-
tion will cause GI damage and ulceration. A possible
dissociation of antiinflammatory effects from GI toxicity
is suggested by the recent discovery that COX exists in
two isoforms, COX-1 and COX-2, which are encoded by
two distinct genes.5 COX-1 is expressed constitutively
and is thought to provide cytoprotection; COX-2 expres-
sion, however, is transiently upregulated by proinflam-
matory mediators and downregulated by corticoster-
oids.6 This regulated expression suggests that a selective
inhibitor of COX-2 might have antiinflammatory prop-
erties and lack GI side effects. This hypothesis has been
supported by several in vivo studies with known selec-
Prior to the discovery of two isoforms of the COX, our
efforts on a dual cyclooxygenase and 5-lipoxygenase
inhibitor program11,12 had identified a number of sub-
stituted di-tert-butylphenols as potent COX inhibitors,
some of which lacked inhibitory activity toward 5-li-
poxygenase and were not fully evaluated under the dual
inhibitor strategy. The possibility that these compounds
would be selective COX-2 inhibitors was investigated
and structure-activity relationships (SAR) for selective
inhibitors of COX-2 were developed in several series.
SAR studies of the chemical series related to 4 have
been reported.10 In this paper we report SAR studies
on the 1,3,4- and 1,2,4-thiadiazole-derived di-tert-bu-
tylphenol series. Also reported are pharmacological
evaluations of selective inhibitors in the carrageenan
footpad edema (CFE) assay, in a hyperalgesia model,
and in a model of GI ulceration.
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 the structures I and II
(Chart 2) were identified as potent and selective inhibi-
tors.
Com p ou n d Eva lu a tion s
† Department of Chemistry.
The primary assays for the compounds described in
this SAR study were purified enzyme assays. Recom-
binant human COX-2 (rhCOX-2) and ram seminal
§ Department of Chemical Development.
| Department of Biochemistry.
‡ Department of Immunopathology.
10.1021/jm980570y CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

1162 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Ch a r t 1. Known Selective COX-2 Inhibitors
Song et al.
Sch em e 1. Synthesis of 1,3,4-Thiadiazole 12
Sch em e 2. 1,3,4-Thiadiazole Synthesis
Ch a r t 2. Mass Screening Hits
vesicle COX-1 (oCOX-1) were used to measure inhibitory
activity against isolated enzymes in a cell-free environ-
ment. 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
model, 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.
Sch em e 3. Synthesis of 1,3,4-Thiadiazoles 20a -d
Ch em istr y
butoxide as the base failed to give the corresponding
hydroxylamine derivative; the 1,2,4-thiadiazolone 22
was isolated instead (Scheme 4). Thiadiazoles 24a ,b
were prepared by acylation and sulfonylation, respec-
tively of 5-aminothiadiazole 2314 (Scheme 5). Condensa-
tion of 3115 with diphenyl cyanocarbonimidate followed
by deprotection with acid gave oxadiazole 8 (Scheme 6).
The preparation of 6a ,12 7a ,15 16c,13 18b,13 19,13 20e,12
23,14 25a ,12b-e,13 26a ,b,13 27,12 28a ,b,16 29,14 and
30a ,15b14 has been reported previously.
The diisopropylphenol-derived thiadiazolethione 12
was prepared according to the procedure developed for
the corresponding di-tert-butylphenol derivative (Scheme
1).12 Condensation of the sodium salt of compound 9
with carbon disulfide followed by alkylation with ethyl
chloroacetate gave thioester 10, which was converted
to thiohydrazide 11 by treatment with hydrazine.
Condensation of 11 with carbon disulfide produced
thiadiazolethione 12 in good yield.
Alkylation of thiadiazolethiones 5,12 12, 13,12 and 1412
with sodium hydroxide and alkyl chloride or alkyl iodide
gave thiadiazoles 6b-j, 15a -d , 16a ,b, and 17 (Table
1, Scheme 2). Compound 18a was obtained by oxidation
of 6b with MCPBA. The choline salt of 6b (6b‚choline)
was made by a standard method.
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 . It is well documentated that
COX enzyme catalysis involves radical intermediates17
such as the phenoxy radical generated on a tyrosine
residue.18 Since our chemical leads are di-tert-butylphe-
nols which can form phenoxy radicals and act as radical
scavengers, it was of interest to define the importance
Treatment of sulfone 1913 with sodium alkoxide
produced the corresponding aryl ethers 20a -d in good
yield (Scheme 3). Reaction of sulfone 2114 with O-
methylhydroxylamine in tert-butyl alcohol using tert-

COX-2 Inhibitors. 2. 1,3,4- and 1,2,4-Thiadiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1163
Sch em e 4. Synthesis of 1,3,4-Thiadiazole 22
SH (5), the compound was active but not very selective,
slightly favoring COX-2. When R3 was changed from SH
to SMe (6a ), more than a 30-fold increase in activity
against both enzymes was observed, leaving the selec-
tivity essentially unchanged. When R3 was changed
from SMe to SEt (6b), the potency against COX-2
remained unchanged (IC50 ) 0.14 µM), but the activity
against COX-1 was reduced more than 200-fold, result-
ing in a potent and selective COX-2 inhibitor. Modifica-
tion of R3 from SEt to S-n-Pr led to 6d , which was
essentially inactive against both enzymes. Other sub-
stitutions, such as S-i-Pr (6e) or SBn (6g), also gave
inactive compounds. Several compounds with ionizable
groups as part of R3 (6h -j) were neither potent nor
selective. Compound 6b (PD 164387) was the most
potent (IC50 ) 0.14 µM) and selective (more than 700-
fold) COX-2 inhibitor in this series.
Sch em e 5. Synthesis of 1,2,4-Thiadiazoles 24a ,b
In this 1,3,4-thiadiazole series the potency and selec-
tivity are very sensitive to minor structural modifica-
tions. For example, the ethyl thioether 6b was the most
potent and selective COX-2 inhibitor in this series, but
the corresponding trifluoroethyl thioether 6c was inac-
tive against both enzymes. The ethyl ether 20b was
more than 40-fold less potent against COX-2 and
essentially nonselective, and the sulfoxide 18a was
inactive against both enzymes. Similar changes in
activity were observed in the comparison of thioether
6a with ether 20a and sulfoxide 18b. In addition,
substitution of NHMe for SMe resulted in a reversal of
selectivity affording 25c as a selective COX-1 inhibitor.
Replacement of two of the aromatic carbon atoms with
nitrogens led to two di-tert-butylpyrimidine analogues
(28a ,b in Table 1). In comparison with the correspond-
ing di-tert-butylphenols, these compounds were more
potent against COX-1 and less potent against COX-2
(6a vs 28a , 6b vs 28b). Di-tert-butylpyrimidine 28a was
a selective COX-1 inhibitor.
With regard to R3 substitution, the SAR trend ob-
served in the 1,3,4-oxadiazole series (13, 16a -c, 26a ,b,
and 27) was very similar to that observed in the 1,3,4-
thiadiazole series discussed above. Again, the SEt
substituent (16a ) was optimal in terms of potency and
selectivity.
In the 1,2,4-thiadiazole series (Table 2) an acidic
proton on the side chain seems to be important for
potency and selectivity (22 vs 29, 7a vs 23). The
substitution on the nitrogen atom is also important.
Among the analogues examined (23, 7a , 24a ,b, and
30a ), compound 7a was exclusively preferred. It is worth
noting that thiadiazole 7a and oxadiazole 8 showed
similar enzyme potency.
of the phenolic hydroxy group for the observed inhibitory
activity toward COX. To address this mechanism-
related SAR question, methyl ethers 17 and 14 (Table
1), in which the exchangeable protons are replaced with
methyl groups, were synthesized and evaluated in the
purified enzyme assays. Compared to the corresponding
di-tert-butylphenols 6a and 5, the methyl ethers 17 and
14 had similar potency against COX-2 but were less
potent in the COX-1 assay, making them more selective
COX-2 inhibitors. These data suggest that the observed
inhibitory activity of the two di-tert-butylphenols 6a and
5 in enzyme assays is not due to antioxidant properties
of the compounds.
The effects of R2 substitution were investigated in the
1,3,4-thiadiazole series (Table 1). Changing the tert-
butyl groups to isopropyl groups abolished the activity
against both enzymes (6a vs 15a , 6b vs 15b). The two
isopropylphenols 15c,d were also inactive against both
enzymes, as were the corresponding di-tert-butylphenols
(6d ,e).
The side chain which is preferred for selective COX-2
inhibition in the 1,3,4-thiadiazole series was not pre-
ferred in the 1,2,4-thiadiazole series (6a in Table 1 vs
30b in Table 2). The side chain which is preferred for
selective COX-2 inhibition in the 1,2,4-thiadiazole series
was not preferred in the 1,3,4-thiadiazole series (7a in
Table 2 vs 25d in Table 1). The 1,3,4- and 1,2,4-
thiadiazole rings clearly are not functionally inter-
changeable.
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, potent compounds
The substitution effects of R3 were explored in detail
in the di-tert-butylphenol-derived 1,3,4-thiadiazole se-
ries. Amines, ethers, and thioethers were examined.
Within the amine series compound 25a was active and
selective for COX-2. Substitutions on the nitrogen all
led to decreased potency (25d , 26a , 25e, 26b ) or
decreased selectivity (25b,c). Compound 25c was shown
to be a selective COX-1 inhibitor. Within the ether series
compounds were either nonselective (20a ,b) or inactive
(20c,d ). The most potent and selective COX-2 inhibitors
were identified among the thioethers. Within this series
SAR studies reveal that the size of the R3 substituent
is important for potency and selectivity. When R3 was

1164 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Sch em e 6. Synthesis of 1,2,4-Oxadiazole 8
Song et al.
DBU
dimethoxyethane
Ta ble 1. Enzyme and Cellular Activity of 1,3,4-Thiadiazoles and 1,3,4-Oxadiazolesa-c,f
IC50 (µM)
compd
R1
R2
X
Y
R3
rhCOX-2
oCOX-1
COX-2 J 774A.1
COX-1 hplatelet
5
6a
6b
6c
6d
6e
6f
6 g
6h
6i
6j
13
14
15a
15b
15c
15d
16a
16b
16c
17
18a
18b
20a
20b
20c
20d
20e
25a
25b
25c
25d
25e
26a
26b
27
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OH
OH
OH
OMe
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
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
i-Pr
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
S
S
S
S
S
S
S
S
S
S
S
O
S
S
S
S
S
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
S
S
SH
SMe
SEt
SCH2CF3
S-n-Pr
S-i-Pr
SCH2CH2CO2Me
SBn
SCH2CH2CO2H
SCH2CH2NH2
SCH2CH2NEt2
SH
SH
SMe
SEt
S-n-Pr
S-i-Pr
SEt
S-n-Pr
SMe
SMe
SOEt
SOMe
OMe
OEt
O-i-Pr
O-n-Pr
OH
NH2
NHNH2
NHMe
NHCN
NHCNHNH2
NHCN
NHCNHNH2
OH
5.5
20
0.35
0.009
0.18
NAd
NAd
8.5
NA
0.18
3.1
NA
4.1
0.15
0.14
NA
100
NA
NA
NA
NA
41
0.51
100
NA
NA
NA
NA
NA
NA
36
NA
NA
NA
25@10
NA
NA
NA
NA
NA
12
5.8
NA
NA
2.6
3.4
NA
NA
NA
34@30
17
0.52
NA
13.6
9.9
NAd
NA
NA
NA
NA
NA
NA
NA
NT
NA
NA
NT
1.9
NAd
NAd
NA
0.92
0.30
NA
1.3
1.5
NT
0.015
NAd
0.14
0.012
0.23b
NT
19
47
1.5
14@10
NA
NA
NA
6.5
NA
4.1
0.34
NA
NA
3
i-Pr
i-Pr
i-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
1.9
1.2
0.77
14.6
NA
0.20
1.2
4.5
3.6
4.1
0.53
3.2
0.30
NA
2.8
5.8
2.8
NT
6.1
0.07
NAd
NAd
0.24
0.11
3.7
0.69
0.033
0.21
0.39
0.33
NA
0.026
0.018
0.070
0.022
0.0064
NA
NA
NA
3.5
1.3
9.6
NA
17
88
11@10e
23
0@10e
16@30
NA
NA
1.4
2.0
0.31
0.19
5.8
3.2
28a
28b
1
SMe
SEt
0.097
39@10
NA
NA
0.0122
0.20
0.68
NA
3.3
2
indomethacin
0.0082
a
NA, not active (in enzyme assays, NA is defined as IC50 value greater than 100 µM; in cellular assays, NA is defined as IC50 value
greater than 20 µM). NT, not tested. c When compounds were insoluble at a concentration of 100 µM, the percent inhibition at the
b
highest concentration tested is shown. Found inactive in acetylated J 774A.1 cells (Ac-J 7), not further tested. e Found inactive at 10 µM
d
in the oCOX-2 enzyme assays, not further tested. f In the purified enzyme assays, standard errors for the IC50 determinations done at
least in duplicate averaged 30% for rhCOX-2 and 10% for oCOX-1; In the cellular assays, standard errors for the interday IC50
determinations done at least in duplicate averaged 45% for COX-1 and 30% for COX-2.
identified in the COX-2 enzyme assay tended to be more
potent in the COX-2 cellular assay (see 6a , 16a , 28b in
Table 1 and 22 in Table 2 as examples). In the case of
COX-1 there was no clear trend. Consequently, a
prediction of selectivity in the cellular assays could not
be made based on selectivity observed in the isolated
enzyme assays. For example, the most selective COX-2
inhibitor 6b, which was at least 700-fold selective in the
isolated enzyme assays, was only 17-fold selective in
cellular assays. Compound 28a was a selective COX-1
inhibitor in the enzyme assays, but the selectivity was
reversed in cellular assays. Compounds inactive in both
isolated enzyme assays could be very potent in both
cellular assays (18a , 20e, 26a , 6e) or could be potent

COX-2 Inhibitors. 2. 1,3,4- and 1,2,4-Thiadiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1165
Ta ble 2. Enzyme and Cellular Activity of 1,2,4-Thiadiazoles and 1,2,4-oxadiazolesa-d
IC50 (µM)
compd
X
R
rhCOX-2
oCOX-1
COX-2 J 774A.1
COX-1 hplatelet
7a
8
22
S
O
S
S
S
S
S
S
S
NHCN
NHCN
OH
0.047
0.064
1.1
NA
NA
NA
100
27
20@10
32
51
83
NA
NA
NA
24
24
16@10
0.026
1.4
0.018
NT
NA
NT
NT
NA
0.26
1.2
1.2
0.43
NT
NT
NT
NT
1.7
3.6
23
NH2
24a
24b
29
30a
30b
NHAc
NHSO2Me
OMe
NHCNHNH2
SMe
a
NA ) Not Active (in enzyme assays, NA is defined as IC50 value greater than 100 µM; in cellular assays, NA is defined as IC50 value
greater than 20 µM). NT, not tested. c When compounds were insoluble at a concentration of 100 µM, the percent inhibition at the
highest concentration tested is shown. In the purified enzyme assays, standard errors for the IC50 determinations done at least in
b
d
duplicate averaged 30% for rhCOX-2 and 10% for oCOX-1; In the cellular assays, standard errors for the interday IC50 determinations
done at least in duplicate averaged 45% for COX-1 and 30% for COX-2.
Ta ble 3. In Vivo Activity of Thiadiazoles 6b‚choline, 7a , and 25d and Standards 1, 2, and Indomethacina
ED40 (mg/Kg)
lesion
frequency (%)
damaged
area (%)
ulcer
index
PGE2
inhibition (%)
compd
CFEb
hyperalgesiac
6b‚ch olin e
7.1
5.8d
7.7
NT
0.2
1.3
7.4
0.3
0.6
12.5
87.5
12.5
0
0
75
0.05
4.91
0.02
0
0
0.18
0.64
430
0.25
0
77.4 ( 3.75
87.7 ( 7.52
22.0 ( 8.00
30.7 ( 12.1
-1.58 ( 13.5
96.5 ( 0.470
7a
25d
2
1
0.61
>30
1.6
0
indomethacin
13.36
a
b
In GI safety studies, the rats were dosed at 100 mg/kg. NT, not tested. Compounds dosed orally, 10 rats/test group. c Compounds
dosed orally, 8 mice/test group. The compound was tested as its choline salt.15
d
only against COX-2 (25d , 15b,c). The lack of correlation
between isolated enzyme assay and cellular assay
results leads to the speculation that these compounds
may possess other pharmacological activity besides
inhibition of COX-1 and COX-2.
of 6b) and 7a were orally active in CFE with ED40
values of 7.1 and 5.8 mg/kg, respectively. Compound 7a
also provided potent analgesic activity (ED40 ) 0.2 mg/
kg). Compound 25d was also active in CFE (ED40 ) 7.7
mg/kg) even though it failed to inhibit either COX in
the purified enzyme assays.
Within this chemical series compounds 6b, 7a , and
14 were identified as potent and selective COX-2 inhibi-
tors with consistent data from both isolated enzyme and
cellular assays. Compound 25d was identified as a
potent and selective COX-2 inhibitor based on cellular
assays. The most potent and selective COX-1 inhibitors
based on the isolated enzyme assay are 28a and 25c.
When compared to standard COX-2 inhibitors 1 and
2, compounds 6b and 7a showed weak activity against
COX-1 in the oCOX-1 enzyme assay with IC50 values of
100 and 32 µM, respectively. In cellular assays, com-
pound 6b was less potent and selective against COX-2
than 2, and compound 7a was similar to 2 in terms of
potency and selectivity. Activity toward inhibition of
COX-1 activity was observed with 6b (IC50 ) 3.1 µM),
7a (IC50 ) 1.2 µM), and the standard selective COX-2
inhibitor 2 (IC50 ) 3.3 µM) in the cellular COX-1 assay
using human platelet-rich plasma. Compound 25d ,
similar to the standard selective COX-2 inhibitor 1, was
potent toward inhibition of COX-2 activity (IC50 ) 0.033
µM) and was inactive toward inhibition of COX-1
activity.
Compounds 6b‚choline, 7a , and 25d were further
evaluated for their gastric toxicity and inhibition of
PGE2 synthesis in rat gastric mucosa. Results for these
three compounds and two known selective COX-2
inhibitors are shown in 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 rat gastric mucosa by 31%, whereas a
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. At a dose of 100
mg/kg, compound 25d that was inactive toward COX-1
activity in the cellular COX-1 assay caused only 22%
inhibition of PGE2 production which is about the same
as that observed with 2. As expected, compound 25d
caused minimal GI damage (ulcer index ) 0.25). Com-
pounds 6b‚choline and 7a that had some activity
against COX-1, especially in the cellular COX-1 assay,
caused reduction in PGE2 production at a level similar
to that of indomethacin (77% for 6b‚choline and 88%
for 7a ) in rat gastric mucosa following a dose of 100 mg/
In Vivo Stu d ies. The three compounds 6b, 7a , and
25d were studied in vivo as the parent or their choline
salt (Table 3). Compounds 6b‚choline (the choline salt

1166 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
Ann Arbor, MI). For 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 cells20) 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
to convert 20-30% of added substrate to products during a
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 incubations. The concentration of compound causing
50% inhibition (IC50) was estimated using the software package
KaleidaGraph, 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: % inhibition ) 100/(1 + (inhibitor concentration/
kg. However, compounds 6b‚choline and 7a had very
different effects on gastric damage. Compound 7a
caused more GI damage in rats than indomethacin as
indicated by the ulcer index (ulcer index ) 430), but the
GI damage detected with 6b‚choline was minimal (ulcer
index ) 0.64).
The results obtained with 25d support the hypothesis
that compounds which selectively inhibit COX-2 activity
are antiinflammatory agents and cause less GI damage.
The in vivo data obtained with 6b ‚choline and 7a
suggest that, at least in the rat, the level of reduction
in PGE2 production is not the sole determinant of GI
lesion formation, and other factors must also be involved
in evolution of gastric damage. Additional pharmaco-
logical properties of the substituted di-tert-butylphenols
besides the inhibition of COX isoforms or the pharma-
cological properties of their metabolites may contribute
to these effects observed in vivo. For example, compound
6b‚choline or its metabolites could have other pharma-
cological properties that are beneficial in terms of GI
lesions.
IC50)
slope), and best fits for IC50 and slope coefficient were
determined by least-squares analysis. Standard errors for
replicate determinations averaged 30% for rhCOX-2 and 10%
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 either untreated
or after acetylation with aspirin (Ac-J 7) 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 per-
formed in flat bottom 96-well plates (100-µL cell preparation,
100-µL drug dilution). After a 60-min preincubation of cells
and test compounds, samples were spiked with 3 µg of
arachidonic acid and incubated 30 min, and the reaction was
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). Percent inhibition was determined
using the formula: {1 - [(drug - medium)/(maximum -
medium)]} × 100, where “drug” is 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). Standard errors for interday deter-
minations averaged 45% for COX-1 and 30% for COX-2.
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 21 for the details). A
Student’s t-test was done on each experimental group (com-
pared to vehicle) to evaluate statistical significance.
Con clu sion
SAR studies of these 1,3,4- and 1,2,4-thiadiazole-
derived 2,6-di-tert-butylphenol series have shown that
both in vitro potency and selectivity of COX isoform
inhibition are very sensitive to structural changes.
Compound 6b was identified as the most potent and
selective COX-2 inhibitor in this series with consistent
data from both isolated enzyme assays and cellular
assays. Compound 25d was potent and selective toward
inhibition of COX-2 activity in cellular assays, even
though it failed to inhibit either enzyme in the isolated
enzyme assays. Both 6b‚choline and 25d were orally
active in an acute model of inflammation and caused
minimal GI damage. In vivo studies with 25d support
the hypothesis that the antiinflammatory activity of
NSAIDs results from inhibition of COX-2 and their GI
toxicity from inhibition of COX-1. The in vivo data
obtained with 6b‚choline suggest that additional phar-
macological properties of this compound besides the
inhibition of COX isoforms or the pharmacological
properties of its metabolites may contribute to the GI
safety profiles observed. Further improvements in po-
tency and selectivity of these compounds could lead to
novel and safe antiinflammatory 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.19
An a lgesia . Analgesic activity was measured by the acetic
acid-induced writhing test as previously described.22 Male
Swiss-Webster mice were fasted for 16 h prior to oral admin-
istration of compounds or vehicle (HPMC with Tween 80).
Sixty minutes later, each animal was injected intraperitoneally
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 regres-
sion analysis. A two-way ANOVA was used to determine the
level of statistical significance for each experimental group.
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,

COX-2 Inhibitors. 2. 1,3,4- and 1,2,4-Thiadiazoles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1167
Ta ble 4. Physical Data for Thiadiazoles 6c-j, 15a -d , 16a ,b, 17, and 20a -c
compd
R1
R2
X
R3
method
mp (°C)
formula
recryst solvent
yield (%) analysis
6c
6d
6e
6f
6g
6h
6i
6j
15a
15b
15c
15d
16a
16b
17
20a
20b
20c
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
i-Pr
i-Pr
i-Pr
t-Bu
t-Bu
S
S
S
S
S
S
S
S
S
S
S
S
O
O
S
S
S
S
SCH2CF3
S-n-Pr
S-i-Pr
SCH2CH2CO2Me
SBn
SCH2CH2CO2H
SCH2CH2NH2
SCH2CH2NEt2
SMe
SEt
S-n-Pr
S-i-Pr
SEt
Aa
A
A
Ab
A
Ac
A
A
A
A
A
A
A
A
A
B
100-101 C18H23F3N2OS2 pentane
35
89
97
62
92
62
71
52
67
76
81
82
69
63
83
56
62
67
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
107-108
93-95
C
C
19H28N2OS2
19H28N2OS2
83-85
C20H28N2O3S2
23H28N2OS2
102-104
C
154-156 C19H26N2O3S2
131-133 C18H27N3OS2
85-88
143
124-125
74-75
tert-butyl methyl ether
C22H35N3OS2
C
C
C
C
C
C
C
C
C
C
15H20N2OS2
16H22N2OS2
17H24N2OS2
17H24N2OS2
18H26N2O2S
19H28N2O2S
18H26N2OS2
17H24N2O2S
18H26N2O2S
19H28N2O2S
toluene
toluene
heptane
toluene
hexanes
hexanes
152-153
125-126
88-89
S-n-Pr
SMe
OMe
OEt
OMe t-Bu
85-88
OH
OH
OH
t-Bu
t-Bu
t-Bu
130-132
110-122
100-103
Et2O/hexanes
hexanes
B
B
O-i-Pr
a
b
Reaction mixture was refluxed for 18 h. The choline salt of 5 was used as the starting material; no base was used. c The choline salt
of 5 was used as the starting material; triethylamine was used instead of NaOH.
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
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.
2H, J ) 7.32 Hz, CH2CH3), 3.35-3.38 (m, 2H, HOCH2CH2N),
3.78-3.84 (m, 2H, HOCH2CH2N), 5.35 (bs, 1H, OH), 7.15 (s,
2H, 2 × ArH); MS (CI) m/z 351 (MH+). Anal. (C18H25N2O1S2‚
C5H14NO) C, H, N.
(4-H y d r o x y -3,5-d iis o p r o p y lt h io b e n zo y ls u lfa n y l)-
a cetic Acid Eth yl Ester (10). Sodium hydride (60% in
mineral oil, 4.40 g, 110 mmol) was washed with pentane (2 ×
20 mL) and then mixed with 50 mL of DMF. After the mixture
stirred at ambient temperature for 5 min under nitrogen
atmosphere, 2,4-diisopropylphenol (18.5 mL, 99.8 mmol) was
added dropwise (exothermic reaction and gas evolution). The
reaction mixture was stirred at ambient temperature for 15
min; then carbon disulfide (6.00 mL, 102 mL) was added
dropwise. The resulting reaction mixture was stirred at 75 °C
for 30 min; then ethyl chloroacetate (11.0 mL, 103 mmol) was
added at 75 °C. After the addition, the reaction mixture was
allowed to cool to ambient temperature and was stirred for 16
h at which time TLC showed that no starting material was
left. A mixture of 150 mL of EtOAc and 200 mL of water was
added, and the resulting mixture was stirred for 10 min. Two
layers were separated, the aqueous layer was extracted with
EtOAc (2 × 100 mL), and the combined organic solution was
washed with water (2 × 300 mL) and brine (1 × 300 mL) and
dried over Na2SO4. Concentration in vacuo afforded a viscous
oil which upon standing overnight at 4 °C solidified to an
orange solid. Trituration with petroleum ether/2-propanol (99:
1) gave 21.8 g (64%) of crude product (10). A small amount of
product was recrystallized from tert-butyl methyl ether/hex-
anes to give pure compound 10: mp 88-89 °C; IR 3395, 2969,
Meth od A. 2,6-Di-ter t-bu tyl-4-(5-eth ylsu lfa n yl[1,3,4]-
th ia d ia zol-2-yl)p h en ol (6b). To a solution of 512 (514 mg, 1.59
mmol) in THF (3 mL) cooled at 0 °C was added a solution of
1 N NaOH (1.69 mL) under a nitrogen atmosphere. After the
mixture stirred for 15 min, ethyl iodide (140 µL, 1.75 mmol)
was added. After stirring at 0 °C for additional 1 h, solvents
were stripped off in vacuo affording a yellow solid. Recrystal-
lization from CH3CN/H2O gave 6b as pale-yellow crystals in
79% yield: mp 128-129 °C; IR 3578, 1407, 1378, 1241, 1226,
1
705 cm-1; H NMR (CDCl3) δ 1.48 (s, 18H, 2 × t-Bu), 1.49 (t,
3H, J ) 7.23 Hz, CH3), 3.36 (q, 2H, J ) 7.47 Hz, CH2), 5.56 (s,
1H, OH), 7.70 (s, 2H, 2 × ArH); MS (CI) m/z 351 (MH+). Anal.
(C18H26N2O1S2) C, H, N.
1
1719, 1588, 1292, 1192, 1157, 697 cm-1; H NMR (CDCl3) δ
1.29-1.33 (m, 15H, 2 × CH(CH3)2 and CH2CH3), 3.15 (septet,
2H, J ) 6.75 Hz, 2 × CH(CH3)2), 4.21 (s, 2H, CH2), 4.24 (q,
2H, J ) 6.99 Hz, CH2CH3), 5.27 (s, 1H, OH), 7.26 (s, 2H, 2 ×
Using this method, compounds 6c-j, 15a -d , 16a ,b, and 17
were prepared. The physical data and chemical yields are
summarized in Table 4.
H
arom); MS (APCI) m/z 341.2 (MH+). Anal. (C17H24O3S2) C, H.
2,6-Di-ter t-bu tyl-4-(5-eth ylsu lfa n yl[1,3,4]th ia d ia zol-2-
yl)p h en ol Ch olin e Sa lt (6b‚ch olin e). To a solution of 6b
(2.00 g, 5.71 mmol) in ethanol was added choline carbonate
(80%, 1.00 mL, 5.71 mmol). The resulting reaction misture was
refluxed for 1 h. After cooling to ambient temperature, the
solution was concentrated in vacuo affording a yellow thick
oil. Recrystallization twice from EtOAc/MeO-t-Bu and EtOAc
gave 1.7 g (66%) of pure 6b‚choline as a yellow solid: mp 138-
140 °C dec; IR 3436, 2948, 1586, 1394, 1368, 1353, 1221, 712
4-Hyd r oxy-3,5-d iisop r op ylth ioben zoic Acid Hyd r a zid e
(11). A solution of 10 (5.71 g, 16.8 mmol) in 50 mL of ethanol
at ambient temperature was treated with hydrazine hydrate
(1.60 mL, approximately 27.5 mmol). The reaction solution was
stirred for 1.5 h at which time TLC showed the reaction was
complete. Water (80 mL) was added, and the resulting suspen-
sion was stirred briskly for 15 min. Filtration and water
washing followed by air-drying gave 4.11 g (97%) of 11 as a
white solid: 1H NMR (DMSO-d6) δ 1.11 (d, 12H, J ) 6.84 Hz,
2 × CH(CH3)2), 3.24 (septet, 2H, J ) 6.84 Hz, 2 × CH(CH3)2),
1
cm-1; H NMR (DMSO-d6) δ 1.29 (s, 18H, 2 × t-Bu), 1.30 (t,
3H, J ) 7.16 Hz, CH2CH3), 3.08 (s, 9H, RN(CH3)3), 3.12 (q,

1168 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Song et al.
7.40 (s, 2H, 2 × ArH). The material was used in the next step
mL of toluene was heated at reflux for 16 h. The precipitated
product was filtered and washed with water. Recrystallization
of the crude product from aqueous ethanol gave 0.15 g (33%)
without further purification.
2,6-Diisop r op yl-4-(5-m er ca p t o[1,3,4]t h ia d ia zol-2-yl)-
p h en ol (12). To a solution of 11 (4.11 g, 16.3 mmol) in 20 mL
of ethanol was added carbon disulfide (2.34 mL, 40.0 mmol).
The resulting reaction mixture was refluxed for 1.5 h at which
time TLC showed no starting material left. The reaction
mixture was cooled to ambient temperature and concentrated
in vacuo affording a solid. Recrystallization from toluene gave
3.1 g (65%) of 12 as white fibrous needles: mp 220-221 °C;
IR 3430, 3099, 1479, 1306, 1065, 724 cm-1; 1H NMR (DMSO-
d6) δ 1.18 (d, 12H, J ) 6.75 Hz, 2 × CH(CH3)2), 3.32 (septet,
2H, J ) 6.75 Hz, 2 × CH(CH3)2), 3.34 (s, 2H, CH2), 7.31 (s,
2H, 2 × Harom), 8.97 (s, 1H, exchangeable proton), 14.54 (s,
1H, exchangeable proton); MS (CI) m/z 295 (MH+). Anal.
(C14H18N2O1S2) C, H, N.
of 24a : mp 313-315 °C; IR 3628, 1698, 1662, 1544, 1315 cm-1
;
1H NMR (DMSO-d6) δ 1.43 (s, 18H, t-Bu), 2.25 (s, 3H, Me),
7.41 (s, 1 H, OH), 7.99 (s, 2 H, ArH), 13.0 (br s, 1 H, NH);
EIMS m/z 348 (M+). Anal. (C18H25N3O2S) C, H, N.
N-[3-(3,5-Di-ter t-bu tyl-4-h yd r oxyp h en yl)-[1,2,4]th ia d i-
a zol-5-yl]m eth a n esu lfon a m id e (24b). A solution of 2314
(0.50 g, 1.6 mmol) in 3 mL of pyridine was cooled in ice and
treated dropwise with methanesulfonyl chloride (0.25 mL, 0.37
g, 3.2 mmol). The mixture was stirred at ice-bath temperature
for 45 min and then for 16 h at room temperature. An
additional 0.45 mL (0.67 g, 5.8 mmol) of methanesulfonyl
chloride was added dropwise, and the mixture was stirred for
4 h. The mixture was evaporated, and the residue was
dissolved in water. The solution was acidified with 1.0 N HCl,
and the precipitated product was filtered and washed with
water. Purification of the crude product by flash chromatog-
raphy (elution with 50% ethyl acetate in hexanes) followed by
recrystallization from aqueous ethanol gave 0.20 g (32%) of
2,6-Di-ter t-b u t yl-4-(5-et h ylsu lfin yl[1,3,4]t h ia d ia zol-2-
yl)p h en ol (18a ). A solution of 6b (3.1 g, 8.8 mmol) in 55 mL
of CH2Cl2 was treated with MCPBA (3.5 g, approximately 9.6
mmol) in one charge at ambient temperature under nitrogen
atmosphere. The resulting yellow-amber solution was stirred
for 16 h at which time the reaction solution had become very
cloudy. The reaction mixture was cooled in an ice-salt bath
and filtered; the solids were washed with cold CH2Cl2. TLC
showed two very well separated spots (ether:hexanes ) 3:2).
The filtrate was concentrated in vacuo, and the residue was
purified by flash chromatography (elution with CH2Cl2-1%
CH3OH in CH2Cl2). The product with smaller Rf value was
isolated. Recrystallization from tert-butyl methyl ether/pentane
gave 0.81 g (25%) of 18a : mp 134-135 °C; IR 3520, 2961, 1599,
24b: mp 222-224 °C; IR 3616, 1605, 1559, 1289, 1121 cm-1
;
1H NMR (DMSO-d6) δ 1.41 (s, 18H, t-Bu), 3.06 (s, 3H, Me),
7.67 (s, 1H, OH), 7.79 (s, 2H, ArH); EIMS m/z 384 (M+). Anal.
(C17H25N3O3S2) C, H, N.
[3-[3,5-Bis(1,1-d im e t h yle t h yl)-4-[(m e t h oxye t h oxy)-
m eth oxy]ph en yl]-1,2,4-oxadiazol-5-yl]cyan am ide (32). 1,8-
Diazabicyclo[5.4.0]undec-7-ene (0.64 g, 4.2 mmol) was added
to a stirred solution of 3115 (1.0 g, 2.8 mmol) in 30 mL of
dimethoxyethane under an inert atmosphere at room temper-
ature and was followed after 5 min by diphenyl cyanocarbon-
imidate (1.0 g, 4.2 mmol). After 4 h the solvent was removed
by rotary evaporator and the residue dissolved in 100 mL of
ethyl acetate. The solution was washed with 100 mL of 1 N
HCl and then 100 mL of saturated brine and then dried over
MgSO4. The solvent was removed under reduced pressure to
leave a waxy solid residue which was triturated in cold tert-
butyl methyl ether to afford 0.9 g (79%) of 32: mp 167-168
1
1385, 1240, 1227, 1028, 704 cm-1; H NMR (CDCl3) δ 1.39 (t,
3H, J ) 7.48 Hz, CH3), 1.49 (s, 18H, 2 × t-Bu), 3.21-3.40 (m,
2H, CH2), 5.68 (s, 1H, OH), 7.79 (s, 2H, ArH); MS (CI) m/z
367 (MH+). Anal. (C18H26N2O2S2) C, H, N.
Meth od B.2,6-Di-ter t-bu tyl-4-(5-pr opoxy[1,3,4]th iadiazol-
2-yl)p h en ol (20d ). Sodium metal (1.4 g, 61 mmol) was
dissolved in warm anhydrous 1-propanol (30 mL). To this
freshly made solution of sodium 1-propoxide in 1-propanol was
added 1913 (2.3 g, 6.1 mmol) forming a dark-red solution. After
stirring under nitrogen atmosphere at reflux for 3 min, TLC
(30% EtOAc in hexanes as eluant) indicated that the starting
material had been completely consumed. The reaction solution
was cooled to ambient temperature and mixed with brine
solution and the pH adjusted to 3 with concentrated aqueous
HCl solution. The mixture was extracted with CH2Cl2 (3 × 40
mL); the combined organic phase was dried over Na2SO4 and
then concentrated in vacuo. The crude product was further
purified by flash chromatography (20% EtOAc in hexanes as
eluant) to gave 20d as a brown solid in 84% yield: mp 93-95
°C; IR 3587, 1502, 1230, 1146, 957, 705 cm-1; 1H NMR (CDCl3)
δ 1.05 (t, 3H, J ) 7.47 Hz, CH3), 1.47 (s, 18H, 2 × t-Bu), 1.88
(h, 2H, J ) 6.75 Hz, CH2), 4.50 (t, 2H, J ) 6.51 Hz, CH2), 5.51
(s, 1H, OH), 7.62 (s, 2H, 2 × Harom); MS (CI) m/z 349 (MH+).
Anal. (C19H28N2O2S) C, H, N.
1
°C; IR (KBr) 2219, 1659 cm-1; H NMR (DMSO-d6) δ 1.42 (s,
18H, CH3), 3.28 (s, 3H, OCH3), 3.55 (m, 2H, CH2), 3.89 (m,
2H, CH2), 4.97 (s, 2H, CH2), 7.76 (s, 2H, aromatic); MS (+APCI)
m/z 403 (MH+). Anal. (C21H30N4O4) C, H, N.
[3-[3,5-Bis(1,1-d im eth yleth yl)-4-h yd r oxyp h en yl]-1,2,4-
oxa d ia zol-5-yl]cya n a m id e (8). 30% HBr in acetic acid (18
mL) was added to a round-bottom flask with 32 (6.0 g, 15
mmol) and stirred at room temperature. After 1 h the solution
was poured into 500 mL of ice water, and stirred for 20 min.
The precipitate was filtered off, rinsed three times with water,
and dried. Recrystallization from acetonitrile afforded 2.2 g
(47%) of the crude product. Recrystallization of a sample from
acetonitrile afforded pure 8: mp 275 °C dec; IR 3617, 2960,
2217, 2203, 1669 cm-1 1H NMR (DMSO-d6) δ 1.41 (s, 18H,
;
CH3), 7.62 (s, 2H, ArH), 7.81 (bs, 1H, OH); MS (+APCI) m/z
315 (MH+). Anal. (C17H22N4O2) C, H, N.
Using method B, compounds 20a -c were prepared. The
physical data and chemical yields are summarized in Table 4.
Ack n ow led gm en t. We thank Dr. Gary McClusky
and staff for microanalytical and spectral data and
Godwin Okonkwo, Keith Laemont, and Mark Lesch for
animal testing.
3-(3,5-Di-ter t-b u t yl-4-h yd r oxyp h en yl)-4H -[1,2,4]t h ia -
d ia zol-5-on e (22). To a mixture of O-methylhydroxylamine
hydrochloride (75.5 mg, 0.904 mmol) and potassium tert-
butoxide in 15 mL of dry tert-butyl alcohol was added com-
pound 2114 (134 mg, 0.301 mmol). The resulting reaction
mixture was refluxed for 5 min. The deep-red color disap-
peared, and a white cloudy solution was obtained. The reaction
mixture was diluted with 10 mL of CH2Cl2, washed with brine
(2 × 20 mL), dried over Na2SO4, and concentrated in vacuo.
The solid was triturated with Et2O/hexanes to gave 63 mg
(68%) of 22 as a white solid: mp 251-252 °C; IR 3611, 3588,
Refer en ces
(1) Presented in part: (a) Song, Y.; Connor, D. T.; Sercel, A. D.;
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1669, 1427, 1243, 792 cm-1; H NMR (CDCl3) δ 1.49 (s, 18H,
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