Design, synthesis, and biological evaluation of potent thiazine- and thiazepine-based matrix metalloproteinase inhibitors

J . Med. Chem. 1999, 42, 4547-4562

4547

Design , Syn th esis, a n d Biologica l Eva lu a tion of P oten t Th ia zin e- a n d

Th ia zep in e-Ba sed Ma tr ix Meta llop r otein a se In h ibitor s

Neil G. Almstead,* Rimma S. Bradley, Stanislaw Pikul, Biswanath De, Michael G. Natchus, Yetunde O. Taiwo,

Fei Gu, Lisa E. Williams, Barbara A. Hynd, Michael J . J anusz, C. Michelle Dunaway, and Glen E. Mieling

Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040

Received J une 25, 1999

The synthesis and enzyme inhibition data for a series of thiazine- and thiazepine-based matrix

metalloproteinase (MMP) inhibitors are described. The thiazine- and thiazepine-based inhibitors

were discovered by optimization of hetererocyclic sulfonamide-based inhibitors. The most potent

series of inhibitors was obtained by modification of the amino acid ^D-penicillamine. This amino

acid provides a gem-dimethyl group on the thiazine or thiazepine ring which has a dramatic

effect on the in vitro potency of this series. In particular, the sulfide 4a and the sulfone 5a

were potent, broad-spectrum inhibitors of the MMPs with IC₅₀’s against MMP-1 of 0.8 and 1.9

nM, respectively. The binding mode of this novel thiazepine-based series of MMP inhibitors

was established based on X-ray crystallography of the complex of stromelysin and 4a .

Ch a r t 1. Selected MMP Inhibitors

In tr od u ction

The matrix metalloproteinases (MMPs) are a struc-

turally related class of enzymes that are responsible for

the metabolism of the extracellular matrix proteins.^1,2

Members of this family, which include the collagenases,

stromelysins, and gelatinases, are involved in the

normal tissue remodeling processes such as wound

healing, angiogenesis, and pregnancy. In these physi-

ological processes the MMP activity is tightly regulated.

The aberrant expression of these zinc- and calcium-

dependent enzymes has been linked to the accelerated

breakdown of connective tissues associated with patho-

logical disease states including arthritis,³tumor inva-

sion and metastasis,^4-7periodontal disease,⁸and mul-

tiple sclerosis.^9-11The MMPs and the inhibition of these

enzymes have therefore become attractive targets for

structure-based drug design.^12-15

At the time we began to work in the field, few articles

or patents had been published which described the

design and synthesis of cyclic, sulfonamide-based in-

hibitors. Previous work in the field had primarily been

focused on the development of acylic based MMP inhibi-

tors (marimastat,¹⁵Ro 31-9790,¹⁶and CGS-27023A¹⁷).

Our interest was in the design of molecules which

possess some conformational rigidity,¹⁸possibly leading

to increased in vitro potency and enhanced pharmaco-

logical properties. This structural rigidity could be

introduced by the addition of a linker group (or ring)

between the sulfonamide nitrogen atom and the C-2

carbon on the amino acid. An investigation of hetero-

cyclic based sulfonamides was therefore undertaken

with the goal to discover potent cyclic small molecule

inhibitors of the MMPs. Recently, articles have been

published which describe the design, synthesis, and SAR

of heterocyclic inhibitors.¹⁹In addition, Agouron Phar-

maceuticals is developing the thiazine analogue AG-

3340 (Chart 1) as a disease-modifying agent for the

treatment of cancer.²⁰This manuscript provides an

account of the discovery of a novel series of MMP

inhibitors and describes the synthesis, initial structure-

activity relationship (SAR) studies, and in vitro activity

of this series of compounds.

Ch em istr y

Synthesis of the thiazine- and thiazepine-based in-

hibitors was accomplished using one of two methods.

In the first method, the methyl ester of D-penicillamine

was allowed to react with 3-bromopropanol or 2-bromo-

ethanol in the presence of DBU in DMF (Scheme 1). The

resulting sulfide amino ester was then treated with the

appropriate sulfonyl chloride and triethylamine to afford

the desired sulfonamide 1 or 6. Ring closure of the

sulfonamide intermediate to produce the thiazepine 2

or thiazine 7 was accomplished under Mitsunobu²¹

conditions with diethyl azodicarboxylate and triphenyl-

phosphine. Hydrolysis of the methyl ester with lithium

iodide/pyridine provided the carboxylic acids 3 and 8.²²

The carboxylic acid was then treated with oxalyl chlo-

ride and DMF to generate the acid chloride in situ which

was then treated with hydroxylamine to afford the

hydroxamic acids 4 and 9. The corresponding sulfones

Published on Web 10/15/1999

4548 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Sch em e 1. Procedure A^a

Almstead et al.

Sch em e 3^a

a

Reagents and conditions: (a) 1. oxalyl chloride, DMF, CH₂Cl₂,

2. MeNHOH, water, THF; (b) 1. oxalyl chloride, DMF, CH₂Cl₂, 2.

NH₂OMe, water, THF.

Sch em e 4^a

a

Reagents and conditions: (a) 1,8-diazabicyclo[5.4.0]undec-7-

ene (2 equiv), Br(CH₂)₃OH (n ) 1) or Br(CH₂)₂OH (n ) 0), DMF,

0 °C; (b) ArSO₂Cl, Et₃N, 1,4-dioxane, water; (c) diethyl azodicar-

boxylate, Ph₃P, THF; (d) LiI, pyridine, reflux; (e) 1. oxalyl chloride,

DMF, CH₂Cl₂, 2. NH₂OH, water, THF; (f) CH₃CO₃H, CHCl₃.

Sch em e 2. Procedure B^a

a

Reagents and conditions: (a) NaN₃, MeSO₃H; (b) LiAlH₄; (c)

4-MeOPhSO₂Cl, Et₃N, dioxane, water; (d) J ones reagent, acetone;

(e) 1. oxalyl chloride, DMF, CH₂Cl₂, 2. NH₂OH, water, THF.

The carboxylic acids could also be converted to the

corresponding N-methyl- and O-methylhydroxamide

derivatives. The carboxylic acid 3 was treated with

oxalyl chloride to generate the acid chloride intermedi-

ate which was subsequently treated with either N-

methyl- or O-methylhydroxylamine to produce 12 or 13,

respectively (Scheme 3).

a

Reagents and conditions: (a) 1,8-diazabicyclo[5.4.0]undec-7-

ene (2 equiv), DMF, 0 °C; (b) 1,8-diazabicyclo[5.4.0]undec-7-ene

(1 equiv), DMF, 80 °C; (c) ArSO₂Cl, 4-methylmorpholine, CHCl₃,

reflux.

5 and 10 could be readily prepared by treatment of the

hydroxamic acid with peracetic acid in chloroform. The

hydroxamic acids were obtained as crystalline com-

pounds which could be purified by recrystallization from

the appropriate solvent.

The azepine 17 was synthesized in racemic form as

shown in Scheme 4. This compound was prepared to

assess the importance of the sulfur atom in the thi-

azepine ring for enzyme inhibition. Ring expansion via

Schmidt rearrangement of an appropriately substituted

â-keto ester appeared to be the most appealing route to

this molecule. The Schmidt rearrangement was carried

out with the keto ester 14²³by treatment with meth-

anesulfonic acid followed by sodium azide to afford

amide 15.²⁴The amide was reduced with lithium

aluminum hydride to afford the amino alcohol which

was subsequently treated with 4-methoxyphenylsulfonyl

chloride and triethylamine to generate sulfonamide 16.

Oxidation of the primary alcohol with the J ones reagent

provided the carboxylic acid which could be transformed

to the desired hydroxamic acid 17 by treatment with

oxalyl chloride followed by the addition of hydroxyl-

amine. This methodology was also used to prepare the

corresponding azepine 21.

In the second method, the thiazepine ring was pre-

pared in one step from the methyl ester of D-penicill-

amine and 3-bromo-1-chloropropane (Scheme 2). The

penicillamine methyl ester was allowed to react with

the alkyl bromide in the presence of DBU to produce

the intermediate chloropropyl sulfide. The addition of

1 more equiv of DBU and then heating the mixture

afforded the thiazepine 11. The thiazepine methyl ester

was then treated with the corresponding sulfonyl chlo-

ride and N-methylmorpholine to produce the desired

sulfonamide. This method provided the desired sulfon-

amides in high yield, although the reaction of the

corresponding sulfonyl chloride with the secondary

amine was extremely slow. The remaining transforma-

tions leading to the hydroxamic acids were performed

in a manner analogous to that described in Scheme 1.

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4549

Sch em e 5^a

were synthesized by a modification of procedure A

(Scheme 1) from the corresponding amino acid deriva-

tive and (R)- or (S)-3-chloro-1-phenyl-1-propanol.

Resu lts a n d Discu ssion

Develop m en t of th e Heter ocylic Ba sed MMP

In h ibitor s. 1. Effect of Rin g Size a n d Rin g Heter o-

a tom . All compounds were tested in vitro for the

inhibition of truncated collagenase-1 (MMP-1),²⁶stro-

melysin (MMP-3),²⁷matrilysin (MMP-7),²⁶and colla-

genase-3 (MMP-13).²⁶Selected molecules have also been

tested for inhibition of gelatinase-A (MMP-2),²⁶neutro-

phil collagenase (MMP-8),²⁶gelatinase-B (MMP-9),²⁶

and MT-MMP-1.²⁸Initially, the effect of both ring size

and presence of a ring heteroatom on enzyme inhibition

was examined (Table 1). The inhibitors based on either

the six- or seven-membered rings were quite potent

against collagenase-1 (MMP-1) and stromelysin-1 (MMP-

3). The corresponding five-membered proline- and cys-

teine-based inhibitors were determined to be almost 1

order of magnitude less potent than the larger rings.

Because of the lower potency observed with the five-

membered heterocycles, we chose to concentrate our

efforts on the six- and seven-membered heterocycles.

The presence of a ring heteroatom in both the six- and

seven-membered series did not significantly influence

the potency or selectivity of the inhibitor (compare

compounds 39-41). In the six-membered ring series,

substitution of the ring carbon with either an oxygen

or sulfur atom had only a negligible effect on enzyme

inhibition. The piperazine-based inhibitor (compound

43, substitution with -NH) was determined to be

somewhat less potent, although it has been observed

that further substitution of the ring nitrogen in the

piperazine series can lead to a significant increase in

potency.²⁹The results obtained with the sulfide 41 and

the sulfone 42 were somewhat surprising. Oxidation of

the sulfur atom in compound 41 to the corresponding

sulfone 42 led to a significant decrease in potency.

Further examination of the sulfur-based inhibitors

was initiated because of the ease of synthesis of these

compounds and the ready availability of starting ma-

terials in the correct D-configuration for this series.³⁰

2. Effect of C-2 gem -Dim eth yl Gr ou p on in Vitr o

In h ibition . When we began to work in the area of

heterocyclic MMP inhibitors, we proposed that a gem-

dimethyl group, when introduced adjacent to the hy-

droxamic acid functionality, may reduce the potential

for metabolic degradation of the hydroxamic acid. It was

hypothesized that steric shielding of the hydroxamic

acid group could prevent or hinder this metabolic

pathway. An examination of the effect a C-2 gem-

dimethyl group might have on in vitro potency was

undertaken. After installation of the gem-dimethyl

group on the heterocyclic inhibitors, a dramatic increase

in potency was observed in both the thiazine (six-

membered ring) and thiazepine (seven-membered ring)

series (compare values in Table 2 versus Table 1). The

thiazine-based inhibitors containing the gem-dimethyl

group were extremely potent inhibitors of both MMP-1

and MMP-3. Almost a 2 orders of magnitude increase

in potency was observed against MMP-1 with the

addition of the gem-dimethyl group (compare compound

41, Table 1, with compound 9, Table 2). A significant

a

Reagents and conditions: (a) 1,8-diazabicyclo[5.4.0]undec-7-

ene (2 equiv), Cl(CH₂)₅OH (for 25) or Cl(CH₂)₂O(CH₂)₂OH (for 26),

DMF, 0 °C; (b) 4-CH₃OPhSO₂Cl, Et₃N, 1,4-dioxane, water; (c)

diethyl azodicarboxylate, Ph₃P, THF; (d) LiI, pyridine, reflux; (e)

1. oxalyl chloride, DMF, CH₂Cl₂, 2. NH₂OH, water, THF; (f)

CH₃CO₃H, CHCl₃.

Large sulfur-containing heterocycles were prepared

including the thiazonine 22 and the oxathiazonine 23

as shown in Scheme 5. The D-penicillamine methyl ester

was treated with DBU and the corresponding alkyl

chloride to provide the intermediate sulfide which was

subsequently converted to the 4-methoxyphenylsulfon-

amide (25 or 26) by treatment with 4-methoxyphenyl-

sulfonyl chloride and triethylamine. Mitsunobu cycliza-

tion produced the thiazonine (27 or 28) in moderate

yield. The methyl ester was removed with lithium iodide

in refluxing pyridine to give the carboxylic acid. Treat-

ment of the carboxylic acid with oxalyl chloride followed

by the addition of hydroxylamine yielded the desired

hydroxamic acid (22 or 23). Oxidation of the oxazathi-

azonine to the corresponding sulfone 24 was accom-

plished by treatment with peracetic acid.

Hydroxyl- and alkoxyl-substituted thiazepines were

synthesized as shown in Scheme 6. The synthesis begins

with the addition of (S)-4-(bromomethyl)-2,2-dimethyl-

1,3-dioxolane²⁵to D-penicillamine in the presence of

dilute sodium hydroxide. Addition of 4-methoxyphenyl-

sulfonyl chloride to the amino acid followed by treat-

ment of the acid with ethereal diazomethane generated

the methyl ester 29. Conversion of 29 to the correspond-

ing diol 30 was achieved by hydrolysis with dilute HCl.

Differential protection of the diol was accomplished by

the addition of t-BuMe₂SiCl and triethylamine to pro-

duce the silyl ether. The secondary alcohol was then

protected with MOMCl to give 31. Removal of the silyl

ether with fluoride provided primary alcohol 32 which

was subjected to Mitsunobu cyclization conditions to

give the thiazepine 33. The methyl ester was hydrolyzed

with LiI in pyridine, and the resulting carboxylic acid

was treated with oxalyl chloride followed by hydroxyl-

amine to afford the desired hydroxamic acid 34. Oxida-

tion of the sulfide to the sulfone and concomitant

removal of the MOM group was accomplished by treat-

ment with peracetic acid to generate 35.

The thiazepine-based hydroxamic acids 36a and 36b

containing an aryl substituent on the thiazepine ring

4550 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

Sch em e 6^a

a

Reagents and conditions: (a) 2 N NaOH, EtOH; (b) 4-MeOPhSO₂Cl, Et₃N, 1,4-dioxane, water; (c) CH₂N₂, Et₂O; (d) 1 N HCl, THF; (e)

TBDMSCl, Et₃N, CH₂Cl₂; (f) NaI, MOMCl, i-Pr₂EtN, DME; (g) n-Bu₄NF, THF; (h) diethyl azodicarboxylate, Ph₃P, THF; (i) LiI, pyridine,

reflux; (j) 1. oxalyl chloride, DMF, CH₂Cl₂, 2. NH₂OH, water, THF; (k) CH₃CO₃H, CHCl₃.

Ta ble 1. Effect of Ring Size and Ring Heteroatom on MMP

gem-dimethyl group has a dramatic effect on the potency

Inhibition

of this series.

The thiazepines containing the C-2 gem-dimethyl

group were determined to be as potent inhibitors of both

MMP-1 and MMP-3 as the corresponding thiazine-based

compounds. The major disparity between the thiazine-

and thiazepine-based inhibitors was observed upon

oxidation of the ring sulfur to the corresponding sulfone.

Oxidation of the ring sulfur in the thiazepine series did

not result in any significant decrease in potency (com-

pare thiazines 9 and 10 with thiazepines 4a and 5a ).

This result was especially gratifying, as metabolic

oxidation of a sulfide to the corresponding sulfoxide and

sulfone is a well-known process.³¹It was also deter-

mined that the sulfone analogues were significantly

more water soluble than the corresponding sulfide

analogues (compound 4a , 0.1 mg/mL, and compound 5a ,

0.8 mg/mL, at pH 7.4). We chose to concentrate our

studies on further elaboration of the thiazepine (seven-

membered ring)-based inhibitors which retain almost

all potency even when the ring sulfur has been oxidized.

The corresponding azepine analogue 17 was also

tested for in vitro inhibition. The in vitro profile of

compound 17 was almost identical to the profile ob-

served with the thiazepine 4a . Apparently the sulfur

atom does not significantly influence the potency of the

molecule even in the gem-dimethyl series.

The nine-membered thiazonine 22 was prepared to

determine if the enzyme active site could accommodate

the large rings. This compound was determined to be

as potent an inhibitor as the corresponding thiazepine

a

See Experimental Section for details of the enzyme assays.

nd, not determined.

4a .

increase (at least 1 order of magnitude) in potency was

also observed against MMP-3 and MMP-7 when the

gem-dimethyl group was introduced. Interestingly, the

influence of the gem-dimethyl group on the inhibition

of MMP-13 was much less significant. The effect on in

vitro inhibition was also observed in the sulfone series

(compare compound 42, Table 1, with compound 10,

Table 2). The sulfone 10 with the gem-dimethyl group

was still an extremely potent inhibitor of MMP-1 and

MMP-3, although it was significantly less potent than

the parent thiazine 9. It is readily apparent that the

The enantiomer of 4a , R-4a , was prepared from

L-penicillamine and tested for in vitro activity in order

to examine the importance of amino acid configuration

on enzyme inhibition. This compound (R-4a ) was a very

poor inhibitor of the MMPs, with potencies 3-4 orders

of magnitude lower than that of the corresponding

enantiomer 4a (vide infra).

Str u ctu r e of Str om elysin -In h ibitor Com p lex.³²

To better understand the binding interactions between

the thiazepine based hydroxamic acids and the MMP

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4551

Ta ble 2. Effect of gem-Dimethyl Group and Ring Size on MMP Inhibition

a

See Experimental Section for details of the enzyme assays. nd, not determined. ^bK_ivalues reported for AG3340 are given in parentheses.

^cK_ivalues reported for CGS27023A are given in parentheses. R-Enantiomer of 4a .

d

F igu r e 1. Binding interactions between 4a and stromelysin.

enzymes, we obtained a crystal of the truncated stro-

melysin-4a complex and solved its structure using the

molecular replacement method. The catalytic site of

stromelysin (MMP-3) with the bound inhibitor is shown

in Figure 1. In general, interactions of the hydroxamic

acid with stromelysin were consistent with those al-

ready reported in the literature.^18,33-35The hydroxamic

acid acted as a bidentate ligand with the two oxygen

atoms in binding distance of 2.0 and 2.6 Å from the

active site Zn²⁺ion. The terminal OH oxygen and the

nitrogen atom of the hydroxamic acid formed additional

hydrogen bonds with the oxygen Oꢀ2 (2.4 Å) of the

carbonyl group of Glu-202 and the carbonyl group (2.7

Å) of Ala-165, respectively. The methoxyphenylsulfon-

amide group is clearly directed toward the S1′ site, and

a hydrogen bond between Leu-164 and the sulfonamide

oxygen may develop.

F igu r e 2. Structure of stromelysin-4a complex.

The most interesting feature of the enzyme-inhibitor

interactions was revealed by inspection of the thiazepine

ring. The thiazepine was oriented in a pseudo-chair

conformation with the ring sulfur and one of the methyl

groups directed toward Val-163. It is clear from inspec-

tion of the crystal structure that near the S2′ site, the

inhibitor-binding cleft is large and relatively open. This

cleft can accommodate P2′ groups of various sizes, which

may explain the flat SAR observed after modification

of the ring size and adding substituents to the thiaze-

pine ring. Some of the side chains at this site vary

among the different MMPs, but Pro-221 which is closest

to the P2′ group is conserved among all the MMPs. The

4552 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Ta ble 3. In Vitro Profile of Thiazepine MMP Inhibitors

Almstead et al.

a

See Experimental Section for details of the enzyme assays. nd, not determined.

thiazepine ring reaches close to the Pro-221 ring struc-

ture in the observed crystal structure. The stronger

hydrophobic interaction that results probably contrib-

utes to increased potency with this series, and this

interaction with Pro-221 is conserved for all MMPs. The

high potency observed with the thiazepine series may

also be influenced by a second strong hydrophobic

interaction between one of the methyl groups and Val-

163.

The significant reduction in potency observed with

compounds derived from L-penicillamine (see compound

R-4a , Table 2) was quite surprising and deserves further

comment. The inhibition of the MMPs with acyclic

L-amino acid-derived sulfonamides has previously been

described by Ciba (Novartis).¹⁷These inhibitors were

observed to be approximately 2 orders of magnitude less

potent than the corresponding compounds derived from

D-amino acids. When the thiazepine-based inhibitors

were prepared from L-penicillamine, almost a 4 orders

of magnitude decrease in potency was observed. The

lack of potency observed with the L-penicillamine-

derived analogues is likely due to poor interactions

between the hydroxamic acid group and the active site

zinc atom. The thiazepines which possess the L-config-

uration at the R-carbon would not be well-accom-

modated in the active site (see Figure 2). Due to these

unfavorable interactions, the pseudo-chair structure

observed in Figure 2 is much more difficult to adopt with

the L-configured thiazepines, thus requiring significant

reorganization of the enzyme and substrate for maximal

inhibition to occur. The influence of configuration on

inhibitor potency may be less significant with the acyclic

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4553

Ta ble 4. In Vitro Profile of Thiazepine MMP Inhibitors

a

See Experimental Section for details of the enzyme assays. nd, not determined.

sulfonamides due to the conformational flexibility that

these compounds already possess.

MMPs, but again, these compounds did not show any

selectivity for MMP-3 over MMP-1. The aryl ethers 4h

and 4j, the heterocyclic sulfonamide 4i, and the bi-

phenylsulfonamide 4k were all broad-spectrum inhibi-

tors and demonstrated extremely potent inhibition

against MMP-13. The unexpectedly high potency of

inhibitors with large P1′ groups toward MMP-1 has also

been observed by Agouron Pharmaceuticals with the

thiazine-based inhibitor AG3340.³⁷

Based on the results obtained with the thiazepines,

it is apparent that the addition of the gem-dimethyl

group to the ring produces a potent, broad-spectrum

inhibitor which is not significantly influenced by the P1′

(sulfonamide) region of the molecule. The addition of

longer or bulkier substituents on the aryl ring (P1′

group) which are not usually tolerated by the shallow

pocket metalloproteinases (MMP-1 and MMP-7) only

had a moderate effect on enzyme selectivity with the

thiazepine series.

SAR of th e Th ia zep in e Rin g. Modifications were

made to the thiazepine ring to determine if further

substitution of the thiazepine ring or a change in the

ring size would alter the enzyme inhibition profile

(Table 4). The first analogues tested (34 and 35) contain

either a hydroxyl group or a methoxymethyl ether at

C-6. Both of these compounds were potent broad-

spectrum inhibitors and exhibited almost an identical

inhibition profile to the parent compound 4a . The nine-

membered oxathiazonines 23 and 24 were also ex-

tremely potent MMP inhibitors. The effect of ring size

Effect of Su lfon a m id e Gr ou p on in Vitr o In h ibi-

tion . The sulfonamide portion of the molecule and the

influence of this group on enzyme inhibition were also

examined (Table 3). The p-methoxyphenylsulfonamide

4a , one of the first compounds we examined, was

determined to be an extremely potent and broad-

spectrum inhibitor. The corresponding sulfone analogue,

5a , was also prepared and tested for in vitro activity.

This compound possessed a broad spectrum of activity

against all of the MMPs we tested including matrilysin

(MMP-7) and MT1-MMP³⁶(MMP-14, IC₅₀2.2 nM).

Replacement of the p-methoxy group with a p-bromo

substituent led to a significant shift in enzyme selectiv-

ity. Both the sulfide 4b and the sulfone 5b showed

increased selectivity for MMP-1 over MMP-3. The

o-methyl-p-bromophenylsulfonamide 4c was found to be

even more selective for MMP-1. Clearly, the bromo-

phenylsulfonamides are well-tolerated by MMP-1, but

the decrease in potency for MMP-3 remains hard to

rationalize.

The results obtained with the p-butoxy substituent

were somewhat surprising. On the basis of previous

observations, compounds which possess a long P1′

substituent were expected to be more potent for MMP-3

while sparing MMP-1.³⁵Instead, a slight decrease in

potency was observed for both MMP-1 and MMP-3 with

compounds 4d and 5d . The alkylated-phenylsulfon-

amides 4e and 4f were also potent inhibitors of the

4554 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

by preparative reverse-phase high-pressure liquid chromatog-

raphy (RP-HPLC) using a Waters model 4000 Delta Prep

instrument equipped with a Waters Symmetry preparative

steel column (C-18, 19 mm × 300 mm) as the stationary phase.

The mobile phase employed 0.1% aqueous trifluoroacetic acid

with acetonitrile as the organic modifier. Linear gradients were

used in all cases, and the flow rate was 20 mL/min. Analytical

purity was assessed by RP-HPLC using a Waters 600 system

equipped with a diode array spectrometer (λ range 200-400

nm). The stationary phase was a Waters Symmetry C-18

column (4.6 mm × 200 mm). The mobile phase employed 0.1%

aqueous trifluoroacetic acid with acetonitrile as the organic

modifier and a flow rate of 1.0 mL/min.

¹H NMR spectra were recorded on a Varian Unity-300

instrument. Chemical shifts are reported in parts per million

(ppm, δ units). Coupling constants are reported in units of

hertz (Hz). Splitting patterns are designated as s, singlet; d,

doublet; t, triplet; q, quartet; m, multiplet; br, broad. Low-

resolution mass spectra (MS) were recorded on a Micromass

Platform quadrupole mass spectrometer. Mass spectra were

acquired in either the positive or negative ion mode under

electrospray ionization (ESI). Combustion analyses were per-

formed internally.

Human synovial proMMP-3 was obtained from Dr. Hideaki

Nagase, University of Kansas Medical Center, Kansas City,

KS. Human fibroblast proMMP-1, human MMP-9, and human

recombinant MMP-7 catalytic domain were obtained from Dr.

Howard Welgus, J ewish Hospital, St. Louis, MO. Human

recombinant MMP-8 catalytic domain was obtained from Dr.

Harald Tschesche, University Bielefeld, Bielefeld, Germany.

Human recombinant proMMP-2 was purified from CHO cells

as described below. Human recombinant truncated MMP-3

and truncated MMP-1 were purified from E. coli cells as

described below. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂was

purchased from Bachem Bioscience, King of Prussia, PA.

Phenoxyphenylsulfonyl chloride³⁸and (pyrid-4-yl)oxyphenyl-

sulfonyl chloride³⁹were prepared following literature proce-

dures.

P r oced u r e A for F or m a tion of Th ia zep in es 4a -4h .

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2-h yd r oxyp r o-

p yl)-^D-p en icilla m in e (1a ). A solution of 2-bromopropanol

(3.50 g, 24.1 mmol) in DMF (40 mL) was stirred at room

temperature, and then a solution of ^D-penicillamine methyl

ester hydrochloride (5.0 g, 25.1 mmol), 1,8-diazabicyclo[5.4.0]-

undec-7-ene (7.64 g, 50.2 mmol, 2 equiv), and DMF (40 mL)

was slowly added by dropwise addition over a 30 min period.

The resulting mixture was stirred at room temperature

overnight. The solvent was then removed to leave a thick oil.

The oil was dissolved in dioxane (75 mL) and water (75 mL)

and then triethylamine (6.32 g, 62.5 mmol, 2.5 equiv) followed

by 4-methoxyphenylsulfonyl chloride (5.42 g, 26.3 mmol, 1.05

equiv) were added at room temperature. The resulting mixture

was stirred for 18 h at room temperature and then acidified

with 1 N HCl. The mixture was extracted with methylene

chloride (2 × 250 mL) and the organic extracts were dried

(MgSO₄) and then concentrated to an oil. Purification of the

resulting methyl ester was accomplished by chromatography

on silica gel using 1/1 hexane/EtOAc as the eluent. The desired

product (8.0 g, 82%) was obtained as a clear, colorless oil: ¹H

NMR (CDCl₃) δ 7.71 (d, J ) 8.6, 2 H), 6.88 (d, J ) 8.6, 2 H),

5.62 (d, J ) 11, 1 H), 3.82 (s, 3 H), 3.78 (d, J ) 11, 1 H), 3.66

(m, 2 H), 3.40 (s, 3 H), 2.59 (br m, 2 H), 2.05 (br s, 1 H), 1.64

(m, 2 H), 1.36 (s, 3 H), 1.34 (s, 3 H); ¹³C NMR (CDCl₃) δ 170.13,

162.86, 130.69, 129.30, 113.89, 62.91, 60.81, 55.44, 51.91,

46.54, 31.31, 25.73, 24.40.

and the inclusion of another ring heteroatom had only

a minimal effect on the enzyme inhibition profile. The

introduction of a C-5 phenyl substituent resulted in

significantly lower potency for MMP-1 and MMP-3. The

â-phenyl compound 36b was more potent than the

corresponding R-phenyl inhibitor 36a for MMP-13.

Finally, the results obtained from in vitro testing of the

methylcysteine analogue 45 indicated that introduction

of a C-3 methyl group only had a modest effect on the

inhibitory profile (compare compound 44, Table 1, with

compound 45, Table 4).

Im p or ta n ce of th e Hyd r oxa m ic Acid Moiety for

in Vitr o Activity. Modifications were also made to the

hydroxamic acid portion of the molecule in order to

determine if either a carboxylic acid or a substituted

hydroxamic acid derivative would retain any in vitro

activity. The corresponding carboxylic acids 3a and 3h

were found to be inactive against all of the MMPs (IC₅₀’s

> 10 µM). The N-methylhydroxamic acids 12a and 12h

and the N-methoxyhydroxamic acid derivative 13h were

also tested for in vitro inhibition. The N-methylhydrox-

amic acids were more than 3 orders of magnitude less

potent than the parent hydroxamic acid. The N-meth-

oxyhydroxamic acid 13h was also completely inactive

against the MMPs. These results clearly demonstrate

the importance of the hydroxamic acid group in the

thiazepine series for in vitro inhibition. Any modification

of this group leads to a dramatic and almost complete

loss of activity.

Con clu sion

A potent series of thiazepine-based hydroxamic acid

metalloproteinase inhibitors was discovered and opti-

mized. The compounds which possess a gem-dimethyl

group at C-2 were determined to be at least 1 order of

magnitude more potent against MMP-1 (collagenase-1)

than the corresponding dihydro analogues. A significant

boost in potency was also observed against MMP-3

(stromelysin-1) when the gem-dimethyl group was added.

The most potent inhibitors were readily prepared from

the amino acid D-penicillamine which contains the

requisite gem-dimethyl group. The compounds demon-

strated broad-spectrum inhibition of the MMPs ir-

respective of the structure of the P1′ group. Even the

large phenoxyphenyl- and butoxyphenylsulfonamides

were potent inhibitors of MMP-1. Increasing the ring

size and adding substituents to the thiazepine ring also

did not result in any appreciable change in potency for

the series. This broad-spectrum activity may be due to

strong hydrophobic interactions of the thiazepine ring

with Pro-221.

Exp er im en ta l Section

Gen er a l. All commercial chemicals and solvents are reagent

grade and were used without further purification unless

otherwise specified. The following solvents and reagents have

been abbreviated: tetrahydrofuran (THF), ethyl ether (Et₂O),

dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dimethyl-

formamide (DMF), methanol (MeOH). All reactions except

those in aqueous media were carried out with the use of

standard techniques for the exclusion of moisture. Reactions

were monitored by thin-layer chromatography on 0.25-mm

silica gel plates (60F-254, E. Merck) and visualized with UV

light, iodine vapors, or 5% phosphomolybdic acid in 95%

ethanol. Final compounds were typically purified either by

flash chromatography on silica gel (E. Merck, 40-63 µm) or

Met h yl 4-[(4-Met h oxyp h en yl)su lfon yl]-2,2-d im et h yl-

h exah ydr o-1,4-th iazepin e-3(S)-car boxylate (2a). The meth-

yl ester 1a (30.0 g, 76.6 mmol) in THF (400 mL) was stirred

at room temperature and then triphenylphosphine (24.1 g, 91.9

mmol, 1.2 equiv) followed by diethyl azodicarboxylate (14.7 g,

84.3 mmol, 1.1 equiv) were added. The resulting solution was

stirred at room temperature for 2 h. The solvent was removed;

then the thick yellow oil was diluted with methylene chloride

and silica gel (60 g) was added. The solvent was removed to

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4555

leave a white powder. This powder was placed upon a chro-

matography column and eluted with 8/2 hexane/EtOAc. The

desired product (25.0 g, 87%) was obtained as a colorless oil:

¹H NMR (CDCl₃) δ 7.68 (d, J ) 8.6, 2 H), 6.91 (d, J ) 8.6, 2

H), 3.99 (m, 1 H), 3.83 (s, 3 H), 3.42 (m, 1 H), 3.38 (s, 3 H),

2.82 (br m, 1 H), 2.63 (m, 1 H), 2.22 (m, 1 H), 1.87 (m, 1 H),

1.61 (s, 3 H), 1.32 (s, 3 H); ¹³C NMR (CDCl₃) δ 168.94, 162.63,

130.83, 129.17, 113.67, 67.03, 55.38, 51.19, 46.93, 43.82, 31.45,

29.40, 26.18; MS (ESI) 374 (M⁺), 391 (MNH₃⁺).

4-[(4-Meth oxyph en yl)su lfon yl]-2,2-dim eth yl-h exah ydr o-

1,4-th ia zep in e-3(S)-ca r boxylic Acid (3a ). The methyl ester

2a (26.7 g, 71.5 mmol) in pyridine (400 mL) was stirred at

room temperature under an argon atmosphere. Lithium iodide

(115 g, 858 mmol, 12 equiv) was added and the resulting

solution was heated to reflux for 3 h. The reaction mixture

was cooled to room temperature and then the solution was

acidified with 1N HCl. The mixture was extracted with

methylene chloride and then the organic extracts were dried

(Na₂SO₄) and concentrated to an oil under reduced pressure.

The oil was purified by column chromatography using 1/1

hexane/EtOAc as the eluent to provide the desired product (18

g, 70%) as a light yellow oil: ¹H NMR (CDCl₃) δ 7.76 (d, J )

8.7, 2 H), 6.93 (d, J ) 8.7, 2 H), 3.88 (s, 3 H), 3.83 (m, 1 H),

3.64 (m, 1 H), 2.80 (br m, 2 H), 2.22 (m, 1 H), 1.90 (m, 1 H),

1.61 (s, 3 H), 1.42 (s, 3 H); ¹³C NMR (CDCl₃) δ 173.47, 162.75,

130.63, 129.38, 113.81, 67.44, 55.40, 47.04, 44.05, 31.29, 29.61,

26.42, 26.19; MS (ESI) 360 (M⁺), 377 (MNH₃⁺).

33.19, 30.36, 27.12, 26.99, 20.62; MS (ESI) 437, 439 (M⁺), 454,

456 (MNH₃⁺). Anal. (C₁₅H₂₁BrN₂O₄S₂) C, H, N.

N-Hyd r oxy-4-[(4-bu toxyp h en yl)su lfon yl]-2,2-d im eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e (4d ). Follow-

ing procedure A described above, 1.65 g (4.15 mmol) of

carboxylic acid 3d was converted to 1.4 g (80%) of the desired

product 4d (white powder, recrystallized from CH₃CN/H₂O):

¹H NMR (CDCl₃) δ 7.76 (d, J ) 8.6, 2 H), 6.98 (d, J ) 8.6, 2

H), 4.88 (s, 1 H), 4.22 (s, 1H), 4.01 (m, 3 H), 3.32 (m, 2 H),

2.84 (m, 1 H), 2.63 (m, 1H), 2.22 (m, 1 H), 1.78 (m, 3 H), 1.56

(m, 3 H), 1.45 (m, 2 H), 1.13 (m, 3 H), 1.01 (m, 3 H): ¹³C NMR

(CDCl₃) δ 170.12, 167.89, 164.57, 130.40, 115.72, 69.19, 65.05,

44.52, 32.85, 32.25, 30.24, 27.15, 26.53, 20.21, 14.10; MS (ESI)

417 (M⁺), 434 (MNH₃⁺). Anal. (C₁₈H₂₉N₂O₅S₂‚0.5 H₂O) C, H,

N.

N-Hyd r oxy-4-[[4-(2-m eth oxyeth oxy)p h en yl]su lfon yl]-

2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m -

id e (4 g). Following procedure A described above, 4.27 g (10.6

mmol) of carboxylic acid 3g was converted to 3.57 g (80%) of

the desired product 4g (white powder, solid was triturated

from toluene): ¹H NMR (DMSO-d₆) δ 10.76 (br s, 1 H), 8.91

(s, 1 H), 7.72 (d, J ) 8.8, 2 H), 7.07 (d, J ) 8.8, 2 H), 4.75 (m,

1 H), 4.19 (m, 2 H), 4.15 (s, 3 H), 3.68 (m, 2 H), 3.31 (s, 3 H),

3.13 (m, 1 H), 2.70 (m, 2 H), 2.03 (m, 1 H), 1.75 (m, 1 H), 1.45

(s, 3 H), 1.20 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 165.08, 162.27,

131.54, 129.68, 115.44, 70.80, 68.09, 63.10, 58.87, 47.78, 43.22,

31.86, 30.03, 26.47, 26.24; MS (ESI) 419 (M⁺), 436 (MNH₃⁺).

Anal. (C₁₇H₂₆N₂O₆S₂‚H₂O) C, H, N.

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

h exah ydr o-1,4-th iazepin e-3(S)-car boxam ide (4a). The car-

boxylic acid 3a (14.9 g, 41.6 mmol) in dichloromethane (200

mL) was stirred at room temperature and then oxalyl chloride

(10.8 g, 85.2 mmol, 2.05 equiv) and DMF (3.04 g, 41.6 mmol)

were added. The resulting solution was stirred at room

temperature for 30 min. In a separate flask, hydroxylamine

hydrochloride (11.6 g, 166 mmol, 4 equiv) in THF (50 mL) and

water (10 mL) was stirred at 0 °C. Triethylamine (25.3 g, 249.6

mmol, 6 equiv) was added and the resulting solution was

stirred at 0 °C for 15 min. The acid chloride solution was next

added to the hydroxylamine solution at 0 °C and the resulting

mixture was allowed to stir overnight at room temperature.

The reaction mixture was next acidified with 1 N HCl and then

extracted with dichloromethane. The organic extracts were

dried (Na₂SO₄) and concentrated to a solid under reduced

pressure. The solid was recrystallized from CH₃CN to provide

a white powder (11.4 g, 73%): ¹H NMR (CD₃OD) δ 7.42 (d, J

) 8.6, 2 H), 6.65 (d, J ) 8.6, 2 H), 3.91 (s, 1 H), 3.56 (s, 3 H),

2.98 (m, 1 H), 2.52 (m, 1 H), 2.38 (m, 1 H), 1.85 (m, 1 H), 1.52

(m, 1 H), 1.22 (s, 3 H), 0.98 (s, 3 H); MS (ESI) 392 (MNH₃+),

375 (M⁺). Anal. (C₁₅H₂₂N₂O₅S₂) C, H, N.

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

h exa h yd r o-1,4-th ia zep in e-3(R)-ca r boxa m id e (R-4a ). Fol-

lowing procedure A described above, the title compound was

prepared from ^L-penicillamine as a white powder. Anal.

(C₁₅H₂₂N₂O₅S₂) C, H, N.

N-Hyd r oxy-4-[(4-br om op h en yl)su lfon yl]-2,2-d im eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e (4b). Follow-

ing procedure A described above, 14.65 g (36.0 mmol) of

carboxylic acid 3b was converted to 13.2 g (87%) of the desired

product 4b (white powder, recrystallized from acetonitrile): ¹H

NMR (DMSO-d₆) δ 10.10 (s, 1H) 7.73 (m, 4 H), 4.89 (s, 1 H),

4.65(br m, 1 H), 4.10 (s, 1 H), 3.35 (m, 2 H), 2.91 (br m, 1 H),

2.64 (br m, 1 H), 2.25 (m, 1 H), 1.89 (m, 1 H), 1.57 (s, 3 H),

1.36 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 168.91, 138.08, 133.55,

130.00, 128.62, 65.38, 47.08, 44.59, 32.95, 30.44, 27.28, 26.74;

MS (ESI) 423, 425 (M⁺). Anal. (C₁₄H₁₉BrN₂O₄S₂) C, H, N.

4-[(4-P h en oxyph en yl)su lfon yl]-2,2-dim eth yl-h exah ydr o-

1,4-th ia zep in e-3(S)-ca r boxylic Acid (3h ). Following pro-

cedure A described above, 5.03 g (11.5 mmol) of methyl ester

2h was converted to 4.51 g (92%) of carboxylic acid 3h : ¹H

NMR (CDCl₃) δ 7.75 (d, J ) 8.8, 2 H), 7.42 (m, 2 H), 7.23 (m,

1 H), 7.01 (m, 4 H), 4.58 (s, 1 H), 3.84 (m, 1 H), 3.61 (m, 1 H),

2.83 (m, 1 H), 2.66 (m, 1 H), 2.20 (m, 1 H), 1.90 (m, 1 H), 1.61

(s, 3 H), 1.42 (s, 3 H); ¹³C NMR (CDCl₃) δ 173.36, 161.69,

155.40, 143.20, 133.06, 130.39, 129.94, 126.68, 125.10, 120.35,

117.71, 67.99, 47.49, 44.64, 31.78, 30.10, 26.84, 26.67; MS (ESI)

422 (M⁺).

N-Hydr oxy-4-[(4-ph en oxyph en yl)su lfon yl]-2,2-dim eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e (4h ). Follow-

ing procedure A described above, 0.40 g (0.95 mmol) of

carboxylic acid 3h was converted to 0.18 g (44%) of hydroxamic

acid 4h : ¹H NMR (DMSO-d₆) δ 10.88 (br s, 1 H), 8.90 (s, 1 H),

7.78 (d, J ) 8.8, 2 H), 7.55 (m, 2 H), 7.25-7.05 (m, 7 H), 4.78

(m, 1 H), 4.12 (s, 1 H), 3.18 (m, 2 H), 2.62 (m, 2 H), 2.04 (m, 1

H), 1.78 (m, 1 H), 1.45 (s, 3 H), 1.22 (s, 3 H); ¹³C NMR (DMSO-

d₆) δ 165.00, 161.30, 155.48, 133.09, 131.07, 130.04, 125.62,

120.82, 118.20, 63.20, 47.75, 43.48, 31.96, 30.04, 26.48, 26.25;

MS (ESI) 437 (M⁺). Anal. (C₂₀H₂₄N₂O₅S₂) C, H, N.

P r oced u r e B for F or m a tion of Th ia zep in es 4i-4k .

Meth yl 2,2-Dim eth yl-h exa h yd r o-1,4-th ia zep in e-3(S)-ca r -

boxyla te (11). ^D-Penicillamine methyl ester (25.0 g, 125.2

mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 38.1 g, 250.4

mmol, 2.0 equiv), and DMF (80 mL) were added to a solution

of 3-bromo-1-chloropropane (20.7 g, 131.4 mmol, 1.05 equiv)

in DMF (45 mL) at 0 °C. The mixture was added slowly over

a 1-h period. The resulting mixture was heated to 80 °C and

additional DBU (19.1 g, 125.2 mmol, 1 equiv) was added. The

reaction was complete after 5 h at 80 °C. The reaction was

cooled to room temperature and then poured into dilute sodium

bicarbonate solution. The resulting mixture was extracted with

EtOAc. The organic extracts were dried (MgSO₄) and then

concentrated to an oil under reduced pressure. Purification of

the oil was accomplished by chromatography on silica gel using

85/15 hexane/EtOAc as the eluent to provide 10.5 g (41%) of

the desired product: ¹H NMR (DMSO-d₆) δ 3.73 (s, 3 H), 3.41

(s, 1 H), 3.31 (ddd, J ) 2.8, 6.5, 14.2, 1 H), 2.88 (m, 2 H), 2.61

(ddd, J ) 5.4, 5.4, 15.3, 1 H), 1.91 (m, 2 H), 1.42 (s, 3 H), 1.30

(s, 3 H); MS (ESI) 204 (M⁺).

N-Hyd r oxy-4-[(4-br om o-2-m eth ylp h en yl)su lfon yl]-2,2-

dim eth yl-h exah ydr o-1,4-th iazepin e-3(S)-car boxam ide (4c).

Following procedure A described above, 1.65 g (3.9 mmol) of

carboxylic acid 3c was converted to 1.35 g (75%) of the desired

product 4c (white powder, recrystallized from CH₃CN/H₂O):

¹H NMR (CDCl₃) δ 7.56 (m, 3 H), 4.18 (s, 1 H), 3.44 (m, 1H),

3.33 (s, 1 H), 2.81 (m, 3 H), 2.62 (m, 4 H), 2.19 (m, 1 H), 1.90

(m, 1 H), 1.58 (s, 3 H), 1.34 (s, 3 H): ¹³C NMR (CDCl₃) δ 167.04,

142.64, 138.79, 136.66, 131.05, 130.47, 128.67, 65.22, 45.60,

Meth yl 4-[5-(P yr id -2-yl)th ien ylsu lfon yl]-2,2-d im eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxyla te (2i). The thi-

azepine (2.0 g, 9.83 mmol) in chloroform (25 mL) was stirred

at room temperature and then N-methylmorpholine (2.20 g,

4556 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

21.6 mmol, 2.2 equiv), N,N-(dimethylamino)pyridine (25 mg),

and 5-(pyrid-2-yl)thiophene-2-sulfonyl chloride (2.68 g, 10.3

mmol, 1.05 equiv) were added. The resulting mixture was

heated to reflux for 5 days until almost no starting material

remained. The reaction was cooled to room temperature and

then poured into dilute sodium bicarbonate solution. The

mixture was extracted with ethyl acetate. The organic extracts

were dried (Na₂SO₄) and then concentrated to an oil under

reduced pressure. The oil was purified by chromatography on

silica gel using 3/2 hexane/EtOAc as the eluent. The product

(3.0 g, 72%) was obtained as a light yellow oil: ¹H NMR

(DMSO-d₆) δ 8.59 (ddd, J ) 1.0, 1.7, 4.9, 1 H), 7.79-7.66 (br

m, 2 H), 7.52 (d, J ) 3.9, 1 H), 7.49 (d, J ) 4.0, 1 H), 7.25

(ddd, J ) 1.1, 4.8, 7.4, 1 H), 4.60 (s, 1 H), 4.07 (ddd, J ) 3.5,

12.0, 15.6, 1 H), 3.71 (m, 1 H), 3.49 (s, 3 H), 2.88 (m, 1 H),

2.72 (m, 1 H), 2.28 (m, 1 H), 1.95 (m, 1 H), 1.64 (s, 3 H), 1.37

(s, 3 H); ¹³C NMR (DMSO-d₆) δ 168.81, 151.03, 150.81, 149.76,

140.40, 136.86, 132.52, 123.29, 123.16, 118.97; MS (ESI) 427

(M⁺).

4-[5-(P yr id -2-yl)th ien ylsu lfon yl]-2,2-d im eth yl-h exa h y-

d r o-1,4-th ia zep in e-3(S)-ca r boxylic Acid (3i). The methyl

ester (1.48 g, 3.47 mmol) was stirred with 6 N HCl (50 mL) at

reflux for 16 h. The reaction mixture was cooled to room

temperature and then neutralized to pH ∼ 6 with 50% NaOH

solution. The resulting yellow precipitate was filtered off and

then washed with water. The solid was dried under vacuum

to provide 1.20 g (84%) of the desired product.

N -H yd r oxy-4-[5-(p yr id -2-yl)t h ie n ylsu lfon yl]-2,2-d i-

m eth yl-h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e (4i).

The carboxylic acid (1.10 g, 2.66 mmol) in dichloromethane

(20 mL) was stirred at room temperature and then oxalyl

chloride (0.41 g, 3.20 mmol, 1.2 equiv) was added. The

resulting solution was stirred at room temperature overnight.

In a separate flask, hydroxylamine (50% solution in water, 1.76

mL, 26.6 mmol, 10 equiv) in THF (7 mL) and tert-butyl alcohol

(4 mL) were stirred at 0 °C. The acid chloride solution was

next added to the hydroxylamine solution at 0 °C and the

resulting mixture was allowed to stir 2 h at room temperature.

The reaction mixture was poured into water and then ex-

tracted with dichloromethane. The organic extracts were dried

(Na₂SO₄) and concentrated to an oil under reduced pressure.

The oil was crystallized from CH₃CN to provide 0.65 g (57%)

of the desired product as a white powder: ¹H NMR (DMSO-

d₆) δ 9.79 (s, 1 H), 8.96 (s, 1 H), 8.59 (m, 1 H), 8.06 (m, 1 H),

7.93 (m, 1 H), 7.86 (d, J ) 4.0, 1 H), 7.62 (d, J ) 4.0, 1 H),

7.40 (ddd, J ) 1.1, 5.0, 8.8, 1 H), 4.83 (m, 1 H), 4.19 (s, 1 H),

3.42 (m, 1 H), 2.63 (m, 2 H), 2.10 (m, 1 H), 1.92 (m, 1 H), 1.42

(s, 3 H), 1.22 (s, 3 H); MS (ESI) 428 (M⁺). Anal. (C₁₇H₂₁N₃O₄S₃)

C, H, N.

N-H yd r oxy-4-[4-((p yr id -4-yl)oxy)p h en ylsu lfon yl]-2,2-

dim eth yl-h exah ydr o-1,4-th iazepin e-3(S)-car boxam ide (4j).

Following procedure B described above, 2.74 g (6.47 mmol) of

carboxylic acid 3j was converted to 1.70 g (60%) of the desired

hydroxamic acid 4j as a white powder: ¹H NMR (DMSO-d₆) δ

10.75 (s, 1 H), 8.86 (s, 1 H), 8.57 (d, J ) 6.0, 2 H), 7.88 (d, J )

8.6, 2 H), 7.25 (d, J ) 8.6, 2 H), 7.04 (d, J ) 6.0, 2 H), 4.76 (m,

1 H), 4.10 (s, 1 H), 3.23 (m, 1 H), 2.64 (m, 2 H), 2.04 (m, 1 H),

1.83 (m, 1 H), 1.44 (s, 3 H), 1.22 (s, 3 H); MS (ESI) 438 (M⁺).

was added and the mixture was then stirred at room temper-

ature. An additional 3 equiv of 32% peracetic acid was added

after 3 h and the resulting mixture was stirred overnight. Once

the reaction was complete, the solvent was removed under

reduced pressure. The resulting white solid was recrystallized

with acetonitrile to provide a white powder: ¹H NMR (CD₃OD)

δ 7.42 (d, J ) 8.6, 2 H), 6.65 (d, J ) 8.6, 2 H), 3.91 (s, 1 H),

3.56 (s, 3 H), 2.98 (m, 1 H), 2.52 (m, 1 H), 2.38 (m, 1 H), 1.85

(m, 1 H), 1.52 (m, 1 H), 1.22 (s, 3 H), 0.98 (s, 3 H); MS (ESI)

407 (M⁺). Anal. (C₁₅H₂₂N₂O₇S₂) C, H, N.

N-Hyd r oxy-4-[(4-br om op h en yl)su lfon yl]-2,2-d im eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e 1,1-Dioxid e

(5b). Following the above procedure, the hydroxamic acid 4b

(4.2 g, 9.9 mmol) was converted to 4.1 g (91%) of the desired

product 5b (white solid, recrystallized from acetonitrile): ¹H

NMR (DMSO-d₆) δ 10.10 (s, 1H), 7.78 (m, 4 H), 4.92 (s, 1 H),

4.59 (s, 1 H), 3.91 (m, 1 H), 3.74 (m, 1 H), 2.91 (br m, 1 H),

2.64 (br m, 1 H), 2.25 (m, 1 H), 1.89 (m, 1 H), 1.57 (s, 3 H),

1.36 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 168.91, 138.08, 133.55,

130.00, 128.62, 65.38, 47.08, 44.59, 32.95, 30.44, 27.28, 26.74;

MS (ESI) 455, 457 (M⁺). Anal. (C₁₄H₁₉BrN₂O₆S₂) C, H, N.

N-Hyd r oxy-4-[(4-bu toxyp h en yl)su lfon yl]-2,2-d im eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e 1,1-Dioxid e

(5d ). Following the above procedure, the hydroxamic acid 4d

(0.50 g, 1.2 mmol) was converted to 0.44 g (88%) of the desired

product 5d as a white solid (recrystallized from acetonitrile):

¹H NMR (CDCl₃) δ 7.76 (d, J ) 8.6, 2 H), 6.98 (d, J ) 8.6, 2

H), 4.91 (s, 1 H), 4.46 (s, 1H), 4.01 (m, 3 H), 3.91 (m, 1 H),

3.72 (m, 1 H), 3.51 m, 1 H), 3.32 (m, 1 H), 2.18 (m, 1 H), 1.78

(m, 3 H), 1.56 (m, 3 H), 1.45 (m, 2 H), 1.13 (m, 3 H), 1.01 (m,

3 H); ¹³C NMR (CDCl₃) δ 184.76, 164.25, 131.55, 130.61,

115.74, 69.21, 66.67, 60.06, 32.25, 24.95, 23.28, 21.96, 20.21,

14.12; MS (ESI) 449 (M⁺), 466 (MNH₃⁺). Anal. (C₁₈H₂₈N₂O₇S₂‚

0.5 H₂O) C, H, N.

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2-h yd r oxy-

eth yl)-^D-p en icilla m in e (6a ). Following procedure A above

(for the preparation of compound 1a ), the title compound was

prepared from d-penicillamine methyl ester hydrochloride

(19.9 g, 0.1 mol) and 2-bromoethanol (15 g, 0.12 mol, 1.2 equiv).

The desired product 6a was obtained as a clear, colorless oil:

¹H NMR (CDCl₃) δ 7.71 (d, J ) 8.6, 2 H), 6.88 (d, J ) 8.6, 2

H), 5.83 (d, J ) 11, 1 H), 3.78 (s, 3 H), 3.77 (m, 1 H), 3.63 (br

m, 2 H), 3.38 (s, 3 H), 2.75 (br m, 2 H), 2.42 (br s, 1 H), 1.38

(s, 6 H); ¹³C NMR (CDCl₃) δ 170.17, 162.86, 130.71, 129.29,

113.91, 63.57, 61.14, 55.46, 51.99, 46.70, 31.53, 26.31, 26.03.

Met h yl 4-[(4-Met h oxyp h en yl)su lfon yl]-2,2-d im et h yl-

3(S)-th iom or p h olin eca r boxyla te (7a ). Following procedure

A above (for the preparation of compound 2a ) the title

compound was prepared. The desired product 7a was obtained

as a colorless oil: ¹H NMR (CDCl₃) δ 7.61 (d, J ) 8.6, 2 H),

6.92 (d, J ) 8.6, 2 H), 4.38 (s, 1 H), 4.01 (m, 1 H), 3.83 (s, 3 H),

3.72 (dt, J _d) 2.9, 12.5, 1 H), 3.39 (s, 3 H), 3.10 (dt, J _d) 3, J _t)

13, 1 H), 2.42 (m, 1 H), 1.59 (s, 3 H), 1.24 (s, 3 H); ¹³C NMR

(CDCl₃) δ 168.48, 162.73, 130.29, 128.92, 113.85, 62.55, 55.40,

51.13, 40.94, 39.87, 28.21, 27.01, 24.40; MS (ESI) 360 (M⁺),

377 (MNH₃⁺).

4-[(4-Meth oxyp h en yl)su lfon yl]-2,2-d im eth yl-3(S)-th io-

m or p h olin eca r boxylic Acid (8a ). Following procedure A

above (for the preparation of compound 3a ) the title compound

8a was prepared as a light yellow oil: ¹H NMR (CDCl₃) δ 7.67

(d, J ) 8.8, 2 H), 6.91 (d, J ) 8.8, 2 H), 4.44 (s, 1 H), 3.99 (m,

1 H), 3.81 (s, 3 H), 3.68 (dt, J _d) 3, J _t) 12.5, 1 H), 3.08 (dt, J _d

) 3.6, J _t) 12.7, 1 H), 2.40 (m, 1 H), 1.58 (s, 3 H), 1.36 (s, 3

H); ¹³C NMR (CDCl₃) δ 172.92, 162.78, 130.41, 129.06, 113.91,

62.75, 55.39, 40.90, 39.76, 28.39, 27.23, 24.31. MS (ESI) 346

(M⁺), 363 (MNH₃⁺).

N-H yd r oxy-4-[4-(4-flu or op h en yl)p h en ylsu lfon yl]-2,2-

d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m id e

(4k ). Following procedure B described above, 2.40 g (5.66

mmol) of carboxylic acid 3k was converted to 1.20 g (48%) of

the desired product 4k as a white powder: ¹H NMR (DMSO-

d₆) δ 10.80 (s, 1 H), 8.88 (s, 1 H), 7.86 (s, 4 H), 7.82 (m, 2 H),

7.36 (m, 2 H), 4.80 (m, 1 H), 4.22 (s, 1 H), 3.22 (m, 1 H), 2.64

(m, 2 H), 2.08 (m, 1 H), 1.83 (m, 1 H), 1.44 (s, 3 H), 1.22 (s, 3

H); MS (ESI) 439 (M⁺). Anal. (C₂₀H₂₃FN₂O₄S₂) C, H, N.

Gen er a l P r oced u r e for Oxid a tion of Th ia zep in es to

t h e Cor r esp on d in g Su lfon es 5a -5g. N-H yd r oxy-4-[(4-

m et h oxyp h en yl)su lfon yl]-2,2-d im et h yl-h exa h yd r o-1,4-

th ia zep in e-3(S)-ca r boxa m id e 1,1-Dioxid e (5a ). The hy-

droxamic acid 4a (4.7 g, 12.55 mmol) was dissolved in

chloroform (50 mL) at 0 °C in an ice bath. A solution of 32%

peracetic acid (7.9 mL, 37.65 mmol, 3.0 equiv in acetic acid)

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

3(S)-th iom or p h olin eca r boxa m id e (9a ). Following proce-

dure A above (for the preparation of compound 4a ) the title

compound 9a was prepared as a solid which was recrystallized

from CH₃CN/H₂O to provide a white powder: ¹H NMR

(CD₃OD) δ 7.68 (d, J ) 8.8, 2 H), 7.03 (d, J ) 8.8, 2 H), 4.08

(s, 1 H), 3.88 (m, 1 H), 3.86 (s, 3 H), 3.02 (m, 1 H), 2.44 (m, 1

H), 1.57 (s, 3 H), 1.22 (s, 3 H); ¹³C NMR (CD₃OD) δ 167.01,

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4557

164.61, 132.01, 130.30, 115.38, 61.01, 56.14, 42.75, 40.64,

29.12, 27.27, 25.54; MS (ESI) 378 (MNH₃⁺), 361 (M⁺). Anal.

(C₁₄H₂₀N₂O₅S₂) C, H, N.

4.70 (m, 1 H), 4.05 (s, 1 H), 3.40 (m, 4 H), 2.74 (m, 2 H), 2.08

(m, 1 H), 1.83 (m, 1 H), 1.47 (s, 3 H), 1.22 (s, 3 H); ¹³C NMR

(DMSO-d₆) δ 164.63, 161.14, 155.16, 133.69, 130.78, 129.80,

125.35, 120.40, 117.95, 63.30, 62.60, 47.45, 43.47, 31.88, 29.71,

26.03, 25.94; MS (ESI) 451 (M⁺). Anal. (C₂₁H₂₆N₂O₅S₂) C, H,

N.

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

3(S)-th iom or p h olin eca r boxa m id e 1,1-Dioxid e (10a ). The

hydroxamic acid 9a (0.30 g, 0.83 mmol) was dissolved in

chloroform (10 mL) to form a suspension. Peracetic acid (0.20

g, 2.5 mmol, 3.0 equiv) was then added and the resulting

mixture was stirred overnight at room temperature. The

solvent was then removed under reduced pressure. Purification

was achieved through crystallization with acetonitrile to form

a white powder: ¹H NMR (CDCl₃) δ 7.68 (d, J ) 8.6, 2 H),

6.90 (d, J ) 8.6, 2 H), 4.60 (m, 1 H), 4.49 (m, 1 H), 3.85 (s, 3

H), 3.21 (m, 1 H), 3.24 (m, 4 H), 1.53 (s, 3H), 1.30 (s, 3 H); MS

(ESI) 393.0 (M⁺). Anal. (C₁₄H₂₀N₂O₇S₂) C, H, N.

N-Hyd r oxy-N-m eth yl-4-[(4-m eth oxyp h en yl)su lfon yl]-

2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m -

id e (12a ). The carboxylic acid 3a (1.10 g, 3.06 mmol) in

dichloromethane (25 mL) was stirred at room temperature and

then oxalyl chloride (0.80 g, 6.27 mmol, 2.05 equiv) and DMF

(0.22 g, 3.06 mmol) were added. The resulting solution was

stirred at room temperature for 30 min. In a separate flask,

N-methylhydroxylamine hydrochloride (1.02 g, 12.24 mmol, 4

equiv) in THF (8 mL) and water (2 mL) were stirred at 0 °C.

Triethylamine (1.85 g, 18.4 mmol, 6 equiv) was added and the

resulting solution was stirred at 0 °C for 15 min. The acid

chloride solution was next added to the hydroxylamine solution

at 0 °C and the resulting mixture was allowed to stir overnight

at room temperature. The reaction mixture was acidified with

1 N HCl and then extracted with dichloromethane. The organic

extracts were dried (Na₂SO₄) and concentrated to a solid under

reduced pressure. The solid was purified by reverse phase (C₁₈)

HPLC chromatography to provide a white powder (0.69 g,

58%): ¹H NMR (CD₃OD) δ 7.7842 (d, J ) 8.6, 2 H), 7.03 (d, J

) 8.6, 2 H), 5.41 (s, 1 H), 4.64 (m, 1 H), 3.83 (s, 3 H), 3.31 (m,

1 H), 3.02 (s, 3 H), 2.82 (m, 1 H), 2.70 (m, 1 H), 2.16 (m, 1 H),

1.79 (m, 1 H), 1.53 (s, 3 H), 1.24 (s, 3 H); ¹³C NMR (CD₃OD) δ

170.76, 164.50, 132.44, 130.43, 115.09, 59.93, 56.18, 44.46,

36.11, 33.21, 30.39, 27.35, 26.01; MS (ESI) 389 (M⁺). Anal.

(C₁₆H₂₄N₂O₅S₂) C, H, N.

6,6-Dim eth yl-7-ca r bom eth oxy-tetr a h yd r o-2(3H)-a zep i-

n on e (15). The methyl 2,2-dimethyl-6-oxocyclohexanecarbox-

ylate²³(14) (7.3 g, 39.7 mmol) was dissolved in chloroform (180

mL) and cooled to 0 °C. Methanesulfonic acid (38.1 g, 397

mmol) was added followed by the addition of sodium azide.

The reaction mixture was stirred at room temperature for 30

min and then heated to reflux for 5 h. Ice was added to the

reaction mixture and stirred for several minutes; this was

followed by the addition of ammonium hydroxide until the

reaction became basic. The mixture was then extracted with

methylene chloride, and the organic layers were dried (MgSO₄)

and concentrated under reduced pressure to an oil (6.13 g, 68%

yield): ¹H NMR (CDCl₃) δ 6.04 (br s, 1 H), 3.64 (s, 3 H), 2.43

(m, 2 H), 1.71 (m, 2 H), 1.50 (m, 2 H), 0.98 (s, 3 H), 0.96 (s, 3

H); MS (ESI) 200 (M⁺).

2-H yd r oxym e t h yl-3,3-d im e t h yl-h e xa h yd r o-1H -a ze -

p in e. The amide 15 (3.75 g, 18.9 mmol) was dissolved in THF

(100 mL) under an argon atmosphere at room temperature.

Lithium aluminum hydride (1.5 g, 37.7 mmol, 2 equiv) was

next added slowly to the reaction mixture. The reaction

mixture was heated to reflux for 5 h. The mixture was

quenched by the slow, dropwise addition of ethyl acetate until

bubbling of the reaction mixture ceased. Then, water (1.5 mL)

was added to the solution. A 15% NaOH solution (1.5 mL) was

next added followed by water (3.0 mL). The resulting hetero-

geneous mixture was then filtered, and the remaining organic

layer was diluted with water and extracted with ether. The

organic layers were dried over sodium sulfate and concentrated

under reduced pressure: ¹H NMR (CDCl₃) δ 4.08 (m, 1 H),

3.83 (m, 1 H), 3.58 (m, 2 H), 3.15 (m, 1 H), 2.66 (m, 1 H), 2.40

(m, 1 H), 1.70 (m, 1 H), 1.47 (m, 1 H), 1.19 (m, 1 H), 0.93 (s, 3

H), 0.80 (s, 3 H); MS (ESI) 158 (M⁺).

1-[(4-Met h oxyp h en yl)su lfon yl]-2-h yd r oxym et h yl-3,3-

d im et h yl-h exa h yd r o-1H -a zep in e (16). The 2-hydroxy-

methyl-3,3-dimethyl-hexahydro-1H-azepine (2.9 g, 18.9 mmol)

was dissolved in a 1:1 mixture of water and p-dioxane (100

mL), followed by the addition of 4-methoxyphenylsulfonyl

chloride (4.7 g, 22.6 mmol) and triethylamine (7.86 mL, 56.5

mmol). The reaction mixture was stirred overnight. The

reaction was quenched and acidified with 1 N HCl to a pH ∼

2. The mixture was then diluted with water and extracted with

methylene chloride. The organic layers were dried over

magnesium sulfate and concentrated under reduced pressure.

The compound was purified by silica gel chromatography using

hexane:ethyl acetate (3:2) as the eluent: ¹H NMR (CDCl₃) δ

7.78 (d, J ) 8.6, 2 H), 6.92 (d, J ) 8.6, 2 H), 3.85 (s, 3 H), 3.71

(m, 2 H), 3.60 (m, 2 H), 3.31 (m, 1 H), 3.05 (m, 2 H), 1.85 (m,

2 H), 1.52 (m, 4 H), 0.98 (s, 3 H), 0.91 (s, 3 H); MS (ESI) 328

(M⁺), 345 (MNH₃⁺).

N-Hyd r oxy-N-m eth yl-4-[(4-p h en oxyp h en yl)su lfon yl]-

2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m -

id e (12h ). Following the procedure for the preparation of 12a ,

the carboxylic acid 3h (0.42 g, 0.98 mmol) was converted to

the N-methylhydroxamic acid 12h (0.22 g, 55%) as a colorless

solid (purified by reverse phase (C₁₈) HPLC chromatogra-

phy): ¹H NMR (DMSO-d₆) δ 10.23 (s, 1 H), 7.77 (d, J ) 8.8, 2

H), 7.49 (t, J ) 7.9, 2 H), 7.28 (t, J ) 7.3, 1 H), 7.09 (d, J )

8.8, 2 H), 5.28 (s, 1 H), 4.67 (m, 1 H), 3.21 (m, 1 H), 2.94 (s, 3

H), 2.74 (m, 2 H), 2.05 (m, 1 H), 1.83 (m, 1 H), 1.49 (s, 3 H),

1.23 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 168.50, 161.26, 155.58,

133.80, 131.10, 129.95, 125.62, 120.69, 118.13, 58.70, 48.22,

43.70, 36.05, 32.45, 30.43, 26.59, 26.03; MS (ESI) 451 (M⁺).

Anal. C₂₁H₂₆N₂O₅S₂(C, H, N).

N-Meth oxy-4-[(4-ph en oxyph en yl)su lfon yl]-2,2-dim eth yl-

h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e (13f). The

carboxylic acid 3f (0.43 g, 1.02 mmol) in dichloromethane (10

mL) was stirred at room temperature and then oxalyl chloride

(0.27 g, 2.09 mmol, 2.05 equiv) and DMF (75 mg, 1.02 mmol)

were added. The resulting solution was stirred at room

temperature for 30 min. In a separate flask, O-methylhydroxyl-

amine hydrochloride (0.34 g, 4.08 mmol, 4 equiv) in THF (8

mL) and water (2 mL) were stirred at 0 °C. Triethylamine (0.62

g, 6.12 mmol, 6 equiv) was added and the resulting solution

was stirred at 0 °C for 15 min. The acid chloride solution was

next added to the hydroxylamine solution at 0 °C and the

resulting mixture was allowed to stir overnight at room

temperature. The reaction mixture was acidified with 1 N HCl

and then extracted with dichloromethane. The organic extracts

were dried (Na₂SO₄) and concentrated to a solid under reduced

pressure. The solid was purified by reverse-phase (C₁₈) HPLC

chromatography to provide a white powder (0.25 g, 60%): ¹H

NMR (DMSO-d₆) δ 11.32 (s, 1 H), 7.83 (d, J ) 8.8, 2 H), 7.49

(t, J ) 7.4, 2 H), 7.27 (t, J ) 7.3, 1 H), 7.11 (d, J ) 8.8, 2 H),

1-[(4-Meth oxyph en yl)su lfon yl]-3,3-dim eth yl-h exah ydr o-

1H-a zep in e-2-ca r boxylic Acid . The alcohol 16 (0.40 g, 1.22

mmol) was dissolved in acetone (50 mL) and freshly prepared

8 N J ones reagent was added until the solution sustained an

orange/brown color as opposed to a green color. The reaction

mixture was then stirred overnight. 2-Propanol was added to

quench the excess J ones reagent and a green solid precipitated

out of solution. The solid was filtered through Celite, and the

liquid was concentrated under reduced pressure. The residue

was then dissolved in chloroform and washed with water

several times. The organic layer was dried over magnesium

sulfate and concentrated under reduced pressure. The product

was carried forward with no further purification: ¹H NMR

(CDCl₃) δ 7.78 (d, J ) 8.6, 2 H), 6.92 (d, J ) 8.6, 2 H),4.23 (br

s, 1 H), 3.82 (s, 3 H), 3.71 (m, 2 H), 3.60 (m, 2 H), 3.05 (m, 2

H), 1.60 (m, 2 H), 1.32 (m, 3 H), 1.08 (s, 3 H), 0.90 (s, 3 H); MS

(ESI) 342 (M⁺), 359 (MNH₃⁺).

N-Hydr oxy-1-[(4-m eth oxyph en yl)su lfon yl]-3,3-dim eth yl-

h exa h yd r o-1H -a zep in e-2-ca r b oxa m id e (17). The 1-[(4-

4558 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

methoxyphenyl)sulfonyl]-3,3-dimethyl-hexahydro-1H-azepine-

2-carboxylic acid (0.37 g, 1.07 mmol) was converted to the

desired hydroxamic acid 17 following procedure A above (for

the preparation of compound 4a ). The resulting solid was

recrystallized from CH₃CN/H₂O to provide the desired product

as a white powder: ¹H NMR (CDCl₃) δ 7.78 (d, J ) 8.6, 2 H),

7.01 (d, J ) 8.6, 2 H),4.90 (s, 1 H), 4.03 (s, 1 H), 3.85 (s, 3 H),

3.72 (m, 2 H), 3.55 (m, 1 H), 3.28 (m, 1 H), 2.00 (m, 1 H),1.80

(m, 2 H), 1.60 (m, 2 H), 1.38 (m, 2 H), 1.08 (s, 3 H), 0.98 (s, 3

H); MS (ESI) 357 (M⁺). Anal. (C₁₆H₂₄N₂O₅S) C, H, N.

7-Car bom eth oxy-tetr ah ydr o-2(3H)-azepin on e (18). Eth-

yl 2-cyclohexanonecarboxylate (15.0 g, 88.12 mmol) was dis-

solved in chloroform (200 mL) and cooled to 0 °C. Methane-

sulfonic acid (84.7 g, 881.2 mmol) was added followed by the

addition of sodium azide. The reaction mixture was stirred at

room temperature for 30 min and then heated to reflux for 5

h. Ice was added to the reaction mixture and the resulting

solution was stirred for several minutes. Ammonium hydroxide

was added until the reaction was made basic. The mixture was

extracted with methylene chloride, and the organic layers were

dried (MgSO₄) and concentrated under reduced pressure to an

oil: ¹H NMR (CDCl₃) δ 6.41 (br s, 1 H), 4.20 (m, 1 H), 4.03 (m,

1 H), 2.38 (m, 2 H), 2.19 (m, 1 H), 1.83 (m, 1 H), 1.56 (m, 4H),

1.20 (m, 4 H); ¹³C NMR (CDCl₃) δ 175.96, 171.11, 61.87, 55.61,

36.75, 33.49, 29.34, 22.63, 13.83; MS (ESI) 186 (M⁺).

2-Hyd r oxym eth yl-h exa h yd r o-1H-a zep in e. The amide 18

(5.0 g, 27.0 mmol) was dissolved in THF (100 mL) under an

argon atmosphere at room temperature. Lithium aluminum

hydride (2.0 g, 54.0 mmol, 2 equiv) was then cautiously added.

The reaction mixture was heated to reflux for 5 h. The mixture

was quenched by the slow, dropwise addition of ethyl acetate

until bubbling of the reaction mixture ceased. Then, water (2.0

mL) was added to the solution. A 15% NaOH (2.0 mL) was

next added followed by water (5.0 mL). The resulting homo-

geneous solution was filtered, and the remaining organic layer

was diluted with water and extracted with ether. The organic

layers were dried over sodium sulfate and concentrated under

reduced pressure.

1-[(4-Me t h o x y p h e n y l)s u lfo n y l]-2-h y d r o x y m e t h y l-

h exa h yd r o-1H-a zep in e (20). The 2-hydroxymethyl-hexahy-

dro-1H-azepine (3.0 g, 23.5 mmol) was dissolved in a 1:1

mixture of water and p-dioxane (100 mL) followed by the

addition of 4-methoxyphenylsulfonyl chloride (5.8 g, 28.2

mmol) and triethylamine (9.8 mL, 70.5 mmol). The reaction

mixture was left to stir overnight. The reaction was quenched

and acidified with 1 N HCl to a pH ∼ 2. The reaction mixture

was then diluted with water and extracted with methylene

chloride. The organic layers were dried over magnesium

sulfate and concentrated under reduced pressure. The com-

pound was purified by silica gel chromatography using hexane:

ethyl acetate (2:1) as the eluent: ¹H NMR (CDCl₃) δ 7.81 (d,

J ) 8.6, 2 H), 6.88 (d, J ) 8.6, 2 H),3.91 (m, 1 H), 3.82 (s, 3 H),

3.78 (m, 1 H), 3.46 (m, 2 H), 3.04 (m, 1 H), 2.05 (m, 2 H), 1.64

(br m, 4 H), 1.22 (m, 2 H), 0.86 (m, 1 H); ¹³C NMR (CDCl₃) δ

162.45, 133.10, 129.33, 113.90, 66.71, 58.85, 55.36, 42.60,

29.37, 28.14, 26.75, 23.83; MS (ESI) 300 (M⁺), 317 (MNH₃⁺).

N-Hyd r oxy-1-[(4-m eth oxyp h en yl)su lfon yl]-h exa h yd r o-

1H-a zep in e-2-ca r boxa m id e (21). The 1-[(4-methoxyphenyl)-

sulfonyl]-hexahydro-1H-azepine-2-carboxylic acid (1.11 g, 3.56

mmol) was converted to the desired hydroxamic acid 21

following procedure A above (for the preparation of compound

4a ). The resulting solid was recrystallized from CH₃CN/H₂O

to provide a white powder: ¹H NMR (CDCl₃) δ 10.89 (s, 1 H),

7.74 (d, J ) 8.6, 2 H), 6.90 (d, J ) 8.6, 2 H), 4.34 (m, 1H), 3.84

(s, 3 H), 3.69 (m, 1 H), 3.32 (m, 1 H), 2.18 (m, 2 H), 1.71 (br m,

4 H), 1.30 (m, 2 H), 1.15 (m, 2 H); ¹³C NMR (CDCl₃) δ 185.85,

180.89, 170.06, 129.34, 114.24, 55.45, 45.33, 30.13, 29.01,

28.35, 24.22; MS (ESI) 429 (M⁺); HRMS calcd 329.1171, found

329.1171.

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2-h yd r oxy-

p en tyl)-D-p en icilla m in e (25). Following procedure A above,

D-penicillamine (5.0 g, 33.5 mmol) and 1-chloropentanol (4.92

g, 40.2 mmol, 1.2 equiv) were converted to the title compound

which was obtained as a colorless oil. Purification of the methyl

ester was accomplished by chromatography on silica gel using

6/4 hexane/EtOAc as the eluent. The desired product (2.20 g,

15%) was obtained as a clear, colorless oil: ¹H NMR (CD₃OD)

δ 7.74 (d, J ) 8.2, 2 H), 6.92 (d, J ) 8.2, 2 H), 5.42 (d, J ) 8.2,

1 H), 3.81 (s, 3 H), 3.76 (d, J ) 8.1, 1 H), 3.61 (t, J ) 6.1, 2 H),

3.41 (s, 3 H), 2.40 (m, 2 H), 1.60-1.38 (m, 6 H), 1.38 (s, 3 H),

1.26 (s, 3 H); ¹³C NMR (CD₃OD) δ 186.05, 170.13, 162.85,

130.64, 129.33, 113.89, 62.84, 62.39, 55.46, 51.89, 46.45, 32.00,

28.70, 27.78, 25.80, 25.62, 24.96; MS (ESI) 420 (M⁺), 437

(MNH₃⁺).

Meth yl N-Hyd r oxy-4-[(4-m eth oxyp h en yl)su lfon yl]-2,2-

dim eth yl-octah ydr o-1,4-th iazon in e-3(S)-car boxylate (27).

The methyl ester 25 (2.1 g, 5.0 mmol) was converted to the

title compound as a colorless oil (0.50 g, 25%) following

procedure A (for the preparation of compound 2a ): ¹H NMR

(CD₃OD) δ 7.74 (d, J ) 8.2, 2 H), 6.94 (d, J ) 8.2, 2 H), 4.56

(s, 1 H), 3.83 (m, 1 H), 3.81 (s, 3 H), 3.30 (s, 3 H), 2.84 (m, 2

H), 2.56 (m, 1 H), 2.30 (m, 1 H), 2.03 (m, 1 H), 1.63 (m, 2 H),

1.53 (s, 3 H), 1.33 (s, 3 H); ¹³C NMR (CD₃OD) δ 167.74, 162.83,

130.48, 128.26, 113.30, 68.32, 55.41, 50.88, 48.21, 45.85, 31.42,

30.59, 29.44, 29.11, 27.81, 24.53, 22.41, 20.81, 13.90; MS (ESI)

402 (M⁺), 419 (MNH₃⁺).

4-[(4-Meth oxyph en yl)su lfon yl]-2,2-dim eth yl-octah ydr o-

1,4-th ia zon in e-3(S)-ca r boxylic Acid . The methyl ester 27

(0.44 g, 1.10 mmol) was converted to the desired carboxylic

acid following procedure A (for the preparation of compound

3a ). The resulting oil was purified by column chromatography

using 1/1 hexane/EtOAc as the eluent to provide the desired

product as a light yellow oil (0.395 g, 93%): MS (ESI) 388 (M⁺),

405 (MNH₃⁺).

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

octah ydr o-1,4-th iazon in e-3(S)-car boxam ide (22). The 4-[(4-

methoxyphenyl)sulfonyl]-2,2-dimethyl-octahydro-1,4-thiazonine-

3(S)-carboxylic acid (392 mg, 1.01 mmol) was converted to the

desired hydroxamic acid 22 following procedure A (for the

preparation of compound 4a ). The resulting solid was recrys-

tallized from CH₃CN to provide a white powder (0.25 g, 62%):

¹H NMR (CD₃OD) δ 7.72 (d, J ) 8.2, 2 H), 6.98 (d, J ) 8.2, 2

H), 4.62 (m, 1 H), 4.05 (s, 1 H), 3.81 (s, 3 H), 2.84 (m, 2 H),

2.58 (m, 1 H), 2.15 (m, 1 H), 2.02 (m, 1 H), 1.63 (m, 4 H), 1.45

(s, 3 H), 1.23 (s, 3 H); ¹³C NMR (CD₃OD) δ 166.21, 164.76,

131.44, 129.91, 115.40, 65.75, 55.54, 47.64, 32.79, 30.87, 30.77,

30.45, 29.19, 26.23; MS (ESI) 420 (MNH₃⁺), 403 (M⁺). Anal.

(C₁₇H₂₆N₂O₅S₂) C, H, N.

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

octa h yd r o-7-oxa -1,4-th ia zon in e-3(S)-ca r boxa m id e (23).

Following the procedure described above for the preparation

of 22, the 4-[(4-methoxyphenyl)sulfonyl]-2,2-dimethyl-octahy-

dro-7-oxa-1,4-thiazonine-3(S)-carboxylic acid (1.95 g, 5.0 mmol)

was converted to the desired hydroxamic acid 23 (0.25 g, 62%)

as a white powder (recrystallized from CH₃CN): ¹H NMR

(DMSO-d₆) δ 10.61 (s, 1 H), 8.85 (s, 1 H), 7.75 (d, J ) 8.9, 2

H), 7.07 (d, J ) 8.9, 2 H), 5.10 (m, 1 H), 4.05 (m, 2 H), 4.01 (s,

1 H), 3.85 (s, 3 H), 3.40 (m, 3 H), 2.83 (m, 2 H), 2.55 (m, 1 H),

1.34 (s, 3 H), 1.31 (s, 3 H); MS (ESI) 405 (MH⁺). Anal.

(C₁₆H₂₄N₂O₆S₂) C, H, N.

1-[(4-Meth oxyph en yl)su lfon yl]-h exa h yd r o-1H-a zep in e-

2-ca r b oxylic Acid . The alcohol 20 (1.3 g, 4.35 mmol) was

dissolved in acetone (100 mL) at 0 °C and freshly prepared 8

N J ones reagent was added until the solution sustained an

orange/brown color as opposed to a green color. The resulting

mixture was then stirred overnight. 2-Propanol was added to

quench the excess J ones reagent and a green solid precipitated

out. The solid was filtered through Celite, and the liquid was

concentrated under reduced pressure. The residue was then

dissolved in chloroform and washed with water several times.

The organic layer was dried over magnesium sulfate and

concentrated under reduced pressure. The product was carried

forward with no further purification: ¹H NMR (CDCl₃) δ 7.71

(d, J ) 8.6, 2 H), 6.91 (d, J ) 8.6, 2 H), 4.52 (m, 1H), 3.91 (m,

1 H), 3.82 (s, 3 H), 3.78 (m, 1 H), 3.26 (m, 1 H), 2.25 (m, 1 H),

1.64 (br m, 4 H), 1.22 (m, 4 H); ¹³C NMR (CDCl₃) δ 176.95,

162.58, 131.77, 129.19, 113.79, 58.36, 55.37, 44.93, 31.60,

29.54, 29.06, 24.76; MS (ESI) 314 (M⁺), 331 (MNH₃⁺).

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4559

N-Hydr oxy-4-[(4-m eth oxyph en yl)su lfon yl]-2,2-dim eth yl-

oct a h yd r o-7-oxa -1,4-t h ia zon in e-3(S)-ca r b oxa m id e 1,1-

Dioxid e (24). Following the procedure described above for the

preparation of 5a , the N-hydroxy-4-[(4-methoxyphenyl)sulfon-

yl]-2,2-dimethyl-octahydro-7-oxa-1,4-thiazonine-3(S)-carbox-

amide (23) (0.70 g, 1.73 mmol) was converted to the desired

hydroxamic acid 24 (0.60 g, 79%) as a white powder (triturated

from CH₃CN/H₂O): ¹H NMR (DMSO-d₆) δ 11.11 (s, 1 H) 8.95

(s, 1 H), 7.78 (d, J ) 9.0, 2 H), 7.07 (d, J ) 9.0, 2 H), 4.74 (s,

1 H), 4.23 (m, 2 H), 3.98 (m, 2 H), 3.86 (s, 3 H), 3.76 (m, 2 H),

3.40 (m, 1 H), 3.12 (m, 1 H), 1.73 (s, 3 H), 1.33 (s, 3 H); MS

(ESI) 435 (MH^-). Anal. (C₁₆H₂₄N₂O₈S₂‚0.25H₂O) C, H, N.

0.04 (s, 6 H); ¹³C NMR (CDCl₃) δ 170.12, 162.83, 130.72,

129.32, 113.87, 70.55, 65.38, 63.22, 55.44, 51.94, 46.67, 31.31,

26.21, 25.72, 18.04.

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2(S)-m eth -

oxym et h oxy-3-ter t-b u t yld im et h ylsiloxyp r op yl)-^D-p en i-

cilla m in e (31). Sodium iodide (12.0 g, 80 mmol, 4 equiv) was

stirred in dimethoxyethane (120 mL) at room temperature and

then chloromethyl methyl ether (8.2 g, 102 mmol, 5.1 equiv)

was added. A brown suspension formed which became hot to

touch. The resulting mixture was stirred for 10 min and the

methyl N-[(4-methoxyphenyl)sulfonyl]-S-(2(S)-hydroxy-3-tert-

butyldimethylsiloxypropyl)-^D-penicillamine (10.5 g, 20 mmol)

and diisopropylethylamine (14.3 g, 110 mmol, 5.5 equiv) in

dimethoxyethane (30 mL) were added. The mixture was stirred

at room temperature for 1 h and then heated to reflux for 4 h.

The reaction mixture was poured into saturated sodium

bicarbonate solution and then extracted with methylene

chloride. The organic extracts were dried (Na₂SO₄) and then

concentrated to an oil under reduced pressure. The oil was

purified by chromatography on silica gel using 8/2 hexane/

EtOAc as the eluent. The product (8.1 g, 71%) was obtained

as a yellow oil: ¹H NMR (CDCl₃) δ 7.68 (d, J ) 8.5, 2 H), 6.89

(d, J ) 8.5, 2 H), 5.49 (d, J ) 11, 1 H), 4.61 (m, 3 H), 3.81 (s,

3 H), 3.60 (m, 3 H), 3.40 (s, 6 H), 2.59 (br m, 2 H), 1.36 (s, 3

H), 1.34 (s, 3 H), 0.83 (s, 9 H), 0.03 (s, 6 H); ¹³C NMR (CDCl₃)

δ 170.01, 162.81, 130.81, 129.31, 113.84, 96.00, 77.50, 64.27,

63.08, 55.42, 51.82, 46.66, 29.85, 26.26, 25.65 18.04.

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2(S)-m eth -

oxym eth oxy-3-h yd r oxyp r op yl)-^D-p en icilla m in e (32). The

silyl ether 31 (3.16 g, 5.58 mmol) in THF (25 mL) was cooled

to 0 °C and then tetrabutylammonium fluoride (1.0 M in THF,

14 mL, 2.5 equiv) was added. The resulting mixture was

stirred at 0 °C for 30 min and warmed to room temperature.

The reaction mixture was stirred for an additional 3 h. The

reaction mixture was poured into saturated sodium bicarbon-

ate solution and then extracted with methylene chloride. The

organic extracts were dried (Na₂SO₄) and then concentrated

to an oil under reduced pressure. The oil was purified by

chromatography on silica gel using 1/1 hexane/EtOAc as the

eluent. The product (1.96 g, 78%) was obtained as a clear,

colorless oil: ¹H NMR (CDCl₃) δ 7.67 (d, J ) 8.6, 2 H), 6.83 (d,

J ) 8.6, 2 H), 5.64 (d, J ) 11, 1 H), 4.61 (m, 2 H), 3.82 (s, 3 H),

3.58 (m, 3 H), 3.38 (s, 3 H), 3.36 (s, 3 H), 2.81 (m, 1 H), 2.61

(br m, 2 H), 1.31 (s, 3 H), 1.27 (s, 3 H); ¹³C NMR (CDCl₃) δ

169.97, 162.83, 130.68, 129.28, 113.87, 96.27, 79.00, 63.77,

63.07, 55.57, 55.45, 51.92, 46.62, 29.28, 25.96, 25.65.

Meth yl N-[(4-Meth oxyph en yl)su lfon yl]-S-[(2,2-dim eth yl-

1,3-d ioxola n -4-yl)m et h yl]-^D-p en icilla m in e (29). D-Peni-

cillamine (9.56 g, 64.1 mmol) in 2 N NaOH (41.7 mL, 83.3

mmol, 1.3 equiv) was stirred at 0 °C under an argon atmo-

sphere. A solution of (S)-4-(bromomethyl)-2,2-dimethyl-1,3-

dioxolane²⁵(15.0 g, 76.9 mol, 1.2 equiv) in ethanol (30 mL)

was slowly added dropwise to the reaction mixture at 0 °C.

The resulting solution was stirred overnight at room temper-

ature and then the mixture was acidified to pH ∼ 6 with 1 N

HCl. The solvent was removed under reduced pressure to leave

a thick oil. The penicillamine adduct was then dissolved in

dioxane (75 mL) and water (75 mL) and stirred at room

temperature. Triethylamine (19.5 g, 192.3 mmol, 3 equiv) was

then added to the reaction mixture followed by 4-methoxy-

phenylsulfonyl chloride (15.9 g, 76.9 mmol). The resulting

homogeneous solution was stirred at room temperature for 18

h and then acidified to pH ∼ 2 with 1 N HCl. The solution

was poured into water and extracted with methylene chloride.

The organic extracts were dried (MgSO₄) and concentrated to

an oil under reduced pressure. The resulting oil was diluted

in methanol (30 mL) and enough diazomethane in diethyl

ether was added to form a yellow solution. The mixture was

concentrated under reduced pressure to leave a colorless oil.

Purification of the resulting methyl ester was accomplished

by chromatography on silica gel using 8/2 hexane/EtOAc as

the eluent. The desired product (11.2 g, 43%) was obtained as

a clear, colorless oil: ¹H NMR (CDCl₃) δ 7.72 (d, J ) 8.6, 2 H),

6.91 (d, J ) 8.6, 2 H), 5.44 (d, J ) 11, 1 H), 4.04 (m, 3 H), 3.82

(s, 2 H), 3.78 (d, J ) 11, 1 H), 3.63 (m, 2 H), 3.40 (s, 3 H), 2.59

(m, 2 H), 2.44 (m, 2 H), 1.39 (s, 3 H), 1.36 (s, 3 H), 1.34 (s, 3

H), 1.32 (s, 3 H); ¹³C NMR (CDCl₃) δ 170.00, 162.88, 130.61,

129.32, 113.89, 109.39, 74.64, 68.51, 62.83, 60.16, 55.44, 51.93,

46.68, 31.44, 26.62, 25.84, 25.43, 25.25; MS (ESI) 448 (M⁺),

465 (MNH₃⁺).

Meth yl 2(S)-N-[(4-Meth oxyp h en yl)su lfon yl]-S-(2,3-d i-

h yd r oxyp r op yl)-^D-p en icilla m in e (30). The acetonide 29

(11.1 g, 24.8 mmol) in THF (50 mL) was stirred at room

temperature and the 1 N HCl was added The resulting mixture

was stirred overnight at room temperature until all of the

starting material was consumed. The reaction mixture was

concentrated to remove the THF and then the aqueous layer

was extracted with methylene chloride. The organic extracts

were dried (Na₂SO₄) and concentrated to an oil under reduced

pressure. No further purification was performed. The product

(10.0 g, 99%) was obtained as a colorless oil.

Meth yl N-[(4-Meth oxyph en yl)su lfon yl]-S-(2(S)-h ydr oxy-

3-ter t-bu tyld im eth ylsiloxyp r op yl)-^D-p en icilla m in e. The

diol 30 (10.0 g, 24.5 mmol) in methylene chloride (150 mL)

was stirred at room temperature and then triethylamine (2.73

g, 27 mmol, 1.1 equiv) and (dimethylamino)pyridine (100 mg,

0.04 equiv) were added. The tert-butyldimethylsilyl chloride

(3.7 g, 24.5 mmol) was added and the resulting mixture was

stirred at room temperature overnight. The reaction mixture

was poured into dilute sodium bicarbonate solution and

extracted with methylene chloride. The organic extracts were

dried (Na₂SO₄) and then concentrated to an oil under reduced

pressure. Purification of the oil was accomplished by chroma-

tography on silica gel using 7/3 hexane/EtOAc as the eluent.

The product (10.5 g, 82%) was obtained as a clear, colorless

oil: ¹H NMR (CDCl₃) δ 7.65 (d, J ) 8.6, 2 H), 6.85 (d, J ) 8.6,

2 H), 5.62 (d, J ) 11, 1 H), 3.81 (s, 3 H), 3.60 (m, 3 H), 3.37 (s,

3 H), 2.59 (m, 2 H), 1.30 (s, 3 H), 1.28 (s, 3 H), 0.84 (s, 9 H),

Meth yl 6(S)-Meth oxym eth yl-4-[(4-m eth oxyp h en yl)su l-

fon yl]-2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r -

boxyla te (33). The methyl ester 32 (1.9 g, 4.3 mmol) in THF

(15 mL) was stirred at room temperature followed by the

addition of triphenylphosphine (1.35 g, 5.16 mmol, 1.2 equiv)

and diethyl azodicarboxylate (0.82 g, 4.73 mmol, 1.1 equiv).

The resulting solution was stirred at room temperature for 2

h. The solvent was removed and then the thick yellow oil was

diluted with methylene chloride and silica gel (20 g) was added.

The solvent was removed to leave a white powder. This powder

was placed upon a chromatography column and eluted with

8/2 hexane/EtOAc. The desired product (1.60 g, 86%) was

obtained as a colorless oil: ¹H NMR (CDCl₃) δ 7.77 (d, J )

8.8, 2 H), 6.95 (d, J ) 8.8, 2 H), 4.74 (AB, J ) 6.7, 42.3, 2 H),

4.40 (m, 2 H), 4.00 (m, 2 H), 3.86 (s, 3 H), 3.42 (s, 3 H), 3.41 (s,

3 H), 2.80 (m, 2 H), 1.55 (s, 3 H), 1.24 (s, 3 H); ¹³C NMR (CDCl₃)

δ 169.07, 163.19, 131.19, 130.12, 114.09, 95.13, 77.71, 67.29,

55.85, 51.69, 46.91, 46.73, 31.21, 29.43, 26.54; MS (ESI) 434

(M⁺), 451 (MNH₃⁺).

6(S)-Met h oxym et h yl-4-[(4-m et h oxyp h en yl)su lfon yl]-

2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxylic

Acid . The methyl ester 33 (2.2 g, 5.07 mmol) in pyridine (40

mL) was stirred at room temperature under an argon atmo-

sphere. Lithium iodide (8.15 g, 61.9 mmol, 12 equiv) was added

and the resulting solution was heated to reflux for 3 h. The

reaction mixture was cooled to room temperature and then

the solution was acidified with 1 N HCl. The mixture was

extracted with methylene chloride and then the organic

4560 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

extracts were dried (Na₂SO₄) and concentrated to an oil under

reduced pressure. The oil was purified by column chromatog-

raphy using 1/1 hexane/EtOAc as the eluent to provide the

desired product (1.98 g, 93%) as a light yellow oil: ¹³C NMR

(CDCl₃) δ 176.59, 163.66, 132.54, 132.22, 115.22, 95.30, 79.35,

56.47, 56.27, 46.22, 31.09, 29.33, 27.41; MS (ESI) 420 (M⁺),

437 (MNH₃⁺), 442 (MNa⁺).

N-Hydr oxy-6(S)-m eth oxym eth yl-4-[(4-m eth oxyph en yl)-

su lfon yl]-2,2-d im et h yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-

ca r boxa m id e (34). The 6(S)-methoxymethyl-4-[(4-methoxy-

phenyl)sulfonyl]-2,2-dimethyl-hexahydro-1,4-thiazepine-3(S)-

carboxylic acid (2.35 g, 5.6 mmol) was converted to the desired

hydroxamic acid 34 following procedure A above (for the

preparation of compound 4a ). The solid was purified by

reverse-phase HPLC using 60/40 water/acetonitrile as the

eluent to provide a white powder (1.35 g, 56%): ¹H NMR

(DMSO-d₆) δ 10.57 (s, 1 H), 8.88 (br s, 1 H). 7.79 (d, J ) 9.2,

2 H), 7.09 (d, J ) 9.2, 2 H), 4.95 (dd, J ) 3.7, 16.1, 1 H), 4.62

(dd, J ) 7.0, 34.6, 2 H), 3.99 (s, 1 H), 3.84 (s, 3 H), 3.67 (m, 1

H), 3.34 (m, 1 H), 3.28 (s, 3 H), 2.78 (m, 1 H), 2.57 (m, 1 H),

1.26 (s, 3 H), 1.12 (s, 3 H); ¹³C NMR (CD₃OD) δ 169.97, 164.76,

132.54, 131.08, 115.24, 96.09, 79.81, 64.68, 47.51, 47.30, 31.37,

29.90, 26.60; MS (ESI) 373 (M⁺). Anal. (C₁₇H₂₆N₂O₇S₂) C, H,

N.

an argon atmosphere. Lithium iodide (5.35 g, 40 mmol, 12

equiv) was added and the resulting solution was heated to

reflux for 4 h. The reaction mixture was cooled to room

temperature and then the solution was acidified with 1 N HCl.

The mixture was extracted with methylene chloride and then

the organic extracts were dried (MgSO₄) and concentrated to

an oil under reduced pressure. The product was a colorless oil

(1.40 g, 95%), which required no further purification: MS (ESI)

436 (M⁺).

N-Hydr oxy-4-[(4-Meth oxyph en yl)su lfon yl]-2,2-dim eth yl-

5(S)-p h en yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m -

id e (36a ). The 4-[(4-methoxyphenyl)sulfonyl]-2,2-dimethyl-

5(S)-phenyl-hexahydro-1,4-thiazepine-3(S)-carboxylic acid (0.28

g, 0.64 mmol) was converted to the desired hydroxamic acid

36a following procedure A above (for the preparation of

compound 4a ). The resulting solid was triturated from toluene

to provide the desired product (0.25 g, 86%) as a white

powder: ¹H NMR (DMSO-d₆) δ 11.07 (s, 1 H), 9.17 (s, 1 H),

7.00 (m, 7 H), 6.60 (d, J ) 7.7, 2 H), 5.02 (m, 2 H), 3.73 (s, 3

H), 3.69 (m, 1 H), 2.91 (m, 1 H), 2.63 (m, 1 H), 2.02 (m, 1 H),

1.59 (s, 3 H), 1.40 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 164.46,

161.67, 142.67, 133.89, 128.60, 128.15, 127.77, 126.57, 113.56,

69.47, 66.93, 55.88, 46.74, 28.83, 27.46; MS (ESI) 451 (M⁺),

468 (MNH₃⁺). Anal. (C₂₁H₂₆N₂O₅S₂) C, H, N.

N-Hydr oxy-4-[(4-Meth oxyph en yl)su lfon yl]-2,2-dim eth yl-

5(S)-p h en yl-h exa h yd r o-1,4-t h ia zep in e-3(S)-ca r b oxa m -

id e (36b). Following the procedure described above for the

preparation of 36a with (S)-3-chloro-1-phenyl-1-propanol, the

carboxylic acid 39b (1.0 g, 2.30 mmol) was converted to 0.80 g

(79%) of the hydroxamic acid 36b as a white powder (recrys-

tallized from acetonitrile/water): ¹H NMR (DMSO-d₆) δ 11.07

(s, 1 H), 9.10 (s, 1 H), 7.10-6.85 (m, 7 H), 6.58 (d, J ) 7.7, 2

H), 6.67 (m, 1 H), 4.42 (s, 1 H), 3.73 (s, 3 H), 2.92 (m, 1 H),

2.86 (m, 1 H), 2.02 (m, 1 H), 1.59 (s, 3 H), 1.32 (s, 3 H); ¹³C

NMR (DMSO-d₆) δ 173.16, 161.75, 146.95, 139.22, 136.73,

134.52, 130.03, 128.62, 127.78, 127.55, 125.10, 113.29, 67.96,

57.68, 55.62, 47.33, 35.08, 30.36, 26.55, 26.08; MS (ESI) 451

(M⁺). Anal. (C₂₁H₂₆N₂O₅S₂) C, H, N.

N-H yd r oxy-4-[(4-Met h oxyp h en yl)su lfon yl]-3-m et h yl-

h exah ydr o-1,4-th iazepin e-3(S)-car boxam ide (45). The title

compound was prepared from 2-methyl-^D-cysteine⁴⁰following

procedure A to provide 45 as a white solid: ¹H NMR (CD₃OD)

δ 7.88 (d, J ) 8.4, 2 H), 7.06 (d, J ) 8.4, 2 H), 3.82 (s, 3 H),

3.67 (m, 1 H), 3.57 (m, 2 H), 2.98 (m, 3 H), 2.52 (m, 2 H), 1.59

(s, 3 H); ¹³C NMR (CD₃OD) δ 173.14, 164.67, 133.82, 131.31,

115.23, 68.47, 56.24, 46.97, 45.47, 34.83, 33.30, 23.27. Anal.

(C₁₄H₂₀N₂O₅S₂‚H₂O) C, H, N.

N-6-Dih yd r oxy-4-[(4-m et h oxyp h en yl)su lfon yl]-2,2-d i-

m eth yl-h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxa m id e 1,1

Dioxid e (35). The hydroxamic acid 34 (1.30 g, 2.99 mmol) was

dissolved in chloroform (50 mL) and the mixture was stirred

at room temperature. A 32% solution of peracetic acid (3.03

mL, 11.97 mmol, 4.0 equiv) was added and the resulting

mixture was stirred at room temperature. The solvent and

remaining peracetic acid was removed under reduced pressure

to leave the desired product as a white solid. The solid was

recrystallized from acetonitrile to provide the product (0.76 g,

60%) as a white crystalline solid: ¹H NMR (CD₃OD) δ 7.82

(d, J ) 9.2, 2 H), 7.09 (d, J ) 9.2, 2 H), 4.44 (m, 2 H), 4.31 (m,

1 H), 3.89 (s, 3 H), 3.77 (m, 1 H0, 3.52 (m, 2 H), 1.61 (s, 3 H),

1.46 (s, 3 H); ¹³C NMR (CD₃OD) δ 163.57, 129.56, 129.16,

113.94, 65.35, 63.85, 60.50, 54.71, 54.17, 49.79, 22.51, 17.98;

MS (ESI) 423 (M⁺). Anal. (C₁₅H₂₂N₂O₈S₂) C, H, N.

Meth yl N-[(4-Meth oxyp h en yl)su lfon yl]-S-(3-h yd r oxy-

3(R)-p h en ylp r op yl)-^D-p en icilla m in e. Following procedure

A described above, (R)-3-chloro-1-phenyl-1-propanol (4.27 g,

25.0 mmol, 1.0 equiv) and ^D-penicillamine methyl ester

hydrochloride (5.0 g, 25.1 mmol) in 1,8-diazabicyclo[5.4.0]-

undec-7-ene (7.64 g, 50.2 mmol, 2 equiv) were converted to

the desired product as a colorless oil (11.6 g, 99%) under

reduced pressure. No further purification was performed: ¹H

NMR (CDCl₃) δ 7.73 (d, J ) 9.0, 2 H), 7.32 (m, 5 H), 6.94 (d,

J ) 9.0, 2 H), 5.73 (d, J ) 8.9, 1 H), 4.79 (dd, J ) 4.6, 8.6, 1

H), 3.84 (s, 3 H), 3.80 (d, J ) 9.9, 1 H), 3.37 (s, 3 H), 3.23 (br

s, 1 H), 2.58 (dd, J ) 6.9, 7.7, 2 H), 1.98 (m, 1 H), 1.72 (m, 2

H), 1.35 (s, 3 H), 1.32 (s, 3 H); MS (ESI) 468 (M⁺).

Compounds 37-44 were prepared in two steps from the

corresponding amino acid and 4-methoxyphenylsulfonyl chlo-

ride.¹⁷The carboxylic acid was converted to the desired

hydroxamic acid following procedure A for the preparation of

4a .

Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t

Tr u n ca ted MMP -1. Briefly, DNA sequence coding for Val82-

Pro249 of proMMP-1 was amplified by polymerase chain

reaction from a commercially available plasmid, p35-1 (ATCC,

Rockville, MD; Templeton et al. Cancer Res. 1990, 50, 5431-

5437) encoding human interstitial MMP-1. The PCR fragment

was ligated into the expression vector, pET-11a (Novagen,

Madison, WI) and expressed in E. coli BL21(DE3) cells. The

protein was solubilized from inclusion bodies in 6 M urea, 0.15

M NaCl (pH 7.5), refolded in 50 mM Tris-HCl (pH 7.5), 10 mM

CaCl₂, 0.1 mM zinc acetate, and then purified to homogeneity

over a hydroxamic acid inhibitor affinity column as previously

described (Moore and Spilburg Biochemistry 1986, 25, 5189-

5195). N-Terminal sequence analysis confirmed the presence

of three N-terminal sequences of Val-Leu-Thr-Glu-Gly-Asn,

Met-Val-Leu-Thr-Glu-Gly-Asn, and Leu-Thr-Glu-Gly-Asn (mi-

nor).

Meth yl 4-[(4-Meth oxyp h en yl)su lfon yl]-2,2-d im eth yl-

5(S )-p h e n y l-h e x a h y d r o -1,4-t h ia ze p in e -3(S )-c a r b o x -

yla t e. Following procedure A, the methyl N-[(4-methoxy-

phenyl)sulfonyl]-S-(3-hydroxy-3(R)-phenylpropyl)-^D-penicill-

amine (11.7 g, 25.1 mmol) was converted to the desired product

as a thick yellow oil which was subsequently diluted with

methylene chloride and silica gel (50 g) was added. The solvent

was removed to leave a white powder. This powder was placed

upon a chromatography column and eluted with 4/1 hexane/

EtOAc. The desired product (3.7 g, 33%) was obtained as a

colorless oil. The major product from the reaction resulted from

dehydration of the benzyl alcohol to form the undesired styrene

derivative: ¹H NMR (CDCl₃) δ 7.23 (m, 2 H), 6.97 (m, 5 H),

6.48 (d, J ) 9.0, 2 H), 5.64 (br m, 1 H), 5.01 (br m, 1 H), 3.83

(s, 3 H), 3.76 (s, 3 H), 3.00 (m, 1 H), 2.82 (br m, 1 H), 2.58 (br

m, 1 H), 2.11 (m, 1 H), 1.70 (m, 6 H); MS (ESI) 450 (M⁺).

4-[(4-Meth oxyph en yl)su lfon yl]-2,2-d im eth yl-5(S)-ph en -

yl-h exa h yd r o-1,4-th ia zep in e-3(S)-ca r boxylic Acid . The

methyl 4-[(4-methoxyphenyl)sulfonyl]-2,2-dimethyl-5(S)-phen-

yl-hexahydro-1,4-thiazepine-3(S)-carboxylate (1.5 g, 3.33 mmol)

in pyridine (50 mL) was stirred at room temperature under

Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t

P r oMMP -2. A partial cDNA clone for MMP-2 known as K-121

(Tryggvason J . Biol. Chem. 1990, 265, 11077-11082), was

obtained from ATCC and subcloned into the pBlueScript SK^-

(pBS) plasmid. Sanger dideoxy sequencing revealed that the

Thiazine and Thiazepine MMP Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4561

first 134 bp of the coding sequence were missing from the 5′

end of K-121. To restore the missing sequence, two overlapping

90+-mer oligonucleotides were designed and synthesized.

These, along with a 3′ antisense oligonucleotide, were used as

primers to the K-121 template in a series of polymerase chain

reaction (PCR) experiments to synthesize a full-length MMP-2

cDNA. The PCR product was then subcloned into pBS. To

express MMP-2 in the mammalian CHO D^-cell system, it was

first subcloned into the mammalian expression vector pJ T1

(J . Ting, CRD), which contains the DHFR gene. Recombinant

MMP-2/pJ T1 was Polybrene transfected into CHO D^-cells.

Clonal cell populations were isolated and screened for produc-

tion of MMP-2 mRNA using specific oligonucleotide primers

and reverse transcription PCR. Ten clones producing the

highest levels of MMP-2 mRNA were selected, and the DHFR/

MMP-2 construct was amplified by gradually increasing media

methotrexate (MTX, a DHFR inhibitor) concentration. Clonal

selection was further narrowed after assessing MMP bio-

activity in an MMP fluorescence assay (M. Anastasio, CRD)

and on zymogram gels. Those clones showing activity were

tested for MMP-2 protein production by sequential Edman

degradation protein sequencing (F. Wang, P&GP Cell &

Molecular Biology Core) and Western blot. Based on all results,

one clone was expanded for growth in roller bottles. Ap-

proximately 9 L of serum-free conditioned media with an

MMP-2 concentration of ∼35 mG/L were generated. ProMMP-2

was purified from conditioned media as previously described

(Crabbe et al. Eur. J . Biochem. 1993, 218, 431-438) with the

following modifications. Conditioned serum-free media was

reduced in volume with an Amicon S1Y30 spiral-wound

cartridge prior to chromatography on a gelatin-Sepharose 4B

column equilibrated in 25 mM Tris/HCl, 30 mM NaCl, 10 mM

CaCl₂(pH 7.5) (TNC buffer). The column was washed with

equilibration buffer before elution of the bound protein by TNC

buffer containing 1 M NaCl followed by 1 M NaCl and 10%

(by vol) dimethyl sulfoxide (DMSO) in TNC buffer. ProMMP-2

fractions were concentrated in an Amicon ultrafiltration cell

with a YM30 membrane and diafiltered against 25 mM MES/

NaOH, 30 mM NaCl, 10 mM CaCl₂(pH 6.0) (MNC buffer).

The concentrate was then chromatographed over a second

gelatin-Sepharose 4B column equilibrated in MNC buffer,

washed with equilibration buffer to remove nonbinding pro-

teins, and eluted with MNC buffer containing 0.3 M NaCl and

10% (by vol) DMSO. Elution fractions containing progelatinase

A were pooled, diluted, and loaded onto an S-Sepharose Fast

Flow column equilibrated in MNC buffer and eluted with MNC

buffer containing 0.3 M NaCl. The concentrated fractions of

purified proMMP-2 were combined and stored in MNC buffer

containing 0.3 M NaCl at -70 °C. N-Terminal sequence, amino

acid, and mass spectrophotometric analyses confirmed the

identity of the purified protein with the expected sequence for

progelatinase A.

Colla gen a se (MMP -1) In h ibition Assa y. ProMMP-1 was

activated prior to assay by treatment with trypsin. MMP-1

activity was monitored using a fluorescence assay previously

described.²⁶In a Dynatech MicroFLUOR plate, 10 nM acti-

vated collagenase was incubated with 8 µM Mca-Pro-Leu-Gly-

Leu-Dpa-Ala-Arg-NH₂in 50 mM Tris-HCl, 10 mM CaCl₂, 0.15

M NaCl, 0.05% Brij for 20-30 min at 37 °C in the presence of

varying concentrations of inhibitor. The reaction was then

quenched with 50 mM EDTA and the fluorescence increase

monitored on a Perkin-Elmer LS50B spectrofluorimeter (λ_ex

328 nm, λ_em393 nm). Activity was measured as a percentage

of control activity in the absence of inhibitor. Inhibitor

concentrations were run in triplicate, and IC₅₀determinations

were calculated from a 4-parameter logistic fit of the data

within a single experiment.

Str om elysin (MMP -3) In h ibition Assa y. ProMMP-3 was

activated prior to assay by treatment with p-aminophenyl-

mercuric acetate or trypsin. MMP-3 activity was measured by

3

following the degradation of H-reduced, carboxymethylated

transferrin.²⁷In a Multiscreen DP filtration plate (Millipore),

50 ng of activated MMP-3, 30 µg of [³H]transferrin, and

varying concentrations of inhibitor were incubated in a buffer

of 50 mM Tris-HCl, 10 mM CaCl₂, 0.15 M NaCl, 0.05% Brij

(pH 7.5) at 37 °C for 3 h. The reaction was quenched by

addition of 4.4% TCA, and TCA-soluble fragments were

counted for radioactivity. Activity was measured as a percent-

age of control activity in the absence of inhibitor. Inhibitor

concentrations were run in triplicate, and IC₅₀determinations

were calculated from a 4-parameter logistic fit of the data

within a single experiment.

Gela tin a se A (MMP -2), Ma tr ilysin (MMP -7), Neu tr o-

p h il Colla gen a se (MMP -8), Gela tin a se B (MMP -9), a n d

Colla gen a se-3 (MMP -13) In h ibition Assa y. Inhibition of

all enzymes was measured according to the representative

procedure described below for MMP-2. ProMMP-2 was acti-

vated prior to assay by treatment with 1 mM p-aminophenyl-

mercuric acetate for 45 min at 37 °C. ProMMP-9 was activated

with MMP-3 (1:20 MMP-3:MMP-9) and stored at -80 °C until

use. MMP-7, MMP-8, and MMP-13 were all supplied as active

enzymes and stored frozen until use. MMP activity was

monitored using a fluorescence assay previously described,²⁶

modified for a microtiter plate format. In a Dynatech Micro-

FLUOR plate, active enzyme was incubated with 8 µM Mca-

Pro-Leu-Dpa-Ala-Arg-NH₂in 50 mM Tris-HCl (pH 7.5), 10 mM

CaCl₂, 0.15 M NaCl, 0.05% Brij for 20-30 min at 37 °C in the

presence of varying concentrations of inhibitor. The reaction

was then quenched with 50 mM EDTA, and the relative

fluorescence was monitored on a Perkin-Elmer LS50B spec-

trofluorimeter (λ_ex328 nm, λ_em393 nm) fitted with a microplate

reader attachment. Activity was measured as a percentage of

control activity in the absence of inhibitor. Inhibitor concentra-

tions were run in triplicate, and IC₅₀determinations were

calculated from a 4-parameter logistic fit of the data within a

single experiment.

Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t

Tr u n ca ted MMP -3. Briefly, DNA sequence coding for Ala1-

Thr255 of proMMP-3 was amplified by polymerase chain

reaction from a recombinant plasmid encoding human synovial

preproMMP-3 kindly provided by Dr. Hideaki Nagase (Uni-

versity of Kansas Medical Center, Kansas City, KS) and Dr.

Markku Kurkinen (Department of Medicine, UMDNJ -Robert

Wood J ohnson Medical School). After DNA sequence verifica-

tion, this DNA fragment was ligated into the expression vector

pET-3d (Novagen, Madison, WI) and expressed in E. coli BL21

(DE3) cells as previously described (Marcy et al. Biochemistry

1991, 30, 6476-6483). ProMMP-3 was solubilized form inclu-

sion bodies in 8 M guanidine-HCl, refolded in 100 mM Tris-

HCl (pH 7.5), 10 mM CaCl₂, 0.5 mM zinc acetate, and activated

overnight at 37 °C with 1.5 mM p-aminophenylmercuric

acetate. Active, truncated MMP-3 was purified to homogeneity

over a hydroxamic acid inhibitor affinity column as described

(Moore and Spilburg Biochemistry 1986, 25, 5189-5195).

N-Terminal sequence analysis confirmed the sequence to be

consistent with the catalytic domain, Phe83-Thr255, of proMMP-

3.

MT1-MMP (MMP -14) In h ibition Assa y. MT1-MMP was

purified according to the methods reported by Ohuchi et al.²⁸

The inhibitory activity of the test compound to MT1-MMP was

determined by using the synthetic protein substrate, the

coumarinyl peptide derivative Mca-Pro-Leu-Gly-Leu-Dpa-Ala-

Arg-NH₂. A mixture of MT-MMP1 (6 nM) and substrate (4 µM)

in a total volume of 200 µL of 50 mM Tris-HCl (pH 7.5), 10

mM CaCl₂, 0.15 M NaCl, 0.05% Brij was incubated for 20-30

min at 37 °C in the presence of varying concentrations of

inhibitor. The reaction was then quenched with 50 mM EDTA,

and the relative fluorescence was monitored on a Perkin-Elmer

LS50B spectrofluorimeter (λ_ex328 nm, λ_em393 nm) fitted with

a microplate reader attachment. Activity was measured as a

percentage of control activity in the absence of inhibitor.

Inhibitor concentrations were run in triplicate, and IC₅₀

determinations were calculated from a 4-parameter logistic fit

of the data within a single experiment.

Ack n ow led gm en t. The authors thank the numer-

ous P&GP scientists associated with the MMP project

4562 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22

Almstead et al.

(21) Mitsunobu, O. The Use of Diethyl Azodicarboxylate and Tri-

phenylphosphine in Synthesis and Transformation of Natural

Products. Synthesis 1981, 1-28.

whose hard work and dedication over many years have

contributed toward the completion of this work.

(22) When the ester 3a was treated with aqueous hydroxide, some

decomposition of the desired thiazepinecarboxylic acid was

observed.

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J M990330Y

Article Doi

Design, synthesis, and biological evaluation of potent thiazine- and thiazepine-based matrix metalloproteinase inhibitors

DOI: 10.1021/jm990330y

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Article abstract of DOI:10.1021/jm990330y

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