Acyl sulfonamide anti-proliferatives: Benzene substituent structure-activity relationships for a novel class of antitumor agents

Article abstract of DOI:10.1021/jm030594r

Two closely related diaryl acylsulfonamides were recently reported as potent antitumor agents against a broad spectrum of human tumor xenografts (colon, lung, breast, ovary, and prostate) in nude mice. Especially intriguing was their activity against colorectal cancer xenografts. In this paper, rapid parallel synthesis along with traditional medicinal chemistry techniques were used to quickly delineate the structure-activity relationships of the substitution patterns in both phenyl rings of the acylsufonamide anti-proliferative scaffold. Although the molecular target of the compounds remains unclear, we determined that the vascular endothelial growth factor-dependent human umbilical vein endothelial cells assay in combination with a soft agar disk diffusion assay allowed for optimization of potency in the series. The pharmacokinetic properties and in vivo activity in an HCT116 xenograft model are reported for representative compounds.

Full text of DOI:10.1021/jm030594r

J . Med. Chem. 2004, 47, 5367-5380
5367
Acyl Su lfon a m id e An ti-P r olifer a tives: Ben zen e Su bstitu en t Str u ctu r e-Activity
Rela tion sh ip s for a Novel Cla ss of An titu m or Agen ts
Karen L. Lobb,*,† Philip A. Hipskind,† J ames A. Aikins,† Enrique Alvarez,†,‡ Yiu-Yin Cheung,§
Eileen L. Considine,† Alfonso De Dios,| Gregory L. Durst,† Rafael Ferritto,| Cora Sue Grossman,†
Deborah D. Giera,† Beth A. Hollister, Zhongping Huang,§ Philip W. Iversen,† Kevin L. Law,† Tiechao Li,†
Ho-Shen Lin,† Beatriz Lopez,| J ose E. Lopez,† Luisa M. Martin Cabrejas,| Denis J . McCann,† Victoriano Molero,|
J ohn E. Reilly,§ Michael E. Richett,† Chuan Shih,† Beverly Teicher,†,# J ames H. Wikel,† Wesley T. White,† and
Mary M. Mader*,†
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, Albany Molecular Research Inc.,
Albany, New York 12212, Centro de Investigacio´n Lilly, S. A., Avda. de la Industria 30, 28108 Alcobendas, Madrid, Spain,
and Piedmont Research Center, 3300 Gateway Centre, Morrisville, North Carolina 27560
Received November 25, 2003
Two closely related diaryl acylsulfonamides were recently reported as potent antitumor agents
against a broad spectrum of human tumor xenografts (colon, lung, breast, ovary, and prostate)
in nude mice. Especially intriguing was their activity against colorectal cancer xenografts. In
this paper, rapid parallel synthesis along with traditional medicinal chemistry techniques were
used to quickly delineate the structure-activity relationships of the substitution patterns in
both phenyl rings of the acylsufonamide anti-proliferative scaffold. Although the molecular
target of the compounds remains unclear, we determined that the vascular endothelial growth
factor-dependent human umbilical vein endothelial cells assay in combination with a soft agar
disk diffusion assay allowed for optimization of potency in the series. The pharmacokinetic
properties and in vivo activity in an HCT116 xenograft model are reported for representative
compounds.
Sch em e 1
In tr od u ction
Every year, colorectal cancer (CRC) leads to the
deaths of approximately 400,000 people worldwide. In
2004, the American Cancer Society predicts that in the
United States alone approximately 57,000 people will
die from CRC and over 106,000 new cases of CRC will
be diagnosed.1 Thirty percent of CRC patients will have
regional or distal metastases at the time of diagnosis.
Distal metastasis of CRC almost uniformly predicts
eventual death, and well over half of the patients
diagnosed with regional metastases will die from the
disease. Before the 1980s, 5-fluorouracil (5-FU) was the
only treatment option for advanced CRC. At present,
several new chemotherapeutic agents (irinotecan, capecit-
abine, and oxaliplatin) have been developed that offer
an improved outcome for metastatic CRC patients.
Nevertheless, even with the best combination chemo-
therapy regimens available, median survival for ad-
vanced CRC patients is still only about 16 months.2
Recently, we reported the discovery of two closely
related diaryl acylsulfonamides (1 and 2, Scheme 1) that
have the potential to produce a new treatment for
CRC.3,4 Historically, sulfonamides have been associated
with antibacterialchemotherapy;however,theseacylsulfon-
amides demonstrated no activity when tested against
a variety of bacterial strains.4 Compounds 1 and 2 were
discovered in a collaborative drug discovery effort be-
tween Wayne State University (WSU) and Lilly Re-
search Laboratories. In this collaboration, a diverse set
of over 14,000 compounds computationally selected5
from Lilly’s chemical archives was screened using an
in vitro soft agar disk diffusion (SADD) assay developed
by Dr. Thomas Corbett of WSU.6-9 The SADD assay was
designed to identify small molecules with selective anti-
proliferative activity against solid tumor cell lines (such
as the CRC cell lines HCT116, HCT15, and C38) vs leu-
kemia cells (L1210) and normal human fibroblast cells.
In vivo evaluation of 1 and 2 revealed remarkable
antitumor effects against a broad range of human tumor
xenografts in SCID mice.4 Especially intriguing was
their activity against human colon cancer xenografts
(colon H116, colon 15, and colon CX1 H29), which are
known to exhibit resistance to 5-FU and other marketed
oncolytics. At this time, a molecular target for these
compounds remains undetermined. Fortunately, early
in our assessment of 1 and 2, it was discovered that both
compounds potently inhibit vascular endothelial growth
factor (VEGF)-stimulated proliferation of human um-
bilical vein endothelial cells (HUVEC)10 while producing
no cell cycle specific effects at 2-fold IC50 in the HCT116
* To whom correspondence should be addressed. (K.L.L.) Tel: 317-
276-6894. Fax: 317-433-1685. E-mail: Lobb_Karen@Lilly.com. (M.M.M.)
Tel: 317-277-3400. Fax: 317-277-2035. E-mail: Mader_Mary@Lilly.com.
† Lilly Research Laboratories.
‡ Current address: CuraGen Corporation, 322 East Main Street,
Branford, CT 06405.
§ Albany Molecular Research Inc.
| Centro de Investigacio´n Lilly.
Piedmont Research Center.
Current address: Genzyme Corporation, One Kendall Square,
#
Cambridge, MA 02139.
10.1021/jm030594r CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/23/2004

5368 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Sch em e 2. Synthesis of Substituted Acylsulfonamidesa
Lobb et al.
a
Conditions: (a) Two equiv of PS-carbodiimide, 1.5 equiv of
DMAP, in CH2Cl2, 72 h at room temperature followed by 3.0 equiv
of MP-TsOH, 12 h at room temperature. (b) EDC, DMAP, CH2Cl2.
(c) CDI, EtOAc, 60 °C.
cell assay.11 With this information in hand, we sought
to explore the structure-activity relationships (SAR) of
these leads with the aim of optimizing potency and
antitumor activity, especially activity against colorectal
xenografts in vivo.
F igu r e 1. Accumulation of actives vs the rank order of the
compounds assayed for a set of compounds never seen by the
model. The first line is the upper bound (i.e., ideal or perfect
ranking), the last line is the random rate, and the middle line
is the model accumulation rate.
This article describes the trends for substituent effects
on each of the two phenyl rings in our initial leads,
named the acyl sulfonamide anti-proliferative (ASAP)
compounds. The VEGF-HUVEC assay was used to
evaluate the anti-proliferative potencies of the com-
pounds and drive the SAR. In addition, the compounds
were screened in the SADD assay to ensure that the
series continued to exhibit selective activity toward CRC
cell lines vs leukemia and normal fibroblast cell lines.
The results of the National Cancer Institute’s (NCI) 60
cell line COMPARE analysis are reported for compounds
1 and 2. Both compounds demonstrated a negative
correlation toward all other known oncolytics, indicating
a unique mechanism of action.
vided a product that could be purified by recrystalliza-
tion rather than column chromatography.
Use of a n in Silico Qu a n tita tive
Str u ctu r e-Activity Rela tion sh ip (QSAR) Mod el
to P r ior itize Ta r gets
After the SAR effort began, a QSAR model was built
to analyze the VEGF-HUVEC activity data and to aid
in the prioritization of target molecules. The preliminary
model was built from 135 HUVEC IC50 values, and as
the effort progressed, the model was refined by the
inclusion of new SAR data. A final set of 710 compounds
was used in the last iteration of the model. In all
iterations, the IC50 values ranged in activity from 0.02
to 20 µM.
Ch em istr y
The initial exploration of the SAR of compounds 1 and
2 was performed via rapid parallel synthesis (RPS)
using commercially available starting materials (Scheme
2). We used a modification of a published coupling
As an example of the model’s predictivity, Figure 1
shows the accumulation of actives vs rank order for a
set of 570 molecules not used to build the model. This
example is from the model built with 710 IC50 values
(five way cross-validated spearman F ) 0.57). The plot
shows that the model predicts the top 114 compounds
(<1 µM) out of 570 at a better rate than random. A
measure of this predictivity can be described by a metric
called an A50, where the A50 is the number of com-
pounds that must be tested to find 50% of the designated
actives. The A50 for this model is 120 vs 283 for
randomly assigning activity, an enrichment greater
than 2. In the absence of information regarding the
identity or structure of our biological target, this model
provided initial direction to the SAR effort. With a large
number of possible combinations of sulfonamides and
benzoic acids available to us, the model indicated that
the best compounds should be found in the top 20% of
a ranked list. We were, therefore, able to focus our
efforts on a more specific set of substituents and sub-
stitution patterns; although to test the model’s validity,
we also made selected examples that were predicted to
be less active.
procedure for sulfonamides and carboxylic acids.12-14
A
resin-bound carbodiimide was employed as the coupling
reagent,15 along with 4-(dimethylamino)pyridine (DMAP)
as the catalyst and methylene chloride as the solvent.
A tosic acid resin was added to scavenge the DMAP and
protonate the acidic16 acylsulfonamide final products
after a 72 h reaction time.17-20 To isolate the final
products, the two resins were simply filtered off, and
the solvent was removed. Crude reaction mixtures were
purified via ultraviolet (UV)-guided high-performance
liquid chromatography (HPLC).
Derivatives requiring starting materials that were not
commercially available were synthesized using tradi-
tional medicinal chemistry techniques. The required
benzenesulfonamides were generally synthesized from
benzenenesulfonyl chlorides, and the benzoic acids were
prepared using standard literature procedures. Con-
densation of a sulfonamide with a benzoic acid remained
the preferred mode of synthesis, using 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
as the coupling agent. This method was found to be
cleaner and higher yielding than the reaction of sulfonic
acids with benzamides or sulfonyl chlorides with ben-
zamides. Alternatively, on a larger scale, the use of
carbonyldiimidazole (CDI) as the coupling agent pro-
Biologica l Assa ys
VEGF -HUVEC Assa y. The final compounds were
assayed in the VEGF-HUVEC assay to assess their anti-

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5369
Ta ble 1. SAR of Various Substituents on Benzoyl Ring
proliferative potencies.10 This assay measures the in-
hibition of the VEGF-stimulated proliferation of
HUVEC. The minimum significant ratio (MSR) for
comparing average VEGF-HUVEC IC50 values with n
g 3 was calculated to be 2.6.21 That is, the average IC50
values of two compounds can be considered statistically
significantly different if the ratio of the two averages is
greater than 2.6.
compd
no.
average VEGF-HUVEC
R′
2,4-diCl
H
2-Cl
3-Cl
4-Cl
2-CH3
3-Br
4-NO2
IC50 (µM)a
2
3
4
5
6
7
8
9
0.17 ( 0.03
22 ( 0.9
14 ( 3
SADD Assa y. Compounds were also evaluated in the
SADD assay. The SADD assay was designed to identify
small molecules with selective anti-proliferative activity
toward solid tumor cell lines (such as the CRC cell lines
HCT116, HCT15, and C38) vs leukemia cells (L1210)
and normal human fibroblast cells. The SADD assay
was run as described by Corbett et al.6-9 Tumor cells,
leukemia cells, and normal cells were seeded in soft agar
in separate Petri dishes. A round filter paper, on which
the compound had been applied, was placed on top of
the agar. The activity was determined by measuring the
magnitude of the zone of inhibition for cell colony
formation created by the filter paper disk in the various
Petri dishes. A zone of inhibition measuring 6.5 mm was
equal to 200 units. A zero zone meant no inhibition. The
limit of the dish (total inhibition) was 950 zone units.
Ideally, an active compound would show little or no
inhibition of the normal cells, would minimally inhibit
the colony formation of the leukemia cells, and would
maximally inhibit the colony formation of the various
solid tumor cells.
14 ( 2
6.3 ( 0.8
15 ( 3
15b ( 3
11 ( 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34c
35
4-N(CH3)2
3,4-diCl
2,5-diCl
16 ( 5
5.9 ( 0.8
13b ( 7
3,5-diCl
18b ( 4
2-Cl, 4-Br
2-Br, 4-Cl
2,4-diBr
0.20 ( 0.06
0.24 ( 0.04
0.25 ( 0.03
4.6 ( 1.8
0.30 ( 0.04
0.61 ( 0.09
0.62 ( 0.18
0.63 ( 0.17
1.3 ( 0.5
2.7 ( 0.3
2.0 ( 0.1
3.2 ( 0.8
1.4 ( 0.4
5.9b ( 3
11 ( 3
2-F, 4-Br
2-CH3, 4-Cl
2-CH3, 4-Br
2-Cl, 4-NO2
2-Et, 4-Cl
2-Pr, 4-Cl
2,4-diCH3
2-OCH3, 4-Cl
2-NH2, 4-Cl
2,4-bis(CF3)
2-NO2, 4-Cl
2-NO2, 4-CF3
2-NO2, 4-NO2
2-Cl, 4-CN
2-Cl, 4-SO2CH3
2-Cl, 4-OCH3
2-Cl, 4-OH
2-Cl, 4-NH2
2,4-diOCH3
13 ( 2
10 ( 4
Resu lts a n d Discu ssion
>20
9.9 ( 2.1
>15
Compound 2 was chosen as the benchmark compound
for our SAR because it was slightly more potent in the
VEGF-HUVEC assay (IC50 ) 0.17 µM). First, we
investigated the substitutent patterns on the benzoyl
side of the molecule by coupling a variety of carboxylic
acids to 4-chlorobenzenesulfonamide (Scheme 2). Sec-
ond, the substituents on the benzenesulfonamide ring
were examined by coupling an assortment of benzene-
sulfonamides to 2,4-dichlorobenzoic acid. To ensure that
a wide variety of lipophilic, electronic, and steric pa-
rameters were explored, many of the benzoic substitu-
tions found in the Topliss Decision Tree were included
in the SAR.22,23
Ben zoyl SAR. As we investigated the substituent
requirements of the benzoyl ring (Table 1), we found
that the unsubstituted derivative (3) and various mono-
substituted analogues [4, 2-Cl; 5, 3-Cl; 6, 4-Cl; 7, 2-CH3;
8, 3-Br; 9, 4-NO2; and 10, 4-N(CH3)2], all exhibited
substantially reduced potencies compared to the bench-
mark compound (2), which was 2,4-disubstituted. In-
deed, the 2,4-disubstitution pattern found on the ben-
zoyl ring of the lead compounds, 1 and 2, generally
achieved the highest potencies (compounds 14-21).
Other disubstitution patterns, such as in 11 (3,4-diCl),
12 (2,5-diCl), and 13 (3,5-diCl), produced compounds
that were significantly less active than those with the
2,4-disubstitution pattern.
>15
>20
a
b
Shown with standard error. n g 3 unless indicated. n ) 2.
c Trifluoroacetic salt.
allowed substituents. For example, compounds 18 (2-
CH3, 4-Cl), 19 (2-CH3, 4-Br), 20 (2-Cl, 4-NO2), and 21
(2-CH2CH3, 4-Cl) were nearly as active as the dihalide
series. The 2-fluoro-4-bromo analogue (17, IC50 ) 4.6
µM) proved to be an exception to the general success of
halogens, but this may be due to the fluorine atom’s
small size and the decreased polarizability of the
fluoride relative to chloride and bromide.
Small alkyl groups were well-tolerated if placed at the
2-position of the 2,4-disubstituted pattern. The 2-meth-
yl-4-chloro derivative (18) and the 2-methyl-4-bromo
derivative (19) demonstrated anti-proliferative IC50
values of less than 0.65 µM. The influence of steric
interactions was apparent within a series of 2-alkyl-
substituted analogues where the activity declined as the
total number of carbons increased (cf. 18, 2-CH3, 4-Cl;
21, 2-CH2CH3, 4-Cl; and 22, 2-CH2CH2CH3, 4-Cl).
Derivatives with alkyl groups at the 2- and 4-positions,
such as compound 23 (IC50 ) 2.7 µM), were not quite
as potent as the 2-alkyl derivatives with a halogen group
at the 4-position (18, 19, and 21).
Halogen groups were among the favored substituents
at both the 2- and the 4-positions of the disubstituted
pattern. The most potent compounds of the series were
the variously substituted dihalides (2, 2,4-diCl; 14, 2-Cl,
4-Br; 15, 2-Br, 4-Cl; and 16, 2,4-diBr). In addition, the
halogens fared well when in combination with other
Strongly electron-donating groups were generally
detrimental at the 2-position. Compound 24 (2-OCH3,
4-Cl) demonstrated an IC50 of 2.0 µM while its corre-
sponding alkyl analogue, 21 (2-CH2CH3, 4-Cl), had an
IC50 of 0.63 µM, and compound 25 (2-NH2, 4-Cl) was
over 10-fold less active than either 18 (2-CH3, 4-Cl) or

5370 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Lobb et al.
Ta ble 2. SAR of Various Substituents on Benzenesulfonamide
Ring
Halogens at the 3- and/or 4-positions (2, 37, 38,
42-45, and 49) led to numerous examples of potent
compounds. Alkyl groups, as in the 4-methyl derivative
(36), the 3-methyl derivative (48), and the 4-tert-butyl
derivative (40), were also acceptable. A library of 3- and
4-substituted biaryl analogues was prepared, with 53
and 56 shown as representative examples. These com-
pounds were active, consistent with the sulfonamide
half of the molecule apparently having less demanding
steric requirements. Electron-donating groups at the 3-
or 4-position produced excellent to good anti-prolifera-
tive potencies. For instance, compounds 39 (4-OCH3),
46 (4-SCH3), 51 (3-OCH3), and 52 [4-N(CH3)2] exhibited
IC50 values between 0.35 and 0.93 µM. Analogues with
electron-withdrawing groups at these positions (47,
4-acetyl; 50, 3-NO2; 54, 4-CO2CH3; and 55, 4-NO2) were
slightly less potent and demonstrated IC50 values
between 0.56 and 1.3 µM.
Generally, the 4-substituted benzenesulfonamide de-
rivatives were 2-3-fold more potent than those substi-
tuted at the 3-position with the same atom or functional
group. For example, the IC50 of the 4-chloro analogue
(2) was 0.17 µM, whereas that for the 3-chloro compound
(44) was 0.44 µM. This phenomenon was observed for
the 4- vs 3-methyl analogues (36, 4-CH3, IC50 ) 0.21
µM vs 48, 3-CH3, IC50 ) 0.75 µM) as well as the methoxy
pair (39, 4-OCH3, IC50 ) 0.35 µM vs 51, 3-OCH3, IC50
) 0.91 µM). However, the bromide pair, 37 and 38, did
not follow this trend, having essentially identical IC50
values of 0.24 and 0.25 µM, respectively.
compd
no.
average VEGF-HUVEC
R
IC50 (µM)a
1
2
H
0.53 ( 0.02
0.17 ( 0.03
0.21 ( 0.04
0.24 ( 0.07
0.25 ( 0.08
0.35 ( 0.07
0.35 ( 0.12
0.36 ( 0.19
0.41 ( 0.12
0.41 ( 0.08
0.44 ( 0.14
0.46 ( 0.07
0.53 ( 0.23
0.56 ( 0.12
0.75 ( 0.03
0.91 ( 0.22
0.91 ( 0.16
0.91 ( 0.29
0.93 ( 0.38
0.97 ( 0.18
1.2 ( 0.1
4-Cl
4-CH3
4-Br
3-Br
4-OCH3
4-tert-butyl
3-Cl, 4-CH3
3-Cl, 4-Fl
4-F
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
3-Cl
3,4-diCl
4-SCH3
4-acetyl
3-CH3
3,4-diBr
3-NO2
3-OCH3
4-N(CH3)2
3-Ph
4-CO2CH3
4-NO2
4-Ph
3,4-diOCH3
2-Cl
2-Cl, 5-NO2
2-CH3, 5-NO2
2-Br, 4-CH3
3,5-diCl
1.3 ( 0.4
1.4b ( 0.6
3.9b ( 0.3
14b ( 2
>20
16b ( 0.04
>20
1.4 ( 0.4
Synergistic increases in activity were not found for
3,4-disubstitution of the benzenesulfonamide. The di-
substituted analogues were either comparable in activ-
a
b
Shown with standard error. n g 3 unless indicated. n ) 2.
ity to the 3-substituted analogues (cf. 44, 3-Cl, IC50
0.44 µM vs 45, 3,4-diCl, IC50 ) 0.46 µM and 42, 3-Cl,
4-F, IC50 ) 0.41 µM) or less active (cf. 38, 3-Br, IC50
)
2 (2, 4-diCl). The strongly electron-withdrawing nitro
group also decreased potency when placed at the 2-posi-
tion. Compounds 27 (2-NO2, 4-Cl), 28 (2-NO2, 4-CF3),
and 29 (2-NO2, 4-NO2) were all less active than the
corresponding analogues, 2 (2,4-diCl), 26 [2,4-bis(CF3)],
and 20 (2-Cl, 4-NO2). The bis(trifluoromethyl) derivative
(26) exhibited an IC50 of 1.4 µM. Although trifluoro-
methyl groups are electron-withdrawing (σp ) 0.54, π
) 0.88), they are similar in lipophilicity to the favored
halogens (Cl, π ) 0.71; Br, π ) 0.86), which might
explain this compound’s relative potency.24
At the 4-position, the nitro group was better tolerated
than other strongly electron-withdrawing groups. For
example, compounds 30 (2-Cl, 4-CN) and 31 (2-Cl,
SO2CH3) exhibited potencies 15-fold less than compound
20 (2-Cl, 4-NO2, IC50 ) 0.62 µM). Electron-donating
groups, however, were universally unfavorable at the
4-position as can be seen by comparing compounds 32
(2-Cl, 4-OCH3), 33 (2-Cl, 4-OH), and 34 (2-Cl, 4-NH2)
with compound 2 (2,4-diCl) and by comparing compound
35 (2,4-diOCH3) with compound 24 (2-OCH3, 4-Cl).
Ben zen esu lfon a m id e SAR . The benzenesulfon-
amide side of the molecule was less stringent in the
substituent requirements needed to produce active
compounds (Table 2). Compound 1, one of our initial
leads, bears no substituents and was quite potent with
an IC50 of 0.53 µM. Our SAR demonstrates that mono-
substitution at the 3- or 4-position along with the 3,4-
disubstitution pattern usually produced the most potent
analogues (36-57). A variety of substituents at these
positions worked well.
)
0.25 µM vs 49, 3,4-diBr, IC50 ) 0.91 µM and 51, 3-OCH3,
IC50 ) 0.91 µM vs 57, 3,4-diOCH3, IC50 ) 3.9 µM). The
reasons for this phenomenon are not obvious. The
decreased activity of the disubstituted analogues could
possibly be attributed to the greater hydrophobicity of
these compounds relative to the monosubstituted ana-
logues and a subsequent decrease in the ability to
penetrate the HUVEC cell membrane. Examination of
clogP25,26 for representative compounds (Table 3) indi-
cates that this parameter also plays a role in the activity
of the compounds. Hydrophilic compounds with a clogP
less than 2.5 tended to be less potent (cf. 9 and 10), and
a clogP ranging from 3.0 to 4.0 is observed for the more
potent compounds (cf. 2, 14, 36, 37, and 39). However,
a clogP between 3.0 and 4.0 does not ensure activity in
the HUVEC assay (cf. 58, 8, 59, and 13).
Substitution at the 2-position of the benzenesulfon-
amide ring was not tolerated and substantially dimin-
ished the anti-proliferative activity. For example, the
2-chloro derivative (58, Table 2) was over 25-fold less
potent than compound 1, which has no substituents. In
addition, the 2-chloro-5-nitro derivative (59) and the
2-methyl-5-nitro derivative (60) were over 10-fold less
active than the 3-nitro derivative (50), and the 2-bromo-
4-methyl derivative (61) was almost 100-fold less active
than the 4-methyl derivative (36). Last, the activity of
the 3,5-dichloro derivative (62, IC50 ) 1.4 µM) was
unremarkable as compared to the 3-chloro derivative

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5371
Ta ble 3. Correlation of VEGF-HUVEC and SADD Data
SADD assay
zone of inhibitions in various cell lines
compd
no.
VEGF-HUVEC
average IC50 (µM)
solid tumor
HCT116a
leukemia
L1210a
normal
dose
(µg/disk)
R
R′
clogP
fibroblasta,b
2
4-Cl
4-Cl
4-CH3
4-Br
4-OCH3
3-Cl, 4-CH3
4-F
3-Cl
3,4-diCl
4-Cl
3-CH3
3,4-diBr
4-N(CH3)2
3-Ph
4-CO2CH3
4-NO2
4-Cl
3,5-diCl
4-Ph
4-Cl
3,4-diOCH3
4-Cl
4-Cl
4-Cl
2-Cl
4-Cl
4-Cl
2,4-diCl
2-Cl, 4-Br
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2-CH3, 4-Br
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-diCl
2,4-bis(CF3)
2,4-diCl
2,4-diCl
2-NH2, 4-Cl
2,4-diCl
3,4-diCl
4-Cl
3.8
3.9
3.4
3.9
3.1
4.1
3.3
3.8
4.4
3.5
3.4
4.6
3.2
4.8
3.0
3.0
4.3
4.4
4.8
2.7
3.1
3.8
3.1
2.3
3.8
3.2
2.5
3.6
3.8
0.17
0.20
0.21
0.24
0.35
0.36
0.41
0.44
0.46
0.61
0.75
0.91
0.93
0.97
1.2
1.3
1.4
1.4
1.4
3.2
3.9
5.9
6.3
660
460c,d
840
620
650
570
880
515
560
200
200
0-350
0-350
0-510
0-430
0-420
0-380
0-520
0-200
0-360
0-300c
0-200
0-270
0-400c
0-370
0-280
0-310
0-360c
0-200c
0-290
270
200
210
190
210
190
190
200
190
200
190
210
190
210
190
200
190
200
210
210
210
210
200
190
190
210
190
190
200
210
14
36
37
39
41
43
44
45
19
48
49
52
53
54
55
26
62
56
25
57
11
6
0-320
100-170
0-170
200-350
0-280
100-150
40-180
0c
700-750c
220-250
425
100
0-210
0-350c
0
900c
600
650c
540
0-200
0-110
0c
520c
380c
500
0-170c
220-250
0-250
0
610
500c
400c
330c
270c
0c
0-200
0-150c
0-70c
0c
200c
180c
9
58
8
10
59
13
4-NO2
2,4-diCl
3-Br
4-N(CH3)2
2,4-diCl
3,5-diCl
11
14
15
16
>20
18
200c
0c
0c
320c
360c
0
210c
0-30c
180c
0c
2-Cl, 5-NO2
4-Cl
0
0
330c
150c
0-80c
a
Zone of inhibition units (200 units ) 6.5 mm). A zero zone means no inhibition. The limit of the dish (total inhibition) was 950 zone
units. In cases where a zone range was recorded, the colonies were reduced in size at the beginning of the zone, increasing in size to the
top of the zone range, and from there to the edge of the plate, the colonies were uniformly large. n > 2 unless otherwise indicated. See
b
refs 4, 7, and 8 for examples of other agents tested in the SADD assay. The fibroblast cells were viable, but the replication of colonies
d
was inhibited as indicated by their zone ranges. Many in vivo active agents display microcolony zones of this type. c n ) 1. Human
tumor H15 (colon).
(44, IC50 ) 0.44 µM) indicating that the 3,5-disubstitu-
tion pattern does not offer any improvements in potency
for the benzenesulfonamide ring.
HUVEC assay is an appropriate predictor of the ASAP
compounds’ oncolytic activity in vivo.
P h a r m a cok in etic (P K) P r op er ties. Compounds 1
and 14 were pursued in further in vivo studies based
on their potency in the HUVEC and SADD assays.
Because of the acidity of the acylsulfonamide moiety (1,
pKa ) 2.8; 14, pKa ) 2.7), the compounds were formu-
lated in 1-1.5% sodium bicarbonate in phosphate-
buffered saline solution, and exposure was observed by
both oral and iv administration (Table 4). Exposure by
oral administration was observed in rats for both 1 and
14, and no significant accumulation of 1 was observed
after 7 days of dosing relative to a single dose. The doses
for the two compounds were those determined to be
optimal with a minimal toxicity for each. Analysis of
rat plasma showed no metabolites after 7 days of dosing
compound 1, indicating 1 to be metabolically stable.
Compound 1 was excreted unchanged in rat urine and
feces, with a total 29.1% of a single 100 mg/kg dose
recovered in urine after 48 h and a total of 8.4% found
in feces. Oral dosing in mice over 10 days resulted in a
2-4-fold higher exposure and a longer half-life for both
compounds. The administration of 1 and 14 to mice by
iv resulted in shorter half-lives and lower exposures
relative to oral dosing.
SADD a n d VEGF -HUVEC Da ta . The results from
the semiquantitative SADD assay demonstrated that,
in general, the ASAP derivatives continued to exhibit
selective inhibition toward CRC cell lines, such as
HCT116, vs leukemia (L1210) and normal fibroblast
cells just as the lead compounds, 1 and 2, had shown
(Table 3). In addition, upon analysis of the VEGF-
HUVEC data and the SADD data, the minimum VEGF-
HUVEC potency necessary for significant activity in the
SADD assay was delineated. Derivatives with a VEGF-
HUVEC IC50 value of less than 1.0 µM were consider-
ably more likely to exhibit substantial inhibitions of the
HCT116 cell line’s colony formation in the SADD assay.
For example, out of 187 compounds tested at 200 ( 10
µg/disk, 79% of the compounds with a VEGF-HUVEC
IC50 of less than 1 µM produced a zone of inhibition of
500 zone units (16.2 mm) or better in the HCT116 cell
line. Of those compounds with a VEGF-HUVEC IC50 of
greater than 1 µM, only 12% generated a zone of
inhibition of greater than 500 zone units in the HCT116
cell line. This agreement between the SADD data and
the VEGF-HUVEC data indicates that the VEGF-

5372 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Ta ble 4. PK Properties of ASAP Compounds in Rodents
Lobb et al.
compd
no.
dose
(mg/kg)
days of
dosing
species
(route)
AUC (0-24 h)
(µg h/mL)
T1/2
(h)
Cmax
(µg/mL)
R1
H
R2
Cl
R3
Cl
1
100
100
80
80
10
1
7
10
10
7
rata (po)
2023c
2917
11300
10100
4200
3
4
239
221
568
628
200
547
262
rata (po)
mouseb (po)
mouseb (iv)
rata (po)
27
13
59
132
28
14
Cl
Cl
Br
80
20
10
10
mouseb (po)
mouseb (iv)
10700
4670
a
For female F344 rats; the compounds were formulated in 1-1.5% sodium bicarbonate in phosphate-buffered saline; n ) 3; once daily
b
dosing as indicated; blood drawn at t ) 0.5, 1, 2, 4, 6, 8, and 24 h after the final dose. For BALBc mice; the compounds were formulated
in 1-1.5% sodium bicarbonate in phosphate-buffered saline; n ) 3 BALBc mice/time point; blood was drawn at t ) 0.5, 1, 2, 4, 6, 8, and
24 h after the final dose. c AUC (0-infinity).
Ta ble 5. TGD of Selected Compounds in HCT116 Xenograft
Model in Nude Mice
dose (iv)
(mg/kg)
TGD
(days ( SE)
compd
CPT11
100
64
85
128
32
63
15 ( 8
14 ( 7
15 ( 7
22 ( 6
12 ( 6
23 ( 6
1
14
HCT116 Tu m or Xen ogr a ft Mod el. Compounds 1
and 14 were evaluated in a nude mouse xenograft model
bearing human HCT116 colorectal carcinoma (Table 5
and Figure 2). After tumors had reached an average
volume of 100 mm3, the compound was administered
intravenously as its sodium salt in a saline solution (pH
7.4) from days 1-5 and days 8-12. A marketed oncolytic
approved for the treatment of CRC, CPT 11 (irinotecan),
was employed as a positive control, at 100 mg/kg, and
was dosed according to its reported optimal route and
schedule (ip, days 1, 8, and 15).27 Compound 1 was
observed to show activity at three doses (64, 85, and
128 mg/kg), and the plot of the data appears to show a
dose response (Figure 2), which is not reflected in Table
5 data due to the interanimal variability. Compound 14
showed a significantly different tumor growth delay
(TGD) at two doses (32 and 63 mg/kg). In addition, 14
exhibited TGD at the 63 mg/kg dose, which was greater
than what CPT-11 produced when dosed at its MTD of
100 mg/kg. Both 1 and 14 demonstrated tumor regres-
sion in the early phase postdosing at all doses. No body
weight loss was observed in any of the treatment groups.
NCI COMP ARE An a lysis. A COMPARE analy-
sis28,29 at the NCI of compound 1 showed that no
compound in the database had a Pearson correlation
coefficient (PCC) of >0.7 relative to 1 at a high concen-
tration of 100 µM against a panel of 60 cell lines in vitro
(Table 6). The analysis compares a complete set of cell
sensitivities to those of standard agents or other agents
present in the NCI database. A correlation of >0.7 is
considered significant for this type of analysis. With one
exception, chloroquinoxaline sulfonamide (PCC ) 0.589),
no compound in the database was found to have a PCC
> 0.4. These weakly correlated compounds are cytotoxic
through diverse mechanisms of action, including inhibi-
tion of thymidylate synthase (5-FU), DNA synthase
(tetraplatin), topoisomerase (pyrazoloacridine, chloro-
F igu r e 2. Effect of 1 and 14 on the growth of human
colorectal cell line HCT116 in nude mice. Groups of five female
mice were injected with HCT116 cells. Animals bearing tumors
(100 mm3) were treated iv on days 1-5 and 8-12 at the doses
indicated. The relative tumor growth was determined every 4
days for a period of 63 days. Data are means and standard
errors determined from repeated measures of the analysis of
variance model that accounted for within animal correlation
and removal from the study of animals with large tumors.
quinoxaline sulfonamide), and pyrimidine synthetase
(PALA). However, in this analysis, the ASAP series
tended to show their greatest activity in solid tumors
rather than leukemia cell lines (Table 7), consistent with
Corbett’s early observations in the SADD assay with 1
and 2. Additionally, as a group, colorectal cell lines were
most sensitive to the ASAPs. On the basis of these data,
it is difficult to speculate on the molecular target for
the ASAP compounds. Investigations are currently
underway to identify a molecular target, and these
studies will be reported in due course.

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5373
Ta ble 6. Mean Panel GI50 Values and COMPARE Correlation
Coefficients (PCC) Using Compound 1 (NCS 716877) as Seed,
Tested in the U.S. NCI 60 Cell Lines in Vitro Screena
The starting materials were obtained from commercial sources
or synthesized using standard literature procedures. Experi-
mental details for those starting materials that required
synthesis are described in the Supporting Information. The
abbreviations ACN, DMSO, and THF refer to acetonitrile,
dimethyl sulfoxide, and tetrahydrofuran, respectively.
high concn
rank
1
NSC
(M)
PCC
compd
339004
0.0001
0.589
chloroquinoxaline
sulfonamide
1
The H NMRs were taken on a Bruker 250 MHz NMR or a
Bruker 300 MHz NMR or a Mercury 400 MHz NMR. Chemical
shifts were reported in ppm relative to tetramethylsilane or
solvent. The exact masses were determined on a Micromass
Q-TOF II time-of-flight mass spectrometer using negative
mode electrospray ionization unless otherwise indicated. Mid-
west Microlab performed the elemental analyses after the
samples had been blocked at 120 °C unless otherwise noted.
The melting points were determined on a Buchi apparatus and
are not corrected. Analytical thin-layer chromatography (TLC)
was performed on Merck TLC glass plates precoated with F254
silica gel 60 (UV, 254 nm, and iodine).
Except when noted otherwise, LC/MS analytical spectra
were performed on a Waters Alliance 2690 HPLC system (auto
sampler, quaternary pump, DAD-UV, heated column compart-
ment) with a Micromass ZMD single quadrupole mass spec-
trometer. The column was an Xterra MS, C18, 2.1 mm × 30
mm, 3.5 µM column. The solvent conditions were 5-100% ACN
with 0.2% ammonium hydroxide in 3 min and held at 100%
ACN with 0.2% NH4OH for 0.5 min. The flow rate was 1
mL/min. Approximately 5 µL of a ∼0.5 mg/mL sample was
injected. Negative mode electrospray ionization was used. The
reported retention times were based on a UV chromatogram
at 214 nM.
The pKa values were determined using the following pro-
cedure. Three pK/UV titrations were performed in the same
vial using the Sirius GLpKa/DPAS instrument. The first
titration was done by weighing ∼1 mg of material and adding
10 mL of 60% dioxane/40% ionic strength adjusted (ISA) water
(0.15 M KCl) and 3 mL of ISA water. The second titration was
done by adding 2 mL of ISA water to the first titration upon
its completion. Similarly, the third titration added 3 mL of
ISA water to the previous two titrations upon completion. For
each titration, the initial pH was taken and then the solution
was adjusted to pH 12 using 0.5 M KOH. The titrations were
run from pH 12 to pH 1.8, titrating with 0.5 M HCl. The results
were then interpreted via pKa/UV Version 1.001 software
(Sirius Analytical Instruments Ltd., East Sussex, RH18 5DW,
England) and extrapolation to pure water using the Yasuda-
Shedlovsky plot.30,31
RP S Cou p lin g P r oced u r e A. The reactions were per-
formed in 8 mL glass vials fitted with caps lined with poly-
(tetrafluoroethylene). Each reaction vial was charged with the
appropriate carboxylic acid (0.39 mmol, 1.5 equiv) and CH2-
Cl2 (2.0 mL). DMAP (48 mg, 0.39 mmol, 1.5 equiv) and
4-chlorobenzenesulfonamide (50 mg, 0.26 mmol, 1 equiv) were
delivered to each reaction vial in 4.0 mL of a CH2Cl2 stock
solution. N-Cyclohexylcarbodiimide, N′-methyl polystyrene HL
(0.261 g, 0.52 mmol, 2.0 equiv, loading ) 2.0 mmol/g) was
added to each vial. The vials were rotated for 72 h. A
sulfonated polystyrene resin, the resin-bound equivalent of
p-toluenesulfonic acid, was then added to scavenge the DMAP.
After the reaction vials were rotated for another 12 h, both
resins were filtered off using a 0.8 cm × 4.0 cm polypropylene
fritted funnel. A stream of nitrogen evaporated the solvent
from each reaction vial. Crude reaction mixtures were purified
using UV-guided HPLC purification.
2
3
4
5
6
7
8
9
10
11
19893
363812
366140
740
141537
755
375575
224131
339638
368390
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.397
0.393
0.392
0.378
0.378
0.363
0.359
0.358
0.350
0.350
5-FU
tetraplatin
pyrazoloacridine
methotrexate
anguidine
6-mercaptopurine
cyclopentyl cytosine
PALA
phosphotrienin
DUP785 (brequinar)
a
For definitions and methods of calculation of the correlation
coefficient from the COMPARE analysis, see ref 29.
Con clu sion s
ASAPs, such as compounds 1 and 2, represent a novel
series of antitumor agents. RPS along with traditional
medicinal chemistry techniques were used to quickly
delineate the benzene substituent SAR trends of the two
phenyl rings in the ASAP scaffold. We employed the
VEGF-HUVEC assay and the SADD assay to evaluate
the anti-proliferative potencies of the compounds and
to drive the SAR. The benzoyl side of the ASAP
molecules required 2,4-disubstitution for optimal po-
tency. Only limited variations to the benzoyl substitu-
ents were allowed with halides and small alkyl groups
being preferred. On the benzenesulfonamide ring, a
variety of substitutions at the 3- and/or 4-positions were
well-tolerated, but substitution at the 2-position dimin-
ished activity. Data from the SADD assay showed that
the ASAP analogues exhibited selective activity toward
CRC cell lines vs leukemia and normal fibroblast cell
lines. Furthermore, the SADD data agreed well with the
VEGF-HUVEC data indicating that the HUVEC assay
was an appropriate predictor of the ASAP compounds’
oncolytic activity. Key compounds from the series
demonstrated exposure in PK studies performed in rats
and mice. In a nude mouse HCT116 xenograft model
using iv adminstration, compounds 1 and 14 exhibited
tumor growth inhibition and TGD. The molecular target
of the ASAP compounds remains to be elucidated, as
an NCI COMPARE analysis showed no matches. Efforts
to determine the mechanism of action are ongoing.
Additional details regarding the SAR of these com-
pounds will be reported in the future.
Exp er im en ta l Section
Gen er a l Meth od s. All solvents and reagents were used as
obtained from commercial sources unless otherwise indicated.
Ta ble 7. Cytotoxicitya of ASAP Analogues toward Human Cancer Cells
colon
lung
breast
MCF7
renal
leukemia
compd no.
NSC
HCT116
HT29
SW620
NCI-H522
RXF 393
HL-60 (TB)
MGMb
1
2
39
18
716877
716876
724610
724611
5.4
5.3
3.5
9.7
52.0
54.8
41.5
8.6
39.6
9.5
18.7
8.6
39.6
100.0
18.9
1.5
23.9
17.7
4.3
11.0
6.4
32.9
34.7
38.9
49.5
33.1
26.3
28.8
41.7
100.0
37.9
8.6
a
b
Cytotoxicity: GI50 values are the concentrations corresponding to 50% growth inhibition. MGM is the mean graph midpoint for
growth inhibition of all human cancer cell lines successfully tested.

5374 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Lobb et al.
Preparative UV-guided HPLC purifications were performed
a Gilson HPLC system. The reaction mixtures were
332. HRMS obsd mass (M - H)-, 327.9614; calcd mass (C13H9-
Cl2NO3S-1H)-, 327.9602. Anal. (C13H9Cl2NO3S) C, H, N.
4-C h lo r o -N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (2). Compound 2 was synthesized from 2,4-dichloro-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 30%. 1H NMR (250 MHz,
DMSO-d6): δ 7.97 (d, J ) 8 Hz, 2H), 7.74 (d, J ) 8 Hz, 2H),
7.71 (m, 1H), 7.50 (m, 2H). LC/MS studies were performed as
described for 1. LC/MS tr ) 1.76 min, m/z 364, 362, 366
(M - H)-. HRMS obsd mass (M - H)-, 361.9212; calcd mass
(C13H8Cl3NO3S-1H)-, 361.9212. Anal. (C13H8Cl3NO3S) C, H, N
(blocked 60 °C).
N-Ben zoyl-4-ch lor oben zen esu lfon a m id e (3). Compound
3 was synthesized from benzoic acid and 4-chlorobenzene-
sulfonamide using RPS coupling procedure A. Yield ) 33%.
1H NMR (250 MHz, DMSO-d6): δ 7.99 (d, J ) 8 Hz, 2H), 7.86
(d, J ) 7 Hz, 2H), 7.72 (d, J ) 8 Hz, 2H), 7.62 (t, J ) 7 Hz,
1H), 7.46 (t, J ) 7 Hz, 2H). LC/MS tr ) 1.78 min (>99%), m/z
294, 296 (M-H)-. HRMS obsd mass (M-H)-, 294.0009; calcd
mass (C13H10ClNO3S-1H)-, 293.9992. Anal. (C13H10ClNO3S) C,
H, N.
on
dissolved in DMSO to a concentration of ∼0.25 g/mL. Two
injections were made for each sample. The column was a YMC-
ODSA, C18, 5 µM, 120 Å pore size. The solvent conditions were
a gradient over 12 min starting at 5% ACN/H2O, 0.1% HCl
going to 95% ACN/H2O, 0.1% HCl. The flow rate was 20 mL/
min. Fractionation was achieved based on UV absorbance at
214 nM.
RP S Cou p lin g P r oced u r e B. Coupling procedure B was
performed in a manner similar to procedure A except the order
of the starting material addition was reversed. The reaction
vials were first charged with the appropriate sulfonamide (0.26
mmol, 1.0 equiv) and CH2Cl2 (2.0 mL). Then, 2,4-dichloroben-
zoic acid (75 mg, 0.39 mmol, 1.5 equiv) and DMAP (48 mg,
0.39 mmol, 1.5 equiv) were delivered in 4.0 mL of a stock
solution made in CH2Cl2.
Gen er a l Solu tion Cou p lin g P r oced u r e C. To a stirring
solution of the benzoic acid (1.2 equiv) in dry CH2Cl2 (10 mL/
mmol), the phenylsulfonamide (1.0 equiv) was added in one
portion followed by EDC (1.2-1.5 equiv) and then DMAP (1.2
equiv). The mixture was vigorously stirred under nitrogen for
16 h and concentrated under reduced pressure, and the residue
was partitioned between EtOAc and H2O. The organic layer
was washed with 1 N HCl (four times, 20 mL/mmol), and then,
the combined aqueous phases were extracted with EtOAc (2
× 20 mL/mmol). The combined organic layers were washed
with H2O and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was subjected to silica gel chromatography
or crystallization if necessary.
La r ge Sca le Cou p lin g P r oced u r e D. To a reaction
mixture of arylsulfonamide (81.4 mmol), CDI (15.8 g, 97.7
mmol), and EtOAc (300 mL) at room temperature was added
a slurry of substituted benzoic acid (97.7 mmol) in EtOAc (100
mL) over a period of 15 min. Gas evolution was observed but
could be controlled by the rate of slurry addition. The reaction
mixture went into solution by the end of the slurry addition.
Progress of the reaction was monitored by TLC with 1:1
EtOAc/heptane eluent or by HPLC. The reaction was stirred
at room temperature for 30 min and then heated at 60 °C for
90 min or until no gas evolution was observed. It was cooled
to 40 °C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (14.6 mL) was
added all at once. The mixture was stirred until it reached
room temperature and then was quenched with deionized
water (400 mL). The top organic layer was separated, washed
with 1 N HCl (300 mL), dried with anhydrous MgSO4, and
filtered. The cake was washed with EtOAc (20 mL). The filtrate
was concentrated to ca. 50 g of a syrupy solution, and then,
heptane (250 mL) was added with vigorous stirring. The
mixture was heated, a white slurry was formed, refluxed, and
then allowed to equilibrate to room temperature. The white
precipitate was filtered, and the cake was washed with
heptane (20 mL). To recrystallize the crude, a mixture of ca.
20 g of the product and 1:2 EtOAc/heptane (150 mL) was
heated at reflux for 30 min and then cooled to room temper-
ature. The off-white precipitate was filtered, and the cake was
washed with heptane (50 mL). The precipitate was dried under
vacuum at 50 °C for 18 h.
4-Ch lor o-N-(2-ch lor oben zoyl)ben zen esu lfon a m id e (4).
Compound 4 was synthesized from 2-chlorobenzoic acid and
4-chlorobenzenesulfonamide using RPS coupling procedure
A. Yield ) 37%. 1H NMR (250 MHz, DMSO-d6): δ 7.97 (d,
J ) 8 Hz, 2H), 7.73 (d, J ) 8 Hz, 2H), 7.52-7.43 (m, 3H),
7.42-7.34 (m, 1H). LC/MS tr ) 1.85 min, m/z 328, 330
(M - H)-. HRMS obsd mass (M - H)-, 327.9623; calcd mass
(C13H9Cl2NO3S-1H)-, 327.9602. Anal. (C13H9Cl2NO3S) C, H, N.
4-Ch lor o-N-(3-ch lor oben zoyl)ben zen esu lfon a m id e (5).
Compound 5 was synthesized from 3-chlorobenzoic acid and
4-chlorobenzenesulfonamide using RPS coupling procedure A.
1
Yield ) 51%. H NMR (250 MHz, DMSO-d6): δ 7.97 (d, J ) 8
Hz, 2H), 7.92 (s, 1H), 7.80 (d, J ) 8 Hz, 1H), 7.70 (d, J ) 8 Hz,
2H), 7.66 (s, 1H), 7.50 (t, J ) 7 Hz, 1H). LC/MS tr ) 2.02 min,
m/z 328, 330 (M - H)-. HRMS obsd mass (M - H)-, 327.9613;
calcd (C13H9Cl2NO3S-1H)-, 327.9602. Anal. (C13H9Cl2NO3S) C,
H, N.
4-Ch lor o-N-(4-ch lor oben zoyl)ben zen esu lfon a m id e (6).
Compound 6 was synthesized from 4-chlorobenzoic acid and
4-chlorobenzenesulfonamide using RPS coupling procedure
A. Yield ) 49%. 1H NMR (250 MHz, DMSO-d6): δ 7.99 (d,
J ) 8 Hz, 2H), 7.88 (d, J ) 8 Hz, 2H), 7.71 (d, J ) 8 Hz,
2H), 7.56 (d, J ) 8, 2H). LC/MS tr ) 2.03 min, m/z 328, 330
(M - H)-. HRMS obsd mass (M - H)-, 327.9629; calcd mass
(C13H9Cl2NO3S-1H)-, 327.9602. Anal. (C13H9Cl2NO3S) C, H, N.
4-Ch lor o-N-(2-m eth ylben zoyl)ben zen esu lfon a m id e (7).
Compound 7 was synthesized from 2-methylbenzoic acid and
4-chlorobenzenesulfonamide using RPS coupling procedure A.
1
Yield ) 47%. H NMR (250 MHz, DMSO-d6): δ 8.0 (d, J ) 8
Hz, 2H), 7.74 (d, J ) 8 Hz, 2H), 7.36-7.46 (m, 2H), 7.26 (t, J
) 6 Hz, 2H), 2.19 (s, 3H). LC/MS tr ) 1.87min (99%), m/z 308,
310 (M - H)-. HRMS obsd mass (M - H)-, 308.0159; calcd
mass (C14H12ClNO3S-1H)-, 308.0148. Anal. (C14H12ClNO3S) C,
H, N.
N-(3-Br om oben zoyl)-4-ch lor oben zen esu lfon a m id e (8).
Compound 8 was synthesized from 3-bromobenzoic acid and
4-chlorobenzenesulfonamide using RPS coupling procedure A.
1
Yield ) 46%. H NMR (250 MHz, DMSO-d6): δ 8.05 (m, 1H),
N-(2,4-Dich lor oben zoyl)ben zen esu lfon a m id e (1). Com-
pound 1 was synthesized by refluxing 2,4-dichlorobenzoyl
chloride (0.01 mol, 1.0 equiv), benzenesulfonamide (0.012 mol,
1.2 equiv), and K2CO3 (0.015 mol, 1.5 equiv) in dioxane for 4
h. The reaction mixture was diluted with H2O, acidified with
aqueous HCl solution, and extracted with EtOAc. The EtOAc
extracts were washed with brine, dried over Na2SO4, and
evaporated under reduced pressure. The crude material was
recrystallized in toluene and then chromatographed using a
silica gel column and CH2Cl2 as solvent. Yield ) 80%. 1H NMR
(400 MHz, DMSO-d6): δ 12.88 (br s, 1H), 7.99 (d, J ) 8 Hz,
2H), 7.78-7.63 (m, 4H), 7.50 (m, 2H). The LC/MS was
performed on a Waters Micromass ZQ with a Leap Technolo-
gies HTS PAL autosampler and an Agilent 1100 Series Diode
Array Detector with the same column and method as described
in the General Methods section. LC/MS tr ) 1.38, m/z 328, 330,
7.97 (d, J ) 8 Hz, 2H), 7.82 (t, J ) 9 Hz, 2H), 7.69 (d, J ) 8
Hz, 2H), 7.44 (t, J ) 8 Hz, 1H). The second HPLC was run on
a Shimadzu 10ADvc HPLC system (autosampler, heated
column compartment). The column was a Symmetry C18, 4.6
mm × 50 mm, 5 µM column heated at 50 °C. The solvent
conditions were 5-90% ACN with 0.05% TFA in 4 min and
held at 90% ACN with 0.05% TFA for 1.0 min. The flow rate
was 2 mL/min. Approximately 10 µL of a ∼0.5 mg/mL sample
was injected. The reported retention times and purities were
based on a Polymer Lab PL-ELS1000 light scattering detector.
LC/MS tr ) 2.09 min (>99%), m/z 374, 372, 376 (M - H)-.
Second HPLC tr ) 3.08 min (98%). HRMS obsd mass (M -
H)-, 371.9090; calcd mass (C13H9BrClNO3S-1H)-, 371.9097.
4-Ch lor o-N-(4-n it r ob en zoyl)ben zen esu lfon a m id e (9).
Compound 9 was synthesized from 4-nitrobenzoic acid and

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5375
4-chlorobenzenesulfonamide using RPS coupling procedure A.
Yield ) 33%. H NMR (250 MHz, DMSO-d6): δ 8.28 (d, J ) 8
Hz, 2H), 8.08 (d, J ) 8 Hz, 2H), 8.00 (d, J ) 8 Hz, 2H), 7.69
(d, J ) 8 Hz, 2H). Second HPLC performed as described for 8.
LC/MS tr ) 1.86 min (>99%), m/z 339, 341, 340 (M - H)-.
Second HPLC tr ) 2.78 min (97%). HRMS obsd mass (M -
H)-, 338.9853; calcd mass (C13H9ClN2O5S-1H)-, 338.9843.
4-Ch lor o-N-(4-ch lor o-2-m eth ylben zoyl)ben zen esu lfon -
a m id e (18). Compound 18 was synthesized from 4-chloro-2-
methylbenzoic acid and 4-chlorobenzenesulfonamide using
1
1
RPS coupling procedure A. Yield ) 57%. H NMR (250 MHz,
DMSO-d6): δ 7.99 (d, J ) 9 Hz, 2H), 7.74 (d, J ) 9 Hz, 2H),
7.47 (d, J ) 8 Hz, 1H), 7.39-7.30 (m, 2H), 2.19 (s, 3H). LC/
MS tr ) 1.97 min, m/z 342, 344, 346 (M - H)-. HRMS obsd
mass (M - H)-, 341.9776; calcd mass (C14H11Cl2NO3S-1H)-,
341.9758. Anal. (C14H11Cl2NO3S) C, H, N.
4-Ch lor o-N-(4-(d im et h yla m in o)b en zoyl)b en zen esu l-
fon a m id e (10). Compound 10 was synthesized from 4-(di-
methylamino)benzoic acid and 4-chlorobenzenesulfonamide
N-(4-Br om o-2-m eth yl-ben zoyl)-4-ch lor oben zen esu lfon a-
m id e (19). Compound 19 was synthesized from 4-bromo-2-
methylbenzoic acid and 4-chlorobenzenesulfonamide using
1
using RPS coupling procedure A. Yield ) 22%. H NMR (250
MHz, DMSO-d6): δ 12.10 (br s, 1H), 7.97 (d, J ) 8 Hz,
2H), 7.77 (d, J ) 5 Hz, 2H), 7.71 (d, J ) 5 Hz, 2H), 6.67 (d,
J ) 8 Hz, 2H), 3.34 (s, 6H). LC/MS tr ) 1.76 min (>99%)
m/z 337, 339, 340 (M - H)-. HRMS obsd mass (M - H)-,
337.0419; calcd mass (C15H15ClN2O3S-1H)-, 337.0414. Anal.
(C15H15ClN2O3S) C, H, N.
1
RPS coupling procedure A. Yield ) 67%. H NMR (400 MHz,
DMSO-d6): δ 7.98 (dd, J ) 9 Hz, 2 Hz, 2H), 7.74 (dd, J ) 9
Hz, 2 Hz, 2H), 7.51 (d, J ) 1 Hz, 1H), 7.47 (dd, J ) 8 Hz, 2
Hz, 1H), 7.37 (d, J ) 8 Hz, 1H), 2.17 (s, 3H). LC/MS tr ) 2.01
min, m/z 386, 388, 390 (M - H)-. HRMS obsd mass (M - H)-,
385.9258; calcd mass (C14H11BrClNO3S-1H)-, 385.9253. Anal.
(C14H11BrClNO3S) C, H, N.
4-C h lo r o -N -(3,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (11). Compound 11 was synthesized from 3,4-dichloro-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 28%. 1H NMR (250 MHz,
DMSO-d6): δ 8.12 (s, 1H), 7.97 (d, J ) 8 Hz, 2H), 7.81 (d, J )
8 Hz, 1H), 7.73 (d, J ) 8 Hz, 1H), 7.69 (d, J ) 8 Hz, 2H). Second
HPLC performed as described for 8. LC/MS tr ) 2.19 min
(99%), m/z 364, 362, 366 (M - H)-. Second HPLC tr ) 3.24
min (95%). HRMS obsd mass (M - H)-, 361.9218; calcd mass
(C13H8Cl3NO3S-1H)-, 361.9212.
4-C h lo r o -N -(2,5-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (12). Compound 12 was synthesized from 2,5-dichloro-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 28%. 1H NMR (250 MHz,
DMSO-d6): δ 7.99 (d, J ) 9 Hz, 2H), 7.74 (d, J ) 9, 2H), 7.65
(d, J ) 2, 1H), 7.51 (m, 2H). LC/MS tr ) 2.18 min, m/z
362, 364, 366 (M - H)-. HRMS (M - H)- obsd mass,
361.9217; calcd mass (C13H8Cl3NO3S-1H)-, 361.9212. Anal.
(C13H8Cl3NO3S) C, H, N.
4-Ch lor o-N-(2-ch lor o-4-n it r ob en zoyl)b en zen esu lfon -
m id e (20). Compound 20 was synthesized from 2-chloro-4-
nitrobenzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 71%. 1H NMR (400 MHz,
DMSO-d6): δ 8.30 (s, 1H), 8.18 (dd, J ) 8 Hz, 2 Hz, 1H), 7.97
(d, J ) 9 Hz, 2H), 7.75 (d, J ) 8 Hz, 1H), 7.72 (d, J ) 8 Hz,
1H). LC/MS tr ) 1.71 min, m/z 373, 375, 377 (M - H)-. HRMS
obsd mass (M - H)-, 372.9448; calcd mass (C13H8Cl2N2O5S-
1H)-, 372.9453. Anal. (C13H8Cl2N2O5S) C, H, N.
4-Ch lor o-N-(4-ch lor o-2-et h ylb en zoyl)b en zen esu lfon -
a m id e (21). Compound 21 was synthesized from 4-chloro-2-
ethylbenzoic acid and 4-chlorobenzenesulfonamide using cou-
pling procedure C. Yield ) 61%. 1H NMR (400 MHz,
DMSO-d6): δ 8.01 (d, J ) 8.4 Hz, 2H), 7.62 (d, J ) 8.4 Hz,
2H), 7.32-7.45 (m, 3H), 2.47-2.54 (m, 2H), 0.91 (t, J ) 7.7
Hz, 3H). HRMS obsd mass, 355.9944 (M - H)-; calcd
mass (C15H13Cl2NO3S-1H)-, 355.9915. Anal. (C15H13Cl2NO3S)
C, H, N.
4-C h lo r o -N -(3,5-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (13). Compound 13 was synthesized from 3,5-dichloro-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 40%. 1H NMR (250 MHz,
DMSO-d6): δ 7.99 (d, J ) 8 Hz, 2H), 7.89 (s, 3H), 7.70 (d,
J ) 8, 2H). LC/MS tr ) 2.22 min, m/z 364, 362, 366 (M - H)-.
4-Ch lor o-N-(4-ch lor o-2-p r op ylben zoyl)ben zen esu lfon -
a m id e (22). Compound 22 was synthesized from 4-chloro-2-
propylbenzoic acid and 4-chlorobenzenesulfonamide using
coupling procedure C. Yield ) 39%. 1H NMR (400 MHz,
DMSO-d6): δ 8.00 (d, J ) 8.8 Hz, 2H), 7.77 (d, J ) 8.8 Hz,
2H), 7.44 (d, J ) 9.1 Hz, 1H), 7.32-7.35 (m, 2H), 2.44 (t,
J ) 7.9 Hz, 2H), 1.12-1.24 (m, 2H), 0.61 (t, J ) 7.3 Hz, 3H).
LC/MS performed as described for 1. Second HPLC performed
as described for 8. LC/MS tr ) 1.63 min (>99%), m/z 370, 372
(M - H)-. Second HPLC tr ) 4.01 min (99%). HRMS obsd mass
(M - H)-, 370.0094; calcd mass (C16H15Cl2NO3S-1H)-, 370.0072.
4-C h lo r o -N -(2,4-d im e t h y lb e n zo y l)b e n ze n e s u lfo n -
a m id e (23). Compound 23 was synthesized from 2,4-dimethyl-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 59%. 1H NMR (400 MHz,
DMSO-d6): δ 8.02 (dd, J ) 8, 2 Hz, 2H), 7.77 (dd, J ) 9, 3 Hz,
2H), 7.38 (d, J ) 8 Hz, 1H), 7.09-7.10 (m, 2H), 2.31 (s, 3H),
2.19 (s, 3H). LC/MS tr ) 1.90 min, m/z 322, 324 (M - H)-.
HRMS obsd mass (M
-
H)-, 361.9208; calcd mass
(C13H8Cl3NO3S-1H)-, 361.9212. Anal. (C13H8Cl3NO3S) C, H, N.
N-(4-Br om o-2-ch lor oben zoyl)-4-ch lor oben zen esu lfon -
a m id e (14). Compound 14 was synthesized from 4-bromo-2-
chlorobenzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 33%. 1H NMR (250 MHz,
DMSO-d6): δ 7.98 (d, J ) 8 Hz, 2H), 7.84 (s, 1H), 7.76 (d, J )
8 Hz, 2H), 7.63 (d, J ) 7 Hz, 1H), 7.45 (d, J ) 7 Hz, 1H). LC/
MS tr ) 2.24 min, m/z 406, 407, 410 (M - H)-. HRMS obsd
mass (M - H)-, 405.8708; calcd mass (C13H8BrCl2NO3S-1H)-,
405.8707. Anal. (C13H8BrCl2NO3S) C, H, N.
4-Ch lor o-N-(2-br om o-4-ch lor oben zoyl)ben zen esu lfon -
a m id e (15). Compound 15 was synthesized from 2-bromo-4-
chlorobenzoic acid and 4-chlorobenzenesulfonamide using
coupling procedure C. Yield ) 55%. 1H NMR (400 MHz,
DMSO-d6): δ 7.38 (dd, J ) 8.4 Hz, J ) 2.0, 1H), 7.56 (m,
3H), 7.61 (s, 1H), 8.09 (d, J ) 8.8 Hz, 2H), 8.67 (s, 1H). Anal.
(C13H8BrCl2NO3S) C, H, N (vacuum-dried).
HRMS obsd mass (M
-
H)-, 322.0299; calcd mass
(C15H14ClNO3S-1H)-, 322.0305. Anal. (C15H14ClNO3S) C, H, N.
4-Ch lor o-N-(4-ch lor o-2-m et h oxyb en zoyl)b en zen esu l-
fon a m id e (24). Compound 24 was synthesized from 4-chloro-
2-methoxybenzoic acid and 4-chlorobenzenesulfonamide using
1
RPS coupling procedure A. Yield ) 34%. H NMR (400 MHz,
4-C h lo r o -N -(2,4-d ib r o m o b e n zo y l)b e n ze n e s u lfo n -
a m id e (16). Compound 16 was synthesized from 2,4-dibromo-
benzoic acid and 4-chlorobenzenesulfonamide using coupling
DMSO-d6): δ 8.00 (d, J ) 9 Hz, 2H), 7.78 (d, J ) 9 Hz, 2H),
7.41 (d, J ) 8 Hz, 1H), 7.25 (d, J ) 2 Hz, 1H), 7.07 (dd, J ) 8
Hz, 2 Hz, 1H), 3.87 (s, 3H). LC/MS tr ) 2.02 min, m/z 358,
360, 362 (M - H)-. HRMS obsd mass (M - H)-, 357.9725;
calcd mass (C14H11Cl2NO4S-1H)-, 357.9708. Anal. (C14H11Cl2-
NO4S) C, H, N.
4-Ch lor o-N-(2-a m in o-4-ch lor oben zoyl)ben zen esu lfon -
a m id e (25). Compound 25 was synthesized from 2-amino-4-
chlorobenzoic acid and 4-chlorobenzenesulfonamide using
coupling procedure C. Yield ) 8%. 1H NMR (400 MHz,
DMSO-d6): δ 6.43 (d, J ) 8.5 Hz, 1H), 6.77 (s, 1H), 7.57 (d, J
) 8.5 Hz, 2H), 7.67 (d, J ) 8.5 Hz, 1H), 7.87 (d, J ) 8.5 Hz,
2H), 8.00 (br s, 2H). LC/MS performed as described for 1. A
1
procedure C. Yield ) 76%. H NMR (400 MHz, DMSO-d6): δ
7.39 (d, J ) 8.4 Hz, 1H), 7.64 (d, J ) 10.0 Hz, 1H), 7.73 (d, J
) 8.8 Hz, 2H), 7.94-7.99 (m, 3H). Anal. (C13H8Br2ClNO3S) C,
H, N.
4-Ch lor o-N-(4-br om o-2-flu or oben zoyl)ben zen esu lfon -
a m id e (17). Compound 17 was synthesized from 4-bromo-2-
fluorobenzoic acid and 4-chlorobenzenesulfonamide using cou-
1
pling procedure C. Yield ) 64%. H NMR (400 MHz, DMSO-
d6): δ 7.47-7.54 (m, 2H), 7.69-7.75 (m, 3H), 7.96 (d, J ) 8.8
Hz, 2H). Anal. (C13H8BrClFNO3S) C, H, N.

5376 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Lobb et al.
second HPLC was performed on a YMC ODS-A, 2.0 mm × 50
mm, S3 µm column. The solvent conditions were 5-100% ACN
with 0.2% formic acid in 3 min and held at 100% ACN with
0.2% formic acid for 0.5 min. The flow rate was 1.0 mL/min,
and 5 µL was injected. Light scattering detection was used.
LC/MS tr ) 1.68 min (>99%), m/z 343, 345 (M - H)-. Second
HPLC tr ) 2.40 min (>99%). HRMS (positive mode) obsd mass
(M + H)+, 344.9860; calcd mass (C13H10Cl2N2O3S+1H)+,
344.9867.
N-(2,4,-Bis-tr iflu or om eth yl-ben zoyl)-4-ch lor oben zen e-
su lfon a m id e (26). Compound 26 was synthesized from 2,4-
bis(trifluoromethyl)benzoic acid and 4-chlorobenzenesulfon-
amide using RPS coupling procedure A. Yield ) 49%. 1H NMR
(400 MHz, DMSO-d6): δ 8.10-8.13 (m, 2H), 7.95 (d, J ) 8
Hz, 2H), 7.84 (d, J ) 8 Hz, 1H), 7.72 (d, J ) 8 Hz, 2H). LC/MS
tr ) 2.01 min, m/z 430, 432 (M - H)-. HRMS obsd mass (M -
H)-, 429.9720; calcd mass (C15H8ClF6NO3S-1H)-, 429.9739.
Anal. (C15H8ClF6NO3S) C, H, N.
4-Ch lor o-N-(4-ch lor o-2-n it r ob en zoyl)b en zen esu lfon -
a m id e (27). Compound 27 was synthesized from 4-chloro-2-
nitrobenzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 76%. 1H NMR (400 MHz,
DMSO-d6): δ 8.24 (d, J ) 2 Hz, 1H), 7.99 (d, J ) 9 Hz, 2H),
7.94 (dd, J ) 8, 2 Hz, 1H), 7.78 (d, J ) 9 Hz, 2H), 7.75 (d, J )
8 Hz, 1H). LC/MS tr ) 1.78 min, m/z 373, 375, 377 (M - H)-.
HRMS obsd mass (M -H)-,372.9443;calcd mass (C13H8Cl2N2O5S-
1H)-, 372.9453. Anal. (C13H8Cl2N2O5S) C, H, N (vacuum-dried
at 80 °C).
3.82 (s, 3H), 6.91 (dd,
J
)
2,
9
Hz, 1H), 6.99 (d,
J ) 2.4 Hz, 1H), 7.40 (d, J ) 8.6 Hz, 1H), 7.63 (d, J ) 8.6 Hz,
2H), 8.05 (d, J ) 8.6 Hz, 2H). LC/MS was performed as
described for 1. The second HPLC was performed as described
for 25. LC/MS tr ) 1.53 min (>99%), m/z 358, 360 (M - H)-.
Second HPLC tr ) 2.42 min (99%). APCI-MS m/z 358
[C14H11Cl2NO4S-1H]-.
4-Ch lor o-N-(2-ch lor o-4-h ydr oxyben zoyl)ben zen esu lfon -
a m id e (33). The methyl ether derivative, 32 (147 mg; 0.408
mmol), was dissolved in CH2Cl2 and chilled to -20 °C. An
excess of BBr3 (4 mL; 1 M in CH2Cl2) was added, and the
reaction solution was stirred for 1 h at -20 °C, for 1 h at room
temperature, and finally for 2 h at 40 °C. Slow addition of
saturated aqueous NaHCO3 quenched the reaction. The prod-
uct was extracted into CH2Cl2. The organic layer was washed
with 0.5 M HCl, dried with Na2SO4, filtered, and concentrated
in vacuo. Silica chromatography with an isocratic mobile phase
(MeOH/CH2Cl2; 5:95, v/v) purified the desired product (130 mg;
1
90% yield). H NMR (400 MHz, methanol-d4): δ 8.05 (d, 2H,
J ) 8.8 Hz), 7.62 (d, 2H, J ) 8.8 Hz), 7.32 (d, 1H, J ) 8.8 Hz),
6.82 (d, 1H, J ) 2.2 Hz), 6.74 (dd, 1H, J ) 8.4, 2.2 Hz). The
first HPLC was performed on a YMC C18, 2.1 mm × 150 mm,
3.5 µm column with gradient conditions that went from 5 to
100% ACN/MeOH (50/50) with 0.2% NH4CO2H in 7.0 min and
then were held at 100% for 1.0 min. The flow rate was 0.7
mL/min. Approximately 5 µL was injected. The column tem-
perature was 60 ( 10 °C. UV detection was used. The second
HPLC was performed as described for 25. First HPLC tr
)
5.36 min (>99%). Second HPLC tr ) 2.18 min (>99%). HRMS
(positive mode) obsd mass (M)+, 345.9724; calcd mass (C13H9-
Cl2NO4S)+, 345.9707.
4-Ch lor o-N -(2-n it r o-4-t r iflu or om e t h ylb e n zoyl)b e n -
zen esu lfon a m id e (28). Compound 28 was synthesized from
2-nitro-4-trifluoromethylbenzoic acid and 4-chlorobenzene-
sulfonamide using RPS coupling procedure A. Yield ) 57%.
1H NMR (400 MHz, DMSO-d6): δ 9.82 (s, 1H), 8.24 (d, J ) 8
Hz, 1H), 8.00 (d, J ) 9 Hz, 2H), 7.96 (d, J ) 8 Hz, 1H), 7.78
(d, J ) 9 Hz, 2H). LC/MS tr ) 1.85 min, m/z 407, 409 (M -
H)-. HRMS obsd mass (M - H)-, 406.9734; calcd mass
(C14H8ClF3N2O5S-1H)-, 406.9716. Anal. (C14H8ClF3N2O5S) C,
H, N.
4-C h lo r o -N -(2,4-d i n i t r o b e n z o y l)b e n z e n e s u lfo n -
a m id e (29). Compound 29 was synthesized from 2,4-dini-
trobenzoic acid and 4-chlorobenzenesulfonamide using
RPS coupling procedure A. Yield ) 47%. 1H NMR (400
MHz, DMSO-d6): δ 8.75 (d, J ) 2 Hz, 1H), 8.60 (dd, J ) 8, 2
Hz, 1H), 7.99 (d, J ) 8 Hz, 1H), 7.98 (d, J ) 9 Hz, 2H), 7.76
(d, J ) 9 Hz, 2H). LC/MS tr ) 1.56 min, m/z 384, 386 (M -
H)-. HRMS obsd mass (M - H)-, 383.9705; calcd mass
(C13H8ClN3O7S-1H)-, 383.9693. Anal. (C13H8ClN3O7S) C, H, N.
4-Ch lor o-N-(2-ch lor o-4-cya n oben zoyl)ben zen esu lfon -
a m id e (30). Compound 30 was synthesized from 2-chloro-4-
cyanobenzoic acid and 4-chlorobenzenesulfonamide using cou-
pling procedure C. Yield ) 16%. TLC Rf ) 0.20 (9:1
CH2Cl2/MeOH); mp 98-100 °C. 1H NMR (300 MHz, meth-
anol-d4): δ 7.47-7.55 (m, 3H), 7.60 (d, J ) 8.0 Hz, 1H), 7.72
(s, 1H), 7.96 (d, J ) 1.9 Hz, 1H), 7.99 (d, J ) 6.7 Hz,
1H). LC/MS was performed as described for 1. The second
HPLC was performed as described for 8. LC/MS tr ) 1.31 min
(94%), m/z 353, 355 (M - H)-. Second HPLC tr ) 3.67 min
(96%). HRMS obsd mass (M - H)-, 352.9591; calcd mass
(C14H8Cl2N2O3S-1H)-, 352.9554.
4-Ch lor o-N-(2-ch lor o-4-m eth a n esu lfon ylben zoyl)ben -
zen esu lfon a m id e (31). Compound 31 was synthesized from
2-chloro-4-methanesulfonylbenzoic acid and 4-chloroben-
zenesulfonamide using RPS coupling procedure A. Yield )
66%. 1H NMR (400 MHz, DMSO-d6): δ 8.08 (d, J ) 2 Hz,
1H), 8.03 (dd, J ) 9, 2 Hz, 2H), 7.96 (dd, J ) 8, 2 Hz, 1H),
7.79-7.81 (m, 3H), 3.35 (s, 3H). LC/MS tr ) 1.57 min, m/z 406,
408, 410 (M - H)-. HRMS obsd mass (M - H)-, 405.9381;
calcd mass (C14H11Cl2NO5S2-1H)-, 405.9377. Anal. (C14H11Cl2-
NO5S2) C, H, N.
4-Ch lor o-N-(2-ch lor o-4-m eth oxyben zoyl)ben zen esu lfon -
a m id e (32). Compound 32 was synthesized from 2-chloro-4-
methoxybenzoic acid and 4-chlorobenzenesulfonamide using
coupling procedure C. Yield ) 98%. Rf ) 0.51 (94:6 CH2Cl2/
MeOH); mp 42-47 °C. 1H NMR (300 MHz, methanol-d4): δ
4-Ch lor o-N-(4-a m in o-2-ch lor oben zoyl)ben zen esu lfon -
a m id e (34). The starting material, 4-chloro-N-(4-(t-butoxy-
carbonyl)amino-2-chlorobenzoyl)benzenesulfonamide, was pre-
pared by the reaction of N-(4-(tert-butoxycarbonyl)amino-2-
chlorobenzoic acid and 4-chlorobenzenesulfonamide and
was isolated in 81% yield following silica gel chromatography.
ESI-MS m/z 443.0 (C18H18Cl2N2O5S - 1H)-. The Boc-pro-
tected material (130 mg, 0.3 mmol) was treated with 1:1
TFA:CH2Cl2 (2 mL) at room temperature for 40 min. The
reaction mixture was concentrated in vacuo and then lyoph-
ilized from 1:1 MeOH:H2O (5 mL) to provide the title com-
pound (103 mg; 0.21 mmol, 70%) as its TFA salt. 1H NMR (400
MHz, methanol-d4): δ 8.03 (d, 2 H, J ) 8.4 Hz), 7.61 (d, 2 H,
J ) 8.4 Hz), 7.26 (d, 1 H, J ) 2.2 Hz), 6.60 (d, 1 H, J ) 8.4
Hz), 6.59 (dd, 1 H, J ) 8.4 Hz, 2.2 Hz), 4.92 (br s, 3 H). 13C
NMR (75 MHz, methanol-d4): δ 165.9, 151.8, 139.9, 138.5,
133.5, 131.4, 129.9, 129.1, 120.3, 115.5, 112.5. LC/MS was
performed as described for 1. The second HPLC was performed
as described for 25. LC/MS tr ) 1.23 min (91%), m/z 343, 345
(M - H)-. Second HPLC tr ) 2.14 min (93%). HRMS (posi-
tive mode) obsd mass (M + H)+, 344.9873; calcd mass
(C13H10Cl2N2O3S+1H)+, 344.9867.
4-Ch lor o-N -(2,4-d im e t h oxyb e n zoyl)b e n ze n e su lfon -
am ide (35). Compound 35 was synthesized from 2,4-dimethoxy-
benzoic acid and 4-chlorobenzenesulfonamide using RPS
coupling procedure A. Yield ) 84%. 1H NMR (250 MHz,
DMSO-d6): δ 11.49 (br. s, 1H), 7.99 (d, J ) 8 Hz, 2H), 7.73 (d,
J ) 8 Hz, 2H), 7.48 (d, J ) 8 Hz, 1H), 6.64 (d, J ) 2 Hz, 2H),
6.57 (dd, J ) 8, 2 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H).
LC/MS tr ) 2.15 min, m/z 354, 356 (M - H)-. HRMS obsd mass
(M - H)-, 354.0193; calcd mass (C15H14ClNO5S-1H)-, 354.0203.
Anal. (C15H14ClNO5S) C, H, N.
4-Me t h y l-N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (36). Compound 36 was synthesized from 2,4-dichloro-
benzoic acid and 4-methylbenzenesulfonamide using RPS
coupling procedure B. Yield ) 73%. 1H NMR (300 MHz,
DMSO-d6): δ 12.76 (br. s, 1H), 7.85 (2, J ) 7 Hz, 2H), 7.68 (s,
1H), 7.47 (s, 2H), 7.43 (d, J ) 7 Hz, 2H), 2.43 (s, 3H). LC/MS
tr ) 1.87 min, m/z 342, 344, 346 (M - H)-. HRMS obsd mass
(M - H)-, 341.9748; calcd mass (C14H11Cl2NO3S-1H)-, 341.9758.
Anal. (C14H11Cl2NO3S) C, H, N.
4-B r o m o -N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (37). Compound 37 was synthesized from 2,4-dichloro-

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5377
benzoic acid and 4-bromobenzenesulfonamide using RPS
coupling procedure B. Yield ) 66%. 1H NMR (250 MHz,
DMSO-d6): δ 7.90(s, 4H), 7.71 (d, J ) 2 Hz, 1H), 7.54 (d, J )
8 Hz, 1H), 7.48 (dd, J ) 8, 2 Hz, 1H) ppm. LC/MS tr ) 1.94
min, m/z 408, 406, 410 (M - H)-. HRMS obsd mass (M - H)-,
405.8693; calcd mass (C13H8BrCl2NO3S-1H)-, 405.8707. Anal.
(C13H8BrCl2NO3S) C, H, N.
benzoic acid and 3,4-dichlorobenzenesulfonamide using RPS
coupling procedure B. Yield ) 61%. 1H NMR (250 MHz,
DMSO-d6): δ 8.11 (d, J ) 2 Hz, 1H), 7.98-7.89 (m, 2H), 7.71
(d, J ) 2 Hz, 1 H), 7.58 (d, J ) 8 Hz, 1H), 7.49 (dd, J ) 8, 2
Hz, 1H). LC/MS tr
) 2.13 min, m/z 398, 396, 400
(M - H)-. HRMS obsd mass (M - H)-, 395.8827; calcd mass
(C13H7Cl4NO3S-1H)-, 395.8823. Anal. (C13H7Cl4NO3S) C, H, N.
3-B r o m o -N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (38). Compound 38 was synthesized from 2,4-dichloro-
benzoic acid and 3-bromobenzenesulfonamide using RPS
coupling procedure B. Yield ) 22%. 1H NMR (250 MHz,
DMSO-d6): δ 8.06 (t, J ) 2 Hz, 1H), 7.96 (dt, J ) 8 Hz, 2H),
7.71 (d, J ) 2 Hz, 1H), 7.67-7.45 (m, 4H). LC/MS tr ) 1.90
min (>99%), m/z 408, 406, 410 (M - H)-. HRMS obsd mass
(M - H)-, 405.8695; calcd mass (C13H8BrCl2NO3S-1H)-,
405.8707. Anal. (C13H8BrCl2NO3S) C, H, N (vacuum-dried at
80 °C).
N -(2,4-Dich lor o-b e n zoyl)-4-m e t h oxyb e n ze n e su lfon -
a m id e (39). Compound 39 was synthesized from 2,4-dichloro-
benzoic acid and 4-methoxybenzenesulfonamide using RPS
coupling procedure B. Yield ) 68%. 1H NMR (300 MHz,
DMSO-d6): δ 12.67 (br s, 1H), 7.91 (d, J ) 9 Hz, 2H), 7.69 (s,
1H), 7.48 (s, 2H), 7.16 (d, J ) 9 Hz, 2H), 3.86 (s, 3H). LC/MS
tr ) 1.77 min, m/z 358, 360, 362 (M - H)-. HRMS obsd mass
(M - H)-, 357.9695; calcd mass (C14H11Cl2NO4S-1H)-, 357.9708.
Anal. (C14H11Cl2NO4S) C, H, N.
4-ter t-Bu t yl-N-(2,4-d ich lor ob en zoyl)b en zen esu lfon -
a m id e (40). Compound 40 was synthesized from 2,4-dichloro-
benzoic acid and 4-tert-butylbenzenesulfonamide using RPS
coupling procedure B. Yield ) 70%. 1H NMR (250 MHz,
DMSO-d6): δ 12.67 (br s, 1 H), 7.91 (d, J ) 9 Hz, 2 H), 7.69 (s,
1H), 7.48 (s, 2H), 7.16 (d, J ) 9 Hz, 2H), 3.86 (s, 3H). LC/MS
tr ) 1.53 min, m/z 385, 387, 389 (M)-. HRMS obsd mass (M -
H)-, 384.0247; calcd mass (C17H17Cl2NO3S-1H)-, 384.0228.
Anal. (C17H17Cl2NO3S) C, H, N.
N -(2,4-Dich lor ob e n zoyl)-4-m e t h ylsu lfa n ylb e n ze n e -
su lfon a m id e (46). Compound 46 was synthesized from 2,4-
dichlorobenzoic acid and 4-methylsulfanylbenzenesulfon-
amide using RPS coupling procedure B. Yield ) 78%. 1H
NMR (300 MHz, DMSO-d6): δ 7.85 (d, J ) 7 Hz, 2H), 7.68 (s,
1H), 7.50 (s, 2H), 7.47 (d, J ) 7 Hz, 2H). LC/MS tr ) 1.91 min,
m/z 374, 376, 378 (M - H)-. HRMS obsd mass (M - H)-,
373.9474; calcd mass (C14H11Cl2NO3S2-1H)-, 373.9479. Anal.
(C14H11Cl2NO3S2) C, H, N.
4-Ac e t y l-N -(2,4-d i c h lo r o b e n z o y l)b e n z e n e s u lfo n -
a m id e (47). Compound 47 was synthesized from 2,4-dichloro-
benzoic acid and 4-acetylbenzenesulfonamide using RPS cou-
1
pling procedure B. Yield ) 78%. H NMR (400 MHz, DMSO-
d6):
δ 8.23 (d, J ) 8 Hz, 2H), 8.14 (d, J ) 8 Hz,
2H), 7.76 (d, J ) 2 Hz, 1H), 7.52-7.58 (m, 2H), 2.69 (s, 3H).
LC/MS tr ) 1.65 min, m/z 370, 372, 374 (M - H)-. HRMS obsd
mass (M - H)-, 369.9713; calcd mass (C15H11Cl2NO4S-1H)-,
369.9708. Anal. (C15H11Cl2NO4S) C, H, N.
N -(2,4-D ic h lo r o b e n zo y l)-3-m e t h y lb e n ze n e s u lfo n -
a m id e (48). Compound 48 was synthesized from 2,4-dichloro-
benzoic acid and 3-methylbenzenesulfonamide using coupling
1
procedure C. Yield ) 35%. H NMR (400 MHz, methanol-d4):
δ 7.87 (m, 2H), 7.54 (m, 2H), 7.49 (m, J ) 7.7 Hz, 1H), 7.40
(m, 2H), 2.46 (s, 3H). LC/MS was performed as described
for 1. The second HPLC was performed as described for 8.
LC/MS tr ) 1.37 min (>99%), m/z 342, 344 (M - H)-. Second
HPLC tr ) 3.71 min (98%). HRMS obsd mass (M - H)-,
341.9756; calcd mass (C14H11Cl2NO3S-1H)-, 341.9759.
3-Ch lor o-N-(2,4-d ich lor o-b en zoyl)-4-m et h ylb en zen e-
su lfon a m id e (41). Compound 41 was synthesized from 2,4-
dichlorobenzoic acid and 3-chloro-4-methylbenzenesulfon-
amide using RPS coupling procedure B. Yield ) 56%. 1H
NMR (300 MHz, DMSO-d6): δ 7.90 (s, 1H), 7.82 (d, J ) 7 Hz,
1H), 7.70 (s, 1H), 7.63 (d, J ) 7 Hz, 1H), 7.53 (d, J ) 6 Hz,
1H), 7.46 (d, J ) 7 Hz, 1H), 2.44 (s, 3H). LC/MS tr ) 2.04 min,
m/z 378, 376, 380 (M - H)-. HRMS obsd mass (M - H)-,
375.9382; calcd mass (C14H10Cl3NO3S-1H)-, 375.9369. Anal.
(C14H10Cl3NO3S) C, H, N.
3-Ch lor o-N-(2,4-d ich lor o-b en zoyl)-4-flu or ob en zen e-
su lfon a m id e (42). Compound 42 was synthesized from 2,4-
dichlorobenzoic acid and 3-chloro-4-fluorobenzenesulfon-
amide using RPS coupling procedure B. Yield ) 84%. 1H NMR
(400 MHz, DMSO-d6): δ 8.11 (dd, J ) 7, 2 Hz, 1H), 7.98-8.02
(m, 1H), 7.70-7.74 (m, 2H), 7.55 (d, J ) 8 Hz, 1H), 7.49 (dd,
J ) 8, 2 Hz, 1H). LC/MS tr ) 1.96 min, m/z 380, 382, 384 (M
- H)-. HRMS obsd mass (M - H)-, 379.9128; calcd mass
(C13H7Cl3FNO3S-1H)-, 379.9118. Anal. (C13H7Cl3FNO3S) C, H,
N.
3,4-Dib r om o-N-(2,4-d ich lor ob en zoyl)b en zen esu lfon -
a m id e (49). Compound 49 was synthesized from 2,4-dichloro-
benzoic acid and 3,4-dibromobenzenesulfonamide using cou-
pling procedure C. Yield ) 34%. 1H NMR (400 MHz, methanol-
d4): δ 8.32 (d, J ) 2.2 Hz, 1H), 7.97 (d, J ) 8.8 Hz, 1H), 7.93
(d, J ) 2.2 Hz, 1H), 7.54 (d, J ) 1.8 Hz, 1H), 7.43 (s, 1H), 7.42
(d, J ) 1.3 Hz, 1H). LC/MS was performed as described for 1.
The second HPLC was performed as described for 8. LC/MS tr
) 1.63 min (>99%), m/z 486, 488, 484 (M - H)-. Second HPLC
tr ) 3.89 min (95%). HRMS obsd mass (M - H)-, 483.7822;
calcd mass (C13H7Br2Cl2NO3S-1H)-, 483.7812.
N -(2,4-D i c h lo r o b e n z o y l)-3-n i t r o b e n z e n e s u lfo n -
a m id e (50). Compound 50 was synthesized from 2,4-dichloro-
benzoic acid and 3-nitrobenzenesulfonamide using RPS
coupling procedure B. Yield ) 78%. 1H NMR (250 MHz,
DMSO-d6): δ 8.66 (t, J ) 2 Hz, 1H), 8.56 (dd, J ) 8, 2 Hz,
1H), 8.39 (d, J ) 8 Hz, 1H), 7.96 (t, J ) 8 Hz, 1H), 7.70 (d, J
) 2 Hz, 1H), 7.57 (d, J ) 8 Hz, 1H), 7.49 (dd, J ) 8, 2 Hz, 1H).
LC/MS tr ) 1.75 min, m/z 373, 375, 377 (M - H)-. HRMS obsd
mass (M - H)-, 372.9472; calcd mass (C13H8Cl2N2O5S-1H)-,
372.9453. Anal. (C13H8Cl2N2O5S) C, H, N.
N -(2,4-D ic h lo r o -b e n zo y l)-4-flu o r o b e n ze n e s u lfo n -
a m id e (43). Compound 43 was synthesized from 2,4-dichloro-
benzoic acid and 4-fluorobenzenesulfonamide using RPS cou-
N -(2,4-Dich lor ob e n zoyl)-3-m e t h oxyb e n ze n e su lfon -
a m id e (51). Compound 51 was synthesized from 2,4-dichloro-
benzoic acid and 3-methoxybenzenesulfonamide using coupling
procedure C. Yield ) 41%. 1H NMR (400 MHz, CDCl3): δ 7.65
(d, J ) 7.9 Hz, 1H), 7.60 (dt, J ) 2.2 Hz, 1H), 7.53 (d, J ) 8.3
Hz, 1H), 7.44 (dd, J ) 7.9, 8.3 Hz, 1H), 7.33 (d, J ) 2.2 Hz,
1H), 7.26 (dd, J ) 2.2, 8.3 Hz, 1H), 7.16 (dd, J ) 2.6, 8.3 Hz,
1H). 13C NMR (75 MHz, CDCl3): δ 163.0, 159.9, 139.3, 138.9,
132.3, 131.8, 130.6, 130.4, 130.3, 128.0, 121.2, 120.8, 113.2,
56.0. LC/MS was performed as described for 1. The second
HPLC was performed as described for 8. LC/MS tr ) 1.56 min
(>99%), m/z 358, 360 (M - 1)-. Second HPLC tr ) 2.83 min
(98%). ESI-MS m/z: 358.0 (C14H11Cl2NO4S - 1H)-. HRMS
(positive mode) obsd mass (M + H)+, 359.9886; calcd mass
(C14H11Cl2NO4S+1H)+, 359.9859.
1
pling procedure B. Yield ) 61%. H NMR (250 MHz, DMSO-
d6): δ 8.10-8.00 (m, 2H), 7.71 (d, J ) 1.6 Hz, 1H), 7.56-7.44
(m, 4H). LC/MS tr ) 1.80 min, m/z 346, 348, 350 (M - H)-.
HRMS obsd mass (M - H)-, 345.9497; calcd mass (C13H8Cl2-
FNO3S-1H)-, 345.9508. Anal. (C13H8Cl2FNO3S) C, H, N.
3-C h lo r o -N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (44). Compound 44 was synthesized from 2,4-dichloro-
benzoic acid and 3-chlorobenzenesulfonamide using RPS
coupling procedure B. Yield ) 82%. 1H NMR (250 MHz,
DMSO-d6): δ 7.97-7.91 (m, 2H), 7.84 (d, J ) 8 Hz, 1H),
7.74-7.66 (m, 2H), 7.56 (d, J ) 9 Hz, 1H), 7.49 (dd, J ) 8, 2
Hz, 1H). LC/MS tr ) 1.94 min, m/z 364, 362, 366 (M - H)-.
HRMS obsd mass (M
-
H)-, 361.9197; calcd mass
(C13H8Cl3NO3S-1H)-, 361.9212. Anal. (C13H8Cl3NO3S) C, H, N.
3,4-Dich lor o-N-(2,4-d ich lor ob en zoyl)b en zen esu lfon -
a m id e (45). Compound 45 was synthesized from 2,4-dichloro-
N-(2,4-Dich lor ob en zoyl)-4-(d im et h yla m in o)b en zen e-
su lfon a m id e (52). Compound 52 was synthesized from 2,4-

5378 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Lobb et al.
dichlorobenzoic acid and 4-(dimethylamino)benzenesulfon-
amide using RPS coupling procedure B. Yield ) 16%. 1H
NMR (400 MHz, DMSO-d6): δ 7.74 (d, J ) 9 Hz, 2H), 7.69 (d,
J ) 2 Hz, 1H), 7.48 (dd, J ) 8 Hz, 2 H), 7.43 (d, J ) 8 Hz,
1H), 6.81 (d, J ) 9 Hz, 2H), 3.03 (s, 6H). LC/MS studies
were performed as described for 1 except that positive mode
ES ionization was used. LC/MS tr ) 1.51 min, m/z 373
(M + 1)+. HRMS (negative mode ES) obsd mass (M - H)-,
371.0045; calcd mass (C15H14Cl2N2O3S-1H)-, 371.0024. Anal.
(C15H14Cl2N2O3S) C, H, N.
DMSO-d6): δ 8.17 (d, J ) 8 Hz, 1H), 7.72 (d, J ) 2 Hz, 3H),
7.66-7.58 (m, 1H), 7.53 (s, 2H) ppm. LC/MS tr ) 1.81 min,
m/z 364, 362, 366 (M - H)-. HRMS obsd mass (M - H)-,
361.9223; calcd mass (C13H8Cl3NO3S-1H)-, 361.9212. Anal.
(C13H8Cl3NO3S) C, H, N.
2-Ch lor o-N -(2,4-d ich lor ob e n zoyl)-5-n it r ob e n ze n e -
su lfon a m id e (59). Compound 59 was synthesized from 2,4-
dichlorobenzoic acid and 2-chloro-5-nitrobenzenesulfonamide
1
using RPS coupling procedure B. Yield ) 62%. H NMR (300
MHz, DMSO-d6): δ 8.81 (d, J ) 2 Hz, 1H), 8.49 (dd, J ) 9 Hz,
1 H), 7.99 (d, J ) 9 Hz, 1H), 7.70 (d, J ) 2 Hz, 1H), 7.60 (d, J
) 8 Hz, 1H), 7.51 (dd, J ) 8, 2 Hz, 1H). LC/MS tr ) 1.77 min,
m/z 409, 407, 411 (M - H)-. HRMS obsd mass (M - H)-,
406.9070; calcd mass (C13H7Cl3N2O5S-1H)-, 406.9063. Anal.
(C13H7Cl3N2O5S) C, H, N.
Bip h en yl-3-su lfon ic Acid 2,4-Dich lor ob en zoyla m id e
(53). To a solution of 3-bromo-N-(2,4-dichlorobenzoyl)ben-
zenesulfonamide (38) (0.10 mmol) in toluene/ethanol 20/1 (3
mL) were added arylboronic acid (0.18 mmol, 0.18 mL, 1.0 M
in DMF) and tetrakis(triphenylphosphine)palladium(0) (10 mol
%). Then, 2 M aqueous Na2CO3 was added (0.3 mL) and the
stirred mixture was heated to 100 °C overnight (17 h). The
reaction mixture was concentrated. H2O (2.5 mL) and EtOAc
(5 mL) were added. The phases were separated, and the
aqueous layer was extracted with EtOAc (3 × 5 mL). The
solvents were evaporated, and the corresponding crude product
was purified by HPLC to give the title compound. Yield )
N -(2,4-Dich lor ob e n zoyl)-2-m e t h yl-5-n it r ob e n ze n e -
su lfon a m id e (60). Compound 60 was synthesized from 2,4-
dichlorobenzoic acid and 2-methyl-5-nitrobenzenesulfon-
amide using RPS coupling procedure B. Yield ) 84%. 1H NMR
(250 MHz, DMSO-d6): δ 8.72 (d, J ) 3 Hz, 1H), 8.44 (dd, J )
8, 2 Hz, 1H), 7.76 (d, J ) 8 Hz, 1H), 7.71 (d, J ) 2 Hz, 1H),
7.57 (d, J ) 8 Hz, 1 H), 7.50 (dd, J ) 8, 2 Hz, 1H), 2.73 (s,
3H). LC/MS tr ) 1.89 min (>99%), m/z 387, 389, 391 (M -
1
14%. H NMR (200 MHz, methanol-d4): δ 7.40-7.55 (m, 5H),
7.62-7.77 (m, 4H), 7.90-8.04 (m, 2H), 8.17 (m, 1H). 13C NMR
(50 MHz, methanol-d4): δ 125.8; 126.7; 127.2 (2C); 127.8;
128.7; 129.6 (2C); 129.8; 130.3; 130.8; 131.6; 132.2; 133.4;
136.2; 138.3; 138.8; 140.5 and 141.3. ESI-MS m/z 404.0
(C19H13Cl2NO3S-1H)-.
H)-. HRMS obsd mass (M - H)-, 386.9627; calcd mass (C14H10
-
Cl2N2O5S-1H)-, 386.9609. Anal. (C14H10Cl2N2O5S) C, H, N.
2-Br om o-N-(2,4-d ich lor ob en zoyl)-4-m et h ylb en zen e-
su lfon a m id e (61). Compound 61 was synthesized from 2,4-
dichlorobenzoic acid and 4-methylbenzenesulfonamide
4-(2,4-Dich lor oben zoylsu lfa m oyl)ben zoic Acid Meth yl
Ester (54). Compound 54 was synthesized from 2,4-dichloro-
benzoic acid and 4-sulfamoyl-benzoic acid methyl ester using
coupling procedure C. Yield ) 75%. 1H NMR (400 MHz,
DMSO-d6): δ 3.97 (s, 3 H), 7.35 (dd, J ) 8.4, 2.0 Hz, 1H), 7.44
(d, J ) 2.0 Hz, 1H), 7.70 (d, J ) 8.4 Hz, 1H), 8.23 (s, 4H).
LC/MS was performed as described for 1. The second HPLC
was performed as described for 8. LC/MS tr ) 1.65 min (>99%),
m/z 386, 388 (M - H)-. Second HPLC tr ) 2.85 min (99%).
HRMS (positive mode) obsd mass (M)+, 386.9735; calcd mass
(C15H11Cl2NO5S)+, 386.9735. Anal. (C15H11Cl2NO5S‚0.15 CH2-
Cl2) C, H, N.
N -(2,4-D i c h lo r o b e n z o y l)-4-n i t r o b e n z e n e s u lfo n -
a m id e (55). Compound 55 was synthesized from 2,4-dichloro-
benzoic acid and 4-nitrobenzenesulfonamide using RPS
coupling procedure B. Yield ) 60%. 1H NMR (300 MHz,
DMSO-d6): δ 8.46 (d, J ) 9 Hz, 2H), 8.22 (d, J ) 9 Hz, 2H),
7.70 (d, J ) 2 Hz, 1H), 7.56 (d, J ) 8 Hz, 1H), 7.49 (dd, J ) 8,
2 Hz, 1H). LC/MS tr ) 1.80 min, m/z 373, 375, 377 (M - H)-.
HRMS obsd mass (M - H)-, 372.9468; calcd mass (C13H8-
Cl2N2O5S-1H)-, 372.9453. Anal. (C13H8Cl2N2O5S) C, H, N.
1
using RPS coupling procedure B. Yield ) 79%. H NMR (400
MHz, DMSO-d6): δ 7.96 (d, J ) 2 Hz, 1H), 7.75 (d, J ) 8 Hz,
1H), 7.71 (d, J ) 1 Hz, 1H), 7.49-7.54 (m, 2H), 7.43 (dd,
J
) 8, 1 Hz, 1H), 2.29 (s, 3H). LC/MS tr ) 1.91 min,
m/z 420, 422, 424 (M - H)-. HRMS obsd mass (M - H)-,
419.8880; calcd mass (C14H10BrCl2NO3S-1H)-, 419.8864. Anal.
(C14H10BrCl2NO3S) C, H, N.
3,5-Dich lor o-N-(2,4-d ich lor ob en zoyl)b en zen esu lfon -
a m id e (62). Compound 62 was synthesized from 2,4-dichloro-
benzoic acid and 3,5-dichlorobenzenesulfonamide using RPS
coupling procedure B. Yield ) 73%. 1H NMR (250 MHz,
DMSO-d6): δ 8.02 (t, J ) 2 Hz, 1H), 7.88 (d, J ) 2 Hz, 2H),
7.68 (d, J ) 2 Hz, 1H), 7.59 (d, J ) 8 Hz, 1H), 7.48 (dd, J ) 8,
2 Hz, 1H). LC/MS tr ) 2.13 min, m/z 398, 396, 400 (M - H)-.
HRMS obsd mass (M - H)-, 395.8852; calcd mass (C13H7Cl4-
NO3S-1H)-, 395.8823. Anal. (C13H7Cl4NO3S) C, H, N.
HUVEC P r olifer a tion Assa y.10 HUVECs (Clonetics/Bio-
whittaker) were maintained in EGM media (endothelial cell
growth medium, Clonetics/Biowhittaker). On the day of the
assay, the cells were trypsinized and harvested using basal
medium (EBM) with 0.5% FBS (Clonetic/Biowhittaker). The
cells were seeded into 96 well plates at a concentration of 5000
cells/well at a volume of 160 µL. The plates were incubated in
the low serum media overnight at 37 °C, 5% CO2, to quiesce
the cells. The next day, serial dilutions of the compounds were
prepared for a final dosage range of 0.027-20 µM. Twenty
microliters of compounds was added, followed by the addition
of 20 µL of 100 µg/mL VEGF stock in phosphate-buffered
saline/0.1% BSA (R&D Systems), for a final concentration of
20 ng/mL. The cells were placed back in the incubator. After
72 h, 20 µL of WST-1 proliferation reagent (Roche Diagnostics
Corporation) was added to the medium. After 1 h of incubation
at 37 °C, the absorbances were read at 440 nM with a
Spectramax Spectrophotometer (Molecular Devices). The growth
fraction was determined from the absorbance of treated wells
with and without VEGF divided by the absorbance obtained
from control wells set to zero and 1.0. The IC50 values were
calculated using a two parameter logistic curve fit program,
Y ) 1/[1 + (X/c)]b, to the dose-response data where Y )
fraction activity, X ) concentration, c ) IC50, and b ) slope.
The MSR for comparing average IC50 values with n > 3 was
2.6 for this assay.21 That is, the average IC50 values of two
compounds were considered statistically significantly different
if the ratio was more than 2.6. For IC50 values with n ) 2, the
MSR was 3.3.
Bip h en yl-4-su lfon ic Acid 2,4-Dich lor oben zoyla m id e
(56). Compound 56 was synthesized from 4-bromo-N-(2,4-
dichlorobenzoyl)benzenesulfonamide (37) using a procedure
1
similar to that described for 53. Yield ) 32%. White solid. H
NMR (200 MHz, DMSO-d6): δ 7.88 (d, J ) 8.1 Hz, 2H), 7.69
(br d, J ) 7.8 Hz. 4H), 7.51-7.29 (m, 6H). 13C NMR (50 MHz,
DMSO-d6): δ 169.6, 145.3, 141.9, 139.8, 132.8, 131.6, 131.3,
129.6, 129.3, 129.2, 128.1 (2C), 127.7, 127.2 (2C), 126.8 (2C),
126.3 (2C). LC/MS was performed as described in 1. The second
HPLC was performed as described in 25. LC/MS tr ) 1.88
(>99%) m/z 404, 406 (M - H)-. Second HPLC tr ) 2.70 min
(>99%).
N-(2,4-Dich lor oben zoyl)-3,4-d im eth oxyben zen esu lfon -
a m id e (57). Compound 57 was synthesized from 2,4-dichloro-
benzoic acid and 3,4-dimethoxybenzenesulfonamide using RPS
1
procedure B. Yield ) 39%. H NMR (400 MHz, DMSO-d6): δ
7.75 (s, 1H), 7.62 (dd, J ) 8, 2 Hz, 1H), 7.52 (d, J ) 1 Hz, 2H),
7.45 (d, J ) 2 Hz, 1H), 7.23 (d, J ) 9 Hz, 1H), 3.89 (s, 3H),
3.85 (s, 3H). LC/MS tr ) 1.63 min, m/z 388, 390, 392 (M
- H)-. HRMS obsd mass (M - H)-, 387.9830; calcd mass
(C15H12Cl2NO5S)-, 387.9813. Anal. (C15H13Cl2NO5S) C, H, N.
2-C h lo r o -N -(2,4-d ic h lo r o b e n zo y l)b e n ze n e s u lfo n -
a m id e (58). Compound 58 was synthesized from 2,4-dichloro-
benzoic acid and 2-chlorobenzenesulfonamide using gen-
eral RPS procedure B. Yield ) 75%. 1H NMR (250 MHz,

Acyl Sulfonamide Anti-Proliferatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5379
SADD Assa y. The SADD assay to differentiate the anti-
proliferative activity of an agent against normal cells (fibro-
blasts cells, CFU-GM), leukemia cells (L1210), and solid tumor
cells (including multidrug resistant solid tumors) was per-
formed as described by Corbett et al.8,9 The leukemia cells,
solid tumor cells, and normal cells were seeded in soft agar in
separate 60 mm Petri dishes. The drug was placed on a filter
paper disk (standard hole punch of Whatman no. 1) that was
then placed on top of the soft agar. The drug diffused off the
disk as the cancerous cells and normal cells were replicating,
creating a zone of inhibition of colony formation. The zones
were recorded after 6-7 days of incubation at 37 °C. Cytotox-
icity was measured by the zone of inhibition, with 200 zone
units ) 6.5 mm. In general, a 330 zone unit change was
effected by a one log dilution in the concentration of a cytotoxic
agent. A 950 zone was the limit of the dish (total inhibition).
A zero zone meant no inhibition. In cases where a zone range
was recorded, the colonies were reduced in size at the begin-
ning of the zone, increasing in size to the top of the zone range,
and from there to the edge of the plate, the colonies were
uniformly large. Ideally, an active compound would have little
or no activity against the normal cells, would inhibit to some
extent the colony formation of the leukemia cells, and would
inhibit greatly the colony formation of the various solid tumors.
P K Stu d ies. For the study in female F344 rats (n ) 3),
compounds were formulated in 1-1.5% sodium bicarbonate
in phosphate-buffered saline and dosed as indicated (oral
gavage or iv bolus injection). For the seven day study, the
animals were treated once daily. Blood was drawn at t ) 0.5,
1, 2, 4, 6, 8, and 24 h after the final dose. For the study in
BALBc mice (n ) 3 mice/time point), compounds were formu-
lated in 1-1.5% sodium bicarbonate in phosphate-buffered
saline. The compounds were dosed as indicated (oral gavage
or iv bolus injection) and administered once daily from days
1-5 and 8-12. Blood was drawn at t ) 0.5, 1, 2, 4, 6, 8, and
24 h after the final dose.
HCT116 Tu m or Xen ogr a ft Mod el. Female nude mice
were housed in microisolator cages and received food and water
ad libitum. In each experiment, test mice were implanted with
HCT116 human colon tumor cells by transferring tissue from
donor mice. When tumors reached an average volume of 100
mm3, animals were randomized and separated into treatment
groups of 10 animals per group and dosing initiated. The
compound dissolved in saline was administered daily from
days 1-5 and 8-12 by iv bolus injection. Body weights and
tumor volumes were recorded two times weekly. Calculations
of TGD were performed as below.
Qu a n tified En d P oin ts for Assessin g An titu m or Activ-
ity in Solid Tu m or s. The following quantified end points
were used to assess antitumor activity. (i) Tumor volume
(mm3) ) (a × b2)/2 where a ) tumor length (mm) and b )
tumor width (mm). (ii) TGD (T - C value) ) T - C where T
is the median time (days) required for the treatment group
tumors to reach a predetermined size and C is the median time
(in days) for the control group tumors to reach the same size.
QSAR data sets.35 The data set was split into five random
groups for cross-validation such that four groups were used
as a training set and the fifth group was left out, to be
predicted as a test set. This was done five times so that each
compound was eventually left out of the training set and
predicted once. Spearman’s F statistic was used to evaluate
the model ability to rank order the test sets. A final model
with no cross-validation was then built and used in subsequent
work to predict and prioritize new series and ideas for the
project members. The five way cross-validated model gave a
Spearman F ) 0.61, and the final model with no cross-
validation gave a Spearman F ) 0.95.
Ack n ow led gm en t. We thank Paul A. Lee, Scott
Norman, Richard M. Schultz, J ack Dempsey, the DCRT
Analytical Technologies group, Walter Krueger of Pied-
mont Research Center, the Barbara Ann Karmanos
Cancer Institute at WSU, the NCI, and Midwest Mi-
crolab for their excellent technical assistance. We also
gratefully acknowledge Dr. J ames. R. McCarthy and Dr.
Michael R. Wiley for reviewing the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Procedures and
characterization data for benzoic acid starting materials (2-
bromo-4-chlorobenzoic acid, 2,4-dibromobenzoic acid, 2-chloro-
4-methoxybenzoic acid, and 2-chloro-4-cyanobenzoic acid) and
for benzenesulfonamide starting materials (3-methyl-
benzenesulfonamide, 3,4-dibromobenzenesulfonamide, 3-meth-
oxybenzenesulfonamide, and 4-sulfamoylbenzoic acid methyl
ester) and elemental analyses. This material is available free
of charge via the Internet at http://pubs.acs.org.
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