N-O-isopropyl sulfonamido-based hydroxamates: Design, synthesis and biological evaluation of selective matrix metalloproteinase-13 inhibitors as potential therapeutic agents for osteoarthritis

J. Med. Chem. 2009, 52, 4757–4773 4757

DOI: 10.1021/jm900261f

N-O-Isopropyl Sulfonamido-Based Hydroxamates: Design, Synthesis and Biological Evaluation of

Selective Matrix Metalloproteinase-13 Inhibitors as Potential Therapeutic Agents for Osteoarthritis

Elisa Nuti,^†Francesca Casalini,^†Stanislava I. Avramova,^†Salvatore Santamaria,^†Giovanni Cercignani,^‡Luciana Marinelli,^§

Valeria La Pietra,^§Ettore Novellino,^§Elisabetta Orlandini,^†Susanna Nencetti,^†Tiziano Tuccinardi,^{†,^}Adriano Martinelli,^†

Ngee-Han Lim, Robert Visse, Hideaki Nagase, and Armando Rossello*^,†

†

‡

Dipartimento di Scienze Farmaceutiche, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Biologia, Unita di Biochimica,

ꢀ

§

ꢀ

Universita di Pisa, Via San Zeno 51, 56127 Pisa, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli “Federico

II”, Via Domenico Montesano 49, 80131 Napoli, Italy, Department of Matrix Biology, The Kennedy Institute of Rheumatology Division, Imperial

College, London W6 8LH, United Kingdom, and ^{^}Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology,

College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122

Received March 2, 2009

Matrix metalloproteinase-13 (MMP-13) is a key enzyme implicated in the degradation of the extracellular

matrix in osteoarthritis (OA). For this reason, MMP-13 synthetic inhibitors are being sought as potential

therapeutic agents to prevent cartilage degradation and to halt the progression of OA. Herein, we report the

synthesis and in vitro evaluation of a new series of selective MMP-13 inhibitors possessing an arylsulfon-

amidic scaffold. Among these potential inhibitors, a very promising compound was discovered exhibiting

nanomolar activity for MMP-13 and was highly selective for this enzyme compared to MMP-1, -14, and

TACE. This compound acted as a slow-binding inhibitor of MMP-13 and was demonstrated to be effective

in an in vitro collagen assay and in a model of cartilage degradation. Furthermore, a docking study was

conducted for this compound in order to investigate its binding interactions with MMP-13 and the reasons

for its selectivity toward MMP-13 versus other MMPs.

1. Introduction

morphogenesis, bone remodeling, wound healing, and angio-

genesis. Furthermore, the overexpression of MMPs is asso-

ciated with several pathologies, including cancer-cell invasion

and metastasis, the loss of cartilage in osteoarthritis, rheuma-

toid arthritis, cardiovascular diseases, chronic obstructive

pulmonary disease, and periodontitis.⁴

Osteoarthritis (OA^a) is characterized by the destruction of

articular cartilage, chronic joint pain, and inflammation, and it

is estimated to affect about 20 million people in the United

States alone. The destruction of articular cartilage is largely due

to the elevated activities of proteolytic enzymes that degrade the

extracellular matrix (ECM) within the cartilage, whose main

constituents are aggrecan and type II collagen.^1-3Collagen is a

triple helical protein that is resistant to most proteases but is

efficiently recognized and degraded by collagenase 3, known as

matrix metalloproteinase-13 (MMP-13). MMP-13 catalyzes

the hydrolysis of type II collagen at a unique site resulting

in 3/4- and 1/4-length polypeptide products. This enzyme

belongs to a family of enzymes termed matrix metalloprotei-

nases (MMPs), which are zinc-dependent endopeptidases

involved in the remodeling of extracellular matrix proteins.

These endopeptidases have important roles in development,

In osteoarthritis, chondrocytes stimulated by inflammatory

cytokines, injury, or mechanical stimuli produce many

MMPs, the most important of which is MMP-13, which is

considered the principal target in arthritis. MMP-13 is not

found in normal adult tissues but is expressed in the joints and

articular cartilage of osteoarthritis patients.⁵Furthermore, a

MMP inhibitor that preferentially inhibits MMP-13 has been

shown to block the degradation of explanted human osteoar-

thritic cartilage.⁶On the basis of these findings, MMP-13

synthetic inhibitors are being sought as potential therapeutic

agents to prevent cartilage degradation and to slow down the

progression of OA.³Unfortunately, few MMP inhibitors

(MMPIs) have been examined in clinical trials for OA and

none have been successfully utilized as therapeutic agents so

far. In fact, many broad spectrum MMP inhibitors have been

found to display a dose-limiting toxicity in the form of

musculoskeletal side effects including joint stiffness and in-

flammation.⁷Although the human joint side effects are rever-

sible upon withdrawal of the drug, musculoskeletal syndrome

(MSS) has halted clinical trials of many nonselective MMPIs.

While the inhibition of specific MMPs such as MMP-1 or

MMP-14 has been postulated to be responsible for MSS, the

exact cause of this pathology is not yet clear. Others have

speculated that sheddase inhibition, specifically inhibition of

tumor necrosis factor-R converting enzyme (TACE), could be

*To whom correspondence should be addressed. Phone: þ39 050

2219562. Fax: þ39 050 2219605. E-mail: aros@farm.unipi.it.

^aAbbreviations: OA, osteoarthritis; ECM, extracellular matrix; MMP,

matrix metalloproteinase; MSS, musculoskeletal syndrome; TACE, tumor

necrosis factor-R converting enzyme; MMPI, MMP inhibitor; ZBG, zinc-

binding group; DIAD, diisopropyl azodicarboxylate; SDS-PAGE, sodium

dodecyl sulfate-polyacrylamide gel electrophoresis; IL-1R, interleukin-1R;

GAG, glycosaminoglycan; ADAMTS, a disintegrin and metalloproteinase

with thrombospondin type I repeats; Hypro, hydroxyproline; OSM,

oncostatin M; PDB, protein data bank; TIMP, tissue inhibitor of

metalloproteinase; EDC, N-(3-Dimethylaminopropyl)-N⁰-ethylcarbo-

diimide hydrochloride; APMA, p-aminophenylmercuric acetate; DMMB,

dimethylmethylene blue; DMBA, dimethylaminobenzaldehyde; GALS,

genetic algorithm local search; RESP, restrained electrostatic potential;

rmsd, root-mean square deviation; SAR, structure-activity relationship.

r

2009 American Chemical Society

Published on Web 07/16/2009

pubs.acs.org/jmc

4758 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

responsible for the observed side effects. The underlying

mechanisms may possibly be mediated through the failure to

downregulate receptor signaling by proteolytic release of the

extracellular domain.⁸Therefore, trying to reduce the like-

lihood of MSS is an important therapeutic consideration when

treating osteoarthritis. Hence, pharmaceutical research has

mainly focused on the discovery of potent inhibitors of

MMP-13 displaying a high degree of selectivity over other

MMPs.^9-12Almost all MMPIs are based on a chelating moiety

interacting with a catalytic zinc ion and a hydrophobic portion

protruding into the hydrophobic S1⁰subsite, which is a deep

pocket situated in proximity to the catalytic zinc ion. These

compounds behave as competitive inhibitors because the zinc-

binding group (ZBG) binding mode can mimic one of the

transition states occurring during the substrate hydrolysis.¹³

Widely utilized ZBGs include: hydroxamic acids, carboxylic

acids, and thiols. The most used of these is the hydroxamate

group because it has excellent chelating properties.

MMPs have been classified on the basis of their substrate

specificity into the five following classes: gelatinases (MMP-2

and MMP-9), collagenases (MMP-1, -8, -13), stromelysins

(MMP-3, -10), membrane types (MMP-14, -16, -17), and

other related enzymes such as matrilysin (MMP-7).¹⁴There

is a shared similarity in the overall three-dimensional fold

for the MMPs, which is consistent with the relatively high

sequence homology (>40%), which makes the design of a

selective MMPI a difficult task. The major structural differ-

ence observed between the MMP enzymes is the relative size

and shape of the S1⁰pocket. This site is also the major

substrate determinant that has been exploited for designing

selective inhibitors.¹⁵For instance, non-zinc-chelating inhibi-

tors that are highly selective for MMP-13 and that exclusively

bind the S1⁰pocket have recently been reported.¹⁶

In the course of our research on sulfonylated MMP in-

hibitors, we have previously reported compound 1a¹⁷

(Figure 1), which is an arylsulfonamido hydroxamic acid¹⁸

that showed good selectivity for MMP-2 over MMP-1 and

MMP-14. Compound 1a represented the first example

of a new family of tertiary sulfonamides (the N-O-alkyl

sulfonamides) possessing a small O-alkyl group on their

sulfonamido nitrogen. This type of inhibitors resulted in

highly selective inhibition for MMP-2, even with respect to

other potent sulfonamido-based analogues that were later

developed,¹⁹belonging to the well-known family of N-alkyl

tertiary sulfonamides. In an effort to shift the activity and

selectivity toward MMP-13 as target for OA, we aimed to

modify the arylsulfonyl moiety that interacts with the S1⁰

subsite. The active-site structure of MMP-13 is well-known

in the literature because many X-ray crystallography and

NMR studies have been reported on complexes of this enzyme

with different inhibitors.^20-23Bearing in mind that the

MMP-13 S1⁰subsite is an unusually large and deep pocket,

we designed a series of arylsulfonamido hydroxamates ana-

logues of 1a that incorporate bulky and long P1⁰substituents.

A comparison of the inhibitory activity of 1a with those of

previously reported 4⁰-biphenyl sulfonylated analogues (2¹⁷

and 3²⁴) devoid of the oxygen atom linked to the sulfonamide

moiety, which is present in 1a, prompted us to maintain the

N-O-isopropyl group in the new series of compounds. This

substituent was demonstrated to be important in order to

achieve selectivity over MMP-1 and MMP-14, and it mini-

mally affected the activity on MMP-2 and MMP-13 (Table 1).

Therefore, in the present paper, we report the synthesis and

preliminary biological evaluation of a new series of N-iso-

propoxy-arylsulfonamides, 4a-l (Figure 1), bearing different

aryl substituents on the sulfonamidic portion. These com-

pounds were assayed in vitro using recombinant MMPs, and

the most promising scaffold was modified by introducing an

alkyl group in the R position relative to the hydroxamate in

order to increase its inhibitory activity. Compound 5

(Figure 1) was found to be a potent and highly selective

slow-binding inhibitor of MMP-13 in the enzymatic assay

and was successively tested in vitro on collagen and cartilage

explants. Finally, molecular modeling studies were performed

with the aim of revealing, at an atomic level, the interactions

Figure 1. Progression of the SAR: from the lead compound 1a to

the MMP-13 selective inhibitor 5.

Table 1. In Vitro^aActivity (IC₅₀nM Values) of Biphenylsulfonamide Hydroxamic Acids 1a-3

compd

R

MMP-1

MMP-2

MMP-3

MMP-8

MMP-9

MMP-13

MMP-14

1a

2

O-i-Pr

i-Bu

H

12000 ( 1000

4800 ( 300

610 ( 50

12 ( 2

2.0 ( 0.2

5900 ( 340

920 ( 50

320 ( 25

260 ( 10

48 ( 3

7.0 ( 0.7

200 ( 60

34 ( 2

34 ( 1

45 ( 2

39 ( 2

2.8 ( 0.3

2300 ( 170

530 ( 40

70 ( 3

3

^aAssays were run in triplicate. The final values given here are the mean ( SD of three independent experiments.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4759

Scheme 1. Synthesis of N-Isopropoxy-arylsulfonamide Hydro-

xamates 4a-f ^a

Scheme 2. Synthesis of N-Isopropoxy-arylsulfonamide Hydro-

xamates 4g-l^a

^aReagents and conditions: (i) BrCH₂COOt-Bu, Cs₂CO₃, Bu₄NH-

SO₄, DMF; (ii) RB(OH)₂, Pd(PPh₃)₄, K₃PO₄, dioxane/H₂O, 70 ꢀC;

(iii) TFA, CH₂Cl₂, 0 ꢀC; (iv) TBDMSiONH₂, EDC, CH₂Cl₂; (v) TFA,

CH₂Cl₂, 0 ꢀC.

that govern the recognition and the binding of 5 to the target

MMP-13. These findings are a prerequisite for understanding

this compound’s therapeutic activity toward OA as well as

shedding light onto the structural features responsible for the

lack of activity toward MMP-1, -14, and TACE.

^aReagents and conditions: (i) 5-bromothiophene-2-sulfonyl chloride,

NMM, THF; (ii) BrCH₂COOt-Bu, Cs₂CO₃, Bu₄NHSO₄, DMF;

(iii) RB(OH)₂, Pd(PPh₃)₄, K₃PO₄, dioxane/H₂O, 70 ꢀC; (iv) 1-butyl-

4-ethynyl benzene, Pd(PPh₃)₂Cl₂, CuI(I), TEA, DMF; (v) TFA,

CH₂Cl₂, 0 ꢀC; (vi) TBDMSiONH₂, EDC, CH₂Cl₂; (vii) TFA, CH₂Cl₂,

0 ꢀC.

2. Chemistry

N-Isopropoxy-arylsulfonamide hydroxamates 4a-f were

prepared as described in Scheme 1. 4-Bromo-N-isopropoxy-

benzenesulfonamide 6, synthesized as previously reported,²⁵

was reacted with tert-butyl bromoacetate in the presence of

cesium carbonate to generate ester 7. Similarly, sulfonamides

8b,f²⁵were converted to tert-butyl esters 9b,f. Arylsulfon-

amide esters 9a,c-e were obtained by Suzuki coupling of

aryl bromide 7 with the appropriate arylboronic acid using

Pd(PPh₃)₄as the catalyst. Acid hydrolysis of esters 9a-f

yielded carboxylates 10a-f, which were finally converted to

their corresponding hydroxamates by condensation with

O-(tert-butyldimethylsilyl)hydroxylamine and acid cleavage

by TFA.²⁶

Scheme 3. Preparation of Compound 5^a

The thiophene derivatives 4g-l were synthesized by follow-

ing an analogous route, as reported in Scheme 2. Commer-

cially available 5-bromothiophene-2-sulfonyl chloride was

reacted with O-isopropylhydroxylamine hydrochloride

(12)¹⁷in THF, yielding 5-bromo-N-isopropoxythiophene-

2-sulfonamide 13, which was alkylated with tert-butyl bromo-

acetate to give ester 14. This ester was successively converted

by Suzuki coupling to para-fluoro, para-ethyl, and para-

methoxy biaryl derivatives 15h-l, which finally generated

hydroxamates 4h-l. The same 5-bromothiophene intermedi-

ate 14 was submitted to the Sonogashira reaction²⁷in the

presence of Pd(II), CuI, and triethylamine to produce the

acetylenic derivative15g, which was converted into the corres-

ponding hydroxamic acid 4g as described above.

^aReagents and conditions: (i) PPh₃, DIAD, THF_.; (ii) 4-(4⁰-chloro-

benzyloxy)-phenylboronic acid, Pd(PPh₃)₄, K₃PO₄, dioxane/H₂O, 70 ꢀC;

(iii) TFA, CH₂Cl₂, 0 ꢀC; (iv) TBDMSiONH₂, EDC, CH₂Cl₂; (v) TFA,

CH₂Cl₂, 0 ꢀC.

Optically active compound 5 (Scheme 3) was prepared

starting from the (S )-R-hydroxy-tert-butyl-ester 17, which

was synthesized as previously described.²⁸A Mitsunobu

reaction of sulfonamide 6 with the alcohol 17 in the presence

4760 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

Table 2. In Vitro^aActivity (IC₅₀nM Values) of N-Isopropoxy-arylsulfonamides, 4a-l

^aAssays were run in triplicate. The final values given here are the mean ( SD of three independent experiments. ^bIC₅₀values in the high micromolar

range could not be accurately estimated due to fluorescence emission from the compound.

of diisopropyl azodicarboxylate (DIAD) and triphenylphos-

phine gave the tert-butyl ester (R)-18. Thearylintermediate 18

was then converted into 19 by Pd-catalyzed Suzuki coupling

with 4-(4⁰-chlorobenzyloxy)-phenylboronic acid in the pre-

sence of K₃PO₄. Acid cleavage of 19 yielded the carboxylic

acid 20, which was finally converted to its corresponding

hydroxamate 5 by condensation with O-(tert-butyldimethyl-

silyl)hydroxylamine, followed by acid hydrolysis with TFA.

MMP-13 with an IC₅₀=7.2 nM. The introduction of a phe-

noxybenzenesulfonyl substituent (as in compound 4f) caused

a decrease in inhibitory activity for MMP-13 and MMP-2

relative to 1a, which was accompanied by a subsequent

decrease in selectivity. The replacement of a benzene ring

with a 2,5-disubstituted thiophene ring in the sulfonyl group,

as in the 4g-l derivatives, led to a reduction of activity

against MMP-13 and MMP-2 while only slightly affecting

the inhibitory activity on MMP-1 and MMP-14. This reduc-

tion in inhibitory activity was probably due to a loss in

linearity of the arylsulfonyl moiety, which adversely affected

the binding affinity for the S1⁰pocket.

The best results were observed for derivatives 4d,e, which

allowed the curved portion in the arylsulfonyl moiety to

better fit with MMP-13 than with MMP-2. In fact, it is well-

known from the literature that the main difference in the S1⁰

pocket between MMP-13 and MMP-2 is that the MMP-2 S1⁰

loop is two amino acids shorter.⁹In MMP-2, the absence of

residues analogous to G248 and S250 causes the loop to be

displaced by 4 A proximal to S250 of MMP-13. For this

reason, the S1⁰pocket of MMP-13 was probably more

suitable than that of MMP-2 to accommodate the curved-

shaped arylsulfonyl moiety of 4d,e. In particular, com-

pound 4d appeared to be the best of the series with a

3. Results and Discussion

MMP Inhibition. The new hydroxamic acids synthesized

(4a-l and 5) were tested in vitro on human recombinant

MMPs by a fluorometric assay, which used a fluorogenic

peptide²⁹as the substrate. Results are reported in Tables 2

and 3, together with those already obtained for the pre-

viously described analogues 1a-3 (Table 1).

As can be seen from data in Table 2, changing from a

biphenyl (as in compound 1a) to a 4-substituted biphenyl

group in P1⁰(as in 4a-c) caused a general increase in the

inhibitory activity of all tested enzymes without affecting the

selectivity profile. In fact, these derivatives were better

inhibitors for MMP-2 than for MMP-13, although they

remained poor inhibitors for MMP-1 and -14. In particular,

the methylthio derivative 4a was the most active inhibitor for

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4761

Table 3. In Vitro^aInhibitory Activity (IC₅₀nM Values) of Compound 5 Compared to Those of the Previously Described Compounds 1a, 1b, and 4d

compd

MMP-1

MMP-2

MMP-3

MMP-8

MMP-9

MMP-13

MMP-14

MMP-16

TACE

1a

1b

4d

5

12000 ( 1000

490 ( 76

13000 ( 1100

48000 ( 2800

12 ( 2

0.80 ( 0.20

40 ( 3

5900 ( 340

50 ( 2

1800 ( 120

180 ( 13

260 ( 10

1.6 ( 0.1

1700 ( 100

63 ( 3

200 ( 60

6.7 ( 1.6

1100 ( 100

69 ( 10

45 ( 2

4.1 ( 0.2

19 ( 2

2300 ( 170

9.8 ( 0.2

3400 ( 360

14000 ( 1300

900 ( 48

51 ( 3

15000 ( 1200

5000 ( 440

>100000

14000 ( 1000

35000 ( 3400

>100000

9.6 ( 0.4

3.0 ( 0.2

^aAssays were run in triplicate. The final values given here are the mean ( SD of three independent experiments.

Chart 1

Figure 2. Progress curves relative to MMP-13 inhibition by com-

pound 5, in the first phase (15 min) after mixing, at four different

concentrations in duplicate. Two control reaction mixtures (without

inhibitor) are also shown. Dots represent fluorescence readings,

continuous lines are best fits to the kinetic model of Duggleby.³²

nanomolar activity for MMP-13 (IC₅₀= 19 nM). This

compound exhibited a slight selectivity over MMP-2

(IC₅₀=40 nM), a 1000-fold selectivity over MMP-1 and 200-

fold selectivity over MMP-14. In addition, this derivative

showed good selectivity for MMP-13 over MMP-3, -16, and

TACE (all IC₅₀s were in the micromolar range) (Table 3).

On the basis of these results and in order to increase its

inhibitory potency, we decided to further modify 4d by

introducing an alkyl substituent in position R of the hydro-

xamate. In fact, the introduction of an isopropyl group of the

(R) configuration had already yielded good results when

applied to the scaffold of compound 1a, thereby leading to

the discovery of compound 1b (Chart 1) that was very active

against MMP-2.^28,30Therefore, derivative 5, bearing the

same arylsulfonyl moiety of 4d and an isopropyl group in

P1, was synthesized to verify this hypothesis.

As expected, compound 5 (Table 3) proved to be the best

of the series exibiting a 6-fold increase in inhibitory potency

against MMP-13 (IC₅₀=3.0 nM) and a lower activity for

MMP-1, MMP-14, and TACE relative to 4d. In addition, 5

displayed a 16000-fold selectivity for MMP-13 over MMP-1,

a 5000-fold selectivity over MMP-14, and no measurable

inhibitory activity toward TACE (IC₅₀>100 μM). As shown

in Table 3, compound 5 was more selective than 1a and 1b

(in fact, the last was highly potent against MMP-2 but quite

unselective for the other tested enzymes).

Inset: plot of k_obsvs [I] used to calculate k_onand k_off

.

enzymes. Figure 2 shows the reaction progress curves in the

absence and presence of inhibitor 5 for MMP-13 inhibition.

In the absence of inhibitor, the steady-state rate of hydro-

lysis of the fluorogenic substrate was reached immediately

and the reaction progress curve was essentially linear. In

addition, straight lines were also observed in the presence of

fast-binding inhibitors. However, in the presence of a slow-

binding inhibitor, there was a slow decrease in reaction rate,

which varied as a function of the inhibitor concentration.

Depending on the inhibitor, different concentration ranges

had to be used in order to obtain experimental progress

curves that could be analyzed according to Duggleby³²(see

Experimental Section). Nevertheless, preincubation of the

enzyme with the inhibitor for an appropriate period of time

(4 h) gave rise to linear reaction courses (final steady-state

rates). The K_ivalues of the slow-binding component for

inhibition against 5, 4d, and 1b are listed in Table 4.

Compounds 4b and 1a did not show slow-binding behavior

for either MMP-13 or MMP-2. Thus, their K_ivalues were

determined from the slopes of the progress curves because

k_onand k_offvalues could not be evaluated under our assay

conditions. The slow-binding behavior of compounds 5, 4d,

and 1b (k_onof the order 10⁴-10⁵M^{-1 -1}

s ) were matched by

Slow-Binding Inhibition Assays. For the most active com-

pound of the series, 5, and its analogue devoid of the

R-substituent, 4d, a kinetic characterization was carried out in

comparison with the 4-methoxybiphenyl derivative 4b and with

the biphenyl analogues 1a and 1b. To evaluate the kinetics of

inhibition toward MMP-13 and MMP-2, an assay suited for

the kinetic characterization of slow-binding inhibitors was

used.³¹The enzyme was added to a solution of fluorogenic

substrate and increasing concentrations of the inhibitor. Slow-

binding behavior was observed for 5, 4d, and 1b with both

nanomolar range K_ivalues due to comparatively low values for

the offset rate constants (k_offof the order 10^-4-10^-3s^-1).

Taking into account the results shown in Table 4, it

could be suggested that the presence of a P1 substituent

(the isopropyl group of the (R) configuration) might be

responsible for the slow-binding behavior of the inhibitor.

However, a bent and extended P1⁰substituent, such as

the p-chlorobenzyloxy moiety present in 4d and 5, probably

contributed to this property. In fact, the R-unsubstituted

4762 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

Table 4. Kinetic Parameters^afor Compounds 5, 4b,d, and 1a,b Calculated for Inhibition of MMP-13 and MMP-2

MMP-13

MMP-2

compd

k_offꢀ 10^-4(s^-1

)

k_onꢀ 10⁴(M^{-1 -1}

s

)

K_i(nM)

r²

k_offꢀ 10^-4(s^-1

)

k_onꢀ 10⁴(M^{-1 -1}

s

)

K_i(nM)

r²

5

4.0 ( 3.0

6.0 ( 2.0

N/A^b

1.4 ( 0.1

7.3 ( 0.3

N/A

29 ( 1

0.9794

0.9875

N/A

6.0 ( 3.0

22 ( 7

N/A^b

1.1 ( 0.1

8.7 ( 1.0

N/A

54 ( 2

25 ( 1

3.8 ( 0.2

13 ( 2

0.9707

0.8805

N/A

4d

4b

1a

1b

8.2 ( 0.3

5.8 ( 0.2

20 ( 1

N/A^b

N/A

40 ( 2

N/A

0.9756

N/A^b

N/A

33 ( 2

N/A

0.9634

6.0 ( 3.0

1.5 ( 0.5

3.3 ( 2.0

1.0 ( 0.1

^aThe kinetic parameters were evaluated as described in the Experimental Section. ^bN/A, not applicable (no slow-binding behavior).

Figure 3. Effect of inhibitors on collagen degradation by MMP-1 and MMP-13. (A) SDS-PAGE analysis of the reactions for the effect of 5 on

MMP-1 (left) and MMP-13 (right) cleavage of collagen, with arrows indicating the intact and cleaved collagen R2 chain used for densitometric

analysis. (B) Graphs representing the remaining % activity of the MMPs after incubation with the various inhibitors 1a (black closed circles,

solid line), 2 (dark-gray squares, solid line), 3 (light-gray triangles, solid line), and 5 (black triangles, dotted line).

derivative 4d also showed a slow-binding behavior. The lowest

association constant (k_on) for inhibition against MMP-13 was

measured for compound 5, which bears both of these moieties.

The difference in association constants was 5-fold between 5

and 4d and 30-fold between 5 and 1b. Moreover, compound 5

exhibited the slowest dissociation constant (k_off). The slow

dissociation rate of this compound would predictably result

in a long duration of action and, consequently, it would not be

necessary to maintain high systemic levels of drug to continu-

ously inhibit the target enzyme. Importantly, no relevant

difference in the slow-binding kinetic constants between

MMP-2 and MMP-13 was observed.

Collagenase Assays. Having identified compound 5 with a

high affinity and promising selectivity for MMP-13, the

activity of this analogue on MMP-13 and MMP-1 was

assessed by a collagenase assay in comparison to the pre-

viously described compounds 1a-3. IC₅₀s were determined

against full-length MMP-1 and MMP-13 with type I collagen

as a substrate. Samples were run on sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE), and the

amount of collagen cleavage was determined by densito-

metry (Figure 3).

Table 5. In Vitro Activity (IC₅₀, nM) of 1a-3 and 5 by Collagenase

Assay

compd

MMP-1 (collagen)^a

MMP-13 (collagen)^a

1a

2

2900 ( 1100

4100 ( 1600

970 ( 290

25 ( 4

9.4 ( 0.2

2.4 ( 0.1

1.4 ( 0.2

3

5

15000 ( 8200

^aCollagen was used as substrate.

explant model in order to verify the activity of these inhibi-

tors on collagen and aggrecan degradation occurring during

osteoarthritis. The explants were cultured under the stimuli

of interleukin-1R (IL-1R) and oncostatin M (OSM), which

cause aggrecan degradation for the first 7-10 days and then

collagen degradation after approximately 20 days.³³As

reported in Figure 4A, the inhibitors were not effective at

blocking aggrecan breakdown, represented by glycosamino-

glycan (GAG) release, at any of the doses tested. These data

indicate that these compounds were probably not active

against the aggrecanases, members of the disintegrin and

metalloproteinase with thrombospondin type I repeats

(ADAMTS) family, which are the enzymes considered to

be responsible for aggrecan degradation.³⁴

The IC₅₀values obtained for collagen were in agreement with

those obtained using the peptide substrate (Table 5), with 5

being the worst inhibitor for MMP-1 and the best for MMP-13.

Cartilage Degradation Assays. Compound 5 and its pro-

genitor 1a were also tested in a porcine articular cartilage

In contrast, collagen degradation measured by hydroxy-

proline (Hypro) release was inhibited in a concentration-

dependent manner by both inhibitors, with 1a being less

effective than 5 (Figure 4B). Compound 1a was only effective

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4763

Figure 4. Effect of inhibitors on aggrecan and collagen degradation in long-term IL-1R-stimulated porcine articular cartilage explants

cultures. Cartilage was stimulated with IL-1R (10 ng/mL) in the presence or absence of the inhibitor compounds 1a and 5 at various

concentrations (0.1, 1, and 10 μM). The medium was replenished every 3 or 4 days. (A) The amount of GAG (dashed lines) and Hypro (solid

lines) released into each medium was determined in the control (top), compound 1a treated (middle), and compound 5 treated (bottom)

cartilage. (B) The cumulative amount of Hypro released into the medium with the background release of Hypro (without stimulation)

subtracted of compound 1a (top) and compound 5 (bottom) treated cartilage.

in blocking collagen degradation at 10 μM, whereas 5 was

effective at 1 μM. This finding very roughly corresponds to

the fact that 5 was a 10-fold better inhibitor for MMP-13

than 1a in both the fluorometric and in the collagenase assay.

Hence, in the IL-1R/OSM stimulated model of cartilage

degradation, it appeared that the enzyme responsible for

collagenolysis was MMP-13, as compound 5 was highly

selective for that enzyme.

tional changes, which may occur upon binding of our particular

ligand. However, this strategy did allow the exploitation of the

crystallographic data collected to date on MMPs.

X-ray Structures Selection for Docking Studies. As for the

MMP-1 X-ray structures, an apo form (PDB code: 1SU3³⁵),

a full length form (PDB code: 2CLT³⁶), a form bound to

TIMP-1 (PDB code: 2J0T³⁷), and three forms that were

complexed to organic inhibitors (PDB code: 1HFC,³⁸

2TCL,³⁹966C²⁰) have been reported in the RCSB Protein

Data Bank (PDB). A superposition on the R carbon atoms

and using 1HFC as the reference indicated that the protein

folding was highly preserved. However, in 966C, the R214

side chain was displaced from its lowest energy position upon

inhibitor binding, thereby opening the S1⁰pocket. Thus, the

1HFC, where the S1⁰was closed, and the 966C, where it was

open, were both selected for ligand docking studies.

4. Molecular Modeling

Molecular modeling was performed with the aim of reveal-

ing, at an atomic level, the potential interactions that govern the

recognition and the binding of 5to MMP-13 as well as shedding

light on the structural features responsible for the apparent

inactivity toward MMP-1, -14, and TACE. In the attempt to

overcome the rigidity of the enzymes during the ligand docking

process, a detailed comparison among the X-ray structures

currently available was performed for each MMP, and those

that were most diverse were selected for docking experiments.

Obviously, this approach does not ensure representation of all

energetically accessible conformations of MMPs or conforma-

Thirteen MMP-13 X-ray structures have been released in

the PDB. Besides that cocrystallized with TIMP-2, all the

others were cocrystallized with organic inhibitors such as the

4-[4-(4-chloro-phenoxy)-benzenesulfonylmethyl]-tetrahydro-

pyran-4-carboxylic acid hydroxyamide⁴⁰(RS-130830) (PDB

4764 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

Figure 5. Docked structure of 5 into the MMP-13, where a portion of the binding site is visible as gold surface (left side). Detailed interaction

mode of 5 with MMP-13 active site residues (right side). H-Bonds are displayed as gray lines.

code: 830C²⁰). A superposition of all X-ray structures on the

R carbon atoms using 830C as the reference structure showed

that the protein folding and the shape of the catalytic loops

were highly superimposable. In addition, the large majority

of the residues were all preserved in the side chain conforma-

tions in the catalytic site. Thus, in this case, only 830C with

the lower resolution (1.60 A) was selected for our docking

studies.

Several TACE X-ray structures are available cocrystal-

lized with TIMP-3 as well as with peptidic or organic

inhibitors. A superposition, performed as before with the

other MMPs, revealed a strict conservation of the protein

folding and noteworthy flexibility of the S1⁰specificity loop.

Thus, the three structures (3E8R,⁴¹2I47,⁴²2OI0⁴³) that were

the most divergent in the S1⁰loop shape were considered for

docking of compound 5.

With regard to the MMP-14 X-ray structures, we used

1BUV because only two identical structures, complexed with

TIMP-2, were available in the PDB (1BUV, 1BQQ).⁴⁴

Docking of 5 on MMP-13. In the lowest energy docked

conformation of 5, the hydroxamic acid moiety coordinated

the catalytic zinc atom in a bidentate fashion with the two

oxygen atoms and established two hydrogen bonds with the

carbonyl oxygen of A186 and with the E223 side chain.

The N-O-isopropyl sulfonamide group was in a conforma-

tion in which the N-lone pair bisected the OdSdO angle.

According to search results from the Cambridge Structural

Databases, this conformation was the most common one in

the case of acyclic N-O sulfonamides.

The N-O-isopropyl group of 5 was between L184 and

I243 side chains with which hydrophobic interactions were

formed (the distances between the isopropyl central carbon

and the L184 and L185 Cγ were 6.7 and 6.6 A, respectively).

Furthermore, other lipophilic contacts were detectable be-

tween the isopropyl group bound to the inhibitor’s CR and

the L184 side chain.

Docking of 5 on MMP-1, -14, and TACE. Currently, there

are only two well-defined structures of MMP-1 available in

the PDB. These forms mainly differ in the S1⁰pocket, which

can be in a “closed” (low energy) or in an “open” (inhibitor

induced) conformation. Specifically, upon binding of the N-

hydroxy-2-[4-(4-phenoxy-benzenesulfonyl)-tetrahydro-pyr-

an-4-yl]-acetamide (RS-104966),⁴⁰multiple residue side

chains (e.g., L181, R214, V215, S239, Y240, and F242)

change in their conformation. In particular, the R214 side

chain was demonstrated to be forced to adopt a new position,

which enlarged the S1⁰pocket.²⁰Docking of 5 in the “open”

conformation of MMP-1 revealed a binding mode perfectly

superimposable to that obtained for MMP-13 when a high

inhibitory activity was found. However, two interactions

between MMP-13 and 5 at residues F252 and L184 are lost in

MMP-1 that has V246 and N180, respectively. Nevertheless,

the loss of these two interactions does not fully explain

the experimentally observed poor inhibition of MMP-1 by

5 (IC₅₀= 48.3 μM). It is possible that 5 was not able to

induce the “open” conformational change observed upon

RS-104966 binding. Thus, docking calculations in the

MMP-1 “closed” conformation were also performed. In

the lowest energy binding position, 5 was positioned in a

way that the metal ion was coordinated by its hydroxamate

moiety and the isopropyl group was partially inserted in the

S1⁰pocket. Thus, the hydrophobic contacts were still visible,

but the remainder of 5 was largely solvent exposed. Notably,

the aromatic branch of 5 was too long to insert into the closed

conformation of the S1⁰pocket. In light of the experimental

data, the results obtained using MMP-1 in the “closed”

conformation appear more reasonable.

In regard to the low activity of 5 toward MMP-14, only

two out of 100 docked conformations showed the occurrence

of metal coordination and occupancy of the S1⁰pocket at the

same time. However, a careful inspection of the binding

mode revealed that in order to fit into the narrow S1⁰pocket,

the biphenyl moiety must have adopted a deeply unfavorable

geometry where the torsion between the two rings was 90ꢀ

(Car1-Car1-Car2-Car2). Thus, the low activity of 5 toward

MMP-14 can be mainly attributed to the variations in the

S1⁰pockets. Indeed, although MMP-13 and -14 possess a S1⁰

specificity loop of the same length, the latter has a narrower

shaped S1⁰pocket. The partially closed fold of the S1⁰

specificity loop in MMP-14 seems to be because the sV-hB

connecting loop, which is immediately below the S1⁰specifi-

city loop, is three amino acids longer. Moreover, the

sequence identity between the two S1⁰specificity loops is

rather low (25%) and the substitution of T245 and T247 in

MMP-13 with Q262 and M264 in MMP-14 leads to a further

From these docking results, it is clear that the sulfonamide

moiety was particularly important. This group formed a

bifurcated hydrogen bond with L185 and the A186 backbone

amine, thereby enabling the p-Cl-benzyloxy-diphenyl moiety

to plunge deep into the unusually large S1⁰-specificity pocket

(Figure 5). In the S1⁰, the p-Cl-benzyl ring of 5 established a

chlorine-reinforced T-shaped interaction with the aromatic

ring of F252 and formed hydrophobic interactions with L218

and L239 side chains and with hydrophobic portions of T245

and T247. In 5, the phenyl ring next to the sulfonamide group

was flanked by lipophilic residues such as L185 and V219.

Clearly, all these interactions endowed 5 with a low nano-

molar inhibitory activity toward MMP-13.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4765

restriction of the S1⁰channel. This comparative analysis once

more strengthens the concept that the differential substitu-

tions of the S1⁰pocket can be successfully used to design

selective inhibitors. Another remarkable difference between

MMP-13 and -14, which could have affected our ligand

binding process and could be corroborated by the SARs

herein developed, resides in the sIII-sIV connecting loop. In

this instance, (MMP-13)-L184 is substituted by (MMP-14)-

F198 and this effectively reduces the available room in the S1

pocket. Notably, only the membrane-type MMP-14 and -16

contain a Phe residue at this position, while the following

smaller amino acids are present in other MMPs: N (MMP-1),

L (MMP-2, -9, -13), I (MMP-8, -11, -12), V (MMP-3), T

(MMP-7), and S (MMP-10). Thus, these described struct-

ural variations of MMP-14 can be taken into account in the

fine-tuning of a specific inhibitor’s activity.

The docking of 5 into the three most divergent TACE

X-ray structures furnishes a clear explanation of the struct-

ural features responsible for the observed inactivity against

this enzyme. In fact, despite the substantial structural simi-

larities in the active sites between TACE and MMP-13, the

unique shape of the S1⁰specificity pocket of TACE does not

allow the full occupancy by the long and semirigid P1⁰

substituent of 5 and the simultaneous metal coordination

by the hydroxamate moiety. Indeed, it is well established^45,46

that TACE inhibitors must have a bent shaped P1⁰group in

order to properly adapt to the curved contour of the TACE

S1⁰tunnel.

optimization of its selectivity profile. In this respect, it is not

clear whether the dual specificity of compound 5 for MMP-13

and MMP-2 will be beneficial or not in terms of protecting

cartilage degradation. In fact, the role of MMP-2 activityitself

in the pathogenesis of OA is unclear. Interestingly, mRNA

levels of MMP-2 are increased in OA patients compared to

normal controls, suggesting that MMP-2 may play a negative

role in this disease.⁴⁷On the other hand, MMP-2-null mice

exhibit a more severe arthritic phenotype than wild type mice

in antigen-induced arthritis, suggesting that the total loss of

MMP-2 activity is unfavorable.⁴⁸Further modifying com-

pound 5 to reduce MMP-2-cross reactivity will bypass these

concerns.

6. Experimental Section

Chemistry. Melting points were determined on a Kofler hot-

stage apparatus and are uncorrected. ¹H and ¹³C NMR spectra

were determined with a Varian Gemini 200 MHz spectrometer.

Chemical shifts (δ) are reported in parts per million downfield

from tetramethylsilane and referenced from solvent references.

Coupling constants J are reported in hertz; ¹³C NMR spectra

were fully decoupled. The following abbreviations are used:

singlet (s), doublet (d), triplet (t), double-doublet (dd), broad

(br), and multiplet (m).Where indicated, the elemental composi-

tions of the compounds agreed to within (0.4% of the calcu-

lated value. Chromatographic separations were performed on

silica gel columns by flash column chromatography (Kieselgel

40, 0.040-0.063 mm; Merck) or using ISOLUTE Flash Si II

cartridges (STEPBIO). Reactions were followed by thin-layer

chromatography (TLC) on Merck aluminum silica gel (60 F254)

sheets that were visualized under a UV lamp, and hydroxamic

acids were visualized with FeCl₃aqueous solution. Evaporation

was performed in vacuo (rotating evaporator). Sodium sulfate

was always used as the drying agent. Commercially available

chemicals were purchased from Sigma-Aldrich. The purity of

the final compounds was determined by reverse-phase HPLC on

a Merck Hitachi D-7000 liquid chromatograph. HPLC purity

was determined to be >95% for all final products using a

Discovery C18 column (250 mm ꢀ 4.6 mm, 5 μm, Supelco),

with a gradient of 20% water/80% methanol at a flow rate of

1.4 mL/min, with UV monitoring at the fixed wavelength of

256 nm. See the Supporting Information for compound

purity analysis data for final compounds. Optical rotation were

obtained on a Perkin-Elmer 343 polarimeter with a continuous

Na lamp (589 nm).

tert-Butyl2-(4-Bromo-N-isopropoxyphenylsulfonamido)acetate

(7). A solution of sulfonamide 6 (2.0 g, 6.8 mmol) in anhydrous

DMF (14.4 mL) was treated with tert-butyl bromoacetate

(1.2 mL, 8.16 mmol), cesium carbonate (2.2 g, 6.8 mmol), and

tetrabutylammonium hydrogen sulfate (2.3 g, 6.8 mmol). The

reaction mixture was stirred for 3 days at room temperature,

diluted with H₂O, and extracted with EtOAc. The combined

organic extracts were dried over anhydrous Na₂SO₄, filtered, and

evaporated under reduced pressure to afford 7 as a white solid

(2.68 g, 96.4% yield); mp 89-90 ꢀC. ¹H NMR (CDCl₃) δ: 1.24

(d, J=6.4 Hz, 6H), 1.45 (s, 9H), 3.58 (br s, 2H), 4.53 (septet, J=

6.2 Hz, 1H), 7.67-7.76 (m, 4H).

Although the molecular modeling studies presented herein

surely provide valuable explanations for the ligand activity

and selectivity, X-ray crystallography would be needed to

finally prove these predictions.

5. Conclusions

A new series of N-isopropoxy-arylsulfonamide inhibitors

for MMP-13, selective over MMP-1 and MMP-14, was

designed and synthesized by optimizing the structural char-

acteristics of the previously described derivative 1a. Among

the novel analogues, a very promising compound was dis-

covered (5), which showed nanomolar activity for MMP-13

and high selectivity over MMP-1, -14, -16, and TACE. A

kinetic characterization of 5 showed a slow-binding behavior

of this inhibitor toward MMP-13 and MMP-2. The slow

dissociation of this compound would predictably result in a

long duration of action and, consequently, it would not be

necessary to maintain high systemic levels of drug to con-

tinuously inhibit the target enzyme. Because of its promising

properties, 5 was chosen for further studies examining col-

lagen degradation that confirmed the data obtained with

isolated enzymes by a fluorometric assay. Finally, hydroxa-

mates 5 and 1a were assayed in the IL-1R/OSM stimulated

model of cartilage degradation, and both compounds showed

no significant effect on aggrecan degradation, thereby sug-

gesting to be inactive toward aggrecanases. The inhibitory

activity of 5 on cartilage breakdown was 10-fold better than

1a, which very roughly corresponded to the fact that 5 was a

10-fold better inhibitor for MMP-13 than 1a. Thus, on the

basis of the effectiveness of the MMP-13 selective inhibitor 5,

it can be deduced that the major collagenase involved in the

IL-1R stimulated porcine cartilage degradation model is

probably MMP-13.

General Procedure for the Preparation of esters 9a,c-e. A

solution of aryl bromide 7 (500 mg, 1.22 mmol) in anhydrous

dioxane (12.2 mL)/H₂O (2.7 mL) was sequentially treated under

nitrogen with K₃PO₄(596.5 mg, 2.81 mmol), the appropri-

ate arylboronic acid (2.00 mmol), and Pd(PPh₃)₄(141 mg,

0.12 mmol). The resulting mixture was stirred for 20 min at

70 ꢀC. After being cooled to RT, the mixture was treated with

NaHCO₃and extracted with EtOAc The combined organic

extracts were dried over anhydrous Na₂SO₄, filtered, and eva-

porated under reduced pressure. The crude was purified by flash

chromatography on silica gel column.

The next steps of our research program will be the evalua-

tion of the in vivo efficacy of 5 in OA models and a further

4766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

tert-Butyl 2-(N-isopropoxy-4⁰-(methylthio)biphenyl-4-ylsulfo-

namido)acetate (9a). The title compound was prepared from aryl

bromide 7 and 4-(methylthio)phenylboronic acid following the

general procedure. Purified with n-hexane/EtOAc 7:1 (25%) as

white solid; mp 115-116 ꢀC. ¹H NMR (CDCl₃) δ: 1.26 (d, J=

6.4 Hz, 6H), 1.46 (s, 9H), 2.53 (s, 3H), 3.63 (br s, 2H), 4.58 (septet

J=6.2 Hz, 1H), 7.32-7.36 (m, 2H), 7.53-7.56 (m, 2H), 7.71-

7.75(m, 2H), 7.89-7.93 (m, 2H).

6H), 2.54 (s, 3H), 3.81 (br s, 2H), 4.57 (septet, J=6.2 Hz, 1H),

7.33-7.37 (m, 2H), 7.53-7.58 (m, 2H), 7.73-7.77 (m, 2H),

7.90-7.95 (m, 2H).

2-(N-Isopropoxy-4⁰-methoxybiphenyl-4-ylsulfonamido)acetic Acid

(10b). The title compound was prepared from ester 9b following

the general procedure. White solid (77% yield); mp 110-111 ꢀC.

¹H NMR (CDCl₃) δ: 1.26 (d, J=6.2 Hz, 6H), 3.82 (br s, 2H), 3.87

(s, 3H), 4.57 (septet, J=6.2 Hz, 1H), 6.99-7.04 (m, 2H), 7.55-

7.60 (m, 2H), 7.71-7.75 (m, 2H), 7.88-7.93 (m, 2H).

tert-Butyl 2-(4⁰-Butoxy-N-isopropoxybiphenyl-4-ylsulfonami-

do)acetate (9c). The title compound was prepared from aryl

bromide 7 and 4-butoxyphenylboronic acid following the gen-

eral procedure. Purified with n-hexane/EtOAc 10:1 (25%) as a

colorless oil. ¹H NMR (CDCl₃) δ: 0.99 (t, J=7.3 Hz, 3H), 1.26

(d, J=6.2 Hz, 6H), 1.42-1.53 (m, 11H), 1.73-1.87 (m, 2H), 3.62

(br s, 2H), 4.02 (t, J=6.6 Hz, 2H), 4.58 (septet, J=6.2 Hz, 1H),

6.97-7.02 (m, 2H), 7.53-7.57 (m, 2H), 7.69-7.73 (m, 2H),

7.87-7.91 (m, 2H).

2-(4⁰-Butoxy-N-isopropoxybiphenyl-4-ylsulfonamido)acetic Acid

(10c). The title compound was prepared from ester 9c following

the general procedure. White solid (79% yield); mp 79-80 ꢀC.

¹H NMR (CDCl₃) δ: 0.99 (t, J=7.3 Hz, 3H), 1.25 (d, J=6.2 Hz,

6H), 1.41-1.61 (m, 2H), 1.73-1.85 (m, 2H), 3.81 (br s, 2H), 4.02

(t, J=6.4 Hz, 2H), 4.57 (septet, J=6.4 Hz, 1H), 6.98-7.02 (m,

2H), 7.54-7.58 (m, 2H), 7.71-7.75 (m, 2H), 7.88-7.92 (m, 2H).

2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphenyl-4-ylsulfon-

amido)acetic Acid (10d). The title compound was prepared from

ester 9d following the general procedure. Yellow oil (88% yield).

¹H NMR (DMSO-d₆) δ: 1.17 (d, J=6.2 Hz, 6H), 3.67 (s, 2H),

4.41 (septet, J=6.4 Hz, 1H), 5.19 (s, 2H), 7.13-7.17 (m, 2H),

7.48 (s, 4H), 7.73-7.77 (m, 2H), 7.85-7.91 (m, 4H).

(E)-2-(4-(2-(Biphenyl-4-yl)vinyl)-N-isopropoxyphenylsulfon-

amido)acetic Acid (10e). The title compound was prepared from

ester 9e following the general procedure. Solid (70% yield); mp

185-186 ꢀC. ¹H NMR (DMSO-d₆) δ: 1.17 (d, J=6.2 Hz, 6H),

3.66 (s, 2H), 4.40 (septet, J=6.2 Hz, 1H), 7.34-7.54 (m, 5H),

7.71-7.75 (m, 6H), 7.82-7.94 (m, 4H).

2-(N-Isopropoxy-4-phenoxyphenylsulfonamido)acetic Acid (10f).

The title compound was prepared from ester 9f following the

general procedure. Pale yellow oil (90% yield). ¹H NMR

(CDCl₃) δ: 1.23 (d, J=5.6 Hz, 6H), 3.77 (s, 2H), 4.27 (br s,

1H), 4.54 (septet, J=6.2 Hz, 1H), 7.05-7.12 (m, 4H), 7.21-7.29

(m, 1H), 7.40-7.47 (m, 2H), 7.79-7.83 (m, 2H).

General Procedure for the Preparation of O-TBDMS Acid

Hydroxyamides (11a-f). 1-[3-(Dimethylamino)propyl]-3-ethyl

carbodiimide hydrochloride (EDC) was added portionwise

(34.5 mg, 0.18 mmol) to a stirred and cooled solution (0 ꢀC) of

the appropriate carboxylic acid 10a-f (0.12 mmol), and O-(tert-

butyldimethylsilyl)hydroxylamine (17.7 mg, 0.12 mmol) in

freshly distilled CH₂Cl₂(2.5 mL). After stirring at room tem-

perature overnight, the mixture was washed with water and the

organic phase was dried and evaporated in vacuo to afford the

O-silylate intermediates 11a-f.

tert-Butyl 2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphenyl-

4-ylsulfonamido)acetate (9d). The title compound was prepared

from aryl bromide 7 and 4-(4-chlorobenzyloxy)phenylboronic

acid following the general procedure. Purified with n-hexane/

EtOAc 5:1 (25%) as a solid. ¹H NMR (CDCl₃) δ: 1.26 (d, J=

6.2 Hz, 6H), 1.46 (s, 9H), 3.63 (br s, 2H), 4.58 (septet J=6.0 Hz,

1H), 5.09 (s, 2H), 7.03-7.08 (m, 2H), 7.38 (s, 4H), 7.54-7.59

(m, 2H), 7.69-7.73 (m, 2H), 7.87-7.92 (m, 2H).

(E)-tert-Butyl 2-(4-(2-(Biphenyl-4-yl)vinyl)-N-isopropoxyphe-

nylsulfonamido)acetate (9e). The title compound was prepared

from aryl bromide 7 and (E)-2-(biphenyl-4-yl)vinylboronic acid

following the general procedure. Purified with n-hexane/EtOAc

1

9:1 (25%) as a yellow solid. H NMR (CDCl₃) δ: 1.25 (d, J=

6.2 Hz, 6H), 1.47 (s, 9H), 3.61 (br s, 2H), 4.56 (septet, J=6.2 Hz,

1H), 7.20 (s, 1H), 7.34-7.50 (m, 5H), 7.61-7.70 (m, 7H), 7.83-

7.87 (m, 2H).

General Procedure for the Preparation of Esters 9b,f. A

solution of sulfonamide 8b or 8f (0.62 mmol) in anhydrous

DMF (1.3 mL) was treated with tert-butyl bromoacetate

(0.11 mL, 0.74 mmol), cesium carbonate (202 mg, 0.62 mmol),

and tetrabutylammonium hydrogen sulfate (210.5 mg, 0.62 mmol).

The reaction mixture was stirred for 3 days at room tempera-

ture, diluted with H₂O, and extracted three times with EtOAc.

The combined organic extracts were dried over anhydrous

Na₂SO₄, filtered, and evaporated under reduced pressure to

afford 9b and 9f. The crude products were purified by flash

chromatography.

tert-Butyl 2-(N-Isopropoxy-4⁰-methoxybiphenyl-4-ylsulfona-

mido)acetate (9b). The title compound was prepared from

sulfonamide 8b following the general procedure. Purified with

n-hexane/EtOAc 8:1 using a ISOLUTE FLASH Si II cartridge

N-(tert-Butyldimethylsilyloxy)-2-(N-isopropoxy-4⁰-(methylthio)-

biphenyl-4-ylsulfonamido)acetamide (11a). The title compound

was prepared from carboxylic acid 10a following the general

procedure. The crude product was purified by flash chromato-

graphy with n-hexane/EtOAc 6:1 using a ISOLUTE FLASH

Si II cartridge (STEPBIO). White solid (26% yield). ¹H NMR

(CDCl₃) δ: 0.21 (s, 6H), 0.97 (s, 9H), 1.25 (d, J=6.2 Hz, 6H), 2.53

(s, 3H), 3.71 (br s, 2H), 4.47 (septet, J=6.2 Hz, 1H), 7.32-7.37

(m, 2H), 7.53-7.57 (m, 2H), 7.73-7.77 (m, 2H), 7.89-7.93 (m,

2H), 8.60 (br s, 2H).

N -(tert-Butyldimethylsilyloxy)-2-(N-isopropoxy-4⁰-methoxy-

biphenyl-4-ylsulfonamido)acetamide (11b). The title compound

was prepared from carboxylic acid 10b following the general

procedure. The crude product was purified by flash chromato-

graphy with n-hexane/EtOAc 4:1 using a ISOLUTE FLASH Si

II cartridge (STEPBIO). White solid (64% yield). ¹H NMR

(CDCl₃) δ: 0.21 (s, 6H), 0.97 (s, 9H), 1.26 (d, J=6.2 Hz, 6H), 3.71

(br s, 2H), 3.87 (s, 3H), 4.46 (septet, J=6.2 Hz, 1H), 6.99-7.04

(m, 2H), 7.55-7.59 (m, 2H), 7.71-7.75 (m, 2H), 7.88-7.92 (m,

2H), 8.57 (br s, 1H).

1

(STEPBIO) (66%) as a white solid. H NMR (CDCl₃) δ: 1.26

(d, J=6.2 Hz, 6H), 1.46 (s, 9H), 3.62 (br s, 2H), 3.87 (s, 3H), 4.58

(septet, J=6.2 Hz, 1H), 6.97-7.05 (m, 2H), 7.53-7.61 (m, 2H),

7.69-7.73(m, 2H), 7.87-7.92 (m, 2H).

tert-Butyl 2-(N-Isopropoxy-4-phenoxyphenylsulfonamido)acetate

(9f). The title compound was prepared from sulfonamide 8f

following the general procedure. Purified with n-hexane/EtOAc

1

15:1 (45%) as a yellow oil. H NMR (CDCl₃) δ: 1.23 (d, J=

6.2 Hz, 6H), 1.46 (s, 9H), 3.59 (br s, 2H), 4.55 (septet, J=6.2 Hz,

1H), 7.03-7.12 (m, 4H), 7.24-7.27 (m, 1H), 7.39-7.47 (m, 2H),

7.76-7.82 (m, 2H).

General Procedure for the Preparation of Carboxylic Acids

10a-f. TFA (14.82 mmol, 1.14 mL) was added dropwise to a

stirred, ice-chilled solution of tert-butyl esters 9a-f (0.26 mmol)

in freshly distilled dichloromethane (2.0 mL). The mixture was

stirred under these reaction conditions for 5 h, and the solvent

was removed in vacuo to give the carboxylic acids 10a-f. The

crude products were purified by trituration with n-hexane/Et₂O.

2-(N-Isopropoxy-4⁰-(methylthio)biphenyl-4-ylsulfonamido)acetic

Acid (10a). The title compound was prepared from ester 9a

following the general procedure. White solid (98% yield);

mp 116-117 ꢀC. ¹H NMR (CDCl₃) δ: 1.27 (d, J=6.2 Hz,

2-(4⁰-Butoxy-N-isopropoxybiphenyl-4-ylsulfonamido)-N-(tert-butyl-

dimethylsilyloxy)acetamide (11c). The title compound was prepared

from carboxylic acid 10c following the general procedure. The crude

product was purified by flash chromatography on silica gel column

(n-hexane/EtOAc 4:1). Colorless oil (25% yield). ¹H NMR

(CDCl₃) δ: 0.21 (s, 6H), 0.91-1.02 (m, 12H), 1.25 (d, J=5.6 Hz,

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4767

6H), 1.45-1.57 (m, 2H), 1.73-1.83 (m, 2H), 3.71 (s, 2H), 4.02 (t, J=

6.6 Hz, 2H), 4.45 (septet, J=5.6 Hz, 1H), 6.98-7.02 (m, 2H), 7.53-

7.57 (m, 2H), 7.71-7.75 (m, 2H), 7.87-7.91 (m, 2H), 8.57 (br s, 1H).

N-(tert-Butyldimethylsilyloxy)-2-(4⁰-(4-chlorobenzyloxy)-N-

isopropoxybiphenyl-4-ylsulfonamido)acetamide (11d). The title

compound was prepared from carboxylic acid 10d following

the general procedure. The crude product was purified by flash

chromatography on silica gel column (n-hexane/EtOAc 3:1).

Yellow oil (25% yield). ¹H NMR (CDCl₃) δ: 0,21 (s, 6H), 0.97

(s, 9H), 1.26 (d, J=6.2 Hz, 6H), 3.71 (s, 2H), 4.46 (septet, J=

6.2Hz, 1H), 5.09(s, 2H), 7.04-7.08 (m, 2H), 7.38 (s, 4H), 7.54-7.59

(m, 2H), 7.71-7.75 (m, 2H), 7.88-7.92 (m, 2H), 8.56 (br s, 1H).

(E)-2-(4-(2-(Biphenyl-4-yl)vinyl)-N-isopropoxyphenylsulfonamido)-

N-(tert-butyldimethylsilyloxy)acetamide (11e). The title compound

was prepared from carboxylic acid 10e following the general proce-

dure. The crude product was purified by flash chromatography on

silica gel column (n-hexane/EtOAc 3.5:1). Yellow solid (35% yield).

¹H NMR (CDCl₃) δ: 0.22 (s, 6H), 0.98 (s, 9H), 1.25 (d, J=6.0 Hz,

6H), 3.72 (s, 2H), 4.45 (septet, J=6.2 Hz, 1H), 7.20 (s, 1H), 7.28 (s,

1H), 7.36-7.50 (m, 4H), 7.61-7.72 (m, 7H), 7.83-7.88 (m, 2H), 8.56

(br s, 1H).

(septet, J=5.8 Hz, 1H), 5.10 (s, 2H), 7.04-7.09 (m, 2H), 7.39 (s,

4H), 7.55-7.59 (m, 2H), 7.72-7.76 (m, 2H), 7.88-7.92 (m,

2H). ¹³C NMR (CDCl₃) δ: 21.27, 69.51, 80.29, 115.61, 127.28,

128.78, 128.89, 128.98, 129.76, 130.36, 131.69, 134.07, 135.15,

147.17, 159.38. Anal. (C₂₄H₂₅ClN₂O₆S) C, H, N.

(E)-2-(4-(2-(Biphenyl-4-yl)vinyl)-N-isopropoxyphenylsulfona-

mido)-N-hydroxyacetamide (4e). The title compound was pre-

pared from O-silylate intermediate 11e following the general

procedure. White solid (73% yield). ¹H NMR (CDCl₃) δ: 1.25

(d, J=5.8 Hz, 6H), 3.80 (s, 2H), 4.46 (septet, J=6.2 Hz, 1H), 7.20

(s, 1H), 7.28 (s, 1H), 7.36-7.49 (m, 4H), 7.63-7.72 (m, 7H),

7.84-7.88 (m, 2H). ¹³C NMR [(CD₃)₂CO] δ: 21.39, 56.26,

79.67, 127.50, 128.03, 128.41, 129.71, 131.13, 131.97, 133.04,

135.84, 136.66, 141.03, 141.78, 144.15. Anal. (C₂₅H₂₆N₂O₅S)

C, H, N.

N-Hydroxy-2-(N-isopropoxy-4-phenoxyphenylsulfonamido)-

acetamide (4f). The title compound was prepared from O-silylate

intermediate 11f following the general procedure. Pale-yellow solid

(75% yield); mp 112-113 ꢀC. ¹H NMR (CDCl₃) δ: 1.22 (d, J=

5.8 Hz, 6H), 3.74 (s, 2H), 4.44 (m, 1H), 7.05-7.11 (m, 4H), 7.21-

7.28 (m, 1H), 7.39-7.47 (m, 2H), 7.78-7.82 (m, 2H). ¹³C NMR

[(CD₃)₂CO] δ: 21.66. 79.96. 118.20. 121.62. 126.38. 131.49. 133.37.

156.02. 163.90. Anal. (C₁₇H₂₀N₂O₆S) C, H, N.

5-Bromo-N-isopropoxythiophene-2-sulfonamide (13). A solu-

tion of 5-bromothiophene-2-sulfonyl chloride (2.0 g, 8.9 mmol)

in anhydrous THF (20 mL) was added dropwise to a stirred

and cooled (0 ꢀC) solution of O-isopropylhydroxylamine

hydrochloride 12 (1.0 g, 8.9 mmol) and N-methylmorpholine

(1.9 mL, 17.9 mmol) in anhydrous THF (20 mL). After stirring

at rt for 4 days, the reaction mixture was diluted with EtOAc and

washed with H₂O. The organic phase was dried over anhydrous

Na₂SO₄, filtered, and evaporated under reduced pressure to

afford 13 as a yellow solid (2.17 g, 80.7% yield); mp 118-119 ꢀC.

¹H NMR (CDCl₃) δ: 1.24 (d, J=6.2 Hz, 6H), 4.31 (septet,

J=6.0, 1H), 6.80 (s, 1H), 7.12 (d, J=3.8 Hz, 1H), 7.46 (d, J=

4.0 Hz, 1H).

N-(tert-Butyldimethylsilyloxy)-2-(N-isopropoxy-4-phenoxy-

phenylsulfonamido)acetamide (11f). The title compound was

prepared from carboxylic acid 10f following the general pro-

cedure. The crude product was purified by flash chromatog-

raphy on silica gel column (n-hexane/EtOAc 4:1). White solid

1

(25% yield); mp 131-132 ꢀC. H NMR (CDCl₃) δ: 0.21 (s,

6H), 0.98 (s, 9H), 1.24 (d, J=5.6 Hz, 6H), 3.67 (s, 2H), 4.43

(septet, J=6.0 Hz, 1H), 7.05-7.11 (m, 4H), 7.21-7.28 (m, 1H),

7.40-7.50 (m, 2H), 7.78-7.82 (m, 2H), 8.56 (br s, 1H).

General Procedure to Synthesize Hydroxamic Acids 4a-f.

TFA (0.31 mL, 3.99 mmol) was added dropwise to a stirred

and ice-chilled solution of the appropriate O-silylate 11a-f

(0.07 mmol) in CH₂Cl₂(0.5 mL). The solution was stirred under

these reaction conditions for 5 h, and the solvent was removed

in vacuo. The crude products were purified by trituration with

n-hexane/ Et₂O.

tert-Butyl 2-(5-Bromo-N-isopropoxythiophene-2-sulfonamido)-

acetate (14). A solution of sulfonamide 13 (1.0 g, 3.4 mmol) in

anhydrous DMF (7.2 mL) was treated with tert-butyl bromoa-

cetate (0.6 mL, 4.08 mmol), cesium carbonate (1.1 g, 3.4 mmol),

and tetrabutylammonium hydrogen sulfate (1.15 g, 3.4 mmol).

The reaction mixture was stirred for 3 days at room temperature,

diluted with H₂O, and extracted with EtOAc. The combined

organic extracts were dried over anhydrous Na₂SO₄, filtered, and

evaporated under reduced pressure. The crude product was

purified by flash chromatography (n-hexane/ EtOAc 30:1) using

a ISOLUTE FLASH Si II cartridge (STEPBIO) to afford 14 as a

yellow solid (1.13 g, 79.4% yield). ¹H NMR (CDCl₃) δ: 1.27 (d,

J=6.4 Hz, 6H), 1.47 (s, 9H), 3.63 (br s, 2H), 4.56 (septet, J=

6.2 Hz, 1H), 7.14 (d, J=3.4 Hz, 1H), 7.41 (d, J=3.4 Hz, 1H).

tert-Butyl 2-(5-((4-Butylphenyl)ethynyl)-N-isopropoxythiophene-

2-sulfonamido)acetate (15g). To a solution of 14 (500 mg,

1.21 mmol) in 4.3 mL of DMF were added 1-butyl-4-ethynyl-

benzene (0.25 mL, 1.45 mmol), copper(I) iodide (11.5 mg,

0.06 mmol), dichlorobis(triphenylphosphine)palladium(II) (21 mg,

0.03 mmol), and triethylamine (0.34 mL, 2.42 mmol) at room

temperature. The mixture was degassed and stirred under an

argon atmosphere at rt for 30 min. The mixture was poured into

water and extracted with EtOAc. The organic solution was

washed with 1 N HCl, 5% NaHCO₃solution, and brine, dried,

and evaporated. The residue was purified by flash chromatog-

raphy (n-hexane/EtOAc 30:1) using a ISOLUTE FLASH Si II

cartridge (STEPBIO) to give 15g as a yellow oil (26% yield). ¹H

NMR (CDCl₃) δ: 0.93 (t, J=6.9 Hz, 3H), 1.28 (d, J=6.2 Hz,

6H), 1.33-1.41 (m, 2H), 1.47 (s, 9H), 1.53-1.68 (m, 2H), 2.63

(t, J=7.8 Hz, 2H), 3.66 (br s, 2H), 4.59 (septet, J=6.2 Hz, 1H),

7.17-7.23 (m, 3H), 7.42-7.46 (m, 2H), 7.55 (d, J=4.0 Hz, 1H).

2-(5-((4-Butylphenyl)ethynyl)-N-isopropoxythiophene-2-sulfon-

amido)acetic Acid (16g). TFA (0.9 mL, 11.74 mmol) was added

dropwise to a stirred, ice-chilled solution of tert-butyl ester 15g

N-hydroxy-2-(N-isopropoxy-4⁰-(methylthio)biphenyl-4-ylsulfon-

amido)acetamide (4a). The title compound was prepared from

O-silylate intermediate 11a following the general procedure. White

solid (95% yield); mp 134-135 ꢀC. ¹H NMR (CDCl₃) δ: 1.25 (d,

J=6.2 Hz, 6H), 2.53 (s, 3H), 3.79 (s, 2H), 4.48 (septet, J=6.0 Hz,

1H), 7.33-7.37 (m, 2H), 7.53-7.57 (m, 2H), 7.73-7.78 (m, 2H),

7.89-7.94 (m, 2H). ¹³C NMR (CDCl₃) δ: 15.63, 21.33, 80.23,

126.74, 127.37, 127.76, 130.40, 135.33, 140.27, 146.87. Anal.

(C₁₈H₂₂N₂O₅S₂) C, H, N.

N-Hydroxy-2-(N-isopropoxy-4⁰-methoxybiphenyl-4-ylsulfon-

amido)acetamide (4b). The title compound was prepared from

O-silylate intermediate 11b following the general procedure.

1

Solid (97% yield); mp 141-142 ꢀC. H NMR (DMSO-d₆) δ:

1.15 (d, J=6.2 Hz, 6H), 3.82 (s, 3H), 4.34 (septet, J=6.2 Hz,

1H), 7.06-7.10 (m, 2H), 7.73-7.77 (m, 2H), 7.86-7.97 (m, 4H),

10.71 (s, 1H). ¹³C NMR (DMSO-d₆) δ: 20.91, 55.28, 78.39,

114.59, 126.57, 128.39, 129.99, 130.12, 130.21, 145.30, 159.89.

Anal. (C₁₈H₂₂N₂O₆S) C, H, N.

2-(4⁰-Butoxy-N-isopropoxybiphenyl-4-ylsulfonamido)-N-hydro-

xyacetamide (4c). The title compound was prepared from

O-silylate intermediate 11c following the general procedure.

1

Yellow solid (78% yield); mp 73-74 ꢀC. H NMR (CDCl₃) δ:

0.99 (t, J=7.5 Hz, 3H), 1.25 (br s, 6H), 1.41-1.60 (m, 2H), 1.73-

1.87 (m, 2H), 3.80 (br s, 2H), 4.01 (t, J=6.4 Hz, 2H), 4.46 (br s,

1H), 6.97-7.02 (m, 2H), 7.53-7.57 (m, 2H), 7.68-7.75 (m, 2H),

7.87-7.93 (m, 2H). ¹³C NMR (CDCl₃) δ: 14.05, 19.44, 21.31,

31.43, 68.01, 80.20, 115.23, 127.14, 128.63, 129.69, 130.32, 130.98,

147.26. Anal. (C₂₁H₂₈N₂O₆S) C, H, N.

2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphenyl-4-ylsulfon-

amido)-N-hydroxyacetamide (4d). The title compound was

prepared from O-silylate intermediate 11d following the gen-

1

eral procedure. Yellow solid (78% yield); mp 88-89 ꢀC. H

NMR (CDCl₃) δ: 1.26 (d, J=6.0 Hz, 6H), 3.83 (s, 2H), 4.47

4768 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

(101.1 mg, 0.21 mmol) in freshly distilled dichloromethane

(1.6 mL). The mixture was stirred under these reaction condi-

tions for 5 h, and the solvent was removed in vacuo. The crude

product was purified by trituration with n-hexane/Et₂O to give

16g as a yellow solid (90 mg, 95% yield). ¹H NMR (CDCl₃) δ:

0.92 (t, J=7.1 Hz, 3H), 1.24-1.40 (m, 8H), 1.54-1.66 (m, 2H),

2.62 (t, J=7.3 Hz, 2H), 3.94 (s, 2H), 4.50 (septet, J=6.4 Hz, 1H),

7.16-7.20 (m, 3H), 7.40-7.44 (m, 2H), 7.49 (d, J=3.4 Hz, 1H).

2-(5-((4-Butylphenyl)ethynyl)-N-isopropoxythiophene-2-sulfon-

amido)-N-hydroxyacetamide (4g). 1-[3-(Dimethylamino)propyl]-

3-ethyl carbodiimide hydrochloride (EDC) was added portion-

wise (60.4 mg, 0.31 mmol) to a stirred and cooled solution

(0 ꢀC) of the carboxylic acid 16g (92.4 mg, 0.21 mmol) and

O-(tert-butyldimethylsilyl)hydroxylamine (30.9 mg, 0.21 mmol)

in freshly distilled CH₂Cl₂(4.5 mL). After stirring at room

temperature overnight, the mixture was washed with water and

the organic phase was dried and evaporated in vacuo. The crude

was purified by flash chromatography on silica gel (n-hexane/

EtOAc 4:1) to afford the O-silylate intermediate. ¹H NMR

(CDCl₃) δ: 0.21 (s, 6H), 0.89- 0.92 (m, 3H), 0.97 (s, 9H), 1.27

(d, J=6.2 Hz, 6H), 1.33-1.40 (m, 2H), 1.53-1.67 (m, 2H), 2.63

(t, J=7.8 Hz, 2H), 3.74 (s, 2H), 4.48 (septet, J=6.4 Hz, 1H),

7.17-7.24 (m, 3H), 7.42-7.46 (m, 2H), 7.57 (d, J=4.0 Hz, 1H),

8.45 (br s, 1H).

6.2 Hz, 6H), 1.47 (s, 9H), 3.85 (s, 3H), 4.60 (septet, J=6.2 Hz,

1H), 6.91-6.98 (m, 2H), 7.22 (d, J=4.0 Hz, 1H), 7.51-7.57 (m,

2H), 7.60 (d, J=4.0 Hz, 1H).

General Procedure for the Preparation of Carboxylic Acids

16h-l. TFA (0.8 mL, 10.26 mmol) was added dropwise

to a stirred, ice-chilled solution of tert-butyl esters 15h-l

(0.18 mmol) in freshly distilled dichloromethane (1.4 mL). The

mixture was stirred under these reaction conditions for 5 h, and

the solvent was removed in vacuo to give the carboxylic acids

16h-l. The crude products were purified by trituration with

n-hexane/ Et₂O.

2-(5-(4-Fluorophenyl)-N-isopropoxythiophene-2-sulfonamido)-

acetic Acid (16h). The title compound was prepared from tert-

butyl ester 15h following the general procedure. Pale-yellow oil

(90% yield). ¹H NMR (CDCl₃) δ: 1.28 (d, J=6.4 Hz, 6H), 3.86

(br s, 2H), 4.60 (septet, J=6.2 Hz, 1H), 5.87(br s, 1H), 7.11(d, J=

8.4 Hz, 1H), 7.16 (d, J=8.6 Hz, 1H), 7.27 (d, J=4.0 Hz, 1H),

7.56-7.60 (m, 2H), 7.64 (d, J=4.0 Hz, 1H).

2-(5-(4-Ethylphenyl)-N-isopropoxythiophene-2-sulfonamido)-

acetic Acid (16i). The title compound was prepared from tert-

butyl ester 15i following the general procedure. Pale-yellow

1

solid (88% yield); mp 90-91 ꢀC. H NMR (CDCl₃) δ: 1.22-

1.30 (m, 8H), 2.69 (q, J=7.5 Hz, 2H), 3.87 (s, 2H), 4.60 (septet,

J=6.2 Hz, 1H), 7.25-7.31 (m, 3H), 7.52-7.56 (m, 2H), 7.64 (d,

J=3.8 Hz, 1H).

TFA (0.2 mL, 2.85 mmol) was added dropwise to a stirred and

ice-chilled solution of O-silylate (26 mg, 0.05 mmol) in CH₂Cl₂

(0.5 mL). The solution was stirred under these reaction condi-

tions for 5 h, and the solvent was removed in vacuo. The crude

product was purified by trituration with n-hexane/Et₂O to give

the hydroxamate 4g as a solid (16 mg, 71% yield); mp 66-67 ꢀC.

¹H NMR [(CD₃)₂CO] δ: 0.92 (t, J=7.1 Hz, 3H), 1.24 (d, J=

6.0 Hz, 6H), 1.33-1.44 (m, 2H), 1.54-1.69 (m, 2H), 2.67 (t, J=

7.8 Hz, 2H), 3.58 (s, 2H), 4.40-4.60 (m, 1H), 7.29-7.33 (m, 2H),

7.47-7.53 (m, 3H), 7.71 (d, J=3.6 Hz, 1H), 8.27 (br s, 1H), 10.30

(br s, 1H). ¹³C NMR [(CD₃)₂CO] δ: 14.16, 21.31, 22.93, 34.15,

36.14, 56. 36, 80.22, 80.65, 98.04, 119.51, 129.67, 132.37, 132.68,

133.02, 136.41, 145.84. Anal. (C₂₁H₂₆N₂O₅S₂) C, H, N.

General Procedure for the Preparation of Esters 15h-l. A

solution of aryl bromide 14 (800 mg, 1.93 mmol) in anhydrous

dioxane (19.3 mL)/H₂O (4.2 mL) was sequentially treated under

nitrogen with K₃PO₄(942.5 mg, 4.44 mmol), the appropriate

arylboronic acid (3.28 mmol), and Pd(PPh₃)₄(223 mg, 0.19

mmol). The resulting mixture was stirred for 20 min at 70 ꢀC.

After being cooled to RT, the mixture was treated with NaHCO₃

and extracted with EtOAc The combined organic extracts were

dried over anhydrous Na₂SO₄, filtered, and evaporated under

reduced pressure.

2-(N-Isopropoxy-5-(4-methoxyphenyl)thiophene-2-sulfonami-

do)acetic Aacid (16l). The title compound was prepared from

tert-butyl ester 15l following the general procedure. Pale-yellow

solid (90% yield); mp 135-137 ꢀC. H NMR (CDCl₃) δ: 1.29

(d, J=6.2 Hz, 6H), 3.85 (s, 5H), 4.59 (septet, J=6.2 Hz, 1H),

6.93-6.97 (m, 2H), 7.23 (d, J=3.8 Hz, 1H), 7.52-7.57 (m, 2H),

7.62 (d, J=3.8 Hz, 1H).

General Procedure to Synthesize Hydroxamic Acids 4h-l.

1-[3-(Dimethylamino)propyl]-3-ethyl carbodiimide hydrochlor-

ide (EDC) was added portionwise (72 mg, 0.37 mmol) to a stirred

and cooled solution (0 ꢀC) of the appropriate carboxylic acid

16h-l (0.25 mmol) and O-(tert-butyldimethylsilyl)hydroxylamine

(36.8 mg, 0.25 mmol) in freshly distilled CH₂Cl₂(5.3 mL). After

stirring at room temperature overnight, the mixture was washed

with water and the organic phase was dried and evaporated

in vacuo to afford the O-silylate intermediate. TFA (6.5 mL,

0.5 mmol) was added dropwise to a stirred and ice-chilled solution

of the appropriate O-silylate (0.11 mmol) inCH₂Cl₂(0.9mL). The

solution was stirred under these reaction conditions for 5 h, and

the solvent was removed in vacuo. The crude products were

purified by trituration with n-hexane/Et₂O to give the desired

hydroxamates 4h-l.

1

2-(5-(4-Fluorophenyl)-N-isopropoxythiophene-2-sulfonamido)-

N-hydroxyacetamide (4h). The title compound was prepared

from the carboxylic acid 16h following the general procedure.

White solid (89% yield); mp 75-76 ꢀC. ¹H NMR (CDCl₃) δ: 1.27

(d, J=6.0 Hz, 6H), 3.83 (s, 2H), 4.50 (septet, J=6.4 Hz, 1H), 7.11

(d, J=8.2 Hz, 1H), 7.16 (d, J=8.2 Hz, 1H), 7.25-7.29 (m, 1H),

7.55-7.62 (m, 2H), 7.65 (d, J=3.8 Hz, 1H). ¹³C NMR [(CD₃)₂-

CO] δ: 21.35, 56.36, 80.04, 117.12 (d, J_C-F=21.9 Hz), 124.75,

129.30 (d, J_C-F=8.2 Hz), 129.83, 137.48, 153.07, 161.68, 163.36,

166.62. Anal. (C₁₅H₁₇FN₂O₅S₂) C, H, N.

tert-Butyl 2-(5-(4-Fluorophenyl)-N-isopropoxythiophene-2-

sulfonamido)acetate (15h). The title compound was prepared

from aryl bromide 14 and 4-fluorophenylboronic acid follow-

ing the general procedure. The crude was purified by flash

chromatography on silica gel (n-hexane/EtOAc 6:1) to give a

pale-yellow oil (25% yield). H NMR (CDCl₃) δ: 1.29 (d, J=

6.4 Hz, 6H), 1.47 (s, 9H), 3.68 (br s, 2H), 4.60 (septet, J=6.2 Hz,

1H), 7.09-7.17 (m, 2H), 7.56-7.63 (m, 2H).

1

tert-Butyl 2-(5-(4-Eethylphenyl)-N-isopropoxythiophene-2-sulfon-

amido)acetate (15i). The title compound was prepared from aryl

bromide 14 and 4-ethylphenylboronic acid following the general

procedure. The crude was purified by flash chromatography

(n-hexane/EtOAc 30:1) using a ISOLUTE FLASH Si II cartridge

2-(5-(4-Ethylphenyl)-N-isopropoxythiophene-2-sulfonamido)-

N-hydroxyacetamide (4i). The title compound was prepared

from the carboxylic acid 16i following the general procedure.

White solid (89.6% yield); mp 113-114 ꢀC. ¹H NMR (CDCl₃)

δ: 1.22-1.29 (m, 8H), 2.69 (q, J=7.5 Hz, 2H), 3.83 (s, 2H), 4.50

(septet, J=5.6 Hz, 1H), 7.25-7.31 (m, 3H), 7.51-7.55 (m, 2H),

7.65 (d, J=3.3 Hz, 1H). ¹³C NMR [(CD₃)₂CO] δ: 15.82, 21.35,

56.36, 79.96, 124.06, 127.07, 129.65, 130.38, 130.75, 137.46,

146.88, 154.60, 163.34. Anal. (C₁₇H₂₂N₂O₅S₂) C, H, N.

N-Hydroxy-2-(N-isopropoxy-5-(4-methoxyphenyl)thiophene-

2-sulfonamido)acetamide (4l). The title compound was prepared

from the carboxylic acid 16l following the general procedure.

1

(STEPBIO) to give 15i as a yellow oil (26% yield). H NMR

(CDCl₃) δ: 1.22-1.30 (m, 8H), 1.47 (s, 9H), 2.68 (q, J=7.6 Hz,

2H),3.68(brs,2H),4.59(septet,J=6.2 Hz, 1H), 7.23-7.29(m,3H),

7.51-7.55 (m, 2H), 7.61 (d, J=3.8 Hz, 1H).

tert-Butyl 2-(N-Isopropoxy-5-(4-methoxyphenyl)thiophene-2-

sulfonamido)acetate (15l). The title compound was prepared

from aryl bromide 14 and 4-methoxyphenylboronic acid follow-

ing the general procedure. The crude was purified by flash

chromatography on silica gel (n-hexane/EtOAc 6:1) to give 15l

as a pale-yellow oil (54% yield). ¹H NMR (CDCl₃) δ: 1.29 (d, J=

1

White solid (71% yield); mp 138-140 ꢀC. H NMR (CDCl₃)

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4769

δ: 1.27 (d, J=6.2 Hz, 6H), 3.82 (s, 2H), 3.86 (s, 3H), 4.49 (septet,

J=6.2 Hz, 1H), 6.93-6.97 (m, 2H), 7.23 (d, J=4.0 Hz, 1H),

7.52-7.56 (m, 2H), 7.63 (d, J=4.0 Hz, 1H). ¹³C NMR (CDCl₃)

δ: 21.31, 55.62, 56.67, 80.32, 114.79, 122.37, 125.06, 127.54,

127.96, 137.08, 155.20, 160.88, 164.70. Anal. (C₁₆H₂₀N₂O₆S₂)

C, H, N.

(R)-tert-Butyl 2-(4-Bromo-N-isopropoxyphenylsulfonamido)-

3-methylbutanoate (18). Diisopropyl azodicarboxylate (DIAD)

(2.5 mL, 12.5 mmol) was added dropwise to a solution contain-

ing (S)-tert-butyl 2-hydroxy-3-methylbutanoate 17 (0.87 g,

5.0 mmol), the sulfonamide 6 (2.2 g, 7.5 mmol) ,and triphenyl-

phosphine (3.9 g, 15 mmol) in anhydrous THF (60 mL) under

nitrogen atmosphere at 0 ꢀC. The resulting solution was stirred

for 5 h at RT and evaporated under reduced pressure to afford a

crude product, which was purified by flash chromatography

on silica gel (n-hexane/EtOAc=40:1) to yield 18 (1.9 g, 84%) as

pure-yellow oil. ¹H NMR (CDCl₃) δ: 0.88 (d, J=6.4 Hz, 3H),

1.15 (br s, 9H), 1.25 (t, J=6.7 Hz, 9H), 2.10-2.36 (m, 1H), 3.67

(d, J=10.6 Hz, 1H), 4.42 (septet, J=6.2 Hz, 1H), 7.63-7.68 (m,

2H), 7.75-7.80 (m, 2H).

give the hydroxamate 5 as a white solid (78 mg, 80% yield); mp

94-95 ꢀC; [R]²⁰_Dþ65ꢀ (c 0.1, CHCl₃). ¹H NMR (CDCl₃) δ: 0.92

(d, J=6.4 Hz, 3H), 0.96-1.22 (m, 3H), 1.30 (d, J=6.4 Hz, 3H),

1.36 (d, J=6.2 Hz, 3H), 1.92-2.12 (m, 1H), 3.92 (br s, 1H), 4.53

(septet, J=6.4 Hz, 1H), 5.09 (s, 2H), 7.02-7.06 (m, 2H), 7.37 (s,

4H), 7.53-7.57 (m, 2H), 7.67-7.71 (m, 2H), 7.87-7.91 (m, 2H).

¹³C NMR (CDCl₃) δ: 19.47, 20.76, 21.66, 69.47, 70.25, 81.58,

115.54, 126.97, 128.72, 128.85, 128.94, 130.07, 131.89, 132.05,

134.02, 135.22, 146.67, 159.24. Anal. (C₂₇H₃₁ClN₂O₆S) C, H, N.

MMP Inhibition Assays.⁴⁹Recombinant human MMP-14

and MMP-16 catalytic domains were a kind gift of Prof. Gillian

Murphy (Department of Oncology, University of Cambridge,

UK). pro-MMP-1, pro-MMP-2, pro-MMP-3, pro-MMP-8,

pro-MMP-9, pro-MMP-13, and TACE (ADAM-17) were pur-

chased from Calbiochem.

Proenzymes were activated immediately prior to use with

p-aminophenylmercuric acetate (APMA 2 mM for 1 h at 37 ꢀC

for MMP-2, MMP-1, and MMP-8, 1 mM for 1 h at 37 ꢀC for

MMP-9 and MMP-13). Pro-MMP-3 was activated with trypsin

5 μg/mL for 30 min at 37 ꢀC, followed by soybean trypsin

inhibitor 62 μg/mL. For assay measurements, the inhibitor

stock solutions (100 mM in DMSO) were further diluted, at

seven different concentrations (0.01 nM-300 μM) for each

MMP in the fluorimetric assay buffer (FAB: Tris 50 mM,

pH=7.5, NaCl 150 mM, CaCl₂10 mM, Brij 35 0.05%, and

DMSO 1%). Activated enzyme (final concentration 2.9 nM for

MMP-2, 2.7 nM for MMP-9, 1.5 nM for MMP-8, 0.3 nM for

MMP-13, 5 nM for MMP-3, 1 nM for MMP-14 cd, 15 nM

for MMP-16 cd, 2.0 nM for MMP-1, and 7.5 nM for TACE)

and inhibitor solutions were incubated in the assay buffer

for 4 h at 25 ꢀC. TACE was incubated for 20 min at 25 ꢀC.

After the addition of 200 μM solution of the fluorogenic

substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys-

(Dnp)-NH₂(Sigma) for MMP-3 and Mca-Lys-Pro-Leu-Gly-

Leu-Dap(Dnp)-Ala-Arg-NH₂(Bachem) for all the other en-

zymes in DMSO (final concentration 2 μM), the hydrolysis was

monitored every 15 s for 15 min recording the increase in

fluorescence (λ_ex=325 nm, λ_em=395 nm) using a Molecular

Devices SpectraMax Gemini XS plate reader. The assays were

performed in triplicate in a total volume of 200 μL per well in

96-well microtiter plates (Corning, black, NBS). The MMP

inhibition activity was expressed in relative fluorescent units

(RFU). Percent of inhibition was calculated from control reac-

tions without the inhibitor. IC₅₀was determined using the

formula: V_i/V_o=1/(1 þ [I]/IC₅₀), where V_iis the initial velocity

of substrate cleavage in the presence of the inhibitor at concen-

tration [I] and V_ois the initial velocity in the absence of the

inhibitor. Results were analyzed using SoftMax Pro software⁵⁰

and GraFit software.⁵¹

(R)-tert-Butyl 2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphe-

nyl-4-ylsulfonamido)-3-methylbutanoate (19). A mixture of aryl

bromide 18 (300 mg, 0.66 mmol), 4-(4⁰-chlorobenzyloxy)-

phenylboronic acid (295 mg, 1.12 mmol), Pd(PPh₃)₄(38 mg,

0.03 mmol), and K₃PO₄(322 mg, 1.5 mmol) in dioxane (6.6 mL)/

H₂O (1.5 mL) was heated at 70 ꢀC under nitrogen. After 1 h, the

reaction mixture was diluted with sat. solution of NaHCO₃,

extracted with EtOAc, and dried over Na₂SO₄. The organic

layer was concentrated in vacuo, and the crude product was

purified by flash chromatography (n-hexane/EtOAc =20:1)

using a ISOLUTE FLASH Si II cartridge (STEPBIO) to give

1

19 as a white solid (230 mg, 67% yield). H NMR (CDCl₃) δ:

0.89 (d, J=6.6 Hz, 3H), 1.10 (br s, 9H), 1.27 (t, J=6.4 Hz, 9H),

2.12-2.40 (m, 1H), 3.75 (d, J=10.6 Hz, 1H), 4.45 (septet, J=

6.4 Hz, 1H), 5.09 (s, 2H), 7.03-7.07 (m, 2H), 7.38 (s, 4H), 7.51-

7.56 (m, 2H), 7.64-7.69 (m, 2H), 7.93-7.97 (m, 2H).

(R)-2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphenyl-4-ylsulfon-

amido)-3-methylbutanoic Acid (20). TFA (1.75 mL, 22.8 mmol)

was added dropwise to a stirred, ice-chilled solution of tert-butyl

ester 19 (230 mg, 0.4 mmol) in freshly distilled dichloromethane

(4 mL). The mixture was stirred under these reaction conditions

for 5 h, and the solvent was removed in vacuo. The crude

product was purified by trituration with n-hexane/Et₂O to give

the carboxylic acid 20 (167 mg, 78% yield) as a white solid;

mp 92-93 ꢀC; [R]²⁰_Dþ83ꢀ (c 0.07, CHCl₃). ¹H NMR (CDCl₃) δ:

0.91 (d, J=6.6 Hz, 3H), 1.02-1.22 (m, 3H), 1.24 (d, J=6.2 Hz,

3H), 1.30 (d, J=6.2 Hz, 3H), 2.08-2.22 (m, 1H), 3.90 (d, J=

10.2 Hz, 1H), 4.48 (septet, J=6.2 Hz, 1H), 5.09 (s, 2H), 7.03-

7.07 (m, 2H), 7.38 (s, 4H), 7.54-7.58 (m, 2H), 7.63-7.67 (m,

2H), 7.88-7.92 (m, 2H).

Slow-Binding Inhibition Assay. For slow-binding inhibition

measurements, the inhibitor stock solutions (10 mM in DMSO)

were further diluted at four different concentrations (50-

480 nM for 5, 10-80 nM for 4d, 2-40 nM for 4b, 5-150 nM

for 1a, and 2-20 nM for 1b). After the addition of the enzyme

(the final concentration was 1.3 nM for MMP-13 and 1.4 nM for

MMP-2) to the solution containing substrate and inhibitor, the

hydrolysis of the fluorogenic substrate was monitored every 3 s

for 15 min at 25 ꢀC as described above. The control reactions do

not contain inhibitor. Data were fitted to the following equa-

tion³²by SigmaPlot 9.0 Software: ΔF=ν_ft þ [(ν₀- ν_f)1 -

e^-kobst]/k_obswhere ΔF is the variation of fluorescence, ν_fis the

steady state velocity, ν₀is the initial velocity, and k_obsis the

apparent first-order rate constant for slow-binding. Association

and dissociation rate constants (k_onand k_off, respectively) were

obtained from the slope and intercept, respectively, of plots of

(R)-2-(4⁰-(4-Chlorobenzyloxy)-N-isopropoxybiphenyl-4-ylsulfon-

amido)-N-hydroxy-3-methylbutanamide (5). 1-[3-(Dimethylamino)-

propyl]-3-ethyl carbodiimide hydrochloride (EDC) was added

portionwise (86.2 mg, 0.4 mmol) to a stirred and cooled solution

(0 ꢀC) of the carboxylic acid 20 (160 mg, 0.3 mmol) and O-(tert-

butyldimethylsilyl)hydroxylamine (44.2 mg, 0.3 mmol) in freshly

distilled CH₂Cl₂(7 mL). After stirring at room temperature

overnight, the mixture was washed with water and the organic

phase was dried and evaporated in vacuo. The crude was purified by

flash chromatography (n-hexane/EtOAc 6:1) using a ISOLUTE

FLASH Si II cartridge (STEPBIO) to afford the O-silylate inter-

mediate (121 mg, 61% yield) as a yellow oil. ¹H NMR (CDCl₃) δ:

0.06 (s, 6H), 0.84-0.93 (m, 12H), 1.22-1.26 (m, 9H), 2.05-2.27

(m, 1H), 4.33-4.40 (m, 1H), 5.09 (s, 2H), 7.02-7.06 (m, 2H), 7.38

(s, 4H), 7.55-7.59 (m, 2H), 7.69-7.73 (m, 2H), 7.90-7.95 (m, 2H).

TFA (0.8 mL, 10.26 mmol) was added dropwise to a stirred

and ice-chilled solution of O-silylate (120 mg, 0.18 mmol) in

CH₂Cl₂(2 mL). The solution was stirred under these reaction

conditions for 5 h, and the solvent was removed in vacuo. The

crude product was purified by trituration with n-hexane/Et₂O to

the k_obsvalues versus the inhibitor concentration according to

the equation:

32

k

=k_offþ k_on[I]/(1 þ [S]/K_M).

obs

This equation describes a one-step association mechanism

(Scheme 4) where S is the fluorogenic substrate used and EI is

the complex between the enzyme and the inhibitor. Under our

4770 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15

Nuti et al.

conditions, the concentration of substrate is well below K_M, thus the

term [S]/K_Mcould be neglected. The k_off/k_onratio gives the K_ivalue.

Cambridge Structural Database was searched to find the pre-

ferred torsion values for the N-isopropoxy aryl sulfonamide. As

no one example for acyclic N-isopropoxy aryl sulfonamide was

found, we approached the problem in two steps by assessing first

the Car-Car-S-N torsion of the aryl solfonamides devoid of any

ortho substituents, and a clear preference for a 90ꢀ dihedral

angle has been found regardless the substituents on the nitrogen

atom. On the basis of these findings, we simplified the query

excluding the aryl moiety founding 14 acyclic N-O sulfona-

mides. Now, plotting the torsion O-S-N-X or O-S-N-O

(data not shown), it resulted that independently from the

substituents on the N, two preferred conformations have been

found. The most populated one is that in which the N-lone pair

bisects the OdSdO angle, while the other one occurs when one

N-substituent bisects the OdSdO angle.

Because Autodock force fields have no parameters describing

the lowest energy conformations of groups of atoms and in the

case of N-bisubstituted arylsolfonamides, it distorted the low-

energy conformations, giving rise to conformations which are

unlikely to exist, we decided to keep the torsions involving the

Car-S and S-N bond fixed using the values found as preferred

in the CSD. Thus, the ligand in the two possible conformers was

docked, considering also the symmetric geometries and the

N-pyramidal inversion.

The hydroxamate moiety was considered in its singly deproto-

nated form (hydroxamato), in line with previous experimental

studies.⁵⁶

The parametrization of 5 for the docking experiments was

performed using the Gaussian03 package⁵⁷by applying a full

geometry optimization at Hartree-Fock level of theory and

calculating the charges using the restrained electrostatic poten-

tial (RESP) fitting procedure.^58,59For both the optimization

and the electrostatic potential calculation, the locally dense

6-31G* basis set was used.

Scheme 4

Collagenase Assays. MMP-1 (0.5 nM) or MMP-13 (1 nM)

was preincubated with the different inhibitors at a range of

concentrations (0-10 μM) for 2 h at 25 ꢀC in collagenase buffer

(50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05%

Brij-35, 0.02% NaN₃). Acid soluble collagen purified from

guinea pig skin neutralized with collagenase buffer was added

to bring the final concentration of substrate to 1.35 mg/mL. The

reactions were incubated for a further 16 h at 25 ꢀC, before the

reactions were stopped with SDS-PAGE loading buffer contain-

ing 50 mM EDTA. The amount of collagen degradation was

determined by densitometric quantification of the full length

and 3/4 cleaved fragment of the R2- chain of collagen following

SDS-PAGE separation (7.5% acrylamide) and staining with

Coomassie Brilliant Blue-R250 using the Biorad GS-710 cali-

brated scanning densitometer and analyzed using the Phoretix

1D software (Nonlinear Dynamics, Newcastle Upon Tyne,

UK). The percentage of collagen cleavage was calculated ac-

cording to the method of Welgus et al.⁵²

Cartilage Degradation Assays. Porcine articular cartilage

from the metacarpophalangeal joints of 3-9 month old pigs

were dissected and cut into pieces approximately 0.5 mmꢀ

3 mm ꢀ 3 mm. After dissection, cartilage explants were allowed

to rest for 24 h at 37 ꢀC under 5% CO₂in DMEM containing

100 U/mL penicillin, 100 mg/mL streptomycin, 10 mM HEPES,

and 10% fetal calf serum (FCS) before switching to media

without FCS for a further 3 days. Rested explants were stimu-

lated with 10 ng/mL IL-1R with the inhibitors at 0.1, 1, and

10 μM, respectively, in serum free medium. The medium was

changed every 3-4 days for the duration of the experiment.

Glycosaminoglycan (GAG) and hydroxyproline (Hypro) re-

leased into the medium was taken as measures of aggrecan

and collagen degradation, respectively. The amount of GAG

released was standardized against shark/whale chondroitin

sulfate (Sigma) using the dimethylmethylene blue (DMMB)

assay modified for use in a 96-well plate.³⁴Hypro released into

the culture media was determined using a modification of the

assay as described by Bergman and Loxley.⁵³Briefly, condi-

tioned media (50 μL) was acid hydrolyzed by the addition of an

equal volume of 6 N HCl for 20 h at 95 ꢀC in a microcentrifuge

tube. The acid was removed from the hydrolyzed residue by

drying in a vacuum centrifuge for 4 h. The dried residue was fully

resuspended in 40 μL dH₂O. The sample was reacted with 25 μL

chloramine T reagent (1.4% (w/v) chloramine T, 30.8% (v/v)

isopropanol, 335 mM NaOAc, 100 mM trisodium citrate,

23 mM citric acid) for 4 min at room temperature before addition

of 150 μL of dimethylaminobenzaldehyde (DMBA) reagent

(16.7% (w/v) DMBA, 75% (v/v) isopropanol, 52.5% (v/v) per-

chloric acid). The mixture was incubated at 70 ꢀC for 40 min and

left to cool before the absorbance at 560 nm was read. The

amount of hydroxyproline was calculated by comparison to a

standard curve (0-1.2 μg ^L-hydroxyproline).

With regard to the protein setup, to create initial coordinates

for docking studies, calcium ions, cocrystallized inhibitors, and

all water molecules of the crystal structures were removed and

excluded from calculations. For the zinc atom, an ion para-

metrization, specific for the hydroxamic chelators and based on

quantum chemistry calculations, was used as suggested by

Cheng et al.⁶⁰

Docking Setup. The docking area has been defined by a box,

centered on the catalytic zinc atom. Grids points of 65 ꢀ 60 ꢀ 60

with 0.375 A spacing were calculated around the docking area

for all the ligand atom types using AutoGrid4. For inhibitor 5,

100 separate docking calculations were performed. Each dock-

ing calculation consisted of 25ꢀ10⁶energy evaluations using the

Lamarckian genetic algorithm local search (GALS) method. A

low-frequency local search according to the method of Solis and

Wets was applied to docking trials to ensure that the final

solution represents a local minimum. Each docking run was

performed with a population size of 150, and 300 rounds of Solis

and Wets local search were applied with a probability of 0.06. A

mutation rate of 0.02 and a crossover rate of 0.8 were used to

generate new docking trials for subsequent generations. The

docking results from each of the 100 calculations were clus-

tered on the basis of root-mean-square deviation (rmsd=2.0 A)

between the Cartesian coordinates of the ligand atoms and were

ranked on the basis of the free energy of binding.

Energy Minimization of the Docked Complex. The lowest

energy binding pose of 5 with MMP-13, as resulted from

docking calculations, was subjected to an energy minimiza-

tion by means of the AMBER 9.0 package software.⁶¹Ligand

force-field parameters were derived using the Antechamber

program, and partial charges were calculated using the RESP

methodology as described above. For the purpose of the energy

minimization, the complex was first inserted in a box (68.524 A ꢀ

65.510 A ꢀ71.425 A) of TIP3P⁶²pre-equilibrated water mole-

cules. Then, four Na^þions were randomly placed by means of

the add ions routine of Xleap to maintain neutrality in the

Molecular Modeling. Docking Simulations. Molecular dock-

ing of compound 5 into the three-dimensional X-ray structure of

the metalloenzymes was carried out using the AutoDock soft-

ware package (version 4.0) as implemented through the graphi-

cal user interface AutoDockTools (ADT 1.5.1).⁵⁴

Ligands and Protein Setup. The inhibitor structure was first

generated through the Dundee PRODRG2 Server.⁵⁵Then the

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4771

system. First, water molecules and counterions were minimized

using steepest descent followed by conjugated gradient. Second,

a minimization of the entire system was performed by means of

2000 steps of steepest descent followed by 3000 steps of con-

jugated gradient. The energy optimization was run for water

solution using the particle mesh Ewald (PME) method⁶³for the

treatment of the long-range electrostatics; a 8.0 A distance

cutoff was used for direct space nonbonded calculations and a

0.00001 Ewald convergence tolerance for the inclusion of long-

range electrostatic contributions; the SHAKE option (tolerance

0.00005 A) was used for constraining bonds involving hydrogen

atoms. Visual inspection of docking results were made with

MGL-Tools package,⁶⁴and Figure 5 was rendered by the

Chimera software package.⁶⁵

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Supporting Information Available: A table reporting the

HPLC purity analysis and the combustion analysis data of the

final products. This material is available free of charge via the

Internet at http://pubs.acs.org.

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Article Doi

N-O-isopropyl sulfonamido-based hydroxamates: Design, synthesis and biological evaluation of selective matrix metalloproteinase-13 inhibitors as potential therapeutic agents for osteoarthritis

DOI: 10.1021/jm900261f

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