Investigating chelating sulfonamides and their use in metalloproteinase inhibitors

Article abstract of DOI:10.1039/c2dt12373h

Matrix metalloproteinase inhibitors (MMPi) utilize zinc-binding groups (ZBGs) to chelate the catalytic Zn(ii) ion resulting in enzyme inhibition. Adapting findings from the literature of Zn(ii) ion sensors, we previously reported chelating sulfonamide inh

Full text of DOI:10.1039/c2dt12373h

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PAPER
Investigating chelating sulfonamides and their use in metalloproteinase
inhibitors†
Alisa Tanakit, Matthieu Rouffet, David P. Martin and Seth M. Cohen*
Received 8th December 2011, Accepted 10th February 2012
DOI: 10.1039/c2dt12373h
Matrix metalloproteinase inhibitors (MMPi) utilize zinc-binding groups (ZBGs) to chelate the catalytic
Zn(II) ion resulting in enzyme inhibition. Adapting findings from the literature of Zn(II) ion sensors, we
previously reported chelating sulfonamide inhibitors of MMP-2, some of which showed excellent
selectivity over other gelatinases (MMP-9). Herein, we greatly expand our investigation of chelating
sulfonamides as MMP inhibitors (MMPi) with the synthesis and screening of several new libraries
consisting of 2-phenyl-7-sulfonamidobenzimidazole, 2-phenyl-7-sulfonamidobenzoxazole, 7-
sulfonamidobenzimidazole, 7-sulfonamidobenzoxazole, and 2-(2-sulfonamidophenyl)-quinoline ZBG
derivatives. A novel microwave irradiation synthetic procedure was utilized to rapidly and efficiently
prepare these molecules. To better understand the coordination chemistry underlying these ZBGs, crystal
structures of representative molecules with several first row transition metals were determined and
differences in coordination preferences were considered. Surprisingly, only compounds with the 2-phenyl-
7-sulfonamidobenzimidazole ZBG showed inhibition of MMP-2, suggesting that the specific structure of
the ZBG can have a pronounced effect of inhibitory activity.
interest from this study was the observation that some sulfona-
mides displayed selectivity for MMP-2 over MMP-9, an unusual
finding because these MMPs both belong to the gelatinase sub-
Introduction
Matrix metalloproteinases (MMPs) comprise a family of highly
homologous Zn(II)-dependent endopeptidases involved in many
important physiological processes, specifically the cleavage of
extracellular proteins.1–3 MMPs are able to degrade proteins
from the extracellular matrix (ECM) and the overexpression and
misregulation of MMPs have been associated with a variety of
pathologies including cardiovascular disease, arthritis, and
inflammation.4–7 The role of MMPs in these diseases has made
them therapeutic targets and for over three decades, various
research groups have developed small molecules that show selec-
tive MMP inhibition.8,9 Most MMPi employ a zinc-binding
group (ZBG) to bind the active site metal ion.10,11 The vast
majority of MMPi use a hydroxamic acid as the ZBG, which is a
strong chelator, but is limited by poor pharmacokinetics, low
oral bioavailability, and inadequate selectivity for zinc.12
class of MMPs. Moreover, when comparing 8-sulfonamidoqui-
noline and 2-sulfonamidophenylbenzimidazole derivatives
(Fig. 1), the 2-sulfonamidophenylbenzimidazole compounds
were more selective, which again clearly demonstrates the role of
the ZBG in the selectivity of the inhibitors.
Herein, we further explore the effect of the ZBG on the
activity and selectivity of chelating sulfonamide-based MMPi.
Five related sulfonamide libraries based on 7-sulfonamidoben-
zoxazole (ZBG1), 2-phenyl-7-sulfonamidobenzoxazole (ZBG2),
7-sulfonamidobenzimidazole (ZBG3), 2-phenyl-7-sulfonamido-
benzimidazole (ZBG4), and 2-(2-sulfonamidophenyl)-quinoline
(ZBG5) were synthesized. The coordination chemistry of these
In an effort to identify new scaffolds that could act as strong
and potentially more selective ZBGs, we reported that chelators
found in Zn(II) sensors, such as 8-sulfonamidoquinoline13,14 and
2-sulfonamidophenyl-benzimidazole (Fig. 1),15–17 could be used
effectively as ZBGs for MMPi development.18 Of particular
Department of Chemistry and Biochemistry, University of California,
San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
E-mail: scohen@ucsd.edu; Fax: (+858) 822-5598; Tel: (+858) 822-
5596
†Electronic supplementary information (ESI) available. CCDC
840783–840794. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c2dt12373h
Fig. 1 Structure and IC50 values of select chelating 2-sulfonamidophe-
nylbenzimidazole (BISL-1, left) and 8-sulfonamidoquinoline (QSL-1,
middle) inhibitors. The BISL-1 inhibitor displays better overall selectiv-
ity for MMP-2 versus other MMPs. The proposed mode of binding for
these chelating sulfonamide inhibitors is shown on the right.
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6507–6515 | 6507

7-Sulfonamidobenzoxazole (ZBG1)
ligands was explored and the libraries were screened against
MMP-2 and MMP-9. Overall, we find that even these relatively
small changes to the ZBG have a pronounced effect on the
inhibitory ability of these compounds, with the majority of com-
pounds being far less effective than the chelating sulfonamides
we previously identified. We have examined the coordination
chemistry of these new ligands to gain insight into their metal-
binding behavior to facilitate further studies on their use in
metalloenzyme inhibitors.
To a solution of 4-aminobenzoxazole (50 mg, 0.37 mmol) in
pyridine (1 mL) was added 1.5 eq (0.56 mmol) of sulfonyl
chloride. The transparent solution was heated in the microwave
at 130 °C for 3 min (Power = 300 W). The solution was then
poured into 5 mL of water and the precipitate was filtered off,
rinsed with water, and dried under vacuum. If no precipitate
formed, the aqueous phase was extracted twice with chloroform,
the combined organic layers were washed with a solution of HCl
(1 M), dried with MgSO4, filtered, and evaporated under reduced
pressure. The crude product was either purified by column of
chromatography or recrystallized from an appropriate solvent.
Experimental methods
General
2-Phenyl-7-nitrobenzoxazole (3)
Starting materials and solvents were purchased from commercial
suppliers (Sigma-Aldrich, Alfa Aesar, Fisher, etc.) and used as
received. Microwave synthesis reactions were performed in
10 mL or 35 mL microwave vials using a CEM Discover S
reactor. UV-visible spectra were collected on a Perkin Elmer
Lambda 25 spectrophotometer in DMSO with 0.1% triethyl-
amine. Column chromatography was performed using a Tele-
dyne ISCO Combiflash system with prepacked silica cartridges.
1H/13C NMR spectra were recorded at ambient temperature on a
400 Varian FT-NMR instrument located in the Department of
Chemistry and Biochemistry at the University of California,
San Diego. Mass spectra were obtained at the Molecular Mass
Spectrometry Facility in the Department of Chemistry and Bio-
chemistry at the University of California, San Diego.
To a solution of 2-amino-4-nitrophenol (1) (1.0 g, 6.48 mmol) in
trimethylorthobenzoate (3.4 mL, 19.5 mmol) and a catalytic
amount of p-toluenesulfonic acid (61 mg, 0.32 mmol). The sol-
ution was then heated in a microwave reactor at 130 °C for
15 min. Toluene (10 mL) was added to the reaction mixture and
the precipitate was vacuum filtered and washed with toluene
1
(1.35 g, 87%). H NMR (CDCl3, 500 MHz) δ 7.49 (dt, J1 = 8.0
Hz, J2 = 1.0 Hz, 1H), 7.57 (t, J = 7.0 Hz, 2H), 7.62 (dt, J1 = 7.0
Hz, J2 = 1.0 Hz, 1H), 7.92 (dd, J1 = 8.0 Hz, J2 = 0.5 Hz, 1H),
8.20 (dd, J1 = 8.0 Hz, J2 = 0.5 Hz, 1H), 8.37 (d, J = 8.5 Hz,
2H). 13C NMR (CDCl3, 100 MHz) 166.44, 152.46, 137.03,
132.86, 129.06, 128.52, 125.78, 124.31, 120.98, 116.55.
ESI-MS m/z 241.21 (M + H)+.
2-Phenyl-7-aminobenzoxazole (5)
7-Nitrobenzoxazole (2)
To a solution of 2-phenyl-7-nitrobenzoxazole (3) (1.2 g,
5.0 mmol) in 400 mL methanol was added 10% Pd/C (527 mg,
0.5 mmol) portionwise. The suspension was placed under H2(g)
atmosphere for 30 min (P = 40 psi) and then filtered through a
pad of celite. The transparent solution is evaporated to yield a
black solid that was recrystallized from ethanol (850 mg, 71%).
1H NMR (CD3OD, 500 MHz) δ 6.65 (dd, J1 = 8.0 Hz, J2 = 1.0
Hz, 1H), 6.91 (dd, J1 = 8.5 Hz, J2 = 1.0 Hz, 1H), 7.21 (t, J = 8.0
Hz, 1H), 7.56 (m, 3H), 8.19 (m, 2H). 13C NMR (CD3OD,
100 MHz) δ 159.72, 151.70, 141.59, 131.66, 129.71, 129.23,
127.37, 127.18, 126.78, 108.53, 97.99. ESI-MS m/z 211.23 (M
+ H)+.
To a toluene (25 mL) suspension of 3-nitro-2-aminophenol (1)
(1 g, 6.5 mmol) in a 35 mL microwave tube was added triethy-
lorthoformate (3.2 mL, 19.4 mmol) and a catalytic amount of p-
toluenesulfonic acid monohydrate (62 mg, 0.3 mmol). The red
suspension was stirred at 130 °C for 2 min in a microwave
reactor, after which the reaction was complete by TLC. The
orange solution was then cooled to −20 °C and the precipitate
was isolated by filtration as a light pink solid (991 mg, 93%). 1H
NMR (CD3OD, 400 MHz) δ 7.67 (t, J = 8.4 Hz, 1H), 8.12 (dd,
J1 = 8.0 Hz, J2 = 0.8 Hz, 1H), 8.26 (dd, J1 = 8.4 Hz, J2 = 0.8
Hz, 1H), 8.79 (s, 1H). ESI-MS m/z 165.06 (M + H)+, 186.91 (M
+ Na)+.
2-Phenyl-7-sulfonamidobenzoxazole (ZBG2)
To
a solution of 2-phenyl-7-aminobenzoxazole (50 mg,
7-Aminobenzoxazole (4)
0.24 mmol) in pyridine (1 mL) was added 1.5 eq (0.36 mmol)
of sulfonyl chloride. The transparent solution was heated in the
microwave at 130 °C for 3 min (Power = 300 W). The solution
was then poured into 5 mL of water and the precipitate was
filtered off, rinsed with water and dried under vacuum. If no pre-
cipitate was formed, the aqueous phase was extracted twice with
chloroform. The combined organic layers were washed with a
solution of HCl (1 M), dried and evaporated under reduced
pressure. The crude product was either purified by column of
chromatography or recrystallized from an appropriate solvent.
To a solution of 4-nitrobenzoxazole (2) (1.91 g, 11.6 mmol) in
160 mL of methanol was added 10% Pd/C (1.24 g, 1.16 mmol)
portionwise. The suspension was placed under H2(g) atmosphere
for 1 h (P = 40 psi) and then filtered through a pad of celite. The
transparent solution was evaporated to yield a black solid
1
(1.42 g, 91%). H NMR (CD3OD, 400 MHz) δ 6.63 (dd, J1 =
8.0 Hz, J2 = 0.8 Hz, 1H), 6.87 (dd, J1 = 8.4 Hz, J2 = 0.8 Hz,
1H), 7.12 (t, J = 8.4 Hz, 1H), 8.25 (s, 1H). ESI-MS m/z 135.22
(M + H)+.
6508 | Dalton Trans., 2012, 41, 6507–6515
This journal is © The Royal Society of Chemistry 2012

7-Nitrobenzimidazole (7)
To solution of 3-nitro-1,2-phenylenediamine (6) (1 g,
through a pad of celite. The transparent solution is evaporated to
yield a black solid (258 mg, 81%). 1H NMR (DMSO, 500 MHz)
δ 5.24 (s, NH, 2H), 6.37 (d, J = 7.5, Hz 1H), 6.75 (brs, 1H),
6.90 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.0 Hz, 1H), 7.53 (t, J = 7.5
Hz, 2H), 8.12 (d, J = 8.0 Hz, 2H). ESI-MS m/z 210.33
(M + H)+.
a
6.53 mmol) in toluene (25 mL) was added triethylorthoformate
(3.25 mL, 19.59 mmol) with a catalytic amount of p-toluenesul-
fonic acid (60 mg, 0.38 mmol). The solution was heated in the
microwave at 120 °C for 1 min, followed by cooling to −20 °C
to produce a precipitate. The precipitate was isolated by vacuum
filtration (1.0 g, 94%). 1H NMR (CD3OD, 400 MHz) δ 7.46 (t, J
= 8.0 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 7.6 Hz,
1H), 8.46 (s, 1H). ESI-MS m/z 164.22 (M + H)+.
2-Phenyl-7-sulfonamidobenzimidazole (ZBG4)
To a solution of 2-phenyl-7-aminobenzimidazole (50 mg,
0.24 mmol) in pyridine (1 mL) was added 2 eq (0.48 mmol) of
sulfonyl chloride. The transparent solution was heated in the
microwave at 130 °C for 3 min (Power = 300 W). The solution
was then poured into 5 mL of water and the precipitate was
filtered off, rinsed with water, and dried under vacuum. If no pre-
cipitate was formed, the aqueous phase was extracted twice with
chloroform. The combined organic layers were washed with a
solution of 1 M HCl, dried with MgSO4, filtered, and evaporated
under reduced pressure. The crude product was either purified by
column chromatography or recrystallized from an appropriate
solvent.
7-Aminobenzimidazole (9)
To a solution of 7-nitrobenzimidazole (7) (495 mg, 3.03 mmol)
in 50 mL of methanol was added 10% Pd/C (322 mg, 0.3 mmol)
portionwise. The suspension was placed under H2(g) atmosphere
for 3.5 h (P = 40 psi) and then filtered through a pad of celite.
The transparent solution was evaporated to yield a black solid
1
(403 mg, 99%). H NMR (CD3OD, 400 MHz) δ 6.55 (dd, J1 =
7.6 Hz, J2 = 0.8 Hz, 1H), 6.89 (dd, J1 = 8.0 Hz, J2 = 0.8 Hz,
1H), 7.02 (t, J = 8.0 Hz, 1H), 8.03 (s, 1H). ESI-MS m/z 134.21
(M + H)+.
7-Sulfonamidobenzimidazole (ZBG3)
tert-Butyl-(2-(quinolin-2-yl)phenyl)carbamate (12)
To a solution of 7-aminobenzimidazole (50 mg, 0.37 mmol) in
pyridine (2 mL) was added 1.5 eq (0.37 mmol) of sulfonyl
chloride. The transparent solution was heated at 100 °C for ∼4 h
(until complete by TLC). The pyridine was then evaporated off
and 15 mL of water was added to the remaining solution and
stirred for 15 min. The precipitate was filtered off and rinsed
with water and recrystallized from an appropriate solvent. If no
precipitate was formed, the aqueous phase was extracted three
times with chloroform. The organic layer was then washed with
a solution of 1 M HCl, dried with MgSO4 and evaporated under
vacuum to obtain an oil that was either purified by column of
chromatography or recrystallized from an appropriate solvent.
To a solution of 2-bromoquinoline (500 mg, 2.40 mmol) in
9 mL of toluene and 9 mL of 2 M K2CO3 was added 2-(N-Boc-
amino)phenylboronic acid (11) (1.15 g, 3.6 mmol), and a cataly-
tic amount of triphenylphosphine (277 mg, 0.24 mmol). The sol-
ution was heated to reflux at 100 °C overnight. Then water was
added and the solution was extracted three times with dichloro-
methane. The organic layer was dried with anhydrous mag-
nesium sulfate and was purified by column chromatography
(Hex–EtOAc, 100 : 0 to 96 : 4) to isolate the desired product
1
(761 mg, 99%). H NMR (CHCl3, 400 MHz) δ 7.13 (td, J1 =
6.0 Hz, J2 = 0.8 Hz, 1H), 7.42 (td, J1 = 6.4 Hz, J2 = 0.8 Hz,
1H), 7.58 (t, J = 6.0 Hz, 1H), 7.77 (td, J1 = 6.8 Hz, J2 = 0.8 Hz,
1H), 7.82 (dd, J1 = 6.4 Hz, J2 = 0.8 Hz, 1H), 7.85 (d, J = 6.4
Hz, 1H), 7.89 (d, J = 6.4 Hz, 1H), 8.08 (d, J = 6.4 Hz, 1H), 8.27
(d, J = 6.8 Hz, 1H), 8.42 (d, J = 6.4 Hz, 1H). ESI-MS m/z
320.98 (M + H)+, 320.98 (M + Na)+.
2-Phenyl-7-nitrobenzimidazole (8)
To a solution of 3-nitro-1,2-phenylenediamine (6) (100 mg,
0.65 mmol) in toluene (25 mL) was added trimethylorthobenzo-
ate (336 μL, 1.9 mmol) and a catalytic amount of p-toluenesulfo-
nic acid (6.2 mg, 0.03 mmol). The solution was heated in the
microwave at 130 °C for 1 min. The solution was then cooled
down to −20 °C. The precipitate was vacuum filtered, rinsed
2-(2-Aminophenyl)quinoline (13)
In a dry round bottom flask was placed tert-butyl (2-(quinolin-2-
yl)phenyl)carbamate (12) (300 mg, 0.93 mmol) and dry dichlor-
omethane was added under nitrogen atmosphere. Trifluoroacetic
acid was then added and the solution was stirred at room temp-
erature for 3.5 h. The solution was evaporated to dryness and the
precipitate was dissolved in dichloromethane and washed with 1
M NaOH. The product was then purified by column chromato-
graphy (Hex–EtOAc, 100 : 0 to 98 : 2). 1H NMR (CHCl3,
400 MHz) δ 6.20 (brs, 2H, NH2), 6.80–6.84 (m, 2H), 7.21 (td,
J1 = 7.6 Hz, J2 = 1.2 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H),
7.68–7.72 (m, 2H), 7.80–7.85 (m, 2H), 8.04 (d, J = 8.8 Hz, 1H),
8.20 (d, J = 8.8 Hz, 1H). ESI-MS m/z 221.34 (M + H)+.
1
with diethyl ether, and then collected (78 mg, 50%). H NMR
(DMSO, 400 MHz) δ 8.12–8.15 (m, 6H), 8.16 (s, 1H), 8.18 (d,
J = 0.8 Hz, 1H), 8.19 (d, J = 0.8 Hz, 1H). ESI-MS m/z 240.25
(M + H)+.
2-Phenyl-7-aminobenzimidazole (10)
To a solution of 2-phenyl-7-nitrobenzimidazole (8) (300 mg,
1.25 mmol) in 140 mL methanol was added 10% Pd/C (133 mg,
0.12 mmol) portionwise. The suspension was placed under
H2(g) atmosphere for 30 min (P = 40 psi) and then filtered
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6507–6515 | 6509

2-(2-Sulfonamidophenyl)quinoline (ZBG5)
To solution of 2-(2-aminophenyl)quinoline (50 mg,
The synthesis of the 2-phenyl-7-sulfonamidobenzoxazole
(ZBG2) and 2-phenyl-7-sulfonamidobenzimidazole derivatives
(ZBG4) was performed using the same general synthetic route
(Scheme 1); however, the reactivity of 2-amino-3-nitrophenol (1)
and 3-nitro-1,2-phenylenediamine (6) with trimethylorthobenzo-
ate were slightly different. Using the aforementioned microwave
condensation, the conversion of the 3-nitro-1,2-phenylenedia-
mine (6) into the 2-phenyl-7-nitrobenzimidazole (8) was com-
plete within 1 min at 130 °C with isolated yields of ∼50%. In
contrast, 2-amino-3-nitrophenol (1) required longer reaction
times (15 min) in order to achieve full conversion to 2-phenyl-7-
nitrobenzoxazole (3) in good yields (87%). Subsequent
reduction of the nitro groups of compounds 3 and 8 gave the free
amines (5, 10) in >81% yield. The sulfonamide couplings were
performed using a microwave procedure generating the desired
sulfonamide inhibitors.18
a
0.23 mmol) in pyridine (2 mL) is added 2 eq (0.45 mmol) of
sulfonyl chloride. The transparent solution was heated in the
microwave at 130 °C for 3 min (Power = 300 W). The solution
was then poured into 5 mL of water and the precipitate was
filtered off, rinsed with water and dried under vacuum. If no pre-
cipitate was formed, the aqueous phase was extracted twice with
chloroform. The combined organic layers were washed with a
solution of 1 M HCl, dried with MgSO4, filtered, and evaporated
under reduced pressure. The crude product was either purified by
column of chromatography or recrystallized from an appropriate
solvent.
Inhibition assays
Finally, 2-(2-sulfonamidophenyl)-quinoline derivatives (ZBG5,
Scheme 1) were synthesized in three steps via a Suzuki coupling
between the 2-bromoquinoline and N-Boc-phenylboronic acid
(11). After the Suzuki coupling (12), removal of the Boc group
with TFA and microwave-mediated coupling with sulfonyl chlor-
ides yielded the desired compounds. For all of the aforemen-
tioned chelating sulfonamide ZBGs, eight derivatives were
synthesized in order to produce small fragment libraries suitable
for in vitro screening against MMPs (Scheme 1).
MMP activity assays were carried out in white NUNC 96-well
plates as previously described.19–21 Each well contained a total
volume of 100 μL : 60 μL of buffer (50 mM HEPES, 10 mM
CaCl2, 0.05% Brij-35, pH 7.5), 20 μL of human recombinant
MMP-2, or -9 (1.16 U or 0.9 U, respectively per well, BIOMOL
International), and 10 μL of the inhibitor solution (50 μM final
concentration). After a 30 min incubation period at 37 °C, the
reaction was initiated by the addition of 10 μL fluorogenic MMP
substrate (4 μM final concentration, Mca-Pro-Leu-Gly-Leu-Dpa-
Ala-Arg-NH2·AcOH, BIOMOL International). Kinetic measure-
ments were recorded using a Bio-Tek Flx 800 fluorescence plate
reader every min for 20 min with excitation and emission wave-
lengths at 320 and 400 nm, respectively. IC50 values were calcu-
lated using GraphPad Prism 5 software. All assays were run in
duplicate.
Coordination chemistry of ZBGs
In order to gain a better fundamental understanding into the
coordination chemistry of these ZBGs, which is directly relevant
to understanding their ability or inability to inhibit
metalloproteinases,22–24 metal complexes with these ligands
were prepared and their structures determined by single-crystal
X-ray diffraction methods. Complexes were generally formed
between the tosylated ligand and transition metal salts with the
addition of triethylamine as a base. Most of the complexes could
be crystallized by diffusing diethyl ether into a solution of the
complex dissolved in a mixture of CH2Cl2 and MeOH (see ESI
and Table S1† for details). Surprisingly, not all of the chelating
sulfonamides formed the expected, simple bidentate chelates,
and others even showed unexpected reactivity in the presence of
metal ions.
Upon reaction with ZnCl2, ZBG1a undergoes a chemical
rearrangement to 3-tosyl-7-hydroxybenzimidazole (THBI). The
structure of the THBI product was confirmed by X-ray crystallo-
graphy (Fig. 2). The C–O bond length is 1.358(2) Å, consistent
with an aromatic alcohol. The heterocycle C–N bond lengths are
1.304(3) and 1.388(3) Å for the imine and amine nitrogen
atoms, respectively. It is important to note that under similar
crystallization conditions but in the absence of Zn(II), ZBG1a
readily crystallizes without undergoing any rearrangement
(Fig. 2). However, ZBG1a does transform to THBI in the pres-
ence of Zn(NO3)2, further confirming the role of Zn(II) in this
rearrangement.
Results
Synthesis
The synthesis of the 7-sulfonamidobenzoxazole (ZBG1,
Scheme 1) and 7-sulfonamidobenzimidazole derivatives (ZBG3)
began with an acid-catalyzed condensation of triethylorthofor-
mate with either 2-amino-3-nitrophenol (1) or 3-nitro-1,2-pheny-
lenediamine (6), respectively. The reported procedures for this
type of reaction usually involve refluxing conditions in toluene
for >1.5 h.2,4,7 By exploring a variety of microwave irradiation
reaction conditions, it was determined that the transformation
could be completed in 2 min at 130 °C. In addition, the products
2, 7 were easily isolated by filtration in >90% yield and did not
require further purification. Reduction of the nitro group by
hydrogenation gave the primary amines 4, 9 in >90% yield. For
the final step, amine 4 was combined with a sulfonyl chloride in
neat pyridine for 3 min using a previously reported microwave
procedure to generate the different chelating sulfonamide frag-
ments.18 In the case of 9, the microwave-mediated sulfonamide
coupling procedure generated the disubstituted sulfonamides on
the 3- and 7-position even when only one equivalent of sulfonyl
chloride was utilized under microwave conditions. In order to
obtain the monosubstituted ZBG3 compounds on the 7-position,
a previously reported conventional heating procedure was
utilized.5
The rearranged THBI ligand binds to Zn(II) ions by forming a
neutral Zn4L6Cl2 cluster (Fig. 3). The cluster consists of two
pairs of symmetry-related Zn(II) ions. The first type of Zn(II) ion
is coordinated by four different THBI ligands, adopting a
6510 | Dalton Trans., 2012, 41, 6507–6515
This journal is © The Royal Society of Chemistry 2012

Scheme 1 Synthesis of new inhibitors using a three-step microwave procedure.
of the ZBG1a ligand was observed. In all of the complexes, the
sulfonamide group was not deprotonated and the ligand was
found to bind only through imine nitrogen atom (Fig. 4). The Co
(II) and Ni(II) complexes form mononuclear complexes where
the metal ions adopt a distorted octahedral geometry with the
equatorial plane comprised of two chelating acac ligands and the
axial positions occupied by two monodentate ZBG1a molecules.
The M–N bond lengths are 2.223(2) and 2.158(2) Å for the Co
(II) and Ni(II) complexes, respectively. The Cu(II) complex is
similar to the Co(II) and Ni(II) compounds, in that the ZBG1a
binds in the same manner (Cu–N = 2.203 Å); however, the
overall structure of the complex is dinuclear, maintaining the
Cu2(OAc)4 paddlewheel structure (Fig. 4).
Fig. 2 ZBG1a (left) undergoes a rearrangement to THBI (right) in the
presence of Zn(II). Thermal ellipsoids are shown at 50% probability and
most hydrogen atoms have been omitted for clarity.
The reaction between ZBG2a and ZnCl2 yielded a mono-
nuclear complex with a slightly distorted tetrahedral Zn(II) ion
coordinated by two chloride ligands and one ZBG2a molecule
through its imine and deprotonated sulfonamide nitrogen atoms
(Fig. 5). The Zn–N bond distances are 2.116(3) and 2.038(3) Å
for the imine and sulfonamide donors, respectively. The charge
of the anionic complex is balanced by a protonated triethylamine
molecule in the crystal lattice. Attempts to crystallize free
ZBG2a or other metal complexes with this ligand, including Co
(II), Ni(II), and Cu(II) were unsuccessful.
distorted octahedral geometry. The Zn(II) ion is chelated by two
THBI ligands, as well as being bridged to its symmetric equival-
ent by the hydroxy groups of two other symmetry-equivalent
THBI molecules. The second type of zinc ions are 4-coordinate,
bound by the imide nitrogen of two THBI and two deprotonated
hydroxy groups of two more THBI ligands. The fourth coordi-
nation site on each tetrahedral Zn(II) ion is occupied by a chlor-
ide ion.
The coordination chemistry of ZBG1a with Co(acac)2, Ni
(acac)2 (acac = acetylacetonate), and Cu2(OAc)4 were also
explored. Interestingly, with these metal ions, no rearrangement
In contrast to the other ZBGs studied here, ZBG3a readily
forms complexes and crystallizes with late first-row transition
metals. Co(II) and Zn(II) yielded isostructural complexes where
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6507–6515 | 6511

Fig. 3 Asymmetric unit of the Zn4(THBI)6Cl2 cluster (left). Hydrogen atoms and a diethyl ether molecule have been excluded for clarity. Thermal
ellipsoids are shown at 50% probability and hydrogen atoms have been removed for clarity. The full cluster (middle) is shown with sulfonamide substi-
tuents removed for clarity. The complex connectivity (right) is highlighted by showing each unique ligand in a different color with Zn(II) ions in gray
and chloride ions removed for clarity.
Fig. 4 Complexes of ZBG1a (left to right) with Co(II), Ni(II), and Cu(II). Thermal ellipsoids are shown at 50% probability and most hydrogen atoms
have been removed for clarity. In the case of the Cu(II) complex the asymmetric unit contains two independent molecules, but only one is shown.
(II), but the Cu(II) center has a distorted square pyramidal geome-
try (Fig. 6). The axial ligand is the imine nitrogen atom from one
of the chelating ZBG3a ligands. The Cu–N bond distances for
equatorial imine nitrogen atoms are 1.993(4) and 1.978(4) Å,
while the axial Cu–N distance is much longer at 2.249(4) Å due
to Jahn–Teller distortion. The two sulfonamide nitrogen atoms
bind to copper at distances of 2.075(4) and 2.131(4) Å. Finally,
the Ni(II) complex with ZBG3a displayed the most distinct
structure, with the Ni(II) ion coordinated in a distorted octahedral
geometry by two chelating ZBG3a ligands and two water
molecules (Fig. 6). The two sulfonamide nitrogen donor atoms
Fig. 5 Structure of Zn(ZBG2a)Cl2. The asymmetric unit contains two
are positioned trans to each other on the coordination sphere.
anionic complexes, two protonated triethylamine molecules, and a
While the water ligands are held in a cis arrangement. The nickel
methanol molecule. Thermal ellipsoids are shown at 50% probability
ion binds to the sulfonamide nitrogen atoms at distances of
2.177(5) and 2.239(5) Å, while the Ni–N imine bond distances
and hydrogen atoms have been omitted for clarity.
are 2.031(5) and 2.069(5) Å. The bite angle for the chelating
the metal ion adopts a distorted trigonal bipyramidal geometry.
In both complexes, two chelating ZBG3a ligands occupy both
equatorial and axial sites, with the third equatorial position occu-
pied by an monodentate ZBG3a ligand coordinated via a single
imine nitrogen atom (Fig. 6). The axial Zn–N bond lengths are
2.261(3) and 2.319(3) Å while the corresponding Co–N dis-
tances are slightly shorter at 2.239(3) and 2.258(3) Å. The equa-
torial Zn–N bond lengths are 2.005(3) and 2.008(3) Å while the
Co(II) complex displays slightly longer distances of 2.021(3) and
2.026(3) Å. The ZBG3a molecule binds with a M–N distance of
2.023 and 2.047 Å for Zn(II) and Co(II), respectively. ZBG3a
also readily forms a complex with Cu(II), with three ZBG3a
ligands bound in a similar fashion as found with Co(II) and Zn
ZBG3a ligands in all of these complexes are very similar
at ∼81–83°.
To date, attempts to isolate and crystallize complexes with
ZBG4 or ZBG5 under a variety of reaction conditions have not
been successful. However, the free ZBG4a ligand was crystal-
lized as a protonated cation, which served to confirm the connec-
tivity of this compound (Fig. S1†).
In vitro activity against MMPs
The five new libraries (i.e. 40 compounds, each based on one of
the five ZBGs, Table 1) were screened for inhibitory activity
6512 | Dalton Trans., 2012, 41, 6507–6515
This journal is © The Royal Society of Chemistry 2012

Fig. 6 Transition metal complexes of ZBG3a. Asymmetric units of Zn(II), Co(II), Cu(II), and Ni(II) complexes (top left to bottom right). Thermal
ellipsoids are shown at 50% probability and hydrogen atoms and solvent molecules have been removed for clarity.
against MMP-2 and MMP-9. A standard in vitro fluorescent
substrate assay was utilized, and each compound was screened
at a fixed concentration of 50 μM. In the case of MMP-2,
the derivatives of ZBG1, ZBG2, and ZBG3 showed little or
no inhibition at 50 μM. All of the ZBG5 compounds showed
some activity against both MMP-2 and MMP-9, and three inhibi-
tors (ZBG5d, ZBG5f, ZBG5i) gave nearly 30% inhibition
against both MMPs at 50 μM (Table 1). However, the 2-phenyl-
7-sulfonamidobenzimidazole (ZBG4) derivatives showed the
best overall activity at 50 μM, with five of eight compounds
showing >40% inhibition. Because all of the ZBG derivatives
in this study utilize the same sulfonamide ‘backbones’
(R groups, Table 1), the assay results indicate that modest
Discussion
Previous studies have shown that sulfonamide compounds such
as 1 form stable chelates upon binding Zn(II) and other metal
ions.13,25 Our earlier studies show that such molecules can bind
to the active site Zn(II) ions in MMPs eliciting good inhibition of
these enzymes.18 In this study, we sought to develop a better
structure–activity relationship with respect to the nature of these
chelating sulfonamide ZBGs. ZBG1 and ZBG3 were designed
to examine relatively conservative structural and electronic
changes, looking at the impact of the quinoline ring on the
activity of these compounds. ZBG1 and ZBG3 change the
fused, 6-membered ring quinoline to a fused 5-membered ben-
zoxazole and benzimidazole ligand, respectively. However,
when exploring the coordination chemistry of ZBG1, it was
found that the benzoxazole ring was unstable in the presence of
Zn(II) and rearranges to yield 7-hydroxylbenzimidazole deriva-
tives (Fig. 2). A similar rearrangement of this heterocycle has
been reported in the literature upon treatment with a reducing
agent such as NaBH4.26 Therefore, we attribute the poor inhi-
bition activity of ZBG1 derivatives to this chemical rearrange-
ment, which destroys the desired chelator shortly after binding to
Zn(II). In contrast, the benzoimidazole analog (ZBG3) did not
rearrange upon binding Zn(II) and readily formed stable com-
plexes with a variety of transition metal ions. Nonetheless, these
compounds did not show activity against MMP-2. The coordina-
tive ability of ZBG3 and compound 1 are very similar,18 but
they do provide slightly different bite angles (79° for ZBG3
compared to 83° for compound 1) upon binding Zn(II), which
changes to the ZBG are playing
a significant role in
generating effective MMP inhibition. This is rather remarkable
considering the structural similarity of all the ZBGs presented
here, in addition to the related sulfonamides that were
already reported.18 Among the ZBG4 derivatives, the best hit
was the 4-trifluoromethylphenyl derivative (ZBG4b) that was
determined to have an IC50 value of 40 μM against MMP-2
(Table 1). This is consistent with our earlier studies (see com-
pounds BISL-1 and QSL-1, Fig. 1),18 where the 4-trifluoro-
methylphenyl derivatives were also found to be the most active
compounds against MMP-2 (out of ∼40 different sulfonamide
substituents). Clearly, the inhibitory activity of these compounds
is surprisingly sensitive to the nature of the ZBG. This is likely
due to a combination of effects, such as sterics, but may also
reflect differences in the coordination chemistry of these ligands
as well (vide supra).
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6507–6515 | 6513

Table 1 Summary of MMP inhibition results. The compound name is listed above the percent inhibition obtain at a ligand concentration of 50 μM
against MMP-2 (top) and MMP-9 (bottom). Note that fewer ZBG1 derivatives were prepared because of observed ligand instability (see text)
R =
ZBG1b
0
0
n.d.
ZBG1d
0
0
n.d.
n.d.
ZBG1g
0
0
n.d.
ZBG1i
0
0
ZBG2b
0
0
ZBG2c
0
0
ZBG2d
0
0
ZBG2e
0
0
ZBG2f
0
0
ZBG2g
0
0
ZBG2h
7
0
ZBG2i
0
0
ZBG3b
0
0
ZBG3c
5
0
ZBG3d
0
0
ZBG3e
0
0
ZBG3f
0
0
ZBG3g
10
0
ZBG3h
0
0
ZBG3i
0
1
ZBG4b
69a
26
ZBG4c
0
11
ZBG4d
50
14
ZBG4e
51
26
ZBG4f
10
20
ZBG4g
50
23
ZBG4h
41
17
ZBG4i
0
0
ZBG5b
10
5
ZBG5c
5
18
ZBG5d
27
26
ZBG5e
18
18
ZBG5f
27
28
ZBG5g
9
9
ZBG5h
10
3
ZBG5i
27
23
a An IC50 value of 40 μM was determined against MMP-2.
may produce different binding orientations in the MMP active
that results in the observed loss of activity. Regardless, by exam-
ining the simple coordination chemistry of these ligands, one
can a priori rule out the use of ZBG1 in metalloprotein inhibi-
tors, but conclude that ZBG3 is a feasible candidate for inhibitor
development.
4-trifluoromethylphenyl derivatives of these ZBGs highlights the
subtle nature of identifying effective scaffolds for developing
metalloprotein inhibitors.
While ZBG1 and ZBG3 were synthesized as analogues to
compound QSL-1, ZBG2 and ZBG4 were synthesized to gain
more insight around the phenylbenzimidazole sulfonamide
ZBG found in compound BISL-1.27 ZBG2 and ZBG4 essen-
tially preserve the steric and electronic features of compound
BISL-1, but place the sulfonamide substituent in a position
on the chelator that is more similar to that found in ZBG1,
ZBG3, and compound QSL-1. The result is that while the
sulfonamide moiety of compound BISL-1 generates a 6-mem-
bered chelate ring when bound to a metal ion, ZBG2 and ZBG4
will form a 5-membered chelate similar to that for ZBG3
or QSL-1. Thus, ZBG2 and ZBG4 are structurally related to
compound BISL-1 but certain features similar to QSL-1,
making these ZBGs a hybrid our two earlier scaffolds.18,27
Although the ZBG2 derivatives did not show any activity
against MMP-2 or MMP-9 at 50 μM, most of the ZBG4 deriva-
tives were active against both MMP-2 and MMP-9, with one
compound (ZBG4b) giving an IC50 of 40 μM against MMP-2.
ZBG4b possesses a 4-trifluoromethylphenyl sulfonamide substi-
tuent, which is consistent with the best hits from our earlier
study.18 Interestingly, the only difference between ZBG3
and ZBG4 is the added phenyl ring on the 2-position of the
ZBG. The significant difference in the activity between the
Conclusions
In summary, we have developed an efficient microwave pro-
cedure to synthesize benzimidazole and benzoxazole compounds
and their sulfonamide derivatives. The resulting compounds,
inspired from our previous findings18,27 and work on biocompa-
tible Zn(II) sensors,15–17,25,28 were screened against the Zn(II)-
dependent MMP-2 and MMP-9 metalloenzymes to identify com-
pounds with good inhibitory activity. Much to our surprise, none
of the new ZBGs were more potent than our original hits
(BISL-1 and QSL-1, Fig. 1) and indeed most displayed very
poor activity, despite the high similarity of the ZBGs. By exam-
ining the coordination chemistry and reactivity of these ligands
with various transition metal ions, we identified unexpected reac-
tivity that in some cases explains the poor inhibition activity of
the compounds. Specifically, ZBG1 was found to be reactive in
the presence of Zn(II), and several other ZBGs did not readily
form metal complexes under standard conditions. The work here
highlights how even subtle changes to the ZBG of a metallopro-
tein inhibitor can have a pronounced effect on activity, which
suggests to us that more studies are required to better define,
understand, and exploit the coordination chemistry of these
ligands for use against important medicinal targets.
6514 | Dalton Trans., 2012, 41, 6507–6515
This journal is © The Royal Society of Chemistry 2012

13 K. M. Hendrickson, J. P. Geue, O. Wyness, S. F. Lincoln and A.
D. Ward, Coordination and fluorescence of the intracellular Zn2+ probe
[2-methyl-8-(4-toluenesulfonamido)-6-quinolyloxy]acetic acid (zinquin
A) in ternary Zn2+ complexes, J. Am. Chem. Soc., 2003, 125, 3889–
3895.
14 R. McRae, P. Bagchi, S. Sumalekshmy and C. J. Fahrni, In situ
imaging of metals in cells and tissues, Chem. Rev., 2009, 109 (10), 4780–
4827.
15 M. M. Henary and C. J. Fahrni, Excited state intramolecular proton trans-
fer and metal ion complexation of 2-(2′-hydroxyphenyl)benzazoles in
aqueous solution, J. Phys. Chem. A, 2002, 106 (21), 5210–5220.
16 M. M. Henary, et al., Excited-state intramolecular proton transfer in 2-
(2′-arylsulfonamidophenyl)benzimidazole derivatives: the effect of donor
and acceptor substituents, J. Org. Chem., 2007, 72 (13), 4784–4797.
17 M. M. Henary, Y. G. Wu and C. J. Fahrni, Zinc(II)-selective ratiometric
fluorescent sensors based on inhibition of excited-state intramolecular
proton transfer, Chem.–Eur. J., 2004, 10 (12), 3015–3025.
18 B. Macías, et al., Synthesis and structural characterization of zinc com-
plexes with sulfonamides containing 8-aminoquinoleine, Z. Anorg. Allg.
Chem., 2003, 629 (2), 255–260.
19 D. T. Puerta, et al., Heterocyclic zinc-binding groups for use in next-gen-
eration matrix metalloproteinase inhibitors: potency, toxicity, and reactiv-
ity, JBIC, J. Biol. Inorg. Chem., 2006, 11, 131–138.
20 D. T. Puerta, J. A. Lewis and S. M. Cohen, New beginnings for matrix
metalloproteinase inhibitors: identification of high-affinity zinc-binding
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21 D. T. Puerta, J. Mongan, B. L. Tran, J. A. McCammon and S. M. Cohen,
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Acknowledgements
We thank Dr Y. Su for performing mass spectrometry exper-
iments. This work was supported by a grant from the National
Institutes of Health (R21 HL094571; R01 GM098435). D.P.M.
is supported by a SMART scholarship from the Office of Sec-
retary Defense - Test and Evaluation (Grant No. N00244-09-1-
0081).
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