Design and synthesis of piperazine-based matrix metalloproteinase inhibitors

Article abstract of DOI:10.1021/jm990366q

A new generation of cyclic matrix metalloproteinase (MMP) inhibitors derived from dl-piperazinecarboxylic acid has been described. The design involves: incorporation of hydroxamic acid as the bidentate chelating agent for catalytic Zn²⁺, placement of a sulfonamide group at the 1N-position of the piperazine ring to fill the S1' pocket of the enzyme, and finally attachment of diverse functional groups at the 4N-position to optimize potency and peroral absorption. A unique combination of all three elements produced inhibitor 20 with high affinity for MMPs 1, 3, 9, and 13 (24, 18, 1.9, and 1.3 nM, respectively). X-ray crystallography data obtained for MMP-3 cocrystallized with 20 gave detailed information on key binding interactions defining an overall scaffold geometry for piperazine-based MMP inhibitors.

Full text of DOI:10.1021/jm990366q

J . Med. Chem. 2000, 43, 369-380
369
Design a n d Syn th esis of P ip er a zin e-Ba sed Ma tr ix Meta llop r otein a se In h ibitor s
Menyan Cheng, Biswanath De,* Stanislaw Pikul, Neil G. Almstead, Michael G. Natchus, Melanie V. Anastasio,
Sara J . McPhail,† Catherine E. Snider,† Yetunde O. Taiwo, Longyin Chen,‡ C. Michelle Dunaway, Fei Gu,
Martin E. Dowty, Glen E. Mieling, Michael J . J anusz, and Sherry Wang-Weigand
Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040, and
Procter and Gamble, Corporate Research Division, Miami Valley Laboratories, Cincinnati, Ohio 45253
Received J uly 16, 1999
A new generation of cyclic matrix metalloproteinase (MMP) inhibitors derived from dl-
piperazinecarboxylic acid has been described. The design involves: incorporation of hydroxamic
acid as the bidentate chelating agent for catalytic Zn2+, placement of a sulfonamide group at
the 1N-position of the piperazine ring to fill the S1′ pocket of the enzyme, and finally attachment
of diverse functional groups at the 4N-position to optimize potency and peroral absorption. A
unique combination of all three elements produced inhibitor 20 with high affinity for MMPs 1,
3, 9, and 13 (24, 18, 1.9, and 1.3 nM, respectively). X-ray crystallography data obtained for
MMP-3 cocrystallized with 20 gave detailed information on key binding interactions defining
an overall scaffold geometry for piperazine-based MMP inhibitors.
Ch a r t 1. Hydroxamic Acid-Based MMPIs
In tr od u ction
Matrix metalloproteinases (MMPs) constitute a family
of zinc-dependent endoproteinases that are involved in
extracellular matrix remodeling. These enzymes are
secreted as zymogens which are subsequently processed
by other proteolytic enzymes to generate the active
forms. Under normal physiological conditions, the pro-
teolytic activity of the MMPs is controlled at any of the
following three known stages: transcription, activation
of the zymogens, and inhibition of the active forms by
various tissue inhibitors of MMPs (TIMPs).1 In patho-
logical conditions this equilibrium is shifted toward
increased MMP activity leading to tissue degradation.2
Consequently a considerable amount of effort has been
invested in designing orally active MMP inhibitors
(MMPIs) with the understanding that such agents will
be able to either halt or slow the progression of diseases
such as osteoarthritis, tumor metastasis, and corneal
ulceration.3
and (c) conformational stability of the cyclic backbone.
To impart a higher proportion of nonpeptidic character,
a decision was made to incorporate a sulfonamide unit
at the 1N-position of piperazinecarboxylic acid (see CGS-
27023A).6 The distal nitrogen atom (4N) of the pipera-
zine ring provided the desired site for SAR work
reported in this publication.
Ch em istr y. The piperazine-based MMPIs were syn-
thesized from commercially available dl-piperazinecar-
boxylic acid following the general procedure detailed in
Scheme 1. Selective protection of piperazinecarboxylic
acid with di-tert-butyl dicarbonate under optimized
conditions produced the desired monoamine 5. The
crude amine 5 was subsequently treated with p-meth-
oxyphenylsulfonyl chloride, and then the resulting sul-
fonamide was treated with methanolic HCl to provide
the desired common intermediate 7 in good overall yield.
For SAR studies, intermediate 7 was treated with
various reagents to provide the desired substitued
piperazine derivatives 8. The methyl ester 8 was then
treated with hydroxylamine in KOH-MeOH to provide
the desired hydroxamic acid 9. For example, treatment
of 7 with methanesulfonyl chloride in Et3N afforded the
Design Str a tegy for th e Cyclic MMP Is
Previous work from our laboratory on both SAR
studies and crystallography with partially cyclic and
acyclic MMPIs (see 1 and 2, Chart 1) indicated that the
hydroxamic acid unit was critical in terms of preserving
high potency.4 As indicated in Figure 2, with a 4-point
attachment to the active surface, this critical unit
behaves like a molecular magnet. As part of our long-
term interests in the design of potent MMPIs containing
a central, conformationally constrained, heterocycle
ring,4,5 dl-piperazinecarboxylic acid was chosen to pro-
vide the central backbone. Convenient features of this
starting material include: (a) commercial availability,
(b) convenient synthetic access to a variety of analogues,
* To whom correspondence should be addressed. Tel: 513-622-3641.
Fax: 513-622-3681. E-mail: de.b@pg.com.
† Corporate Research Division, Procter and Gamble Co.
‡ Present address: Department of Molecular Pharmacology
&
Biological Chemistry, Northwestern University Medical School, Chi-
cago, IL 60611-3008.
10.1021/jm990366q CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/19/2000

370 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
Sch em e 1a
a
Reagents: (a) 1.1 equiv (t-BOC)2O, 2 equiv NaOH, dioxane-water; (b) 1 equiv p-MeOPhSO2Cl, Et3N/cat. DMAP; (c) SOCl2, MeOH;
(d) sulfonyl chlorides or alkyl halides or isocyanates or acyl halides, etc.; (e) NH2OH in KOH and MeOH.
Ta ble 1. In Vitro Profile of Substituted Piperazine-Based
Ta ble 2. In Vitro Profile of Sulfonamide- and
MMPIs
Carbamate-Substituted Piperazine-Based MMPIs
a
See Experimental Section for details of the enzyme assays;
nd ) not determined.
bis-sulfonamide which, upon treatment with hydroxyl-
amine in KOH-MeOH, provided the hydroxamic acid
15 (see Table 2). In a similar fashion, treatment of 7
with benzyloxycarbonyl (CBz) chloride in dioxane-
water afforded the desired carbamate methyl ester
which was then converted to the desired hydroxamic
acid 20 (Table 2). Additional details for the remaining
analogues are provided in the Experimental Section.
a
See Experimental Section for details of the enzyme assays;
nd ) not determined.
the N-alkyl substituent. Even substitution with a large
group such as the heterocycle in 14 did not significantly
alter the in vitro profile of the simple N-alkylamines.
Su lfon a m id e- a n d Ca r ba m a te-Su bstitu ted P ip -
er a zin es. The bis-sulfonamide- and carbamate-substi-
tuted piperazines all showed enhanced potency for
MMPs 3, 9, and 13 relative to the N-alkyl derivatives
(Tables 1 and 2). Many of these compounds were also
quite potent inhibitors of MMP-1. Increasing the steric
bulk of the bis-sulfonamide appeared to have very little
influence on the in vitro profile of these compounds. The
heterocyclic sulfonamides 17 and 18 were only slightly
less potent for MMP-1 than the methanesulfonamide
15. The in vitro profile of the carbamate-based MMPIs
was also quite insensitive to the changes in both steric
and electronic environments.
Resu lts a n d Discu ssion
N-Alk ylp ip er a zin es. All compounds were tested for
the inhibition of MMPs 1, 3, 7, 9, and 13.7 The SAR work
presented herein was performed with the p-methoxy-
phenylsulfonamide moiety appended R with respect to
the hydroxamic acid unless otherwise specified. Initial
SAR studies were performed with alkyl-substituted
piperazine derivatives. The parent compound of the
series (compound 10, Table 1) was a nanomolar inhibitor
of MMPs 1, 3, 9, and 13 but exhibited micromolar
activity against MMP-7. Substitution of the NH in
compound 10 with an alkyl chain did not significantly
alter the potency or selectivity of the series. Like the
parent compound, analogues 11-13 were all potent
inhibitors of MMPs 9 and 13, and they were all less
potent against MMP-7. All of the compounds in the
series demonstrated a preference for MMP-3 vs MMP-1
although the extent of this selectivity varied based upon
Am id e-Su bstitu ted P ip er a zin es. The amide-sub-
stituted piperazines possessed a consistent in vitro
profile with potent inhibition of MMPs 9 and 13 and
weak inhibition of MMP-7 (Table 3). Some variation was
observed with the inhibition of MMPs 1 and 3. The steric

Piperazine-Based MMPIs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 371
Ta ble 3. In Vitro Profile of Amide-Substituted
Piperazine-Based MMPIs
almost an identical in vitro profile even though these
compounds are significantly different in steric require-
ments. The pyridylamide 29 was significantly less
potent than the corresponding benzamide 28. The
presence of a basic nitrogen in this position is clearly
not well-tolerated by the MMPs.
Ur ea -Su bstitu ted P ip er a zin es. The urea-substi-
tuted piperazines demonstrated a similar in vitro profile
to the corresponding amides in Table 3. These com-
pounds were potent inhibitors of MMPs 9 and 13 and
weak inhibitors of MMP-7 (Table 4). The parent com-
pound in the series, 36, was a potent MMPI which only
spared MMP-7. This compound possessed moderate
selectivity for MMP-3 vs MMP-1. The corresponding
n-hexylurea 37 possessed little to no selectivity (MMP-1
vs MMP-3), but the corresponding N-methyl-N-hexyl-
urea 38 was surprisingly selective for MMP-3. Cyclic
urea-based inhibitors were also prepared including the
morpholino- and N-methylpiperazine-based inhibitors
47 and 48. The inclusion of a ring at this position did
not result in a dramatic shift in either potency or
selectivity for this series.
Replacement of the p-methoxyphenylsulfonamide with
a p-bromophenylsulfonamide (compound 39) resulted in
a reversal in the in vitro selectivity profile which was
generally observed in this series. The compound was
significantly less potent for MMP-3 than the corre-
sponding p-methoxyphenylsulfonamide 38. The only
other p-bromophenylsulfonamide prepared in this se-
ries, compound 49, was also more potent for MMP-1 vs
MMP-3.
a
See Experimental Section for details of the enzyme assays.
nd ) not determined.
bulk of the amide substituent appears to play little if
any role in determining the in vitro potency of the series.
The acetamide 23 and the biphenylamide 35 exhibited
Absor p tion Stu d ies. As a first step toward under-
standing the overall pharmacokinetic profile of the
Ta ble 4. In Vitro Profile of Urea-Substituted Piperazine-Based MMPIs
a
b
See Experimental Section for details of the enzyme assays; nd ) not determined. Compound 41 is a thiourea.

372 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
Ta ble 5. Predicted Peroral Absorption and ex Vivo Efficacy
Data
compd
absorption (%)a
log Pb
explant IC50 (nM)c
12
13
15
16
17
20
21
23
25
27
30
35
37
38
39
43
44
45
46
49
93-98
98
51-62
5
37
59
10
8-18
13
6
14
3.43
2.63
0.24
1.38
0.57
1.76
1.14
0.12
2.15
2.17
1.18
3.29
2.82
3.16
3.85
2.36
1.85
2.70
2.62
2.03
>1000
>1000
94
<10
57
43
100
nd
nd
37
35
5
100
29-76
44-87
0
3-4
10-23
34-37
35-37
3-6
115
100
nd
108
22
44
370
nd
F igu r e 1. Structure of the stromelysin-13 complex.
a
Data obtained from both single and multiple determinations;
b
see ref 11 for details. Calculated octanol/water partition coef-
ficient (ClogP, by BioByte Corp., Claremont, CA). c Data are
expressed as an IC50 from multiple concentrations of inhibitor each
run in 8 replicate cartilage cultures; nd ) not determined.
piperazine-based MMPIs, an attempt was made to
determine the permeability of a selected number of
analogues. In terms of predicted in vitro peroral absorp-
tion values, most of the compounds evaluated showed
moderate to good absorption potential.8 However, it was
difficult to identify any definitive trends in the absorp-
tion data with physicochemical properties or molecular
structure. For example, while the sulfonamides 15 and
17 demonstrated moderate to good absorption, a close
relative, 16, showed a much lower absorption potential.
Low predicted absorption of these compounds, in some
cases, may have been attributed to low solubility of the
molecule. In addition, there was evidence that some
compounds in this class were substrates for the various
concentration-dependent efflux systems (including P-
glycoprotein), encoded by the MDR1 gene, present in
the enterocytes of the intestinal tract (data not shown).
Nevertheless, the relatively lipophilic analogues 12 and
13 (N-alkyl analogues) and 35 showed the best absorp-
tion values. However, attempts to improve the hydro-
philic properties of the molecules tended to result in a
lower absorption potential in many cases (examples
based on calculated log octanol/water partition coef-
ficients: 15, 16, 17, 20, 21, 27, 44, and 49). In contrast,
compound 39, which had the highest calculated log
octanol/water coefficient of all the analogues (3.85), may
have partitioned into the intestinal cell membranes and
stayed there, resulting in no transport. Indeed, further
experimentation would be necessary to better charac-
terize the intestinal transport properties of these com-
pounds.
F igu r e 2. Binding interactions of 13 bound to truncated
MMP-3.
explant cultures, including potency for the MMPs,
stability of the compounds in the culture medium, and
permeability of the MMPIs into cartilage matrix. Pre-
liminary permeability studies done in-house using
articular cartilage demonstrate that small MMPIs (MW
400-500) of similar types do penetrate the matrix with
ease.10
A close examination of the explant data reveals that
there is agreement between binding potency and efficacy
in this model for a select group of analogues. For
example, most of the broad-spectrum MMPIs (15, 20,
21, etc.) showed efficacy at an IC50 level close to 100
nM or less (see Table 5). Some analogues with high
IC50’s for the inhibition of MMP-1 (e.g. 12 and 13) failed
to demonstrate any efficacy. Although it has been
suggested that MMP-1 plays a prominent role in the
BNC explant model,11 it is difficult to draw any conclu-
sion from these limited examples as to which factor or
factors have an overriding effect in terms of determining
efficacy in this model.
Str u ctu r a l An a lysis. During the progression of this
work, crystallography data for a number of enzyme-
inhibitor complexes were reported.12 Most of these
structures were for acyclic inhibitors. To understand the
binding mode of piperazine-based inhibitors, the crystal
structure of 20 bound to truncated MMP-3 was solved.
As shown in Figures 1 and 2, the hydroxamic acid unit
was tightly anchored through four contact points. Bi-
dentate chelation of Zn2+ was achieved using two nearly
identical bonds (2.12 and 2.14 Å). Glu-202 was properly
poised with two bonding possibilities (2.61 and 3.05 Å).
Ala-165 carbonyl formed the expected hydrogen bond
Ca r tila ge Degr a d a tion . To obtain a preliminary
assessment of the efficacy of the piperazine-based
MMPIs, a cartilage explant cell culture method was
used. The degradation of cartilage matrix was measured
ex vivo using cultures of bovine nasal cartilage stimu-
lated with IL-1.9 An inhibitory effect on matrix degrada-
tion in this model may reflect cartilage-protecting
potential of compounds. A number of factors can affect
the collagen-protecting performance of MMPIs in bovine

Piperazine-Based MMPIs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 373
F igu r e 4. Possible role played by structural Ca2+ to induce a
critical H-bonding.
Con clu sion
The design and synthesis of a new generation of cyclic
MMPIs have been described. Variation of substituents
attached to the 4N-position of a piperazine scaffold has
produced a series of potent inhibitors with varying
degree of selectivity for MMPs reported herein. By
proper optimization of various substituents, a number
of inhibitors with high absorption profiles and desirable
efficacies in the bovine nasal cartilage assay were
identified. A careful analysis of the X-ray data revealed
that certain structural elements (optimized substituents
attached to the distal nitrogen, a bidentate ligand for
catalytic Zn2+, hydrogen bonding with Leu-164, etc.) are
essential for achieving high binding affinity and selec-
tivity. Some explanations, as to why an electron-
donating group attached to the phenylsulfonyl unit
enhances the binding potency for certain MMPs, have
been provided. The required geometry of folding for the
piperazine-based MMPIs has also been delineated.
F igu r e 3. Structure of the stromelysin-13 complex.
with the hydroxamate nitrogen atom (3.28 Å). One of
the oxygen atoms of the sulfonamide unit formed a tight
hydrogen bond with Leu-164 (2.89 Å) and was also found
in hydrogen bond distance (3.22 Å) with the nitrogen
atom of Ala-165. The p-methoxyphenyl unit was found
to be buried in the deep S1′ pocket. Similar interactions
were observed in complexes of 47 with MMPs 1, 3, and
13. These data will be reported in due course.
Exp er im en ta l Section
1
Gen er a l Meth od s a n d Ma ter ia ls. H NMR spectra were
During the SAR work with the piperazine-based
MMPIs, the possible orientation of substituents at-
tached to the distal nitrogen became a major subject of
discussions. SAR derived from a diverse series of sub-
stituents was quite intriguing. The crystallography data
obtained for 20 partly answered this question. The Cbz
group was placed in an intermediate position between
the S1 and S2′ pockets. Some hydrophobic interactions
with Phe-210 and Phe-186 could be observed. Based on
the nature of this site, it is possible that some of the
polar substituents (as in 17 and 21) can shift more
toward the solvent.The importance of Leu-164 in terms
of forming a strong hydrogen bond with the peptidic
backbone, or in this case with one of the oxygen atoms
of the sulfonamide unit, has been a subject of much
discussion.13 One can hypothesize that the proximity of
structural Ca2+ to Val-163 carbonyl polarizes the bond
considerably, leading to lower pK for the Leu-164 amidic
proton (see Figures 3 and 4). This, in turn, can help Leu-
164 form a strong hydrogen bond with a suitably
oriented oxygen atom, supplied by either an amidic
carbonyl group or a sulfonamide, as seen for 20. A
careful survey of many MMPIs reveals that a 1,4-
relationship between the two heteroatoms, as shown in
Figure 2, is generally preserved in order to achieve high
binding potency (also see BB-2516 and BY12-9566 in
Figure 4).14
measured at 300 MHz on either a Bruker or GE instrument
using deuteriochloroform as solvent unless otherwise indi-
cated. Mass spectra were measured at 70 eV in CI (chemical
ionization) or ESI (electrospray ionization). Elemental analyses
(C, H, N) were performed at either Oneida Research Services
or Procter and Gamble Analytical and Stability Laboratories
in Norwich. Analytical thin-layer chromatography (TLC)
analysis was performed using Merck DC-F254 glass-based silica
gel plates. Flash chromatography was performed using
kieselgel 60 (230-400 mesh) silica gel. All organic extracts
were dried over anhydrous MgSO4 or Na2SO4 prior to solvent
removal on a rotary evaporator under reduced pressure. For
the synthesis of a hydroxamic acid from the corresponding
methyl ester, a general procedure, as outlined in Fieser &
Fieser, Vol. 1, p 478, was followed.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon ylp ip er a zin e-2-
ca r boxa m id otr iflu or oa cetic Acid (10). To a solution of Boc-
piperazine (19) (95 mg, 0.23 mmol) in 5 mL of CH2Cl2 was
added trifluoroacetic acid (5 mL) at 0 °C. The reaction was
stirred for 4 h at room temperature, followed by removal of
solvent under reduced pressure. The crude product was
purified by flash silica gel column eluting with CH3OH/EtOAc
(1:9) to give 90 mg (61% yield) of desired product as a foamy
solid: 1H NMR (DMSO-d6) δ 2.63 (td, J ) 12.3, 4.0 Hz, 1H),
2.82 (dd, J ) 13.6, 2.8 Hz, 1H), 3.01 (d, J ) 13.5 Hz, 1H), 3.26-
3.34 (m, 1H), 3.48 (td, J ) 12.2, 3.3 Hz, 1H), 3.61-3.72 (m,
1H), 3.89 (s, 3H), 4.37 (d, J ) 3.5 Hz, 1H), 7.10 (dd, J ) 9.0,
1.2 Hz, 2H), 7.80 (dd, J ) 8.9, 1.2 Hz, 2H); ion spray MS 338
[M + Na]+, 316 [M + H]+; HRMS (M+) calcd for C12H18N3O5S
316.0967, found (M+) 316.0971.

374 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
1-(4-Meth oxyp h en yl)su lfon yl-4-(ter t-bu toxyca r bon yl)-
p ip er a zin e-2-ca r boxylic Acid (6). To a solution of pipera-
zine-2-carboxylic acid dihydrochloride (4) (3 g, 14.8 mmol) in
30 mL of p-dioxane and 15 mL of water at 0 °C was added
slowly aqueous sodium hydroxide (2.3 mL, 50% w/w, 19.4 N,
44.4 mmol), followed by di-tert-butyl dicarbonate (3.6 g, 16.3
mmol). After 5 h at room temperature (or overnight at larger
scale), triethylamine (4.1 mL, 29.6 mmol) and 4-methoxyphe-
nylsulfonyl chloride (3.0 g, 14.8 mmol) were added, and the
reaction was stirred overnight. The reaction mixture was
concentrated under reduced pressure and partitioned between
EtOAc and 1 N HCl. The EtOAc layer (2 × 100 mL) was
washed with brine, dried over MgSO4, filtered, and concen-
trated in vacuo to give the title compound (4.2 g, 80%) as a
solid: 1H NMR (CDCl3) δ 1.38 (s, 9H), 2.78-2.94 (m, 1H),
3.05-3.18 (m, 1H), 3.30-3.44 (m, 1H), 3.54-3.68 (m, 1H), 3.86
(s, 3H), 3.72-3.88 (m, 1H), 4.46-4.62 (m, 2H), 6.95 (dd, J )
9.0, 2.2 Hz, 2H), 7.70 (dd, J ) 9.2, 2.2 Hz, 2H); CI+ MS 418
[M + NH4]+, 401 [M + H]+, 362 [M + NH4 - tBu]+, 345 [M +
H - tBu]+, 301 [M + H - tBoc]+.
Meth yl 1-(4-Meth oxyp h en yl)su lfon ylp ip er a zin e-2-ca r -
boxyla te Hyd r och lor id e (7). To a solution of 1-(4-methoxy-
phenyl)sulfonyl-4-(tert-butoxycarbonyl)piperazine-2-carboxylic
acid (6) (19.6 g, 48.9 mmol) in 100 mL of methanol was added
thionyl chloride (18 mL, 244.5 mmol) dropwise at room
temperature. The reaction was stirred overnight. The reaction
mixture was concentrated under reduced pressure to a solid
residue, which was triturated from 5% methanol/hexane to
give the title compound (13.06 g, 76%) as a solid: 1H NMR
(CDCl3) δ 2.98 (td, J ) 12.8, 3.8 Hz, 1H), 3.15 (dd, J ) 12.5,
4.3 Hz, 1H), 3.42 (d, J ) 9.8 Hz, 1H), 3.52-3.64 (m, 1H), 3.66
(s, 3H), 3.80-3.91 (m, 2H), 3.88 (s, 3H), 4.86 (d, J ) 4.8 Hz,
1H), 6.98 (d, J ) 9.2 Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H); CI+ MS
315 [M + H]+.
1.04-1.36 (m, 8H), 1.58-1.67 (m, 1H), 1.70-1.82 (m, 1H),
1.98-2.12 (m, 2H), 2.52-2.61 (m, 1H), 2.98-3.08 (m, 1H),
3.32-3.59 (m, 2H), 3.82 (s, 3H), 4.22 (s, 1H), 7.08 (d, J ) 9.0
Hz, 2H), 7.76 (d, J ) 9.0 Hz, 2H), 8.80 (s, 1H), 10.48 (s, 1H);
ESI+ MS 400 [M + H]+. Anal. (C18H29N3O5S‚0.25H2O) C, H,
N.
N -H yd r oxy-1-(4-m e t h oxyp h e n yl)su lfon yl-4-p h e n yl-
m eth ylp ip er a zin e-2-ca r boxa m id e (13). The title compound
was prepared from amine hydrochloride 7 following the
procedure described for compound 11: yield 40%; 1H NMR (5%
NaOD) δ 1.45-1.66 (m, 1H), 2.32 (d, J ) 11.9 Hz, 1H), 2.96
(d, J ) 11.6 Hz, 1H), 3.22-3.51 (m, 4H), 3.64 (d, J ) 13.2 Hz,
1H), 3.80 (s, 3H), 4.24 (s, 1H), 6.88 (d, J ) 9.0 Hz, 2H), 6.94-
7.03 (m, 2H), 7.19-7.30 (m, 3H), 7.59 (d, J ) 9.0 Hz, 2H); ion
spray MS 428 [M + Na]+, 406 [M + H]+. Anal. (C19H23N3O5S‚
HCl‚0.5H2O) C, H, N.
Met h yl 4-[4-(4-Br om op h en yl)t h ia zol-2-yl]-1-(4-m et h -
oxyp h en yl)su lfon ylp ip er a zin e-2-ca r boxyla te (14a ). To a
solution of amine hydrochloride 7 (300 mg, 0.86 mmol) and
acetic acid (50 µL, 0.86 mmol) in ethanol (5 mL) was added
4-bromophenacyl thiocyanate (220 mg, 0.86 mmol). The reac-
tion mixture was stirred at room temperature for 6 h, neutral-
ized with triethylamine, and partitioned between ether and
water. The ether layer was washed with water and brine, dried
over MgSO4, and concentrated under reduced pressure. The
crude product was purified by flash chromatography (hex-
anes-ethyl acetate 4/1 v/v) to give 135 mg (28% yield) of the
title compound as colorless solid: mp 68-70 °C; 1H NMR
(CDCl3) δ 3.18 (dt, J ) 11.6, 3.9 Hz, 1H), 3.37 (dd, J ) 13.6,
3.0 Hz, 1H), 3.49 (s. 3H), 3.55 (m, 1H), 3.78 (m, 1H), 3.84 (s,
3H), 4.00 (m, 1H), 4.51 (bd, J ) 13.2 Hz, 1H), 4.76 (m, 1H),
6.78 (s, 1H), 6.95 (d, J ) 8.6 Hz, 2H), 7.47 (d, J ) 8.6 Hz, 2H),
7.64 (d, J ) 8.6 Hz, 2H), 7.73 (d, J ) 8.6 Hz, 2H); 13C NMR
(CDCl3) δ 41.2, 48.0, 50.5, 52.5, 54.7, 55.9, 102.8, 114.2, 121.6,
127.5, 129.4, 131.0, 131.6, 133.6, 150.5, 163.1, 169.3, 170.2.
Meth yl 1-(4-Meth oxyp h en yl)su lfon yl-4-m eth ylp ip er a -
zin e-2-ca r boxyla te (11a ). To a solution of amine hydrochlo-
ride 7 (1.2 g, 3.4 mmol) and sodium acetate (0.84 g, 10.26
mmol) in 10 mL of ethanol was added paraformaldehyde (0.21
g, 6.84 mmol), followed by sodium cyanoborohydride (0.45 g,
6.84 mmol) slowly at room temperature. The reaction was
stirred overnight at room temperature. The reaction mixture
was concentrated under reduced pressure and stirred in 1 N
HCl for 30 min. The mixture was made basic by 1 N NaOH
and extracted with EtOAc (3 × 80 mL) in the presence of some
solid NaCl. The EtOAc layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure. The
crude product was purified by column chromatography on
silica gel using EtOAc as elute to give the title compound (0.68
g, 62%) as an oil: 1H NMR (CDCl3) δ 2.04 (td, J ) 15.4, 3.7
Hz, 1H), 2.24 (s, 3H), 2.21-2.26 (m, 1H), 2.67 (ddd, J ) 11.3,
1.4, 1.7 Hz, 1H), 3.21 (ddd, J ) 11.4, 1.8, 1.9 Hz, 1H), 3.38 (td,
J ) 11.0, 3.2 Hz, 1H), 3.61 (s, 3H), 3.54-3.62 (m, 1H), 3.87 (s,
3H), 4.64 (t, J ) 1.9 Hz, 1H), 6.94 (dd, J ) 8.9, 2.0 Hz, 2H),
7.72 (dd, J ) 8.9, 2.0 Hz, 2H); CI+ MS 329 [M + H]+.
N-Hydr oxy-4-[4-(4-br om oph en yl)th iazol-2-yl]-1-(4-m eth -
oxyp h en yl)su lfon ylp ip er a zin e-2-ca r boxa m id e (14). To
methyl ester 14a (121 mg, 0.22 mmol) was added NH2OK (2
mL of 1.7 M solution in methanol; prepared as described in
Fieser & Fieser, Vol. 1, p 478) and the reaction mixture was
stirred overnight at room temperature. The reaction mixture
was neutralized with 1 N aqueous hydrochloric acid and
concentrated under reduced pressure. The crude product was
purified by flash chromatography (hexanes-ethyl acetate 3/7
v/v) to give 54.6 mg (45% yield) of the title compound as
1
colorless solid: mp 181 °C dec; H NMR (CDCl3) δ 3.00 (dt, J
) 11.6, 5.7 Hz, 1H), 3.24 (dd, J ) 12.6, 5.8 Hz, 1H), 3.68-3.90
(m, 3H), 3.81 (s, 3H), 4.26 (dd, J ) 12.5, 1.9 Hz, 1H), 4.59 (m,
1H), 6.98 (s, 1H), 7.06 (d, J ) 8.7 Hz, 2H), 7.48 (d, J ) 8.7 Hz,
2H), 7.70 (d, J ) 8.7 Hz, 2H), 7.82 (d, J ) 8.7 Hz, 2H); 13C
NMR (CDCl3) δ 43.1, 47.7, 55.6, 56.2, 104.0, 115.7, 122.3, 128.8,
130.7, 132.0, 132.6, 135.4, 151.5, 165.0, 168.0, 171.9; ion spray
MS 553, 555 [M + H]+, 575, 577 [M + Na]+. Anal. (C21H21
BrN4O5S2) C, H, N.
-
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-m eth ylp ip -
er a zin e-2-ca r boxa m id e (11). The methyl ester 11a (0.45 g,
1.37 mmol) was mixed with NH2OK (6 mL, 10.2 mmol, 1.7 M
in methanol; solution prepared as described in Fieser & Fieser,
Vol. 1, p 478) and stirred overnight at room temperature. Silica
(1.5 g) was added to the mixture and the solvent was
evaporated. The dry silica was poured on top of a flash silica
gel column which was subsequently eluted with CH3OH/EtOAc
(1:5) to give 275 mg (61% yield) of desired product as a thick
oil: 1H NMR (DMSO-d6) δ 1.74 (td, J ) 15.3, 3.4 Hz, 1H), 1.86
(s, 3H), 1.90 (dd, J ) 12.1, 4.2 Hz, 1H), 2.36 (d, J ) 10.2 Hz,
1H), 3.12 (d, J ) 10.4, 1H), 3.42 (td, J ) 11.0, 3.2 Hz, 1H),
3.73 (br d, J ) 12.8 Hz, 1H), 3.91 (s, 3H), 4.42 (br s, 1H), 7.08
(dd, J ) 8.9, 2.0 Hz, 2H), 7.80 (dd, J ) 8.9, 2.0 Hz, 2H); ion
spray MS 352 [M + Na]+, 330 [M+ + H]+; HRMS ([M + H]+)
calcd for C13H19N3O5S 330.1124, found ([M + H]+) 330.1116.
Met h yl
1-(4-Met h oxyp h en yl)su lfon yl-4-m et h ylsu l-
fon ylp ip er a zin e-2-ca r boxyla te (15a ). To a solution of amine
hydrochloride 7 (1.5 g, 4.3 mmol), triethylamine (1.8 mL, 12.7
mmol), and DMAP (52 mg, 0.43 mmol) in 2 mL of water and
8 mL of p-dioxane was added methanesulfonyl chloride (0.43
mL, 5.56 mmol) dropwise at room temperature. The reaction
was stirred overnight at room temperature. The reaction
mixture was concentrated under reduced pressure and parti-
tioned between EtOAc and water. The EtOAc layer was
washed with 1 N HCl, water, and brine, dried over MgSO4,
and concentrated under reduced pressure to give the title
compound (0.8 g, 50%): 1H NMR (CDCl3) δ 2.88 (s, 3H), 2.89-
2.97 (m, 1H), 3.01 (dd, J ) 12.1, 4.0 Hz, 1H), 3.40 (td, J )
12.0, 3.4 Hz, 1H), 3.61 (s, 3H), 3.67-3.80 (m, 2H), 3.85 (s, 3H),
4.21 (ddd, J ) 10.3, 1.9, 1.9 Hz, 1H), 4.78-4.81 (m, 1H), 6.97
(d, J ) 9.0 Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H); ion spray MS
410 (100, M + NH4+), 393 (46, M+ + H).
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(n -h exyl)-
p ip er a zin e-2-ca r boxa m id e (12). The title compound was
prepared following the procedure described for compound 11:
yield 38%; 1H NMR (DMSO-d6) δ 0.92 (t, J ) 6.7 Hz, 3H),
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-m eth ylsu l-
fon ylp ip er a zin e-2-ca r boxa m id e (15). The methyl ester 15a
(0.7 g, 1.78 mmol) was mixed with NH2OH (10 mL, 14.2 mmol,

Piperazine-Based MMPIs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 375
1
1.5 M in methanol) and stirred overnight at room temperature.
The reaction mixture was acidified with HCl, silica (2 g) was
added, and the solvent was evaporated. The dry silica was
poured on top of a flash silica gel column which was subse-
quently eluted with 5% CH3OH/EtOAc to give 0.31 g (46%
yield) of the desired product as a white solid: 1H NMR (DMSO-
d6) δ 2.56-2.64 (m, 1H), 2.79 (s, 3H), 2.78-2.88 (m, 1H), 3.40-
3.49 (m, 1H), 3.52-3.70 (m, 2H), 3.73-3.81 (m, 1H), 3.83 (s,
3H), 4.36-4.42 (m, 1H), 7.07 (d, J ) 9.0 Hz, 2H), 7.78 (d, J )
9.0 Hz, 2H), 8.92 (s, 1H), 10.65 (s, 1H); ion spray MS 411 [M
+ NH4]+, 394 [M + H]+. Anal. (C13H19N3O7S2) C, H, N.
N-H yd r oxy-1,4-b is(4-m et h oxyp h en ylsu lfon yl)p ip er a -
zin e-2-ca r boxa m id e (16). The title compound was prepared
from amine hydrochloride 7 following the sequence of reactions
described for compound 15: yield 54%; 1H NMR (DMSO-d6) δ
1.63 (td, J ) 11.5, 3.1 Hz, 1H), 1.82 (dd, J ) 11.8, 4.2 Hz, 1H),
3.22-3.41 (m, 1H), 3.42-3.61 (m, 1H), 3.72-3.88 (m, 2H), 3.81
(s, 3H), 3.89 (s, 3H), 4.47 (br s, 1H), 6.93 (d, J ) 9.0 Hz, 2H),
7.10 (d, J ) 9.0 Hz, 2H), 7.48 (d, J ) 9.0 Hz, 2H), 7.64 (d, J )
9.0 Hz, 2H); ion spray MS 508 [M + Na]+, 503 [M + NH4]+,
486 [M + H]+. Anal. (C19H23N3O8S2 ) C, H, N.
pound 15: yield 60%; H NMR (CDCl3) δ 2.70-2.98 (m, 1H),
3.08 (dd, J ) 11.9, 3.2 Hz, 1H), 3.46-3.65 (m, 2H), 3.78-3.90
(m, 1H), 3.86 (s, 3H), 3.96-4.18 (m, 1H), 4.24 (br s, 1H), 4.93-
5.06 (m, 2H), 7.06 (dd, J ) 8.9, 2.2 Hz, 2H), 7.25-7.40 (m,
5H), 7.72 (dd, J ) 8.9, 2.2 Hz, 2H), 8.89 (s, 1H), 10.72 (s, 1H);
ion spray MS 472 [M + Na]+, 450 [M + H]+. Anal. (C20H23
-
N3O7S) C, H, N.
N -H yd r oxy-1-(4-m e t h oxyp h e n yl)su lfon yl-4-(3-p yr i-
d in ylm eth oxyca r bon yl)p ip er a zin e-2-ca r boxa m id e (21).
The title compound was prepared as a white solid from amine
hydrochloride 7 following the procedure described for com-
pound 15: yield 47%; 1H NMR (DMSO-d6) δ 3.01-3.60 (m,
5H), 3.83 (s, 3H), 4.01-4.13 (m, 1H), 4.21-4.28 (m, 1H), 4.98-
5.08 (m, 2H), 7.08 (d, J ) 9.0 Hz, 2H), 7.34-7.40 (m, 1H),
7.67-7.78 (m, 3H), 8.22 (s, 1H), 8.47-8.56 (m, 2H), 10.77 (s,
1H); ESI+ MS 451 (100, M+ + H). Anal. (C19H22N4O7S‚H2O)
C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(3-eth oxy-
1-p r op oxyca r bon yl)p ip er a zin e-2-ca r boxa m id e (22). The
title compound was prepared from amine hydrochloride 7
following the procedure described for compound 15: yield 51%;
1H NMR (DMSO-d6) δ 1.02 (t, J ) 6.9 Hz, 3H), 1.61-1.72 (m,
2H), 2.62-2.81 (m, 1H), 2.92-3.01 (m, 1H), 3.27-3.38 (m, 5H),
3.44-3.59 (m, 2H), 3.80 (s, 3H), 3.83-4.01 (m, 3H), 4.13-4.19
(m, 1H), 7.05 (d, J ) 9.0 Hz, 2H), 7.66 (d, J ) 9.0 Hz, 2H),
8.79 (s, 1H), 10.63 (s, 1H); ion spray MS 463 [M + NH4]+, 446
M + H]+. Anal. (C18H27N3O8S) C, H, N.
Meth yl 1-(4-Meth oxyp h en yl)su lfon yl-4-a cetylp ip er a -
zin e-2-ca r boxyla te (23a ). To a solution of amine hydrochlo-
ride 7 (2 g, 5.7 mmol), triethylamine (3.2 mL, 22.8 mmol), and
DMAP (70 mg, 0.57 mmol) in 10 mL of p-dioxane was added
acetic anhydride (0.81 mL, 8.5 mmol) dropwise at room
temperature. The reaction was stirred overnight at room
temperature. The reaction mixture was concentrated under
reduced pressure and partitioned between EtOAc and water.
The EtOAc layer was washed with 1 N HCl, water, and brine,
dried over MgSO4, and concentrated under reduced pressure
to give the title compound (1.37 g, 67%). The 1H NMR spectrum
shows two distinct rotamers: 1H NMR (CDCl3) δ 2.04 (d, J )
10.0 Hz, 6H), 2.43 (dd, J ) 12.4, 4.5 Hz, 1H), 2.94 (dd, J )
12.1, 4.0 Hz, 1H), 3.08 (td, J ) 12.6, 3.4 Hz, 1H), 3.21-3.41
(m, 2H), 3.58 (d, J ) 10.0 Hz, 6H), 3.56-3.58 (m, 1H), 3.64-
3.80 (m, 3H), 3.88 (d, J ) 3.4 Hz, 6H), 4.32 (d, J ) 14.6 Hz,
1H), 4.48-4.62 (m, 1H), 4.72-4.77 (m, 1H), 4.92 (d, J ) 14.1
Hz, 1H), 6.92-7.01 (m, 4H), 7.64-7.78 (m, 4H); ion spray MS
374 [M + NH4]+, 357 [M + H]+.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-a cetylp ip -
er a zin e-2-ca r boxa m id e (23). The methyl ester (1.3 g, 3.65
mmol) was mixed with NH2OH (21 mL, 28.8 mmol, 1.5 M in
methanol) and stirred overnight at room temperature. After
adjusting the pH to 5 with acetic acid, the crude product was
mixed with silica gel, and the solvent was evaporated. The dry
silica was poured on top of a flash silica gel column which was
subsequently eluted with EtOAc followed by a mixture of 20%
CH3OH/EtOAc to give 0.7 g (54% yield) of the desired product.
The 1H NMR spectrum shows two distinct rotamers: 1H NMR
(DMSO-d6) δ 1.39 (s, 3H), 2.18-2.26 (m, 1H), 2.28-2.37 (m,
1H), 2.60-2.68 (m, 1H), 2.85-2.91 (m, 1H), 3.03-3.12 (m, 1H),
3.77 (s, 3H), 3.79-4.01 (m, 1H), 6.93 (d, J ) 9.0 Hz, 2H), 7.72
(d, J ) 9.0 Hz, 2H), 8.92 (s, 1H), 10.65 (s, 1H); ion spray MS
375 [M + NH4]+, 358 [M + H]+. Anal. (C14H19N3O6S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-[(2-a m in o-
4-m et h yl-5-t h ia zolyl)su lfon yl]p ip er a zin e-2-ca r b oxa m -
id e (17). The title compound was prepared as a white solid
from amine hydrochloride 7 following the procedure described
1
for compound 15: yield 27%; H NMR (DMSO-d6) δ 2.03 (td,
J ) 13.6, 3.6 Hz, 1H), 2.22 (s, 3H), 2.19-2.30 (m, 1H), 3.25-
3.38 (m, 1H), 3.42-3.60 (m, 1H), 3.74-3.91 (m, 2H), 3.83 (s,
3H), 4.52 (s, 1H), 7.03 (d, J ) 9.0 Hz, 2H), 7.72 (d, J ) 9.0 Hz,
2H), 7.84 (s, 2H), 8.98 (s, 1H), 10.72 (s, 1H); ion spray MS 514
[M + Na]+, 492 [M + H)+. Anal. (C16H21N5O7S3) C, H, N.
N -H yd r oxy-1-(4-m e t h oxyp h e n yl)su lfon yl-4-[(3,5-d i-
m eth yl-4-isoxa zolyl)su lfon yl]p ip er a zin e-2-ca r boxa m id e
(18). The title compound was prepared as a white solid from
amine hydrochloride 7 following the sequence of reactions
described for compound 15: yield 47%; 1H NMR (DMSO-d6) δ
1.92-2.02 (m, 1H), 2.18 (s, 3H), 2.19-2.26 (m, 1H), 2.44 (s,
3H), 3.24-3.36 (m, 1H), 3.39-3.49 (m, 1H), 3.66-3.82 (m, 2H),
3.80 (s, 3H), 4.42-4.48 (m, 1H), 6.98 (d, J ) 9.0 Hz, 2H), 7.64
(d, J ) 9.0 Hz, 2H), 8.92 (s, 1H), 10.73 (s, 1H); ion spray MS
492 [M + NH4]+, 475 [M + H]+. Anal. (C17H22N4O8S2) C, H, N.
Met h yl 1-(4-Met h oxyp h en yl)su lfon yl-4-(ter t-b u t oxy-
ca r bon yl)p ip er a zin e-2-ca r boxyla te (19a ). To a solution of
acid 6 (2 g, 5.61 mmol) in 8 mL of DMF was added potassium
tert-butoxide (1 g, 8.4 mmol) at room temperature. After 30
min, iodomethane (0.42 mL, 6.73 mmol) was added at 0 °C,
and the resulting mixture was stirred at room temperature
overnight. The reaction mixture was quenched with water and
partitioned between EtOAc and H2O. The EtOAc layer was
washed with 1 N HCl, 1 N NaOH, H2O, and brine, dried
(MgSO4), filtered, and concentrated in vacuo. Column chro-
matography of the residue on silica gel using hexane/EtOAc
1
(4:1) provided the title compound as an oil: 1.25 g (54%); H
NMR (CDCl3) δ 1.39 (s, 9H), 2.77-2.98 (m, 1H), 3.03-3.19 (m,
1H), 3.31-3.50 (m, 1H), 3.53 (s, 3H), 3.56-3.68 (m, 1H), 3.86
(s, 3H), 3.92-4.21 (m, 1H), 4.39-4.53 (m, 1H), 4.53-4.61 (m,
1H), 6.96 (dd, J ) 9.0, 2.2 Hz, 2H), 7.70 (dd, J ) 9.1, 2.2 Hz,
2H); CI+ MS 432 [M + NH4]+, 415 [M + H]+, 376 [M + NH4 -
tBu]+, 359 [M + H - tBu]+, 315 [M + H - tBoc]+.
N-H yd r oxy-1-(4-m et h oxyp h en yl)su lfon yl-4-(ter t-b u t -
oxyca r bon yl)p ip er a zin e-2-ca r boxa m id e (19). The methyl
ester 19a (0.33 g, 0.8 mmol) was converted to title compound
following the procedure described for compound 15, providing
the desired product as a white foamy solid: yield 61%; 1H NMR
(CDCl3) δ 1.37 (s, 9H), 2.72-2.99 (m, 1H), 3.05 (dd, J ) 14.6,
3.9 Hz, 1H), 3.54-3.64 (m, 2H), 3.86 (s, 3H), 3.81-3.91 (m,
1H), 4.11-4.30 (m, 2H), 7.04 (dd, J ) 8.8, 1.2 Hz, 2H), 7.74
(dd, J ) 8.9, 1.2 Hz, 2H); ion spray MS 438 [M + Na]+, 433
[M + NH4]+, 416 [M + H]+; HRMS (M+) calcd for C17H26N3O7S
416.1491, found (M+) 416.1501.
Meth yl 1-(4-Meth oxyp h en yl)su lfon yl-4-cycloh exa n e-
ca r bon ylp ip er a zin e-2-ca r boxyla te (25a ). To a solution of
amine hydrochloride 7 (2 g, 5.7 mmol), triethylamine (2.4 mL,
17.6 mmol), and DMAP (70 mg, 0.57 mmol) in 4 mL of water
and 8 mL of p-dioxane was added cyclohexanecarbonyl chloride
(1.0 mL, 7.4 mmol) at 0 °C. The reaction was stirred overnight
at room temperature. The reaction mixture was concentrated
under reduced pressure and partitioned between EtOAc and
water. The EtOAc layer was washed with 1 N HCl, aqueous
NaHCO3, water, and brine, dried over MgSO4, and concen-
trated under reduced pressure to give 1.5 g (62%) of the desired
product: 1H NMR (CDCl3) δ 1.12-1.86 (m, 8H), 2.28-2.50 (m,
1H), 2.58-2.73 (m, 1H), 2.88-2.98 (m, 1H), 3.08-3.39 (m, 2H),
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-ben zyloxy-
ca r bon ylp ip er a zin e-2-ca r boxa m id e (20). The title com-
pound was prepared as an amorphous solid from amine
hydrochloride 7 following the procedure described for com-

376 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
3.42-3.58 (m, 3H), 3.60-3.76 (m, 1H), 3.89 (s, 3H), 4.36-4.47
(m, 1H), 4.48-4.77 (m, 2H), 4.92-5.01 (m, 1H), 6.96 (d, J )
9.0 Hz, 2H), 7.62-7.74 (m, 2H); CI+ MS 425 [M + H]+.
(m, 3H), 8.89 (s, 1H), 10.72 (s, 1H); ion spray MS 448 [M +
Na]+, 443 [M + NH4]+, 426 [M + H]+. Anal. (C17H19N3O6S2) C,
H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-fu r oylp ip -
er a zin e-2-ca r boxa m id e (31). The title compound was pre-
pared as a white solid from amine hydrochloride 7 following
the procedure described for compound 24: yield 36%; 1H NMR
(DMSO-d6) δ 2.92-4.15 (m, 6H), 3.83 (s, 3H), 4.24-4.36 (m,
1H), 6.57-6.64 (m, 1H), 6.91 (d, J ) 3.4 Hz, 1H), 7.06 (d, J )
9.0 Hz, 2H), 7.82 (s, 1H), 8.91 (s, 1H); ion spray MS 432 [M +
Na]+, 427 [M + NH4]+, 410 [M + H]+; HRMS (M+ + H) calcd
for C17H19N3O7S 410.1022, found (M+ + H) 410.1020.
N-H yd r oxy-1-(4-m et h oxyp h en yl)su lfon yl-4-cycloh ex-
a n eca r bon ylp ip er a zin e-2-ca r boxa m id e (25). The methyl
ester 25a (1.3 g, 3.0 mmol) was mixed with NH2OH (16.3 mL,
24.5 mmol, 1.7 M in methanol) and stirred overnight at room
temperature. After adjusting the pH to 5 with acetic acid, the
crude product was mixed with silica gel, and the solvent was
evaporated. The dry silica was poured on top of a flash silica
gel column which was subsequently eluted with 5% CH3OH/
EtOAc to give 0.58 g (46% yield) of the desired product as a
white solid: 1H NMR (DMSO-d6) δ 0.98-1.24 (m, 6H), 1.34-
1.64 (m, 4H), 2.26-2.59 (m, 2H), 2.88-3.14 (m, 2H), 3.21-
3.60 (m, 2H), 3.80 (s, 3H), 4.08-4.34 (m, 2H), 7.06 (d, J ) 9.0
Hz, 2H), 7.60-7.78 (m, 2H), 8.72-8.96 (m, 1H), 10.58-10.74
(m, 1H); ion spray MS 448 [M + Na]+, 443 [M + NH4]+, 426
[M + H]+. Anal. (C19H27N3O6S‚0.5H2O) C, H, N.
N-Hydr oxy-1-(4-m eth oxyph en yl)su lfon yl-4-(1-oxoh exyl)-
p ip er a zin e-2-ca r boxa m id e (24). The title compound was
prepared as a solid from amine hydrochloride 7 following the
procedure described for compound 25: yield 54%; 1H NMR
(DMSO-d6) δ 0.80 (t, 3H), 1.07-1.26 (m, 4H), 1.27-1.41 (m,
2H), 2.02-2.21 (m, 2H), 2.86-3.08 (m, 2H), 3.30-3.93 (m, 3H),
3.78 (s, 3H), 3.92-4.28 (m,3H), 7.04 (d, J ) 8.9 Hz, 2H), 7.68
(t, J ) 10.4 Hz, 2H); ion spray MS 436 [M + Na]+, 414 [M +
H]+; HRMS (M+ + H) calcd for C18H27N3O6S 414.1699, found
(M+ + H) 414.1690.
N-Hydr oxy-1-(4-br om oph en yl)su lfon yl-4(S)-(2-h ydr oxy-
3-m eth yl-1-oxobu tyl)p ip er a zin e-2-ca r boxa m id e (26). The
title compound was prepared as a white foaming solid from
amine hydrochloride 7 following the procedure described for
compound 25: yield 70%; 1H NMR (DMSO-d6) δ 0.63-0.84 (m,
6H), 2.57-2.72 (m, 1H), 2.83-3.04 (m, 2H), 3.10-3.21 (m, 1H),
3.41-3.62 (m, 2H), 3.83 (s, 3H), 3.88-4.06 (m, 2H), 4.18-4.28
(m, 2H), 4.33-4.64 (m, 2H), 7.08 (d, J ) 9.0 Hz, 2H), 7.62-
7.82 (m, 2H), 8.82 (s, 1H), 10.62 (s, 1H); ion spray MS 433 [M
+ NH4]+, 416 [M + H]+. Anal. (C17H25N3O7S) C, H, N.
N-H yd r oxy-1-(4-m et h oxyp h en yl)su lfon yl-4-p h en oxy-
a cetylp ip er a zin e-2-ca r boxa m id e (27). The title compound
was prepared as a white solid from amine hydrochloride 7
following the procedure described for compound 25: yield 40%;
1H NMR (DMSO-d6) δ 2.42-2.60 (m, 1H), 2.90-3.18 (m, 2H),
3.37-3.81 (m, 2H), 3.96 (s, 3H), 3.88-4.18 (m, 1H), 4.23-4.41
(m, 1H), 4.59-4.84 (m, 2H), 6.86 (d, J ) 7.9 Hz, 2H), 6.91 (t,
J ) 8.0 Hz, 2H), 7.12 (d, J ) 8.8 Hz, 2H), 7.23 (t, J ) 8.4 Hz,
2H), 7.66-7.82 (m, 2H); ion spray MS 467 [M + NH4]+, 450
[M + H]+. Anal. (C20H23N3O7S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(isoxa zole-
5-ca r bon yl)p ip er a zin e-2-ca r boxa m id e (32). The title com-
pound was prepared as a white solid from amine hydrochloride
7 following the procedure described for compound 24: yield
1
32%; H NMR (CD3OD) δ 3.59-3.76 (m, 2H), 3.82-4.01 (m,
6H), 4.08-4.80 (m, 2H), 4.78-4.98 (m, 2H), 7.01-7.21 (m, 4H),
7.72-7.86 (m, 2H); ion spray MS 428 [M + NH4]+, 411 [M +
H]+. Anal. (C16H18N4O7S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(4-m eth yl-
1,2,3-th ia d ia zole-5-ca r bon yl)p ip er a zin e-2-ca r boxa m id e
(33). The title compound was prepared as a white solid from
amine hydrochloride 7 following the procedure described for
compound 24: yield 42%; 1H NMR (DMSO-d6) δ 2.41-2.62 (m,
2H), 2.71-2.88 (m, 2H), 2.79 (s, 3H), 2.89 (s, 3H), 3.24-3.42
(m, 1H), 3.78-3.87 (m, 2H), 7.02-7.13 (m, 2H), 7.62-7.78 (m,
2H), 9.59 (s, 1H), 10.54 (s, 1H); ion spray MS 459 [M + NH4]+,
442 [M + H]+; HRMS (M+ + H) calcd for C16H20N5O6S2
442.0855, found (M+ + H) 442.0842.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(5-m eth yl-
3-p h en ylisoxa zole-4-ca r b on yl)p ip er a zin e-2-ca r b oxa m -
id e (34). The title compound was prepared as a white solid
from amine hydrochloride 7 following the procedure described
for compound 24: yield 47%; 1H NMR (DMSO-d6) δ 2.32-2.43
(m, 3H), 2.92-3.16 (m, 2H), 3.35-3.46 (m, 1H), 3.50-3.68 (m,
2H), 3.83 (s, 3H), 4.24-4.40 (m, 1H), 4.57-4.64 (m, 1H), 7.01-
7.13 (m, 2H), 7.40-7.62 (m, 5H), 7.72 (d, J ) 9.0 Hz, 2H),
8.80-8.97 (m, 1H), 10.58-10.80 (m, 1H); ion spray MS 518
[M + NH4]+, 501 [M + H]+; HRMS (M+ + H) calcd for
C
23H24N4O7S 501.1444, found (M+ + H) 501.1455.
N -H y d r o x y -1-(4-m e t h o x y p h e n y l)s u lfo n y l-4-(4-b i-
p h en ylca r bon yl)p ip er a zin e-2-ca r boxa m id e (35). The title
compound was prepared as a white solid from amine hydro-
chloride 7 following the procedure described for compound
24: yield 45%; 1H NMR (DMSO-d6) δ 2.78-4.58 (m, 7H), 3.87
(s, 3H), 7.09 (d, J ) 9.0 Hz, 2H), 7.29-7.41 (m, 3H), 7.50 (t, J
) 7.5 Hz, 2H), 7.64-7.78 (m, 6H), 8.92 (s, 1H); ion spray MS
518 [M + Na]+, 513 [M + NH4]+, 496 [M + H]+. Anal.
(C25H25N3O6S) C, H, N.
Meth yl 1-(4-Meth oxyp h en yl)su lfon yl-4-a m id op ip er a -
zin e-2-ca r boxyla te (36a ). To a solution of amine hydrochlo-
ride 7 (1.5 g, 4.3 mmol), triethylamine (1.8 mL, 12.7 mmol),
and DMAP (52 mg, 0.43 mmol) in 4 mL of water and 8 mL of
p-dioxane was added trimethylsilyl isocyanate (0.89 mL, 4.45
mmol) dropwise at room temperature. The reaction was stirred
overnight at room temperature. The reaction mixture was
concentrated under reduced pressure and partitioned between
EtOAc and water. The EtOAc layer was washed with 1 N HCl,
water, and brine, dried over MgSO4, and concentrated under
reduced pressure. The crude product was purified by column
chromatography (EtOAc) to give the title compound (0.33 g,
23%): 1H NMR (CDCl3) δ 2.88 (td, J ) 14.2, 4.5 Hz, 1H), 3.14-
3.26 (m, 2H), 3.58 (s, 3H), 3.63-3.72 (m, 1H), 3.88 (s, 3H),
3.98-4.06 (m, 1H), 4.24-4.33 (m, 1H), 4.64-4.79 (m, 3H), 6.98
(d, J ) 9.0 Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H); CI+ MS 358 [M
+ H]+.
N-Hydr oxy-1-(4-m eth oxyph en yl)su lfon yl-4-ben zoylpip-
er a zin e-2-ca r boxa m id e (28). The title compound was pre-
pared as a colorless solid from amine hydrochloride 7 following
the procedure described for compound 23: yield 73%; 1H NMR
(CDCl3) δ 3.05-3.80 (m, 6H), 3.90 (s, 3H), 4.60 (m, 1H), 7.01
(d, J ) 9.3 Hz, 2H), 7.40-7.46 (m, 5H), 7.79 (d, J ) 9.3 Hz,
2H); ion spray MS 420 [M + H]+. Anal. (C19H21N3O6S) C, H,
N.
N -H y d r o x y -1-(4-m e t h o x y p h e n y l)s u lfo n y l-4-n ic o -
tin oylp ip er a zin e-2-ca r boxa m id e Hyd r och lor id e (29). The
title compound was prepared as a solid from amine hydro-
chloride 7 following the procedure described for compound
24: yield 31%; 1H NMR (DMSO-d6) δ 2.19-3.45 (m, 6H), 3.89
(s, 3H), 3.96-4.04 (m, 1H), 7.04 (d, J ) 8.9 Hz, 2H), 7.39-
7.46 (m, 1H), 7.64 (d, J ) 8.9 Hz, 2H), 8.01-8.22 (m, 1H),
8.56-8.86 (m, 3H); ion spray MS 443 [M + Na]+, 421 [M +
H]+. Anal. (C18H20N4O6S) C, H, N.
N -H y d r o x y -1-(4-m e t h o x y p h e n y l)s u lfo n y l-4-t h i o -
p h en eca r bon ylp ip er a zin e-2-ca r boxa m id e (30). The title
compound was prepared as a white solid from amine hydro-
chloride 7 following the procedure described for compound
24: yield 30%; 1H NMR (DMSO-d6) δ 2.93-3.12 (m, 1H), 3.50-
3.68 (m, 2H), 3.80 (s, 3H), 3.97-4.07 (m, 1H), 4.12-4.18 (m,
3H), 6.98-7.10 (m, 3H), 7.26 (d, J ) 1.8 Hz, 1H), 7.64-7.72
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-a m id op ip -
er a zin e-2-ca r boxa m id e (36). The methyl ester 36 (0.2 g, 0.56
mmol) was mixed with NH2OK (3.2 mL, 4.5 mmol, 1.5 M in
methanol; solution prepared as described in Fieser & Fieser,
Vol. 1, p 478) and stirred overnight at room temperature. The
solvent was removed under reduced pressure. The mixture was
then dissolved in 1 mL of methanol, acidified with HCl (4 N

Piperazine-Based MMPIs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 377
in dioxane) to pH ∼6. The crude product was subsequently
eluted with 20% CH3OH/EtOAc to give 110 mg (55% yield) of
the title compound: 1H NMR (DMSO-d6) δ 2.66-2.76 (m, 1H),
2.79-2.98 (m, 1H), 3.17-3.21 (m, 1H), 3.44-3.56 (m, 1H),
3.65-3.76 (m, 1H), 3.94 (s, 3H), 4.02-4.08 (m, 1H), 4.19-4.22
(m, 1H), 5.94 (s, 2H), 7.08 (d, J ) 9.0 Hz, 2H), 7.74 (d, J ) 9.0
Hz, 2H), 8.62 (s, 1H), 10.34 (s, 1H); ion spray MS 397 [M +
NH4]+, 359 [M + H]+; HRMS (M+ + H) calcd for C13H18N4O6S
359.1025, found 359.1035.
N-Hydr oxy-1-(4-m eth oxyph en yl)su lfon yl-4-(n -h exylam i-
n oca r bon yl)p ip er a zin e-2-ca r boxa m id e (37). The title com-
pound was prepared as a white solid from amine hydrochloride
7 following the procedure described for compound 36: yield
26%; 1H NMR (CDCl3) δ 0.80-0.93 (m, 3H), 1.20-1.38 (m, 6H),
1.39-1.49 (m, 2H), 2.50 (td, J ) 12.5, 3.0 Hz, 1H), 2.71 (dd, J
) 11.8, 3.2 Hz, 1H), 3.02-3.21 (m, 3H), 3.72 (d, J ) 11.7 Hz,
1H), 3.86-3.96 (m, 1H), 3.89 (s, 3H), 4.22 (d, J ) 13.6 Hz,
1H), 4.53 (d, J ) 1.3 Hz, 1H), 5.17-5.23 (m, 1H), 7.00 (d, J )
8.8 Hz, 2H), 7.78 (d, J ) 8.9 Hz, 2H), 9.77 (br s, 1H); ion spray
MS 465 [M + Na]+, 443 [M + H]+. Anal. (C19H30N4O6S ) C, H,
N.
Meth yl 1-(4-Meth oxyp h en yl)su lfon yl-4-(N-m eth yl-N-
h exyla m in oca r bon yl)p ip er a zin e-2-ca r boxyla te (38a ). To
a solution of phosgene (20% in toluene, 17 mL, 34 mmol) in
dichloroethane (15 mL) at 0 °C was added amine hydrochloride
7 (3 g, 8.5 mmol) slowly. The reaction was stirred for 2 h at 45
°C and overnight at room temperature. The reaction mixture
was concentrated under reduced pressure, dissolved in 15 mL
of dichloroethane, and added to a solution of N-methylhexy-
lamine (2.6 mL, 17.1 mmol) and triethylamine (3.6 mL, 12.6
mmoL) in dichloroethane (15 mL) at 0 °C dropwise. The
resulting mixture was stirred overnight at room temperature.
The reaction was diluted by 1 N HCl and extracted with
EtOAc. The EtOAc layer was washed with 1 N HCl, water,
and brine, dried over MgSO4, and concentrated under reduced
pressure. The crude product was purified by column chroma-
tography (hexane/EtOAc, 1:2) to give the title compound (1.9
g, 49%): 1H NMR (CDCl3) δ 0.84 (t, J ) 6.9 Hz, 3H), 1.13-
1.28 (m, 6H), 1.39-1.48 (m, 2H), 2.72 (s, 3H), 2.81 (td, J )
12.6, 3.4 Hz, 1H), 2.98-3.18 (m, 3H), 3.40-3.50 (m, 2H), 3.54
(s, 3H), 3.59-3.66 (m, 1H), 3.84 (s, 3H), 3.92-3.98 (m, 1H),
4.59-4.61 (m, 1H), 6.92 (d, J ) 9.0 Hz, 2H), 7.67 (d, J ) 9.0
Hz, 2H); ion spray MS 473 [M + NH4]+, 456 [M + H]+.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(N-m eth yl-
N-h exyla m in oca r bon yl)p ip er a zin e-2-ca r boxa m id e (38).
The methyl ester (0.82 g, 1.8 mmol) was mixed with NH2OH
(10 mL, 14.4 mmol, 1.5 M in methanol) and stirred overnight
at room temperature. After adjusting the pH to 5 with acetic
acid, the crude product was mixed with silica gel, and the
solvent was evaporated. The dry silica was poured on top of a
flash silica gel column which was subsequently eluted with
EtOAc to 5% CH3OH/EtOAc to give 0.40 g (49% yield) of the
desired product: 1H NMR (DMSO-d6) δ 0.80 (t, J ) 6.9 Hz,
3H), 1.03-1.23 (m, 6H), 1.28-1.41 (m, 2H), 2.40-2.51 (m, 1H),
2.60 (s, 3H), 2.61-2.70 (m, 1H), 2.82-3.02 (m, 3H), 3.21-3.32
(m, 1H), 3.48-3.59 (m, 2H), 3.81 (s, 3H), 4.21-4.26 (m, 1H),
7.04 (d, J ) 9.0 Hz, 2H), 7.69 (d, J ) 9.0 Hz, 2H), 8.80 (s, 1H),
10.64 (s, 1H); ion spray MS 479 [M + Na]+, 457 [M + H]+.
Anal. (C20H32N4O6S‚0.5H2O) C, H, N.
benzyl isocyanate (0.19 mL, 1.57 mmol) dropwise at room
temperature. The reaction mixture was allowed to stir for 4 h
at room temperature, washed with water, dried over NaSO4,
and concentrated under reduced pressure. The residue was
mixed with NH2OH (3.4 mL, 5.9 mmol, 1.76 M in methanol)
and stirred for 3 h at room temperature. The reaction mixture
was neutralized with 1 N aqueous hydrochloric acid and
concentrated under reduced pressure. The crude product was
purified by flash chromatography (ethyl acetate to ethyl
acetate-ethanol 9/1 v/v) to give 220.5 mg (34% yield) of the
title compound as a colorless glass: 1H NMR (CD3OD) δ 2.92
(m, 1H), 3.10 (m, 1H), 3.61-4.22 (m, 4H), 3.88 (s, 3H), 4.28
(m, 2H), 4.36 (m, 1H), 7.12 (d, J ) 9.0 Hz, 2H), 7.25 (m, 5H),
7.82 (d, J ) 9.0 Hz, 2H); ion spray MS 449 [M + H]+. Anal.
(C20H24N4O6S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-ben zylth io-
ca r ba m oylp ip er a zin e-2-ca r boxa m id e (41). The title com-
pound was prepared as a white solid from amine hydrochloride
7 following the procedure described for compound 40: yield
14%; 1H NMR (CD3OD) δ 3.17-4.82 (m, 9H), 3.92 (s, 3H), 7.11
(d, J ) 9.5 Hz, 2H), 7.24-7.36 (m, 5H), 7.84 (d, J ) 9.5 Hz,
2H); ion spray MS 465 [M + H]+; Anal. (C20H24N4O5S2‚
0.25EtOAc) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(N-m eth yl-
N-p h en ylm eth yla m in oca r bon yl)p ip er a zin e-2-ca r boxa m -
id e (42). The title compound was prepared from piperazine-
2-carboxylic acid dihydrochloride (4) following the procedure
described for compound 38: yield 40%; 1H NMR (DMSO-d6) δ
2.56 (s, 3H), 2.72-2.81 (m, 1H), 3.21-3.41 (m, 3H), 3.46-3.65
(m, 2H), 3.81 (s, 3H), 4.08-4.32 (m, 3H), 7.06 (d, J ) 9.0 Hz,
2H), 7.11-7.18 (m, 2H), 7.20-7.31 (m, 3H), 7.67 (d, J ) 9.0
Hz, 2H), 8.80 (s, 1H), 10.66 (s, 1H); ion spray MS 463 [M +
H]+. Anal. (C21H26N4O6S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(2-p h en yl-
eth yla m in oca r bon yl)p ip er a zin e-2-ca r boxa m id e (43). The
title compound was prepared from piperazine-2-carboxylic acid
dihydrochloride (4) following the procedure described for
1
compound 38: yield 36%; H NMR (CDCl3) δ 2.44-2.58 (m,
1H), 2.70 (dd, J ) 13.8, 4.0 Hz, 1H), 2.97 (t, J ) 5.6 Hz, 2H),
3.01-3.14 (m, 1H), 3.32-3.44 (m, 2H), 3.70 (d, J ) 13.0 Hz,
1H), 3.82-3.92 (m, 1H), 3.90 (s, 3H), 4.18 (d, J ) 12.8 Hz,
1H), 4.52 (br s, 1H), 5.12-5.23 (m, 1H), 7.01 (dd, J ) 9.0, 2.0
Hz, 2H), 7.13-7.32 (m, 5H), 7.78 (dd, J ) 9.0, 2.0 Hz, 2H); ion
spray MS 485 [M + Na]+, 463 [M + H]+. Anal. (C21H26N4O6S)
C, H, N.
N-H yd r oxy-1-(4-m et h oxyp h en yl)su lfon yl-4-(3-m et h -
oxyph en ylam in ocar bon yl)piper azin e-2-car boxam ide (44).
The title compound was prepared as a white solid from
piperazine-2-carboxylic acid dihydrochloride (4) following the
procedure described for compound 38: yield 57%; 1H NMR
(DMSO-d6) δ 2.84-2.92 (m, 1H), 2.98 (dd, J ) 14.2, 3.5 Hz,
1H), 3.51-3.63 (m, 2H), 3.71 (s, 3H), 3.86 (s, 3H), 3.82-3.91
(m, 1H), 4.13 (dd, J ) 13.2, 1.8 Hz, 1H), 4.24-4.30 (m, 1H),
6.52 (dd, J ) 9.1, 2.5 Hz, 1H), 6.98 (dd, J ) 9.0, 2.0 Hz, 2H),
7.02-7.13 (m, 4H), 7.78 (d, J ) 9.0 Hz, 2H), 8.43 (s, 1H), 8.89
(s, 1H), 10.77 (s, 1H); ion spray MS 482 [M + Na]+, 465 [M +
H]+. Anal. (C20H24N4O7S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(N-p r op yl-
N-cyclop r op ylm eth yl a m in oca r bon yl)p ip er a zin e-2-ca r -
boxa m id e (45). The title compound was prepared as a white
solid from piperazine-2-carboxylic acid dihydrochloride (4)
following the procedure described for compound 38: yield 43%;
1H NMR (DMSO-d6) δ 0.04-0.13 (m, 2H), 0.38-0. 42 (m, 2H),
0.78 (t, J ) 7.8 Hz, 3H), 0.80-0.90 (m, 1H), 1.36-1.44 (m, 2H),
2.48-2.58 (m, 1H), 2.68-2.76 (dd, J ) 14.1, 4.1 Hz, 1H), 2.83-
2.92 (m, 2H), 2.99-3.12 (m, 2H), 3.22-3.36 (m, 2H), 3.56-
3.62 (m, 2H), 3.86 (s, 3H), 4.23-4.30 (m, 1H), 7.08 (d, J ) 9.0
Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H), 8.80 (s, 1H), 10.66 (s, 1H);
ion spray MS 455 [M + H]+. Anal. (C20H30N4O6S‚0.25H2O) C,
H, N.
N-Hyd r oxy-1-(4-br om op h en yl)su lfon yl-4-(N-m eth yl-N-
h exyla m in oca r bon yl)pip er a zin e-2-ca r boxa m id e (39). The
title compound was prepared as a white solid from piperazine-
2-carboxylic acid dihydrochloride 4 following the procedure
described for compound 38: yield 51%; 1H NMR (DMSO-d6) δ
0.80 (t, J ) 7.0 Hz, 3H), 1.04-1.23 (m, 7H), 1.27-1.41 (m, 2H),
2.61 (s, 3H), 2.68-2.81 (m, 1H), 2.82-3.02 (m, 2H), 3.25-3.32
(m, 1H), 3.48-3.60 (m, 3H), 4.21-4.27 (m, 1H), 7.64 (d, J )
8.6 Hz, 2H), 7.74 (d, J ) 8.6 Hz, 2H), 8.78 (s, 1H), 10.62 (s,
1H); ion spray MS 507, 505 [M + H]+. Anal. (C19H29N4O5SBr)
C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-ben zylca r -
ba m oylp ip er a zin e-2-ca r boxa m id e (40). To a solution of
amine hydrochloride 7 (500 mg, 1.43 mmol) and triethylamine
(0.4 mL, 2.86 mmol) in 10 mL of dichloromethane was added
N-Hydr oxy-1-(4-m eth oxyph en yl)su lfon yl-4-[(h exah ydr o-
1H-a zep in -1-yl)ca r bon yl]p ip er a zin e-2-ca r boxa m id e (46).
The title compound was prepared as a white solid from
piperazine-2-carboxylic acid dihydrochloride (4) following the

378 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
procedure described for compound 38: yield 62%; 1H NMR
(DMSO-d6) δ 1.31-1.42 (m, 4H), 1.45-1.60 (m, 4H), 2.36-2.48
(m, 1H), 2.58-2.67 (m, 1H), 3.12 (t, J ) 5.5 Hz, 4H), 3.28-
3.30 (m, 1H), 3.44-3.57 (m, 3H), 3.80 (s, 3H), 4.20-4.23 (m,
1H), 7.05 (d, J ) 9.0 Hz, 2H), 7.67 (d, J ) 9.0 Hz, 2H), 8.80 (s,
1H), 10.64 (s, 1H); ion spray MS 441 [M + H]+. Anal.
(C19H28N4O6S) C, H, N.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(4-m or p h o-
lin ylca r bon yl)p ip er a zin e-2-ca r boxa m id e (47). The title
compound was prepared from piperazine-2-carboxylic acid
dihydrochloride (4) following the procedure described for
compound 38: yield 87%; 1H NMR (DMSO-d6) δ 2.50-2.61 (m,
1H), 2.79 (dd, J ) 13.5, 4.4 Hz, 1H), 2.90-3.06 (m, 6H), 3.28-
3.64 (m, 6H), 3.79 (s, 3H), 4.22 (t, J ) 3.5 Hz, 1H), 7.03 (d, J
) 8.9 Hz, 2H), 7.68 (d, J ) 8.9 Hz, 2H), 8.81 (s, 1H), 10.66 (s,
1H); ion spray MS 451 [M + Na]+, 429 [M + H]+; HRMS (M+
+ H) calcd for C17H24N4O7S 429.1444, found (M+ + H)
429.1446.
use. Cartilage degradation assays were performed in 96-well
microtiter plates with each well containing 2 cartilage disks
in 200 mL of DMEM media supplemented with ascorbic acid
and antibiotics as described above but without ITS and 50 ng/
mL interleukin-1-R (IL-1-R; Genzyme Inc., Cambridge, MA).
Unstimulated control cultures were treated identically except
IL-1 was omitted. MMPIs were evaluated in cultures contain-
ing DMEM with 1 mM, 500 nM, 250 nM, 125 nM, or 62.5 nM
of test compound. Supernatants were removed from the plates
at 7-day intervals and stored at -70 °C until analysis for
collagen degradation. Fresh media with or without inhibitor
was added to the plates and the cultures continued for 21 days.
Collagen degradation was measured by the release of
hydroxyproline into the culture media as previously described
(Ellis, A. J .; et al. Biochem. Biophys. Res. Commun. 1994, 201,
94-101). Collagen degradation was measured in 8 replicate
cartilage cultures, and the data are expressed as the mean (
SD.
N-Hyd r oxy-1-(4-m eth oxyp h en yl)su lfon yl-4-(4N-m eth -
ylpiper azin e-1N-car bon yl)piper azin e-2-car boxam ide Hy-
d r och lor id e (48). The title compound was prepared from
piperazine-2-carboxylic acid dihydrochloride (4) following the
procedure described for compound 38. The hydrochloride
product was recrystallized from from EtOAc/methanol to give
X-r a y Cr yst a llogr a p h y w it h Tr u n ca t ed MMP -3. A
truncated form of MMP-3 containing the catalytic domain with
residues 83-255 was crystallized as described.15 1 mM com-
pound 20 was added to the crystal hanging drop for 3 h. Data
were then collected on a Mar-Research 180-mm image plate
detector system. These data were processed using the HKL
program. The structure was solved and refined to 2.4 Å using
XPLOR. The MMP-3-20 complex structure has been deposited
with the Protein Data Bank (PDB code 1D8F).
1
a white solid: yield 40%; H NMR (DMSO-d6) δ 2.03 (s, 3H),
2.08-2.20 (m, 5H), 2.19-2.50 (m, 1H), 2.63 (dd, J ) 14.1, 4.2
Hz, 1H), 2.94-3.08 (m, 5H), 3.18-3.42 (m, 2H), 3.50-3.64 (m,
2H), 3.79 (s, 3H), 4.07-4.13 (m, 1H), 7.01 (d, J ) 9.0 Hz, 2H),
7.66 (d, J ) 9.0 Hz, 2H); ion spray MS 480 [M + K]+, 442 [M
+ H]+; HRMS (M+ + H) calcd for C18H28N5O6S 442.1760, found
(M+ + H) 442.1752.
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
Tr u n ca ted MMP -1. Briefly, the DNA sequence coding for Val-
82-Pro-249 of proMMP-1 was amplified by polymerase chain
reaction from a commercially available plasmid, p35-1 (ATCC,
Rockville, MD; Templeton, N. S.; et al. Cancer Res. 1990, 50,
5431-5437) encoding human interstitial MMP-1. The PCR
fragment was ligated into the expression vector, pET-11a
(Novagen, Madison, WI), and expressed in E. coli BL21(DE3)
cells. The protein was solubilized from inclusion bodies in 6
M urea, 0.15 M NaCl (pH 7.5), refolded in 50 mM Tris-HCl
(pH 7.5), 10 mM CaCl2, 0.1 mM zinc acetate, then purified to
homogeneity over a hydroxamic acid inhibitor affinity column
as previously described (Moore, W. A.; Spilburg, C. A. Bio-
chemistry 1986, 25, 5189-5195). N-Terminal sequence analy-
sis confirmed the presence of three N-terminal sequences of
Val-Leu-Thr-Glu-Gly-Asn, Met-Val-Leu-Thr-Glu-Gly-Asn, and
Leu-Thr-Glu-Gly-Asn (minor).
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
p r oMMP -2. A partial cDNA clone for MMP-2 known as K-121
(Tryggvason, K. J . Biol. Chem. 1990, 265, 11077-11082) was
obtained from ATCC and subcloned into the pBlueScript SK-
(pBS) plasmid. Sanger dideoxy sequencing revealed that the
first 134 bp of the coding sequence were missing from the 5′
end of K-121. To restore the missing sequence, two overlapping
90+-mer oligonucleotides were designed and synthesized.
These, along with a 3′ antisense oligonucleotide, were used as
primers to the K-121 template in a series of polymerase chain
reaction (PCR) experiments to synthesize a full-length MMP-2
cDNA. The PCR product was then subcloned into pBS. To
express MMP-2 in the mammalian CHO D- cell system, it was
first subcloned into the mammalian expression vector pJ T1
(J . Ting, CRD), which contains the DHFR gene. Recombinant
MMP-2/pJ T1 was Polybrene transfected into CHO D- cells.
Clonal cell populations were isolated and screened for produc-
tion of MMP-2 mRNA using specific oligonucleotide primers
and reverse transcription PCR. Ten clones producing the
highest levels of MMP-2 mRNA were selected, and the DHFR/
MMP-2 construct was amplified by gradually increasing media
methotrexate (MTX, a DHFR inhibitor) concentration. Clonal
selection was further narrowed after assessing MMP bioac-
tivity in an MMP fluorescence assay (M. Anastasio, CRD) and
on zymogram gels. Those clones showing activity were tested
for MMP-2 protein production by sequential Edman degrada-
tion protein sequencing (F. Wang, P&GP Cell & Molecular
Biology Core) and Western blot. Based on all results, one clone
was expanded for growth in roller bottles. Approximately 9 L
of serum-free conditioned media with an MMP-2 concentration
N-Hydr oxy-1-(4-br om oph en yl)su lfon yl-4-[N-bis(2-m eth -
oxyeth yl)a m in oca r bon yl]p ip er a zin e-2-ca r boxa m ide (49).
The title compound was prepared from piperazine-2-carboxylic
acid dihydrochloride (4) following the procedure described for
1
compound 38: yield 45%; H NMR (CDCl3) δ 3.01-3.19 (m,
3H), 3.36 (s, 6H), 3.38-3.42 (m, 4H), 3.44-3.57 (m, 5H), 3.59-
3.68 (m, 2H), 4.17-4.22 (m, 1H), 4.66-4.71 (m, 1H), 7.60-
7.67 (m, 3H), 7.79 (d, J ) 9.0 Hz, 2H); ion spray MS 525, 523
[M + H]+. Anal. (C18H27N4O7SBr) C, H, N.
In Vitr o In testin a l P er m ea bility Stu d ies. (Performed as
described in: Dowty, M. E.; Dietsch, C. R. Pharm. Res. 1997,
14, 1792-1797.) Briefly, a portion of rat ileum tissue was
freshly excised and mounted in an in vitro diffusion chamber
system (NaviCyte, San Diego, CA). The amount of drug
transported across the tissue with time at various donor
concentrations of drug was determined at 37 °C and pH 7.4.
Permeability coefficients, kp (cm/min) were calculated at steady
state with the following equation from Fick’s laws of diffusion:
kp ) [S]/[ACo]
where S is the slope of the linear (steady state) portion of the
cumulative amount transported through the membrane versus
time plot in µg/min, A is the cross-sectional area of the tissue
surface exposed to solute transport in cm2 (0.636 cm2), and Co
is the initial concentration of solute in the donor chamber in
µg/mL (20-90 µg/mL). A predicted percent absorption, Abs,
is obtained from the ratio of the kp value for the test compound
divided by the kp of the mannitol internal standard (kp ratio)
from the following equation:
Abs ) [100]/[1 + exp{-7.42(kp ratio - 0.812)}]
Ca r tila ge Exp la n t Cu ltu r es. Bovine nasal cartilage ob-
tained the same day from a local abattoir was sliced and placed
in F-12 media containing ascorbic acid (50 mg/mL) and 2%
penicillin-streptomycin-fungizone. Cartilage disks were pre-
pared (7-12 mg wet weight) using a sterile belt punch.
Cartilage explants were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing glutamine (2 mM), ascor-
bic acid (50 mg/mL), 2% penicillin-streptomycin-fungizone,
and 1% ITS universal culture supplement (Collaborative
Biomedical Products, Bedford, MA) for at least 3 days before

Piperazine-Based MMPIs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 379
of ∼35 mg/L were generated. proMMP-2 was purified from
conditioned media as previously described (Crabbe, T.; et al.
Eur. J . Biochem. 1993, 218, 431-438) with the following
modifications. Conditioned serum-free medium was reduced
in volume with an Amicon S1Y30 spiral-wound cartridge prior
to chromatography on a gelatin-Sepharose 4B column equili-
brated in 25 mM Tris/HCl, 30 mM NaCl, 10 mM CaCl2, pH
7.5 (TNC buffer). The column was washed with equilibration
buffer before elution of the bound protein by TNC buffer
containing 1 M NaCl followed by 1 M NaCl and 10% (by vol)
dimethyl sulfoxide (DMSO) in TNC buffer. proMMP-2 fractions
were concentrated in an Amicon ultrafiltration cell with a
YM30 membrane and diafiltered against 25 mM MES/NaOH,
30 mM NaCl, 10 mM CaCl2, pH 6.0 (MNC buffer). The
concentrate was then chromatographed over a second gelatin-
Sepharose 4B column equilibrated in MNC buffer, washed
with equilibration buffer to remove nonbinding proteins, and
eluted with MNC buffer containing 0.3 M NaCl and 10% (by
vol) DMSO. Elution fractions containing progelatinase A were
pooled, diluted, and loaded onto an S-Sepharose Fast Flow
column equilibrated in MNC buffer and eluted with MNC
buffer containing 0.3 M NaCl. The concentrated fractions of
purified proMMP-2 were combined and stored in MNC buffer
containing 0.3 M NaCl at -70 °C. N-Terminal sequence, amino
acid, and mass spectrophotometric analyses confirmed the
identity of the purified protein with the expected sequence for
progelatinase A.
age of control activity in the absence of inhibitor. Inhibitor
concentrations were run in triplicate, and IC50 determinations
were calculated from a 4-parameter logistic fit of the data
within a single experiment.
Gela tin a se A (MMP -2), Ma tr ilysin (MMP -7), Neu tr o-
p h il Colla gen a se (MMP -8), Gela tin a se B (MMP -9), a n d
Colla gen a se 3 (MMP -13) In h ibition Assa ys. Inhibition of
all enzymes was measured according to the representative
procedure described below for MMP-2. proMMP-2 was acti-
vated prior to assay by treatment with 1 mM p-aminophe-
nylmercuric acetate for 45 min at 37 °C. proMMP-9 was
activated with MMP-3 (1:20 MMP-3:MMP-9) and stored at -80
°C until use. MMPs 7, 8, and 13 were all supplied as active
enzymes and stored frozen until use. MMP activity was
monitored using a fluorescence assay previously described,7
modified for a microtiter plate format. In a Dynatech MicroF-
LUOR plate, active enzyme was incubated with 8 µM Mca-
Pro-Leu-Dpa-Ala-Arg-NH2 in 50 mM Tris-HCl (pH 7.5), 10 mM
CaCl2, 0.15 M NaCl, 0.05% Brij for 20-30 min at 37 °C in the
presence of varying concentrations of inhibitor. The reaction
was then quenched with 50 mM EDTA, and the relative
fluorescence was monitored on a Perkin-Elmer LS50B spec-
trofluorimeter (λex 328 nm, λem 393 nm) fitted with a microplate
reader attachment. Activity was measured as a percentage of
control activity in the absence of inhibitor. Inhibitor concentra-
tions were run in triplicate, and IC50 determinations were
calculated from a 4-parameter logistic fit of the data within a
single experiment.
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
Tr u n ca ted MMP -3. Briefly, the DNA sequence coding for Ala-
Ack n ow led gm en t. A number of dedicated scientists
contributed toward the success of this project. The
following is a partial list: Lisa Williams, Richard Bohne,
Sean Peng, Tim Baker, Vikram Patel, J ohn McIver, and
Randy Matthews.
1-Thr-255 of proMMP-3 was amplified by PCR from
a
recombinant plasmid encoding human synovial preproMMP-3
kindly provided by Dr. Hideaki Nagase (University of Kansas
Medical Center, Kansas City, KS) and Dr. Markku Kurkinen
(Department of Medicine, UMDNJ -Robert Wood J ohnson
Medical School). After DNA sequence verification, this DNA
fragment was ligated into the expression vector, pET-3d
(Novagen, Madison, WI), and expressed in E. coli BL21 (DE3)
cells as previously described (Marcy, A. I.; et al. Biochemistry
1991, 30, 6476-6483). proMMP-3 was solubilized form inclu-
sion bodies in 8 M guanidine-HCl, refolded in 100 mM Tris-
HCl (pH 7.5), 10 mM CaCl2, 0.5 mM zinc acetate, and activated
overnight at 37 °C with 1.5 mM p-aminophenylmercuric
acetate. Active, truncated MMP-3 was purified to homogeneity
over a hydroxamic acid inhibitor affinity column as described
(Moore, W. A.; Spilburg, C. A. Biochemistry 1986, 25, 5189-
5195). N-Terminal sequence analysis confirmed the sequence
to be consistent with the catalytic domain, Phe-83-Thr-255,
of proMMP-3.
Refer en ces
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Colla gen a se (MMP -1) In h ibition Assa y. proMMP-1 was
activated prior to assay by treatment with trypsin. MMP-1
activity was monitored using a fluorescence assay previously
described.7 In a Dynatech MicroFLUOR plate, 10 nM activated
collagenase was incubated with 8 µM Mca-Pro-Leu-Gly-Leu-
Dpa-Ala-Arg-NH2 in 50 mM Tris-HCl, 10 mM CaCl2, 0.15 M
NaCl, 0.05% Brij for 20-30 min at 37 °C in the presence of
varying concentrations of inhibitor. The reaction was then
quenched with 50 mM EDTA and the fluorescence increase
monitored on a Perkin-Elmer LS50B spectrofluorimeter (λex
328 nm, λem 393 nm). Activity was measured as a percentage
of control activity in the absence of inhibitor. Inhibitor
concentrations were run in triplicate, and IC50 determinations
were calculated from a 4-parameter logistic fit of the data
within a single experiment.
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Str om elysin (MMP -3) In h ibition Assa y. proMMP-3 was
activated prior to assay by treatment with p-aminophenyl-
mercuric acetate or trypsin. MMP-3 activity was measured by
3
following the degradation of H-reduced, carboxymethylated
transferrin.16 In a Multiscreen DP filtration plate (Millipore),
50 ng of activated MMP-3, 30 µg of [3H]transferrin, and
varying concentrations of inhibitor were incubated in a buffer
of 50 mM Tris-HCl, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij
(pH 7.5) at 37 °C for 3 h. The reaction was quenched by
addition of 4.4% TCA, and TCA-soluble fragments were
counted for radioactivity. Activity was measured as a percent-
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G.; Taiwo, Y. O.; Gu, F.; Williams, L. E.; Hynd, B. A.; J anusz,
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MMP Inhibitors. J . Med. Chem. 1999, 42, 4547-4562.

380 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Cheng et al.
(6) (a) MacPherson, L. J .; Bayburt, E. K.; Capparelli, M. P.; Carroll,
B. J .; Goldstein, R.; J ustice, M. R.; Ziu, L. J .; Hu, S.-i.; Melton,
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Orally Active Stromelysin Inhibitor That Blocks Cartilage
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MacPherson, L. J .; Parker, D. T. Arylsulfonamido-substituted
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C.; O’Byrne, E. M.; Goldberg, R. L.; Ganu, V. S. CGS27023A: A
Novel Potent, and Orally Active Matrix Metalloprotease Inhibi-
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Type II Collagen, But Not Proteoglycan, Correlates with Matrix
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(13) See Babine and Bender review (p 1436), ref 14.
(14) (a) Hibner, B.; Card, A.; Flynn, C.; Housley, T.; Wihelm, S.;
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Angiogenic Properties. American Association for Cancer Re-
search, 89th Annual Meeting, New Orleans, 1998; Poster 2063.
(b) See the following for BB-2516 and other similar MMPIs:
White, A. D.; Bocan, T. M. A.; Boxer, P. A.; Peterson, J . T.;
Schrier, D. Emerging Therapeutic Advances for the Development
of Second generation Matrix Metalloproteinase Inhibitors. Curr.
Pharm. Des. 1997, 3, 45-58.
(15) Chen, L.; Rydel, T. J .; Gu, F.; Dunaway, C. M.; Pikul, S.;
Dunham, K. M.; Barnett, B. L. Crystal Structure of the Strome-
lysin Catalytic Domain at 2 Å Resolution: Inhibitor-induced
Conformational Changes. J . Mol. Biol. 1999, in press.
(16) Okada, Y.; Nagase, H.; Harris, E. D. A Metalloproteinase from
Human Rheumatoid Synovial Fibroblasts that Digests Connec-
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(7) (a) Knight, C. G.; Wilenbrock, F.; Murphy, G. A. A Novel
Coumarin-labeled Peptide for Sensitive Continuous Assays of
the Matrix Metalloproteinases. FEBS Lett. 1992, 296, 263-266.
(b) Also see Experimental Section for further details.
(8) See Experimental Section for details of the in vitro intestinal
permeability studies.
(9) See Experimental Section for details of the cartilage explant
assay.
(10) Peng, S. X.; VonBargen, E. C.; Bornes, D. M.; Pikul, S. Perme-
ability of Articular Cartilage to Matrix Metalloprotease Inhibi-
tors. Pharm. Res. 1998, 15, 1414-1418. Additional note: It is
understood that the physicochemical properties of bovine nasal
cartilage and articular cartilage are different. This work is cited
in order to convey a sense that small MMPIs are capable of
penetrating cartilage matrix.
J M990366Q