Acyl sulfonamide anti-proliferatives. Part 2: Activity of heterocyclic sulfonamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.11.041

The anti-proliferative activity of acylated heterocyclic sulfonamides is described in Vascular Endothelial Growth Factor-dependent Human Umbilical Vascular Endothelial Cells (VEGF-HUVEC) and in HCT116 tumor cells in a soft agar diffusion assay.

Full text of DOI:10.1016/j.bmcl.2004.11.041

Bioorganic & Medicinal Chemistry Letters 15 (2005) 617–620
Acyl sulfonamide anti-proliferatives. Part 2: Activity of
heterocyclic sulfonamide derivatives
Mary M. Mader,a,* Chuan Shih,a Eileen Considine,a Alfonso De Dios,b
Cora Sue Grossman,a Philip A. Hipskind,a Ho-Shen Lin,a Karen L. Lobb,a Beatriz Lopez,b
a
b
a
a
´
Jose E. Lopez, Luisa M. Martin Cabrejas, Michael E. Richett, Wesley T. White,
Yiu-Yin Cheung,c Zhongping Huang,c John E. Reillyc and Sean R. Dinnc
aLilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
b
´
Centro de Investigacion Lilly, S. A., Avda. De La Industria 30, 28108-Alcobendas, Madrid, Spain
cAlbany Molecular Research Inc., Albany, NY 12212-5098, USA
Received 17 September 2004; revised 12 November 2004; accepted 16 November 2004
Available online 13 December 2004
Abstract—The anti-proliferative activity of acylated heterocyclic sulfonamides is described in Vascular Endothelial Growth Factor-
dependent Human Umbilical Vascular Endothelial Cells (VEGF-HUVEC) and in HCT116 tumor cells in a soft agar diffusion assay.
ꢀ 2004 Elsevier Ltd. All rights reserved.
We have recently reported on the in vitro activity of
acylsulfonamide anti-proliferative agents 1, demonstrat-
ing a correlation between cytoxicity as determined in
Vascular Endothelial Growth Factor-dependent Human
Umbilical Vascular Endothelial Cells (VEGF-HUVEC)
and anti-tumor activity as measured in a soft-agar disk
diffusion (SADD) assay versus HCT116 colon carci-
noma cells.1,2 The mechanism of action of the com-
pounds remains under investigation, but the series
shows efficacy in vivo in the tumor xenograft model,
HCT 116, with no correlation to known drugs in the
NCI COMPARE analysis. In these studies, 3,4-disubsti-
tuted benzenesulfonamides were found to be highly ac-
tive, and it was therefore of interest to determine the
activity of a variety of heterocyclic sulfonamide deriva-
tives (Table 1).
Table 1. VEGF-HUVEC inhibition by previously reported com-
pounds
Compound R1
Ave. VEGF- HCT116,
HUVEC
Dose
(lg, disk)
SADD zone
of inhibitionb
a
IC50 (lM)
1a
1b
1c
(H)
4-Cl
0.53
0.17
700
660
560
115
200
200
3,4-diCl 0.46
a Values are means of at least three experiments.
b Zone of inhibition units (200 units = 6.5 mm).
ring (2a and 3a). The least active compounds are those
substituted by the sulfonamide at the 4- or 7- site in
these 5/6 fused ring systems (2d,e, 3c, 4b). This finding
was consistent with the SAR of parent series 1. Place-
ment of the sulfonyl in the ortho position resulted in
the least active members of that family, and the 4- and
7-substitution of 2, 3, and 4 is a pseudo-ortho position
for these heterocycles.
We found that fused bicyclic heterocyclic derivatives
that mimic the 3,4-disubstitution pattern of benzene–
sulfonamide are comparably active to 1c (Table 2).
Thus, the attachment of the sulfonamide to the aromatic
six-membered ring at position 5 or 6 (e.g., 2b,c, 3b, 4a) is
preferred approximately twofold over to attachment of
the sulfonamide to the 2-position of the five-membered
Fused heterocycles containing a saturated ring are also
active, and oxygen-containing analogs attached through
the aromatic ring have IC50 values <0.5 lM (Table 3;
Keywords: Cytotoxic; Oncolytic.
*
Corresponding author. Tel.: +1 317 2773400; fax: +1 317 2772035;
e-mail: mader_mary@lilly.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.041

618
M. M. Mader et al. / Bioorg. Med. Chem. Lett. 15 (2005) 617–620
Table 2. VEGF-HUVEC inhibition by 5/6 fused heterocyclic sulfon-
amide analogsa
Table 4. VEGF-HUVEC inhibition by five-membered ring hetero-
cyclic sulfonamide analogsa
Compound R1
R2
X
VEGF-HUVEC c Log P
IC50 (lM)
Compound
Y
Point of sulfonamide
attachment
VEGF-HUVEC
IC50 (lM)
6a
6b
6c
6d
6e
6f
H
H
––
––
––
––
––
––
––
––
0.54
0.25
0.60
0.22
0.76
0.36
0.39
0.60
0.70
0.31
0.68
1.39
3.16
1.27
1.09
0.54
2.24
1.18
0.60
3.23
4.19
4.19
4.89
4.80
4.04
4.04
3.73
3.73
4.26
3.31
3.51
3.10
3.23
3.30
3.37
2.58
3.08
3.60
2.88
H
Br
H
2a
2b
2c
2d
2e
3a
3b
3c
4a
4b
S
2-
6-
5-
4-
7-
2-
6-
7-
6-
7-
0.96
0.64
0.62
3.30
4.16
0.97
0.59
5.21
1.87
3.04
Br
Br
Cl
H
S
Br
Br
Cl
S
S
S
6g
6h
6i
Cl
H
H
O
O
O
NH
NH
Me
H
Me
H
6j
Et
MeO
––
––
6k
6l
H
H
MeO2C ––
a Values are means of at least three experiments.
6m
7
H
NC
––
––
––
S
––
––
––
––
––
––
––
8a
8b
8c
8d
8e
8f
Cl
OMe
H
S
S
Me
Et
S
Table 3. VEGF-HUVEC inhibition by fused heterocyclic sulfonamide
analogsa
S
Et
NH >20
a Values are means of at least three experiments.
6a–m appears to be independent of the substituentsÕ
electronic effects, but more closely correlated to their
lipophilicity as predicted by c log P,3 with a value of
>4.0 tending to prevail in the most potent thiophene
analogs. A loss of potency relative to 6a is observed if
the thiophene itself is attached as a 3-sulfonamido deriv-
ative (e.g., 7), but in the absence of a molecular target,
the reasons for this deviation are not clear.
Compound
Ar
VEGF-HUVEC IC50 (lM)
5a
5b
0.43
0.48
5c
5d
1.54
0.89
Addition of nitrogen within the five-membered ring re-
sulted in decreased activity. Thiazoles 8a–e were two-
to fivefold less potent than the corresponding thioph-
enes, with one exception (8b), and imidazole analog 8f
was inactive under the assay conditions. More generally,
inclusion of nitrogen in the ring systems in Tables 2–4
results in a decreased activity relative to the oxygen
and sulfur analogs with sulfonamide attached at the
same position (e.g., 4a vs 2b and 3b). The indole, thia-
zole, and imidazole analogs presumably are more hydro-
philic than the thiophene series because of the ring
nitrogen, and may not penetrate the HUVEC cell mem-
brane as efficiently as the thiophenes.1 An alternative
explanation may be that the decrease in activity of the
nitrogen heterocycles reflects an unfavorable interaction
with the receptor.
5e
5f
0.40
0.80
a Values are means of at least three experiments.
5a,b,e). The least active within this set is the naphthyl
derivative 5f, but the observation of decreased activity
when the distal ring is the six-membered aromatic is
consistent with the activity of the 2-benzofuranyl and 2-
benzothienyl analogs 2a and 3a, respectively. The
benzodioxinyl analog 5e was more active than 5f, but
the saturated heterocycle 5e may allow for greater flexi-
bility in fitting into the target active site.
Monoheterocyclic sulfonamides were also highly active
in the primary assay (Table 4). The thiophene analogs
6a–m, whether substituted at the 4- or 5-position of
the thiophene, showed similar potency to the substituted
phenyl analogs 1. Optimal cases were the thiophenes
with small substituents, such as halo or alkyl examples
6a–j, with IC50 values <1 lM. The activity of thiophenes
Representative potent compounds from each series were
additionally assayed against HCT116 tumor cells in a
SADD assay that provided a qualitative measure of
the compounds oncolytic, cytotoxic activity. We earlier
observed that approx. 80% of compounds with IC50 val-
ues <1 lM in the VEGF-HUVEC assay were also active
in HCT116, with lesser effect on normal fibroblasts and

M. M. Mader et al. / Bioorg. Med. Chem. Lett. 15 (2005) 617–620
619
Table 5. SADD activity of compounds with VEGF-HUVEC IC50 < 1
lMa
aminopyridine (DMAP) to provide the acylsulfonamide
products.
Compound
HCT116
L1210
Normal
fibroblasts
Dose
(lg/disk)
Preparation of the 4-substituted thiophene-2-sulfon-
amides presented the greatest challenge, as deprotona-
tion/sulfonylation of 9a or 9b would have yielded a
2,3-disubstitution pattern. Thus, a bromine blocking
group was first introduced at the 2-position (Scheme
2). Sulfonylation by PCl5 and chlorosulfonic acid fol-
lowed by treatment in concentrated ammonium hydrox-
ide gave the trisubstituted sulfonamides 11a and 11b.
Removal of the blocking group was accomplished by
treatment of the bromothiophenes with zinc dust to
yield 12a and 12b.
2a
5a
5b
5c
5e
5f
500
680
0–100
0
0–370
0–400
0–400
0–330
0–400
0–270
0–350
0–390
210
210
200
210
200
210
190
190
610
630
0
0–60
0–250
200–270
0
580
470–530
490
600
6a
6b
0–180
a SADD Zone of Inhibition reported, zone of inhibition units: 200
units = 6.5 mm. A zero zone means no inhibition.
Thiophene derivatives were also prepared from commer-
cially available 5-bromothiophene-2-sulfonamide 13
(Scheme 3). Palladium-catalyzed methoxy-carbonyl-
ation provided ester 14,8 and palladium-catalyzed cyana-
tion of the bromide resulted in nitrile 15.9
L1210 leukemia cells.1 The heterocyclic sulfonamide
derivatives possessed a similar profile as shown in Table
5, indicating the heterocyclic analogs are likely to be
active at the same receptor.
The primary synthetic focus was the preparation of the
heterocyclic sulfonamides, and a variety of methods
were employed. In the preparation of 4-, 5-, 6-, and 7-
sulfonamido 5/6 fused heterocycles, the key precursors
were the brominated analogs A,4 which were subjected
to strong base to effect lithium–halogen exchange. The
bromoindole precursors for products 4a and 4b required
no protection, but were first deprotonated by KH at
0 ꢁC, then reacted with an excess of t-BuLi at À78 ꢁC
to effect the halogen/metal exchange. In all cases, the
lithiated intermediate was treated with SO2 gas, fol-
lowed by oxidation in situ of the lithium sulfinate inter-
mediates B by N-chlorosuccinimide (NCS). Stirring the
resultant sulfonyl chlorides with ammonium hydroxide
provided the desired sulfonamides D (Scheme 1).5 Prep-
aration of 2-sulfonamido-benzothiophene and 2-sulfon-
amido-benzofuran was accomplished respectively by
deprotonation of benzothiophene and benzofuran with
n-BuLi, followed by treatment with SO2, NCS, and
NH4OH as above.6 The saturated, oxygenated bicyclic
heterocyclic sulfonamides giving rise to products 5a,b,
and 5e were prepared by literature methods.7 The sul-
fonamides were reacted with 2,4-dichlorobenzoic acid
in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC) and N,N-dimethyl-
Thiazolyl sulfonamides 18 were prepared by deprotona-
tion/sulfonylation, followed by oxidation of the lithium
sulfinate salt by one of two methods (Scheme 4). Alkyl
and halo substituted thiazole sulfinate derivatives were
treated with N-chlorosuccinimide followed by aqueous
ammonium hydroxide.6,10 Alternatively, the lithium
sulfinate of an alkoxy or unsubstituted thiazole was ami-
nated by treatment with hydroxylamine-O-sulfonic acid
to provide the sulfonamides.
Finally, the imidazole analog was prepared from 2-ethyl-
imidazole as shown in Scheme 5. This route required
protection of N-1 in compound 19 by [2-(trimethylsil-
yl)-ethoxy]-methyl chloride (SEM chloride), and then
proceeded as above, with a sequence of a deprotonation,
SO2 quench, NCS oxidation, and treatment with aque-
ous concentrated ammonium hydroxide, yielding sul-
fonamide 22. Following the EDC coupling of 22 to
Scheme 2. Reagents and conditions: (a) NBS, CHCl3, AcOH, rt; (b) (i)
PCl5, ClSO3H; (ii) NH4OH, acetone; (c) Zn dust, AcOH.
Scheme 1. Reagents and conditions: (a) (i) base, THF, À78 ꢁC; (ii)
SO2, À78 ꢁC to rt; (b) NCS; (c) NH4OH; (d) 2,4-dichlorobenzoic acid,
EDC, DMAP, CH2Cl2, rt.
Scheme 3. Reagents and conditions: (a) CO, Pd(OAc)2, Ph2P(CH2)3-
PPh2, Et3N, DMF, MeOH; (b) Zn(CN)2, Pd(PPh3)4, DMF, l-wave.

620
M. M. Mader et al. / Bioorg. Med. Chem. Lett. 15 (2005) 617–620
McCann, D. J.; Molero, V.; Reilly, J. E.; Richett, M. E.;
Shih, C.; Teicher, B.; Wikel, J. H.; White, W. T.; Mader,
M. M. J. Med. Chem. 2004, 47, 5367–5380.
2. The minimum significant ratio (MSR) for comparing
average VEGF-HUVEC IC50Õs with n P 3 was calculated
to be 2.6.
3. Daylight CLOGP v. 4.72; Daylight Chemical Information
Systems, Inc.: Mission Viejo, CA.
Scheme 4. Reagents and conditions: (a) (i) n-BuLi, THF, À78 ꢁC; (ii)
SO2, À78 ꢁC to rt; (b) for R = Me, Et, or Cl: (i) NCS; (ii) NH4OH; (c)
for R = OMe or H: H2NOSO3H, NaOAc.
4. 6-Bromobenzothiophene: (a) Briner, K.; Burkholder, T.
P.; Conway, R. G.; Cunningham, B. E.; Finley, D. R.;
Heinz, L. J.; Jesudason, C. D.; Kohlman, D. T.; Liang, S.
X.; Xu, Y.-c. PCT Int. Appl. WO 2001 09126 A1, 2001;
Chem. Abstr. 2001, 134, 162912; (b) 6- and 7-Bro-
mobenzofuran: Briner, K.; Burkhart, J. P.; Burkholder,
T. P.; Cunningham, B. E.; Fisher, M. J.; Gritton, W. H.;
Jesudason, C. D.; Miller, S. C.; Mullaney, J. T.; Reinhard,
M. R.; Rothhaar, R. R.; Stevens, F. C.; Winneroski, L. L.,
Jr.; Xu, Y.; Xu, Y.-c. PCT Int. Appl. WO 2001 09122 A2,
2001; Chem. Abstr. 2001, 134, 162921; (c) A general
method for preparation of benzofurans: Barker, P.; Finke,
P.; Thompson, K. Synth. Commun. 1989, 19, 257–265.
5. A representative example for the conversion of a hetero-
cyclic bromide to a sulfonic acid amide is as follows: To a
solution of 4-bromobenzo[b]thiophene (1.43 g, 6.71 mmol)
in anhydrous Et2O (31 mL) at À78 ꢁC was added t-BuLi
(9.1 mL of 1.7 M in pentane, 15.4 mmol) dropwise. After
stirring for 40 min at À78 ꢁC, the reaction was warmed to
0 ꢁC for 5 min then re-cooled to À78 ꢁC. Sulfur dioxide
was bubbled over the solution for 5 min then the reaction
was warmed to room temperature overnight. N-Chloro-
succinimide (2.69 g, 20.1 mmol) was added to the reaction
and the mixture was stirred for 1.5 h. The reaction was
filtered under vacuum and the precipitate was washed with
Et2O. The filtrate was concentrated under vacuum to give
the crude sulfonyl chloride (1.07 g), which was dissolved in
acetone (50 mL) and was added to a stirred solution of
concentrated NH4OH (20 mL) in acetone (20 mL) at 0 ꢁC
and the reaction was stirred for 1 h. The reaction was
partitioned between EtOAc and H2O and the aqueous
layer was separated and extracted with EtOAc (2 · ). The
combined organic extracts were dried (MgSO4), filtered,
and concentrated under vacuum to give the crude product.
Flash chromatography on silica gel eluting with hexane–
CH2Cl2 (4:1) afforded the benzo[b]thiophene-4-sulfon-
amide (281 mg, 20%).
Scheme 5. Reagents and conditions: (a) SEMCl, NaH, THF, 0 ꢁC; (b)
(i) n-BuLi, THF, À78 ꢁC, (ii) SO2; (c) (i) NCS, (ii) NH4OH; (d) 2,4-
dichlorobenzoic acid, EDC, DMAP, CH2Cl2, rt; (e) HCl, EtOH,
50 ꢁC.
2,4-dichlorobenzoic acid, removal of the SEM protect-
ing group under acidic conditions provided 8f.
Thus, several mono- and bicyclic heterocyclic sulfon-
amides were found to possess activity in VEGF-HU-
VEC and HCT116 SADD assay comparable to the
parent biaryl series 1. The preferred point of sulfon-
amide substitution for 5/6 fused bicyclic compounds is
at the 5- or 6-position, and for thiophenes is at the 2-po-
sition. The in vivo activity of these analogs in tumor
xenograft models is under investigation.
Acknowledgements
6. (a) Mohamadi, F.; Spees, M. M. U.S. Patent 5,169,860,
1992; Chem. Abstr. 1993, 118, 94316; (b) Besterman, J. M.;
Delorme, D.; Rahil, J. WO Patent 2001 02411, 2001;
Chem. Abstr. 2001, 134, 95480.
7. Tao, E. V. P.; Miller, W. D. Eur. Patent Appl. EP 583,960
B1, 1994; Chem. Abstr. 1994, 120, 245066.
The contributions of Prof. Thomas Corbett of the Bar-
bara Ann Karmanos Cancer Institute at Wayne State
University are gratefully acknowledged.
8. Drent, E. Brit. UK Patent Appl. GB 2,261,662 A1, 1993;
Chem. Abstr. 1993, 119, 159881.
References and notes
9. Tschaen, D. M.; Desmond, R.; King, A. O.; Fortin, M. C.;
Pipik, B.; King, S.; Verhoeven, T. R. Synth. Commun.
1994, 24, 887–890.
10. Howbert, J. J.; Mohamadi, F.; Spees, M. M. Eur. Patent
Appl. EP 467,613 A1, 1992; Chem. Abstr. 1992, 116,
194142.
1. Lobb, K. L.; Hipskind, P. A.; Aikins, J. A.; Alvarez, E.;
Cheung, Y.-Y.; Considine, E. L.; De Dios, A.; Durst, G.
L.; Ferritto, R.; Grossman, C. S.; Giera, D. D.; Hollister,
B. A.; Huang, Z.; Iversen, P. W.; Law, K. L.; Li, T.; Lin,
H.-S.; Lopez, B.; Lopez, J. E.; Martin Cabrejas, L. M.;