2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides as ocular hypotensive agents: Synthesis, carbonic anhydrase inhibition and evaluation in the rabbit

Bioorganic & Medicinal Chemistry 8 (2000) 957±975

2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide

1,1-Dioxides as Ocular Hypotensive Agents: Synthesis, Carbonic

Anhydrase Inhibition and Evaluation in the Rabbit

Hwang-Hsing Chen, ^aSharon Gross, ^aJohn Liao, ^aMarsha McLaughlin, ^aTom Dean, ^a

William S. Sly ^band Jesse A. May ^a,*

^aOphthalmic Products Research, Alcon Research, Ltd., Fort Worth, TX 76134, USA

^bDepartment of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO 63104, USA

Received 11 November 1999; accepted 6 December 1999

AbstractÐNovel non-chiral 2H-thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides were synthesized for evaluation as

potential candidates for the treatment of glaucoma. All of the compounds prepared were potent high anity inhibitors of human

carbonic anhydrase II, K_i<0.5 nM. Additionally, inhibition of recombinant human carbonic anhydrase IV was determined for

selected compounds; these were shown to be moderate to potent inhibitors of this isozyme with IC₅₀values ranging from 4.25 to

73.6 nM. Of the compounds evaluated for their ability to lower intraocular pressure in naturally hypertensive Dutch-belted rabbits,

5a, 17a3, 17b1, 17b2, 17h2 and 17i1 showed signi®cant ecacy (>20% decrease) in this model following topical ocular adminis-

Introduction

Topical ocular administration of a carbonic anhydrase

inhibitor has long been viewed as an attractive alter-

native to oral therapy because of the expectation that it

would reduce the incidence and magnitude of side

eects, and thereby improve patient compliance. This

interest in the development of a topical ocular CA inhi-

bitor has lead to the recent introduction of dorzola-

mide⁵(A), from the laboratories of Merck & Co., and

brinzolamide⁶(B), from our own laboratories. These

CA inhibitors are both topically eective for lowering

intraocular pressure in glaucoma therapy. Both of these

compounds display a pronounced stereoselectivity with

regard to enzyme inhibition and subsequent in vivo

ecacy. Dorzolamide, with two chiral centers (4S, 6S),

is the one diastereomer that is therapeutically eective.

Similarly, brinzolamide (4R) is the therapeutically

eective enantiomer of this structure.

Elevated intraocular pressure (ocular hypertension) is

believed to lead to damage and eventual death of nerve

®bers within the optic nerve. The resulting progressive

reduction of the ®eld of vision ultimately leads to

blindness. Current therapeutic intervention for glau-

coma is primarily directed toward physiological

mechanisms that lead to the reduction of intraocular

pressure in an eort to prevent nerve ®ber damage.¹

Carbonic anhydrase (CA) is intimately involved in the

production of aqueous humor, and inhibitors of this

enzyme are eective in lowering intraocular pressure by

reducing the production of aqueous humor. Agents

such as Diamox and Neptazane have been used sys-

temically for the treatment of ocular hypertension for

over 40 years.^{2 4}Unfortunately, patient compliance for

this therapy has generally been poor due to a variety of

non-life threatening, but discomforting, systemic side

eects resulting from the inhibition of extraocular

enzyme.

Because of the considerable disadvantages associated

with the preparation of a single enantiomer, either by a

stereoselective synthesis or resolution, it was of interest

to identify non-chiral analogues of brinzolamide (B)

that possessed the desired physicochemical pro®le and

requisite biological activity. We had previously

observed that 2-substituted 3,4-dihydro-2H-thieno[3,2-

e]-1,2-thiazine-6-sulfonamide 1,1-dioxides (such as C)

*Corresponding author. Tel.: +1-817-551-8150; fax: +1-817-568-

7661; e-mail: jesse.may@alconlabs.com

PII: S0968-0896(00)00026-2

958

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

demonstrated potent inhibition of human CA-II

(K_i=0.490.15 nM, IC₅₀=2.51 nM), indicating that a

substituent at position four of this structure was not

required to achieve the desired level of enzyme inhibi-

tion.⁷This observation suggested that appropriately

substituted derivatives of the parent heterocyclic ring,

devoid of any chiral centers, would provide an attractive

series of molecules for evaluation as potential ther-

apeutic agents (Fig. 1).

Though substitution at C4 proved to be quite bene®cial

in enhancing biological activity in the dihydro-

thienothiazine series of compounds, including brinzola-

mide, such substitution in the parent heterocycle did not

appear desirable. The orientation of such a substituent

would be anticipated to result in unfavorable interac-

tions with the wall of the active site pocket. Substitution

at ring positions two and three appeared more attrac-

tive, since such substituents would be oriented toward

the top of the active site pocket and would provide an

opportunity to form favorable binding interactions with

residues on the surface of this portion of the enzyme.

Therefore, the synthesis of a few examples of 2-ami-

noalkyl substituted analogues and an extended series of

2-substituted 3-(4-morpholinylmethyl)-2H-thieno[3,2-e]-

and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides was

pursued in order to evaluate these compounds as poten-

tial candidates for topical ocular glaucoma therapy.

Scheme 1. Reagents: (a) 2-Bromoethyl acetate, NaH/DMF; (b) i.

Ms₂O and triethylamine or 2,6-lutidine/THF; ii. DBN/DMF; (c)

NaOH/MeOH; (d) i. Ms₂O/THF; ii. alkylamine; (e) i. NaH, 1,4-

dibromo-2-butene/DMF; ii. Morpholine; (f) TFA.

The synthetic procedures used to prepare 2,3-di-

substituted 2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide

1,1-dioxides are shown in Schemes 2±5. Compound 9

was initially envisioned as a desirable intermediate for

the introduction of a variety of substituents on the

nitrogen atom at position two. Lithiation of the ethyl-

ene glycol acetal of 3-thiophenecarboxaldehyde⁹(6)

followed by reaction with sulfur dioxide and N-chloro-

succinimide provided the intermediate 2-sulfonyl

chloride, which was reacted with glycine ethyl ester to

give 7. Acidic hydrolysis of 7 provided the aldehyde 8,

but all attempts to prepare 9 directly from 8 were

unsuccessful. When 8 was treated with DBU, the

cyclized product, 10, was obtained in modest yield.

Attempts to aect dehydration of 10 under a variety of

conditions all resulted in the consumption of 10 while

producing no identi®able products. Since 9 was un-

available as a strategic intermediate, it was necessary to

introduce the desired nitrogen substituent prior to

cyclization. This approach required the use of inter-

mediate 12, which could be prepared from either 7 or

11, and which readily cyclized upon treatment with

DBU to give the desired 2-substituted 3-carbethoxy

intermediates 13 (Scheme 3). Incorporation of an aryl

group at position two of 13 required the preparation of

the appropriate intermediate 11. Reduction of the ester

group of 13 with DIBAL-H gave the primary alcohols,

14, which were sulfamoylated (n-butyllithium, sulfur

dioxide, hydroxylamine-O-sulfonic acid) to give sulfona-

mides 15 in good yield (Scheme 4).

Chemistry

The sequence used for the preparation of 2-substituted

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides

is shown in Scheme 1. Dehydration of 2, prepared by

alkylation of 1⁸with 1-acetoxy-2-bromoethane, was

accomplished by initial formation of the mesylate fol-

lowed by elimination in the presence of DBU to give the

thieno[3,2-e]-1,2-thiazine ring, and subsequent depro-

tection with aqueous base provided the intermediate

alcohol, 3. Conversion of 3 to the mesylate followed by

treatment with the appropriate amine aorded com-

pounds 4a±d. Deprotection of the sulfonamide by

treatment with tri¯uoroacetic acid provided 5a±d. The

preparation of 5e followed a similar sequence, namely

alkylation of 1 with 1,4-dibromo-2-butene followed by

treatment with morpholine, and ®nally dehydration to

give 4e. Deprotection of 4e, as before, gave 5e.

Mesylation of the alcohol followed by reaction with the

appropriate amine provided the desired compounds, 17.

Figure 1.

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

959

Scheme 4. Reagents: (a) DIBAL-H/THF; (b) i. n-BuLi/THF; ii. SO₂;

iii. HOSA, NaOAc/H₂O; (c) i. Ms₂O/THF; ii. Alkyl amine/THF.

Scheme 2. Reagents: (a) i. n-BuLi/THF; ii. SO₂; iii. NCS/CH₂Cl₂; iv.

glycine ethyl ester; (b) HCl/THF; (c) DBU, molecular seives/EtOAc.

Scheme 3. Reagents: (a) i. n-BuLi/THF; ii. SO₂; iii. NCS/CH₂Cl₂; iv.

primary or secondary amine; (b) i. Ethyl bromoacetate/DMF; ii. t-

BuOK/DMF; (c) i. Alkyl halide/DMF; ii. t-BuOK/DMF; (d) i. p-

TsOH/acetone; ii. DBN/EtOAc.

Scheme 5. Reagents: (a) i. n-BuLi/THF; ii. SO₂; iii. NCS/CH₂Cl₂; iv.

t-BuNH₂; (b) i. p-TsCl/THF; ii. Amine/DMF; (c) Borontribromide/

CH₂Cl₂.

Alternately, by reversing the order of these last two

steps, one could initially obtain the amine 16, which

gave 17 upon sulfamoylation. Of these two sequences,

the former, proceeding through 15, was the preferred

route. A variation of this approach, involving the

incorporation of a t-butyl protected sulfonamide group,

was used for the preparation of the 2-(3-hydroxyphenyl)

substituted compounds (Scheme 5). Reaction of 14k by

following a sequence utilizing n-butyllithium, sulfur

dioxide, N-chlorosuccinimide, and t-butylamine readily

provided the sulfonamide with concomitant chlorina-

tion of the alcohol to give 18. Reaction of 18 with

the desired amine gave 19k1 and 19k8, and these, fol-

lowing treatment with boron tribromide, provided the

desired phenolic compounds 17p1 and 17p8, respectively

(Table 1).

The synthesis of a representative member of the iso-

meric thieno[2,3-e]-1,2-thiophene ring system was

accomplished by a sequence similar to that used for the

preparation of the thieno[3,2-e]-1,2-thiazine-6-sulfona-

mides (Scheme 6). The readily available ethylene glycol

acetal of 3,5-dibromo-2-thiophenecarboxaldehyde¹⁰(20)

served as starting material. Halogen to hydrogen

exchange of the bromine atom at ring position ®ve of

compound 20 was accomplished by metallation with

n-butyllithium followed by treatment with 1-propanol

to give 3-bromo-2-thiophenecarboxaldehyde acetal.

This compound was not isolated, but was sulfamoylated

960

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

directly to give 21 by using the procedure described for

the preparation of 18, though sarcosine ethyl ester was

used as the amine. Cleavage of the acetal group of 21

with tri¯uoroacetic acid gave the aldehyde, which

underwent facile cyclization upon treatment with DBN

to give 22. Compound 22 was converted to 25 through a

series of steps similar to those described in Scheme 4.

It is informative to note that attempts to prepare the

desirable synthetic intermediate 28 by cyclization of the

thiophene-2,4-disulfonamide 27, where the protected

primary sulfonamide was introduced into the thiophene

ring prior to ring closure, were unsuccessful (Scheme 7).

Treatment of 20 with n-butyllithium followed by reac-

tion with sulfur dioxide and then treatment with N-

chlorosuccinimide resulted in exchange of the bromine

at position ®ve to give the intermediate sulfonyl

chloride. This product was reacted with t-butylamine to

give the protected sulfonamide 26. The structure of

26 was con®rmed by a subsequent halogen±proton

exchange reaction; the coupling constants for the two

thiophene protons in the product of this reaction were

consistent for a 2,5-disubstituted thiophene. Treatment

of 26 with a second metallation sequence (n-butyl-

lithium/sulfur dioxide/N-chlorosuccinimide) resulted in

exchange of the bromine at position three to give the

sulfonyl chloride intermediate. Reaction of the sulfonyl

chloride with sarcosine ethyl ester followed by hydro-

lysis of the acetal, which required prolonged treatment

with tri¯uoroacetic acid and resulted in concomitant

cleavage of the t-butyl group, provided 27. All attempts

to obtain the desired intermediate 28 by cyclization of

27 produced decomposition, most probably by way of a

nucleophilic attack on the electron de®cient thiophene

ring.

Table 1. Substituent legend for compounds 11±17

R

NR²R³

a

b

c

d

e

f

g

h

i

Me

Et

nPr

iPr

allyl

CH₂cPr

cHexyl

(CH₂)₂OMe

(CH₂)₃OMe

CH₂±C₆H₄±(4-OMe)

C₆H₄±(3-OMe)

C₆H₃±(3,4-OMe)

C₆H₄±4-(4-Morpholinyl)

(CH₂)₃OH

C₆H₄±(3-OH)

1

2

3

4

5

6

7

8

9

4-morpholinyl

CH₂CH₂OMe CH₂CH₂OMe

CH₂CH₂OMe CH₂CH₂CH₂OMe

CH₂CH₂OH

H

CH₂CH₂OMe Me

CH₂CH₂OMe allyl

CH₂CꢀCH

H

cPr

j

10 1-imidazoyl

k

m

n

o

p

Results and Discussion

Biological assays

The ability of the target compounds to inhibit human

carbonic anhydrase II (hCA-II) was determined using a

pH-stat assay similar to that previously described; this

assay measures the ability of a compound to inhibit the

carbonic anhydrase catalyzed hydration of carbon

Scheme 6. Reagents: (a) i. n-BuLi, 1-propanol/Et₂O; ii. n-BuLi; iii.

SO₂; iv. NCS/CH₂Cl₂; v. Sarcosine ethyl ester; (b) i. TFA/acetone; ii.

DBN, molecular seives/EtOAc; (c) DIBAL-H/THF; (d) i. n-BuLi/

THF; ii. SO₂; iii. HOSA, NaOAc/H₂O; (e) i. p-TsCl/THF; ii. Mor-

pholine/THF.

Scheme 7. Reagents: (a) i. n-BuLi/THF; ii. SO₂; iii. NCS/CH₂Cl₂; (b)

t-BuNH₂; (c) NaH/THF; (d) i. Sarcosine ethyl ester; ii. TFA.

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

961

dioxide (IC₅₀).^11,12Inhibitor binding to hCA-II was

determined using a ¯uorescence competition assay that

monitors the decrease in ¯uorescence resulting from the

displacement of dansylamide from the enzyme by an

inhibitor (K_i).^12,13The ability of compounds to lower

intraocular pressure was evaluated by testing in a colony

of naturally ocular hypertensive Dutch-belted rabbits.

ecacy in lowering intraocular pressure have been pro-

posed.¹⁴These criteria were modi®ed during the devel-

opment of dorzolamide to include the requirement that

the molecule possess an amphoteric character, e.g., that

it has a weakly acidic primary sulfonamide and a

weakly basic amine.¹⁵It has been suggested that this

amphoteric property facilitates the sequestering of the

compound in ocular tissues such as the stroma, which

would provide a depot of the inhibitor from which to

maintain the relatively high concentration of inhibitor

required to achieve the level of enzyme inhibition

necessary to reduce aqueous humor formation. Com-

pounds 5a±c,e, containing weakly basic alkylamines

(pK_a5.34±6.69) at ring position two (Table 2), were

particularly potent inhibitors of hCA-II, while 5d, which

incorporates a signi®cantly more basic amine (pK_a8.27),

was a weaker inhibitor with a lower anity for the

enzyme. The 4-morpholinyl-2-butenyl group introduced

As illustrated in Table 2, compounds 5, 17 and 25 all

showed high anities for hCA-II and were all potent

inhibitors of the enzyme, with inhibition constants in

the low nanomolar range. This level of CA-II inhibition

is well within the range required to achieve a decrease in

intraocular pressure, provided, of course, that the phy-

sicochemical properties of a particular compound are

satisfactory to permit access to the target tissue, the

ciliary body. A series of speci®c, predictive criteria that

a molecule must satisfy to demonstrate topical ocular

Table 2. In vitro enzyme data and physicochemical data for compounds 5, 17 and 25

c

Compound

hCA-II

rhCA-IV

Solubility pH 5.0 (%)

DC pH 7.4

pK_a

IC₅₀(nM)^a

K_i(nM)^b

IC₅₀(nM)^a

5a

5b

5c

5d

5e

17a1

17a3

17a6

17a7

17b1

17b2

17b4

17c1

17c2

17c8

17d1

17d8

17e1

17f1

17g1

17h1

17h2

17i1

17i9

17i10

17j1

17k1

17k5

17m1

17n1

17o1

17p1

0.91

0.82

0.96

2.25

Ð

1.66

Ð

1.17

1.11

1.04

0.96

1.24

1.48

1.52

1.46

1.60

0.99

1.41

0.79

5.58

4.41

2.63

3.12

2.23

2.01

1.27

1.16

1.05

0.34 (0.03)

0.32 (0.08)

0.25 (0.02)

0.52 (0.05)

0.24 (0.06)

0.26 (0.03)

0.08 (0.01)

0.15 (0.04)

0.10 (0.01)

0.21 (0.03)

0.21 (0.02)

0.33 (0.06)

0.27 (0.02)

0.15 (0.01)

0.10 (0.01)

0.33 (0.03)

0.47 (0.10)

0.16 (0.06)

0.30 (0.08)

0.31 (0.01)

0.22 (0.02)

0.21 (0.01)

0.25 (0.02)

0.26 (0.05)

0.12 (0.01)

0.11 (0.04)

0.15 (0.01)

0.11 (0.01)

0.08 (0.01)

0.12 (0.01)

0.20 (0.03)

0.15 (0.05)

0.16 (0.01)

0.21 (0.01)

0.13 (0.01)

0.51 (0.09)

Ð

15.2 (0.80)

Ð

73.6

Ð

16.9 (1.3)

0.442

0.201

0.067

2.52

Ð

0.03

17.0

4.97

1.91

2.3

6.09

12

52.6

18.2

172

19.3

53.5

2.55

55.9

177

42.5

61

5.90; 8.45

5.34; 8.27

5.49; 8.43

8.27^d

6.69; 8.56

5.01; 8.34

6.13; 8.53

6.45; 8.34

5.95; 8.37

5.20; 8.49

5.46; 8.40

6.28; 8.39

5.26; 8.36

5.52; 8.61

5.68; 8.47

5.33^e

5.64; 8.46

5.09; 8.44

5.87; 8.23

5.64; 8.24

5.93; 8.17

5.53; 8.47

5.24; 8.43

6.36; 8.46

6.08; 8.37

5.33, 8.49

5.30; 8.27

6.61; 8.32

5.24; 8.43

5.45; 8.22

5.39; 8.46

5.22, 8.51

5.71; 8.23

8.38^f

Ð

1.24

0.634

0.028

0.323

0.451

4.41+

0.007

0.024

0.0076

0.113

0.041

0.081

0.002

0.218

2.54

0.768

3.61

0.156

0.0027

0.013

0.27

0.055

0.034

0.40

0.014

Ð

16.9 (1.4)

Ð

29.7

42.5

57

23.3 (0.9)

Ð

49.5

39.9

Ð

23.6

322

3.85

13.5

14.8

19.8

6.32

222

148

10.1

40.1

68.8

3.16

57.1

39.1

8.7

Ð

5.45 (0.25)

7.41 (0.60)

Ð

10.1 (0.99)

8.65 (0.35)

Ð

4.25 (0.37)

6.25 (1.66)

Ð

44.9

32.0 (0.7)

17.5

1.50

1.62

1.15

0.60 (0.11)

2.52

3.19 (0.30)

3.74 (0.02)

8.88

17p8

25

0.026

0.4

Ð

Brinzolamide

Dorzolamide

Acetazolamide

Methazolamide

Methylbenzolamide

6.56

15.2

Ð

5.9; 8.3

Ð

12.5

3.90

Ð

205

5.6

Ð

^aSingle determinations except duplicates as noted with standard deviation.

^bMean of at least two determinations with standard deviation.

^cAmine and sulfonamide values, respectively.

^dValue includes both the amine and the sulfonamide ionization, 2 equivalents.

^eUnable to obtain satisfactory in¯ection for sulfonamide pK.

^fUnable to obtain satisfactory in¯ection for amine pK.

962

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

into compound 5e provided a very potent inhibitor of

hCA-II; the inhibition was suciently beyond the limits

of the current assay that a reliable inhibition constant

could not be determined for this compound.

group for this compound might assume a more desir-

able orientation than when directly attached to the

nitrogen atom.

As observed with other sulfonamide inhibitors of CA-II,

compounds 5 and 17 displayed a higher value for the

inhibition constant (IC₅₀) for rhCA-IV than for hCA-II

(2.7- to 33-fold higher). With the exception of the com-

pounds bearing polar substituents or a cyclohexyl moi-

ety as noted above, the potencies of the compounds

evaluated were more comparable to acetazolamide

(IC₅₀=17.5 nM) than methazolamide (IC₅₀=206 nM).

Further, the potency of several of the compounds was

comparable to that of methylbenzolamide²¹(IC₅₀=

5.6 nM), a compound which has been reported to have a

higher anity for bovine CA-IV than benzolamide.²²In

general, compounds 17 showed a greater inhibition of

rhCA-IV than that observed for the 3,4-dihydro-2H-

thieno[3,2-e]-1,2-thiazines, such as brinzolamide.²³An

X-ray crystallographic analysis of the enzyme±inhibitor

complexes formed upon co-crystallization of 17k1 and

17p1 with hCA-II and murine CA-IV is in progress and

will be reported in due course.²⁴

Incorporation of the weakly basic 4-morpholinylmethyl

group into position C3 and a variety of alkyl and aryl

substituents at N2 provided compounds 17a1±17p1, all

of which displayed a high anity (K_i) as well as good

inhibition for hCA-II (Table 2). Branching at the a-

carbon of an N2 alkyl substituent was observed to be

detrimental to enzyme anity as demonstrated by the

reduction in anity of 17d1 compared to that of com-

pounds 17a±c1. Interestingly, 17g1, with a cyclohexyl

substituent, showed very good inhibition of hCA-II.

The ethers 17h1 and 17i1 showed a modest decrease in

inhibition in spite of a high anity for the enzyme.

Incorporation of an aromatic moiety such as 4-methoxy-

phenylmethyl (17j1) or 3-methoxyphenyl (17k1) into the

molecule at ring position two provided a modest

improvement in inhibitor anity for the enzyme, rela-

tive to aliphatic substituents. The high anity observed

for 17n1 suggests that a region of considerable bulk

tolerance exists beyond the phenyl group. A comparison

of the in vitro enzyme data for 17a1 and 25 reveals that

the orientation of the sulfur atom in the thiophene ring

relative to the thiazine ring has no apparent eect on

enzyme binding or inhibition.

In view of our concern regarding the eect that basicity

might have on the in vivo availability of these com-

pounds, it was of interest to evaluate compounds that

incorporate a variety of other weakly basic amines into

the molecule at position C3. These compounds were

modestly stronger bases (pK 5.51 to 6.64) than the

compounds bearing the 4-morpholinylmethyl group (pK

5.01 to 5.33). This modest increase in basicity was not

detrimental for inhibitor binding to the enzyme, since all

of these compounds were also potent high anity inhi-

bitors of hCA-II (Table 2). For example, modi®cation

of 17a1 by the incorporation of N-(2-methoxyethyl)-

N-(3-methoxypropyl)amine, N-(2-methoxyethyl)methyl-

amine, or N-(2-methoxyethyl)-N-allylamine (17a3, a6,

a7) resulted in an increase in enzyme anity and inhi-

bition, particularly for 17a3 with an approximately

3-fold increase in anity as the pK increased from 5.01

to 6.13. The inhibition constant of 17a3 appeared to be

beyond the limits of the assay since a reliable value

could not be obtained. Replacement of the 4-morpholi-

nylmethyl group of 17b1 with bis(2-methoxyethyl)amine

resulted in no change in the enzyme anity or inhibition

and only a minimal increase in the basicity of 17b2;

however, the distribution coecient increased 3-fold.

Incorporation of diethanolamine as the base resulted in

a modest decrease in enzyme anity; however, the

basicity of 17b4 increased by one pK unit and the dis-

tribution coecient decreased 20-fold relative to 17b2,

providing an increase of more than 10-fold in solubility.

Inclusion of the heteroaromatic amine imidazole into

17i10 provided a 2-fold increase in anity relative to

17i1 with a modest increase in inhibition.

Since recent observations^{15 18}suggest a signi®cant role

for CA isozyme IV in the control of intraocular pres-

sure, representative examples of structures 5 and 17

were tested for their ability to inhibit isozyme IV.

Recombinant human CA-IV (rhCA-IV), which lacks the

trans-membrane glycosylphosphatidylinositol anchor

portion of the native enzyme, has been shown to be

functionally equivalent to CA-IV isolated from human

lung.^19,20The compounds that were evaluated against

rhCA-IV were also shown to be moderate to potent

inhibitors of this isoform, with IC₅₀values from 73.6

nM to 4.25 nM. A comparison of the inhibition con-

stants of 5b and 5d suggests that, as with hCA-II, the

presence of a charged species, such as a protonated

amine, within the substituent on N2 is detrimental to

rhCA-IV binding. Additionally, compounds 17h1 and

17i1, bearing methoxyalkyl groups at N2, show rela-

tively low inhibition, again suggesting, as with hCA-II,

the detrimental nature of polar functionality in this

region of the molecule. Interestingly, 17g1, with an N2

cyclohexyl substituent, also displays a relatively low

level of inhibition of rhCA-IV, in contrast to the very

good inhibition of hCA-II. Incorporation of a phenyl

substituent at N2 provided the most potent inhibitors of

rhCA-IV. A comparison of 17k1 and 17p1 suggests that

a hydroxyl group on the phenyl ring provides a modest

binding advantage over the methoxy group, while the

presence of a second methoxy group in the para position

(17m1) does not appear to be detrimental to inhibition.

The activity of 17n1 suggests that, as observed for hCA-

II, signi®cant bulk can be tolerated at the para position

of the phenyl group while maintaining good inhibition

of rhCA-IV. Additionally, the high level of enzyme

inhibition provided by 17j1 implies that the phenyl

Representative compounds were evaluated for their

ability to lower intraocular pressure (IOP) in naturally

hypertensive Dutch-belted rabbits. The response of a

compound in this model is considered to be a rather

good predictor of the IOP response of the same com-

pound in primate models of glaucoma and in man. A

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

963

test compound that decreases the baseline intraocular

pressure by at least 4 mmHg or a decrease of greater

than 15% is considered to provide a favorable response.

However, compounds which decrease intraocular pres-

sure by more than 20% are of primary interest for sub-

sequent evaluation in primate models of glaucoma.

Table 3. Reduction of IOP in naturally hypertensive Dutch-Belted

rabbits

Compound^a

Á^maxmm Hg^b

Max IOP dec (%)

5a

5b

7.0 (0.47)

5.2 (0.70)

3.8 (1.19)*

10.4 (0.89)

4.1 (0.61)

4.9 (0.58)

5.6 (0.77)

9.6 (0.86)

2.1 (0.36)

2.5 (0.48)

2.6 (0.37)

2.9 (0.87)**

2.6 (0.75)**

4.9 (0.95)

5.6 (0.61)

3.5 (0.52)

3.0 (0.50)

4.1 (1.04)**

3.5 (0.69)

7.0 (0.56)

9.7 (1.05)

7.2 (0.92)

21.6

18.2

11

36.1

15.8

19

22.8

32.5

9.1

17a1

17a3

17a6

17a7

17b1

17b2

17b4

17d8

17e1

In the present study, the maximum decrease in IOP was

observed within the ®rst 2 h after dosing each of the

compounds (Table 3). All of the compounds were

administered as suspensions with the exception of 17b4

and 17h2, which were suciently soluble to provide

solutions. It was previously observed that brinzolamide

eectively lowered IOP in the rabbit and the monkey

when topically administered as a suspension.²⁵The

2-substituted compounds 5a and 5b both showed a

favorable reduction of pressure in this model, 21.6

and 18.2%, respectively. Within the series of 2,3-di-

substituted compounds that were evaluated, four com-

pounds (17a6, 17a7, 17m1, 25) provided a reduction in

pressure that was in the 15±20% range, while ®ve com-

pounds (17a3, 17b1, 17b2, 17h2, 17i1) lowered pressure

greater than 20%. Further, two compounds, 17a3 and

17b2, provided an IOP reduction comparable to that of

brinzolamide and dorzolamide in this model of ocular

hypertension. The remainder of the compounds that

were evaluated provided no signi®cant reduction of

intraocular pressure at the test dose.

9.1

10.7

11

17f1

17g1

17h2

17i1

9.8

20.2

21.2

12.6

11.7

15.7

13.7

19.7

30.2

29.6

17j1

17k1

17m1

17o1

25

Brinzolamide

Dorzolamide^c

^apH 5.0, 1 mg total dose.

^bMean (SEM). Signi®cance of dierence from control group P<0.001,

except *P<0.05 and **P<0.01.

^cpH 7.4.

The compounds that provided a 15±20% reduction in

IOP displayed a broad range in lipophilicity, with DC

values ranging from 5 for 5b (18% reduction) to 172 for

17a7 (19% reduction). However, those compounds that

lowered pressure greater than 20% fell within a much

narrower lipophilicity range, with DC values between

13.5 and 53.5, suggesting that this lower range is pre-

ferred for this class of compounds. Though all of the

compounds (except 17h2) that lowered pressure greater

than 20% were dosed as suspensions, it is of interest to

note that there is an overlap in solubility for the mem-

bers of these two groups of active compounds. The

more active compounds generally had the greater

intrinsic solubility (Table 2). A comparison of 17b2 and

17b4 illustrates that high solubility alone does not cor-

relate with ecacy, and further suggests a lower limit

for lipophilicity within this class of inhibitors. The

basicity of the amine did not appear to have a sig-

ni®cant impact on in vivo ecacy in Dutch-belted rab-

bits, since there was essentially no dierence in the pK

range between the compounds that produced a 15±20%

reduction and those that resulted in a >20% reduction

(5.24 to 6.45 and 5.20 to 6.13, respectively). Further-

more, the inactive compounds had a similar basicity

range, 5.01 to 6.28 (Table 3).

rhCA-IV (73.6 to 4.25 nM). Selected compounds within

these series were shown to lower IOP in naturally

hypertensive Dutch-belted rabbits, demonstrating that

non-chiral derivatives of brinzolamide are ecacious in

lowering IOP in the rabbit when topically administered

as suspensions. Compounds 5a, 17a3, 17b1, 17b2, 17h2,

and 17i1 were identi®ed as candidates for subsequent

evaluation in primate models of glaucoma.

Experimental

Melting points were determined in open capillaries

using a Thomas-Hoover Uni-Melt Apparatus and are

uncorrected. Organic extracts were dried over MgSO₄

(unless otherwise noted) which was removed by ®ltra-

tion and washed with the appropriate dry solvent.

Chromatography refers to low pressure column chro-

matography conducted on 230±400 mesh silica gel from

E. Merck. Evaporations were performed under reduced

pressure on a rotary evaporator at 40 ^ꢁC unless other-

wise indicated. ¹H NMR spectra were determined at

200 MHz with a Varian Model VXR-200 spectrometer

and ¹³C NMR were determined at 50.3 MHz with the

Varian instrument. Spectra were recorded in CDCl₃or

Me₂SO-d₆, and chemical shifts are reported in parts per

million (d) relative to tetramethylsilane as internal stan-

dard. Elemental analyses were performed by Atlantic

Microlabs, Norcross, Georgia and are within 0.4% of

the theoretical values.

Conclusions

A facile approach to the synthesis of non-chiral 2- and

2,3-substituted 2H-thieno[3,2-e]-1,2-thiazine-6-sulfona-

mide 1,1-dioxides has been provided. These compounds

were shown to be potent high anity inhibitors of hCA-

II (K_i<0.5 nM) and moderate to potent inhibitors of

2-[2-(Acetoxy)ethyl]-N-(1,1-dimethylethyl)-4-hydroxy-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

(2). Sodium hydride (60% dispersion in mineral oil,

964

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

0.42 g, 10.6 mmol) was added to a solution of N-(1,1-

dimethylethyl)-4-hydroxy-2H-thieno[3,2-e]-1,2-thiazine-

6-sulfonamide 1,1-dioxide (1)⁸(3.00 g, 8.82 mmol) in

anhydrous DMF (50 mL) which was under a nitrogen

atmosphere and at ambient temperature. After stirring

for 20 min the mixture was cooled to 0 ^ꢁC and 2-bro-

moethyl acetate (2.21 g, 13.2 mmol, 1.46 mL) was added;

stirring continued at this temperature for 2 h followed

by stirring for 18 h at ambient temperature. The reac-

tion mixture was added to 5% aqueous NaHCO₃

(100 mL) and this mixture was extracted with EtOAc

(2Â200 mL). The combined extracts were dried and

evaporated to an oil which was puri®ed by chromato-

graphy (EtOAc/hexane, 1:1) to give a foam (3.36, 89%):

¹H NMR (DMSO-d₆) d 8.17 (s, 1H), 7.59 (s, 1H), 6.15

(br s, 1H), 4.82 (t, J=6 Hz, 1H), 4.21 (t, J=6 Hz, 2H),

3.99 (dd, J=6 and 14 Hz, 1H), 3.80 (dd, J=6 and

14 Hz, 1H), 3.59 (t, J=6 Hz, 2H), 2.02 (s, 3H), 1.02 (s,

9H).

2,6-lutidine (0.431 g, 4.02 mmol). After 30 min, addi-

tional methanesulfonic anhydride (0.349 g, 2.00 mmol)

and 2,6-lutidine (0.431 g, 4.02 mmol) were added and

the reaction mixture was stirred for 30 min. Evaporation

of the solvent provided a residue which was dissolved in

anhydrous DMF (50 mL) and DBN (1 mL) was added.

This mixture was heated at re¯ux temperature for 1 h,

cooled, poured into 5% aqueous NaHCO₃(100 mL)

and extracted with EtOAc (2Â100 mL). The combined

extracts were dried and evaporated to a residue which

was puri®ed by chromatography (5% MeOH/CH₂Cl₂)

to give a viscous oil (0.35 g, 57%): ¹H NMR (DMSO-d₆)

d 8.22 (s, 1H), 7.73 (s, 1H), 7.05 (d, J=7.7 Hz, 1H), 6.53

(d, J=7.7 Hz, 1H), 5.66 (m, 2H), 4.36 (m, 2H), 3.52 (m,

4H), 2.89 (m, 2H), 2.27 (m, 4H), 1.19 (s, 9H).

2-[2-[Bis(2-methoxyethyl)amino]ethyl]-2H-thieno[3,2-e]-

1,2-thiazine-6-sulfonamide 1,1-dioxide hydrochloride (5a).

To a solution of 3 (1.02 g, 2.79mmol) and triethylamine

(0.84 g, 8.36 mmol) in anhydrous THF (50 mL) under

nitrogen was added methanesulfonic anhydride (0.80 g,

4.18 mmol). This mixture was stirred at ambient tem-

perature for 30 min followed by evaporation to a residue

which was dissolved in EtOAc (80 mL) and washed with

5% aqueous NaHCO₃(50 mL). The organic phase was

dried and evaporated to give a solid (1.06 g) which was

dissolved in anhydrous DMF (50 mL) and bis(2-meth-

oxyethyl)amine (5 mL) was added. This mixture was

heated at re¯ux temperature for 1 h, cooled and poured

into 5% aqueous NaHCO₃(100 mL). The solution was

extracted with EtOAc (2Â100 mL) and the combined

extracts were dried and evaporated to give a crude oil

which was puri®ed by chromatography (gradient, 50%

to 100% EtOAc/hexane) to give 4a as a viscous oil

(0.89 g, 66%): ¹H NMR (DMSO-d₆) d 8.22 (s, 1H), 7.70

(s, 1H), 7.12 (d, J=7.8 Hz, 1H), 6.47 (d, J=7.8 Hz, 1H),

3.78 (t, J=5.5 Hz, 2H), 3.25 (t, J=6.0 Hz, 4H), 3.14 (s,

6H), 2.71 (t, J=5.5 Hz, 2H), 2.58 (t, J=6.0 Hz, 4H),

1.19 (s, 9H). A solution of this oil in tri¯uoroacetic acid

(8 mL) was stirred at ambient temperature for 18 h.

Evaporation gave a residue which was mixed with 5%

aqueous NaHCO₃(50 mL) and extracted with EtOAc

(2Â80 mL). The combined extracts were dried and eva-

porated to a residue which was puri®ed by chromato-

graphy (gradient, 3 to 5% MeOH/CH₂Cl₂) to give an

oil (0.74 g) which was converted to the HCl salt by

treatment with 2 N HCl in EtOH (0.63 g, 79%): mp 60±

2-(2-Hydroxyethyl)-N-(1,1-dimethylethyl)-2H-thieno[3,2-

e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (3). To a solu-

tion of 2 (3.36 g, 7.89 mmol) and 2,6-lutidine (3.00 mL,

25.7 mmol) in anhydrous THF (30 mL) under nitro-

gen was added methanesulfonic anhydride (2.06 g,

11.8 mmol). This mixture was stirred for 30 min at

ambient temperature followed by evaporation to a resi-

due. Anhydrous DMF (50 mL) and DBU (1 mL) were

added to the residue and this mixture was heated at

165 ^ꢁC (bath temperature) for 20 min and evaporated to

dryness. Methanol (50 mL) and 2 N NaOH (20 mL)

were added to the residue and this mixture was stirred

for 2 h at ambient temperature. Methanol was evapo-

rated and the aqueous mixture was extracted with

EtOAc (2Â100 mL). The combined extracts were dried

and evaporated to give the desired product as an oil

(2.78 g, 96%): ¹H NMR (DMSO-d₆) d 8.21 (s, 1H), 7.71

(s, 1H), 7.09 (d, J=7.8 Hz, 1H), 6.44 (d, 1H, J=7.8 Hz),

5.00 (t, 1H), 3.79 (t, J=5.4 Hz, 2H), 3.60 (q, J=5.4 Hz,

2H), 1.19 (s, 9H).

N-(1,1-Dimethylethyl)-2-[4-(4-morpholinyl)-2-butenyl]-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

(4e). Sodium hydride (60% dispersion in mineral oil,

0.113 g, 2.82 mmol) was added to a solution of 1 (0.80 g,

2.35 mmol) in anhydrous DMF (50 mL) under nitrogen.

After 20 min, the reaction mixture was cooled (ice bath),

1,4-dibromo-2-butene (0.754 g, 3.53 mmol) was added

and the mixture stirred for 2 h. Morpholine (5 mL) was

added and the reaction mixture was stirred at ambient

temperature for 18 h. DMF was evaporated under

reduced pressure and the residue was mixed with 5%

aqueous NaHCO₃(100 mL) and extracted with EtOAc

(2Â100 mL). The combined extracts were dried and

evaporated to a residue which was puri®ed by chroma-

tography (EtOAc) to give the desired product as a vis-

65 ^ꢁC; H NMR (DMSO-d₆) d 10.90 (br s, 1H), 8.16 (s,

1

2H), 7.71 (s, 1H), 7.17 (d, J=7.7 Hz, 1H), 6.60 (d,

J=6.9 Hz, 1H), 4.28 (br s, 2H), 3.69 (br s, 4H), 3.5±3.7

(6 H), 3.25 (s, 6H); ¹³C NMR (DMSO-d₆) d 149.8,

141.6, 134.0, 133.9, 127.5, 126.3, 66.2, 66.1, 66.0, 58.2,

.

52.7. Anal. (C₁₄H₂₃N₃O₆S₃HCl) C, H, N.

2-[2-(4-Morpholinyl)ethyl]-2H-thieno[3,2-e]-1,2-thiazine-

6-sulfonamide 1,1-dioxide hydrochloride (5b). Treatment

of 3 in a manner similar to that described for the pre-

paration of 5a, but using morpholine, gave, after chro-

matography (gradient, 70% to 100% EtOAc in hexane),

the free base (mp 135±137 ^ꢁC) which was converted to

1

cous oil (0.65 g, 58%): H NMR (DMSO-d₆) d 8.18 (s,

1H), 7.61 (s, 1H), 6.13 (d, J=7.6 Hz, 1H), 5.71 (m, 1H),

4.86 (m, 1H), 3.91 (t, J=6.4 Hz, 2H), 3.79 (m, 2H), 3.57

(t, J=4.5 Hz, 4H), 2.94 (m, 2H), 2.31 (m, 4H), 1.21 (s,

9H). To a solution of this oil (0.64 g, 1.34 mmol) in

anhydrous THF (30 mL) under nitrogen was added

methanesulfonic anhydride (0.349 g, 2.00 mmol) and

the HCl salt (67%): mp 234±236 ^ꢁC; H NMR (DMSO-

1

d₆) d 8.15 (s, 2H), 7.71 (s, 1H), 7.18 (d, J=7.8 Hz, 2H),

6.62 (d, J=7.8 Hz, 2H), 4.25 (br s, 2H), 3.94 (br s, 2H),

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

965

3.71 (br s, 2H), 3.30 (m, 4H), 3.13 (br s, 2H); ¹³C NMR

(DMSO-d₆) d 149.9, 141.6, 133.8, 127.5, 126.4, 103.6,

63.2, 54.1, 51.3; MS (EI) m/z 380 (M⁺). Anal. (C₁₂H₁₇

for 18 h, gradually warming to ambient temperature.

The reaction mixture was ®ltered through a ®lter aide

that was rinsed with CH₂Cl₂(150 mL). To the cooled

(0 ^ꢁC) ®ltrate was added 5% aqueous NaHCO₃

(200 mL) followed by the addition of glycine ethyl ester

hydrochloride in two portions (26.8 g, 192 mmol). The

mixture was stirred at ambient temperature for 3 h and

the aqueous layer was separated and extracted with

CH₂Cl₂(2Â100 mL). The combined extracts were

washed with water (120 mL), dried and evaporated to a

residue which was puri®ed by chromatography (40%

EtOAc in hexane) to give an oil (16.5 g, 61%): ¹H NMR

(DMSO-d₆) d 8.48 (br s, 1H), 7.82 (d, J=4.9 Hz, 1H),

7.20 (d, J=5.2 Hz, 1H), 6.18 (s, 1H), 4.10±3.90 (m, 4H),

4.01 (q, J=7.2 Hz, 2H), 3.77 (s, 2H), 1.12 (t, J=7.1 Hz,

3H). Anal. (C₁₁H₁₅NO₆S₂) C, H, N.

.

N₃O₅S₃HCl) C, H, N.

2-[2-[4-Acetyl-(1-piperazinyl)]ethyl]-2H-thieno[3,2-e]-1,2-

thiazine-6-sulfonamide 1,1-dioxide (5c). Treatment of 3

in a manner similar to that described for the prepara-

tion of 5a, but using 1-acetylpiperazine, gave, after

chromatography (gradient, EtOAc to 10% EtOH/

EtOAc), a solid which was recrystallized from MeOH/

CH₂Cl₂to give 5c (52%): mp 180±183 ^ꢁC; ¹H NMR

(DMSO-d₆) d 8.08 (s, 2H), 7.67 (d, J=1.4 Hz, 1H), 7.11

(d, J=7.6 Hz, 1H), 6.53 (d, J=7.6 Hz, 1H), 3.86 (t,

J=5.8 Hz, 2H), 3.31 (4H, m), 2.55 (2 H), 2.32 (m, 4H),

1.94 (s, 3H); ¹³C NMR (DMSO-d₆) d 168.1, 148.9,

141.2, 134.3, 127.5, 126.5, 103.1, 56.8, 52.8, 52.2, 45.6,

45.1, 21.1; IR (KBr) 3095, 2954, 1613, 1448, 1349, 1319,

1161. Anal. (C₁₄H₂₀N₄O₅S₃) C, H, N.

N-[(3-Formyl-2-thienyl)sulfonyl]-glycine ethyl ester (8).

To a solution of 7 (31.0 g, 97.0 mmol) in THF (100 mL)

was added 3 N HCl and this mixture was stirred for 6 h

at room temperature. Brine (100 mL) was added and the

mixture was extracted with EtOAc (3Â100 mL). The

combined extracts were washed with brine (20 mL),

dried and evaporated to give a colorless glass (22.0 g,

2-[2-(Propylamino)ethyl]-2H-thieno[3,2-e]-1,2-thiazine-6-

sulfonamide 1,1-dioxide hydrochloride (5d). Treatment

of 3 in a manner similar to that described for the pre-

paration of 5a, but using propylamine, provided 5d

(50%): mp 208±210 ^ꢁC; ¹H NMR (DMSO-d₆) d 9.21 (br

s, 2H), 8.16 (br s, 2H), 7.71 (s, 1H), 7.19 (d, J=7.7 Hz,

1H), 6.58 (d, J=7.8 Hz, 1H), 4.16 (t, J=7.0 Hz, 2H),

3.19 (br s, 2H), 2.85 (br s, 2H), 1.60 (t, J=8.0 Hz,

2H);¹³C NMR (DMSO-d₆) d 149.8, 141.6, 134.00, 127.5,

126.3, 103.1, 48.3, 45.6, 42.7, 18.9, 10.9. Anal. (C₁₁H₁₇

1

82%): H NMR (DMSO-d₆) d 10.33 (s, 1H), 9.01 (br s,

1H), 7.90 (d, J=5.2 Hz, 1H), 7.53 (d, J=5.2 Hz, 1H),

3.91 (s, 2H), 3.90 (q, J=7.2 Hz, 2H), 1.07 (t, J=7.2 Hz,

3H).

Ethyl 3,4-dihydro-4-hydroxy-2H-thieno[3,2-e]-1,2-thia-

zine-3-carboxylate 1,1-dioxide (10). To a solution of 8

(4.50 g, 16.2 mmol) in EtOAc (150 mL) was added DBU

(1 mL) and molecular sieves (5 g). The mixture was

heated at re¯ux temperature for 1 h, cooled to room

temperature, and 1 N HCl (100 mL) was added. The

organic layer was separated, dried, and evaporated

to give a residue (3.10 g) which solidi®ed from EtOAc/

hexane to give recovered starting material (1.45 g);

evaporation of the ®ltrate gave 10 (1.78 g, 40%) as a

.

N₃O₄S₃HCl) C, H, N.

2-[4-(4-Morpholinyl)-2-butenyl]-2H-thieno[3,2-e]-1,2-thi-

azine-6-sulfonamide 1,1-dioxide hydrochloride (5e). A

solution of 4e (0.35 g, 0.82 mmol) in tri¯uoroacetic acid

(5 mL) was stirred at ambient temperature for 3 days

and evaporated to dryness. The residue was combined

with 5% aqueous NaHCO₃(50 mL) and this mixture

was extracted with EtOAc (2Â80 mL). The combined

extracts were dried and evaporated to a residue which

was puri®ed by chromatography (6% MeOH/CH₂Cl₂)

to give a viscous oil (0.21 g, 68%). To a solution of this

oil in MeOH (5 mL) was added a 2 N solution of HCl in

EtOH (2 mL); this mixture was evaporated to give the

hydrochloride salt_ꢁas an amorphous solid (0.152 g,

1

viscous oil: H NMR (DMSO-d₆) d 7.91 (d, J=4.8 Hz,

1H), 7. 86 (brs, 1H), 7.19 (d, J=5.0 Hz, 1H), 5.79 (d,

J=8.2 Hz, 1H), 4.88 (dd, 1H), 4.72 (dd, 1H), 4.20 (q,

J=7.1 Hz, 2H), 1.24 (t, J=7.1 Hz, 3H).

3-(1,3-Dioxolan-2-yl)-N-(1-methylethyl)-2-thiophene-

sulfonamide (11d). Prepared by using the procedure

described for the preparation of 7, but isopropylamine

(26.1 g, 441 mmol) and triethylamine (10 mL) were

added to the intermediate sulfonyl chloride prepared

from 6 (23.0 g, 147 mmol). Puri®cation by chromato-

graphy (30% EtOAc in hexane) gave a colorless oil

1

50%): mp 108±112 C; H NMR (DMSO-d₆) d 10.3 (br

s, 1H), 8.15 (br s, 2H), 7.71 (s, 1H), 7.13 (d, J=7.7 Hz,

1H), 6.57 (d, J=7.7 Hz, 1H), 6.15±5.75 (m, 2H), 4.80 (d,

J=5.5 Hz, 2H), 4.05±3.60 (m, 4H), 3.34 (br s, 4H), 2.99

.

(br t, 2H). Anal. (C₁₂H₁₉N₃O₅S₃HCl 0.5 H₂O) C, H, N.

1

N-[[3-(1,3-Dioxolan-2-yl)-2-thienyl]sulfonyl]-glycine ethyl

ester (7). To a solution of 3-thiophenecarboxaldehyde

ethylene glycol acetal (6)⁹(10.0 g, 64.0 mmol) in THF

(100 mL) at 70 ^ꢁC and under nitrogen was added n-

butyllithium (28.2 mL of a 2.5 M solution in hexane)

and the mixture was maintained at this temperature for

50 min. Sulfur dioxide gas was passed over the reaction

mixture (10 min) and stirring continued for 1 h at ambi-

ent temperature. Evaporation provided a residue which

was dissolved in CH₂Cl₂(150 mL) and cooled on an

ice/MeOH bath. Following the addition of N-chloro-

succinimide (11.11g, 83.0 mmol), this mixture was stirred

(25.3 g, 62%): H NMR (DMSO-d₆) d 7.88 (br s, 1H),

7.81 (d, J=5.2 Hz, 1H), 7.20 (d, J=5.2 Hz, 1H), 6.21 (s,

1H), 4.01 (m, 5H), 0.99 (d, J=2.6 Hz, 6H).

Compounds 11e±h and 11k±n were prepared from 7 and

the appropriate amine using a procedure similar to that

described for the synthesis of 11d.

3-(1,3-Dioxolan-2-yl)-N-(2-propenyl)-2-thiophenesulfon-

amide (11e). Allylamine; puri®ed by chromatography

(gradient, 10 to 40% EtOAc in hexane) to give an oil

1

(12%): H NMR (DMSO-d₆) d 8.12 (br s, 1H), 7.84 (d,

966

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

J=5.2 Hz, 1H), 7.22 (d, J=5.2 Hz, 1H), 6.21 (s, 1H), 5.8

(m, 1H), 5.1 (m, 2H), 4.15±3.95 (m, 4H), 3.52 (m, 2H).

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-ethyl-glycine

ethyl ester (12b). A solution of 7 (7.5 g, 23.0 mmol) in

anhydrous DMF (35 mL) was added to a cold (ice bath)

suspension of 60% sodium hydride in mineral oil

(1.02 g, 26.0 mmol) in DMF (25 mL) under nitrogen.

After stirring this mixture for 30 min ethyl bromide

(1.92 mL, 26.0 mmol) was added and after stirring for

2 h additional sodium hydride (0.51 g, 13.0 mmol) and

ethyl bromide (1 mL) were added. The reaction mixture

was stirred for 18 h at ambient temperature and evapo-

rated to a residue which was suspended in 5% aqueous

NaHCO₃(150 mL). This mixture was extracted with

EtOAc (3Â50 mL) and the combined extracts were

washed with brine (70 mL), dried, and evaporated to an

amber oil (6.46 g, 80% crude yield) which was used in

the next step without further puri®cation (88% pure by

NMR): ¹H NMR (DMSO-d₆) d 7.90 (d, J=5.2 Hz, 1H),

7.25 (d, J=5.2 Hz, 1H), 6.15 (s, 1H), 4.15±3.90 (m, 8H),

3.20 (q, J=7.2 Hz, 2H), 1.56 (t, J=7.2 Hz, 3H), 1.05 (t,

J=7.0 Hz, 3H).

N-Cyclopropylmethyl-3-(1,3-dioxolan-2-yl)-2-thiophene-

sulfonamide (11f). Cyclopropylmethylamine; puri®ed

by chromatography (gradient, hexane to 50% EtOAc

1

in hexane) to give an oil (63%): H NMR (DMSO-d₆)

d 8.01 (br s, 1H), 7.82 (d, J=5.2 Hz, 1H), 7.20 (d,

J=5.2 Hz, 1H), 6.21 (s, 1H), 4.09 (m, 4H), 2.74 (d,

J=6.9 Hz, 2H), 0.82 (m, 1H), 0.39 (m, 2H), 0.10 (m,

2H).

3-(1,3-Dioxolan-2-yl)-N-cyclohexyl-2-thiophenesulfonamide

(11g). Cyclohexylamine; puri®ed by chromatography

1

(25% EtOAc in hexane) to give an oil (72%): H NMR

(DMSO-d₆) d 7.92 (s, 1H), 7.81 (d, J=5.0 Hz, 1H), 7.20

(d, J=5.0 Hz, 1H), 6.22 (s, 1H), 4.15±3.90 (m, 4H), 3.05

(m, 1H), 1.62 (m, 5H), 1.14 (m, 5H).

3-(1,3-Dioxolan-2-yl)-N-(2-methoxyethyl)-2-thiophene-

sulfonamide (11h). 2-Methoxyethylamine; puri®ed by

chromatography (50% EtOAc in hexane) to give an oil

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-propyl-

glycine ethyl ester (12c). From 7 and bromopropane/

NaI (oil, 69%): ¹H NMR (DMSO-d₆) d 7.90 (d,

J=5.0 Hz, H1), 7.23 (d, J=5.0 Hz), 6.15 (s, 1H), 4.15±

3.90 (m, 8H), 3.19 (t, J=7.5 Hz, 2H), 1.48 (q, J=7.5 Hz,

2H), 1.15 (t, J=7.5 Hz, 3H), 0.79 (t, J=7.5 Hz, 3H).

1

(79%): H NMR (CDCl₃) d 7.45 (d, J=4.9 Hz, 1H),

7.26 (d, J=4.9 Hz, 1H), 6.29 (s, 1H), 4.06 (br s, 4H),

3.43 (m, 2H), 3.28 (s, 3H), 3.17 (m, 2H).

3-(1,3-Dioxolan-2-yl)-N-(3-methoxyphenyl)-2-thiophene-

sulfonamide (11k). m-Anisidine; puri®ed by chromato-

graphy (30% EtOAc in hexane) to give a solid (62%):

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-(1-methyl-

ethyl)-glycine ethyl ester (12d). To a solution of 11d

(25.0 g, 90.3 mmol) in anhydrous DMF (350 mL) at 0^ꢁ

was added a 1 M solution of potassium t-butoxide in t-

butanol (99.3 mL, 99.3 mmol) followed by the addition

of ethyl bromoacetate (12 mL, 18.1 g, 108.4 mmol); this

mixture was stirred for 1 h and then allowed to stand at

5 ^ꢁC for 18 h. The reaction mixture was diluted with 5%

aqueous NaHCO₃(600 mL) and extracted with ether

(3Â300 mL). The combined extracts were dried and

evaporated to a syrup (36.25 g, 99%) which was used in

the next reaction without further puri®cation: ¹H NMR

(DMSO-d₆) d 7.89 (d, J=5.6 Hz, 1H), 7.24 (d, J=

5.5 Hz, 1H), 6.14 (s, 1H), 4.2±3.9 (m, 8H), 1.19 (t,

J=7.0 Hz, 3H), 0.92 (d, J=5.0 Hz, 6H).

mp 112±114 ^ꢁC; H NMR (DMSO-d₆) d 10.52 (s, 1H),

1

7.81 (d, J=5.0 Hz, 1H), 7.19 (d, J=5.0 Hz, 1H), 7.15

(m, 1H), 6.8±6.6 (m, 3H), 6.17 (s, 1H), 4.15±3.85 (m,

4H), 3.67 (s, 3H).

3-(1,3-Dioxolan-2-yl)-N-(3,4-dimethoxyphenyl)-2-thio-

phenesulfonamide (11m). 3,4-Dimethoxyaniline; puri®ed

by chromatography (EtOAc:hexane, 1:1) to give a

dark syrup (58%): ¹H NMR (DMSO-d₆) d 10.12 (s,

1H), 7.79 (d, J=5.2 Hz, 1H), 7.17 (d, J=5.2 Hz, 1H),

6.85 (d, J=8.6 Hz, 1H), 6.67 (m, 2H), 6.10 (s, 1H), 4.1±

3.8 (m, 4H), 3.68 (s, 3H), 3.63 (s, 3H); MS (CI) m/z 372

(M+1).

3-(1,3-Dioxolan-2-yl)-N-[(4-(4-morpholinyl)phenyl]-2-thio-

phenesulfonamide (11n). 4-Morpholinophenyl; puri®ed

by recrystallization (57%, EtOAc/hexane): mp 171±

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-(2-propenyl)-

glycine ethyl ester (12e). Prepared from 11e and ethyl

bromoacetate as described for the preparation of 12d

(amber oil, 76%): ¹H NMR (DMSO-d₆) d 7.93 (d,

J=5.2 Hz, 1H), 7.26 (d, J=5.2 Hz, 1H), 6.16 (s, 1H),

5.65 (m, 1H), 5.3±5.1 (m, 2H), 4.1±3.9 (m, 11H), 1.14 (t,

J=7.0 Hz, 3H); MS (CI) m/z 362 (M+1).

174 ^ꢁC; H NMR (DMSO-d₆) d 10.00 (s, 1H), 7.78 (d,

1

J=5.2 Hz, 1H), 7.16 (d, J=5.2 Hz, 1H), 6.95 (d,

J=9.1 Hz, 2H), 6.82 (d, J=9.1 Hz, 2H), 6.08 (s, 1H),

4.15±3.85 (m, 4H), 3.69 (t, J=5.0 Hz, 4H), 3.02 (t,

J=4.9 Hz, 4H).

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-methyl-

glycine ethyl ester (12a). Prepared by using the proce-

dure described for the preparation of 7, but a solu-

tion of sarcosine ethyl ester hydrochloride (15.0 g,

97.6 mmol) was mixed with the intermediate sulfonyl

chloride (prepared from 37.3 mmol of 6). Puri®cation by

chromatography (30% to 50% EtOAc/hexane) gave an

oil (8.95 g, 72%): ¹H NMR (DMSO-d₆) d 7.92 (d,

J=5.2 Hz, 1H), 7.26 (d, J=5.2 Hz, 1H), 6.13 (s, 1H),

4.10±3.90 (m, 8H), 2.87 (s, 3H), 1.13 (t, J=7.1 Hz, 3H);

MS (CI) m/z 336 (M+1).

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-methyl-

cyclopropyl-glycine ethyl ester (12f). Prepared from 11f

and ethyl bromoacetate as described for the preparation

of 9d (oil, 97%): ¹H NMR (DMSO-d₆) d 7.91 (d,

J=5.2 Hz, 1H), 7.22 (d, J=5.2 Hz, 1H), 6.14 (s, 1H),

4.18 (s, 2H), 4.1±3.9 (m, 6H), 3.13 (d, J=7.0 Hz, 2H),

1.15 (t, J=7.12 Hz, 3H), 0.85 (m, 1H), 0.42 (m, 2H),

0.13 (m, 2H); MS (CI) m/z 376 (M+1).

N-Cyclohexyl-N-[(3-formyl-thien-2-yl)sulfonyl]-glycine

methyl ester (12g). Prepared from 11g and methyl

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

967

bromoacetate as descibed for the preparation of 12d,

but isolated as the aldehyde following treatment with

aqueous 2 N HCl for 4 h at ambient temperature and

subsequent extraction with EtOAc (2Â100 mL), drying,

and evaporation to give an oil (94%): ¹H NMR

(DMSO-d₆) d 7.97 (d, J=5.2 Hz, 1H), 7.56 (d, J=

5.2 Hz, 1H), 4.16 (s, 2H), 3.65 (m, 1H), 3.62 (s, 3H),

1.75±1.05 (m, 10H).

(50 mL). The mixture was extracted with EtOAc

(2Â200 mL) and the combined extracts were dried and

evaporated to give the crude aldehyde which was dis-

solved in EtOAc (150 mL) and DBN (0.5 g) was added.

After heating at re¯ux temperature for 2 h, the mixture

was cooled to ambient temperature, washed with 5%

aqueous NaHCO₃(50 mL), dried, and evaporated to a

residue which was puri®ed by chromatography (gra-

dient, 30% to 50% EtOAc in hexane) to give unreacted

1

N-[[(3-Formyl-thien-2-yl)sulfonyl]-N-(2-methoxyethyl)-

glycine methyl ester (12h). Prepared from 11h as descri-

bed for the preparation of 12g, but puri®ed by chroma-

tography (EtOAc/hexane, 2:1) to give a syrup (87%):

¹H NMR (CDCl₃) d 10.35 (s, 1H), 7.60 (d, J=5.2 Hz,

1H), 7.50 (d, J=5.4 Hz, 1H), 4.30 (s, 2H), 3.63 (s, 3H),

3.55 (s, 4H), 3.26 (s, 3H).

12a (1.39 g, 16%) and a gummy solid (5.05 g, 70%): H

NMR (DMSO-d₆) d 8.06 ((d, J=5.2 Hz, 1H), 7.73 (s,

1H), 7.48 (d, J=5.2 Hz, 1H), 4.31 (q, J=7.2 Hz, 2H),

3.15 (s, 3H), 1.31 (t, J=7.2 Hz, 3H); MS (CI) m/z 274

(M+1).

Compounds 13b±n were prepared using a procedure

similar to that described for the synthesis of 13a. See

Table 4.

N-[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-(3-methoxy-

propyl)-glycine ethyl ester (12i). From 7 and 1-bromo-3-

1

methoxypropane/NaI (oil, 90%): H NMR (DMSO-d₆)

2-Methyl-2H-thieno[3,2-e]-1,2-thiazine-3-methanol 1,1-

dioxide (14a). To a solution of 13a (1.00 g, 3.66 mmol)

in anhydrous THF (20 mL) at 70 ^ꢁC was added

DIBAL-H (1.0 M, 7.69 mL, 7.69 mmol). The mixture

was warmed to ambient temperature and stirred for 2 h;

additional DIBAL-H (20 mL, 20 mmol) was added and

the reaction mixture was stirred for 18 h. Methanol

(100 mL) was added and the mixture evaporated to a

residue to which 2 N HCl (50 mL) was added followed

by extraction with EtOAc (2Â80 mL). The combined

extracts were dried and evaporated to give a solid which

was recrystallized (EtOAc/hexane) to give a colorless

d 7.91 (d, J=5.0 Hz, 1H), 7.27 (d, J=5.0 Hz, 1H), 6.13

(s, 1H), 4.10 (s), 4.05 (q, J=7.2 Hz, 2H), 4.01 (m, 4H),

3.30 (m, 4H), 3.18 (s, 3H), 1.70 (m, 2H), 1.14 (t,

J=7.2 Hz, 3H); MS (CI) m/z 394 (M+1).

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-[(4-meth-

oxyphenyl)methyl]-glycine ethyl ester (12j). From 7 and

4-methoxybenzyl chloride with t-BuOK/t-BuOH (oil,

1

98%): H NMR (DMSO-d₆) d 7.92 (d, J=5.2 Hz, 1H),

7.27 (d, J=5.2 Hz, 1H), 7.11 (d, J=6.7 Hz, 2H), 6.86 (d,

J=6.7 Hz, 2H), 6.19 (s, 1H), 4.38 (s, 2H), 4.09±3.86 (m,

6H), 3.90 (s, 2H), 3.71 (s, 3H), 1.06 (t, J=7.1 Hz, 3H).

solid (0.80 g, 95%): mp 128±130 ^ꢁC; H NMR (DMSO-

1

d₆) d 7.95 (d, J=5.0 Hz, 1H), 7.21 (d, J=5.0 Hz, 1H),

6.51 (s, 1H), 5.57 (t, J=4.8 Hz, 1H), 4.34 (d, J=4.8 Hz,

2H), 3.32 (s, 3H).

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-(3-methoxy-

phenyl)-glycine ethyl ester (12k). Prepared from 11k as

described for the preparation of 12d (oil, 95%): ¹H

NMR (DMSO-d₆) d 7.90 (d, J=5.4 Hz, 1H), 7.27 (d,

J=5.4 Hz, 1H), 7.25 (s, 1H), 6.89 (m, 2H), 6.74 (m, 1H),

5.86 (s, 1H), 4.51 (s, 2H), 4.09 (q, J=7.2 Hz, 2H), 3.67

(s, 3H), 1.15 (t, J=7.2 Hz, 3H).

Compounds 14b±n were prepared using a procedure

similar to that described for the synthesis of 14a. See

Table 5.

3-Hydroxymethyl-2-methyl-2H-thieno[3,2-e]-1,2-thiazine-

6-sulfonamide 1,1-dioxide (15a). To a solution of 14a

(3.20 g, 13.8 mmol) in anhydrous THF (50 mL) under

nitrogen at 70 ^ꢁC was added n-butyllithium (12.7 mL

of a 2.5 M solution, 31.9 mmol) over 5 min. The mixture

was stirred for 10 min before a stream of sulfur dioxide

was passed over the surface of the reaction mixture for

5 min. The reaction mixture was warmed to ambient

temperature and then evaporated to a residue which was

suspended in water (200 mL) and cooled (0 ^ꢁC). Hyd-

roxylamine-O-sulfonic acid (4.70 g, 41.6 mmol) and

sodium acetate trihydrate (7.53 g, 55.4 mmol) were

added and the mixture was stirred at ambient tempera-

ture for 18 h followed by neutralization with 5% aqu-

eous NaHCO₃(50 mL). This mixture was extracted with

EtOAc (2Â200 mL) and the combined extracts were

dried and evaporated to a residue which was recrys-

tallized (EtOAc/hexane) to give a solid (3.55 g, 83%):

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-(3,4-dimeth-

oxyphenyl)-glycine ethyl ester (12m). Prepared from 11m

as described for the preparation of 12d (oil, 99%): H

NMR (CDCl₃) d 7.46 (d, J=5.0 Hz, 1H), 7.25 (d,

J=5.0 Hz, 1H), 6.84 (m, 2H), 6.75 (d, J=8.7 Hz, 1H),

6.02 (s, 1H), 4.46 (s, 2H), 4.25±3.95 (m, 6H), 3.86 (s,

3H), 3.77 (s, 3H), 1.25 (t, J=7.1 Hz, 3H).

1

N-[[3-(1,3-Dioxolan-2-yl)thien-2-yl]sulfonyl]-N-[4-(mor-

pholin-4-yl)phenyl]-glycine methyl ester (12n). Prepared

from 11n as described for the preparation of 12g (oil,

1

99%): H NMR (DMSO-d₆) d 7.88 (d, J=5.0 Hz, 1H),

7.27 (d, J=5.0 Hz, 1H), 7.03 (d, J=9.1 Hz, 2H), 6.88 (d,

J=9.1 Hz, 2H), 5.89 (s, 1H), 4.46 (s, 2H), 4.15±3.85 (m,

4H), 3.69 (m, 4H), 3.63 (s, 3H), 3.11 (m, 4H).

Ethyl 2-Methyl-2H-thieno[3,2-e]-1,2-thiazine-3-carboxy-

late 1,1-dioxide (13a). A solution of 12a (8.80 g,

26.3 mmol) and p-toluenesulfonic acid (1.0 g) in acetone

(250 mL) at ambient temperature was stirred for 18 h.

Water (0.5 mL) was added and the mixture stirred for

4 h followed by the addition of 5% aqueous NaHCO₃

ꢁ

1

mp 144±146 C; H NMR (DMSO-d₆) d 8.08 (s, 2H),

7.69 (s, 1H), 6.64 (s, 1H), 5.65 (t, J=5.4 Hz, 1H), 4.37

(d, J=5.4 Hz, 2H), 3.51 (s, 3H); ¹³C NMR (DMSO-d₆)

d 149.1, 145.4, 140.8, 127.6, 125.6, 101.9, 60.5, 29.5.

Anal. (C₈H₁₀N₂O₅S₃) C, H, N.

968

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

Table 4. NMR data for compounds 13

¹H NMR (DMSO-d₆) d

mp (^ꢁC)

Oil

Yield (%)

61

13b

13c

13d

13e

13f

13g

13h

13i

8.07 (d, J=5.1 Hz, 1H), 7.82 (s, 1H), 7.49 (d, J=5.1 Hz, 1H), 4.33 (q, J=7.2 Hz, 2H), 3.74 (q,

J=7.1 Hz, 2H), 1.33 (t, J=7.0 Hz, 3H), 0.95 (t, J=7.0 Hz, 3H)

7.91 (d, J=5.2 Hz, 1H), 7.25 (d, J=5.2 Hz, 1H), 6.15 (s, 1H), 4.03 (m, 2H), 1.48 (m, 2H), 1.15 (t,

J=7.0 Hz, 3H), 0.79 (t, J=7.3 Hz, 3H)

8.02 (d, J=5.0 Hz, 1H), 7.79 (s, 1H), 7.48 (d, J=5.0 Hz, 1H), 4.31 (q, J=7.1 Hz, 2H), 4.01 (m, 1H),

1.32 (t, J=7.1 Hz, 3H), 1.21 (d, J=6.8 Hz, 6H)

8.06 (d, J=5.0 Hz, 1H), 7.81 (s, 1H), 7.48 (d, J=5.0 Hz, 1H), 5.6±5.4 (m, 1H), 5.05 (d, J=8.5 Hz,

2H), 4.34 (q, J=7.1 Hz, 4H), 1.32 (t, J=7.1 Hz, 3H)

8.07 (d, J=5.2 Hz, 1H), 7.86 (s, 1H), 7.50 (d, J=5.2 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 3.67 (d,

J=6.9 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H), 0.63 (m, 1H), 0.18 (m, 2H), 0.05 (m, 2H)

8.04 (d, J=5.0 Hz, 1H), 7.82 (s, 1H), 7.45 (d, J=5.1 Hz, 1H), 3.86 (s, 3H), 3.61 (m, 1H), 1.80±1.45

(m, 7H), 1.30±0.86 (m, 3H)

7.62 (s, 1H), 7.57 (d, J=5.1 Hz, 1H), 7.15 (d, J=5.1 Hz, 1H), 4.13 (t, J=4.9 Hz, 2H), 3.91 (s, 3H),

3.27 (t, J=4.9 Hz, 2H), 2.97 (s, 3H) (CDCl₃)

8.06 (d, J=5.0 Hz, 1H), 7.77 (s, 1H), 7.48 (d, J=5.0 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H), 3.83 (t,

J=7.0 Hz, 2H), 3.05 (s, 3H), 2.99 (t, J=6.0 Hz, 2H), 1.59 (m, 2H), 1.31 (t, J=7.0 Hz, 3H)

7.98 (d, J=5.8 Hz, 1H), 7.66 (d, J=0.8 Hz, 1H), 7.29 (d, J=5.8 Hz, 1H), 6.84 (d, J=8.2 Hz, 2H),

6.67 (d, J=8.8 Hz, 2H), 4.91 (s, 2H), 4.31 (q, J=7.1 Hz, 2H), 3.63 (s, 3H), 1.29 (t, J=7.1 Hz, 3H)

8.13 (d, J=5.0 Hz, 1H), 7.96 (s, 1H), 7.60 (d, J=5.0 Hz, 1H), 7.31 (t, J=8.1 Hz, 1H), 6.99 (m, 1H),

6.62 (m, 2H), 4.09 (q, J=5.1 Hz, 2H), 3.74 (s, 3H), 1.02 (t, J=7.1 Hz, 3H)

7.69 (s, 1H), 7.64 (d, J=5.1 Hz, 1H), 7.27 (d, J=5.1 Hz, 1H), 6.84 (d, J=1.6 Hz, 1H), 6.75 (d,

J=8.7 Hz, 1H), 6.54 (dd, J=8.7 and 1.6 Hz, 1H), 4.14 (q, J=7.1 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H),

1.09 (t, J=7.1 Hz, 3H) (CDCl₃)

Oil

52

97±99

Oil

50 (from 11d)

60

Oil

63

155±157

61±64

82±83

Oil

67 (from 11g)

94

54 (from 11i)

13j

55

46

75

13k

13m

107±109

Foam

13n

8.11 (d, J=5.1 Hz, 1H), 7.92 (s, 1H), 7.57 (d, J=5.1 Hz, 1H), 6.88 (s, 4H), 3.71 (m, 4H), 3.66 (s,

3H), 3.12 (m, 4H)

220±222

54

Table 5. NMR data for compounds 14

¹H NMR (DMSO-d₆) d

mp (^ꢁC)

Yield (%)

94

14b

14c

14d

14e

14g

14h

14i

7.94 (d, J=5.0 Hz, 1H), 7.23 (d, J=5.0 Hz, 1H), 6.64 (s, 1H), 5.60 (t, J=5.4 Hz, 1H, exchange), 4.35 (d,

J=5.4 Hz, 2H), 3.84 (q, J=7.1 Hz, 2H), 1.10 (t, J=7.1 Hz, 3H)

7.89 (d, J=5.0 Hz, 1H), 7.23 (d, J=5.0 Hz, 1H), 6.67 (s, 1H), 5.6 (br s, 1H), 4.35 (s, 2H), 3.76 (t,

J=7.3 Hz, 2H), 1.6±1.4 (m, 2H), 0.72 (t, J=7.4 Hz, 3H)

7.91 (d, J=5.0 Hz, 1H), 7.22 (d, J=5.0 Hz, 1H), 5.59 (t, J=4.6 Hz, 1H), 6.74 (s, 1H), 4.32 (m, 3H), 1.29

(d, J=6.9 Hz, 6H)

7.96 (d, J=5.0 Hz, 1H), 7.24 (d, J=5.0 Hz, 1H), 6.65 (s, 1H), 5.9±5.7 (m, 1H), 5.62 (t, J=5.6 Hz, 1H), 5.07

(d, J=11 Hz, 1H), 4.92 (d, J=18 Hz, 1H), 4.45 (m, 2H), 4.30 (d, J=5.6 Hz, 2H); MS (CI) m/z 257 (M+1).

7.48 (d, J=5.0 Hz, 1H), 6.98 (d, J=5.0 Hz, 1H), 6.69 (s, 1H), 4.49 (d, J=5.9 Hz, 2H), 3.89 (m, 1H), 2.12

(t, J=5.9 Hz, 1H), 1.78 (m, 7H), 1.25 (m, 3H) (CDCl₃)

7.52 (d, J=5.1 Hz, 1H), 7.01 (d, J=5.1 Hz, 1H), 6.47 (s, 1H), 4.43 (d, J=6.8 Hz, 2H), 3.97 (t, J=4.9 Hz,

2H), 3.69 (t, J=4.9 Hz, 2H), 3.36 (s, 3H) (CDCl₃)

7.92 (d, J=5.0 Hz, 1H), 7.22 (d, J=5.0 Hz, 1H), 6.63 (s, 1H), 5.56 (t, J=4.7 Hz, 1H), 4.32 (d, J=4.7 Hz,

2H), 3.83 (t, J=7.4 Hz, 2H), 3.17 (t, J=6.0 Hz, 2H), 3.13 (s, 3H), 1.68 (m, 2H)

7.95 (d, J=5.0 Hz, 1H), 7.21 (d, J=5.0 Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 6.62 (s,

1H), 5.61 (t, J=4.6 Hz, 1H), 4.96 (s, 2H)

Oil

65±67

67±69

51±53

119±121

Oil

99

63

93

81

88

99

73

97

95

Oil

14j

Oil

14k

14m

14n

8.03 (d, J=5.1 Hz, 1H), 7.37 (d, J=5.1 Hz, 1H), 7.36 (d, J=5.1 Hz, 1H), 7.07 (m, 1H), 6.85±6.75 (m, 3H),

5.48 (t, J=4.6 Hz, 1H), 3.93 (J=4.6 Hz, 2H), 3.77 (s, 3H)

8.02 (d, J=5.0 Hz, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.2 (m, 1H), 7.02 (m, 1H), 6.80 (m, 1H), 6.77 (s, 1H), 5.45

(t, J=4.6 Hz, 1H), 3.94 (d, J=4.6 Hz, 2H), 3.80 (s, 3H), 3.73 (s, 3H)

8.01 (d, J=5.0 Hz, 1H), 7.34 (d, J=5.0 Hz, 1H), 7.06 (d, J=9.1 Hz, 2H), 6.98 (d, J=9.1 Hz, 2H), 6.76 (s,

1H), 5.46 (t, J=4.6 Hz, 1H), 3.91 (d, J=4.6 Hz, 2H), 3.73 (m, 4H), 3.17 (m, 4H)

141±143

Foam

Oil

Compounds 15b±i and 15k±n were prepared using a

procedure similar to that described for the synthesis of

15a. See Table 6.

(80 mL) added. This mixture was extracted with EtOAc

(2Â200 mL) and the combined extracts were dried and

evaporated to an oil which was puri®ed by chromato-

graphy (30 to 50% EtOAc in hexane) to give a colorless

solid (0.82 g, 84%): mp 104±106 ^ꢁC; H NMR (DMSO-

1

2-Methyl-3-[(4-morpholinyl)methyl]-2H-thieno[3,2-e]-1,2-

thiazine 1,1-dioxide (16a1). To a solution of 14a (0.79 g,

3.42 mmol) and triethylamine (1.04 g, 10.3 mmol) in

anhydrous THF (30 mL) at ambient temperature was

added methanesulfonic anhydride (0.89 g, 5.13 mmol).

After 30 min, morpholine (2 mL) was added and the

mixture was stirred for an additional hour at ambient

temperature and then heated at re¯ux temperature for

1 h; solvent was evaporated and 5% aqueous NaHCO₃

d₆) d 7.94 (d, J=5.0 Hz, 1H), 7.18 (d, J=5.0 Hz, 1H),

6.53 (s, 1H), 3.57 (t, J=4.8 Hz, 4H), 3.39 (s, 3H), 3.38

(s, 2H), 2.40 (t, J=4.6 Hz, 4H).

2-Methyl-3-[[N-(2-methoxyethyl)-N-(3-methoxypropyl)-

amino]methyl]-2H-thieno[3,2-e]-1,2-thiazine 1,1-dioxide

(16a3). Reaction of 14a (2.97 g, 12.9 mmol) with 3-

methoxypropylamine as described for the preparation

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

969

Table 6. NMR data for compounds 15

¹H NMR (DMSO-d₆) d

mp (^ꢁC)

163±165

Yield (%)

74

15b

15c

15d

15e

15f

8.07 (s, 2H), 7.70 (s, 1H), 6.75 (s, 1H), 5.68 (t, J=5.1 Hz, 1H), 4.37 (d, J=5.1 Hz, 2H), 3.86 (q,

J=7.0 Hz, 2H), 1.09 (t, J=7.0 Hz, 3H)

8.07 (s, 2H), 7.70 (s, 1H), 6.77 (s, 1H), 5.66 (t, J=5.2 Hz, 1H), 4.36 (d, J=5.4 Hz, 2H), 3.78 (t,

J=7.4 Hz, 2H), 1.49 (q, J=7.3 Hz, 2H), 0.72 (t, J=7.4 Hz, 3H)

8.05 (s, 2H), 7.70 (s, 1H), 6.84 (s, 1H), 5.65 (t, J=4.6 Hz, 1H), 4.33 (m, 3H), 1.31 (d, J=7.0 Hz,

3H)

8.08 (s, 2H), 7.71 (s, 1H), 6.76 (s, 1H), 5.9±5.6 (m, 2H), 5.10 (d, J=11 Hz, 1H), 4.92 (d,

J=18 Hz, 1H), 4.46 (d, J=4.8 Hz, 2H), 4.30 (d, J=4.8 Hz, 2H)

8.07 (s, 2H), 7.71 (s, 1H), 6.79 (s, 1H), 5.67 (t, J=4.8 Hz, 1H), 4.40 (d, J=4.8 Hz, 2H), 3.71 (d,

J=7.1 Hz, 2H), 1.02 (m, 1H), 0.32 (m, 4H)

143±145

Oil

63

98

66

Glass

Oil

35 (from 14f)

15g

15h

15i

8.05 (s, 2H), 7.69 (s, 1H), 6.86 (s, 1H), 5.65 (br s, 1H), 4.35 (s, 2H), 3.89 (m, 1H), 1.75 (m, 7H),

1.22 (m, 3H)

8.06 (s, 2H), 7.69 (s, 1H), 6.74 (s, 1H), 5.65 (t, J=4.5 Hz, 1H), 4.36 (d, J=4.5 Hz, 2H), 3.96 (t,

J=5.3 Hz, 2H), 3.4±3.2 (m, 4H), 3.06 (s, 3H)

8.07 (s, 2H), 7.70 (s, 1H), 6.76 (s, 1H), 5.64 (t, J=4.6 Hz, 1H), 4.35 (d, J=4.6 Hz, 2H), 3.86 (m,

2H), 3.19 (m, 2H), 3.14 (s, 3H), 1.69 (m, 2H)

Glass

Oil

77

68

Oil

86

15k

15m

15n

8.12 (s, 2H), 7.83 (s, 1H), 7.40 (t, J=8.1 Hz, 1H), 7.09 (m, 1H), 6.95 (s, 1H), 6.79 (m, 2H), 5.56

(t, J=4.5 Hz, 1H), 3.96 (d, J=4.5 Hz, 2H), 3.77 (s, 3H)

8.12 (s, 2H), 7.82 (s, 1H), 7.03 (d, J=8.3 Hz, 1H), 6.90±6.75 (m, 3H), 5.55 (t, J=4.5 Hz, 1H),

3.95 (d, J=4.5 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 3H)

8.11 (s, 2H), 7.81 (s, 1H), 7.08 (d, J=9.1 Hz, 2H), 6.88 (s, 1H), 5.53 (t, J=4.6 Hz, 1H), 3.93 (br

s, 2H), 3.73 (m, 4H), 3.18 (m, 4H)

180±182

Foam

Oil

72

77

31 (from 14n)

of 16a1 provided the intermediate secondary amino-

ether as an oil (84%) which was reacted with 2-bro-

moethyl methyl ether (DMF/NaH; 100 ^ꢁC, 18 h).

Puri®cation by chromatography (EtOAc to 10% MeOH

in EtOAc) gave a syrup (0.70 g, 39%): ¹H NMR

(DMSO-d₆) d 7.96 (d, J=5.0 Hz, 1H), 7.19 (d, J=

5.0 Hz, 1H), 6.53 (s, 1H), 3.58 (m, 4H), 3.40 (s, 3H), 3.32

(d, J=4.7 Hz, 2H), 2.40 (m, 4H).

2-Methyl-3-(4-morpholinylmethyl)-2H-thieno[3,2-e]-1,2-

thiazine-6-sulfonamide 1,1-dioxide hydrochloride (17a1).

To a mixture of 16a1 (0.30 g, 1.04 mmol) in anhydrous

THF (30 mL) under nitrogen at 65 ^ꢁC was added n-

butyllithium (2.5 M, 0.625 mL, 1.56 mmol) over a 5 min

period. The mixture was stirred at 50 ^ꢁC for 10 min

and at 70 ^ꢁC for 1 h. Sulfur dioxide was passed over

the reaction mixture for 5 min followed by warming the

mixture to ambient temperature and evaporation to

provide a residue. Cold water (50 mL) and 5% aqueous

NaHCO₃(50 mL) were added to the residue and this

mixture was extracted with EtOAc (100 mL) to give

0.17 g (58%) of unreacted starting material. Hyd-

roxylamine-O-sulfonic acid (0.294 g, 2.60 mmol) was

added to the aqueous solution which was stirred at

ambient temperature for 3 h. The reaction mixture was

extracted with EtOAc (2Â100 mL) and the combined

extracts were dried and evaporated to give the free base

(0.098 g, 26%). The combined crude product from two

batches (0.383 g) was treated with 1.5 M HCl/MeOH

(1 mL) and evaporated to give the HCl salt; recrystalli-

zation from a mixture of EtOH and water gave a yellow

solid (0.172 g, 11%): mp 231±233 ^ꢁC (dec); ¹H NMR

(DMSO-d₆) d 11.56 (br s, 1H), 8.16 (s, 2H), 7.82 (s, 1H),

7.16 (s, 1H), 4.35 (br s, 2H), 3.92 (m, 4H), 3.40 (m, 2H),

3.27 (s, 3H), 3.18 (m, 4H); ¹³C NMR (DMSO-d₆) d

149.7, 138.8, 134.5, 128.1, 116.0, 62.9, 55.6, 50.6, 34.5.

2-Methyl-3-[[N-(2-methoxyethyl)-N-(2-propenyl)amino]-

methyl]-2H-thieno[3,2-e]-1,2-thiazine 1,1-dioxide (16a7).

Reaction of 14a (2.00 g, 8.66 mmol) with 2-methoxy-

ethylamine as described for the preparation of 16a1

provided the intermediate secondary aminoether as an

oil (78%) which was reacted with allyl bromide. Pur-

i®cation by chromatography (25% EtOAc in hexane)

gave an oil (1.6 g, 74%): ¹H NMR (DMSO-d₆) d 7.95 (d,

J=5.0 Hz, 1H), 7.19 (d, J=5.0 Hz, 1H), 6.56 (s, 1H),

5.88 (m, 1H), 5.20 (m, 1H), 3.53 (s, 2H), 3.45 (t, J=

5.9 Hz, 2H), 3.37 (s, 3H), 3.22 (s, 3H), 3.15 (m, 2H), 2.65

(t, J=5.9 Hz, 2H).

2-(3-Methoxypropyl)-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine 1,1-dioxide (16i1). Prepared from 14i

in a manner similar to that described for 16a1 to give

1

a syrup (94%): H NMR (DMSO-d₆) 7.92 (d, J=5.1

Hz, 1H), 7.19 (d, J=5.1 Hz, 1H), 6.64 (s, 1H), 3.91 (t,

J=7.2 Hz, 2H), 3.56 (t, J=4.8 Hz, 4H), 3.36 (s, 2H), 3.16

(m, 2H), 3.13 (s, 3H), 2.39 (t, J=4.2 Hz, 4H), 1.72 (m,

2H).

.

Anal. (C₁₂H₁₇N₃O₅S₃HCl 0.5 H₂O) C, H, N.

3-[[N-(2-Methoxyethyl)-N-(3-methoxypropyl)amino]meth-

yl]-2-methyl-2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide

1,1-dioxide hydrochloride (17a3). Prepared from 16a3 as

described for 17a1 (42%, recrystallized from 2-propa-

nol): mp 173±175 ^ꢁC; ¹H NMR (DMSO-d₆) 8.19 (s, 2H),

7.82 (s, 1H), 7.24 (s, 1H), 4.41 (br s, 2H), 3.76 (m, 4H),

3.45±3.25 (m, 7H), 3.22 (s, 6H), 2.03 (m, 2H); ¹³C NMR

(DMSO-d₆) d 149.8, 138.8, 135.2, 128.1, 116.3, 69.1,

65.8, 58.3, 57.9, 51.0, 50.4, 34.5, 22.9. Anal. (C₁₅H₂₃

2-[(4-Methoxyphenyl)methyl]-3-[(4-morpholinyl)methyl]-

2H-thieno[3,2-e]-1,2-thiazine 1,1-dioxide (16j1). Pre-

pared from 14j in a manner similar to that described for

ꢁ

1

16a1 (88%): mp 112±114 C; H NMR (DMSO-d₆) d

7.97 (d, J=5.1 Hz, 1H), 7.20 (d, J=5.1 Hz, 1H), 6.96 (d,

J=8.7 Hz, 2H), 6.80 (d, J=8.7 Hz, 2H), 6.65 (s, 1H),

5.10 (s, 2H), 3.67 (s, 2H), 3.58 (t, J=4.5 Hz, 4H), 3.21

(s, 2H), 2.38 (s, 4H).

.

N₃O₆S₃HCl) C, H, N.

970

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

3-[N-(2-Methoxyethyl)-N-methylamino]methyl]-2-methyl-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

(17a6). To a solution of 15a (0.80 g, 2.58 mmol) and

methanesulfonic anhydride (0.67 g, 3.87 mmol) in anhy-

drous THF (30 mL) at 0 ^ꢁC was added triethylamine

(0.52 g, 5.16 mmol). After 30 min the reaction mixture

was cooled (ice bath) and N-(2-methoxyethyl)methyl-

amine (1 mL) was added. This mixture was allowed to

warm to ambient temperature, stirred for 2 h and heated

at re¯ux temperature for 10 min followed by evapora-

tion to a residue which was combined with 2 N HCl

(50 mL) and extracted with EtOAc (100 mL). The aqu-

eous layer was separated, combined with 5% aqueous

NaHCO₃(150 mL) and extracted with EtOAc

(2Â100 mL). The combined extracts were dried and

evaporated to an oil which was puri®ed by chromato-

graphy (4% MeOH in CH₂Cl₂) to give a solid (0.635 g,

66%): mp 127±129 ^ꢁC (dec); ¹H NMR (DMSO-d₆) d

8.08 (s, 2H), 7.65 (s, 1H), 6.64 (s, 1H), 3.46 (m, 4H), 3.40

(s, 3H), 3.24 (s, 3H), 2.59 (t, J=5.8 Hz, 3H), 2.21 (s,

3H). Anal. (C₁₂H₁₉N₃O₅S₃) C, H, N.

Prepared from 15c and morpholine as described for

17a6 but converted to the HCl salt (47%): mp 201±

ꢁ

1

203 C; H NMR (DMSO-d₆) d 8.15 (s, 2H), 7.85 (s,

1H), 7.22 (s, 1H), 4.35 (br s, 2H), 4.0±3.8 (m, 6H), 3.22

(br s, 4H), 1.22 (m, 2H), 0.59 (t, J=7.3 Hz, 3H); MS (CI)

.

m/z 408 (M+1). Anal. (C₁₄H₂₁N₃O₅S₃HCl) C, H, N.

3-[[Bis(2-methoxyethyl)amino]methyl]-2-propyl-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydrochloride

(17c2). Prepared from 15c and bis(2-methoxyethyl)-

amine as described for 17a6 but converted to the HCl

salt (42%): mp 201±203 ^ꢁC; ¹H NMR (DMSO-d₆) d 10.7

(br s, 1H), 8.19 (s, 2H), 7.83 (s, 1H), 7.3 (br s, 1H), 4.41

(br s 2H), 4.0±3.6 (m, 8H), 3.38 (br s, 2H), 3.31 (s, 6H),

1.2 (m, 2H), 0.60 (t, J=7.4 Hz, 3H). Anal. (C₁₆H₂₇

.

N₃O₆S₃HCl) C, H, N.

2-Propyl-3-[(2-propynylamino)methyl]-2H-thieno[3,2-e]-

1,2-thiazine-6-sulfonamide 1,1-dioxide (17c8). Prepared

from 15c and propargylamine as described for 17a6

(40%): mp 136±138 ^ꢁC; H NMR DMSO-d₆) d 8.07 (s,

1

2H), 7.69 (s, 1H), 6.76 (s, 1H), 3.83 (t, J=7.0 Hz, 2H),

3.67 (s, 2H), 3.32 (s, 2H), 3.15 (s, 1H), 2.7 (br s, 1H),

1.45 (q, J=7.0 Hz, 2H), 0.69 (t, J=7.5 Hz, 3H). Anal.

(C₁₃H₁₇N₃O₄S₃) C, H, N.

3-[[N-(2-Methoxyethyl)-N-(2-propenyl)amino]methyl]-2-

methyl-2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-

dioxide hydrochloride (17a7). Prepared from 16a7 as

described for 17a1 (58%, recrystallized from CH₂Cl₂/

hexane): mp 107±109 ^ꢁC; H NMR (DMSO-d₆) d 8.07

2-(1-Methylethyl)-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17d1).

Prepared from 15d and morpholine as described for

1

(br s, 2H), 7.65 (s, 1H), 6.67 (s, 1H), 5.98±5.75 (m, 1H),

5.20 (m, 2H), 3.55 (s, 2H), 3.45 (t, J=5.9 Hz, 2H), 3.42

(s, 3H), 3.22 (s, 3H), 3.15 (m, 1H), 2.65 (t, J=5.9 Hz,

2H); ¹³C NMR (DMSO-d₆) d 149.0, 143.0, 140.4, 134.9,

127.5, 126.6, 118.1, 105.5, 70.0, 57.9, 56.2, 56.1, 51.5,

30.3. Anal. (C₁₄H₂₁N₃O₅S₃) C, H, N.

17a6 (32%): mp 196±198 ^ꢁC; H NMR (DMSO-d₆) d

1

8.06 (s, 2H), 7.64 (s, 1H), 6.77 (s, 1H), 4.42 (m, 1H), 3.61

(t, J=4.3 Hz, 4H), 3.45 (s, 2H), 2.42 (br s, 4H), 1.40 (d,

J=6.9 Hz, 6H); ¹³C NMR (DMSO-d₆) d 148.6, 142.2,

139.8, 130.0, 127.5, 108.8, 66.2, 60.4, 53.7, 52.4, 21.7.

Anal. (C₁₄H₂₁N₃O₅S₃) C, H, N.

2-Ethyl-3-[(4-morpholinyl)methyl]-2H-thieno[3,2-e]-1,2-

thiazine-6-sulfonamide 1,1-dioxide hydrochloride (17b1).

Prepared from 15b and morpholine as described for

17a6 but converted to the HCl salt (56%): mp 239±

2-(1-Methylethyl)-3-[(2-propynylamino)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17d8).

Prepared from 15d and propargylamine as described

for 17a6 (39%): mp 133±135 ^ꢁC; ¹H NMR (DMSO-d₆) d

8.06 (s, 2H), 7.68 (s, 1H), 6.82 (s, 1H), 4.38 (m, 1H), 3.69

(s, 2H), 3.33 (d, J=2.5 Hz, 2H), 3.14 (t, J=2.3 Hz, 1H),

1.32 (d, J=6.9 Hz, 6 H); ¹³C NMR (DMSO-d₆) d 148.6,

143.6, 140.0, 130.6, 127.8, 109.5, 82.3, 74.2, 53.7, 49.7,

35.9, 21.7. Anal. (C₁₃H₁₇N₃O₄S₃) C, H, N.

ꢁ

1

241 C; H NMR (DMSO-d₆) d 8.17 (s, 2H), 7.86 (s,

1H), 7.29 (s, 1H), 4.40 (br s, 2H), 3.94 (m, 6H), 3.10 (m,

4H), 0.77 (t, J=6.8 Hz, 3H); MS (CI) m/z 394 (M+1).

.

Anal. (C₁₃H₁₉N₃O₅S₃HCl) C, H, N.

2-Ethyl-3-[[bis(2-methoxyethyl)amino]methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydrochloride

(17b2). Prepared from 15b and bis(2-methoxyethyl)-

amine as described for 17a6 but converted to the HCl

salt (43%): mp 185±186 ^ꢁC; ¹H NMR (DMSO-d₆) d 8.17

(s, 2H), 7.83 (s, 1H), 7.32 (s, 1H), 4.4 (br s, 2H), 3.9±3.6

(m, 6H), 3.4 (br s, 4H), 3.30 (s, 6H), 0.79 (t, J=7.0 Hz,

3-[(4-Morpholinyl)methyl]-2-(2-propenyl)-2H-thieno[3,2-

e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17e1). Pre-

pared from 15e and morpholine as described for 17a6

(64%): mp 136±138 ^ꢁC; H NMR (DMSO-d₆) d 8.06 (s,

1

.

3H). Anal. (C₁₅H₂₅N₃O₆S₃HCl) C, H, N.

2H), 7.65 (s, 1H), 6.75 (s, 1H), 5.6±5.9 (m, 1H), 5.05 (d,

J=11 Hz, 1H), 4.80 (d, J=18 Hz, 1H), 4.56 (d, J=

4.7 Hz, 2H), 3.57 (m, 4H), 3.27 (s, 2H), 2.36 (m, 4H);

MS (CI) m/z 406 (M+1). Anal. (C₁₄H₁₉N₃O₅S₃) C, H, N.

2-Ethyl-3-[[bis(2-hydroxyethyl)amino]methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydrochloride

(17b4). Prepared from 15b and diethanolamine as

described for 17a6 but converted to the HCl salt (66%):

2-Cyclopropylmethyl-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17f1). Prepared from 15f and morpholine as

described for 17a6 but converted to the HCl salt (30%):

mp 211±214 ^ꢁC; H NMR (DMSO-d₆) d 8.19 (s, 2H),

1

7.83 (s, 1H), 7.35 (s, 1H), 5.18 (br s, 2H), 4.47 (br s, 2H),

3.85 (m, 8H), 3.31 (br s, 4H), 0.75 (t, J=6.9 Hz, 3H).

.

Anal. (C₁₃H₂₁N₃O₆S₃HCl) C, H, N.

mp 120±130 ^ꢁC; H NMR (DMSO-d₆) d 8.15 (s, 2H),

1

7.88 (s, 1H), 7.28 (s, 1H), 4.45 (s, 2H), 3.1±4.1 (br m,

.

3-[(4-Morpholinyl)methyl]-2-propyl-2H-thieno[3,2-e]-1,2-

thiazine-6-sulfonamide 1,1-dioxide hydrochloride (17c1).

4H), 0.6 (m, 1H), 0.15 (m, 4H). Anal. (C₁₅H₂₁N₃O₅S₃

.

HCl 0.4 2-propanol) C, H, N.

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

971

2-Cyclohexyl-3-[(4-morpholinyl)methyl]-2H-thieno[3,2-e]-

1,2-thiazine-6-sulfonamide 1,1-dioxide (17g1). Prepared

from 15g and morpholine as described for 17a6 (33%):

for 17a6 (20%): mp 212±214 ^ꢁC; ¹H NMR (DMSO-d₆) d

8.14 (s, 2H), 7.64 (s, 1H), 7.12 (br s), 7.00 (d, J=8.6 Hz,

2H), 6.69 (d, J=8.6 Hz, 2H), 5.04 (s, 2H), 4.43 (br s,

2H), 3.94 (br s, 4H), 3.69 (br s, 2H), 3.41 (br s, 2H), 3.21

mp 200±202 ^ꢁC; H NMR (DMSO-d₆) d 8.05 (s, 2H),

1

.

7.62 (s, 1H), 6.74 (s, 1H), 3.98 (m, 1H), 3.58 (br s, 4H),

3.45 (s, 2H), 2.48 (s, 4H), 2.08±1.50 (m, 7H), 1.40±0.99

(m, 3H); ¹³C NMR (DMSO-d₆) d 148.6, 142.2, 139.9,

129.9, 127.5, 108.7, 66.3, 61.7, 60.5, 52.4, 31.6, 26.0,

24.8. Anal. (C₁₇H₂₅N₃O₅S₃) C, H, N.

(br s, 4H). Anal. (C₁₉H₂₃N₃O₆S₃HCl) C, H, N.

2-(3-Methoxyphenyl)-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17k1). Prepared from 15k and morpholine as

described for 17a6 but converted to the HCl salt (29%):

mp 170±174 ^ꢁC; H NMR (DMSO-d₆) d 8.22 (s, 2H),

1

2-(2-Methoxyethyl)-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17h1).

Prepared from 15h and morpholine as described for

7.95 (s, 1H), 7.59 (s, 1H), 7.35 (t, J=8.1 Hz, 1H), 7.06

(m, 1H), 6.96 (s, 1H), 6.67 (dd, J=1.2 and 7.6 Hz, 1H),

3.91 (br s, 4H), 3.79 (s, 3H), 3.57 (br s, 2H), 3.20 (br s,

4H); ¹³C NMR (DMSO-d₆) d 159.8, 150.3, 139.0, 135.2,

130.3, 128.4, 119.4, 115.2, 114.6, 63.1, 62.4, 55.6, 50.7,

17a6 (32%): mp 164±166 ^ꢁC; H NMR (DMSO-d₆) d

1

8.05 (s, 2H), 7.63 (s, 1H), 6.7 (s, 1H), 4.03 (dd, 2H), 3.55

(m, 4H). Anal. (C₁₄H₂₁N₃O₆S₃) C, H, N.

.

25.6, 10.4. Anal. (C₁₈H₂₁N₃O₆S₃HCl 0.33 1-propanol)

C, H, N.

2-(2-Methoxyethyl)-3-[[bis(2-methoxyethyl)amino]methyl]-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

hydrochloride (17h2). Prepared from 15h and bis(2-

methoxyethyl)amine as described for 17a6 but con-

verted to the HCl salt (31%): mp 180±182 ^ꢁC; ¹H NMR

(DMSO-d₆) d 10.75 (br s, 1H), 8.18 (s, 2H), 7.81 (s, 1H),

7.28 (s, 1H), 4.41 (br s, 2H), 4.00 (m, 2H), 3.78 (m, 4H),

3.39 (m, 4H), 3.90 (s, 6H), 3.17 (m, 2H), 2.87 (s, 3H);

¹³C NMR (DMSO-d₆) d 149.0, 138.3, 132.9, 127.8,

118.4, 69.3, 65.9, 58.2, 57.7, 54.2, 51.5, 47.7. Anal.

2-(3-Methoxyphenyl)-3-[(2-hydroxyethylamino)methyl]-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

hydrochloride (17k5). Prepared from 15k and ethanola-

mine as described for 17a6 but converted to the HCl salt

(26%): mp 137±140 ^ꢁC; H NMR (DMSO-d₆) d 9.48 (s,

1

2H), 8.22 (s, 2H), 7.91 (s, 1H), 7.45 (s, 1H), 7.37 (t,

J=8.1 Hz, 1H), 7.07 (m, 1H), 6.92 (m, 1H), 6.72 (m,

1H), 5.30 (s, 1H), 3.79 (s, 3H) 3.74 (br s, 4H), 3.00 (m,

2H); ¹³C NMR (DMSO-d₆) d 159.8, 150.2, 139.2, 135.2,

135.1, 130.5, 130.4, 128.2, 119.7, 115.3, 115.2, 114.5,

.

(C₁₆H₂₇N₃O₇S₃HCl) C, H, N.

.

2-(3-Methoxypropyl)-3-[(4-morpholinyl)methyl]-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17i1). Prepared from 16i1 as described for

56.3, 55.5, 48.1, 47.1. Anal. (C₁₆H₁₉N₃O₆S₃HCl 0.5

H₂O) C, H, N.

ꢁ

1

17a6 (35%): mp 145±149 C; H NMR (DMSO-d₆) d

8.19 (s, 2H), 7.85 (s, 1H), 7.28 (s, 1H), 4.38 (br s, 2H),

3.97 (4.1±3.6, 8H), 3.5±2.9 (m, 8H), 1.40 (br s, 2H); ¹³C

NMR (DMSO-d₆) d 149.5, 149.5, 138.6, 128.1, 68.5,

2-(3,4-Dimethoxyphenyl)-3-[(4-morpholinyl)methyl]-2H-

thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17m1). Prepared from 15m and morpholine

as described for 17a6 but converted to the HCl salt

(46%): mp 219±221 ^ꢁC; ¹H NMR (DMSO-d₆) d 11.7 (br

s, 2H), 8.21 (s, 2H), 7.94 (s, 1H), 7.5 (br s, 1H), 7.04 (d,

J=1.6 Hz, 1H) 6.96 (d, J=8.7 Hz, 1H), 6.57 (dd, J=8.7

and 1.6 Hz, 1H), 3.91 (br s, 6H), 3.77 (s, 6H), 3.17 (br s,

.

63.1, 57.8, 50.6. Anal. (C₁₅H₂₃N₃O₆S₃HCl) C, H, N.

3-[(Cyclopropylamino)methyl]-2-(3-methoxypropyl)-2H-

thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17i9). Prepared from 15i and cyclopropyl-

amine as described for 17a6 but converted to the HCl

salt (20%): mp 210±211 ^ꢁC; ¹H NMR (DMSO-d₆) d 9.85

(s, 2H), 8.19 (s, 2H), 7.79 (s, 1H), 7.24 (s, 1H), 4.19 (s,

2H), 3.90 (t, J=5 Hz, 2H), 3.5 (m, 2H), 3.10 (s, 3H),

3.07 (t, J=6 Hz, 2H), 2.65 (m, 1H), 1.49 (m, 2H), 1.04

(d, J=6 Hz, 4H); ¹³C NMR (DMSO-d₆) d 146.4, 135.7,

131.6, 127.9, 124.8, 111.7, 65.4, 54.7, 44.1, 41.3, 25.6,

.

4H). Anal. (C₁₉H₂₃N₃O₇S₃HCl) C, H, N.

2-[(4-Morpholinyl)phenyl]-3-[(4-morpholinyl)methyl]-2H-

thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17n1). Prepared from 15n and morpholine

as described for 17a6 but converted to the HCl salt

(32%): mp 230±235 ^ꢁC (dec); H NMR (DMSO-d₆) d

1

8.22 (s, 2H), 7.94 (s, 1H), 7.56 (s, 1H), 7.06 (dd, J=9.1,

18.7 Hz, 2H), 6.96 (d, J=9.1 Hz, 2H), 4.82 (br s, 2H),

3.92 (br s, 6H), 3.70 (br s, 4H), 3.16 (br s, 6H); ¹³C

NMR (DMSO-d₆) d 151.3, 150.2, 139.0, 133.4, 130.8,

128.7, 128.4, 124.6,115.0, 65.9, 63.0, 55.6, 50.6, 47.0.

.

24.9; MS (CI) m/z 408 (M+1). Anal. (C₁₄H₂₁N₃O₅S₃

.

HCl 0.33 2-propanol) C, H, N.

3-[(1-Imidazolyl)methyl]-2-(3-methoxypropyl)-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17i10).

Prepared from 15i and imidazole as described for 17a6

.

Anal. (C₂₁H₂₆N₄O₆S₃HCl H₂O) C, H, N.

(36%): mp 197±199 ^ꢁC; H NMR (DMSO-d₆) d 8.11 (s,

2-(3-Hydroxypropyl)-3-(4-morpholinylmethyl)-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17o1). A

solution of 17j1 (1.00 g, 2.25 mmol) in CH₂Cl₂(20 mL)

was cooled to 0 ^ꢁC and bromodimethylborane (0.82 g,

6.75 mmol) was added. The reaction mixture was

allowed to warm to ambient temperature and, after

stirring for 3 h, was diluted with EtOAc (100 mL) and

water (100 mL). The organic layer was separated, dried

and evaporated to a residue which was puri®ed by

1

2H), 7.74 (s, 1H), 7.72 (s, 1H), 7.16 (s, 1H), 6.96 (s, 1H),

6.81 (s, 1H), 5.21 (s, 2H), 3.80 (t, J=6 Hz, 2H), 3.12 (s,

3H), 1.53 (m, 2H); MS (CI) m/z 419 (M+1). Anal.

(C₁₄H₁₈N₄O₅S₃) C, H, N.

2-[(4-Methoxyphenyl)methyl]-3-[(4-morpholinyl)methyl]-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide

hydrochloride (17j1). Prepared from 16j1 as described

972

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

chromatography (gradient, 50% EtOAc in hexane to

EtOAc) to give a viscous syrup which crystallized

(chlorobutane/hexane) to give a colorless solid (0.76 g,

EtOAc in hexane) to give an oil (4.41 g, 62%): ¹H NMR

(DMSO-d₆) d 8.24 (s, 1H), 7.86 (s, 1H), 7.39 (t, J=

8.2 Hz, 1H), 7.2±7.0 (m, 2H), 6.9±6.7 (m, 2H), 4.32 (s,

2H), 3.74 (s, 3H), 1.20 (s, 9H); MS (CI) m/z 477 (M+1).

79%): mp 135±138 ^ꢁC; H NMR (DMSO-d₆) d 8.09 (s,

1

2H), 7.67 (s, 1H), 6.76 (s, 1H), 4.55 (t, J=4.8 Hz, 1H),

3.97 (t, J=7.2 Hz, 2H), 3.59 (br s, 4H), 3.41 (s, 2H), 3.30

(m, 2H), 2.42 (br s, 4H), 1.63 (m, 2H); ¹³C NMR

(DMSO-d₆) d 148.8, 140.8, 139.9, 128.0, 127.6, 107.9,

66.1, 59.7, 52.6, 42.5, 32.9; MS (CI) m/z 424 (M+1).

Anal. (C₁₄H₂₁N₃O₆S₃) C, H, N.

N-(1,1-Dimethylethyl)-2-(3-methoxphenyl)-3-[(4-morpho-

linyl)methyl]-2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide

1,1-dioxide (19k1). To

a solution of 18 (0.86 g,

1.88 mmol) in anhydrous THF (50 mL) at 0 ^ꢁC was

added p-toluenesulfonyl chloride (0.57 g, 3.0 mmol) and

triethylamine (0.523 mL, 3.76 mol). After stirring for

1 h, morpholine (3 mL) and DMF (30 mL) were added

and the mixture heated at re¯ux temperature for 2 h.

Evaporation of the solvent provided a residue which

was combined with 5% aqueous NaHCO₃(100 mL) and

the mixture was extracted with EtOAc (2Â100 mL). The

combined extracts were dried and evaporated to a syrup

which was puri®ed by chromatography (EtOAc in

hexane, 1:1) to give a glassy solid (0.86 g, 91%): mp 75±

80 ^ꢁC; ¹H NMR (DMSO-d₆) d 8.23 (s, 1H), 7.83 (s, 1H),

7.39 (t, J=8.2 Hz, 1H), 7.09 (m, 1H), 6.93 (s, 1H), 6.81

(m, 2H), 3.76 (s, 3H), 3.52 (m, 4H), 3.30 (d, J=4.7 Hz,

2H), 2.25 (m, 4H), 1.22 (s, 9H).

2-(3-Hydroxyphenyl)-3-(4-morpholinylmethyl)-2H-thieno-

[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (17p1). To

a solution of 19k1 (0.86 g, 1.63 mmol) in CH₂Cl₂

(50 mL) at 0 ^ꢁC was added a 1 M solution of boron

tribromide in CH₂Cl₂over 3 min. The mixture was

warmed to ambient temperature and allowed to stir

for 2 h. The reaction mixture was poured into 5% aqu-

eous NaHCO₃(100 mL) and extracted with EtOAc

(2Â100 mL). The combined extracts were dried and

evaporated to dryness. Chromatography on silica (5%

MeOH/CH₂Cl₂) gave a solid, which was recrystallized

(EtOAc/hexane) to give an o white solid (0.39 g, 52%):

mp 220±222 ^ꢁC; H NMR (DMSO-d₆) d 9.80 (s, 1H),

1

8.10 (s, 2H), 7.67 (s, 1H), 7.22 (t, J=8.1 Hz, 1H), 6.92

(s, 1H), 6.84 (s, 1H), 6.64 (m, 2H), 3.52 (br s, 4H), 3.01

(s, 2H), 2.24 (br s, 4H); ¹³C NMR (DMSO-d₆) d 157.7,

149.4, 141.6, 140.3, 135.0, 129.7, 128.6, 128.0, 119.5,

116.4, 116.2, 108.1, 66.1, 59.2, 52.3. Anal. (C₁₇H₁₉

N₃O₆S₃) C, H, N.

N-(1,1-Dimethylethyl)-2-(3-methoxyphenyl)-3-[(2-propy-

nylamino)methyl-2H-thieno[3,2-e]-1,2-thiazine-6-sulfon-

amide 1,1-dioxide (19k8). To a solution of 18 (1.0 g,

2.1 mmol) in anhydrous DMF (20 mL) was added pro-

pargylamine (1.77 g, 32.1 mmol); this mixture was stir-

red at ambient temperature for 30 min followed by

heating at 80 ^ꢁC for 2 h and evaporation to dryness. The

residue was combined with 5% aqueous NaHCO₃

(100 mL) and extracted with EtOAc (2Â100 mL). The

combined extracts were dried and evaporated to an oil

which was puri®ed by chromatography (EtOAc/hexane,

1:1) to give a syrup (0.53 g, 51%): ¹H NMR (DMSO-d₆)

d 8.24 (s, 1H), 7.86 (s, 1H), 7.37 (t, J=8.3 Hz, 1H), 7.08

(m, 1H), 6.95 (s, 1H), 6.78 (m, 2H), 3.76 (s, 3H), 3.28

(m, 5H), 3.02 (s, 1H), 1.22 (s, 9H).

2-(3-Hydroxyphenyl)-3-[(2-propynylamino)methyl]-2H-

thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide hydro-

chloride (17p8). A solution of 19p8 (0.53 g, 1.2 mmol)

was treated with boron tribromide as described for the

preparation of 17p1; column chromatography (30%

hexane in EtOAc) gave an oil that was converted to the

HCl salt; recrystallization (EtOH/CH₂Cl_ꢁ₂) gave a yel-

1

lowish solid (0.27 g, 57%): mp 195±198 C; H NMR

(DMSO-d₆) d 10.05 (br s, 1H), 8.22 (s, 2H), 7.93 (s, 1H),

7.40 (s, 1H), 7.24 (t, J=8.3 Hz, 1H), 6.89 (m, 1H), 6.64

(m, 2H), 3.86 (s, 2H), 3.77 (s, 2H), 3.68 (s, 1H); ¹³C

NMR (DMSO-d₆) d 158.1, 150.2, 139.1, 135.0, 134.8,

130.7, 130.2, 128.3, 118.3, 116.9, 115.5, 115.0, 79.9, 74.6,

3,5-Dibromo-2-(1,3-dioxolan-2-yl)-thiophene (20). A

mixture consisting of 3,5-dibromo-2-thiophenecarbox-

aldehyde¹⁰(11.0 g, 40.7 mmol), p-toluenesulfonic acid

(0.25 g) and ethylene glycol (5.06 g, 81.5 mmol) in tolu-

ene (150 mL) was heated at re¯ux temperature for 1.5 h;

water was removed by a Dean-Stark trap. The reaction

mixture was cooled and poured into 5% aqueous

NaHCO₃(100 mL). The organic layer was separated,

dried and evaporated to a residue. Puri®cation of this

crude material by chromatography (6% EtOAc in hex-

.

46.1, 34.9. Anal. (C₁₆H₁₅N₃O₅S₃HCl) C, H, N.

3-Chloromethyl-N-(1,1-dimethylethyl)-2-(3-methoxphenyl)-

2H-thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide (18).

To a solution of 14k (4.81 g, 14.9 mmol) in anhydrous

THF (80 mL) at 70^ꢁC was added n-butyllithium (2.5M,

14.9 mL, 37.2 mmol) and the mixture was stirred for 1 h

followed by the introduction of sulfur dioxide gas over

the surface of the reaction mixture for 5 min. The mixture

was warmed to ambient temperature, evaporated to

dryness and the residue was mixed with dichloro-

methane (250mL); this suspension was cooled on an ice

bath and N-chlorosuccinimide (6.96 g, 52.1 mmol) was

added. After stirring for 2 h, t-butylamine (15 mL,

143 mmol) was added and stirring continued for 16h fol-

lowed by the addition of 5% aqueous NaHCO₃(200mL).

This mixture was extracted with EtOAc (2Â200 mL)

and the combined extracts were dried and evaporated to

an oil which was puri®ed by chromatography (40%

1

ane) gave an oil (10.33 g, 81%): H NMR (CDCl₃) d

6.93 (s, 1H), 6.05 (s, 1H), 4.13±4.00 (m, 4H).

N-[2-(1,3-Dioxolan-2-yl)-3-thienyl]sulfonyl]-N-methyl-

glycine ethyl ester (21). To a solution of 20 (10.0 g,

31.8 mmol) in anhydrous ether (150 mL) at 75 ^ꢁC was

added n-butyllithium (2.5 M in hexane, 13.4 mL,

33.4 mmol) while maintaining the temperature below

65 ^ꢁC; a precipitate formed during the addition. After

the addition was complete, 1-propanol (1.91 g, 31.8mmol)

was added to quench the 2-lithio species and provide a

solution of 3-bromo-2-(1,3-dioxolan-2-yl)thiophene. To

this solution was added a solution of n-butyllithium

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

973

(2.5 M in hexane, 13.4 mL, 33.4 mmol) followed by pas-

sing sulfur dioxide over the reaction mixture for about

10 min. This mixture was warmed to ambient tempera-

ture and evaporated to dryness. The residue was mixed

with CH₂Cl₂(150 mL), cooled to 0 ^ꢁC, and N-chloro-

succinimide was added followed by stirring for 40 min.

Saturated aqueous Na₂HCO₃(100 mL) was added to

the mixture followed by sarcosine ethyl ester HCl salt

(7.34 g, 47.8 mmol) and stirring continued for 30 min.

The organic layer was separated, dried, and evaporated

to a residue which was puri®ed by chromatography

(30% EtOAc in hexane) to give a viscous oil (5.96 g,

24 (0.78 g, 2.52 mmol) and triethylamine (1.02 g,

10.1 mmol) in anhydrous THF (30 mL) at ambient tem-

perature was added p-toluenesulfonyl chloride (0.961 g,

5.04 mmol). After stirring for 4.5 h, morpholine (2 mL)

was added and the mixture stirred for 1 h followed by

heating at re¯ux temperature for 10 min. The reaction

mixture was evaporated to a residue which was com-

bined with 5% aqueous NaHCO₃(80 mL). This mixture

was extracted with EtOAc (2Â100 mL) and the com-

bined extracts were dried and evaporated to an oil

which was puri®ed by chromatography (5% to 10%

CH₃OH in CH₂Cl₂) to give an amorphous solid (0.41 g,

43%). Recrystallization from EtOAc/CH₂Cl₂gave a

yellowish solid: mp 192±194 ^ꢁC; ¹H NMR (DMSO-d₆) d

7.92 (s, 2H), 7.72 (s, 1H), 6.77 (s, 1H), 3.62 (m, 4H), 3.45

(s, 2H), 3.43 (s, 3H), 2.41 (m, 4H); ¹³C NMR (DMSO-

d₆) d 144.4, 144.3, 143.7, 142.5, 126.2, 122.2, 102.2, 66.1,

60.1, 52.6, 29.5. Anal. (C₁₂H₁₇N₃O₅S₃) C, H, N.

1

53%): H NMR (DMSO-d₆) d 7.75 (d, J=5.5 Hz, 1H),

7.32 (d, J=5.4 Hz, 1H), 6.41 (s, 1H), 4.1±3.9 (m, 8H),

2.86 (s, 3H), 1.15 (t, J=7.2 Hz, 3H).

Ethyl 2-methyl-2H-thieno[2,3-e]-1,2-thiazine-3-carboxylate

1,1-dioxide (22). A mixture of 21 (5.86 g, 16.5 mmol)

and tri¯uoroacetic acid (8 mL) in acetone (50 mL) was

heated at re¯ux temperature for 1 h, cooled, and poured

into water (100 mL). Acetone was evaporated and the

residue was combined with 5% aqueous NaHCO₃

(50 mL) and this mixture was extracted with EtOAc

(2Â150 mL). The combined extracts were dried and

evaporated to give crude aldehyde which was dissolved

in EtOAc (100 mL) followed by the addition of DBN

(1 mL) and molecular sieves (5 g). This mixture was

heated at re¯ux temperature for 15 min, cooled, and

poured into 2 N HCl (50 mL). The organic layer was

separated, dried, and evaporated to an oil which was

puri®ed by chromatography (30% EtOAc in hexane) to

4-Bromo-5-(1,3-dioxolan-2-yl)-N-(1,1-dimethylethyl)-thio-

phene-2-sulfonamide (26). A solution of 20 (2.09 g,

6.66 mmol) in anhydrous THF (20 mL) was treated as

described for the preparation of 18 to give, after puri-

®cation by chromatography (30% EtOAc in hexane), an

1

oil (1.83 g, 74%); H NMR (DMSO-d₆) d 7.97 (s, 1H),

7.56 (s, 1H), 6.04 (s, 1H), 4.01 (m, 4H), 1.17 (s, 9H).

N-[[5-(Aminosulfonyl)-2-formyl-thiophen-3-yl]sulfonyl]-N-

methyl-glycine ethyl ester (27). To a solution of 26

(5.63 g, 15.21 mmol) in anhydrous THF (60 mL) at

room temperature was added NaH (60% dispersion in

mineral oil, 0.669 g, 16.7 mmol) under nitrogen. After

stirring for 1.5 h, the mixture was cooled to 70 ^ꢁC and

n-butyllithium (9.13 mL, 22.8 mmol) was added. An ali-

quot was removed after 15 min and quenched with

give an o-white solid (3.60 g, 79%): mp 87±89 ^ꢁC: H

1

NMR (DMSO-d₆) d 8.06 (d, J=5.3 Hz, 1H), 7.91 (s,

1H), 7.53 (d, J=5.4 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H),

3.11 (s, 3H), 1.33 (t, J=7.1 Hz, 3H).

1

water; NMR showed that lithiation was complete: H

2-Methyl-2H-thieno[2,3-e]-1,2-thiazine-3-methanol 1,1-

dioxide (23). To a solution of 22 (3.16 g, 11.6 mmol) in

anhydrous THF (30 mL) at 0 ^ꢁC was added DIBAL-H

(1.0 M, 29.0 mL, 29.0 mmol). This mixture was stirred

for 30 min, warmed to ambient temperature, and stirred

for an additional 30 min. The mixture was evaporated

to a residue which was combined with EtOAc (100 mL)

and poured into 2 N HCl (50 mL). The organic layer

was separated, washed with brine, dried and evaporated

to an oil which was puri®ed by chromatography

(EtOAc/hexane, 1/1) to give an oil which solidi®ed upon

standing (2.3 g, 88%): mp 78±80^ꢁC; ¹H NMR (DMSO-

d₆) d 7.64 (d, J=5.4 Hz), 7.40 (d, J=5.4 Hz), 6.61 (s,

1H), 5.61 (t, J=4.8 Hz, 1H), 4.35 (d, J=4.8 Hz, 2H),

3.34 (s, 3H).

NMR (DMSO-d₆) d 7.76 (s, 1H), 7.44 (d, J=5.1 Hz,

1H), 7.19 (d, J=5.1 Hz, 1H), 6.07 (s, 1H), 3.96 (m, 4H),

1.13 (s, 9H). Sulfur dioxide was passed over the reaction

mixture for 5 min followed by warming to room tem-

perature and evaporation. The residue was suspended in

CH₂Cl₂(150 mL), cooled on an ice bath, and N-chloro-

succinimide (4.06 g, 30.4 mmol) was added. After stir-

ring the mixture for 1 h at ambient temperature,

sarcosine ethyl ester HCl salt (7.01 g, 45.6 mmol) and

5% NaHCO₃(100 mL) were added and the mixture stir-

red for 3 h. The organic phase was separated, dried, and

evaporated to a residue which was puri®ed by chroma-

tography (30% EtOAc in hexane) to give a gum (5.07 g,

65%); ¹H NMR (DMSO-d₆) d 8.01 (s, 1H), 7.69 (s, 1H),

6.41 (s, 1H), 4.15 (t, J=7.0 Hz, 2H), 4.06 (m, 6H), 2.90

(s, 3H), 1.19 (s, 9H), 1.15 (t, J=7.0 Hz, 3H). A portion

(1.00 g, 2.13 mmol) of this intermediate was dissolved in

tri¯uoroacetic acid (5 mL) and stirred at room tem-

perature for 18 h. Evaporation of the reaction mixture

provided a residue which was suspended in 5% aqueous

NaHCO₃(150 mL) and extracted with EtOAc

(2Â100 mL). The combined extracts were dried and

evaporated to a residue which was puri®ed by chroma-

tography (40% EtOAc in hexane) to give a viscous oil

which solidi®ed upon standing (0.35 g, 44%): mp 105±

3-Hydroxymethyl-2-methyl-2H-thieno[2,3-e]-1,2-thiazine-

6-sulfonamide 1,1-dioxide (24). A solution of 23 (1.00 g,

4,33 mmol) in anhydrous THF (30 mL) was treated as

described for the preparation of 15a to give, after pur-

i®cation by chromatography (50% to 80% EtOAc in

1

hexane), a viscous oil (0.80 g, 60%): H NMR (DMSO-

d₆) d 7.90 (s, 2H), 7.72 (s, 1H), 6.73 (s, 1H), 5.73 (t,

J=4.7 Hz, 1H), 4.39 (d, J=4.7 Hz, 2H), 3.36 (s, 3H).

ꢁ

1

2-Methyl-3-(4-morpholinylmethyl)-2H-thieno[2,3-e]-1,2-

thiazine-6-sulfonamide 1,1-dioxide (25). To a solution of

107 C; H NMR (DMSO-d₆) d 10.29 (s, 1H), 8.18 (s,

2H), 7.84 (s, 1H), 4.25 (s, 2H), 4.06 (q, J=7.2 Hz, 2H),

974

H.-H. Chen et al. / Bioorg. Med. Chem. 8 (2000) 957±975

2.95 (s, 3H), 1.38 (t, J=7.2 Hz, 3H); HRMS (FAB) m/z

for C₁₀H₁₄N₂O₇S₃: Calcd: 371.0036. Found: 371.0041.

In vitro inhibition of rhCA-IV. Inhibition constants were

determined using the same pH stat assay procedure

described above, but using rhCA-IV (0.26 E.U./mL)

prepared as described previously.^19,20

Aqueous solubility determination. The compound of

interest was added to 0.1 M phosphate buer at pH 7.4

and the pH was adjusted as desired. Samples containing

an excess of undissolved material were stirred at ambi-

ent temperature for a minimum of 18 h. Sample pH was

adjusted as required and the mixture stirred an addi-

tional 15 min and ®ltered through a 0.45 micron nylon

®lter. The ®ltrate was analyzed by RP-HPLC against

concentration standards for the compound of interest.

In vitro binding of inhibitor to hCA-II. Inhibitor binding

to hCA-II was determined using a ¯uorescence compe-

tition assay.^12,13Displacement of dansylamide from

hCA-II was determined at 37 ^ꢁC in cuvettes (3 mL)

8

containing 1Â10 M enzyme (Sigma Chemical Co.),

5

5Â10 M dansylamide, and 100 mM disodium hydro-

gen phosphate at pH 7.4. At time zero the test or con-

trol articles were added in a dose volume of 30 mL and

the mixture was incubated at 37 ^ꢁC for 3 min; the

¯uorescence intensity for each concentration was the

average of triplicate determinations. Fluorescence mea-

surements were made with a Perkin±Elmer LS-50B

luminescence spectrometer using a thermostatically

controlled cell maintained at 37 ^ꢁC with excitation and

emission wavelengths of 280 and 460 nm, respectively.

Relative binding of the inhibitor was determined by

measuring and comparing the average ¯uorescence

intensities of the treated versus control reactions. Bind-

ing constants were estimated from a Dixon plot of the

data (reciprocal of the average relative ¯uorescence

intensity versus inhibitor concentration), for each con-

centration. Reported binding constants are generally the

mean of at least duplicate determinations.

Distribution coecients. Partitioning of compounds

between n-octanol and aqueous buer was determined

at pH 5.0 and 7.4 using 0.1 M phosphate buers. The

initial concentration (C₁) of compound in buer and the

buer concentration following extraction with n-octanol

(C₂) were determined by RP-HPLC analysis against

concentration standards for the speci®c compound.

The distribution coecient (DC) of a compound at a

given pH was calculated using the equation DC_pH

(C₁C₂)/C₂.

=

pK_adetermination. Ionization constants were deter-

mined by potentiometric titration (Kyoto AT-310

Potentiometric Titrator) in water or a mixture of water

and an organic solvent such as methanol, acetone, or

acetonitrile. If a solvent mixture was used, the nominal

pK_avalues were plotted against the percentage of

organic solvent to provide by extrapolation the pK_aof

the compound in water.

Acute intraocular pressure determination. Intraocular

pressure was determined with an applanation pneuma-

tonometer after light corneal anesthesia with 0.1% pro-

paracaine. Following an IOP measurement, residual

anesthetic was washed out of the eyes with saline. After

two subsequent baseline IOP measurements were recor-

ded, one eye of each of the seven to ten Dutch-belted

rabbits in the test group was topically dosed with two 25

mL aliquots of compound (1 mg total dose) and the

contralateral eye was dosed with vehicle. Subsequent

IOP measurements were taken at 0.5, 1, 2, 3, and 4 h.

Compounds were formulated as 2% suspensions, with

the exception of 17b4 and 17h2, which were 2% solu-

tions, in acetate buered mannitol vehicle containing

0.01% benzalkonium chloride, 0.01% disodium EDTA,

0.1% polysorbate 80, 0.8% hydroxypropylmethyl-

cellulose, and adjusted to pH 5.0.

Biological assays

In vitro inhibition of hCA-II. Inhibition of hCA-II

activity was determined using a pH stat assay similar to

that previously described^11,12wherein enzyme activity

is proportional to the volume of an NaOH solution

required to maintain the pH of the reaction mixture at a

predetermined value. A Radiometer VIT 90 Video

titration system jacketed glass reaction vessel was

maintained at 4 ^ꢁC with a circulating water bath. The

assay measured the rate of CO₂hydration by determin-

ing the addition rate of a 0.15 N NaOH solution to the

reaction vessel containing 0.02 M HEPES buer (5 mL),

pH 7.2, such that this value was maintained over a period

of 4 min as CO₂(approximately 13% CO₂in helium)

was bubbled into the buer as a stream of premixed CO₂

(5 mL/min) and helium (35 mL/min). The rate of addi-

tion of NaOH to the buer in the absence of hCA-II

was de®ned as blank. The rate (slope) increase following

the introduction of 10 L of hCA-II (Sigma Chemical

Co.) to the buer (0.35 E.U./mL) re¯ected the control

level of hCA-II activity. Enzyme inhibition was deter-

mined by addition to the enzyme buer mixture of var-

ious aliquots (5 to 100 mL) of a suitable concentration of

inhibitor dissolved in a mixture of ethanol and water

(1:1, volume) followed by stirring for 4 min prior to the

introduction of substrate. IC₅₀, the inhibitor concentra-

tion resulting in 50% inhibition of the enzyme activity,

was obtained from the plot of the net rate (slope) increase

of NaOH addition against log inhibitor volume.

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Article Doi

2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides as ocular hypotensive agents: Synthesis, carbonic anhydrase inhibition and evaluation in the rabbit

DOI: 10.1016/S0968-0896(00)00026-2

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Full text of DOI:10.1016/S0968-0896(00)00026-2

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