Isosteric exchange of the acylsulfonamide moiety in Abbott's Bcl-XL protein interaction antagonist

Article abstract of DOI:10.1016/j.bmcl.2008.05.096

A multi-component reaction strategy was used for the fast and efficient synthesis of amide isosteres of known Bcl-2 inhibitors capable of disrupting protein-protein interactions. Ugi reaction and a subsequent nucleophilic aromatic substitution reaction provide a versatile path to libraries of compounds similar to Abbott's acylsulfonamides. Modeling arguments are used to explain the inferior activity of the amide as opposed to the sulfonamide series.

Full text of DOI:10.1016/j.bmcl.2008.05.096

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4115–4117
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isosteric exchange of the acylsulfonamide moiety in Abbott’s Bcl-XL
protein interaction antagonist
c
c
c
Alexander Dömling a,b,c, , Walfrido Antuch , Barbara Beck , Vesna Schauer-Vukašinovic
*
´
a Department of Pharmaceutical Sciences, University of Pittsburgh, PA 15261, USA
b Department of Chemistry, University of Pittsburgh, PA 15261, USA
c Morphochem AG, Gmunderstrasse 37–37a, 81379 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A multi-component reaction strategy was used for the fast and efficient synthesis of amide isosteres of
known Bcl-2 inhibitors capable of disrupting protein–protein interactions. Ugi reaction and a subsequent
nucleophilic aromatic substitution reaction provide a versatile path to libraries of compounds similar to
Abbott’s acylsulfonamides. Modeling arguments are used to explain the inferior activity of the amide as
opposed to the sulfonamide series.
Received 22 April 2008
Revised 21 May 2008
Accepted 22 May 2008
Available online 29 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Multi-component reaction
Protein–protein interaction
Bcl
Cancer
Apoptosis
Isocyanide
Structure-based design
Human cancers overexpressing Bcl-2 and related survival pro-
teins are often resistant to cytotoxic therapeutics.1 These include
breast, prostate, colorectal, gastric, and (non)-small-cell lung can-
cer, as well as neuroblastomas, B-cell lymphoma, and melanomas.
Acquired resistance toward radiation or chemotherapy often leads
to tumor relapse and aggressive formation of metastasis and finally
to clinical failure of the current therapeutic regiment. Bcl-2 family
members are important regulators of programmed cell death,
belonging to the mitochondrial apoptosis pathway. Proapoptotic
Bcl-2 family members such as Bcl-2, Bcl-XL, and Bclw interact with
antiapoptotic Bcl-2 family members, for example, Bax, Bak, and
Bad and neutralize each other. Several experimental results indi-
cate that Bcl-2 family members are promising new cancer targets.
Thus synthetic cell permeable BH3 peptides2 and newly discovered
small molecules trigger apoptosis in cancer cells and show tumor
regression in xenograft models.3–6
O
WO-2002024636
O
O
O2N
S
N
H
N
H
1
F
O
O
O
O2N
S
2
N
H
HN
N
S
N
N
ABT-737
Cl
In a program to discover and develop Bcl2 family protein inter-
action antagonists Abbott scientists described a series of N-acyl-
sulfonamide-based inhibitors, for example, 1.7,8 These compound
series was developed using a NMR fragment-based approach, to-
gether with parallel chemistry (Fig. 1). Recently several potent in
vitro and in vivo inhibitors together with structural information
about their binding mode into Bcl-XL have been reported and
Figure 1. Examples of Abbott’s acylsulfonamides.
two compounds currently undergo clinical trials. ABT-737, 2 a
pan-Bcl family inhibitor targeting Bcl-2, Bcl-w, and Bcl-XL has been
shown to induce regression of lymphoma and small-cell lung can-
cer in corresponding xenograft models.
As a continuation of our interests to discover novel Bcl-2 family
protein interaction antagonists we present here our attempts to
prepare compounds similar to Abbott’s Bcl-XL inhibitors replacing
the sulfonamide backbone against an Ugi backbone.9
* Corresponding author. Tel.: +1 412 383 5300.
E-mail address: asd30@pitt.edu (A. Dömling).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.096

4116
A. Dömling et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4115–4117
Our structural design of our novel inhibitors is based upon the
O2N
F
NH2
O2N
F
NHCHO
HCOOH
analysis of the binding cleft of Bcl-2 and structural disclosure of
the binding mode of a representative compound (Fig. 2). The Ab-
bott Bcl-2 inhibitors adopt a V-shaped conformation with the
N-acylsulfonamide moiety at the solvent rim of the deep and
hydrophobic binding groove and the biphenyl and 2-amino nitro-
phenyl moieties pointing toward the floor of the groove. Due to
the elongated binding groove which is not completely filled by
the compound we reasoned that an elongation of the inhibitor by
a 2-atom insertion fragment in the backbone would be accepted
by the Bcl-2 protein. From the analysis of X-ray structures of Ugi
backbones it appears that there is sufficient conformational
freedom to readily adopt into a large binding groove. Therefore
we reasoned that the quasi isosteric replacement of the N-acylsulf-
ΔT
3
4
87%
O2N
NC
POCl3
TEA
F
5
68%
Scheme 1. Preparation of 4-fluoro-3-nitro-phenylisocyanide 5 useful for isocya-
nide-based multi-component reactions and subsequent nucleophilic aromatic
substitution reactions.
COOH
onamide against a a-acylaminocarbonamide would be tolerated by
O
the Bcl-2 binding groove (Fig. 3). Although the sulfonamide con-
sists of the three backbone atoms SNC and the Ugi backbone com-
prises the five backbone atoms NCCNC, modeling suggests a similar
3D shape and a reasonable overlap of the terminal residues. An
isosteric replacement would have several advantages; the access
to a novel class of potential Bcl-2 inhibitors by a convergent and
comparatively short synthetic route; the circumvention of intellec-
tual property issues and potentially improved pharmacokinetic
properties.
H
N
6
7
8
O2N
F
N
MeOH
CH2O
+
+
O
MeNH2
O2N
NC
5
+
9
F
O
H
DMF, K2CO3
RT
O2N
N
N
9
+
R1R2NH
To test this idea we had to synthesize a phenyl isocyanide
capable to undergo subsequent nucleophilic aromatic substitution
R1
O
N
R2
reactions.10 Thus starting from 3-nitro-4-fluoroaniline
3 we
assembled the corresponding isocyanide in the classical sequence
formylation, dehydration according to Ugi in overall 60% yield
(Scheme 1). This novel isocyanide allows for U-MCRs and even
more subsequent diversification by nucleophilic aromatic substitu-
tion reactions.
Pursuing the idea of an isosteric replacement of the N-acylsulf-
onamide against an a-acylaminocarbonamide we prepared a larger
Scheme 2. Synthesis of potential Bcl-2 protein interaction antagonists by an initial
Ugi reaction and a second nucleophilic aromatic substitution reaction.
several nucleophilic aromatic substitutions under mild conditions
for the preparation of a small library of potential Bcl-2 inhibitors
(Fig. 4 and Table 1).12
amount of the Ugi intermediate 9 by the reaction of methylamine
8, formaldehyde 7, isocyanide 3, and biphenyl carboxylic acid 6
(Scheme 2).11 This intermediate was used as starting material for
To compare our compounds against authentic Abbott com-
pounds we resynthesized several of those according to the patent
procedures. Screening was performed as recently described using
a FP assay.13 The activities of the
summarized in Table 1.
a-acylaminocarbonamides are
Initial docking studies using MOLOC indicate that our
a
-acyla-
minocarbonamides could indeed potentially bind deep into the
hydrophobic Bcl-2 groove (Fig. 4).14
However, as by the FP assay performed the affinity of our first
generation of compounds is at least 2–3 magnitudes less. This
can be explained in part by the fact that the more active com-
pounds of the Abbott series have a molecular weight which is still
Figure 2. An Abbott sulfonamide-based inhibitor bound onto Bcl-XL (PDB IP: 1YSI).
Pictures generated using PyMol (www.pymol.com).
O
O
O
O
H
N
O2N
R1
O2N
R1
S
N
N
R2
H
R2
O
Figure 3. Structural comparison of the Abbott sulfonamide backbone with an Ugi
Figure 4. Compound 12 (green sticks) docked into the high resolution NMR stru-
backbone.
cture of an Abbott compound (yellow sticks) in Bcl-XL (PDB IP: 1YSI).

A. Dömling et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4115–4117
4117
Table 1
5. Enyedy, P. J.; Ling, Y.; Nacro, K.; Tomita, Y.; Wu, X.; Cao, Y.; Guo, R.; Li, B.; Zhu,
X.; Huang, Y.; Long, Y.-Q.; Roller, P. P.; Yang, D.; Wang, S. J. Med. Chem. 2001, 44,
4313.
6. Wang, S.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Wang, R.; Tang, G.; Guo, J.;
Shangary, S.; Qiu, S.; Gao, W.; Yang, D.; Meagher, J.; Stuckey, J.; Krajewski, K.;
Jiang, S.; Roller, P. P.; Abaan, H. O.; Tomita, Y.; Wang, S. J. Med. Chem. 2006, 49,
6139.
7. Petros, A. M.; Dinges, J.; Augeri, D. J.; Baumeister, S. A.; Betebenner, D. A.; Bures,
M. G.; Elmore, S. W.; Hajduk, P. J.; Joseph, M. K.; Landis, S. K.; Nettesheim, D. G.;
Rosenberg, S. H.; Shen, W.; Thomas, S.; Wang, X.; Zanze, I.; Zhang, H.; Fesik, S.
W. J. Med. Chem. 2006, 49, 656.
8. Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong, R. C.; Augeri, D. J.;
Belli, B. A.; Bruncko, M.; Deckwerth, T. L.; Dinges, J.; Hajduk, P. J.; Joseph, M. K.;
Kitada, S.; Korsmeyer, S. J.; Kunzer, A. R.; Letai, A.; Mitten, M. J.; Nettesheim, D.
G.; ShiChung, Nimmer, P. M.; O’Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed,
J. C.; Shen, W.; Tahir, S. K.; Thompson, C. B.; Tomaselli, K. J.; Wang, B.; Wendt,
M. D.; Stephen, W.; Fesik, S. W.; Rosenberg, S. H. Nature 2005, 435, 677.
9. Antuch, W.; Menon, S.; Chen, Q.-Z.; Lu, Y.; Sakamuri, S.; Beck, B.; Schauer-
Vukasinovic, V.; Agarwal, S.; Hess, S.; Dömling, A. Bioorg. Med. Chem. Lett 2006,
16, 1740.
10. Experimental procedure for 4-fluoro-3-nitro-phenylisocyanide 5: One hundred
millimoles (15.6 g) of 4-fluoro-3-nitro aniline is refluxed in 150 ml formic acid
for 12 h. The residual formic acid is evaporated under reduced pressure to yield
18.4 g (100%) of crude formamide product 4, which can be further purified by
crystallization from ethanol. 1H (400 MHz, d6-DMSO; major and minor
diastereomer; ratio 7.15:1) = 7.51 (m, 1H), 7.82 (m, 1H), 8.48 (d, 1H), 10.61
(br s, 1H); 7.61 (m, 1H), 7.92 (m, 1H), 8.83 (d, 1H), 10.40 (br d, 1H); 13C (100
MHz, d6-DMSO) = 113.8, 115.5, 115.6, 118, 8, 119.0, 119.2, 119.4, 124.4, 124.5,
126.3, 126.4, 134.8, 134.9, 135.4, 135.5, 136.2, 136.3, 149.2, 151.7, 160.1, 162.7.
HPLC–MS 2.73 min; [M+H]+ 185.
In vitro FP data of selected Bcl-2 family protein–protein interrupters (Bcl-w)
Compound
Structure
KI (lM)
O
H
N
O2N
N
10
12.95 0.1
10.80 0.7
O
N
S
O
H
N
O2N
N
N
11
12
O
O
H
O2N
N
N
9.43 1.3
O
N
H
1/3 higher (cf. ABT-737 812 Da vs compound 10 490 Da) as com-
pared to our initial series of compounds. Analyzing the published
molecular structure it appears that the interaction between the
small molecule and the Bcl-2 protein is almost exclusively gov-
erned by van der Waals interactions and shape complementarity.
Moreover the N-acylsulfonamides can undergo a favorable electro-
static interaction with the carboxyl group of the rim amino acid
A solution of 40 mmol (7.36 g) formamide 4 and 100 mmol (10 g, 13.7 ml)
triethylamine in 40 ml DCM is prepared and cooled to 0 °C. To this solution
40 mmol (3.7 ml) POCl3 are added slowly to maintain the temperature at 0 °C.
The mixture is maintained for another hour at 0 °C and then warmed-up to
20 °C and stirred for another 6 h. Thirty-two milliliters of a aqueous solution of
8 g Na2CO3 are carefully added under vigorous stirring for 30 min. The aqueous
phase is extracted 3Â with each 20 ml DCM. The combined organic phases are
dried over K2CO3 and evaporated. The residue is crystallized from ether/DCM
À20 °C and filtered to yield 5.45 g 3-nitro-4-fluorophenylisocyanide 5 (82%).
1H (400 MHz, d6-DMSO) = 7.76 (m, 1H), 8.08 (m, 1H), 8.49 (m, 1H); 13C
(100 MHz, d6-DMSO) = 120.1, 120.3, 124.8, 134.2, 134.3, 153.2, 155.8, 166.2.
HPLC–MS 2.79 min; [M+H]+ 167.
R139, whereas the present
a-acylaminocarbonamides can appar-
ently not undergo such a hydrogen bond interaction. However
based on the promising initial low micromolar activity and the still
rather low molecular weight we are confident that we will be able
to design a second generation of more potent Bcl-2 inhibitors
based on the Ugi backbone in the future.
11. Experimental procedure for biphenyl-4-carboxylic acid [(4-fluoro-3-nitro-
phenylcarbamoyl)-methyl]-methyl-amide 9: One millimole of each of the
starting materials aldehyde, primary amine, carboxylic acid, and isocyanide
are stirred in 1 ml of methanol for 48 h at 20 °C. The solvent is evaporated and
the residue is purified using silica gel chromatography to yield 265 mg 9 (65%
yield). 1H (400 MHz, d6-DMSO, mixture of two rotamers around the tertiary
amide bond) = 3.04 (3H, br s, major), 3.07 (3H, br s, minor), 4.15 (2H, br s,
minor), 4.32 (2H, br s, major); 7.38–7.91 (m, 11H), 8.51 (m, 1H, minor), 8.59
(m, 1H, major), 10.49 (br s, NH, minor), 10.63 (1H, br s, major). HPLC–MS
3.73 min; [M+H]+ 408.
12. Experimental procedure for biphenyl-4-carboxylic acid methyl-[(3-nitro-4-
thiomorpholin-4-yl-phenyl-carbamoyl)-methyl]-amide 10: A solution of K2CO3
(0.246 mmol) and thiomorpholine (0.147 mmol) in DMF was mixed with
50 mg 9 (0.123 mmol). The solution was stirred at 20 °C for 3 days when the
reaction was complete according to HPLC–MS analysis. The solvent was
evaporated under reduced pressure and the residual material was purified
using silicagel chromatography to yield 29 mg 10 (48% yield). HPLC–MS
3.92 min; [M+H]+ 491.
13. A primary screen for Bcl project is based on fluorescence polarization (FP)
technology and is well described in literature (Zhang, H.; Nimmer, P.;
Rosenberg, S.H.; Ng, S.C.; Joseph, M. Anal. Biochem. 2002, 307, 70). We have
established assays for Bcl-2 (SantaCruz Biotech), Bcl-XL (R&D Systems), and
Bcl-w (R&D Systems) using as the binding partner the 5-carboxyfluorescein-
labeled 16-mer peptide tracer Flu-Bak-BH3 (sequence GQVGRQLAIIGDDINR is
derived from the Bak BH3 domain). The assays were performed in a 384-well
format, in 20 mM potassium phosphate buffer pH 7.4, containing 1 mM EDTA,
50 mM NaCl, and 0.05% pluronic F-68. The final concentration of DMSO in all
In summary, we have prepared several potential Bcl-2 protein–
protein interaction antagonists in two steps utilizing an IMCR and
a subsequent aromatic substitution reaction. For this purpose we
developed a versatile bifunctional p-fluorophenyl isocyanide useful
for nucleophilic aromatic substitution reactions. The chemistry is
high yielding and potentially useful to prepare arrays of compounds.
The designed compounds are based upon the described acylsulfona-
mide inhibitors of Abbott. However, in contrast to the acylsulfona-
mides the best inhibitors of the newly described series are at least
2–3 orders of magnitude less active. Possible reasons for this drop
of activity and possible routes towards more potent inhibitors are
discussed. However, regarding the yet small molecular weight of
the described compounds, the current backbone comprises a good
starting point for further medicinal chemistry to yield higher active
compounds.
References and notes
1. O’Neill, J.; Maniom, M.; Schwartz, P.; Hockenbery, D. M. Biochim. Biophys. Acta
2004, 1705, 43.
2. Holinger, E. P.; Chittenden, T.; Lutz, R. J. J. Biol. Chem. 1999, 274, 13298.
3. Tang, G.; Yang, C. Y.; Nikolovska-Coleska, Z.; Guo, Y.; Qiu, S.; Wang, R.; Gao, W.;
Wang, G.; Stuckey, J.; Krajewski, K.; Jiang, S.; Roller, P. P.; Wang, S. J. Med. Chem.
2007, 50, 1723.
assays was 10%. The reaction was carried in a 50-lL volume and the resulting
polarization signal was measured at kex = 485 nm/kem = 535 nm using an
UltraReader (Tecan) after 2 h incubation of the reaction mixture at room
temperature. Validation of the assays was performed using non-labeled Bak-
BH3 peptide as a control inhibitor.
4. Kitada, S.; Leone, M.; Sareth, S.; Zhai, D.; Reed, J. C.; Pellechia, M. J. Med. Chem.
2003, 46, 4559.
14. MOLOC: Paul Gerber, Gerber Molecular Design, http://www.moloc.ch.