Design of selective and soluble inhibitors of tumor necrosis factor-α converting enzyme (TACE)

4252

J . Med. Chem. 2001, 44, 4252-4267

Design of Selective a n d Solu ble In h ibitor s of Tu m or Necr osis F a ctor -r

Con ver tin g En zym e (TACE)⁺

Michael H. Rabinowitz,*^,†Robert C. Andrews,^†,J . David Becherer,^‡D. Mark Bickett,^‡Dulce G. Bubacz,^†

J ames G. Conway,^§David J . Cowan,^†Micheal Gaul,^†,&Kimberly Glennon,^†Millard H. Lambert,^†

M. Anthony Leesnitzer,^‡Darryl L. McDougald,^†Marcia L. Moss,^‡,#David L. Musso,^†and Michele C. Rizzolio^|

GlaxoSmithKline, Five Moore Drive, Research Triangle Park, North Carolina 27709

Received J une 13, 2001

A program to improve upon the in vitro, in vivo, and physicochemical properties of N-

hydroxyformamide TACE inhibitor GW 3333 (1) is described. Using the primary structure of

pro-TNF-R, along with a homology model of the catalytic domain of TACE based on the X-ray

diffraction coordinates of adamalysin, we synthesized N-hydroxyformamide TACE inhibitors

containing a P2′ arginine side chain. Introduction of nitro and sulfonyl electron-withdrawing

groups covalently bound to the P2′ guanidine moiety rendered the inhibitors electronically

neutral at cellular pH and led to potent inhibition of TNF-R release from stimulated

macrophages. Inhibitors containing these arginine mimetics were found to have increased

solubility in simulated gastric fluid (SGF) relative to 1, allowing for the incorporation of

lipophilic P1′ side chains which had the effect of retaining potent TACE inhibition, but reducing

potency against matrix metalloproteases (MMPs) thus increasing overall selectivity against

MMP1, MMP3, and MMP9. Selected compounds showed good to excellent in vivo TNF inhibition

when administered via subcutaneous injection. One inhibitor, 28a , with roughly 10× selectivity

over MMP1 and MMP3 and high solubility in SGF, was evaluated in the rat zymosan-induced

pleuisy model of inflammation and found to inhibit zymosan-stimulated pleural TNF-R elevation

by 30%.

In tr od u ction

blood cells. It is signaling through the p55 receptor that

is thought to be responsible for most of the pro-

inflammatory effects of TNF-R.¹²Both membrane-bound

and soluble forms of each receptor have been found,¹³

and are believed to play important roles in immuno-

regulation in nonpathological states. Binding of the

TNF-R trimer to TNFR leads to the induction of apop-

tosis through tyrosine kinase signaling which activates

transcription factors such as NF-κB.¹⁴

Tumor necrosis factor-R (TNF-R) is a potent proin-

flammatory cytokine that is produced by many different

cell types, including lymphocytes, mast cells, and poly-

morphonuclear cells, but primarily by activated mono-

cytes and macrophages.^1,2Overproduction of TNF-R is

strongly correlated with diverse pathologies such as HIV

infection,³rheumatoid arthritis,⁴septic shock,⁵insulin

resistance,⁶and graft rejection.⁷TNF-R is produced as

a 233 amino acid, 26 kDa membrane-bound pro form

which is found in the plasma membrane as well as

localized in intracellular compartments. It is processed

to a soluble 17 kDa form by proteolysis at Ala⁷⁶-Val⁷⁷

by tumor necrosis factor-R converting enzyme (TACE),

a membrane-bound zinc metalloprotease.^8,9Both the 26

kDa pro form and the 17 kDa mature form are active

only as trimers which are thought to induce signaling

by trimerizing the TNF receptor upon binding.^10,11

Two TNF-R receptors are known: the 55 kDa TNFR1

which is thought to be present on all cell types, and the

75 kDa TNFR2 found predominantly on endothelial and

A number of therapeutic strategies have been pro-

posed for inhibition of the pro-inflammatory TNF-R

signaling pathway. In addition to soluble receptor

therapy, inhibition of protein phosphorylation with

kinase inhibitors¹⁴and antisense treatment^15,16have

been shown to inhibit the activation of NF-κB by TNF-

R. A monoclonal humanized mouse anti-TNF antibody

(Remicade) is currently marketed for the treatment of

rheumatoid arthritis and Crohn’s Disease.¹⁷A dimeric

fusion protein of the soluble p75 receptor and human

IgG1 is currently sold as Enbrel and has proven effective

in treating rheumatoid arthritis.¹⁸A drawback of both

the monoclonal antibody and soluble receptor therapies

is the possibility of serious infections and sepsis result-

ing from complete inhibition of TNF-R signaling.¹⁹Also

potentially problematic is the development of autoan-

tibodies in patients receiving these therapies.^20,21An-

other approach has been the development of TNFR1

antagonists via modeling of the TNF receptor-ligand

complex.²²A final strategy for the intervention of TNF-

promoted inflammation is inhibition of the biosynthesis

of TNF-R either via prevention of its production at the

transcriptional level or by inhibition of its posttransla-

tional processing. Inhibitors of p38 MAP kinase act via

inhibiting the gene expression of a number of cytokines

⁺A preliminary account of this work was presented at the 220th

meeting of the American Chemical Society, August 20-21, 2000,

Washington, D.C.

* Address correspondence to current address: R. W. J ohnson

Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego,

CA92121. Tel: (858)320-3340. Fax: (858)784-3267. E-mail: mrabinow@

prius.jnj.com.

^†Division of Chemistry.

^‡Division of Molecular Biochemistry.

^§Department of Molecular Pharmacology.

^|Department of Pharmaceutical Development.

Current Address: TransTech Pharma Inc., 4170 Mendenhall Oaks

Parkway, Suite 110, High Point, NC 27265.

#

Current Address: Cognosci, Inc., 2 Davis Dr., P. O. Box 12076,

Research Triangle Park, NC 27709.

&

Current Address: 3-Dimensional Pharmaceuticals, Inc., 665

Stockton Drive, Suite 104, Exton, PA 19341-1151.

Published on Web 10/16/2001

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4253

including TNF.²³The approach that we have chosen is

inhibition of the processing of 26 kDa via inhibition of

TACE, which is expected to reduce levels of soluble 17

kDa TNF-R while not interfering with the production

or function of the 26 kDa cell-associated form.

In h ibitor Design

Discoveries made in our laboratories have already

shown that good selectivity for TACE over MMPs could

be obtained with large lipophilic substituents at either

the P1 or P1′ positions of N-hydroxyformamide inhibi-

tors.³⁶Unfortunately, these substitutions had the effect

of further decreasing the already low aqueous solubility

of this class of TACE inhibitor. We reasoned that by

incorporating the P2′ arginine (Arg) residue of 26 kDa

TNF-R into our small molecule inhibitors we could

increase the solubility of the inhibitors such as to allow

the incorporation of lipophilic groups at P1 and P1′.

Because of the expectation that the charged guani-

dinium side chain of arginine would render inhibitor

molecules unable to reach the intracellular enzyme (vide

supra), we turned our attention to a homology model³⁷

of TACE that was created by modeling the primary

sequence of the catalytic domain of TACE onto the

known X-ray structure of the snake venom metallopro-

tease adamalysin³⁸using the MVP program.³⁹(Indeed,

cationic N-hydroxyformamide P2′ Arg inhibitors syn-

thesized in parallel format displayed low nM inhibition

of TACE in the enzyme SPA assay but failed to inhibit

TNF-R release in LPS stimulated Monomac 6 cells even

at the highest doses tested (unpublished results).) Var-

ious N-hydroxyformamide inhibitors were docked into

the model using MVP. Docking calculations with argi-

nine at the P2′ position suggested that the arginine side

chain would extend generally outward from the enzyme

active site cleft. Several side chain conformations are

possible, including conformations where the guani-

dinium group makes hydrogen bonds with the backbone

carbonyl of Met345 or with the side-chain hydroxyl of

Thr347. Similar interactions with the arginine at the

same position in the 26 kDa TNFR substrate could

account for some of the enzyme’s substrate selectivity.

These interactions would depend primarily on the

hydrogen bonding character of the guanidinium group,

rather than its net electrical charge. The model sug-

gested that, even when an inhibitor makes these

hydrogen bonding interactions, there would still be

adequate space available around the guanidinium group

for further functional group substitution. This supported

our belief that small electron-withdrawing groups at-

tached to the guanidinium array of the P2′ residue of

the inhibitors would be sterically tolerated. More im-

portantly, addition of such an electron-withdrawing

group would reduce the basicity of the guanidine,

rendering it electronically neutral at physiological pH.⁴⁰

After this analysis, the X-ray structure of the catalytic

domain of TACE was published,⁴¹supporting the as-

sumptions made using our homology model. The docking

of a P2′-nitroguanidinium inhibitor into the crystal-

lographic structure of TACE is shown in Figure 1.

The suggestion that TACE is a metalloprotease was

first proposed in 1994^24,25,26when it was discovered that

small molecule metalloprotease inhibitors could inhibit

soluble TNF-R production in stimulated macrophages

with little effect on the production of other cytokines.

Subsequently in 1997, TACE was isolated, purified,

cloned, and characterized.^27,28In addition to a Zn-

binding HExxHxxGxxH motif, TACE was found to

contain a signal peptide domain, a “cysteine-switch” pro-

domain, a disintegrin domain, a cysteine-rich domain,

a transmembrane segment, and a cytosolic tail²⁹making

it a member of the ADAM/reprolysin family of metal-

loproteases. Its primary sequence is most closely related

to that of the metalloprotease ADAM-10.⁹TACE is

found to a small degree on the cell surface but is

considered to exist predominantly in the perinuclear

space, and it is hypothesized that the action of TACE

on the ectodomain shedding of TNF-R may occur largely

in an intracellular compartment.³⁰Indeed, we have

observed a lack of correlation between small molecule

TACE inhibitors in the enzyme and whole-cell TNF

release assays supporting the idea that therapeutically

useful small molecule TACE inhibitors not only have

to be potent inhibitors of the free enzyme, but also must

have the appropriate physicochemical properties to

allow cell penetration.³¹

The goals of our inhibitor program were to create a

soluble, orally bioavailable small molecule inhibitor of

TACE. In addition, because of reports of tendonitic

adverse effects leading to joint pain in humans treated

with nonselective MMP inhibitors,^32,33we desired good

selectivity for TACE over MMPs such as collagenase 1

(MMP1), gelatinase B (MMP9), and stromelysin 1

(MMP3). We have developed a number of potent small

molecule N-hydroxyformamide inhibitors with either

2-pyridyl or 2-thiazolyl amides occupying the P3′ posi-

tions, of which 1 is representative. Compound 1 is a

potent inhibitor of TACE that has been shown to com-

pletely inhibit LPS-induced TNF-R production in mice

after oral administration.³¹It has also been shown to

be effective in the peptidoglycan polysaccharide (PGPS)

reactivation arthritis model in the rat.^34,35However, this

compound is not selective for TACE versus other MMPs

leading to the possibility of tendonitic side effects in

vivo. Additionally, 1 was found to have a solubility in

simulated gastric fluid (SGF) of 0.08 mg/mL making

formulation and further compound development poten-

tially problematic. In this article, we disclose our efforts

toward identifying TACE inhibitors with improved

aqueous solubility and metalloprotease selectivity.

Targets chosen to evaluate this hypothesis were

N-hydroxyformamide inhibitors containing nitro-Arg,

methanesulfonyl-Arg, and 2-pyridylsulfonyl-Arg occupy-

ing the P2′ position. Additionally, in hopes of stabilizing

the central amide bond in analogy to P2′ branched side

chains similar to those found in orally bioavailable

inhibitors such as 1 and marimastat, we sought inhibi-

tors containing P2′ â-methyl and â,â-dimethyl substi-

tuted Arg (e.g., 2 and 3, EWG ) electron withdrawing

group).

4254 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

F igu r e 1. Compound 28a docked in the active site binding pocket of the X-ray structure of the TACE catalytic domain.⁴¹The

nitroarginine-containing P2′ side chain can be seen extending into solvent space. Compound 28a was docked and minimized

using the MVP program.³⁹Hydrogen, nitrogen, oxygen, sulfur, and zinc atoms are shown in white, blue, red, yellow, and purple,

respectively. Carbon atoms are blue and green in the protein and inhibitor, respectively. The surface of the protein is depicted

with a Connolly dot surface.

The inhibition of TACE was measured with a scintil-

lation proximity assay (SPA) peptide construct as

described previously.⁴³

Compounds that displayed significant potency in the

SPA assay were subsequently evaluated in a whole-cell

assay to determine their potency of inhibition of TNF-R

release by the action of TACE under more physiologi-

cally relevant conditions. Thus, human Mono Mac-6

mononuclear cells were preincubated with inhibitor and

then stimulated with LPS. Soluble TNF-R released in

the media was quantified via ELISA assay.^26,44

Compounds with sufficient potency in the whole-cell

TNF-R release assay were then evaluated for their in

vivo activity following an inflammatory challenge. TACE

inhibitor was administered by subcutaneous (sc) injec-

tion or oral gavage (40 mg/kg) to female mice 10 min

prior to intraperitoneal (ip) injection of lipopolysaccha-

ride (LPS, 0.2 mg/kg). Plasma levels of TNF-R were

measured at 90 min post LPS injection and quantified

using an ELISA assay.

A zymosan-induced rat pleurisy was used to evaluate

compounds in a model of inflammation. Intrapleural

injection of zymosan produced an inflammatory re-

sponse in the pleural cavity signified by neutrophil

Biology

infiltration and elevated cytokine levels. TNF-R titers

in the pleural exudate were quantified by ELISA assay

after pretreatment with either vehicle or TACE inhibi-

tor.³⁵

Metalloprotease inhibitors 28a -u were evaluated in

vitro to determine their inhibition potency against

matrix metalloproteases MMP1, MMP3, and MMP9

using a fluorescence-based peptide assay.⁴²

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4255

Sch em e 1^a

a

Reagents: (i) LDA, alkyl halide, THF, DMPU, -78 °C to 0 °C, 12-48 h; (ii) LiOH, THF, MeOH, H₂O, 12 h; (iii) THPONH₂, DCC,

EtOAc, 0 °C to RT, 3-6 h; (iv) a. MsCl, pyridine, 0 °C, 6 -12 h; b. Me₂CO, K₂CO₃, reflux, 12 h; (v) NaOH, dioxane, H₂O, RT, 24 h; (vi)

AcOCHO, pyridine, DCM, 0 °C, 1 h; (vii) H₂Pd/C or Pd(OH)₂, MeOH, 3-13 h; (viii) H₂, Rh/Al₂O₃, MeOH, 70 psi, 24-48 h.

Ch em istr y

NaOH in aqueous dioxane followed by formylation with

freshly prepared acetic formic anhydride in pyridine.⁴⁷

Acids 7j-t could then be used either in their crude

forms or purified on silica gel as their pentafluorophenyl

esters (prepared by esterification with pentafluorophe-

nol and DCC).

N^G-Substituted arginine analogues 9, 11, and 15 were

synthesized as shown in Scheme 2. Coupling of com-

mercially available NR-Boc-N^G-nitroArg (8) with 2-ami-

nothiazole followed by Boc removal afforded P2′-P3′

right-hand piece 9. Sulfonylation of NR-Boc Arg (10)

with 2-chlorosulfonylpyridine⁴⁸in water followed by

coupling with 2-aminothiazole and Boc deprotection

provided P2′ N^G-2-pyridylsulfonyl coupling partner 11.

Synthesis of N^G-methanesulfonyl Arg derivative 15

could not be achieved by sulfonylation with methane-

sulfonyl chloride owing to this reagent’s instability in

water. An indirect route involved the construction of the

mesylguanidine moiety by treatment of NR-Boc orni-

thine (12) with dimethyl methylsulfonyldithioimidocar-

Synthesis of the P1-P1′ left-hand portions of the

N-hydroxyformamide inhibitors was carried out as

depicted in Scheme 1. Enantioenriched (R)-â-hydroxy-

esters 4a -g were prepared by Noyori asymmetric

reduction of the corresponding â-ketoesters.⁴⁵Frater-

Seebach alkylation⁴⁶of the lithium enolates of 4a -g

with an appropriate alkyl, allylic, or benzylic halide

resulted in anti aldols 5a -i, 5s, and 5t. Diastereose-

lectivity of 10:1 to 15:1 was typically observed, with the

minor syn diastereomer being removed by silica gel

chromatography either at this stage, or at the azetidi-

none stage. Hydrogenation of olefinic aldol products (H₂,

Pd) and the benzylic aldol products (H₂, Rh) resulted

in fully saturated esters 5j-r . Ester hydrolysis (aqueous

LiOH), hydroxamate formation (THPONH₂, DCC), and

cyclization (MsCl, pyridine then K₂CO₃) gave rise to the

corresponding (3R, 4S) azetidinones 6j-t. The com-

pleted left-hand acid coupling partners were then

prepared from 6j-t through â-lactam hydrolysis with

4256 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

Sch em e 2^a

a

Reagents: (i) 2-Aminothiazole, EDC, DMF, 25-40 °C, 24-48 h; (ii) HCl, dioxane, 25 °C, 0.3-1 h; (iii) 2-pyridylsulfonyl chloride,

NaOH, acetone, H₂O, 0 °C, 5 h; (iv) MsNdC(SMe)₂, NaOH, EtOH, H₂O, 65 °C then reflux, 15 h; (v) i-BuOCOCl, TEA, DMF, -23 °C, 0.75

h., then 2-aminothiazole, -23 °C to RT; (vi) NH₃AgNO₃, MeCN, 0 to 25 °C, 2 h.

bonate to afford N-methanesulfonylisothiourea 13. Cou-

pling with 2-aminothiazole via the mixed anhydride,

guanidine formation (NH₃, AgNO₃), and Boc deprotec-

tion resulted in the desired methanesulfonylguanidine

amino amide 15.

Attention was then turned toward the synthesis of

â-methyl and â,â-gem-dimethyl arginine right-hand

pieces.

facile cyclization in both the mesylation and azide

reduction steps, promoted by the gem-dimethyl effect.⁵¹

(See Supporting Information).

The â-methyl arginine derivative 24 (Scheme 4) was

transformed to N^G-nitro-NR-Boc arginine (25) via the

following four-step sequence: (1) Cbz deprotection (Pd/

C, cyclohexadiene); (2) ester cleavage (TFA); (3) nitration

(fuming HNO₃, fuming H₂SO₄); and (4) amine protection

(Boc₂O, NaHCO₃). Amide bond formation with 2-ami-

nothiazole and Boc deprotection as before provided the

desired â-methylnitroarginine right-hand piece (27).

The desired N-hydroxyformamide inhibitors 28a -u

were prepared by DEPC mediated amide coupling of

THP-protected acids 7j-t with amino amides 9, 11, 15,

23, and 27 in DMF (Scheme 5). Alternatively, the

coupling could be achieved via pentafluorophenyl ester

activation of the carboxylic acid. The crude coupled

products were purified by silica gel chromatography. In

the case of compounds 28q, 28t, and 28u , the minor

diastereomers produced from the coupling of stere-

ochemically impure 23 and 27 could be removed at this

stage by chromatography. Compounds 28p , 28r , and

28s were evaluated as 85:15 to 90:10 mixtures of

diastereomers. Final compounds were then obtained by

removal of the THP protecting group with 80% AcOH

at 40 °C and purified by trituration to provide TACE

inhibitors 28a -u as broad-melting amorphous solids.

Amino acid 16a ⁴⁹(85% ee, 9:1 mixture of diastereo-

mers) was converted to Cbz-protected tert-butyl ester

17 (Scheme 3) by the sequential treatment with aqueous

NaOH, then CbzCl, followed by esterification with

dimethylformamide di tert-butyl acetal. Hydroboration

of alkene 17 with in situ generated thexylborane fol-

lowed by H₂O₂oxidation provided primary alcohol 18

in 88% isolated yield with no evidence of regioisomeric

2° alcohol formation. Mesylation and azide formation

provided 19 without pyrrolidine formation (as was

observed in the attempted synthesis of the â,â-dimethyl

Arg analogue (vide infra). Azide reduction via hydro-

genation over Lindlar catalyst resulted in amine 20

which could be converted to N-mesyl isothiourea 21

through the action of dimethyl methylsulfonyldithio-

imidocarbonate (71% two steps) or guanidinium salt 24

by treatment with 3,5-dimethylpyrazole-1-carboxami-

dine nitrate (72%; Scheme 4). Transformation of 21 to

Cbz protected right-hand coupling fragment 22 was

completed as described for 15 (vide supra).

Resu lts a n d Discu ssion

An attempt to prepare the â,â-dimethyl Arg analogue

of 23 (cf. 3) using a similar sequence, starting with the

known â,â-dimethyl amino acid 16b⁵⁰was hampered by

Inhibitors containing a P2′ nitroarginine side chain

(28a -f, k , l) showed excellent inhibition of TACE in our

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4257

Sch em e 3^a

a

Reagents: (i) NaOH, H₂O, 80 °C, 1 h, then CbzCl, 0 to 23 °C, 30 min; (ii) (t-BuO)₂CHNMe₂, toluene, 80 °C, 1 h; (iii) thexylborane,

THF, 0 °C, 1 h, then H₂O₂, pH 7.0; (iv) a. MsCl, pyridine, DCM, 0 °C, 12 h; b. NaN₃, DMF, 40 °C, 12 h; (v) H₂, Lindlar catalyst, TEA,

MeOH, 1 h; (vi) (MeS)₂CdNMs, NaOAc, EtOH, H₂O, reflux; (vii) TFA, thioanisole, H₂O, DCM (88:5:5:2), 23 °C, 60 min; (viii) a. i-BuOCOCl,

NMM, DMF, -23 °C, 0.5 h; b. 2-aminothiazole, -23 °C to RT, 14 h; (ix) NH₃, AgNO₃, MeOH, 0 to 23 °C, 2 h; (x) HBr, AcOH, 23 °C, 0.5

h.

Sch em e 4^a

a

Reagents: (i) 3,5-Dimethylpyrazole-1-carboxamidine nitrate, TEA, DMF, 23 °C, 72 h; (ii) 10% Pd/C, 1,4-cyclohexadiene, MeOH, reflux,

60 min; (iii) TFA, H₂O, 6 h; (iv) fuming HNO₃, fuming H₂SO₄; (v) di-t-butyldicarbonate, NaHCO₃, H₂O, THF, RT, 18 h; (vi) i-BuOCOCl,

TEA, DMF, -15 °C, then 2-aminothiazole, -15 to 23 °C, 18 h; (x) HCl, dioxane, 23 °C, 2 h.

SPA assay (IC₅₀) 4.3-49 nM; Table 1). Variation of

the P1 substituent between straight chain and branched

aliphatic groups had little effect on enzyme potency.

However, the placement of a benzyl group at this

position reduced affinity for TACE by ca. 10-fold (cf. 28a

and 28l). These results are consistent with the expecta-

tion that the P1 substituent, like the P2′ substituent,

projects out from the enzyme active site into surround-

ing solvent, permitting moderate steric bulk at this

position. With respect to TACE inhibition, substitution

at the P1′ position also seems to be tolerant of a variety

of alkyl and aromatic groups such as isobutyl (28a and

28l), cyclohexyl (28b, 28d , and 28k ), 4-methylcyclohexyl

(28e and f), and simple aromatic groups (5-methylthien-

2-yl, 28c). Selectivity for TACE over MMP1, MMP9, and

MMP3, however, is strongly influenced by the choice of

substituent at this position. Comparing 28a , 28c, 28d ,

and 28f, where the P1 substituent is held constant (n-

Pr), selectivities progress from low TACE selectivity

over MMPs (28a , 28l, P1′ ) isobutyl) to high selectivity

against MMP1 (IC₅₀>2500 nM) (28c, P1′ ) 5-meth-

ylthien-2-yl). In the case of the inhibitors shown in Table

1, three compounds, 28b, 28c, and 28f, display greater

than 10-fold selectivity against MMP9. These represent

some of the most selective TACE inhibitors over MMP9

in the N-hydroxy formamide series.

As mentioned above, N-hydroxyformamide inhibitors

containing the charged guanidinium residue of arginine

in the P2′ position fail to inhibit TNF release from LPS-

stimulated mononuclear cells. However, as Table 1

shows, the electronically neutral nitroarginine inhibitors

show good to excellent inhibition of TNF release from

4258 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

Sch em e 5^a

a

Reagents: (i) DEPC, NMM, DMF, 12-16 h, 23 °C; (ii) AcOH, H₂O, 40 °C, 16-20 h; (iii) pentafluorophenol, DCC, 0-23 °C, 2h. *See

tables 1-3 for the identities of R, R′, R′′, and R′′′.

Ta ble 1. In Vitro and Solubility Profiles of P2′ Nitro-arginine Inhibitors

a

b

IC₅₀(nM). Standard errors for enzyme assays were typically (30% of the mean or less. SPA assay (see text). ^cFluorescence assay

(see text). Inhibition of TNF release from mononuclear cells following LPS stimulation (see text); IC 50 (nM). ^eSolubility in mg/mL in

d

simulated gastric fluid. “>” Indicates full solubility at the highest concentration tested. ^fNot determined.

whole cells (IC₅₀23-420 nM), presumably due to the

neutral arginine mimetic’s ability to diffuse or be

transported through the plasma membrane.

The solubility of P2′ nitro-arginine inhibitors also

displayed, in general, a marked improvement over

earlier compounds such as 1. With the exception of the

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4259

Ta ble 2. In Vitro and Solubility Profiles of P2′ Methanesulfonyl-arginine and 2-Pyridylsulfonyl-arginine Inhibitors

a

b

IC₅₀(nM). Standard errors for enzyme assays were typically (30% of the mean or less. SPA assay (see text). ^cFluorescence assay

(see text). Inhibition of TNF release from mononuclear cells following LPS stimulation (see text); IC₅₀(nM). ^eSolubility in mg/mL in

d

simulated gastric fluid. “>” Indicates full solubility at the highest concentration tested. ^fNot determined.

trifluorinated inhibitor 28k, all compounds tested showed

solubilities in SGF (Table 1) from 2- to over 20-fold

greater than that of 1 (0.08 mg/mL, SGF; Table 1). In

the case of the TACE inhibitors which are selective over

MMP1 (28b, 28d , and 28f) increased solubility was

observed even with lipophilic cyclohexyl and 5-methyl-

cyclohexyl substituents at P2′ (cf. 1).

Evaluation of P2′ sulfonylated arginine derivatives

(Table 2) shows a similar pattern of high activity of

TACE inhibition (IC₅₀6.0-42 nM) and selectivity over

MMPs. As with the P2′ nitroarginine inhibitors, cy-

cloalkyl and thienyl substituents at P1′ appear to

attenuate the inhibition of MMP1, MMP3, and MMP9.

However, in the case of the P2′ methanesulfonyl- and

2-pyridylsulfonylarginine inhibitors, inhibition of TNF-R

release from whole cells appears to be roughly 2-20-

fold less potent then their nitroarginine analogues.

This discrepancy is not accounted for by simply consid-

ering the difference in enzyme inhibition potencies, and

may be due to a difference in the degree of plasma

membrane passive diffusion of the two subtypes of

inhibitor.

Solubility in SGF was measured for three of the

compounds listed in Table 2, and methanesulfonyl- and

2-pyridylsulfonylarginine inhibitors were found to be

2-14 times more soluble than 1, with improved selec-

tivity over MMPs (Table 2).

Because the â-methylated arginine inhibitors were

prepared as chimeras of 1 and Marimastat in hopes of

achieving oral bioavailability in th P2′ Arg series, only

P1′ isobutyl inhibitors were initially evaluated. N^G-

Nitroarginine and methanesulfonylarginine inhibitors

bearing methylation at the â-carbon of the central

arginine (28p -u ; Table 3) were evaluated in enzyme

and whole cell assays. Methylation of 28n , 28m , and

28a (resulting in compounds 28p , 28r , and 28s, respec-

tively) resulted in no loss of potency for TACE inhibition.

Other inhibitors (28u , 28q, and 28t) also showed

excellent inhibition of TACE. For compounds in Table

3, good selectivities over stromelysin-1 (MMP3) were

found when P1 was methyl, suggesting a preference of

MMP3 for larger alkyl groups at this position in the

N-hydroxyformamide series. Selectivities for MMP1 and

MMP9 were modest, supporting our earlier findings that

4260 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

Ta ble 3. In Vitro and Solubility Profiles of P2′ â-Methyl Methanesulfonylarginine and Nitroarginine Inhibitors

a

b

IC50 (nM). Standard errors for enzyme assays were typically (30% of the mean or less. SPA assay (see text). ^cFluorescence assay

(see text). Inhibition of TNF release from mononuclear cells following LPS stimulation (see text); IC₅₀(nM). ^eSolubility in mg/mL in

d

simulated gastric fluid. “>” Indicates full solubility at the highest concentration tested. ^fNot determined.

Ta ble 4. Inhibition of Soluble TNF-R Production in

TACE selectivity over these enzymes is poor for P1′

isobutyl-containing inhibitors. Although inhibition of

TNF-R release for nitroarginine inhibitors 28s and 28t

was about the same as that for the unmethylated

analogues (cf. 28a ), the addition of a â-methyl group to

the sulfonylarginine derivatives appears to enhance

inhibition potency in the whole-cell assay. Methylated

compounds 28p and 28r show 17- and 3-fold increased

potency over their nonmethylated analogues, 28n and

28m , respectively. The solubility in SGF was measured

for two of the compounds (28u and 28t) and found to

be 10- and over 20-fold greater than that of 1 (Table 3).

LPS-Stimulated Mice

%inhibition^a

compound

sc^b

96***

po^c

-29

-3

-27, -39*

-6

-19

-9

-28

-24, -34

32

28

-5

28a

28b

28d

28e

28f

28h

28j

28k

28o

28p

28q

28r

28s

28t

56***

72***, 75***

71***

64***

ND^d

73***

ND^d

In Vivo

ND^d

7

ND^d

-10

Some of the compounds were evaluated for their

efficacy in reducing plasma TNF-R levels in mice after

stimulation with LPS. Nitroarginine inhibitors 28a ,

28b, 28d , 28e, 28f, and 28k were evaluated by both

subcutaneous and oral administration at 40 mg/kg body

weight. After sc injection, P2′ nitroarginine inhibitors

all showed good to excellent inhibition of TNF release

in the LPS-treated mouse (Table 4). The most efficacious

compound, 28a , caused near complete inhibition of

soluble TNF-R production induced by LPS challenge.

These compounds were then evaluated for oral efficacy

ND^d

-2

28u

ND^d

-3

a

Percent inhibition of soluble TNF-R as compared to vehicle-

treated controls. Vehicle-treated compared to compound using

b

2-sided t-test: *, P < 0.05; ***, P < 0.001. sc ) 40 mg/kg

subcutaneous injection 10 minutes prior to ip LPS injection. ^cpo

) 40 mg/kg oral dose given by gavage 10 minutes prior to ip LPS

d

injection. Not determined.

at 40 mg/kg by oral gavage as a suspension. As Table 4

shows, none of the nitroarginine P2′ inhibitors had any

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4261

Ta ble 5. Inhibition of TNF-R Production in Rat Zymosan

Inflammation Model

with significant aqueous solubility to allow for parenter-

al administration in acute settings.

% inhibition

of TNF-R production

compound

total TNF-R^a

Exp er im en ta l Section

vehicle

1442 ( 87.09 (n ) 6)

1004 ( 46.74^c(n ) 6)

Gen er a l. All commercial chemicals and solvents were

reagent grade and were used without further purification

unless otherwise specified. The following abbreviations have

been used: tetrahydrofuran (THF), ethyl acetate (EtOAc),

dimethyl sulfoxide (DMSO), dichloromethane (DCM), trifluo-

roacetic acid (TFA), dimethylformamide (DMF), methanol

(MeOH), lithium diisopropylamide (LDA), 1,3-dimethyl 3,4,5,6-

tetrahydro-2(1H)-pyrimidinone (DMPU), ethylenediamine tet-

racetic acid (EDTA), thin-layer chromatography (TLC), 1,3-

dicylcohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-

3-ethylcarbodiimide hydrochloride (EDC), tetrahydropyran-2-

yl (THP), t-butoxycarbonyl (Boc), methanesulfonyl (Ms),

triethylamine (TEA), diethyl phosphorylcyanide (DEPC), 4-m-

ethylmorpholine (NMM), and acetic acid (AcOH). Details of

the general experimental procedures and instrumentation

employed have been previously described.⁵³

In Vitr o. TACE SP A Assa y.⁴³Inhibition constants for

inhibitors of TACE were determined in 96-well plates using a

scintillation proximity assay (SPA). A tritium labeled, bioti-

nylated peptide based on the cleavage sequence of 26kDa

TNF-R (Biotin-SPLAQAVRSSSRTP*S-NH₂) was synthesized

for substrate in these assays. Human TACE, isolated in a

microsomal preparation from Mono Mac-6 monocytic leukemia

cells, was diluted 1/25 into 10 mM HEPES, pH 7.5, for

incubation with TACE substrate. After 1 h at 37 °C, substrate

turnover was determined in the presence or absence of

inhibitor using streptavidin SPA beads (Amersham). Percent-

age inhibition vs log [inhibitor] was plotted to determine the

IC₅₀where Ki ) IC₅₀/(1 + [S]/Km).

28a ^b

30.%

a

Results are pg in pleural exudate as assayed by ELISA (see

b

text for details). Compound (50 mg/kg) administered sc in

aqueous 5% EtOH, 5% propylene glycol, 10% Cremephore, 10%

Molecusol suspension (5 mL/kg body weight). ^cP < 0.01 (compared

with vehicle control using unpaired two-sided t-test).

effect on TNF-R production when dosed orally. Solubility

was most likely not a major factor in this lack of oral

efficacy as all of the six compounds except for 28k had

greater solubility in simulated gastric fluid than 1,

which shows >90% efficacy at inhibiting TNF-R produc-

tion in the same model when dosed orally at 40 mg/kg.³¹

As we had set improvement over 1 as a goal of this

current study, we evaluated the remaining inhibitors

only via the oral route. The â-methyl nitroarginine

inhibitors, 28s and 28t, did not show inhibition of

TNF-R production when dosed orally in the mouse,

suggesting that the underpinnings of oral bioavailability

in compounds such as Marimastat and 1 are more

complex than the presence of steric bulk at the â-carbon

of the P2′ side chain. Compounds 28o and 28p showed

a nonsignificant 30% and 28% inhibition of TNF-R

production after oral dosing. There may be some specific

metabolism or efflux transport associated with the

nitroarginine and sulfonylarginine groups as incorpo-

rated in the inhibitors studied in the present work.

Efflux transport or low adsorption may be more likely

than metabolic liability owing to the known metabolic

stability of nitroarginine itself.⁵²

MMP Assa ys.⁴²Recombinant catalytic domains of MMP-1

and full length active MMP-3 were expressed and purified from

E. coli. Enzymes were refolded in 200 mM NaCl, 50 mM Tris,

5 mM CaCl₂, 10 µM ZnSO₄, and 0.01% Brij 35, pH 7.6, for 1 h

prior to the assay. The catalytic domain of proMMP-9 was

purified from the media of baculovirus infected T. ni cells.

Assays were run in a total volume of 0.180 mL of assay buffer

containing 200 mM NaCl, 50 mM Tris, 5 mM CaCl₂, 10 µM

ZnSO₄, and 0.01% Brij 35, pH 7.6. MMP-1, MMP-3, and

MMP-9 concentrations were adjusted to 1.0 nM, 5 nM, and

0.1 nM, respectively. Enzymes were preincubated with inhibi-

tor for 20 min at room temperature and the reactions were

initiated with the addition of the fluorogenic substrate, Dnp-

Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH₂. Dose responses

were generated using eleven point 3-fold dilutions of the

inhibitor. Product was measured using excitation and emission

wavelengths of 343 and 450 nm, respectively.

Compound 28a was evaluated in the zymosan-

induced rat pleurisy model of inflammation. When 28a

was administered in a single 50 mg/kg sc bolus dose 10

min prior to zymosan injection, at 4 h a significant 30%

(P < 0.01) reduction in total pleural cavity TNF-R was

found (Table 5). For comparison, 1 at 80 mg/kg po

showed a significant (P < 0.001) 89% inhibition of

pleural TNF-R at 4 h compared to vehicle control.³⁵

Con clu sion s

We have discovered potent and selective inhibitors of

TACE in the N-hydroxyformamide class of prime-side

inhibitors. By incorporating mimetics of the P2′ Arg of

26 kDa TNF-R into this class of TACE inhibitor, greatly

increased aqueous solubility was observed. By selec-

tively blocking the production of soluble 17 kDa TNF-

R, our approach to TNF inhibition may be viewed as

complimentary to that of agents which block both the

17 kDa as well as the membrane-associated 26 kDa

forms. Therapeutic blockade of only the mature 17 kDa

form could offer the advantages of allowing normal

inflammatory responses to progress without potentially

leading to immune suppression. Additionally, a small

molecule approach to TNF blockade could provide better

tolerability and patient compliance than with injectable

therapies. Inhibitors in this class were shown to be

effective in vivo at lowering systemic TNF-R levels in

the LPS-challenged mouse as well as lowering TNF-R

in the pleural cavity of zymosan challenged rats.

Compounds of the general formula 28 may thus be

considered to be potent, selective inhibitors of TACE

Cellu la r Assa y.²⁶Inhibition of lipopolysaccharide (LPS)-

induced TNF release was measured in human Mono Mac-6

cells.⁴⁴Mono Mac-6 cells in RPMI 1640 media with 10% fetal

bovine serum (FBS) were preincubated for 10 min with

compounds 28a -u and then stimulated with 10 ng/mL phorbol

12-myristate 13-acetate (PMA; Sigma Chemical Co.) and 30

ng/mL LPS (Sigma). After 2 h, TNF-R was measured in the

media by ELISA kit (R&D Systems, Minneapolis, MN).

In Vivo. LP S-In d u ced TNF -R Relea se in Mice. Com-

pounds were administered either via sc injection, dissolved in

DMSO and added to a mixture of 0.9% NaCl and 30% Trappsol

HPB-20 (CTD Inc., Gainsville, FL) for a final DMSO concen-

tration of 1%, or by oral gavage (0.5% hydroxypropyl methyl-

cellulose, 0.1% Tween 80 as a homogenized suspension). The

sc dose was administered as a 0.2-mL solution and the oral

suspension was administered by gavage at 5 mL/kg body

weight, both given 10 min prior to LPS injection. C3H/hen

female mice were injected ip with 0.2 mg/kg body weight of

LPS in phosphate buffered saline and were killed 90 min after

LPS injection by carbon dioxide asphyxiation. Blood was taken

immediately from the inferior vena cava, and plasma was

prepared and frozen at -80 °C. Plasma concentrations of

TNF-R were measured by ELISA (R&D Systems, Minneapolis,

4262 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

MN). TNF-R concentrations from n ) 7 mice receiving test

compound were compared to those from a control group

receiving vehicle alone (n ) 7).

(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-5-({(Z)-(m et h yl-

su lfan yl)[(m eth ylsu lfon yl)im in o]m eth yl}am in o)pen tan oic

a cid (13). An aqueous solution of NaOH (3.1 mL, 2.0 N) was

added to a mixture of NR-Boc-^L-ornithine (1.30 g, 5.59 mmol)

and MsNdC(SMe)₂(1.67 g, 8.38 mmol) in 30 mL of absolute

ethanol and stirred at 60-65 °C. After 15 h, the reaction

mixture was refluxed for a further 2 h. The ethanol was

removed in vacuo and the resulting slurry was added to H₂O

and extracted with ether (3×), acidified to pH 1 with 1 N HCl,

and extracted with EtOAc (4×). The combined EtOAc layers

were washed with H₂O and brine and dried over MgSO₄.

Filtration and concentration provided 13 (2.40 g, 112%) as a

white oily foam, contaminated by a small amount of MsNd

C(SMe)₂. ¹H NMR (400 MHz, DMSO-d₆) (major rotamer) δ 12.4

(bs, 1H), 7.8 (bs, 1H), 7.06 (bd, J ) 7.2 Hz, 1H), 3.83 (m, 1H),

3.4 (bs, 1H), 3.24 (m, 2H), 2.86 (s, 3H), 2.44 (s, 3H), 1.54 (m,

4H), 1.34 (s, 9H) ppm. ESIMS m/z (pos. ion) 384 (M+H)⁺, 406

(N+Na)⁺, (neg. ion) 382 (M-H)⁺.

ter t-Bu tyl (1S)-4-({(Z)-(m eth ylsu lfan yl)[(m eth ylsu lfon yl)-

im in o]m eth yl}a m in o)-1-[(1,3-th ia zol-2-yla m in o)ca r bon -

yl]bu tylca r ba m a te (14). Compound 13 (3.03 g, 7.90 mmol)

and TEA (0.799 g, 7.90 mmol, 1.10 mL) were dissolved in DMF

(40 mL) and cooled to -23 °C. i-BuOCOCl (1.08 g, 1.03 mL,

7.90 mmol) was added dropwise with stirring. After 45 min,

2-aminothiazole (0.791 g, 7.9 mmol) was added, and stirring

continued for a further 30 min at -23 °C. The reaction mixture

was allowed to warm to ambient temperature over 15 h then

added to a half-saturated NaCl solution and extracted with

EtOAc (3×). The combined organic layers were washed with

1 N NaHSO₄, 5% NaHCO₃, and brine, and dried over MgSO₄.

Purification on SiO₂(hexanes/EtOAc 4:1, then 100% EtOAc)

provided amide 14 as a white foam (3.30 g, 7.09 mmol, 90.%).

¹H NMR (400 MHz, CDCl₃) δ 7.9 (bs, 1H), 7.54 (d, J ) 3.6

Hz), 7.01 (d, J ) 3.6 Hz, 1H), 5.25 (bs, 1H), 4.45 (m, 1H), 3.30

(m, 2H), 2.98 (s, 3H), 2.35 (s, 3H), 1.95 (m, 1H), 1.73 (m, 2H),

1.65 (m, 2H), 1.44 (s, 9H) ppm. ESIMS m/z 466 (M+H)⁺, 488

(M+Na)⁺.

(2S)-2-Am in o-5-({(E)-a m in o[(m et h ylsu lfon yl)im in o]-

m eth yl}a m in o)-N-(1,3-th ia zol-2-yl)p en ta n a m id e h yd r o-

ch lor id e (15). To a 0 °C solution of isothiourea 14 (3.30 g,

7.09 mmol) in 11 mL of a 2.0 M solution of ammonia in

methanol was added AgNO₃(1.32 g, 7.80 mmol) dissolved in

MeCN (5 mL) over 10 min. A yellow precipitate of AgSMe

formed, and the reaction mixture was stirred as it warmed to

ambient temperature over 2 h. Volatiles were removed in

vacuo and the resulting mixture was triturated in EtOAc. The

AgSMe was removed by filtration through a glass frit. The

filtrate was concentrated and placed under high vacuum

overnight to provide methanesulfonyl arginine derivative NR-

Boc 15 as a white foam (3.04 g, 7.00 mmol, 99%). ¹H NMR

(300 MHz, DMSO-d₆) δ 12.18 (s, 1H), 7.50 (d, J ) 3.6 Hz, 1H),

7.24 (d, J ) 3.6 Hz, 1H), 7.0-6.4 (m, 2H), 4.06 (m, 1H), 3.07

(m, 2H), 2.77 (s, 3H), 1.7-1.4 (m, 4H), 1.39 (s, 9H) ppm. ESIMS

m/z 435 (M+H)⁺, 464 (M+Na)⁺, 335 (MH-Boc)⁺.

Zym osan -In du ced Rat P leu r isy. Male Lewis rats (Charles

River Laboratories, Raleigh, NC) were dosed subcutaneously

with 28a (50 mg/kg; aqueous suspension with 5% EtOH, 5%

propylene glycol, 10% Cremephore, and 10% Molecusol) 10 min

prior to intrapleural injection of zymosan (Sigma Chemical Co.;

0.5 mg in 0.25 mL of H₂O). After 4 h, the pleural exudate was

collected using a 16 ga. lavage needle and placed in a tube

with EDTA, and cells were removed by centrifugation. Total

TNF-R was measured in the pleural exudate by ELISA

(Biosource International, Camarillo, CA) after spinning down

the cells.

L-N^G-Nitr oa r gin in e 2-a m in oth ia zole a m id e d ih yd r o-

ch lor id e (9). To a stirred solution of Na-Boc-^L-Arg (5.00 g,

15.6 mmol) and 2-aminothiazole (1.72 g, 17.2 mmol) in dry

DMF (16 mL) was added EDC (3.30 g, 17.2 mmol). The solution

was stirred for 4 h then added to half-saturated NaCl solution

and extracted with EtOAc (3×). The combined organic extracts

were washed with 1 N NaHSO₄(2×), 5% NaHCO3 (3×), and

brine, and dried over MgSO₄. Filtration and concentration

provided NR-Boc-N^G-L-nitroarginine 2-aminothiazole amide as

a sticky solid (2.28 g, 5.68 mmol, 36%). ¹H NMR (300 MHz,

CD₃OD) δ 7.42 (d, J ) 3.6 Hz, 1H), 7.10 (d, J ) 3.6 Hz, 1H),

4.31 (m, 1H), 3.29 (m, 2H), 1.85 (m, 1H), 1.71 (m, 2H), 1.41 (s,

9H), 1.33 (m, 1H) ppm.

The Boc amino amide (2.28 g, 5.68 mmol) was dissolved in

5 mL of dry THF and treated with 5 mL of 4 N HCl in dioxane.

After 3 h at ambient temperature, DCM was added and the

precipitated product was filtered. The gummy solid was

triturated with methanol/toluene to afford the title compound

as a tan solid (1.63 g, 4.83 mmol, 85%). ¹H NMR (300 MHz,

DMSO-d₆) δ 8.58 (bs, 4H), 7.99 (bs, 1H), 7.52 (d, J ) 3.6 Hz,

1H), 7.31 (d, J ) 3.6 Hz, 1H), 6.4-5.9 (bs, 2H), 4.08 (m, 1H),

3.17 (bs, 2H), 1.83 (m, 2H), 1.53 (m, 2H) ppm. ESIMS m/z (pos.

ion.) 302 (M+H)⁺; (neg. ion) 300 (M-H)⁺. Anal. (C₉H₁₅N₇O₃S‚

2HCl‚0.25C₄H₈O) C, H, N, S.

L-N^G-(2-P yr idylsu lfon yl)ar gin in e 2-am in oth iazole am ide

h yd r och lor id e (11). Boc-^L-Arg (2.00 g, 7.29 mmol) in 40 mL

of acetone/H₂O (80:20) at 0 °C was brought to pH 11-12 by

the addition of 3 N NaOH solution. 2-Pyridylsulfonyl chloride

(3.2 g, 18.2 mmol) in acetone was added dropwise over 30 min

during which time the pH was maintained at 11-12 with

additional 3 N NaOH. After 5 h at 0 °C, the pH was adjusted

to 6 with 8 N HCl, and the acetone was removed in vacuo.

The resulting oily suspension was acidified to pH 3-4 with

0.5 N citric acid and extracted with EtOAc (3×), washed with

ice cold 0.2 N HCl (3×) and brine, and dried over Na₂SO₄.

Filtration and concentration provided NR-Boc-N^G-L-(2-pyridyl-

sulfonyl)arginine as a yellow glass (1.54 g, 3.71 mmol, 51%).

ESIMS m/z 416 (M+H)⁺, 438 (M+Na)⁺, 316 (MH-Boc)⁺. The

amino acid (361 mg, 0.869 mmol) and 2-aminothiazole (174

mg, 1.74 mmol) were dissolved in 1.2 mL of anhydrous DMF.

EDC (183 mg, 0.956 mmol) was added, and the reaction

mixture was heated to 40 °C for 40 h. The cooled reaction

mixture was poured into H₂O and extracted with hexanes/

EtOAc (1:1) (4×) to provide NR-Boc-N^G-L-(2-pyridylsulfonyl)-

arginine 2-aminothiazole amide as a white foam (189 mg, 0.38

mmol, 44%). ¹H NMR (400 MHz, CD₃OD) δ 8.56 (d, J ) 4.8

Hz, 1H), 7.94 (d, J ) 3.6 Hz, 2H), 7.45 (m, 1H), 7.42 (d, J )

3.6 Hz, 1H), 7.11 (d, J ) 3.2 Hz, 1H), 4.3-4.1 (m, 1H), 3.3-

3.2 (m, 2H), 1.8-1.4 (m, 4H), 1.42 (s, 9H) ppm. ESIMS m/z

498 (M+H)⁺, 520 (M+Na)⁺, 398 (MH-Boc)⁺. The Boc amino

amide (189 mg, 0.380 mmol) was taken up in 1 mL of 4 N HCl

in dioxane, and after 1 h it was concentrated in vacuo and

repeatedly stripped from MeOH/toluene to provide the title

compound as a white foam contaminated with a small amount

of dioxane (169 mg, 0.360 mmol, 95%). ¹H NMR (400 MHz,

DMSO-d6) δ 8.54 (d, J ) 4.8 Hz, 1H), 8.50 (bs, 2H), 7.93 (m,

1H), 7.81 (d, J ) 7.6 Hz, 1H), 7.51 (m, 2H), 7.29 (d, J ) 4.0

Hz, 1H), 7.25 (bs, 1H), 7.0-6.6 (bs, 4H), 4.02 (m, 1H), 3.08

(m, 2H), 1.78 (m, 2H), 1.45 (m, 2H) ppm. ESIMS m/z 398

(M+H)⁺, 420 (M+Na)⁺.

The Boc amino amide (3.04 g, 7.00 mmol) was dissolved in

3 mL of methanol and 5 mL of 4 N HCl in dioxane. After 2 h

volatiles were removed in vacuo, and the product was repeat-

edly stripped from a mixture of methanol and toluene to

provide 15 as a white foam containing some toluene (2.83 g,

1

8.47 g, 120%). H NMR (400 MHz, DMSO-d₆) δ 8.58 (bs, 6H),

7.51 (d, J ) 3.6, Hz, 1H), 7.31 (d, J ) 3.6 Hz, 1H), 7.16 (m,

1H), 6.8-6.5 (bs, 1H), 4.09 (m, 1H), 3.15 (s, 3H), 3.0-2.6 (bs,

3H), 1.82 (m, 2H), 1.50 (m, 2H) ppm. ESIMS m/z 335 (M+H)⁺.

ter t-Bu tyl (2S,3R)-2-{[(ben zyloxy)ca r bon yl]a m in o}-3-

m eth yl-4-p en ten oa te (17). Acid 16a (25.2 g, 112 mmol) was

stirred at 80 °C in a solution of 2 N NaOH (112 mL) for 1.5 h.

The reaction mixture was cooled to 0 °C and an additional 112

mL of 2 N NaOH was added followed by benzyl chloroformate

(18 mL, 126 mmol). After 45 min stirring at 0 °C, the reaction

mixture was allowed to warm to ambient temperature over 5

h and the mixture was poured into 150 mL of water. The

solution was basified with additional NaOH solution and

extracted with ether, and the ether layer was discarded. The

aqueous phase was acidified with 6 N HCl to pH 1 and

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4263

extracted with ether (3×). The combined organic extracts were

dried over MgSO₄, filtered, and concentrated in vacuo to afford

22.6 g (86.2 mmol, 77%) of tert-butyl (2S,3R)-2-{[(benzyloxy)-

carbonyl]amino}-3-methyl-4-pentenoic acid as a clear oil. ¹H

NMR (300 MHz, CDCl₃) δ 8.39 (bs, 1H), 7.35 (m, 5H), 5.71

ddd, 1H, 5.13 (m, 2H), 4.43 (dd, 1H), 2.74 (m, 1H), 1.10 (d,

3H) ppm. ESIMS m/z 264 (M+H)⁺, 286 (M+Na)⁺.

tert-Butyl (2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-methyl-

4-pentenoic acid (20.6 g, 78.2 mmol) was dissolved in toluene

(156 mL) and heated to 80 °C. Dimethylformamide di-tert-butyl

acetal (DMF-DBA) was added over 30 min., and the reaction

solution was maintained at 80 °C for a further 45 min. when

an additional 10 mL of DMF-DBA was added. After an

additional 1 h, the mixture was allowed to cool to ambient

temperature and was diluted with ether. The reaction mixture

was washed with water, 5% NaHCO₃, and brine, and dried

(MgSO₄). Filtration and concentration provided 17 as a clear

oil (15.5 g, 48.5 mmol, 62%). ¹H NMR (300 MHz, CDCl₃) δ 7.30

(m, 5H), 5.75 (m, 1H), 5.28 (bd, 1H), 5.07 (m, 2H), 4.27 (bd,

1H), 2.63 (m, 1H), 1.44 (s, 9H), 1.04 (d, 3H) ppm.

(2S,3R)-2-{[(Ben zyloxy)ca r b on yl]a m in o}-3-m et h yl-5-

({(Z)-(m eth ylsu lfa n yl)[(m eth ylsu lfon yl)im in o]m eth yl}-

a m in o)p en ta n oic a cid (21). Amine 20 (0.940 g, 2.80 mmol)

was dissolved in 25 mL of 95% ethanol, and aqueous NaOAc

solution was added (1.0 mL, 3.0 M, 3.0 mmol). Dimethyl

methylsulfonyldithioimidocarbonate (0.612 g, 3.07 mmol) was

added, and the reaction mixture was refluxed for 48 h. The

ethanol was removed in vacuo, and the resulting mixture was

extracted with ether. The ether extracts were washed with

water, 1 M NaHSO₄(2×), 5% NaHCO₃, and brine and dried

over MgSO₄. Filtration and concentration afforded the crude

isothiourea (ESIMS m/z 510 (M+Na)⁺) which was taken up

in 12 mL of acid cocktail (CF₃COOH/thioanisole/water/i-Pr₃-

SiH 88:5:5:2) and allowed to stand at ambient temperature

for 1 h. The reaction mixture was concentrated then placed

under high vacuum for 1 h. The resulting oil was taken up in

50 mL of 5% NaHCO₃and sonicated, then extracted with ether

(3×), acidified with 6 N HCl, extracted with DCM (4×), and

dried over MgSO₄. Filtration provided the desired acid (21)

contaminated by 20% methylsulfonyldithioimidocarbonate

1

(0.943 g containing 1.98 mmol of 21, 71% from 20). H NMR

ter t-Bu tyl ((2S,3R)-2-{[(ben zyloxy)ca r bon yl]a m in o}-5-

h yd r oxy-3-m eth ylp en ta n oa te (18). To a 0 °C solution of 2,3-

dimethyl-2-butene (43.2 mL, 1.0 M in THF) was added

dropwise over 20 min a solution of borane in THF (43.2 mmol,

1.0 M). After the solution was stirred an additional 60 min at

0 °C, 17 (13.1 g, 41.2 mmol) in 20 mL of THF was added

dropwise and the resulting solution was allowed to warm to

ambient temperature over 1 h. The solution was recooled to 0

°C and excess borane was quenched by the cautious addition

of 6 mL of 1:1 THF/ethanol followed by 40 mL of pH 7.00

phosphate buffer then 40 mL of 30% H₂O₂. After the reaction

mixture stirred at ambient temperature for 14 h, it was

partially concentrated, added to brine, and extracted with

ether (3×). The combined organic layers were washed with

ice-cold saturated aqueous Na₂S₂O₅solution (4×), water, and

brine, and dried over MgSO₄. Filtration and concentration

provided a crude product that was purified on SiO₂(hexanes/

EtOAc 3:2) to afford compound 18 (12.3 g, 36.7 mmol, 89%).

¹H NMR (400 MHz, CDCl₃) δ 7.34 (m, 5H), 5.40 (bd, 1H), 5.09

(s, 2H), 4.11 (m, 1H), 3.78 (m, 1H), 3.65 (m, 1H), 2.22 (m, 1H),

1.7-1.6 (bs, 1H), 1.63 (m, 2H), 1.45 (s, 9H), 0.84 (d, 3H) ppm.

ESIMS m/z 338 (M+H)⁺, 360 (M+Na)⁺.

ter t-Bu tyl ((2S,3R)-2-{[(ben zyloxy)ca r bon yl]a m in o}-5-

a zid o-3-m eth ylp en ta n oa te (19). Alcohol 18 (12.3 g, 36.4

mmol) was dissolved in 60 mL of DCM and 8.8 mL of pyridine

and cooled to 0 °C. Methanesulfonyl chloride (4.59 g, 40.1

mmol, 3.10 mL) was added via syringe, and the resulting

solution was allowed to stir for 14 h then washed with 1 N

HCl and brine, and dried over MgSO₄. Filtration and concen-

tration afforded the crude mesylate which was taken up in 50

mL of dry DMF, and LiN₃(5.36 g, 109 mmol) was added and

the reaction mixture was warmed to 50 °C for 3 h. The reaction

mixture was added to water and extracted three times with

ether. The combined organic layers were dried over MgSO₄,

filtered, and concentrated to give a crude azide (10.3 g) which

was purified on SiO₂(hexanes/EtOAc 8:1) to provide azide 19

as an oil (9.40 g, 25.8 mmol, 71%). ¹H NMR (400 MHz, CDCl₃)

δ 7.34 (m, 5H), 5.28 (bd, 1H), 5.09 (s, 2H), 4.31 (m, 1H), 3.39

(m, 2H), 2.18 (m, 1H), 1.84 (d, 3H), 1.72 (m, 1H), 1.45 (s, 9H),

1.40 m, 1H) ppm. ESIMS m/z 385 (M+Na)⁺, 329 (M+Na-

C₄H₈)⁺.

(400 MHz, CDCl₃) δ 8.6 (bs, 2H), 7.95 (bs, 1H), 7.33 (m, 5H),

5.41 (d, J ) 8.8, 1H), 5.09 (m, 2H), 4.47 (dd, J ) 9.2, 3.6 Hz,

1H), 3.5-3.3 (m, 2H), 2.99 (s, 3H), 2.36 (s, 3H), 2.2-2.0 (m,

1H), 1.8-1.7 (m, 1H), 1.6-1.5 (m, 1H), 0.91 (d, J ) 6.8, 3H)

ppm. ESIMS m/z 432 (M+H)+, 863 (2M+H)⁺.

Ben zyl (1S,2R)-4-({(E)-a m in o[(m eth ylsu lfon yl)im in o]-

m eth yl}a m in o)-2-m eth yl-1-[(1,3-th ia zol-2-yla m in o)ca r bo-

n yl]bu tylca r ba m a te (22). Isobutyl chloroformate (0.262 g,

1.92 mmol, 0.25 mL) was added with stirring to a -23 °C

solution of acid 21 (0.828 g, 1.92 mmol) and TEA (0.266 mL,

1.92 mmol) in 5 mL of anhydrous DMF. After 1 h, 2-amino-

thiazole (0.192 g, 1.92 mmol) was added, and the reaction

mixture was allowed to warm to ambient temperature over

16 h. The solution was added to 50%-saturated NaCl solu-

tion and extracted with EtOAc (4×). The combined organic

extracts were washed with 1 N HCl (2×), 5% NaHCO₃, and

brine and dried over MgSO₄. Purification on SiO₂(hexanes/

EtOAc 1:2 then 1:5) afforded benzyl (1S,2R)-2-methyl-4-({(Z)-

(methylsulfanyl)[(methylsulfonyl)imino]methyl}amino)-1-[(1,3-

thiazol-2-ylamino)carbonyl]butylcarbamate as a white glassy

solid free from contamination by methylsulfonyldithioimi-

docarbonate (0.608 g, 1.18 mmol, 44% for three steps). ¹H NMR

(400 MHz, CDCl₃) δ 12.0 (bs, 1H), 7.9 (bs, 1H), 7.56 (d, J )

3.6 Hz, 1H), 7.32 (m, 5H), 6.99 (d, J ) 3.6 Hz, 1H), 5.85 (d, J

) 8.8 Hz, 1H), 5.11 (AB, J ) 12 Hz, 2H), 4.67 (m, 1H), 3.2-

3.2 (m, 2H), 2.96 (s, 3H), 2.31 (s, 3H), 2.10 (m, 1H), 1.8 (m,

1H), 1.5 (m, 1H), 0.92 (d, J ) 6.8 Hz, 3H) ppm. ESIMS m/z

514 (M+H)⁺, 536 (M+Na)⁺.

The isothiourea (0.608 g, 1.18 mmol) was dissolved in 6.0

mL of a 2.0 M methanolic ammonia solution and cooled to 0

°C. A solution of AgNO₃(0.220 g, 1.30 mmol) in methanol (3

mL) was added dropwise over 5 min, and the reaction mixture

was allowed to come to ambient temperature over 1.5 h. Ethyl

acetate was added, and the mixture was filtered through Celite

diatomaceous earth and concentrated in vacuo to provide the

title compound as a glassy solid (0.651 g, 1.35 mmol, 114%).

¹H NMR (400 MHz, CD₃OD) δ 7.45 (d, J ) 3.6 Hz, 1H), 7.4-

7.0 (m, 6H), 5.08 (AB, J ) 12.4 Hz, 2H), 4.43 (m, 1H), 3.3-3.2

(m, 2H), 2.84 (s, 3H), 2.2-2.1 (m, 1H), 1.7-1.6(m, 1H), 1.5-

1.4 (m, 1H), 0.94 (d, J ) 6.0 Hz, 3H) ppm. ESIMS m/z 483

(M+H)⁺, 505 (M+Na)⁺.

ter t-Bu tyl ((2S,3R)-2-{[(ben zyloxy)ca r bon yl]a m in o}-5-

a m in o-3-m et h ylp en t a n oa t e (20). tert-Butyl ((2S,3R)-2-

{[(benzyloxy)carbonyl]amino}-5-azido-3-methylpentanoate (19,

1.02 g, 2.81 mmol) was dissolved in 10 mL of methanol and

TEA (0.39 mL, 2.8 mmol), and hydrogenated at 30 psi over

0.05 g of Lindlar catalyst. After 4 h the catalyst was removed

by filtration, and the filtrate was concentrated to give the

[((3R,4S)-4-{[(Ben zyloxy)ca r bon yl]a m in o}-4-ca r boxy-

3-m et h ylb u t yl)a m in o](im in o)m et h a n a m in iu m n it r a t e

((3R) 3-Meth yl L-a r gin in e n itr ic a cid sa lt, 24). To a

solution of amine 20 (0.906 mg, 2.69 mmol) and TEA (0.8 mL,

5.7 mmol) in DMF (5 mL) was added 3,5-dimethylpyrazole-1-

carboxamidine nitrate (0.542 g, 2.69 mmol). After 72 h at

ambient temperature, the volatiles were removed under high

vacuum. The oily residue was taken up in 40 mL of EtOAc,

sonicated to a fine suspension, and triturated by stirring for 1

h. Filtration through glass provided the title compound as a

white solid displaying very broad peaks in the NMR spectrum

(0.856 g, 1.94 mmol, 72%). ¹H NMR (400 MHz, DMSO-d₆) δ

1

primary amine 20 as a clear oil (0.94 g, 2.80 mmol, 100%). H

NMR (400 MHz, DMSO-d₆) δ 7.53 (bd, 1H), 7.45 (bs, 1H), 7.33

(m, 5H), 7.4-6.5 (bs, 2H), 5.02 (m, 2H), 4.00 (m, 1H), 1.98 (m,

1H), 1.6-1.1 (m, 2H), 1.37 (s, 9H), 0.82 (s, 3H) ppm. ESIMS

m/z 379 (M+H)⁺, 323 (M+H-C₄H₈)⁺.

4264 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

7.52 (bd, J ) 3.6 Hz, 1H), 7.45 (bs, 1H), 7.33 (m, 5H), 7.2-6.6

(bs, 4H), 5.00 (AB, J ) 12 Hz, 2H), 4.00 (m, 1H), 3.2-3.0 (m,

2H), 2.1-1.9 (m, 1H), 1.6-1.4 (m, 1H), 1.37 (s, 9H), 1.2 (m,

1H), 0.82 (bd, J ) 6.4 Hz, 3H) ppm. ESIMS m/z 379 (M+H)⁺,

323 (M+H-C₄H₈)⁺.

combined organic layers were washed with pH 7.0 phosphate

buffer and brine and dried over MgSO₄. Purification on SiO₂

(DCM/MeOH 95:5) resulted in an oil which was taken up in

80% aqueous AcOH and heated at 40 °C for 16-20 h. The

compound was isolated by removal of the volatiles in vacuo

and trituration with DCM and ether followed by filtration and

drying of the amorphous solid under vacuum at 50 °C.

(2S,3R)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-5-{[im in o(2-

oxid o-2-oxoh yd r a zin o)m eth yl]a m in o}-3-m eth ylp en ta n o-

ic a cid ((3R) N^R-Boc-N^γ-n itr o-3-m eth yl L-a r gin in e, 25).

(3R)-3-Methyl l-arginine derivative 24 (0.856 g, 1.94 mmol)

was dissolved in 10 mL of methanol and stirred with 1,4-

cyclobutadiene (2 mL) and 10% Pd/C (80 mg) at reflux for 1 h.

The cooled mixture was filtered through Celite diatomaceous

earth and concentrated in vacuo to provide the free amine

(0.599 g of white solid, ESIMS m/z 245 (M+H)⁺, 189 (M+H-

C₄H₈)⁺which was dissolved in 5 mL of 95% TFA in water. After

1 h the volatiles were removed, and the resulting oil was

repeatedly stripped from ethanol, EtOAc, and toluene to

provide (3R)-3-methyl ^L-arginine as an oil (1.04 g; ESIMS m/z

189 (M+H)⁺, 172 (M+H-NH₃)⁺. The crude oil was dissolved

in 1 mL of concentrated H₂SO₄and cooled in an ice-acetone

bath. A pre-cooled (-15 °C) mixture of fuming H₂SO₄(1.5 mL)

and fuming nitric acid (1.0 mL) was added dropwise to the

stirred solution. After 2 h at -10 to -15 °C, the cooling bath

was removed, and ice was added to the reaction mixture.

Ammonium hydroxide solution (30%) was added cautiously to

bring the reaction mixture to pH 8, then the pH was adjusted

to 5 with glacial acetic acid. The volatiles were removed in

vacuo to provide a yellow solid which was triturated for 1 h

with methanol. Filtration through glass provided a crude

yellow solid (ESIMS m/z 234 (M+H)⁺) which was dissolved in

15 mL of 5% NaHCO₃and concentrated to dryness (2×). The

resulting mixture was taken up in 10 mL of fresh 5% NaHCO₃

and THF (10 mL), and di(tert-butyl) dicarbonate (0.465 g) was

added. After 18 h, 5 g of NaHCO₃was added along with

additional di(tert-butyl) dicarbonate (0.05 g). After an ad-

ditional 72 h, the reaction mixture was extracted with ether

(2×). The aqueous layer was acidified with 6 N HCl to pH 1,

saturated with NaCl, and extracted with DCM (3×). The

combined organic layers were dried over MgSO₄, filtered, and

concentrated to give 25 as a yellow foam (0.125 g, 0.375 mmol,

19% from 24). ¹H NMR (400 MHz, CDCl₃) δ 10.8-10.6 (bs,

2H), 8.68 (bs, 1H), 7.9-7.5 (bs, 1H), 5.38 (d, J ) 8.0 Hz, 1H),

4.45 (d, J ) 7.2 Hz, 1H), 3.8-3.6 (bs, 1H), 3.4-3.2 (bs, 1H),

2.3-2.1 (bs, 1H), 1.7-1.6 (m, 1H), 1.5-1.4 (m, 1H), 1.43 (s,

9H), 0.86 (d, J ) 6.8 Hz, 3H) ppm. ESIMS m/z 334 (M+H)⁺,

356 (M+Na)⁺, 278 (M+H-C₄H₈)⁺.

Alternatively, pentafluorophenyl ester 7n (1 equiv) was

dissolved in DMF and stirred with either 23 or 27 (1.1 equiv)

at 40 °C for 2-5 h. Workup and purification were identical to

those of the DEPC coupling procedure (vide supra).

(2R,3S)-N-{(1R)-4-Nitr ogu a n yl-1-[(1,3-th ia zol-2-yla m i-

n o)ca r b on yl]b u t yl}-3-[for m yl(h yd r oxy)a m in o]-2-isob u -

tylh exa n a m id e (28a ). White amorphous solid, 33%. ¹H NMR

(400 MHz, DMSO-d₆, mixture of formamide rotamers) δ 12.14

(s, 1H), 9.70 (s, 0.33H), 9.44 (s, 0.67H), 8.6-8.4 (bs, 1H), 8.42

(m, 1H), 8.34 (s, 0.33H), 8.2-7.6 (bs, 1H), 7.94 (s, 0.67H), 7.46

(d, J ) 3.2 Hz, 1H), 7.20 (d, J ) 3.2 Hz, 1H), 4.48 (m, 1H),

4.16 (m, 0.33H), 3.47 (m, 0.67H), 3.16 (m, 2H), 2.66 (m, 1H),

1.8-1.0 (m, 11H), 0.85 (d, J ) 6.4 Hz, 3H), 0.83-0.72 (m, 6H)

ppm. ESIMS m/z 515 (M+H)⁺, 537 (M+Na)⁺. λ_max) 269.0 nm.

Anal. (C₂₀H₃₄N₈O₆S) C, H, N.

(2R)-5-Nit r ogu a n yl-2-({(2R,3S)-2-(cycloh exylm et h yl)-

3-cyclop r op yl-3-[for m yl(h yd r oxy)a m in o]p r op a n oyl}-

am in o)-N-(1,3-th iazol-2-yl)pen tan am ide (28b). White amor-

phous solid, 46%. ¹H NMR (400 MHz, CD₃OD, mixture of

formamide rotamers) δ 8.31 (s, 0.15H, partially exchanged

NH), 8.24 (m, 0.1H, partially exchanged NH), 7.89 (s, 0.33H),

7.87 (s, 0.1H, partially exchanged NH), 7.71 (s, 0.67H), 7.41

(m, 1H), 7.10 (m, 1H), 4.64 (m, 1H), 3.6-3.4 (m, 0.33H), 3.25

(m, 0.67H), 3.20 (m, 2H), 2.91 (t, J ) 10 Hz, 0.5H), 2.89 (t, J

) 10 Hz, 0.5H), 2.0-1.4 (m, 12H), 1.3-1.0 (m, 5H), 1.0-0.7

(m, 2H), 1.7-0.3 (m, 2H), 0.3-0.1 (m, 1H) ppm. ESIMS m/z

553 (M+H)⁺, 575 (M+Na)⁺. λ_max269 nm. Anal. (C₂₃H₃₆N₈O₆S‚

¹/₂H₂O) C, H, N, S.

(2R,3S)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m i-

n o)car bon yl]bu tyl}-3-[for m yl(h ydr oxy)am in o]-2-[(5-m eth -

yl-2-th ien yl)m eth yl]h exa n a m id e (28c). Tan solid, 30%. ¹H

NMR (400 MHz, DMSO-d₆, mixture of formamide rotamers)

δ 11.99 (bs, 1H), 9.87 (s, 0.33H), 9.57 (s, 0.67H), 8.6-8.4 (bs,

1H), 8.44 (m, 1H), 8.40 (s, 0.33H), 8.2-7.6 (bs, 2H), 8.01 (s,

0.67H), 7.48 (d, J ) 3.2 Hz, 1H), 7.23 (d, J ) 3.2 Hz, 1H), 6.48

(s, 1H), 6.32 (s, 1H), 4.57 (m, 1H), 4.28 (m, 0.33H), 3.63 (m,

0.67H), 3.14 (m, 2H), 2.9-2.7 (m, 3H), 2.03 (s, 3H), 1.8-1.5

(m, 4H), 1.42 (m, 1H), 1.4-1.0 (m, 3H), 0.78 (m, 3H) ppm.

ESIMS m/z 569 (M+H)⁺, 591 (M+Na)⁺. λ_max269 nm. Anal.

(C₂₂H₃₂N₈O₆S₂‚¹/₃H₂O) C, H, N, S.

(2R,3S)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m i-

n o)ca r b on yl]bu t yl}-2-(cycloh exylm et h yl)-3-[for m yl(h y-

d r oxy)a m in o]h exa n a m id e (28d ). White amorphous solid,

45%. ¹H NMR (400 MHz, DMSO-d₆, mixture of formamide

rotamers) δ 12.11 (bs, 1H), 9.65 (s, 0.33H), 9.39 (s, 0.67H), 8.50

(bs, 1H), 8.44 (m, 1H), 8.30 (s, 0.33H), 8.2-7.6 (bs, 2H), 7.91

(s, 0.67H), 7.44 (d, J ) 3.6 Hz, 1H), 7.18 (d, J ) 3.2 Hz, 1H),

4.53 (m, 1H), 4.13 (t, J ) 11.2 Hz, 0.33H), 3.43 (t, J ) 12 Hz,

0.67H), 3.14 (m, 2H), 2.63 (m, 1H), 1.88 (m, 1H), 1.8-1.3 (m,

11H), 1.3-1.0 (m, 7H), 0.9-0.6 (m, 5H) ppm. λ_max269 nm.

ESIMS m/z 555 (M+H)⁺, 577 (M+Na)⁺. Anal. (C₂₃H₃₈N₈O₆S‚

¹/₄H₂O) C, H, N, S.

ter t-Bu tyl (1S,2R)-4-{[im in o(2-oxid o-2-oxoh yd r a zin o)-

m eth yl]a m in o}-2-m eth yl-1-[(1,3-th ia zol-2-yla m in o)ca r bo-

n yl]bu tylca r ba m a te (26). To a solution of acid 25 (0.123 g,

0.369 mmol) and TEA (0.051 mL, 0.369 mmol) in DMF (1 mL)

at -23 °C was added isobutyl chloroformate (0.048 mL, 0.369

mmol). After 40 min 2-aminothiazole (0.041 g, 0.406 mmol)

was added, and the reaction mixture was allowed to warm to

ambient temperature over 2 h. The reaction mixture was

poured into 50%-saturated NaCl solution and extracted with

EtOAc (4×). The combined organic layers were washed with

1 N HCl (2×), 5% NaHCO₃, and brine, and dried over MgSO₄.

Purification on SiO₂(hexanes/EtOAc 1:5 then 100% EtOAc)

gave the title compound as a yellow foam (0.101 g, 0.243 mmol,

1

66%). H NMR (400 MHz, CD₃OD) δ 7.35 (d, J ) 3.6 Hz, 1H),

(2R,3S)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m i-

n o)ca r bon yl]bu tyl}-3-[for m yl(h yd r oxy)a m in o]-4-m eth yl-

2-[(4-m eth ylcycloh exyl)m eth yl]p en ta n a m id e (28e). White

amorphous solid, 55%. ¹H NMR (400 MHz, DMSO-d₆, mixture

of formamide rotamers and cis/ trans 1,4-dialkylcyclohexane

isomers) δ 12.10 (bs, 1H), 9.60 (m, 0.33H), 9.40 (m, 0.67H),

8.48 (m, 2H), 8.32 (s, 0.2H), 8.29 (s, 0.1H), 8.2-7.6 (m, 2H),

7.90 (s, 0.7H), 7.44 (d, J ) 3.6 Hz, 1H), 7.19 (m, 1H), 4.53 (m,

2H), 4.08 (m, 0.33H), 3.37 (m, 0.67H), 3.12 (m, 2H), 2.91 (m,

1H), 2.0-1.1 (m, 13H), 0.96 (d, J ) 6.4 Hz, 3H), 0.9-0.6 (m,

10H) ppm. λ_max269 nm. ESIMS m/z 569 (M+H)⁺, 591 (M+Na)⁺.

Anal. (C₂₄H₄₀N₈O₆S‚²/₃H₂O) C, H, N, S.

7.03 (d, J ) 3.6 Hz, 1H), 6.85 (bd, J ) 8.0 Hz, 0.7 H (NH

partially exchanged)), 4.31 (m, 1H), 3.25 (M, 2H), 2.1-2.0 (m,

1H), 1.7-1.5 (m, 1H), 1.4 (m, 1H), 1.36 (s, 9H), 0.86 (d, J )

6.8 Hz, 3H) ppm. ESIMS m/z 416 (M+H)⁺, 360 (M+H-C₄H₈)⁺,

316 (M+H-Boc)⁺.

Gen er a l P r oced u r e for th e P r ep a r a tion of N-Hyd r oxy

F or m a m id es 28a -u . Diethyl phosphorylcyanide (DEPC, 1.1

equiv) was added dropwise to a solution of acid 7j-t (1.0 equiv)

and NMM (1.1 equiv) in DMF (0.2-0.5 M) at 0 °C. After 0.5

h, the mixture was allowed to warm to ambient temperature

over 0.5 h. To this solution was added amino amide (9, 11, 15,

23, or 27, 1.1 equiv) and additional NMM (2.2 equiv). After

12-16 h at 23-40 °C the reaction mixture was added to 50%-

saturated NaCl solution and extracted with EtOAc (3×). The

(2R,3S)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m i-

n o)ca r bon yl]bu tyl}-3-[for m yl(h yd r oxy)a m in o]-2-[(4-m e-

th ylcycloh exyl)m eth yl]h exa n a m id e (28f). White amor-

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4265

phous solid, 48%. ¹H NMR (400 MHz, DMSO-d₆, mixture of

formamide rotamers and cis/ trans 1,4-dialkylcyclohexane

isomers) δ 12.10 (bs, 1H), 9.65 (bs, 0.33H), 9.42 (bs, 0.67H),

8.45 (m, 2H), 8.33 (s, 0.2H), 8.30 (s, 0.1H), 8.2-7.6 (bs, 2H),

7.92 (m, 0.67H), 7.44 (s_app, 1H), 7.18 (s_app, 1H), 4.51 (m, 1H),

4.13 (m, 0.33H), 3.35 (m, 0.67H), 3.15 (m, 2H), 2.63 (m, 1H),

1.0-0.9 (m, 16H), 0.9-0.8 (m, 10H) ppm. λ_max269 nm. ESIMS

m/z 569 (M+H)⁺, 591 (M+Na)⁺. Anal. (C₂₄H₄₀N₈O₆S‚²/₃H₂O)

C, H, N, S.

(2R,3S)-N-{(1S)-4-(2-P yr id ylsu lfon yl)gu a n yl-1-[(1,3-th i-

a zol-2-yla m in o)-ca r bon yl]bu tyl}-3-[for m yl(h yd r oxy)a m i-

n o]-2-[(4-m eth ylcycloh exyl)-m eth yl]h exa n a m id e (28g).

White amorphous solid, 35%. ¹H NMR (400 MHz, CD₃OD,

mixture of formamide rotamers and cis/ trans 1,4-dialkylcy-

clohexane isomers) δ 8.57 (d, J ) 4.4 Hz, 1H), 7.95 (m, 2H),

7.47 (d, J ) 4.4 Hz, 1H), 7.42 (d, J ) 3.2 Hz, 1H), 4.59 (m,

1H), 4.35 (m, 0.33H), 3.59 (m, 0.67H), 2.78 (m, 2H), 2.0-1.0

(m, 16H), 0.9-0.6 (m, 10H) ppm (1H not obsvd.). λ_max265 nm.

ESIMS m/z 665 (M+H)⁺, 687 (M+Na)⁺. Anal. (C₂₉H₄₄N₈O₆S₂‚

1H₂O) C, H, N, S.

(2R,3S)-N-{(1S)-4-(2-P yr id ylsu lfon yl)gu a n yl -1-[(1,3-

t h ia zol-2-yla m in o)ca r b on yl]b u t yl}-3-[for m yl(h yd r oxy)-

a m in o]-4-m eth yl-2-[(4-m eth ylcycloh exyl)m eth yl]p en ta n -

a m id e (28h ). White amorphous solid, 32%. ¹H NMR (400

MHz, CD₃OD, mixture of formamide rotamers and cis/ trans

1,4-dialkylcyclohexane isomers) δ 8.56 (d, J ) 4.8 Hz, 1H), 7.95

(m, 2H), 7.47 (m, 1H), 7.42 (m, 1H), 7.11 (m, 1H), 4.59 (m,

1H), 4.25 (m, 0.33H), 3.52 (m, 0.67H), 3.23 (m, 2H), 3.07 (m,

1H), 2.0-1.1 (m, 16H), 1.1-1.0 (m, 4H), 1.0-0.8 (m, 3H), 0.8-

0.7 (m, 3H) ppm. λ_max265 nm. ESIMS m/z 665 (M+H)⁺, 687

(M+Na)⁺. Anal. (C₂₉H₄₄N₈O₆S₂‚1H₂O) C, H, N, S.

(2R,3S)-N-{(1S)-4-(2-P yr id ylsu lfon yl)gu a n yl -1-[(1,3-

t h ia zol-2-yla m in o)ca r bon yl]bu t yl}-3-[for m yl(h yd r oxy)-

a m in o]-2-[(5-m eth yl-2-th ien yl)m eth yl]h exa n a m id e (28i).

White amorphous solid, 33%. ¹H NMR (400 MHz, CD₃OD,

mixture of formamide rotamers) δ 8.55 (d, J ) 4.8 Hz, 1H),

7.94 (m, 2H), 7.47 (m, 1H), 7.45 (m, 1H), 7.13 (m, 1H), 6.45 (d,

J ) 3.0 Hz, 1H), 6.17 (d, J ) 3.2 Hz, 1H), 4.54 (m, 1H), 4.50

(m, 0.33H), 3.73 (m, 0.67H), 3.19 (m, 2H), 2.95 (m, 1H), 2.86

(m, 2H), 2.11 (s, 3H), 1.9-1.1 (m, 8H), 0.84 (m, 3H) ppm. λ_max

265 nm. ESIMS m/z 665 (M+H)⁺, 687 (M+Na)⁺. Anal.

(C₂₇H₃₆N₈O₆S₃‚²/₃H₂O) C, H, N, S.

ESIMS m/z 563 (M+H)⁺. HRMS calcd for [C₂₅H₃₄N₈O₆S]H⁺,

563.2400; found, 563.2409. Anal. (C₂₄H₃₄N₈O₆S‚¹/₂H₂O) C, H,

N, S.

(2R,3S)-N-{(1S)-4-Meth a n esu lfon ylgu a n yl -1-[(1,3-th ia -

zol-2-ylam in o)car bon yl]bu tyl}-3-[for m yl(h ydr oxy)am in o]-

2-isobu tyl-4-m eth ylp en ta n a m id e (28m ). White amorphous

solid, 11%. ¹H NMR (400 MHz, CD₃OD mixture of formamide

rotamers) δ 8.33 (s, 0.33H), 7.93 (s, 0.67H), 7.41 (d, J ) 3.6

Hz, 1H), 7.10 (d, J ) 3.6, 1H), 4.61 (m, 1H), 4.28 (m, 0.33H),

3.46 (m, 0.67H), 3.21 (m, 0.67H), 3.00 (m, 0.33H), 2.86 (s, 3H),

2.0-1.4 (m, 7H), 1.18 (m, 1H), 1.1-1.0 (m, 3H), 0.95-0.90 (m,

6H), 0.81 (d, J ) 6.4 Hz, 3H) ppm. λ_max268 nm. LC R_t) 5.15

min. and 5.53 min. ESIMS m/z 548 (M+H)⁺. HRMS calcd for

[C₂₅H₃₄N₈O₆S]H⁺, 548.2325; found, 548.2319.

(2R,3S)-N-{(1S)-4-Meth a n esu lfon ylgu a n yl -1-[(1,3-th ia -

zol-2-ylam in o)car bon yl]bu tyl}-3-[for m yl(h ydr oxy)am in o]-

2-isobu tylh exa n a m id e (28n ). White amorphous solid, 39%.

¹H NMR (400 MHz, DMSO-d₆, mixture of formamide rotamers)

δ 9.67 (s, 0.33), 9.41 (s, 0.67H), 8.44 (m, 1H), 8.32 (s, 0.33H),

7.91 (s, 0.67H), 7.44 (d, J ) 3.6 Hz, 1H), 7.18 (d, J ) 3.6 Hz,

1H), 7.0-6.6 (m, 1H), 6.47 (bs, 1H), 4.48 (m, 1H), 4.13 (t, J )

10 Hz, 0.33H), 3.45 (t, J ) 10 Hz, 0.67H), 3.04 (m, 2H), 2.71

(s, 3H), 2.67 (m, 1H), 1.7-0.9 (m, 11H), 0.9-0.6 (m, 9H) ppm.

λ_max268 nm. ESIMS m/z 548 (M+H)⁺. Anal. (C₂₁H₃₇N₇O₆S₂‚¹/

₂AcOH) C, H, N, S.

(2R,3S)-N-{(1S)-4-Meth a n esu lfon ylgu a n yl -1-[(1,3-th ia -

zol-2-ylam in o)car bon yl]bu tyl}-3-[for m yl(h ydr oxy)am in o]-

4-m et h yl-2-[(4-m et h ylcycloh exyl)m et h yl]p en t a n a m id e

(28o). White amorphous solid, 48%. ¹H NMR (400 MHz,

DMSO-d₆, mixture of formamide rotamers and cis/ trans

cyclohexane isomers) δ 9.59 and 9.58 (s, 0.33H), 9.39 and 9.35

(s, 0.67H), 8.50 (m, 1H), 8.32 and 8.29 (s, 0.33H), 7.90 (s,

0.67H), 7.44 and 7.43 (d, J ) 3.6 Hz,1H), 7.20 and 7.18 (d, J

) 3.6 Hz, 1H), 7.0-6.6 (bm, 2H), 6.45 (bs, 1H), 4.52 (m, 1H),

4.08 (m, 0.33H), 3.37 (m, 0.67H), 3.04 (m, 2H), 2.91 (m, 1H),

2.17 (s, 3H), 2.0-1.0 (m, 17H), 0.96 (m, 3H), 0.8-0.7 (m, 6H)

ppm (1H not observed). λ_max270 nm. ESIMS m/z 602 (M+H)⁺.

Anal. (C₂₅H₄₃N₇O₆S₂‚²/₃H₂O) C, H, N, S (S calcd 10.45, found

11.08).

(2R,3S)-N-{(1S,2R)-4-Meth a n esu lfon ylgu a n yl -2-m eth -

yl-1-[(1,3-t h ia zol-2-yla m in o)ca r b on yl]b u t yl}-3-[for m yl-

(h ydr oxy)am in o]-2-isobu tylh exan am ide (28p). White amor-

phous solid, 18%. 82:18 mixture of diastereomers. ¹H NMR

(400 MHz, CD₃OD mixture of formamide rotamers) δ 8.37 (s,

0.33H), 7.96 (s, 0.67H), 7.42 (d, J ) 3.6 Hz, 1H), 7.11 (d, J )

3.6 Hz, 1H), 4.60 (m, 1H), 4.36 (m, 0.33H), 3.57 (m, 0.67H),

3.22 (m, 2H), 2.90 (m, 1H), 2.85 (s, 3H), 2.11 (m, 1H), 1.9-1.1

(m, 10H), 1.03 (d, J ) 6.8 Hz, 3H), 0.9-0.8 (m, 6H), 0.8-0.7

(m, 3H) ppm. LC-ESIMS Rt ) 5.17 (82%), m/z 562 (M+H)⁺;

5.74 (18%), m/z 562 (M+H)⁺. λ_max269 nm. HRMS calcd for

[C₂₂H₃₉N₇O₆S₂]H⁺, 562.2482; found, 562.2504.

(2R,3S)-N-{(1S)-4-(2-P yr id ylsu lfon yl)gu a n yl -1-[(1,3-

t h ia zol-2-yla m in o)ca r b on yl]b u t yl}-3-[for m yl(h yd r oxy)-

am in o]-2-isobu tylh exan am ide (28j). White amorphous solid,

40%. ¹H NMR (400 MHz, CD₃OD, mixture of formamide

rotamers) δ 8.57 (d, J ) 4.8 Hz, 1H), 7.95 (s, 2H), 7.48 (m,

1H), 7.46 (d, J ) 3.2 Hz, 1H), 7.10 (d, J ) 3.2 Hz, 1H), 4.56

(m, 1H), 4.33 (m, 0.33H), 3.56 (m, 0.67H), 3.24 (m, 2H), 2.77

(m, 1H), 1.9-1.4 (m, 7H), 1.4-1.3 (m, 2H), 1.1-1.0 (m, 2H),

0.90 (m, 3H), 0.82 (m, 6H) ppm. λ_max265 nm. ESIMS m/z 611

(M+H)⁺, 633 (M+Na)⁺. Anal. (C₂₅H₃₈N₈O₆S₂‚¹/₂AcOH) C, H,

N, S.

(2R,3S)-N-{(1S,2R)-4-Meth a n esu lfon ylgu a n yl -2-m eth -

yl-1-[(1,3-th iazol-2-ylam in o)car bon yl]bu tyl}-6,6,6-tr iflu or o-

3-[for m yl(h yd r oxy)a m in o]-2-isobu tylh exa n a m id e (28q).

White amorphous solid, 26%. ¹H NMR (400 MHz, CDCl₃/CD₃-

OD 1:1, mixture of formamide rotamers) δ 8.38 (s, 0.33H), 7.92

(s, 0.67H), 7.40 (d, J ) 3.6 Hz, 1H), 7.01 (d, J ) 3.6 Hz, 1H),

4.66 (m, 1H), 4.35 (m, 0.33H), 3.62 (m, 0.67H), 3.26 (m, 2H),

2.88 (s, 3H), 2.17 (m, 1H), 2.2-1.9 (m, 4H), 1.7-1.4 (m, 5H),

1.2-1.0 (m, 1H), 0.97 (m, 3H), 0.87 (m, 3H), 0.77 (m, 3H) ppm.

LC-ESIMS Rt ) 5.17 (>98%), m/z 616 (M+H)⁺. Anal.

(C₂₂H₃₆F₃N₇O₆S₂‚³/₄H₂O) C, H, N.

(2R,3S)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m i-

n o)ca r bon yl]bu tyl}-2-(cycloh exylm eth yl)-6,6,6-tr iflu or o-

3-[for m yl(h yd r oxy)a m in o]h exa n a m id e (28k ). White amor-

phous solid, 38%. ¹H NMR (400 MHz, DMSO-d₆, mixture of

formamide rotamers) δ 12.17 (bs, 1H), 9.91 (0.33H), 9.60

(0.67H), 8.53 (m, 1.67H), 8.37 (s, 0.33H), 7.95 (s, 1H), 7.46 (d,

J ) 3.6 Hz, 1H), 7.21 (d, J ) 3.6 Hz, 1H), 4.55 (s, 1H), 4.18

(m, 0.33H), 3.60 (m, 0.67H), 3.29 (m, 2H), 2.75 (m, 1H), 2.15

(m, 2H), 2.0-1.4 (m, 11H), 1.2-1.0 (m, 6H), 0.9-0.6 (m, 2H)

ppm (2 Hs not obsvd). Anal. (C₂₃H₃₅F₃N₈O₆S‚¹/₄Et₂O) C, H, N,

S.

(2R,3S)-N-{(1S,2R)-4-Meth a n esu lfon ylgu a n yl -2-m eth -

yl-1-[(1,3-t h ia zol-2-yla m in o)ca r b on yl]b u t yl}-3-[for m yl-

(h yd r oxy)a m in o]-2-isobu tyl-4-m eth ylp en ta n a m id e (28r ).

White amorphous solid, 17%. 85:15 mixture of diastereomers.

¹H NMR (400 MHz, CD₃OD mixture of formamide rotamers)

δ 8.34 (s, 0.33H), 7.94 (s, 0.67H), 7.42 (d, J ) 3.6 Hz, 1H),

7.10 (d, J ) 3.6 Hz, 1H), 4.60 (m, 1H), 4.27 (m, 0.33H), 3.53

(m, 0.67H), 3.25 (m, 2H), 3.1-3.0 (m, 1H), 2.85 (s, 3H), 2.11

(m, 1H), 1.92 (m, 1H), 1.62 (m, 1H), 1.5-1.3 (m, 3H), 1.1 (m,

3H), 1.0 (m, 3H), 0.9 (m, 6H), 0.77 (d, J ) 6.8 Hz, 3H) ppm.

(2R)-N-{(1S)-4-Nitr ogu a n yl -1-[(1,3-th ia zol-2-yla m in o)-

car bon yl]bu tyl}-2-{(1S)-1-[for m yl(h ydr oxy)am in o]-2-ph e-

n yleth yl}-4-m eth ylp en ta n a m id e (28l). White amorphous

1

solid, 53%. H NMR (400 MHz, DMSO-d₆, mixture of forma-

mide rotamers) δ 12.21 (bs, 1H), 9.83 (s, 0.33H), 9.65 (s, 0.67H),

8.55 (d, J ) 6.4 Hz, 1H), 8.5 (bs, 1H), 8.4-7.8 (bs, 2H), 7.47

(d, J ) 3.2 Hz, 1H), 7.43 (s, 1H), 7.3-7.0 (m, 6H), 4.56 (m,

1H), 4.44 (m, 0.33H), 3.69 (m, 0.67H), 3.18 (m, 2H), 2.84 (m,

2H), 1.8-1.4 (m, 6H), 1.06 (m, 1H), 0.89 (d, J ) 6.4 Hz, 3H),

0.77 (d, J ) 6.4 Hz, 3H) ppm (1H not obsrvd.). λ_max270 nm.

4266 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24

Rabinowitz et al.

LC-ESIMS Rt ) 4.98 (85%), m/z 562 (M+H)⁺; 5.38 (15%), m/z

562 (M+H)⁺. λ_max270 nm. Anal. (C₂₂H₃₉N₇O₆S₂‚³/₄H₂O) C, H,

N.

(8) Becherer, J . D.; Lambert, M. H.; Andrews, R. C. The Tumor

Necrosis Factor-R Converting Enzyme. Handb. Exp. Pharmacol.

2000, 140, 235-58.

(9) Moss, M. L.; White, J . M.; Lambert, M. H.; Andrews, R. C. TACE

and Other ADAM Proteases as Targets for Drug Discovery. Drug

Discovery Today 2001, 6 (8), 417-26.

(10) Banner, D. W.; D’Arcy, A., J .; Gentz, R. W.; Schoenfeld, H.-J .;

Broger, C.; Loetscher, H.; Lesslauer, W. Crystal Structure of the

Soluble Human 55 Kd TNF Receptor-Human TNFâ Complex:

Implications For TNF Receptor Activation. Cell 1993, 73, 431-

45.

(11) Vandenabeele, P.; Declercq, W.; Beyaert, R.; Fiers, W. Two

Tumor Necrosis Factor Receptors: Structure and Function.

Trends Cell. Biochem. 1995, 5, 392-9.

(12) Pfeffer, K.; Matsuyama, T.; Kundig, T. M.; Wakeham, A.;

Kishihara, K.; Shahinian, A.; Wiegmann, K.; Ohashi, P. S.;

Kronke, M.; Mak, T. W. Mice Deficient for the 55 Kd Tumor

Necrosis Factor Receptor are Resistant to Endotoxic Shock, Yet

Succumb to L. monocytogenes Infection. Cell 1993, 73, 457-67.

(13) Selinsky, C. L.; Boroughs, K. L.; Halsey, W. A.; Howell, M. D.

Multifaceted Inhibition of Anti-Tumour Immune Mechanisms

by Soluble Tumour Necrosis Factor Receptor Type I. Immunol-

ogy 1998, 94, 88-93.

(14) Natarajan, K.; Manna, S. K.; Chaturvedi, M. M.; Aggarwal, B.

B. Protein Tyrosine Kinase Inhibitors Block Tumor Necrosis

Factor-Induced Activation of Nuclear Factor-KB, Degradation

of IKBR, Nuclear Translocation of P65, and Subsequent Gene

Expression. Arch. Biochem. Biophys. 1998, 352, 59-70.

(15) Khaled, A. R.; Butfiloski, E. J .; Sobel, E. S.; Schiffenbauer, J .

Use of Phosphorothioate-Modified Oligodeoxynucleotides to

Inhibit NF-κB Expression and Lymphocyte Function. Clin.

Immunol. Immunopathol. 1998, 86, 170-9.

(16) Aggarwal, B. B.; Schwarz, L.; Hogan, M. E.; Rando, R. F. Triple

Helix-Forming Oligodeoxyribonucleotides Targeted to the Hu-

man Tumor Necrosis Factor (TNF) Gene Inhibit TNF Production

and Block the TNF-Dependent Growth of Human Glioblastoma

Tumor Cells. Cancer Res. 1996, 56, 5156-64.

(17) Onrust, S. V.; Lamb, H. M. Infliximab: A Review of its Use in

Crohn’s Disease and Rheumatoid Arthritis. BioDrugs 1998, 10

(5), 397-422.

(2R,3S)-N-{(1S,2R)-4-Nitr ogu a n yl-2-m eth yl-1-[(1,3-th ia -

zol-2-ylam in o)car bon yl]bu tyl}-3-[for m yl(h ydr oxy)am in o]-

2-isobu tylh exa n a m id e (28s). White amorphous solid, 35%.

1

90:10 mixture of diastereomers. H NMR major diastereomer

(400 MHz, CD₃OD mixture of formamide rotamers) δ 8.37 (s,

0.33H), 7.97 (s, 0.67H), 7.42 (d, J ) 3.6 Hz, 1H), 7.11 (d, J )

3.6 Hz, 1H), 4.65 (m, 1H), 4.36 (m, 0.33H), 3.61 (0.67H), 3.36

(m, 2H), 2.92 (m, 0.67H), 2.84 (m, 0.33H), 2.15 (m, 1H), 1.8-

1.1 (m, 8H), 1.03 (d, J ) 6.8 Hz, 3H), 0.95 (m, 1H), 0.91 (m,

6H), 0.78 (m, 3H) ppm. LC-ESIMS Rt ) 5.25 (90%), m/z 529

(M+H)⁺; 5.84 (10%), m/z 529 (M+H)⁺. λ_max270 nm. Anal.

(C₂₁H₃₆N₈O₆S‚³/₄H₂O) C, H, N.

(2S,3R)-5-Nit r ogu a n yl -2-[((2R)-2-{(1S)-1-[for m yl(h y-

d r oxy)a m in o]eth yl}-4-m eth ylp en ta n oyl)a m in o]-3-m eth -

yl-N-(1,3-th ia zol-2-yl)p en ta n a m id e (28t). White amorphous

solid, 43%. ¹H NMR (400 MHz, CD₃OD mixture of formamide

rotamers) δ 8.28 (s, 0.33H), 7.99 (s, 0.67H), 7.42 (d, J ) 3.6

Hz, 1H), 7.11 (d, J ) 3.6 Hz, 1H), 4.68 (m, 1H), 4.46 (m, 0.33H),

3.81 (m, 0.67H), 3.35 (m, 2H), 2.91 (m, 0.67H), 2.88 (m, 0.33H),

2.15 (m, 1H), 1.69 (m, 1H), 1.6-1.3 (m, 3H), 1.28 (d, J ) 6.8

Hz, 2H), 1.20 (d, J ) 6.8 Hz, 1H), 1.1-1.0 (m, 1H), 1.02 (d, J

) 6.8 Hz, 3H), 0.90 (m, 3H), 0.78 (m, 3H) ppm. LC-ESIMS

Rt ) 4.82 (97%) m/z 501 (M+H)⁺. λ_max270 nm. Anal.

(C₁₉H₃₂N₈O₆S‚³/₄H₂O) C, H, N.

(2S,3R)-5-Met h a n esu lfon ylgu a n yl -2-[((2R)-2-{(1S)-1-

[for m yl(h yd r oxy)a m in o]eth yl}-4-m eth ylp en ta n oyl)a m i-

n o]-3-m eth yl-N-(1,3-th iazol-2-yl)pen tan am ide (28u ). White

amorphous solid, 14%. ¹H NMR (400 MHz, CD₃OD mixture of

formamide rotamers) δ 8.28 (s, 0.33H), 7.99 (s, 0.67H), 7.42

(d, J ) 3.6 Hz, 1H), 7.10 (d, J ) 3.6 Hz, 1H), 4.62 (m, 1H),

4,45 (m, 0.33H), 3.81 (m, 0.67H), 3.25 (m, 2H), 2.88 (m, 1H),

2.85 (s, 3H), 2.12 (m, 1H), 1.64 (m, 1H), 1.42 (m, 3H), 1.28 (d,

J ) 6.8 Hz, 2H), 1.18 (d, J ) 6.8 Hz, 1H), 1.1-1.0 (m, 1H),

1.02 (m, 3H), 0.90 (m, 3H), 0.78 (m, 3H) ppm. LC-ESIMS

Rt ) 4.62 (95%) m/z 534 (M+H)⁺. λ_max270 nm. Anal.

(C₂₀H₃₅N₇O₆S₂‚²/₃CH₂Cl₂) C, H, N.

(18) Murray, K. M.; Dahl, S. L. Recombinant Human Tumor Necrosis

Factor Receptor (P75) Fc Fusion Protein (TNFR:Fc) in Rheu-

matoid Arthritis. Ann. Pharmacother. 1997, 31, 1335-8.

(19) Hamilton, K.; Clair, E. W. Tumour Necrosis Factor-Alpha

Blockade: A New Era for Effective Management of Rheumatoid

Arthritis. Expert Opin. Pharmacother. 2000, 1 (5), 1041-52.

(20) Hanauer, S. B. Safety of Infliximab in Clinical Trials. Aliment.

Pharmacol. Ther. 1999, 13 (Suppl. 4), 16-22.

Ack n ow led gm en t. We gratefully acknowledge An-

gela Hassman and Theresa Seaton for TACE assay

development and Robert J ohnson and Manon Villeneuve

for analytical support.

(21) Watts, R. A. Musculoskeletal and Systemic Reactions to Biologi-

cal Therapeutic Agents. Curr. Opin. Rheumatol. 2000, 12 (1),

49-52.

(22) Murali, R.; Hasegawa, A.; Berezov, A.; Kajino, K.; Takasaki, W.

Design and Development of Small-Molecule Inhibitor of Tumor

Necrosis Factor-R. In Cytokine Inhibitors; Ciliberto, G., Savino,

R., Eds.; Marcel Dekker: New York, 2001; pp 133-61.

(23) Gum, R. J .; Young, P. R. p38 Inhibition. In ref 22, pp 329-61.

(24) Gearing, A. J . H.; Beckett, P.; Christodoulou, M.; Churchill, M.;

Clements, J .; Davidson, A. H.; Drummond, A. H.; Galloway, W.

A.; Gilbert, R.; Gordon, J . L.; Leber, T. M.; Mangan, M.; Miller,

K.; Nayee, P.; Owen, K.; Patel, S.; Thomas, W.; Wells, G.; Wood,

L. M.; Wolley, K. Processing of Tumour Necrosis Factor-R

Precursor by Metalloproteinases. Nature 1994, 370, 555-7.

(25) Mohler, K. M.; Sleath, P. R.; Fitzner, J . N.; Cerretti, D. P.;

Alderson, M.; Kerwar, S. S.; Dauphine S. T.; Otten-Evans, C.;

Greenstreet, T.; Weerawarna, K.; Kronheim, S. R.; Petersen, M.;

Gerhart, M. Kozlosky, M.; March, C. J .; Black, R. A. Protection

Against a Lethal Dose of Endotoxin by an Inhibitor of Tumour

Necrosis Factor Processing. Nature 1994, 370, 218-20.

(26) McGeehan, G. M.; Becherer, J . D.; Bast, R. C., J r.; Boyer, C. M.;

Champion, B.; Connolly, K. M.; Conway, J . G.; Furdon, P.; Karp,

S.; Kidao, S.; McElroy, A. B.; Nichols, J .; Pryzwansky, K. M.;

Schoenen, F.; Sekut, L.; Truesdale, A.; Verghese, M.; Warner,

J .; Ways, J . P. Regulation of Tumour Necrosis Factor-R Process-

ing by a Metalloproteinase Inhibitor. Nature 1994, 370, 558-

61.

(27) Black, R. A.; Rauch, C. T.; Kozlosky, C. J .; Peschon, J . J .; Slack,

J . L.; Wolfson, M. F.; Castner, B. J .; Stocking, K. L.; Reddy, P.;

Srinivasan, S.; Nelson, N.; Boiani, N.; Schooley, K. A.; Gerhart,

M.; Davis, R.; Fitzner, J . N.; J ohnson, R. S.; Paxton, R. J .; March,

C. J .; Cerretti, D. P. A Metalloproteinase Disintegrin that

Releases Tumor-Necrosis Factor-R from Cells. Nature 1997, 385,

729-33.

(28) Moss, M. L.; J in, S.-L. C.; Milla, M. E.; Bickett, D. M.; Burkhart,

W.; Carter, H. L.; Chen, W.-J .; Clay, W. C.; Didsbury, J . R.;

Hassler, D.; Hoffman, C. R.; Kost, T. A.; Lambert, M. H.;

Leesnitzer, M. A.; McCauley, P.; McGeehan, G.; Mitchell, J .;

Su p p or tin g In for m a tion Ava ila ble: Experimental pro-

cedures and spectral characterization are provided for com-

pounds 5a -t, 6j-t, and 7j-t. The synthesis of â,â-dimethyl

ornithine derivatives from acid 16b and attempted conversion

to â,â-dimethyl-Arg derivatives is also provided. This material

is available free of charge via the Internet at http://pubs.ac-

s.org.

Refer en ces

(1) Vassalli, P. The Pathophysiology of Tumor Necrosis Factors.

Annu. Rev. Immunol. 1992, 10, 411.

(2) Szatmary, Z. Tumor Necrosis Factor-R: Molecular-Biological

Aspects. Minireview. Neoplasma 1999, 46 (5), 257-66.

(3) Kitano, K.; Rivas, C. I.; Baldwin, G. C.; Vera, J . C.; Golde, D.

W. Tumor Necrosis Factor-Dependent Production of Human

Immunodeficiency Virus 1 in Chronically Infected HL-60 Cells.

Blood 1993, 82, 2742-8.

(4) Elliott, M. J .; Maini, R. N.; Feldmann, M.; Kalden, J . R.; Antoni,

C.; Smolen, J . S.; Leeb, B.; Breedveld, F. C.; Macfarlane, J . D.;

Bijl, H. Randomised Double-Blind Comparison of Chimeric

Monoclonal Antibody to Tumor Necrosis Factor Alpha (Ca2)

Versus Placebo in Rheumatoid Arthritis. Lancet 1994, 344,

1105-10.

(5) Dinarello C. A. The Proinflammatory Cytokines Interleukin-1

and Tumor Necrosis Factor and Treatment of the Septic Shock

Syndrome. J . Infect. Dis. 1991, 163 (6), 1177-84.

(6) Hotamisligil, G. S.; Spiegelman, B. M. Tumor Necrosis Factor

R: A Key Component of the Obesity-Diabetes Link. Diabetes

1994, 43, 1271-8.

(7) Piguet, P. F.; Grau, G. E.; Allet, B.; Vassalli, P. Tumor Necrosis

Factor/Cachectin is an Effector of Skin and Gut Lesions of the

Acute Phase of Graft-Vs.-Host Disease. J . Exp. Med. 1987, 166,

1280-9.

Design of Selective Soluble TACE Inhibitors

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4267

Moyer, M.; Pahel, G.; Rocque, W.; Overton, L. K.; Schoenen, F.;

Seaton, T.; Su, J .-L.; Warner, J .; Willard, D.; Becherer, J . D.

Cloning of a Disintegrin Metalloproteinase That Processes

Precursor Tumor-Necrosis Factor-R. Nature 1997, 385, 733-6.

(29) Black, R. A.; White, J . M. ADAMS: Focus on the Protease

Domain. Curr. Opin. Cell Biol. 1998, 10, 654-9.

(30) Schlondorff, J .; Becherer, J . D.; Blobel, C. P. Intracellular

Maturation and Localization of the Tumour Necrosis Factor R

Convertase (TACE). Biochem. J . 2000, 347 (1), 131-8.

(31) Moss, M. L.; Becherer, J . D.; Conway, J . G.; Warner, J . R.;

Bickett, D. M.; Leesnitzer, M. A.; Seaton, T. M.; Mitchell, J . L.;

McConnell, R. T.; Tippin, T. K.; Whitesell, L. G.; Rizzolio, M.

C.; Hedeen, K. M.; Beaudet, E. J .; Andersen, M.; Lambert, M.

H.; Austin, R.; Stanford, J . B.; Bubacz, D. G.; Chan, J . H.;

Schaller, L. T.; Gaul, M. D.; Cowan, D. J .; Boncek, V. M.;

Rabinowitz, M. H.; Musso, D. L.; McDougald, D. L.; Kaldor, I.;

Glennon, K.; Wiethe, R. W.; Guo, Y.; Andrews, R. C. Screening

and Design of Tumor Necrosis Factor-R: Converting Enzyme

Inhibitors; in ref 22; pp 163-76.

(32) Voss, M. E.; Decicco, C. P.; Wexler, R. R. Preparation of

Arylsulfonylheterocyclylhydroxamic Acids as Metalloproteinase

and TNF Inhibitors for the Treatment of Inflammatory Disor-

ders. PCT Int. Appl. 1999, WO 9958531 A1 19991118.

(33) Bird, J .; Montana, J . G.; Wills, R. E.; Baxter, A. D.; Owen, D. A.

Selective Matrix Metalloproteinase (MMP) Inhibitors Having

Reduced Side-Effects. PCT Int. Appl. 1998, WO 9839024 A1

19980911.

(34) Becherer, J . D. TACE: A Disintegrin Metalloprotease that

Regulates TNF-R Production In vitro and In vivo, New York

Academy of Sciences Conference on Inhibition of Matrix Met-

alloproteinases: Therapeutic Applications, Tampa, FL, Oct.

1998.

(35) Conway, J . G.; Andrews, R. C.; Beaudet, B.; Bickett, D. M.;

Boncek, V.; Brodie, T. A.; Clark, R. L.; Crumrine, R. C.;

Leenitzer, M. A.; McDougald, D. L.; Han, B.; Hedeen, K.; Lin,

P.; Milla, M.; Moss, M.; Pink, H.; Rabinowitz, M. H.; Tippin, T.;

Scates, P. W.; Selph, J .; Stimpson, S. A.; Warner, J .; Becherer,

J . D. Inhibition of tumor necrosis factor-R (TNF-R) Production

and Arthritis in the Rat by GW3333, a Dual Inhibitor of TNF-R

Converting Enzyme and Matrix Metalloproteinases. J . Phar-

macol. Exp. Ther. 2001, 298 (3), 900-8.

(36) Andrews, R. C.; Anderson, M. W.; Stanford, J . B.; Bubacz, D.

G.; Chan, J . H.; Cowan, D. J .; Gaul, M. D.; McDougald, D. L.;

Musso, D. L.; Rabinowitz, M. H.; Wiethe, R. W. Preparation of

N-Formyl-N-Hydroxypeptide Analogues as Metalloprotease In-

hibitors. Brit. UK Pat. Appl. GB 2348198 A1, 2000.

(41) Maskos, K.; Fernandez-Catalan, C.; Huber, R.; Bourenkov, G.

P.; Bartunik, H.; Ellestad, G. A.; Reddy, P.; Wolfson, M. F.;

Rauch, C. T.; Castner, B. J .; Davis, R.; Clarke, H. R. G.; Petersen,

M.; Fitzner, J . N.; Cerretti, D. P.; March, C. J .; Paxton, R. J .;

Black, R. A.; Bode, W. Crystal Structure of the Catalytic Domain

of Human Tumor Necrosis Factor-R-Converting Enzyme. Proc.

Natl. Acad. Sci. U.S.A. 1998, 95 (7), 3408-12.

(42) Bickett, D. M.; Green, M. D.; Berman, J .; Dezube, M.; Howe, A.

S.; Brown, P. J .; Roth, J . T.; McGeehan, G. M. A High Through-

put Fluorogenic Substrate for Interstitial Collagenase (MMP-1)

and Gelatinase (MMP-9). Anal. Biochem. 1993, 212, 58-64.

(43) Leesnitzer, M. A.; Bickett, D. M.; Moss, M. L.; Becherer, J . D. A

High Throughput Assay for the TNF Converting Enzyme. High

Throughput Screening for Novel Anti-inflammatories. Birkhaus-

er Verlag: Basel, Switzerland, 2000; pp 87-100.

(44) Ziegler-Heitbrook, H. W. L.; Thiel, E.; Fuetter, A.; Herzog, V.;

Wirtz, A.; Reithmueller, G. Establishment of a Human Cell Line

(Mono Mac 6) with Characteristics of Mature Monocytes. Int. J .

Cancer 1988, 41, 456-61.

(45) Noyori, R.; Ohkuma, T.; Kitamura, M.; Takaya, H.; Sayo, N.;

Kumobayashi, H.; Akutagawa, S. Asymmetric Hydrogenation of

â-Keto Carboxylic Esters. A Practical, Purely Chemical Access

to â-Hydroxy Esters in High Enantiomeric Purity. J . Am. Chem.

Soc. 1987, 109 (19), 5856-8.

(46) Seebach, D.; Aebi, J .; Wasmuth, D. Diastereoselective R-Alky-

lation of â-Hydroxycarboxylic Esters Through Alkoxide Eno-

lates: (+)-Diethyl (2S, 3R)-3-Allyl-2-Hydroxysuccinate from (-)-

Diethyl S-Malate (Butanedioic Acid, 2-Hydroxy-3-(2-Propenyl)-,

Diethyl Ester, [S-(R, S)]). Org. Synth. 1985, 63, 109-20.

(47) Krimen, L. I. Acetic Formic Anhydride. Org. Synth. 1970, 50,

1-3.

(48) Hanessian, S.; Kagotani, M.; Komaglou, K. Design and Reactivity

of Organic Functional Groups - 2-Pyridylsulfonates as Nucle-

ofugal Esters: Remarkably Mild Transformations into Halides

and Olefins. Heterocycles 1989, 28 (2), 1115-20.

(49) Kazmaier, U.; Krebs, A. Synthesis of Chiral γ,δ-Unsaturated

Amino Acids by Asymmetric Ester Enolate Claisen Rearrange-

ment. Angew. Chem., Int. Ed. Engl. 1995, 34 (18), 2012-4.

(50) Krebs, A.; Kazmaier, U. The Asymmetric Ester Enolate Claisen

Rearrangement as

a Suitable Method for the Synthesis of

Sterically Highly Demanding Amino Acids. Tetrahedron Lett.

1996, 37 (44), 7945-6.

(51) Allinger, N. L.; Zalkow, V. Conformational Analysis. IX. gem-

Dimethyl Effect. J . Org. Chem. 1960, 25, 701-4.

(52) Avontuur, J . A. M.; Buijk, S. L. C. E.; Bruining, H. A. Distribu-

tion and Metabolism of N^G-Nitro-L-Arginine Methyl Ester in

Patients with Septic Shock. Eur. J . Clin. Pharmacol. 1998, 54

(8), 627-31.

(37) Lambert, M. H., unpublished results.

(38) Gomis-Ruth, F. X.; Kress, L. F.; Kellermann, J .; Mayr, I.; Lee,

X.; Huber, R.; Bode, W. Refined 2.0 Å X-ray Crystal Structure

of the Snake Venom Zinc Endopeptidase Adamalysin II. Primary

and Tertiary Structure Determination, Refinement, Molecular

Structure and Comparison with Astacin, Collagenase and Ther-

molysin. J . Mol. Biol. 1994, 239, 513-44.

(39) Lambert, M. H. Docking Conformationally Flexible Molecules

Into Protein Binding Sites. In Practical Application of Computer-

Aided Drug Design; Charifson, P. S., Ed.; Marcel Dekker: New

York, 1997; pp 243-303.

(40) The pK_aof the nitroguanidinium ion in water was measured to

be -0.98. Taylor, P. J .; Wait, A. R. σ₁Values for Heterocycles.

J . Chem. Soc., Perkin Trans. 2, 1986, 1765-70.

(53) Henke, B. R.; Blanchard, S. G.; Brackeen, M. F.; Brown, K. K.;

Cobb, J . E.; Collins, J . L.; Harrington, W. W., J r.; Hashim, M.

A.; Hull-Ryde, E. A.; Kaldor, I.; Kliewer, S. A.; Lake, D. H.;

Leesnitzer, L. M.; Lehmann, J . M.; Lenhard, J . M.; Orband-

Miller, L. A.; Miller, J . F.; Mook, R. A.; Noble, S. A.; Oliver, W.;

Parks, D. J .; Plunket, K. D.; Szewczyk, J . R.; Willson, T. M. N-(2-

Benzoylphenyl)-L-tyrosine PPARγ Agonists. 1. Discovery of a

Novel Series of Potent Antihyperglycemic and Antihyperlipi-

demic Agents. J . Med. Chem. 1998, 41 (25), 5020-36.

J M0102654

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Design of selective and soluble inhibitors of tumor necrosis factor-α converting enzyme (TACE)

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