Endothelin receptor antagonists: Synthesis and structure-activity relationships of substituted benzothiazine-1,1-dioxides

Article abstract of DOI:10.1016/S0968-0896(98)00080-7

The development of benzothiazine-1,1-dioxide derivatives as a new structural class of potent endothelin receptor antagonists is described. Structure-activity relationships (SAR) revealed that PD164800 (1) is a potent antagonist of the ET(A) receptor subtype. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00080-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1447±1456
Endothelin Receptor Antagonists: Synthesis and Structure±
Activity Relationships of Substituted Benzothiazine-1,1-dioxides
K. A. Berryman,a J. J. Edmunds,a,* A. M. Bunker,a S. Haleen,b J. Bryant,b
K. M. Welchb and A. M. Dohertya
aDepartment of Chemistry, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor,
MI 48105, USA
bDepartment of Therapeutics, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor,
MI 48105, USA.
Received 12 December 1997; accepted 10 February 1998
AbstractÐThe development of benzothiazine-1,1-dioxide derivatives as a new structural class of potent endothelin
receptor antagonists is described. Structure±activity relationships (SAR) revealed that PD164800 (1) is a potent
antagonist of the ETA receptor subtype. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
Endothelin-1 (ET-1) is an endogenous peptide that has
been implicated in a number of human diseases including
hypertension, congestive heart failure, renal failure, pul-
monary hypertension, ischemia and cerebral vasospasm.1±9
In humans, ET-1 interacts with least two receptor subtypes
(ETA and ETB).10±13 A third receptor subtype (ETC) has
been identi®ed in the frog, Xenopus laevis; however, this
receptor subtype has not been described in humans.14,15
In our continuing e€orts to design novel endothelin
antagonists we have optimized a weakly active lead, 2
(ETA IC50=16 mM), obtained by screening the Parke-
Davis library of compounds.
A number of nonpeptide ETA/ETB nonselective antago-
nists have been reported including Ro 46-2005,9 Ro 47-
0203 (bosentan),16 CGS 27830,17 and particularly from
these laboratories a series of 1,3-diaryl-2-carboxyindoles.18
In addition, nonpeptide ETA selective antagonists that
have been reported include PD 156707,19 BMS 182874,20
L-749,329,21 SKF 209670,22 and from these laboratories a
series of 1,3-disubstituted-2-carboxyindoles.18,23
Results and Discussion
Chemistry
The endothelin antagonists in this study were prepared
via the intermediacy of a 4-tri¯uoromethanesulphonyl-
oxy-benzothiazine dioxide derivative which underwent:
(i) displacement with a suitably substituted sodium
thiophenoxide, followed by hydrolysis, to a€ord the
compounds of Table 1, or alternatively, (ii) a palladium
mediated aryl cross coupling reaction, followed by
hydrolysis, to a€ord the compounds of Table 2.
Both procedures proved to be particularly general and
were tolerant of a number of thiophenols and boronic
*Corresponding author. Tel.: 734 622 4984; Fax: 734 622 3107.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00080-7

1448
K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
acids. The requisite thiophenols were readily prepared
by treatment of aryl lithiums with elemental sulphur24
or from the reaction of the corresponding aryl iodides
with thiourea and a nickel catalyst.25 The boronic acid
derivatives were generated by trapping the aryl lithium,
or Grignard, with trimethylborate.26
benzylation with a variety of substituted benzyl halides.
With a readily accessible route to compounds of type 5
we treated the 2-benzyl benzothiazine dioxide with
tri¯uoromethanesulphonic anhydride and pyridine in
dichloromethane to a€ord the crucial 4-tri¯uoromethane
sulphonate intermediate 6 which could be isolated, but
was generally used directly. Addition of the sodium salt
of a thiophenol to a solution of the tri¯uoromethane
sulphonate 6 in DMF readily a€orded the addition±
elimination adduct which was hydrolyzed with aqueous
base to a€ord the ®nal target compounds of Table 1.
Alternatively, the tri¯uoromethane sulphonate 6 was
treated with 3,4-methylenedioxyphenyl boronic acid and
catalytic palladium(0) tetrakistriphenylphosphine in
toluene/DMF, which upon re¯uxing a€orded the 4-aryl
adduct in good yield. Hydrolysis with aqueous base
a€orded the target compounds of Table 2.
Scheme 1 depicts an illustrative example for the synth-
esis of the compounds without substitution on the par-
ent benzo-ring. The reaction sequence was initiated by
the alkylation of saccharin, 3, with methyl bromoacetate
followed by ring expansion with sodium methoxide in
dimethylformamide (DMF) to give the benzothiazine
dioxide 4.27 Fortunately, benzylation with 3,4-methyl-
enedioxybenzyl chloride proceeded selectively to a€ord
the derivative 5. In fact, the di€erence in pKa of the
sulfonamide and the enol ester was sucient to allow
Table 1. SAR of pharmacophore 18
Compd
R1
R2
R3
ETA IC50 (nM)
ETB IC50 (nM)
7
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy 5-methoxy
H
3,4-methylenedioxy
3,4-dimethoxy
H
210
230
230
420
440
450
460
540
790
800
580
230
370
460
530
590
520
800
280
100
240
290
530
670
800
2700
6100
8200
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1
H
3,4,5-trimethoxy
3,4-ethylenedioxy
4-methoxy
H
22000
5600
H
H
21000
20000
11000
22000
20000
20000
3900
4-methyl
3-methoxy
H
H
H
4-chloro
3,4-dichloro
H
H
H
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
H
H
16000
5300
4-methyl
3,4,5-trimethoxy
4-chloro
H
H
H
19000
14000
13000
19000
3900
3,4-methylenedioxy 6-chloro
3,4-dichloro
4-methoxy
H
H
H
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
8-methoxy
7,8-dimethoxy
7-methoxy
6,7-methylenedioxy
7-chloro
6-chloro
6,7-dimethoxy
6-methoxy
11000
4000
5300
37
38
39
40
41
42
2100
4500
17000
5200
20000

K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
1449
The synthesis of compounds with substitution on the
parent benzo-ring required signi®cantly more synthetic
steps. The 5-methoxysaccharin derivative that was
required to prepare compounds 42 and 64 was syn-
thesized as described by Shkulev.28 Schemes 2 and 3
illustrate the synthetic routes employed to prepare the
other compounds with substitution on the parent
benzo-ring.
sequence that was used to prepare compounds 36 and 58.
The 7,8-dimethoxy-benzothiazine dioxide 17, which was
used to prepare compound 1, was synthesized as out-
lined in Scheme 3. Starting with 3,4-dimethoxy-2-nitro-
benzoic acid 13, prepared as described by Hess,31 the
substituted methyl anthranilate 14 was isolated after
esteri®cation of the carboxylic acid and reduction of the
nitro group. Diazotization with sodium nitrite and cup-
ric chloride in acetic acid followed by treatment with
sulfur dioxide surprisingly gave the disul®de 15, not the
expected sulfonyl chloride.32 Fortunately, the disul®de
was easily oxidized to the sulfonyl chloride in a manner
similar to that described for the oxidation of 10.30 The
saccharin derivative 16 was isolated after treating the
sulfonyl chloride with glycine methyl ester, triethylamine,
and 4-dimethylaminopyridine (DMAP) in dichloro-
methane. Ring expansion occurred upon exposure to
sodium methoxide in DMF to a€ord the benzothiazine
dioxide 17.
The 8-methoxy-benzothiazine dioxide 12, which was
used to prepare compounds 36 and 58, was synthesized
as outlined in Scheme 2. Esteri®cation of 9 with hydro-
gen chloride gas in methanol followed by Newman
rearrangement29 of the corresponding thiocarbamate
yielded 10. Oxidation with sulfuryl chloride and potas-
sium nitrate30 and addition of glycine methyl ester pro-
duced the saccharin derivative 11. Ring expansion with
sodium methoxide in DMF a€orded the benzothiazine
dioxide 12. Compounds 37 and 59 were prepared from
2-hydroxy-4-methoxy-benzoic acid by the same reaction
Table 2. SAR of pharmacophore 43
Compd
R1
R2
R3
ETA IC50 (nM)
ETB IC50 (nM)
8
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy 5-methoxy
H
3,4-methylenedioxy
3,4-dimethoxy
H
380
2000
15000
630
2500
4400
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
H
3,4,5-trimethoxy
3,4-ethylenedioxy
4-methoxy
H
25000
6500
H
H
5700
1400
11000
540
21000
13000
33000
2900
3-methoxy
4-chloro
H
H
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
H
H
410
900
9300
4-methyl
3,4,5-trimethoxy
4-chloro
H
32000
18000
13000
2700
H
H
530
700
3,4-methylenedioxy 6-chloro
3,4-dichloro
4-methoxy
H
980
H
2300
450
15000
5100
7400
H
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
3,4-methylenedioxy
8-methoxy
7-methoxy
6,7-methylenedioxy
7-chloro
6-chloro
6,7-dimethoxy
6-methoxy
1400
12000
2800
3900
390
8300
1300
8900
3800
830
1800
1700
1700

1450
K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
Similarly, compounds 38 and 60 were prepared from 2-
nitro-4,5-methylenedioxy benzoic acid,33 compounds 41
and 63 were prepared from methyl 2-amino-4,5-dimeth-
oxybenzoate, and compounds 39, 40, 61, and 62 were
prepared from the appropriately substituted disul®de.34
expressing recombinant human receptors (ETA), and
CHO-K1 cells expressing recombinant human receptors
(ETB).19,35
Previously we have demonstrated that 1-benzyl-3-
thioaryl-2-carboxyindoles
and
1-benzyl-3-aryl-2-
carboxyindoles are potent endothelin antagonists.18,23
Therefore, we decided that in the optimization of the
weakly active chemical lead compound 2 we would
incorporate pharmacophores evident from those series
of compounds. This strategy proved to be particularly
successful as judged by the activity of the compounds in
Tables 1 and 2. Typically we investigated the e€ect of
electron donating substituents on ETA receptor binding
anity by comparing binding anity to the appropriate
unsubstituted parent. In Table 1, and in particular
compounds 7, and 19±27, it was apparent that while the
e€ect of the substituent R2 was not particularly striking,
electron donating substituents were preferred. Further-
more multiple electron donating groups appeared to
favorably e€ect binding anity of the compounds. Thus
for further SAR investigations the 3,4-methylenedioxy
substituent was selected as the preferred substituent at
R2. Investigation of the R1 substituent with compounds
7, and 28±35 indicated, in analogy with previous SAR
from the 1,3-disubstituted 2-carboxy indoles, that the
3,4-methylenedioxybenzyl substituent was optimal.
Thus at this point compound 7 appeared most optimal
in terms of binding anity as it relates to substitution
pattern at R1 and R2. Upon investigation of the R3
substituent with compounds 36±42 we observed a
modest increase in binding anity occurred with com-
pound 1. This result stood in stark contrast to 1-benzyl-
3-thioaryl-2-carboxy indoles23 where bis methoxy sub-
stitution on the benzo-ring dramatically improved ETA
receptor binding anity. Clearly there are some subtle
di€erences between the binding mode and associated
protein interaction of the benzothiazine dioxides and the
indole derivatives.
Structure±activity relationships
Structure±activity relationships were investigated using
IC50 values obtained from receptor binding in Ltk-cells
Scheme 1. (a) i.BrCH2CO2CH3, NaH, DMF, rt, 18 h (65%). ii.
NaOCH3, rt, 0.5 h (68%); (b) 3,4-methylenedioxybenzyl chlor-
ide, NaH, DMF, rt, 5 h (69%); (c) tri¯uoromethanesulfonic
anhydride, pyr, CH2Cl2, rt, 0.5 h (95%); (d) i. 3,4-methylene-
dioxythiophenol, NaH, DMF, 2 h, rt, (79%); ii. LiOH, THF,
H2O, CH3OH, rt, 18 h (90%); (e) i. 3,4-methylenedioxy-
phenylboronic acid, Pd(PPh3)4, K2CO3, toluene, DMF, re¯ux,
2 h (75%); ii. LiOH, THF, H2O, CH3OH, rt, 18 h (90%).
Since 1 represented the most active compound from
Table 1, we advanced this compound to some secondary
assays. The ®rst of these assessed the ability of the
compound to inhibit the ET-1 induced release of ara-
chidonic acid in cultured rabbit renal vascular smooth
Scheme 2. (a) i. HCl/CH3OH, re¯ux, 2 h (97%); ii. N,N-dimethylthiocarbamoyl chloride, DABCO, DMF, rt, 18 h (72%); iii. neat,
180 ꢀC, 3 h (85%); iv. NaOCH3, CH3OH, relux, 18 h (95%); (b) i. KNO3, SO2Cl2, CH3CN, rt. 18 h (65%); ii. glycine methyl ester,
Et3N, DMAP, CH2Cl2, 18 h (47%); (c) NaOCH3, DMF, 0.5 h (83%).

K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
1451
Scheme 3. (a) i. CH3I, Cs2CO3, DMF, rt, 18 h (96%); ii. H2 Pd/C, rt. 18 h (quantitative); (b) NaNO2, SO2, CH3CO2H, 0 ꢀC to rt, 18 h
(52%); (c) i. KNO3, SO2Cl2, CH3CN, 5 h (78%); ii. glycine methyl ester, Et3N, DMAP, CH2Cl2, 0 ꢀC, 1 h (69%); (d) NaOCH3, DMF,
0.5 h (35%).
muscle cells which express ETA receptors coupled to
arachidonic acid.35 The IC50 of 150 nM indicated that 1
displayed reasonable antagonistic activity. The second
functional assay then assessed the ability of the test
compound to inhibit the contraction of rabbit femoral
artery rings upon stimulation with ET-1, a functional
response known to be mediated by the agonistic activity
of ET-1 upon ETA receptors.35 A pA2 of 5.8 con®rmed
that 1 was a functional ETA antagonist.
internal tetramethylsilane. Mass spectra were obtained
on a Finnigan 4500 or a VG analytical 7070 E/HF
spectrometer. High Resolution mass spectra were
obtained on a Finnigan MAT 900Q spectrometer.
Elemental analyses were performed on a CEC Model
240 elemental analyzer. Column chromatography was
performed using E. Merck silica gel 60 (230±400 mesh).
Thin-layer chromatography was performed with glass-
backed silica gel (60F254) plates employing UV light, iodine
vapor, or aqueous potassium permanganate/potassium
carbonate stain.
Interestingly, Table 2 shows that pharmacophore 43
a€orded less potent compounds. We explored SAR in a
similar fashion to pharmacophore 18 by observing the
e€ects of substituents at R1, R2, and R3. While similar
trends in activity were seen for these substituents to
pharmacophore 18 none of these compounds were as
potent as compound 1.
The binding assay is explained in detail in reference 35.
Brie¯y, cultured Ltk-cells expressing human cloned ETA
receptors and CHO-K1 cells expressing human cloned
ETB receptors were used. Binding of [125I]ET-1 (ETA) or
[
125I]ET-3 (ETB) to membranes prepared from the cells
above was performed by adding radiolabeled ET-1 or
ET-3 to membranes in the absence or presence of
increasing concentrations of the tested compound. At
the end of the incubation (37 ꢀC, 2 h), free and bound
ligands were separated by ®ltration and counted with a
gamma counter. Nonspeci®c binding was de®ned as the
binding in the presence of 100 nM unlabeled ET-1 or
ET-3. Speci®c binding was computer-analyzed by non-
linear least squares curve ®tting giving the best ®t for a
one-site model. IC50 values were derived from the aver-
age of two to ten competition experiments in which data
points were measured in triplicate.
Conclusion
A new series of endothelin antagonists was designed
and synthesized by incorporating structure±activity
relationships a€orded by a previous series of indole
based endothelin antagonists, in combination with a
chemical lead compound uncovered by screening the
historical library collection of Parke-Davis compounds.
The most potent compound synthesized, 1, was demon-
strated to be a functional antagonists of the ETA recep-
tor. Further in vivo studies are currently underway to
aid in the elucidation of the physiological and patho-
physiological role of endothelin.
1,2-Benzisothiazole-2(3H)-acetic acid, 3-oxo-, methyl
ester, 1,1-dioxide. To a solution of saccharin, 3 (40 g,
0.218 mol) in DMF (100 mL) at 0 ꢀC was added sodium
hydride (8.73 g, 60% in mineral oil, 0.218 mol). After
15 min methyl bromoacetate (20.7 mL, 0.218 mol) was
added and the mixture was stirred at room temperature
for 18 h. The reaction mixture was diluted with
Experimental
Proton NMR spectra were recorded on a Varian Gemini
2000 (300 MHz) or a Varian Unity plus (400MHz)
instruments; shifts are reported in d units relative to

1452
K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
dichloromethane (250 mL) and washed with saturated
sodium bicarbonate (2Â180 mL), water (100 mL), and
brine (2Â150 mL). The organic phase was dried with
magnesium sulfate, evaporated in vacuo, and the pro-
duct crystallized from hot ethanol to give the title com-
pound (36.3 g, 65%). 1H NMR (CDCl3, 300 MHz) d
3.81 (s, 3H), 4.47 (s, 2H), 7.84±7.94 (m, 2H), 7.96 (d,
J=8.0 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H). Anal. calcd for
C10H9N1O5S1: C, 47.06; H, 3.55; N, 5.49; found C,
47.02; H, 3.68; N; 5.37. MS (CI) 256 (M+1).
added tri¯uoromethanesulfonic anhydride (1.56 mL,
9.25 mmol). After 2 h the reaction was diluted with ethyl
acetate (100 mL), washed with 1 N hydrochloric acid
(2Â50 mL), brine (50 mL), dried with magnesium sul-
fate, and then the organic phase was evaporated in
1
vacuo to give 6 (4.0 g, quantitative). H NMR (CDCl3,
400 MHz) d 3.94 (s, 3H), 4.85 (s, 2H), 5.82 (s, 2H), 6.28
(s, 1H), 6.36 (d, J=7.8 Hz, 1H), 6.49 (d, J=7.8 Hz, 1H),
7.66±7.73 (m, 3H), 7.88±7.90 (m, 1H). MS (CI) 521
(M+1).
4-Hydroxy-1,1-dioxo-1,2-dihydro-1ꢀ6-benzo[e][1,2]thiazine-
3-carboxylic acid methyl ester (4). To methanol (100 mL)
was added sodium (5.4 g, 0.23 mol) portionwise. Once
all the sodium had dissolved the solution was con-
centrated in vacuo and the ®nal traces of methanol were
removed under high vacuum. The sodium methoxide
was suspended in dry DMF (65 mL) and then added
to a solution of 1,2-benzisothiazole-2(3H)-acetic acid,
3-oxo-, methyl ester, 1,1-dioxide (20 g, 0.078 mol) in
DMF (30 mL) at 0 ꢀC over 7 min. Upon complete addi-
tion of the sodium methoxide the reaction mixture was
allowed to stir for 30 min more. The product was pre-
cipitated from the reaction mixture with the dropwise
addition of 1 N hydrochloric acid (430 mL), washed
with water (200 mL), and dried at 52 ꢀC under vacuum
overnight to give 4 (13.6 g, 68%). 1H NMR (CDCl3,
400 MHz) d 3.95 (s, 3H), 6.27 (bs, 1H), 7.68±7.77 (m,
2H), 7.90 (d, J=7.5 Hz, 1H), 8.08 (d, J=8.7 Hz, 1H).
Anal. calcd for C10H9N1O5S1: C, 47.06; H, 3.55; N,
5.49; found C, 47.11; H, 3.67; N, 5.16. MS (CI) 256
(M+1).
2-Benzo[1,3]dioxol-5-ylmethyl-4-(benzo[1,3]dioxol-5-yl-
sulfanyl)-1,1-dioxo-1,2-dihydro-1ꢀ6-benzo[e][1,2]thiazine-
3-carboxylic acid methyl ester. A solution of 6 in DMF
(3 mL) was added to sodium 1,3-benzodioxole-5-thiolate
(0.31 g, 2.0 mmol)Ðprepared by dissolving 1,3-benzo-
dioxole-5-thiol (0.31 g, 2.0 mmol) in DMF (3 mL) and
stirring with sodium hydride (0.081 g, 2.0 mmol) for
5 minÐin DMF (3 mL). After stirring at room tem-
perature for 2 h the mixture was diluted with ethyl acet-
ate (100 mL), washed with 1 N sodium hydroxide
(2Â50 mL), brine (50 mL), dried with magnesium sul-
fate, and evaporated in vacuo. Silica gel column chro-
matography eluting with 25% ethyl acetate in hexane
a€orded the title compound as a foam (0.64 g, 79%). 1H
NMR (CDCl3, 300 MHz) d 3.86 (s, 3H), 4.76 (s, 2H),
5.85 (s, 2H), 5.86 (s, 2H), 6.31±6.63 (m, 4H), 6.69±6.73
(m, 2H), 7.43±7.50 (m, 2H), 7.79±7.86 (m, 2H). Anal.
calcd for C25H19N1O8S2: C, 57.14; H, 3.64; N, 2.67;
found C, 56.93; H, 3.93; N; 2.51. MS (CI) 525 (M+1).
2-Benzo[1,3]dioxol-5-ylmethyl-4-(benzo[1,3]dioxol-5-yl-
sulfanyl)-1,1-dioxo-1,2-dihydro-1ꢀ6-benzo[e][1,2]thiazine-
3-carboxylic acid (7). To 2-benzo[1,3]dioxol-5-ylmethyl-
4-(benzo[1,3]dioxol-5-ylsu)-1,1-dioxo-1,2-dihydro-1l6-
benzo[e][1,2]thiazine-3-carboxylic acid methyl ester
(0.467 g, 0.89 mmol) in THF (5 mL), methanol (2 mL),
water (2 mL) was added lithium hydroxide (0.46 g,
19 mmol) and the reaction mixture was stirred at room
temperature. After 5 h the reaction mixture was diluted
with water (80 mL) and 50% hydrochloric acid (10 mL)
and extracted with chloroform (3Â80 mL). The organic
phase was dried with magnesium sulfate and evaporated
2-Benzo[1,3]dioxol-5-ylmethyl-4-hydroxy-1,1-dioxo-1,2-
dihydro-1ꢀ6-benzo[e][1,2]thiazine-3-carboxylic acid methyl
ester (5). To 4 (1.48 g, 5.84 mmol) in DMF (10 mL) was
added sodium hydride (0.257 g, 60% in mineral oil,
6.4 mmol). After 5 min 3,4-methylenedioxybenzyl chlor-
ide (2.2 g, 50 wt.% in dichloromethane, 6.4 mmol) was
added and the mixture stirred at room temperature.
After 18 h the solution was diluted with ethyl acetate
(100 mL), washed with water (2Â80 mL), brine (80 mL),
and dried over magnesium sulfate. The organic phase
was evaporated in vacuo, and the product crystallized
1
in vacuo to a€ord 7 as a foam (0.4 g, 88%). H NMR
1
from ethyl acetate/heptane to give 5 (1.63 g, 72%). H
(CDCl3, 400 MHz) d 4.78 (s, 2H), 5.77 (s, 2H), 5.83 (s,
2H), 6.41 (s, 1H), 6.44±6.50 (m, 2H), 6.57±6.68 (d,
J=11.6 Hz, 1H), 6.69±6.73 (m, 2H), 7.38±7.40 (t, 1H),
7.42±7.46 (t, 1H), 7.74±7.79 (m, 2H) (CO2H absent).
Anal. calcd for C24H17N1O8S2: C, 56.35; H, 3.35; N,
2.74 found C, 56.13; H, 3.49; N, 2.62. MS (CI) 511
(M+).
NMR (CDCl3, 400 MHz) d 3.97 (s, 3H), 4.60 (bs, 2H),
5.76 (s, 2H), 6.33±6.42 (m, 3H), 7.52±7.62 (m, 2H), 7.71±
7.77 (m, 2H), 12.06 (s, 1H). Anal. calcd for
C18H15N1O7S1: C, 55.52; H, 3.88; N, 3.60; found C,
55.45; H, 3.72; N, 3.49. MS (CI) 389 (M+1).
2-Benzo[1,3]dioxol-5-ylmethyl-6,7-dimethoxy-1,1-dioxo-
4-(tri¯uoro-methanesulfonyloxy)-1,2-dihydro-1ꢀ6-benzo[e]-
[1,2]thiazine-3-carboxylic acid methyl ester (6). To a
solution of 5 (3.0 g, 7.71 mmol) in dichloromethane
(15 mL) and pyridine (3.1 mL, 38.6 mmol) at 0 ꢀC was
4-Benzo[1,3]dioxol-5-yl-2-benzo[1,3]dioxol-5-ylmethyl-1,1-
dioxo-1,2-dihydro-1ꢀ6-benzo[e][1,2]thiazine-3-carboxylic
acid methyl ester. To 6 (0.66 g, 1.27 mmol) in toluene
(10 mL) and DMF (2 mL) was added 3,4-methylene-

K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
1453
dioxy-phenylboronic acid (0.35 g, 2.13 mmol), potassium
carbonate (0.29 g, 2.13 mmol), tetrakis(triphenylphos-
phine)palladium(0) (0.16 g, 0.14 mmol) and the mixture
heated at re¯ux. After 2 h the reaction mixture was
cooled to room temperature, diluted with ethyl acetate
(100 mL) washed with sodium bicarbonate (80 mL aq
satd), brine (80 mL), dried with magnesium sulfate, and
evaporated in vacuo. The residue was puri®ed with a
silica gel column eluted with 50% ethyl acetate in hex-
brine (80 mL). The organic phase was dried with mag-
nesium sulfate, evaporated in vacuo, and puri®ed with
silica gel column eluted with 50% ethyl acetate in hex-
ane to give the title compound (1.11 g, 72%) as a white
1
solid. H NMR (CDCl3, 400 MHz) d 3.36 (s, 3H), 3.43
(s, 3H), 3.80 (s, 3H), 3.82 (s, 3H), 7.12 (d, J=8.2 Hz,
1H), 7.22 (t, 1H), 7.53 (d, J=8.2 Hz, 1H). Anal. calcd
for C12H15N1O4S1: C, 53.52; H, 5.61; N, 5.20; found C,
53.60; H, 5.63; N, 5.14. CI (MS) 269 (M+1).
1
ane to give the title compound (0.47 g, 75%). H NMR
(CDCl3, 300 MHz) d 3.59 (s, 3H), 4.83 (s, 2H), 5.80 (s,
2H), 6.00 (s, 2H), 6.52±60 (m, 4H) 6.64 (s, 1H), 6.82 (d,
J=8.0 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.39±7.45 (t,
1H), 7.47±7.53 (t, 1H), 7.84 (d, J=8.0 Hz, 1H) Anal.
calcd for C25H19N1O8S1: C, 60.85; H, 3.88; N, 2.84:
found C, 61.02; H, 3.96; N, 2.71. MS (CI) 494 (M+1).
2-Dimethylcarbamoylsulfanyl-3-methoxy-benzoic
acid
methyl ester. Heated 2-dimethylthiocarbamoyloxy-3-
methoxy-benzoic acid methyl ester (15.0 g, 55.8 mmol)
neat at 180 ꢀC for 3 h. The residue was puri®ed with a
silica gel column eluted with 65% ethyl acetate in hex-
ane to give the title compound (12.78 g, 85%). 1H NMR
(CDCl3, 400 MHz) d 2.95 (bs, 3H), 3.10 (bs, 3H), 3.83 (s,
3H), 3.85 (s, 3H), 7.05 (d, J=8.3 Hz, 1H), 7.29±7.40 (m,
2H). MS (CI) 270 (M+1).
4-Benzo[1,3]dioxol-5-yl-2-benzo[1,3]dioxol-5-ylmethyl-1,1-
dioxo-1,2-dihydro-1ꢀ6-benzo[e][1,2]thiazine-3-carboxylic
acid (8). To 4-benzo[1,3]dioxol-5-yl-2-benzo[1,3]dioxol-
5-ylmethyl-1,1-dioxo-1,2-dihydro-1l6-benzo[e][1,2]thi-
azine-3-carboxylic acid methyl ester (0.47 g, 0.95 mmol)
in THF (10 mL), methanol (3 mL), water (3 mL) was
added lithium hydroxide (0.6 g, 25.1 mmol) and the
reaction mixture was stirred at room temperature. After
18 h 1 N hydrochloric acid (100 mL) was added and the
solution extracted with chloroform (3Â80 mL). The
combined organic layers were washed with brine
(80 mL), dried with magnesium sulfate, and evaporated
in vacuo to give 8 (0.42 g, 90%). 1H NMR (CDCl3,
400 MHz) d 4.85 (s, 2H), 5.79 (s, 2H), 6.03 (s, 2H), 6.48±
6.52 (m, 3H), 6.58±6.61 (m, 2H), 6.82 (d, J=7.8Hz, 1H),
6.92 (d, J=7.8Hz, 1H), 7.38±7.42 (t, 1H), 7.52±7.56 (t,
1H) 7.87 (d, J=7.6 Hz, 1H), (CO2H absent). Anal. calcd
for C24H17N1O8S1: C, 60.12; H, 3.57; N, 2.92: found C,
59.80; H, 3.73; N, 2.86. MS (CI) 479 (M+1).
2-Mercapto-3-methoxy-benzoic acid methyl ester (10).
To
2-dimethylcarbamoylsulfanyl-3-methoxy-benzoic
acid methyl ester: (12.0 g, 44.6 mmol) in methanol
(120 mL) was added sodium hydride (5.4 g of 60%,
133.8 mmol) and the reaction mixture was heated at
re¯ux. After 1.5 h the solvent was evaporated in vacuo.
Then 1 N hydrochloric acid (300 mL) was added and the
solution was extracted with ether (3Â150 mL). The
combined organic phases were washed with brine
(100 mL), dried with magnesium sulfate, and evaporated
in vacuo to give 10 (8.36 g, 95%), which was used with-
out further puri®cation. 1H NMR (DMSO-d6,
400 MHz) d 3.79 (s, 3H), 3.85 (s, 3H), 5.30 (s, 1H), 7.12±
7.31 (m, 2H), 7.53 (d, J=7.7, 1H).
2-Chlorosulfonyl-3-methoxy-benzoic acid methyl ester.
To 10 (8.28 g, 41.8 mmol) in acetonitrile (100 mL) was
added potassium nitrate (10.6 g, 105 mmol). The reac-
tion was cooled to 0 ꢀC and sulfuryl chloride (8.3 mL,
105.0 mmol) was added over 5 min. The reaction mix-
ture was warmed to room temperature and stirred for
18 h. The solvent volume was reduced to 25 mL in vac-
cuo, and sodium bicarbonate (120 mL, aq satd) was
added. The resulting solution was extracted with ethyl
acetate (3Â100 mL) and the combined organic phases
were washed with brine (100 mL), dried with magnesium
sulfate, and evaporated in vacuo to give the title com-
pound (7.0 g, 64%), which was used without further
puri®cation. 1H NMR (CDCl3, 400 MHz) d 3.89 (s, 3H),
4.04 (s, 3H), 7.04 (d, J=7.9 Hz, 1H). 7.15 (d, J=7.9 Hz,
1H), 7.64±7.68 (t, 1H). MS (CI) 264 (M+).
2-Hydroxy-3-methoxy-benzoic acid methyl ester. To 3-
methoxysalicylic acid, 9 (1.0 g, 5.94 mmol) in methanol
(25 mL) was bubbled hydrogen chloride (g) for 10 min.
The reaction mixture was then heated at re¯ux. After 2 h
the solvent was evaporated in vacuo, the residue was
dissolved in chloroform (25 mL), passed through a pad
of silica gel, and evaporated ®ltrate to give the title
compound (1.06 g, 98%) 1H NMR (CDCl3, 400 MHz) d
3.86 (s, 3H), 3.91 (s, 3H), 6.77±6.80 (t, 1H), 7.01 (d,
J=8.1 Hz, 1H), 7.38 (d, J=8.1 Hz, 1H), 10.97 (s, 1H).
2-Dimethylthiocarbamoyloxy-3-methoxy-benzoic
acid
methyl ester. To 2-hydroxy-3-methoxy-benzoic acid
methyl ester (1.05 g, 5.77 mmol) in DMF (5 mL) was
added DABCO (2.59 g, 23.1 mmol) and N,N-dimethyl-
thiocarbamoyl chloride (2.85 g, 23.1 mmol), the reaction
mixture was stirred at room temperature. After 18 h the
reaction mixture was diluted with ethyl acetate
(100 mL), washed with 1 N hydrochloric acid (80 mL),
(7-Methoxy-1,1,3-trioxo-1,3-dihydro-1ꢀ6-benzo[d]iso-
thiazol-2-yl)-acetic acid methyl ester (11). To 2-chloro-
sulfonyl-3-methoxy-benzoic acid methyl ester (6.5 g,
24.5 mmol) in dichloromethane (75 mL) at 0 ꢀC was

1454
K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
added triethylamine (7.5mL, 54.0mmol), glycine methyl
ester hydrochloride (3.4g, 27.0 mmol), 4-dimethylamino-
pyridine (0.1 g, 0.8 mmol) and the reaction mixture stir-
red at room temperature. After 18 h the solvent volume
was reduced to 30 mL in vacuo and ethyl acetate
(120 mL) was added. The organic phase was washed
with 1 N hydrochloric acid (80 mL), brine (80 mL), dried
with magnesium sulfate, and evaporated in vacuo to
give an oil. The oil was dissolved in ethyl acetate and the
product was precipitated with the addition of hexane to
give 12 (3.3 g, 47%). 1H NMR (CDCl3, 400 MHz) d 3.76
(s, 3H), 4.02 (s, 3H), 4.40 (s, 2H), 7.28 (d, J=7.9, 1H), 7.58
(d, J=7.9 Hz, 1H), 7.72±7.59 (t, 1H). MS (CI) 286 (M+1).
water (30 mL). After 30 min sulfur dioxide (g) was bub-
bled through the solution for 15 min, followed by the
addition of cupric chloride dihydrate (5.85 g, 34.3 mmol)
in water (20 mL), and the reaction mixture was warmed
to room temperature. After 8 h, sulfur dioxide (g) was
bubbled through solution for 10 min and the reaction
mixture stirred for 18 h. The reaction mixture was dilu-
ted with water (1000 mL) and extracted with ethyl acet-
ate (3Â600 mL). The combined organic phases were
washed with sodium bicarbonate (2Â400 mL, aq
satd), brine (400 mL), dried with magnesium sulfate,
evaporated in vacuo, and crystallized from dichlor-
omethane/diisopropyl ether to give 15 (8.1 g, 52%).
1H NMR (CDCl3, 400 MHz) d 3.62 (s, 6H), 3.80 (s,
6H), 3.87 (s, 6H), 6.85 (d, J=8.6 Hz, 2H), 7.81 (d,
J=8.6 Hz, 2H). Anal. calcd for C20H22O8S2: C, 52.85;
H, 4.88; found C, 52.55; H, 4.74; MS (CI) 455
(M+1).
4-Hydroxy-8-methoxy-1,1-dioxo-1,2-dihydro-1ꢀ6-benzo[e]-
[1,2]thiazine-3-carboxylic acid methyl ester (12). To 11
(3.0 g, 9.46 mmol) in DMF (10 mL) was added sodium
methoxide (2.0 g, 37.9 mmol). The reaction mixture was
stirred for 30 min at room temperature then quenched
with 1 N hydrochloric acid (100 mL) and 12 (2.24 g,
83%) was collected as a light yellow precipitate. 1H
NMR (DMSO-d6, 400 MHz) d 3.82 (s, 3H), 3.87 (s, 3H),
7.42 (d, J=8.2 Hz, 1H), 7.51 (d, J=8.2 Hz, 1H), 7.69±
7.73 (t, 1H), 9.70 (s, 1H), 11.35 (s, 1H). MS (CI) 286
(M+1).
2-Chlorosulfonyl-3,4-dimethoxy-benzoic acid methyl es-
ter. To 15 (6.55 g, 14.4 mmol) and potassium nitrate
(4.37 g, 43.0 mmol) in acetonitrile (45 mL) was added
sulfuryl chloride (3.45 mL, 43.0 mmol) dropwise over
7 min and the reaction mixture was stirred at room
temperature. After 5 h sodium bicarbonate (500 mL, aq
satd) was added and the solution extracted with ethyl
acetate (600 mL). The organic phase was washed with
brine (400 mL), dried with magnesium sulfate, and eva-
porated in vacuo to give the title compound (6.6 g, 78%)
3,4-Dimethoxy-2-nitro-benzoic acid methyl ester. To 13
(25 g, 110 mmol) in DMF (70 mL) was added cesium
carbonate (71.8 g, 220 mmol) and methyl iodide
(10.3 mL, 165 mmol) and the reaction mixture stirred at
room temperature. After 18 h water (1500 mL) was
added and crystals of the title compound (25.4 g, 96%)
were collected. 1H NMR (CDCl3, 300 MHz) d 3.87 (s,
3H), 3.92 (s, 3H), 3.98 (s, 3H), 7.02 (d, J=8.8 Hz, 1H),
7.81 (d, J=8.8 Hz, 1H). Anal. calcd for C10H11N1O6: C,
49.80; H, 4.60; N, 5.81; found C, 49.76; H, 4.57; N, 5.75.
MS (CI) 242 (M+1).
1
as an oil. Used without further puri®cation. H NMR
(CDCl3, 300 MHz) d 3.90 (s, 3H), 3.96 (s, 3H), 4.08 (s,
3H), 7.21 (s, 2H).
(6,7-Dimethoxy-1,1,3-trioxo-1,3-dihydro-1ꢀ6-benzo[d]iso-
thiazol-2-yl)-acetic acid methyl ester (16). To 2-chloro-
sulfonyl-3,4-dimethoxy-benzoic acid methyl ester (3.4 g,
11.5 mmol) in dichloromethane (130 mL) at room tem-
perature was added triethylamine (6.4 mL, 46.2 mmol)
and glycine methyl ester hydrochloride (1.45 g,
11.5 mmol) and the solution stirred at room tempera-
ture. After 2 h the solution was diluted with dichloro-
methane (300 mL), washed with 1 N hydrochloric acid
(3Â300 mL), brine (300 mL), dried with magnesium sul-
fate, and the solvent evporated in vacuo. The residue
was puri®ed with a silica gel column eluted with 25±
35% ethyl acetate in hexane. The product was then
crystallized from dichloromethane/diisopropyl ether to
give 16 (2.5 g, 69%). 1H NMR (CDCl3, 300 MHz) d 3.80
(s, 3H), 4.00 (s, 3H), 4.16 (s, 3H), 4.42 (s, 2H), 7.25 (d,
J=8.2 Hz, 1H), 7.73 (d, J=8.2 Hz, 1H). Anal. calcd for
C12H13N1O7S1: C, 45.71; H, 4.16; N, 4.44 found C,
45.73; H, 4.12; N, 4.38 MS (CI) 316 (M+1).
2-Amino-3,4-dimethoxy-benzoic acid methyl ester (14).
To 3,4-dimethoxy-2-nitro-benzoic acid methyl ester
(25.3 g, 0.1 mol) in THF (500 mL) and methanol
(500 mL) was added 5%Pd/C (3.0 g). The reaction mix-
ture was then hydrogenated at 50 psi overnight. The
reaction solution was ®ltered through celite and the sol-
vent volume reduced by 50% in vacuo. Hydrochloric
acid (gas) was bubbled through the solution for 10 min,
ether (400 mL) was added, and 14 (25 g, quantitative)
was collected as a light-brown precipitate. 1H NMR
(DMSO-d6, 300 MHz) d 3.66 (s, 3H), 3.76 (s, 3H), 3.82
(s, 3H), 6.38 (d, J=9.2 Hz, 1H), 7.52 (d, J=9.2 Hz, 1H),
(NH2 absent). MS (CI) 242 (M+1).
Methyl-2,20-dithiobis[3,4]-dimethoxybenzoate (15). To 14
(16.94 g, 68.44 mmol) in acetic acid (150 mL) and con-
centrated hydrochloric acid (150 mL) at 0 ꢀC was slowly
added a solution of sodium nitrite (4.73 g, 68.4 mmol) in
4-Hydroxy-7,8-dimethoxy-1,1-dioxo-1,2-dihydro-1ꢀ6-
benzo[e][1,2]thiazine-3-carboxylic acid methyl ester (17).
To 17 (0.23 g, 0.73 mmol) in DMF (1.0 mL) at 0 ꢀC was

K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
1455
added sodium methoxide (0.12 g, 2.22 mmol) and the
reaction mixture stirred for 4 min. The reaction solu-
tion was added to concentrated hydrochloric acid
(2 mL)/ice (2.0 g) and 17 (0.105 g, 46%) was collected
as a white precipitate. 1H NMR (CDCl3, 400 MHz) d
3.79 (s, 3H), 3.80 (s, 3H), 3.90 (s, 3H), 7.45 (d,
J=8.8 Hz, 1H), 7.70 (d, J=9.0 Hz, 1H), 9.65 (s, 1H),
11.48 (s, 1H). Supplementary material. Those interested
in additional compound information may contact the
author.
15. Kumar, C.; Mwangi, V.; Nuthulaganti, P.; Wu, H.-L.;
Pullen, M.; Brunb, K.; Aiyar, H.; Morris R. A.; Naughton, R.;
Nambi, P. J. Biol. Chem. 1994, 269, 1344.
16. Roux, S. P.; Clozel, M.; Sprecher, U.; Gray, G.; Clozel, J.
P. Circulation 1993, 88, I170.
17. Mugrage, B.; Moliterni, J.; Robinson, L.; Webb, R. L.;
Shetty, S. S.; Lipson, K. E.; Chin, M. H.; Neale, R.; Cio, C.
Bioorg. Med. Chem. Lett. 1993, 3, 2099.
18. Bunker, A. M.; Edmunds, J. J.; Berryman, K. A.; Walker,
D. M.; Flynn, M. A.; Welch, K. M.; Doherty, A. M. Bioorg.
Med. Chem. Lett. 1996, 6, 1061.
19. Doherty, A. M.; Patt, W. C.; Edmunds, J. J.; Berryman, K.
A.; Reisdorph, B. R.; Plummer, M. S.; Shahripour, A.; Lee, C.;
Cheng, X.-M.; Walker, D. M.; Haleen, S. J.; Keiser, J. A.;
Flynn, M. A.; Welch, K. M.; Hallak, H.; Taylor, D. G.; Rey-
nolds, E. E. J. Med. Chem. 1995, 38, 1259.
Acknowledgements
The authors would like to thank Dr Gary McClusky and
associates for providing the spectroscopic and analytical
data.
20. Stein, P. D.; Hunt, J. T.; Floyd, D. M.; Moreland, S.;
Dickinson, K. E. J.; Mitchell, C.; Liu, E. C.-K.; Webb, M. L.;
Murugesan, N.; Dickey, J.; McMullen, D.; Zhang, R.; Lee, V.
G.; Sera®no, R.; Delaney, C.; Schae€er, T. R.; Kozlowski, M.
J. Med. Chem. 1994, 37, 329.
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1456
K. A. Berryman et al./Bioorg. Med. Chem. 6 (1998) 1447±1456
34. Theses disul®des were prepared from the appropriately
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