Optimization of heterocyclic substituted benzenesulfonamides as novel carbonic anhydrase IX inhibitors and their structure activity relationship

Article abstract of DOI:10.1016/j.ejmech.2013.01.030

In this study, starting from a lead compound discovered by virtual screening, a series of novel heterocyclic substituted benzenesulfonamides were designed and synthesized as new carbonic anhydrase IX (CA IX) inhibitors. Some compounds exhibited potent inh

Full text of DOI:10.1016/j.ejmech.2013.01.030

European Journal of Medicinal Chemistry 62 (2013) 597e604
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Optimization of heterocyclic substituted benzenesulfonamides as
novel carbonic anhydrase IX inhibitors and their structure
activity relationship
,
,
Rui Gao a 1, Sha Liao b 1, Chen Zhang a, Weilong Zhu a, Liyan Wang b, Jin Huang b,
**
Zhenjiang Zhao b , Honglin Li , Xuhong Qian , Yufang Xu
,
b
a
a,
*
a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology,
Shanghai 200237, China
b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, starting from a lead compound discovered by virtual screening, a series of novel hetero-
cyclic substituted benzenesulfonamides were designed and synthesized as new carbonic anhydrase IX
(CA IX) inhibitors. Some compounds exhibited potent inhibitory effects against CA IX (in the low
nanomolar range) as well as high selectivity against other carbonic anhydrase isozymes (CA I and CA II).
The most potent and selective compound 27 could inhibit CA IX in the subnanomolar level with IC50 of
0.48 nM, which increased the potency by about 40-fold against CA IX compared with the lead compound
26, and presented more than 103 fold selectivity over CA I and CA II. The structureeactivity relationship
(SAR) based on the docking experiments further elucidated the effects of the compounds on the bio-
activity and selectivity.
Received 21 November 2012
Received in revised form
19 January 2013
Accepted 24 January 2013
Available online 4 February 2013
Keywords:
Carbonic anhydrase inhibitors
Benzenesulfonamides
Structureeactivity relationship
Molecular docking
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
extracellular acidification of solid tumors. Thus, CA IX has been
a promising target for hypoxic tumor treatment [10e12].
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metal-
loenzymes, which are the most widely spread biological catalysts
all over the phylogenetic tree and encoded by five evolutionarily
Up to now, many CA IX inhibitors are reported, such as hydrox-
amates, mercaptophenols, metal-complex anions and sulfonamides
[3,13,14]. Among them, the most important class is sulfonamides
derivatives. There are around 30 clinically used drugs (or agents in
clinical development) belonging to the sulfonamide or sulfamate
class, such as clinically used sulfonamides AAZ, MZA, IND, and EZA
(Chart 1) [15,16]. The negative charge of the deprotonated nitrogen in
thesulfonamide group(eSO2NH2) can coordinatewith thepositively
charged Zn2þ; the presence of one proton on the coordinated nitro-
gen atom also satisfies the hydrogen bond donor (Chart 2) [17e19].
However, design of therapeutic agents targeting at CAs is con-
fronted by the presence of a large number of isoforms in humans,
and the selectivity of the existing inhibitors is not optimal for any
specific isozymes [20e23].
Through molecular docking, visual inspection and in vitro inhi-
bition assay, we discovered a heterocyclic substituted benzene-
sulfonamides lead compound 26 against CA IX in our previous work
(paper in submission). It exhibits an IC50 of 18.5 nM and poor inhib-
itory efficiency against CA I, CA II (Chart 2). Based on this lead com-
pound, we designed a series of structurally optimized derivatives,
which were tested to display improved bioactivity and selectivity.
unrelated gene families: a-, b-, g-, d-, and z-CAs [1,2]. These en-
ꢀ
zymes catalyze the reversible hydration of CO2 to HCO3 and Hþ.
Furthermore, CAs are involved in some pathological pathways and
thus are viable targets for the treatment of glaucoma, cancer, obe-
sity, and epilepsy [1e8]. Recently, two tumor-associated membrane
CA isoforms (CA IX and CA XII) have drawn much attention [9].
Many reports are published studying the roles of CA IX in tumor
physiology, such as the control of tumor pH and influence on other
processes in the cell microenvironment that promote cell prolifer-
ation, invasion, or metastasis. The expression of CA IX is remarkably
up-regulated via the hypoxia inducible factor-1 (HIF-1) transcrip-
tion factor. The over-expression of CA IX will induce the pH
imbalance in tumor tissue, and contribute significantly to the
* Corresponding author. School of Pharmacy, East China University of Science and
Technology, 130 Mei Long Road, Shanghai 200237, China. Tel./fax: þ86 21 64251399.
** Corresponding author.
E-mail addresses: zhjzhao@ecust.edu.cn (Z. Zhao), yfxu@ecust.edu.cn (Y. Xu).
Authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.030

598
R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
Compounds 24 and 25 are designed from hit compound 26, by
retaining sulfonamide A and replacing sulfonamide B with amino
(compound 24) and t-butyl (compound 25) respectively. Their CA IX
inhibition IC50 (18.5e28.3 nM) are at the same level with the hit
compound 26. So the substituents R almost doesn’t affect the inhi-
bition. This agrees with the previous observation that sulfonamide A
in these compounds is the key groups coordinated with Zn2þ
.
Compound 27 with the 3-sulfonamide and 4-methyl group at
benzene was synthesized from hit compound 26. It has a high
inhibitory effect against CA IX (IC50 of 0.48 nM) and the selectivity
over cytosolic ones (CA I and CA II) exceeded 103 times. This shows
that 4-methyl contributed deeply to the inhibition and selectivity
against CA IX. Compound 28, with the 4-sulfonamide and 3-methyl
group at benzene, shows about 6 times higher inhibitory activity
toward CA IX (IC50 of 3.3 nM) than the hit 26. But the CA IX
selectivity ratio of compound 28 over the cytosolic ones (CA I and
CA II) is poorer than that of compound 26, which was at the same
level with the clinical drug AZA. From Table 1, the selectivity ratio of
AZA and EZA between CA IX and CA II is in the range of 4.9e19.3,
which prohibited it from clinical trials as CA IX inhibitors.
Chart 1. Clinically used sulfonamides as CAs inhibitors.
2. Results and discussion
2.1. Structure-based SAR and docking analysis
In order to rationalize the affinity between these novel in-
hibitors and Zn2þ in CA IX catalytic sites, we studied their binding
modes within the active binding site of CA IX (PDB: 3IAI) by using
By inspecting the protein structure of CA IX (PDB ID: 3IAI) and the
virtual action mode of the hit compound, several clues were
obtained for the structure optimization. Sulfonamides A of com-
pound 26 can bind with Zn2þ in the active site. Apart from chelating
with Zn2þ, the coordinated sulfonamide nitrogen atom forms
a hydrogen bond with the hydroxyl group of Thr199, and one of the
oxygen atoms of the sulfonamide moiety forms hydrogen bond with
the backbone amide of Thr199 as well. These hydrogen bonds con-
tribute tothe bindingof these inhibitors bind tothe active site. At the
hydrophobic region, the benzene moieties can interact with Val121,
Val143 and Leu198. While at the hydrophilic part of the pocket, the
linker NH could forms hydrogen bonds with the side chains of His64.
Besides the key sulfonamide group for Zn2þ coordination, other
structural modifications could also potentially affect the in-
teractions between the hydrophobic residues and hydrophilic resi-
dues of CA IX and inhibitors, as indicated by molecular modeling.
There are two sulfonamides in the hit compound 26, and the
type of the inhibitors has been reported by C. T. Supuran [24] (Chart
3). Firstly, to identify which sulfonamide coordinated to Zn2þ, the
sulfonamide A was replaced by other substituents such as eCl, eCF3
and eOCH3, while sulfonamide B was retained. This led to com-
pounds 15e18. The position of sulfonamide on benzene might also
affect the activity. Therefore, compounds 19e23 with sulfonamide
B switched to the meta position were developed. Biological activity
evaluation showed that 15e23 had much poor inhibitory activity
against CA IX, CA I and CA II (Table 1), therefore sulfonamide B at
meta or para position couldn’t coordinate with Zn2þ in CAs active
site, and sulfonamide A in hit compound 26 should be the key
sulfonamide. (Chart 4).
molecular docking method. Poor inhibition effect (IC50 > 10 mM) of
compounds 15e23 for CA IX may result from the steric hindrance,
which affects the access of sulfonamides B to the Zn2þ (Table 1).
Instead, sulfonamides A of compounds 24e25 can bind with Zn2þ
in the active site. In common with compound 26, the coordinated
sulfonamide nitrogen atom forms a hydrogen bond with the hy-
droxyl group of Thr199, and one of the oxygen atoms of the sul-
fonamide moiety forms hydrogen bond with the backbone amide of
Thr199 as well. At the hydrophobic region, the benzene moieties
interact with Val121, Val143 and Leu198, and small hydrophobic
groups such as methyl contribute remarkably to the higher affinity.
So, by adding methyl at the ortho position of the left benzene-
sulfonamide, two potent CA IX inhibitors (compounds 27, 28 with
IC50 of 0.48, 3.3 nM) are obtained. While at the hydrophilic part of
the pocket, the linker NH forms hydrogen bonds with the side
chains of His64, Asn62 (compound 27) and leads to the high af-
finities with CA IX (Fig. 1).
The differences of affinity of some inhibitors for the different
isozymes have been evidenced [25]. CA II is the most susceptible to
inhibition by sulfonamides, whereas CA I has generally a lower
affinity for this type of inhibitors and a much larger one for the
inorganic anions, such as cyanide, cyanate, thiocyanate. Therefore,
we focused on the selectivity with CA IX over CA II. As for the high
selectivity of compound 27, superposition of the CA II and CA IX in
Fig. 2 clearly reveals that the amino acid residue 131 of the active
sites is different. This residue is known to be very important for the
binding of sulfonamide inhibitors to CA II [26]. The phenyl group of
phenylalanine locates in the CA II might prevent the interaction
with compound 27, while CA IX has a valine at the position, whose
side chain is much smaller in terms of sterics. Consequently, com-
pound 27 can bind CA IX much better than CA II.
2.2. Synthesis of the target compounds
The target compounds 15e28 were synthesized according to
Scheme 1. The starting material 2,3-dihydrophthalazine-1,4-dione
1 reacted with phosphorus oxybromide in 1,2-dichloroethane to
yield 1,4-dibromophthalazine 2 [27], which further reacted with
different sulfanilamide in the presence of Na2CO3 in DMF to afford
4-((4-bromophthalazin-1-yl)amino)benzenesulfonamide 3 and 4.
By coupling 3 (or 4) with suitable phenylboronic acid derivatives,
the corresponding products 15e23 were obtained. As for the
Chart 2. The binding mode of the benzenesulfonamide with the CA II active site.

R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
599
Chart 3. The structures of lead compound 26 and designed two novel series of compounds.
compounds 24e26, three different aniline derivatives were used to
react with compound 1 in DMF, and the products 12e14 coupled
with the corresponding benzenesulfonamide boronates 10, 11 [28]
and 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfo-
namide in DME and water. Chlorosulfonation of 3-bromotoluene
and 4-bromotoluene at 0 ꢁC followed by ammonolysis using
aqueous ammonium hydroxide in dioxane afforded sulfonamide (8,
9) in good yield. Boronates 10 and 11 were obtained by palladium-
catalyzed borylation of aryl bromide 8, 9 with bis(pinacolato)
diboron. Compounds 27 and 28 were obtained similarly to com-
pounds 24e26 by Suzuki Coupling. Biological activity of com-
pounds 15e28 and positive samples AZA, EZA against CA I, CA II, CA
IX were studied and shown in Table 1.
a Bruker AV-500 spectrometer with chemical shifts reported in
ppm (in Aceton-d6/DMSO-d6/CDCl3, TMS as an internal standard).
Mass spectra were measured on a HP 1100 LCeMS spectrometer.
Melting points were determined by an X-6 micro-melting point
apparatus and uncorrected.
4.1. Synthesis
4.1.1. Synthesis of 1,4-dibromophthalazine (2)
Phosphorus oxybromide (861 mg, 3.0 mmol) was added to
a stirred suspension of 2,3-dihydrophthalazine-1,4-dione (162 mg,
1.0 mmol) in 1,2-dichloroethane (5 mL). The mixture was heated to
100 ꢁC for 18 h and poured into water (100 mL) upon cooling to rt
The aqueous layer was neutralized with saturated Na2CO3 solution
and concentrated to give a residue, which was washed with water
to afford the product as a white solid. Yield: 88%, m.p. 160.1e
3. Conclusion
161.6 ꢁC. 1H NMR (400 MHz, CDCl3)
d (ppm): 8.37e8.33 (m, 2H),
In this paper, some potent CA IX inhibitors with high selectivity
against other carbonic anhydrase isozymes (CA I and CA II) were
designed and synthesized by structural optimization of a previ-
ously discovered lead compound. Compound 27 is the most potent
CA IX inhibitor with an IC50 of 0.48 nM and exhibits higher inhib-
itory effects over CA I and CA II. At the same time, the structuree
activity relationship and CA IX selectivity for the newly synthe-
sized compounds are analyzed by docking analysis, in which the
introduction of methyl group on benzene can improve the activity
and selectivity efficiently due to the favorable interactions with
some specific amino acid residues of CA IX catalytic sites. The hit
compounds with novel scaffold discovered in this work lay the
foundation for further development of therapeutic candidates for
cancer treatment.
8.12e8.08 (m, 2H). GCeMS (EI): m/z calc. 285.8, m/z found 285.8.
4.1.2. Synthesis of 4-((4-bromophthalazin-1-yl)amino)
benzenesulfonamide (3)
To a mixture of 1,4-dibromophthalazine (286 mg, 1.0 mmol) and
4-aminobenzenesulfonamide (172 mg, 1.0 mmol) in DMF (10 mL)
was added potassium carbonate (276 mg, 2.0 mmol). The reaction
was heated at 90 ꢁC overnight. The reaction mixture was distilled
under reduced pressure to remove most of the DMF, and the
remaining residue was partitioned between CH2Cl2 (250 mL) and
saturated ammonium chloride solution (50 mL). The water layer
was extracted with CH2Cl2 (2 ꢂ 100 mL). The combined organic
layers were washed with brine (50 mL), dried over MgSO4, and
evaporated under reduced pressure. Purification was performed by
flash chromatography on silica gel (CH2Cl2) to give the title com-
pound as a yellow crystal. Yield: 20%, 1H NMR (400 MHz, DMSO-d6)
4. Chemistry
d
(ppm): 12.83 (s, 1H), 8.35 (d, J ¼ 8.0 Hz, 1H), 8.10e8.05 (m, 1H),
All starting materials and reagents were of analytic grade and
used without further purification. 1H NMR was measured on
8.00e7.95 (m, 2H), 7.67 (d, J ¼ 8.8 Hz, 2H), 6.60 (d, J ¼ 8.8 Hz, 2H),
5.93 (s, 2H). LC-MS (ESI): C14H12BrN4O2S calc. 379.9, found 380.0.
Table 1
Inhibitory activities of the compounds against CA I, CA II and CA IX.
4.2. General procedure for compounds 15e23
Compounds
IC50(nM)
CA I
Selectivity ratios
4.2.1. 4-((4-(4-Chlorophenyl)phthalazin-1-yl) amino)
CA II
CA IX
I/IX
II/IX
benzenesulfonamide (15)
15
16
17
18
19
20
21
22
23
24
25
26
27
28
EZA
AZA
>104
>104
>104
>104
>104
3930
>104
>104
690
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
>104
>104
A mixture of 4-((4-bromophthalazin-1-yl)amino)benzenesulfo-
namide (189 mg, 0.5 mmol), 4-chlorophenylboronic acid (76 mg,
0.5 mmol) and sodium carbonate (106 mg, 1.0 mmol) in ethanol
(3 mL), water (3 mL) and toluene (10 mL) was degassed with Ar for
10 min. Then tetrakis(triphenylphosphine)palladium (30 mg,
0.025 mmol) was added. The reaction mixture was stirred at 80 ꢁC
under nitrogen for 8 h. After cooling to rt, the reaction mixture was
distilled under reduced pressure to remove the solvent, and
extracted with ethyl acetate (3 ꢂ 5 mL), water (5 mL). The organic
layer was washed with an aqueous saturated solution of NaHCO3
(2 ꢂ 5 mL), dried over Na2SO4, and concentrated to dryness under
reduced pressure. Purification was effected by flash chromatogra-
phy on silica gel (eluted with 0e1% methanol in CH2Cl2) to provide
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
28.3
29.0
18.5
0.48
3.3
220.2 ꢃ 13.1
61.8 ꢃ 3.1
146.5 ꢃ 6.8
>104
455.1 ꢃ 3.2
547.0 ꢃ 34.9
26.8 ꢃ 9.0
4.8
/
/
>104
>102
>102
>103
>104
25.7
270.6
66.0
5.3
>102
24.5
1139.7
8.1
19.3
4.9
>104
>104
>104
85.7 ꢃ 4.1
67.6
0.25
7.7
509.6
37.5

600
R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
Chart 4. Novel designed benzenesulfonamides from structural optimization of the hit compound.
the title compound as a pale yellow solid. Yield: 35%, m.p. 230.8e
231.6 ꢁC. 1H NMR (400 MHz, CDCl3)
(ppm): 12.63 (s, 1H), 8.66e
J ¼ 7.6 Hz, 1H), 7.71 (d, J ¼ 8.8 Hz, 2H), 7.68e7.66 (m, 1H), 6.62e6.60
(m, 2H), 5.93 (s, 2H). 13C NMR (100 MHz, DMSO-d6):
153.08,
d
d
8.64 (m, 1H), 7.87 (d, J ¼ 8.4 Hz, 2H), 7.84 (t, J ¼ 8.4 Hz, 2H), 7.77e
149.44, 138.60, 135.22, 133.44, 132.00, 131.90, 130.97, 129.27, 129.16,
128.62, 128.26, 127.39, 126.95, 126.76, 126.00, 113.01. HRMS (ESI):
C21H16F3N4O2S calc. 445.0946, found 445.0943.
7.75 (m, 1H), 7.55 (s, 4H), 6.70 (d, J ¼ 8.4 Hz, 2H), 5.32 (s, 2H). 13C
NMR (100 MHz, DMSO-d6): d153.06, 149.68, 149.43, 135.16, 134.87,
133.35, 131.84, 129.28, 129.17, 128.60, 128.30, 127.51, 127.04, 126.98,
126.72, 113.01. HRMS (ESI): C20H16ClN4O2S calc. 411.0683, found
411.0676.
4.2.3. 4-((4-Phenylphthalazin-1-yl)amino)benzenesulfonamide (17)
According to the general procedure, compound 3 was treated
with phenylboronic acid and reacted for 4 h. Then the crude
product was purified by flash chromatography on silica gel using
CH2Cl2 to provide compound 17. Pale yellow solid, yield: 33%. 1H
4.2.2. 4-((4-(4-(Trifluoromethyl)phenyl)phthalazin-1-yl)amino)
benzenesulfonamide (16)
According to the general procedure, compound 3 was treated
with 4-(trifluoromethyl)phenylboronic acid and reacted for 10 h.
Then the crude product was purified by flash chromatography on
silica gel (eluted with 0e1% methanol in CH2Cl2) to provide com-
pound 16. Yellow solid, yield: 38%, m.p. 206.1e207.6 ꢁC. 1H NMR
NMR (400 MHz, CDCl3)
d
(ppm): 12.61 (s, 1H), 8.65 (d, J ¼ 8.4 Hz,
1H), 7.86 (d, J ¼ 8.0 Hz, 2H), 7.81 (d, J ¼ 4.8 Hz, 2H), 7.61e7.58 (m,
2H), 7.58e7.55 (m, 3H), 6.69 (d, J ¼ 8.4 Hz, 2H), 4.11 (s, 2H). 13C
NMR (100 MHz, DMSO-d6):
d 153.03, 150.74, 149.48, 135.14, 134.47,
133.70, 133.28, 132.51, 132.00, 129.94, 129.28, 128.59, 127.20,
126.71, 113.03. HRMS (ESI): C20H17N4O2S calc. 377.1072, found
377.1080.
(400 MHz, DMSO-d6)
d (ppm): 12.90 (s, 1H), 8.47e8.45 (m, 1H), 8.09
(t, J ¼ 7.2 Hz, 1H), 8.01e7.98 (m, 2H), 7.96 (d, J ¼ 7.2 Hz, 2H), 7.88 (d,
Fig. 1. The potential binding modes of compounds 26 (left), and 27 (right) against CA IX (PDB code: 3IAI). Protein and the key residues were colored in light pink and green
respectively, and the docked molecules were colored in cyan. The Gray sphere represented the zinc ions. The hydrogen bonds and coordinate interactions were shown with red
dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
601
13C NMR (100 MHz, DMSO-d6):
d
150.19, 148.78, 143.67, 135.45,
134.95, 134.62, 133.51, 133.27, 131.87, 129.89, 129.20, 128.18, 127.64,
127.14, 126.83, 118.23, 114.10, 111.73. HRMS (ESI): C20H16ClN4O2S
calc. 411.0683, found 411.0686.
4.2.7. 3-((4-(4-(Trifluoromethyl)phenyl)phthalazin-1-yl)amino)
benzenesulfonamide (21)
According to the general procedure, compound 4 was treated
with 4-(trifluoromethyl)phenylboronic acid and reacted for 10 h.
Then the crude product was purified by flash chromatography on
silica gel (eluted with 0e1% methanol in CH2Cl2) to provide com-
pound 21. Brown solid, yield: 33%, m.p. 176.7e176.9 ꢁC. 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 13.11 (s, 1H), 8.37 (d, J ¼ 6.8 Hz, 1H),
8.11 (t, J ¼ 7.2 Hz, 1H), 8.03e7.96 (m, 3H), 7.72e7.54 (m, 2H), 7.21e
7.13 (m, 4H), 6.75 (d, J ¼ 7.2 Hz, 1H), 5.55 (s, 2H). 13C NMR (100 MHz,
DMSO-d6):
d 149.71, 143.32, 136.19, 135.14, 134.61, 132.00, 131.90,
131.00, 129.81, 129.29, 129.17, 128.82, 128.17, 127.12, 126.07, 124.41,
124.37, 117.76, 113.37, 111.15. HRMS (ESI): C21H16F3N4O2S calc.
445.0946, found 445.0941.
4.2.8. 3-((4-Phenylphthalazin-1-yl)amino)benzenesulfonamide (22)
According to the general procedure, compound 4 was treated
with phenylboronic acid and reacted for 4 h. Then the crude
product was purified by flash chromatography on silica gel using
CH2Cl2 to provide compound 22. Yellow solid, yield: 26%. 1H NMR
Fig. 2. Superpositions of the crystal structures of the CA II and the CA IX. The binding
poses of the compound 27 (cyan stick) were predicted by the molecular docking. The
pink structure was CA IX (PDB code: 3IAI); the green structure was CA II (PDB code:
3R16); Gray spheres represented the zinc ions. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
(400 MHz, CDCl3)
d (ppm): 12.67 (s, 1H), 8.69e8.66 (m, 1H), 7.87e
7.81 (m, 3H), 7.62e7.56 (m, 6H), 7.45 (d, J ¼ 7.6 Hz, 1H), 7.39 (s, 1H),
6.83 (d, J ¼ 8.0 Hz, 1H), 4.11 (s, 2H). 13C NMR (100 MHz, DMSO-d6):
d
151.19, 150.20, 149.69, 143.67, 135.40, 134.54, 133.90, 133.42,
4.2.4. 4-((4-(3-Methoxyphenyl)phthalazin-1-yl)amino)
benzenesulfonamide (18)
130.47, 129.97, 129.80, 129.09, 127.81, 127.29, 126.84, 117.68, 113.40,
111.19. HRMS (ESI): C20H17N4O2S calc. 377.1072, found 377.1070.
According to the general procedure, compound 3 was treated
with 3-methoxyphenylboronic acid and reacted for 4 h. Then the
crude product was purified by flash chromatography on silica gel
using CH2Cl2 to provide compound 18. Pale yellow solid, yield: 29%,
4.2.9. 3-((4-(3-Methoxyphenyl)phthalazin-1-yl)amino)
benzenesulfonamide (23)
According to the general procedure, compound 4 was treated
with 3-methoxyphenylboronic acid and reacted for 4 h. Then the
crude product was purified by flash chromatography on silica gel
using CH2Cl2 to provide compound 23. Pale yellow solid, yield: 35%,
m.p. 208.5e209.6 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 12.79
(s, 1H), 8.45 (d, J ¼ 7.6 Hz, 1H), 8.00e7.92 (m, 2H), 7.72 (t, J ¼ 8.4 Hz,
3H), 7.50 (t, J ¼ 8.0 Hz,1H), 7.18e7.14 (m, 3H), 6.61 (d, J ¼ 8.8 Hz, 2H),
5.91 (s, 2H), 3.82 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
d 159.71,
m.p. 245.0e245.4 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 12.98
153.05, 150.57, 149.48, 135.76, 135.14, 133.28, 132.48, 132.00, 131.90,
130.21, 129.28, 129.16, 128.60, 127.24, 126.67, 122.17, 113.01,
55.75. HRMS (ESI): C21H19N4O3S calc. 407.1178, found 407.1171.
(s, 1H), 8.48 (d, J ¼ 7.6 Hz, 1H), 8.03e7.95 (m, 2H), 7.76 (d, J ¼ 7.6 Hz,
1H), 7.51 (t, J ¼ 8.0 Hz,1H), 7.20 (d, J ¼ 7.6 Hz, 2H), 7.18e7.15 (m, 4H),
6.76e6.74 (m, 1H), 5.52 (s, 2H), 3.82 (s, 3H). 13C NMR (100 MHz,
DMSO-d6):
d 159.72, 151.05, 150.02, 149.70, 143.65, 135.70, 135.43,
4.2.5. 3-((4-(4-Methoxyphenyl)phthalazin-1-yl)amino)
benzenesulfonamide (19)
133.45, 130.25, 129.80, 127.82, 127.35, 126.80, 122.20, 117.67, 115.71,
115.41, 113.38, 111.17, 55.77. HRMS (ESI): C21H19N4O3S calc. 407.1178,
found 407.1180.
Accordingtothegeneralprocedure, compound4wastreatedwith
4-methoxyphenylboronic acid and reacted for 4 h. Then the crude
product was purified by flash chromatography on silica gel using
CH2Cl2 to provide compound 19. Yellow solid, yield: 20%, m.p. 257.5e
4.2.10. Synthesis of 4-bromo-2-methylbenzenesulfonyl chloride (6)
and 5-bromo-2-methylbenzensulfonyl chloride (7)
258.1 ꢁC.1H NMR (400 MHz, CDCl3)
d (ppm): 12.67 (s,1H), 8.69e8.66
Chlorosulfonic acid (696 mg, 6.0 mmol) was added slowly to
a cold solution (0 ꢁC) of 3-bromotoluene (170 mg, 1.0 mmol) (or 4-
bromotoluene (170 mg, 1.0 mmol)), in CHCl3 (10 mL). The reaction
was allowed to proceed for 4 h with stirring at 0 ꢁC prior to pouring
onto crushed ice (250 g). The resulting mixture was extracted with
CHCl3 (3 ꢂ 150 mL). The combined CHCl3 extracts were washed
with water (3 ꢂ 100 mL) and dried with Na2SO4, and the solvent
was removed in vacuum to yield the arylsulfonyl chloride product
(6 or 7), which was used in next step without purification.
(m,1H), 7.87e7.81 (m, 3H), 7.62e7.56 (m, 6H), 7.45 (d, J ¼ 7.6 Hz,1H),
7.39 (s, 1H), 6.83 (d, J ¼ 8.0 Hz, 1H), 4.11 (s, 2H). 13C NMR (100 MHz,
DMSO-d6):
d 160.28, 150.61, 149.36, 133.91, 132.33, 131.32, 129.30,
127.99, 127.44, 126.53, 125.77, 116.83, 116.46, 114.38, 113.73, 111.56,
55.76. HRMS (ESI): C21H19N4O3S calc. 377.1072, found 377.1070.
4.2.6. 3-((4-(4-Chlorophenyl)phthalazin-1-yl)amino)
benzenesulfonamide (20)
According to the general procedure, compound 4 was treated
with 4-chlorophenylboronic acid and reacted for 8 h. Then the
crude product was purified by flash chromatography on silica gel
(eluted with 0e1% methanol in CH2Cl2) to provide compound 20.
Brown solid, yield: 28%, m.p. 189.1e190.3 ꢁC. 1H NMR (400 MHz,
4.2.11. Synthesis of 4-bromo-2-methylbenzenesulfonamide (8) and
5-bromo-2-methylbenzenesulfonamide (9)
An aqueous solution of NH4OH (15 mL of 30% w/v) was added to
a cold solution of the arylsulfonyl chloride (6 or 7, 268 mg,
1.0 mmol) in dioxane (10 mL) at 0 ꢁC with vigorous stirring, and the
reaction was allowed to proceed for 6 h at 0 ꢁC with stirring. The
insoluble material was removed by filtration, washed with water
DMSO-d6)
d
(ppm): 13.04 (s, 1H), 8.47 (d, J ¼ 7.2 Hz, 1H), 8.37 (d,
J ¼ 8.0 Hz, 1H), 8.12 (t, J ¼ 7.2 Hz, 1H), 8.04e7.96 (m, 3H), 7.72 (d,
J ¼ 7.6 Hz,1H), 7.21e7.13 (m, 4H), 6.75 (t, J ¼ 3.2 Hz,1H), 5.55 (s, 2H).

602
R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
Scheme 1. The route for the synthesis of compounds 15e28. Reagents and reaction conditions: (a) POBr3, C2H4Cl2, 100 ꢁC, 18 h; (b1eb3) 4-Aminobenzenesulfonamide, 3-Amino-
benzene- sulfonamide, aniline derivatives, K2CO3, DMF, 90 ꢁC, 12 h; (c) Arylboronic acid, Na2CO3, ethanol, water, toluene, Pd(PPh3)4, 80 ꢁC, 8 h; (d) Arylboronic acid pinacol ester,
K2CO3, DME, H2O, Pd(PPh3)4, reflux, 16 h; (e) Chlorosulfonic acid, CHCl3, 0 ꢁC, 4 h; (f) NH4OH, dioxane, 0 ꢁC, 6 h; (g) Bis(pinacolato)diboron, KOAc, PdCl2(dppf), dioxane, reflux, 16 h.
(3 ꢂ 10 mL). The organic layer was concentrated and recrystallized
with 50% acetone in hexane to yield the product.
nitrogen. Subsequently the mixture was cooled to room tempera-
ture and filtered through Celite. The filtrate was concentrated and
the residue was purified by flash chromatography on silica gel
eluted with 0e40% ethyl acetate in hexane to give compounds 10
and 11 as a white solid.
4-Bromo-2-methylbenzenesulfonamide (8). White solid, yield:
85%, m.p. 176e177 ꢁC 1H NMR (400 MHz, DMSO-d6)
d (ppm): 7.76
(d, J ¼ 8.4 Hz,1H), 7.65 (s,1H), 7.60 (dd, J1 ¼ 2.0, J2 ¼ 8.8 Hz,1H), 7.52
(s, 2H), 2.58 (s, 3H).
2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benze-
5-Bromo-2-methylbenzenesulfonamide (9). White solid, yield:
nesulfonamide (10). White solid, yield: 62%. 1H NMR (400 MHz,
90%, m.p. 164e165 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 7.95
DMSO-d6)
7.45 (s, 2H), 2.60 (s, 3H), 1.31 (s, 12H).
d
(ppm): 7.86 (d, J ¼ 8.0 Hz, 1H), 7.63 (d, J ¼ 4.8 Hz, 2H),
(d, J ¼ 2.0 Hz,1H), 7.70 (dd, J1 ¼ 2.0, J2 ¼ 8.0 Hz,1H), 7.56 (s, 2H), 7.36
(d, J ¼ 8.0 Hz, 1H), 2.54 (s, 3H).
2-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benze-
nesulfonamide (11). White solid, yield: 66%. 1H NMR (400 MHz,
4.2.12. The synthesis of 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl) benzenesulfonamide(10) and 2-methyl-5-(4,4,5,5
-tetramethyl-1,3,2-dioxaborolan-2-yl) benzenesulfonamide (11)
DMSO-d6)
d
(ppm): 8.18 (s, 1H), 7.74 (d, J ¼ 7.6 Hz, 1H), 7.41 (s, 1H),
7.39 (s, 2H), 2.62 (s, 3H), 1.31 (s, 12H). General Procedure for com-
pounds 24e27.
To
a mixture of 4-bromo-2-methylbenzenesulfonamide 8
(249 mg, 1.0 mmol) or 5-bromo-2-methylbenzenesulfonamide 9
(249 mg, 1.0 mmol), bis(pinacolato)diboron (381 mg, 1.5 mmol),
and KOAc (392 mg, 4 mmol) in 10 mL of dioxane was added
PdCl2(dppf) (40 mg, 0.05 mmol). After bubbling nitrogen for
15 min, the reaction mixture was heated up to reflux for 16 h under
4.2.13. General procedure for the synthesis of 3-(4-((4-
aminosulfamoylphenyl)amino) phthalazin -1-yl)
benzenesulfonamide (24)
A mixture of N-(4-bromophthalazin-1-yl)benzene-1,4-diamine
(157 mg, 0.5 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
603
yl)benzenesulfonamide (142 mg, 0.5 mmol) and potassium car-
4.2.17. 2-Methyl-4-(4-((4-sulfamoylphenyl)amino)phthalazin-1-yl)
benzenesulfonamide (28)
bonate (138 mg, 1.0 mmol) in DME (20 mL), water (10 mL) was
degassed with Ar for 10 min. Then tetrakis(triphenylphosphine)
palladium (30 mg, 0.025 mmol) was added. The mixture was
heated to reflux for 12 h under Ar atmosphere. Subsequently, the
mixture was cooled to rt and diluted with dichloromethane and
washed with brine. The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel eluted with 0e5%
methanol in dichloromethane to give the title compound as
a brown solid. Yield: 73%, m.p. 266.1e267.2 ꢁC. 1H NMR (400 MHz,
According to the general procedure, compound 3 was treated
with compound 10 and reacted for 24 h. Then the crude product
was purified by flash chromatography on silica gel (eluted with 0e
1% methanol in CH2Cl2) to provide compound 28. Yellow solid,
yield: 46%, m.p. >300 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
8.46 (d, J ¼ 7.2 Hz, 1H), 8.03 (d, J ¼ 7.6 Hz, 1H), 7.99e7.96 (m, 1H),
7.70 (d, J ¼ 8.0, 4H), 7.64 (s, 1H) 7.54 (s, 2H), 7.56 (s, 1H), 6.61 (d,
J ¼ 9.6 Hz, 2H), 5.91 (s, 2H), 2.68 (s, 3H). HRMS (ESI): C21H20N5O4S2
calc. 470.0957, found 470.0956.
DMSO-d6)
d
(ppm): 9.02 (s, 1H), 8.63 (d, J ¼ 8.4 Hz, 1H), 8.12 (s,
1H), 7.94e8.02 (m, 2H), 7.90 (t, J ¼ 8.0 Hz, 1H), 7.83 (d, J ¼ 8.0 Hz,
1H), 7.76 (t, J ¼ 7.6 Hz, 1H), 7.49 (s, 1H), 7.47 (s, 2H), 6.62 (d,
J ¼ 8.4 Hz, 2H), 4.92 (s, 2H). 13C NMR (100 MHz, DMSO-d6):
4.3. Biology assay
An Applied Photophysics stopped-flow instrument was used to
test the CA-catalyzed CO2 hydration activity. Phenol red (at a con-
centration of 0.2 mM) has been used as the indicator, working at the
absorbance maximum of 557 nm. The 10e20 mM HEPES (pH 7.5) or
Tris (pH 8.3) buffer solutions with 20 mM Na2SO4 or 20 mM NaClO4
was used as solvents. The rate of the CA-catalyzed CO2 hydration
reaction for a period of 10e100 s was monitored. The CO2 con-
centrations ranged from 1.7 to 17 mM for the determination of the
kinetic parameters and inhibition constants. For each inhibitor, at
least six traces of the initial 5e10% of the reactions have been used
for determining the initial rates. The uncatalyzed rates were
determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (10 mM) were prepared
in distillededeionized water, and diluted up to 0.01 nM were per-
formed thereafter with distillededeionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min at rt prior
to the assay study, in order to allow for the formation of the EeI
complex. The half maximal inhibitory concentration (IC50) was
obtained by nonlinear least-squares methods using PRISM 3 as
reported earlier and represented the mean from at least three dif-
ferent determinations. CA isoforms were recombinant ones
obtained as described previously [29,30].
d
152.92, 151.32, 145.34, 144.85, 138.20, 133.31, 132.59, 132.00,
129.64, 129.53, 127.10, 125.90, 125.84, 125.53, 124.45, 123.22,
118.54, 114.22. HRMS (ESI): C20H18N5O2S calc. 392.1181, found
392.1177.
4.2.14. 3-(4-((4-(tert-Butyl)phenyl)amino)phthalazin-1-yl)
benzenesulfonamide (25)
According to the general procedure, compound 13 was treated
with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfo-
namide and reacted for 24 h. Then the crude product was purified by
flash chromatography on silica gel (eluted with 0e1% methanol in
CH2Cl2) to provide compound 25. Yellow solid, yield: 15%, m.p.
>300 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 9.43 (s,1H), 8.80 (d,
J ¼ 8.4 Hz,1H), 8.14 (s,1H), 8.05 (t, J ¼ 7.2 Hz,1H), 7.95e8.00 (m, 2H),
7.89 (d, J ¼ 8.8 Hz, 2H), 7.78 (t, J ¼ 8.0 Hz, 1H), 7.52 (s, 2H), 7.49 (d,
J¼ 7.2Hz, 2H), 3.99 (s, 2H),1.07(s, 9H).13CNMR(100MHz, DMSO-d6):
d
152.70,152.30,145.24,144.90,138.37,137.95,133.37,132.87,132.20,
129.67, 127.16, 126.08, 125.92, 125.67, 125.43, 123.67, 121.57, 118.81,
34.48, 31.78. HRMS (ESI): C24H25N4O2S calc. 433.1698, found
433.1703.
4.2.15. 3-(4-((4-Sulfamoylphenyl)amino)phthalazin-1-yl)
benzenesulfonamide (26)
Initial rates of 4-nitrophenyl acetate hydrolysis catalyzed by
different CA isozymes were monitored spectrophotometrically, at
400 nm, with a Cary 3 instrument interfaced with an IBM-
compatible PC. Solutions of substrate were prepared in anhydrous
ethanol; the substrate concentrations were 1 mM at 25 ꢁC. A molar
absorption coefficient of 400 Mꢀ1 cmꢀ1 was used for the 4-nitro-
phenolate formed byhydrolysis in the conditions of the experiments
(pH 7.5). Nonenzymatic hydrolysis rates were subtracted from the
observed rates. The experiments were repeated three times at each
inhibitor concentration. Stock solutions of the inhibitor (50 mM)
were prepared (which was not inhibitory at these concentrations)
and diluted up to 0.5 nM with DMSO. At least 8 different inhibitor
According to the general procedure, compound 14 was trea-
ted with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benze-
nesulfonamide and reacted for 12 h. Then the crude product was
purified by flash chromatography on silica gel (eluted with 0e1%
methanol in CH2Cl2) to provide compound 26. Yellow solid,
yield: 39%. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.47 (d,
J ¼ 7.2 Hz, 1H), 8.07 (s, 1H), 8.03 (d, J ¼ 8.0 Hz, 1H), 8.00e7.96
(m, 1H), 7.88 (d, J ¼ 8.0 Hz, 1H), 7.80 (t, J ¼ 7.6 Hz, 1H), 7.73e
7.67 (m, 3H), 7.52 (s, 1H), 6.61 (d, J ¼ 8.8 Hz, 2H), 5.92
(s, 2H). HRMS (ESI): C20H17N5O4NaS2 calc. 478.0620, found
478.0624.
concentrations have been used, ranging from 5 mM, 0.5 mM, 50
mM,
5
m
M, 0.5 M, 50 nM, 5 nM, 0.5 nM. Inhibitor and enzyme solutions
m
4.2.16. 2-Methyl-5-(4-((4-sulfamoylphenyl)amino)phthalazin-1-yl)
benzenesulfonamide (27)
were preincubated together for 15 min at rt prior to the assay study,
to allow for the formation of the EeI complex. Enzyme concentra-
tions were 1 ng/L for CA II, 50 ng/L for CA I [31].
According to the general procedure, compound 3 was treated
with compound 11 and reacted for 24 h. Then the crude product
was purified by flash chromatography on silica gel (eluted with 0e
1% methanol in CH2Cl2) to provide compound 27. Yellow solid,
yield: 53%, m.p. 238.4e239.9 ꢁC. 1H NMR (400 MHz, DMSO-d6)
4.4. Molecular modeling
The lead inhibitor, compound 26, was obtained through virtual
screening in our previous work (unpublished). The protein struc-
tures of CA IX, and CA II were derived from PDB, with the PDB
entries 3IAI and 3R16, respectively. Protein preparation was per-
formed to optimize the protein structures using Glide module in
Maestro 9.0 (Schrödinger Inc), and all the water molecules were
removed. The grid-enclosing box was formed based on the ligand in
the complex structure, and other parameters were set up by
default. These structures of two compounds were’ drawn using the
d
(ppm): 12.87 (s, 1H), 8.46 (d, J ¼ 7.2 Hz, 1H), 8.09 (d, J ¼ 1.6 Hz,
1H), 7.98e7.95 (m, 1H), 7.76 (dd, J1 ¼ 1.6, J2 ¼ 7.6 Hz, 1H), 7.70 (d,
J ¼ 8.4 Hz, 2H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.54 (s, 2H), 6.61 (d,
J ¼ 9.2 Hz, 2H), 5.92 (s, 2H), 5.76 (s, 2H), 2.71 (s, 3H). 13C NMR
(100 MHz, DMSO-d6):
d 153.00, 149.51, 142.93, 137.82, 135.16,
133.38, 133.17, 133.07, 132.38, 128.61, 128.38, 127.42, 126.90,
126.82, 112.95, 20.22. HRMS (ESI): C21H20N5O4S2 calc. 470.0957,
found 470.0959.

604
R. Gao et al. / European Journal of Medicinal Chemistry 62 (2013) 597e604
[13] J.T. Winum, M. Rami, A. Scozzafava, J.L. Montero, C.T. Supuran, Carbonic
anhydrase IX: a new druggable target for the design of antitumor agents, Med.
Res. Rev. 28 (2008) 445e463.
[14] V. Alterio, A.D. Fiore, K.D. Ambrosio, C.T. Supuran, G.D. Simone, Multiple
binding modes of inhibitors to carbonic anhydrases: how to design specific
drugs targeting 15 different isoforms? Chem. Rev. 112 (2012) 4421e4468.
[15] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar,
C.T. Supuran, Ureido-substituted benzenesulfonamides potently inhibit car-
bonic anhydrase IX and show antimetastatic activity in a model of breast
cancer metastasis, J. Med. Chem. 54 (2011) 1896e1902.
Builder tool and then optimized with Ligprep module in Maestro,
both were docked into each protein mentioned above using Glide
with standard precision (SP) approach, and a scaling factor of 1.0
was set to van der Waals (VDW) radii of those protein atoms with
the partial atomic charges less than 0.25. For each protein, the best
20 binding poses of each compound ranked by GlideScore were
remained for further inspection.
[16] Y. Lou, P.C. McDonald, A. Oloumi, S.K. Chia, C. Ostlund, A. Ahmadi, Targeting
tumor hypoxia: suppression of breast tumor growth and metastasis by novel
carbonic anhydrase IX inhibitors, Cancer Res. 8 (2011) 3364.
Acknowledgments
[17 C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for in-
hibitors and activators, Nat. Rev. Drug Discov. 7 (2008) 168.
[18] B.W. Clare, C.T. Supuran, A perspective on quantitative structureeactivity
relationships and carbonic anhydrase inhibitors, Expert Opin. Drug Metab.
Toxicol. 2 (2006) 113.
[19] V. Alterio, A. Di Fiore, K.D. Ambrosio, C.T. Supuran, G. De Simone, in: Drug
Design of Zinc-enzyme Inhibitors: Functional, Structural, and Disease Appli-
cations, Wiley, Hoboken, NJ, 2009, p. 73.
[20] A. Scozzafava, F. Briganti, M.A. Ilies, C.T. Supuran, Carbonic anhydrase in-
hibitors: synthesis of membrane-impermeant low molecular weight sulfon-
amides possessing in vivo selectivity for the membrane-bound versus
cytosolic isozymes, J. Med. Chem. 43 (2000) 292e300.
This work was supported by the Fundamental Research Funds
for the Central Universities, the National Natural Science Founda-
tion of China (grants 21173076, 81102375, 81222046, 81230090 and
81230076), the Shanghai Committee of Science and Technology
(grants 11DZ2260600 and 10431902600), the Special Fund for
Major State Basic Research Project (grant 2009CB918501), and the
863 Hi-Tech Program of China (grant 2012AA020308). Honglin Li is
also sponsored by Program for New Century Excellent Talents in
University (grant NCET-10-0378). We thank Ph.D Houqi Liu at the
Suzhou Institute for Advanced Study, University of Science and
Technology of China (USTC), for providing the Applied Photophysics
SX 20 stopped-flow instrument.
[21] D. Vullo, M. Franchi, E. Gallori, J. Pastorek, A. Scozzafava, S. Pastorekova,
C.T. Supuran, Carbonic anhydrase inhibitors: inhibition of the tumor-
associated isozyme IX with aromatic and heterocyclic sulfonamides, Bioorg.
Med. Chem. Lett. 13 (2003) 1005e1009.
[22] D. Vullo, A. Innocenti, I. Nishimori, J. Pastorek, S. Pastorekova, A. Scozzafava,
C.T. Supuran, Carbonic anhydrase inhibitors. Inhibition of the transmembrane
isozyme XII with sulfonamides-a new target for the design of antitumor and
antiglaucoma drugs? Bioorg. Med. Chem. Lett. 15 (2005) 963e969.
[23] C.T. Supuran, A. Scozzafava, J. Conway, in: Carbonic Anhydrase -Its Inhibitors
and Activators, CRC, Boca Raton, 2004, pp. 67e147.
[24] A. Casini, F. Abbate, A. Scozzafava, C.T. Supuran, Carbonic anhydrase in-
hibitors: X-ray crystallographic structure of the adduct of human isozyme II
with a bis-sulfonamide-two heads are better than one? Bioorg. Med. Chem.
Lett. 13 (2003) 2759e2763.
[25] C.T. Supuran, Carbonic Anhydrase and Modulation of Physiologic and Patho-
logic Processes in the Organism, Helicon, Timisoara, 1994, pp. 29e111.
[26] F. Abbate, A. Casini, A. Scozzafava, C.T. Supuran, Carbonic anhydrase in-
hibitors: X-ray crystallographic structure of the adduct of human isozyme II
with the perfluorobenzoyl analogue of methazolamide. Implications for the
drug design of fluorinated inhibitors, J. Enzym. Inhib. Med. Chem. 18 (2003)
303e308.
References
[1] A. Scozzafava, A. Mastrolorenzo, C.T. Supuran, Carbonic anhydrase inhibitors
and activators and their use in therapy, Expert Opin. Ther. Pat. 16 (2006)
1627e1664.
[2] C.T. Supuran, A. Scozzafava, J. Conway, Carbonic Anhydrase: Its Inhibitors and
Activators, CRC, Boca Raton, 2004, pp. 1e363.
[3] C.T. Supuran, A. Scozzafava, A. Casini, Carbonic anhydrase inhibitors, Med. Res.
Rev. 23 (2003) 146e189.
[4] K.S. Smith, J.G. Ferry, Prokaryotic carbonic anhydrases, FEMS, Microbiol. Rev.
24 (2000) 335e366.
[5] C.T. Supuran, Carbonic anhydrases as drug targets-an overview, Curr. Top.
Med. Chem. 7 (2007) 825e833.
[6] C.T. Supuran, A. Di Fiore, G. De Simone, Carbonic anhydrase inhibitors as
emerging drugs for the treatment of obesity, Expert Opin. Emer. Drugs 13
(2008) 383e392.
[7] C.T. Supuran, Diuretics: from classical carbonic anhydrase inhibitors to novel
applications of the sulfonamides, Curr. Pharm. Des. 14 (2008) 641e648.
[8] G. De Simone, C.T. Supuran, Antiobesity carbonic anhydrase inhibitors, Curr.
Top. Med. Chem. 7 (2007) 879e884.
[9] J. Chiche, K. Ilc, J. Laferriere, E. Trottier, F. Dayan, N.M. Mazure, M.C. Brahimi-
Horn, J. Pouyssegur, Hypoxia-inducible carbonic anhydrase IX and XII pro-
mote tumor cell growth by counteracting acidosis through the regulation of
the intracellular pH, Cancer Res. 69 (2009) 358e368.
[27] A. Hirscfi, D. Orphanos, Quantitative preparation of chloro-and bromoph-
thalazines, Can. J. Chem. 43 (1965) 2708e2710.
[28] X. Li, Y.K. Zhang, Y. Liu, S. Zhang, C.Z. Ding, Y. Zhou, J.J. Plattner, S.J. Baker,
L. Liu, W. Bu, W.M. Kazmierski, L.L. Wright, G.K. Smith, R.L. Jarvest, M. Duan,
J.J. Ji, J.P. Cooper, M.D. Tallant, R.M. Crosby, K. Creech, Z.J. Ni, W. Zou, J. Wright,
Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV
NS3 protease, Bioorg. Med. Chem. Lett. 20 (2010) 7493e7497.
[29] I. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mastrolorenzo, C.T. Supuran,
Carbonic anhydrase inhibitors: inhibition of the transmembrane isozyme XIV
with sulfonamides, Bioorg. Med. Chem. Lett. 15 (2005) 3828e3833.
[30] I. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mastrolorenzo,
C.T. Supuran, Carbonic anhydrase inhibitors. The mitochondrial isozyme VB as
a new target for sulfonamide and sulfamate inhibitors, J. Med. Chem. 48
(2005) 7860e7866.
[10] P. Swietach, R. Vaughan-Jones, A. Harris, Regulation of tumor pH and the role
of carbonic anhydrase 9, Cancer Metastasis Rev. 26 (2007) 299e310.
[11] J. Chiche, M. Brahimi-Horn, J. Pouysségur, Tumour hypoxia induces a meta-
bolic shift causing acidosis: a common feature in cancer, J. Cell. Mol. Med. 14
(2010) 771e794.
[31] E. Truppo, C.T. Supuran, A. Sandomenico, D. Vullo, A. Innocenti, Carbonic
anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity
for sulfonamide inhibitors, Bioorg. Med. Chem. Lett. 22 (2012) 1560e1564.
[12] R. Martinez-Zaguilan, E. Seftor, R. Seftor, Y. Chu, R. Gillies, M. Hendrix, Acidic
pH enhances the invasive behavior of human melanoma cells, Clin. Exp.
Metastasis 14 (1996) 176e186.