Discovery of an orally bioavailable small molecule inhibitor of prosurvival B-cell lymphoma 2 proteins

Article abstract of DOI:10.1021/jm800669s

Overexpression of prosurvival proteins such as Bcl-2 and Bcl-X_L has been correlated with tumorigenesis and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a novel means for the treatment of can

Full text of DOI:10.1021/jm800669s

6902
J. Med. Chem. 2008, 51, 6902–6915
Discovery of an Orally Bioavailable Small Molecule Inhibitor of Prosurvival B-Cell Lymphoma
2 Proteins
Cheol-Min Park,† Milan Bruncko,† Jessica Adickes, Joy Bauch, Hong Ding, Aaron Kunzer, Kennan C. Marsh, Paul Nimmer,
Alexander R. Shoemaker, Xiaohong Song, Stephen K. Tahir, Christin Tse, Xilu Wang, Michael D. Wendt, Xiufen Yang,
Haichao Zhang, Stephen W. Fesik, Saul H. Rosenberg, and Steven W. Elmore*
Cancer Research, Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064
ReceiVed June 3, 2008
Overexpression of prosurvival proteins such as Bcl-2 and Bcl-XL has been correlated with tumorigenesis
and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a
novel means for the treatment of cancer. We recently described the discovery of 1 (ABT-737), which binds
Bcl-2, Bcl-XL, and Bcl-w with high affinity, shows robust antitumor activity in murine tumor xenograft
models, but is not orally bioavailable. Herein, we report that targeted modifications at three key positions
of 1 resulted in a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD)
between oral exposure (AUC) and in vitro efficacy in human tumor cell lines (EC50). The resulting compound,
2 (ABT-263), is orally efficacious in an established xenograft model of human small cell lung cancer, inducing
complete tumor regressions in all animals. Compound 2 is currently in multiple phase 1 clinical trials in
patients with small cell lung cancer and hematological malignancies.
Introduction
proteins in cancer cells leads to a shift in the balance between
anti- and proapoptotic proteins, resulting in the inhibition of
the normal apoptotic pathway. Thus, a small molecule antagonist
of the antiapoptotic proteins may restore normal apoptotic
signaling in tumor cells. Such a molecule not only should be
directly cytotoxic to those cancer cells reliant upon antiapoptotic
proteins for survival but should also enhance the cytotoxicity
of chemotherapeutic agents, such as DNA damaging agents or
taxanes, that exert their effects in part by the up-regulation of
proapoptotic factors.7–9
Apoptosis or programmed cell death is an evolutionarily
conserved process that regulates normal development and tissue
homeostasis by eliminating unwanted and damaged cells.
Alterations in this highly regulated process are implicated in a
number of diseases including cancer.1 Inability to respond to
apoptotic stimuli leads to the accumulation of defective cells
and tumorigenesis. Furthermore, this cellular defect confers
resistance to cytotoxic anticancer agents that typically exert their
effects indirectly through the apoptotic pathway.2
We have recently described our own efforts to discover potent
inhibitors of Bcl-2 family proteins that culminated in the
identification of 1 (ABT-737), which inhibits Bcl-2, Bcl-XL,
and Bcl-w with subnanomolar affinity.10–14 This compound
exhibits robust single agent activity, eliciting complete tumor
regressions in established tumor xenograft models, and potenti-
ates the effects of cytotoxic agents in a variety of settings.10,15–20
However, this compound is not orally bioavailable and its low
aqueous solubility makes formulation for intravenous delivery
extremely difficult. Moreover, oral delivery should allow greater
flexibility with regard to dosing schedules as a single agent or
in combination regimens with parenteral chemotherapeutic
agents in a clinical setting. Herein, we describe our efforts to
impart oral bioavailability to 1 that resulted in the discovery of
2 (ABT-263) (Figure 1).21
Bcl-2a family proteins are the key regulators of apoptosis in
the mitochondria-mediated pathway and comprise both pro- and
antiapoptotic proteins.3–5 This large family of proteins can be
structurally subdivided into three distinct groups based on the
presence of Bcl-2 homology (BH) domains. Direct binding
interactions between these three groups of proteins dictates the
apoptotic fate of cells. The most structurally disparate of the
Bcl-2 family members are those that contain a single BH
domain. These proapoptotic BH3-only proteins (e.g., Bad, Bim,
Noxa) act as molecular sentinels and are activated to propagate
death signaling in response to cellular damage. The multidomain
proapoptotic members, Bax and Bak, contain three BH domains
(BH1-BH3) and respond to these signals by forming membrane
bound oligomers on the mitochondrial surface that results in
loss of membrane integrity, the release of cytochrome C, and
downstream caspase activation.6 The antiapoptotic members
(e.g., Bcl-2, Bcl-XL, Mcl-1) contain four BH domains and serve
to protect cells by directly binding to and sequestering their
proapoptotic counterparts. The overexpression of antiapoptotic
Chemistry
Our strategy for the construction of the acylsulfonamides is
outlined in the retrosynthetic analysis shown in Figure 2.
Carbodiimide-mediated coupling was employed for the forma-
tion of the acylsulfonamides between carboxylic acids and
sulfonamides.22 Introduction of the amino side chains was
achieved either by nucleophilic aromatic substitution prior to
the formation of acylsulfonamides or by palladium-mediated
coupling after acylsulfonamide formation to avoid catalyst
poisoning by the free sulfonamides.
* To whom correspondence should be addressed. Phone: (847) 937-7850.
Fax: (847) 938-1004. E-mail: Steve.elmore@abbott.com.
†
These authors contributed equally to this work.
a Abbreviations: Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 related protein
X; Bak, Bcl-2 antagonist/killer; Bad, Bcl-2 antagonist of cell death; Bik,
bcl-2 interacting killer; Bid, BH3 interacting death domain; Bim, Bcl-2
interacting mediator; Hrk, Harakiri; Bmf, Bcl-2 modifying factor; Bcl-xL,
B-cell lymphoma × long; Mcl-1, myeloid cell leukemia 1; Bcl2-A1, B-cell
lymphoma 2 related protein A1; BH, Bcl homology; IL-3, interleukin 3;
FPA, fluorescence polarization assay.
3-Substituted-4-fluoroarylsulfonamides were either obtained
from commercial sources or prepared via chlorosulfonylation
10.1021/jm800669s CCC: $40.75
2008 American Chemical Society
Published on Web 10/08/2008

Orally BioaVailable Small Molecule Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6903
Likewise, compounds 34 and 2 were prepared by condensation
of 20b/31b and 20b/31e, respectively (Scheme 6).
Results and Discussion
Target affinities (Ki) were determined using fluorescence
polarization assays (FPA), which are competition binding assays
employing a Bad- or Bax-derived BH3 peptide fluorescent
probe.29 Our choice in structural modifications was directed to
those areas that were not likely to disrupt the overall core
binding motif and significantly impact the high binding affinity
displayed by 1. This turned out to be the case, as the vast
majority of compounds presented here exhibited binding af-
finities at or below the detection limit of the Bcl-XL and Bcl-2
FPA (Ki ≈ 1 nM, Supporting Information Table S1). Therefore,
the SAR was driven largely by cellular efficacy. To demonstrate
mechanism-based cellular activity, compounds were first evalu-
ated for their ability to reverse the protection afforded by the
overexpression of human Bcl-2 or Bcl-XL in IL-3 dependent
murine tumor pro-B cell FL5.12 lines in the absence of added
serum proteins. To evaluate their potential relevance to human
cancer, compounds were then evaluated in the human tumor
cell line H146 (small cell lung cancer) in the presence of 10%
human serum. This cell line is known to be dependent upon
Bcl-2 and/or Bcl-xL30 and responsive to 1.10 Those compounds
with promising activity profiles were evaluated for pharmaco-
kinetic properties in rats, while their pharmacokinetic properties
in higher species, combined with efficacy in tumor xenograft
models, were used as the final arbiters for compound selection.
Small molecule drugs that have high molecular weight
(>500), high number of hydrogen bond donors (>5) or
acceptors (>10), and high lipophilicity (clogP) are predicted to
have poor oral absorption and permeability by Lipinski’s rule
of five.31 These predictions hold true for 1. However, many of
the structural features in 1 that impart undesirable drug
properties are important for pharmacological activity. They are
the result of design elements essential for inhibition of a large
surface area, hydrophobic protein-protein interaction, and
the reduction of serum albumin binding.11–13 Thus, to improve
the oral efficacy of this relatively large (MW > 800 Da)
molecule, a careful balance had to be achieved wherein
improved oral pharmacokinetic properties were not deleterious
to target binding affinity or cellular efficacy.32
The low oral bioavailability of 1 was thought to be the
combined result of low aqueous solubility, poor absorption
(permeability) and metabolism. Given the large molecular size
and hydrophobicity of compounds within this series, it seemed
unlikely that significant improvements in aqueous solubility
would be easily obtained by utilizing traditional strategies such
as reducing nonpolar surface area. However, early in these
studies, we uncovered strong evidence that relatively small
modifications that were designed to alter the ionization state
by modulating pKa values of the acylsulfonamide or amine could
significantly enhance oral absorption (Table 1). For example,
simple replacement of the arylnitro group with the less electron
withdrawing trifluoromethyl group (3) imparted a 16-fold
increase in systemic exposure (pharmacokinetic area under the
plasma concentration curve, AUC) after oral dosing in
Sprague-Dawley rats with 24% oral bioavailability. However,
this change was also accompanied by a large decrease in cellular
efficacy. Since N-demethylation was identified as a significant
route of metabolism for 1 and we sought to decrease overall
molecular charge, the highly basic (pKa ≈ 10) dimethylamino
group was also an early focus of our efforts. Removal of the
dimethylaminoethyl side chain (4) altogether resulted in a 37-
Figure 1. Structures of acylsulfonamide Bcl-2 family protein inhibitors.
and subsequent amination (Scheme 1, eq 1). Elaborated sul-
fonamides 10a-d were prepared from the corresponding
arylhalides 8a-d by nucleophilic aromatic substitution with
amine 912 (eq 2). It is of note that the highly electrophilic
trifluoroacetyl group prefers either a keto or a hydrated form
depending on the electronic nature of its ortho substituent. Thus,
while the trifluoroacetyl group in 8a exists as a stable hydrate,
it reverts to the keto form in 10a, which contains an electron
donating ortho amino substituent. This is readily apparent by
the nuclear magnetic resonance signal of the carbonyl carbon.
For the sulfonamide lacking an electron withdrawing group in
the 3-position, palladium mediated coupling23 of the bis-4-
methoxybenzyl protected 4-bromosulfonamide with amine 9
followed by deprotection under acidic conditions smoothly
furnished the desired product 14 (eq 3).
Scheme 2 illustrates the synthesis of trifluoromethylsulfone
analogues 20a and 20b. Alkylation of 2-fluorobenzenethiol 15
with trifluoromethyl iodide under radical conditions24 afforded
the corresponding sulfide.25 Ruthenium catalyzed oxidation of
this highly deactivated sulfide yielded sulfone 17.26 Finally,
sulfonamides 20a and 20b were prepared via nucleophilic
aromatic substitution of aryl fluoride 18 by the chiral amines 9
and 19, respectively. Acylsulfonamide formation between
carboxylic acid 21 and sulfonamides 10a-d, 14, and 20a,b was
achieved in the presence of EDC and DMAP as shown in
Scheme 3.
Compounds 25a and 25b were prepared according to Scheme
4. Sulfonamide 23d was prepared from 4-bromo-3-trifluo-
romethoxyaniline in two steps via diazotization.27 Coupling of
23d or the commercially available 4-bromo-3-methylbenzene-
sulfonamide 23a to carboxylic acid 21 yielded the acylsulfona-
mides 24a and 24b. Palladium mediated coupling of arylbro-
mides 24a,b and amine 9 gave access to the fully elaborated
acylsulfonamide (25a, 25b).
The synthesis of the carboxylic acids (31a-f) containing
cycloalkenyl groups of various ring sizes is outlined in Scheme
5. The intermediates 29a-f were obtained via reductive
amination of piperazine 28 and 2-bromocycloalkene-1-carbox-
aldehydes (27a-f) that were readily accessible from the
corresponding cyclic ketones (26a-f) by treatment with PBr3
and DMF.28
Suzuki cross-coupling of bromides 29a-f with 4-chlorophe-
nylboronic acid and subsequent saponification of the ethyl esters
(30a-f) afforded carboxylic acids 31a-f. Carbodiimide-medi-
ated acylsulfonamide formation of the carboxylic acids and
sulfonamide 3212 yielded the desired compounds 33a-f.

6904 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Park et al.
Figure 2. Retrosynthetic analysis of acylsulfonamides.
Scheme 1. Synthesis of Sulfonamides 10a-d and 14a
(PK/PD) measure. The AUC after oral dosing in rats provides
a measure of whether a given compound can achieve and
maintain circulating concentrations. The EC50 in the H146 small
cell lung cancer human tumor cell line obtained in the presence
of 10% human serum provides a measure of tumor cell killing
in the presence of human serum. Even though 3, 4, and 5 possess
significantly improved oral pharmacokinetic properties, when
compared to that of 1, their AUC/EC50 ratios reveal a less than
optimal tradeoff between cellular potency and exposure. None-
theless, though these early modifications were not effective in
improving the AUC/EC50 ratio, they offered evidence that the
oral pharmacokinetic properties of 1 could be meaningfully
improved.
Encouraged by these findings, key positions along the
backbone of 1 were targeted where changes may impact
molecular charge and/or metabolism. Given the likely effect of
the nitro group on the acylsulfonamide pKa, the known
propensity for arylnitro groups to produce potentially toxic
metabolites, and the dramatic effects of nitro group replacement
outlined above, this position was an obvious choice. Because
oxidative metabolism of the 4-chlorobiphenyl moiety was
another metabolic pathway and little SAR of the internal
aromatic ring had been explored, this area was also targeted.
Each of these areas was initially optimized independently with
the goal of later combining the most desired structural attributes
with the morpholino substituent.
a Reagents and conditions: (a) ClSO3H, 80 °C, 15 h; (b) NH4OH,
THF-MeOH (10:1), -50 to 0 °C, 3 h; (c) 9, DIPEA, THF, 50 °C, 24 h for
10a-c; (d) 9, DIPEA, DMSO, 140 °C, 15 h for 10d; (e) (PMB)2NH, TEA,
DMAP, CH2Cl2, room temp, 15 h; (f) 9, Pd2(dba)3, BINAP, Cs2CO3, toluene,
reflux, 15 h; (g) TFA, CH2Cl2, room temp, 15 h.
Table 2 summarizes the effects of nitro group replacement.
Target affinities of these analogues remained below the FPA
detection limit for both Bcl-2 and Bcl- XL with the exception
of 22d (R ) H), which showed a moderate loss against Bcl-XL
(Ki ) 14 nM). However, modification of the nitroarene had a
dramatic impact on activity against both FL5.12 and H146 cell
lines, where compounds with weaker electron withdrawing
groups (3, 25b, 22c, 22a, and 22b) exhibited a significant loss
of potency. In fact, within this small group of compounds, there
is a good correlation between the aromatic substituent Hammett
σm value,33,34 and thus the acylsulfonamide pKa, and EC50 in
H146 cells. It is not clear if the decreased cellular activity
observed with less acidic cores is driven by reduced cellular
penetration, increased sequestration by the added serum proteins,
or other cellular compartments. The oral pharmacokinetics of
compounds that retained H146 efficacy at or below EC50 ≈ 2
µM were evaluated in Sprague-Dawley rats. Not surprisingly,
oral exposure correlated roughly with the calculated polar
fold increase in oral exposure and 28% oral bioavailability.
Again, this was accompanied by a drastic reduction in cellular
efficacy that is likely driven by high serum binding, since this
substituent was designed specifically to reduce binding to human
serum albumin domain III.12 Replacement of the dimethylamine
with a less basic morpholino group (pKa ≈ 7.5) provided a more
modest 4-fold enhancement in oral exposure with 16% oral
bioavailability; however, the compound retained submicromolar
cellular activity (5). This suggested that the morpholino group
might be a suitable replacement if combined with other changes
that similarly increased oral exposure and/or cellular activity.
Since in vivo antitumor efficacy is ultimately a function of
the ability of a drug to kill tumor cells and to attain adequate
concentrations in the body, these two parameters were quantified
by examining the ratio of the pharmacokinetic AUC to cellular
EC50 (AUC/EC50) as a crude pharmacokinetic/pharmacodynamic

Orally BioaVailable Small Molecule Inhibitor
Scheme 2. Synthesis of Sulfonamides 20a,ba
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6905
a Reagents and conditions: (a) CF3I, TEA, methyl viologen dichloride, DMF, room temp, 15 h; (b) RuCl3, NaIO4, CCl4-acetonitrile-H2O (1:1:2), room
temp, 15 h; (c) (i) ClSO3H, 80-120 °C, 15 h; (ii) NH4OH, iPrOH, -78 °C, 1 h; (d) 9, DIPEA, THF, 50 °C, 15 h for 20a; (e) 19, DIPEA, THF, 50 °C, 15 h
for 20b.
Scheme 3. Synthesis of Target Compounds 3 and 22a-e
net result is approximately 20-fold increase in AUC/EC50 for
34 and 2 relative to 1.
With these two promising candidates in hand, we opted to
further differentiate them by characterization of their pharma-
cokinetic profiles in additional species. As shown in Table 5, 2
displays oral exposure superior to that of 34 in all species tested
(rat, mouse, dog, and monkey). The biological properties and
pharmacokinetic parameters for 2 have been recently reported.35
This compound exhibits low volumes of distribution and plasma
clearance values with an oral elimination half-life from 4.0 to
6.4 h and absolute oral bioavailability of approximately 20%
in all four species above. Figure 3A shows a representative
plasma concentration curve after intravenous or oral dosing in
dog. When examined in a 3-day steady-state pharmacokinetics
study in non-tumor-bearing scid-bg mice, both peak plasma
concentration (Cmax) and AUC increased in a dose proportional
fashion.35 In scid-bg mice bearing H146 flank tumor xenografts,
2 exhibits good tissue distribution after oral dosing with a plasma
to tumor concentration ratio of near unity (Figure 3B).
surface area (PSA) with compounds with lower PSA exhibiting
higher AUC values. The trifluoromethanesulfonyl group (tri-
flone), which has slightly greater electron withdrawing capacity
than the nitro group, gave a correspondingly small increase in
cellular efficacy (22e). However, the lower PSA imparted by
the triflone compared to the nitro group also corresponded to a
modest increase in drug plasma concentrations. The net effect
was a 7-fold increase in AUC/EC50 ratio.
We next evaluated 2 for oral efficacy in an established murine
tumor xenograft model (Figure 4). In order to directly compare
the efficacy of 2 with that of 1, we chose the NCI-H1963 SCLC
cell line. Compound 1 has been reported to produce complete
tumor regression in this model when delivered once daily by
ip injection for 21 days.10 A quantity of 5 × 106 NCI-H1963
cells was inoculated subcutaneously in the flank of scid-bg mice.
The tumors were allowed to grow to an average size of 235
mm3, and mice were size-matched (day 0) into treatment and
control groups. Once daily treatment with 100 mg/kg given
orally for 21 days was well tolerated (<5% weight loss) and
achieved 100% tumor growth inhibition with complete regres-
sion of all tumors (N ) 10) at the end of the dosing period.
These results demonstrate that when given at the same dose
and schedule, oral administration of 2 achieves similar efficacy
to that of 1 when dosed via ip injection.
We next investigated replacements for the 4-chlorobiphenyl
moiety of 1 with the goal of enhancing metabolic stability and
maintaining or improving cellular efficacy. We chose to examine
cycloalkenyl groups of varied ring size as surrogates for the
internal aromatic ring in order to maintain the rigid molecular
geometry essential for the high affinity binding mode of 1.34
These modifications were well-tolerated, as all of the analogues
fell at or below the limits of detection in the FPA assay. Cellular
efficacy better differentiated the compounds, as the six-
membered ring (33b) was more efficacious in the H146 cellular
assay than its five-, seven-, or eight-membered ring counterparts
(33a, 33c, or 33d). Investigation of a variety of six-membered
ring substitutions or replacements identified the gem-dimeth-
ylcyclohexene 33e (Table 3). Although this compound exhibited
slightly lower H146 efficacy compared to the parent cyclohex-
enyl, it displayed subnanomolar EC50 values against both the
Bcl-2 and Bcl-XL dependent FL5.12 cells. The cyclohexene and
gem-dimethylcyclohexene were chosen for further study.
Conclusions
Discovery of small molecule inhibitors of protein-protein
interactions has traditionally been a challenge because of the
requirement to disrupt large, often hydrophobic and seemingly
featureless surface interactions. BH3-mimetics are particularly
interesting from a compound design standpoint. The native
R-helical BH3-domain of proapoptotic proteins derive their high
binding energy through the summation of multiple interactions
with distinct, discontinuous hydrophobic pockets or “hotspots”
(P1-P4) along the surface groove of their binding partners, such
as Bcl-2 and Bcl-xL. Our initial efforts to mimic this interaction
generated compound 1, which efficiently binds two nonadjacent
hot spots of Bcl-xL (P2 and P4)35 and is exquisitely effective
in this regard. Perhaps not surprisingly though, these efforts
The cellular efficacy and oral exposure of compounds
resulting from the combination of modifications identified in
the independent optimization processes are outlined in Table
4. First, incorporation of the cyclohexene alone (33b) resulted
in both improved cellular efficacy and a modest 2-fold increase
in oral exposure relative to 1. The effect of combining the
morpholine and the triflone with either cyclohexene (34) or gem-
dimethylcyclohexene (2) on oral PK was nearly additive,
resulting in a significant enhancement of oral exposure (14- to
22-fold relative to 1) while maintaining cellular efficacy. The

6906 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Scheme 4. Synthesis of Target Compounds 25a,ba
Park et al.
i
a Reagents and conditions: (a) NaNO2, HCl-AcOH, -10 °C, 45 min; SO2, CuCl, AcOH, room temp, 10 min; (b) NH4OH, PrOH, 0 °C to room temp,
15 h; (c) 21, EDC, DMAP, CH2Cl2, room temp, 15 h; (d) 9, Pd2(dba)3, BINAP, Cs2CO3, toluene, reflux, 15 h.
Scheme 5. Synthesis of Target Compounds 33a-fa
a Reagents and conditions: (a) DMF, PBr3, CH2Cl2, room temp, 20 min; (b) Na(CN)BH3, AcOH, EtOH, 15 h; (c) 4-chlorophenylboronic acid, PdCl2(PPh3)2,
Na2CO3, DME-H2O-EtOH (7:3:2), 85 °C, 3 h; (d) LiOH, dioxane-H2O (5:1), 100 °C, 24 h; (e) 32, EDC, DMAP, CH2Cl2, room temp, 15 h.
resulted in a compound with a high molecular weight (813 Da)
and hydrophobicity that has poor solubility and oral absorption.
Three key sites were identified along the backbone of 1 that
were targeted in order to decrease molecular charge and
metabolism while simultaneously minimizing the disruption of
the overall molecular scaffold in order to maintain high target
affinity. Thus, most of the compounds prepared exhibited high
target binding affinity (<1 nM), and the work here was driven
entirely by cell-based activity and pharmacokinetic behavior.
However, relatively minor changes in these large molecules had
a significant impact on oral absorption. For example, replace-
ment of the arylnitro group with trifluoromethyl (3) or removal
of the basic amine (4) yielded a 16- and 37-fold increase in
oral exposure, respectively. As is so often the case when
attempting to optimize multiple variables simultaneously, we
also found these improvements in PK parameters often came
at the expense of other desired properties such as cellular
efficacy. In order to account for cellular efficacy, protein binding,
and oral absorption in a single, semiquantitative measure, we
utilized the PK/PD relationship for AUC/EC50, which has
commonly been employed in other settings to predict in vivo
efficacy.36 Independent optimization of multiple sites led to
modifications at each locale that gave incremental improvements
in the AUC/EC50 ratio. When combined, these effects were
additive and resulted in compound 2, which has roughly 20-
fold improved oral absorption compared to 1. This compound
is orally bioavailable in multiple species, demonstrates good
tissue distribution, and is orally efficacious in tumor xenograft

Orally BioaVailable Small Molecule Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6907
Scheme 6. Synthesis of Target Compounds 34 and 2a
RRMSDK(FITC)FVD, prepared in-house), and the DMSO solution
of compound was shaken for 2 min and then placed in a LJL
Analyst (LJL Bio Systems, CA). A negative control (DMSO, 1
nM BAD peptide, assay buffer) and a positive control (DMSO, 1
nM BAD peptide, 6 nM Bcl-XL, assay buffer) were used to
determine the range of the assay. The effects of 10% human serum
were detected as described above using 30 nM f-Bad peptide and
60 nM Bcl-XL. To determine Bcl-2 Ki values, a mixture totaling
125 µL per well of assay buffer (20 mM phosphate buffer, pH 7.4,
1 mM EDTA, 50 mM NaCl, 0.05% PF-68), 10 nM Bcl-2 protein,39
1 nM fluorescein-labeled Bax peptide (FITC-QDASTKKLSE-
CLKRIGDELDS, prepared in-house), and the DMSO solution of
the test compound was shaken for 2 min and placed in the LJL
Analyst. Polarization was measured at 25 °C using a continuous
fluorescein lamp (excitation 485 nm; emission 530 nm). Percentage
of inhibition was determined by (1 - ((mP value of well-negative
control)/range)) × 100%. Ki and IC50 values were calculated using
Microsoft Excel.
a Reagents and conditions: (a) 31b for 34; (b) 31e for 2.
FL5.12 Cellular Assay. Mouse FL5.12 cells transfected with
Bcl-XL were cultured under standard conditions in RPMI with 2
mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4
µL/L of ꢀ-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS,
and 10% WEHI-3B conditioned media (for IL-3). For assaying the
compound activity, the cells were exchanged into an IL-3-depleted
deprivation media, which was identical to the growth media except
for the absence of FBS and WEHI-3B conditional media, for 2
days. Then the cells were exchanged to either gelatin assay media
(RPMI with 2 mM glutamine, 2% 1 M HEPES, 3.4 mg/mL bovine
gelatin (Sigma)) or 3% FBS assay media (RPMI with 2 mM
glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 µL/L
of ꢀ-mercaptoethanol, 1% penicillin-streptomycin, 3% FBS).
Compounds in series dilutions were added, and the cells were
cultured for 24 h. Cell viability was assayed using the colorimetric
3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium MTS assay or the CellTiter-Glo assay
(Promega Corp., Madison, WI) according to the manufacturer
instructions. Individual determinations were the result of duplicate
values.
Human Tumor Cell Line Viability Assay. The SCLC cell line
NCI-H146 was purchased from American Type Culture Collection,
ATCC, Manassas, VA. Cells were cultured in RPMI 1640 (Invit-
rogen Corp., Grand Island, NY) supplemented with 10% fetal
bovine serum (FBS, Invitrogen), 1% sodium pyruvate, 25 mM
HEPES, 4.5 g/L glucose, and 1% penicillin-streptomycin (Sigma)
and maintained in a humidified chamber at 37 °C containing 5%
CO2. Cells (50 000 per cell) were treated for 48 h in 96-well tissue
culture plates in a total volume of 100 µL of tissue culture medium
supplemented with 10% human serum (Invitrogen). Cell viability
was determined using the CellTiter-Glo luminescent cell viability
assay (Promega Corp., Madison, WI).
Pharmacokinetics. The single dose pharmacokinetics of com-
pounds 1-5, 22a, 22c, 22e, 33b, and 34 were evaluated in
Sprague-Dawley rats (Charles River) after a 5 mg/kg oral dose (n
) 3) (10% DMSO in PEG-400) administered by gavage. The single
dose pharmacokinetics of compounds 34 and 2 were evaluated in
beagle dogs after a 2.5 mg/kg intravenous (n ) 3) or oral (n ) 3)
dose in 10% DMSO in PEG-400. The iv dose was administered as
a slow bolus in a cephalic vein, and oral doses were administered
by gavage followed by 10 mL of water. The single dose pharma-
cokinetics of compounds 34 and 2 were evaluated in cynomolgus
monkeys after a 2.5 mg/kg oral (n ) 3) dose in 10% DMSO in
PEG-400 administered by gastric intubation followed by 5 mL of
water. Heparinized blood samples were obtained from each animal
prior to dosing and at selected time points after compound
administration. The single dose pharmacokinetics of compounds
34 and 2 were evaluated in CD-1 mice after a 50 mg/kg oral dose
(n ) 27) (2% DMSO/5% Tween-80/20% propylene glycol/73%
D5W) administered by gavage. The plasma and tumor concentration
profile of 2 (50 mg/kg) in tumor bearing female scid-bg mice was
examined on the third day of 3 consecutive days of dosing by oral
gavage in 10% EtOH, 30% PEG 400, and 60% Phosal 50 PG (n )
models. More recently, 2 was shown to display dose proportional
oral pharmacokinetics in humans.37
It is interesting to note that the ultimate compound identified
in these studies is far from what is traditionally thought of as
“druglike”. The molecular weight of 2 (974 Da) is actually
greater than that of the prototype compound 1 and violates three
of the four rules of five. To a large degree, the size and
hydrophobicity of this chemical series are due to the nature of
the drug target itself and the requirements for efficiently
mimicking the native BH3 peptide interaction. However, it
became apparent throughout this process that simple changes
that impact its underlying physiochemical properties (pKa of
the charged functionalities or the polar surface area) or its
metabolic potential had a meaningful impact on oral absorption
and exposure, even within this large molecular framework.
Although this is but a single example, it represents progress
toward targeting protein-protein interactions that have typically
been thought of as “undruggable”.
Experimental Section
General Methods. All reactions were carried out under inert
atmosphere (N2) and at room temperature unless otherwise noted.
Solvents and reagents were obtained commercially and were used
without further purification. All reported yields are of isolated
1
products and are not optimized. H NMR spectra were obtained
on a Varian UNITY or Inova (500 MHz), Varian UNITY (400
MHz), or Varian UNITY plus or Mercury (300 MHz) instrument.
Chemical shifts are reported as δ values (ppm) downfield relative
to TMS as an internal standard, with multiplicities reported in the
usual manner. Mass spectra determinations were performed by the
Analytical Research Department, Abbott Laboratories; DCI indi-
cates chemical ionization in the presence of NH3, and ESI indicates
electron spray ionization. Elemental analyses were performed by
Quantitative Technologies, Inc., Whitehouse, NJ. Column chro-
matography was carried out in flash mode on silica gel (Merck
Kieselgel 60, 230-400 mesh). Unless otherwise noted, preparative
HPLC samples were purified on a Zorbax Stable Bond C18 column
(21.4 mm × 250 mm, 7 µm particle size) using a gradient of
20-100% CH3CN/water/0.1% TFA over 45 min at a flow rate of
15 mL/min. HPLC purifications were performed in high-throughput
format and the isolated products concentrated in parallel under
unoptimized conditions using a Savant Speed Vac concentrator to
provide the final products as hydrated trifluoroacetic acid salts.
Fluorescence Polarization Assay. Ki and IC50 values were
determined using a competitive fluorescence polarization assay.
Compounds were serially diluted and added to each well of a 96-
well microtiter plate. To determine Bcl-XL Ki values, a mixture
totaling 125 µL per well of assay buffer (20 mM phosphate buffer,
pH 7.4, 1 mM EDTA, 50 mM NaCl, 0.05% PF-68), 6 nM Bcl-XL
protein,381nMfluorescein-labeledBADpeptide(NLWAAQRYGREL-

6908 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 1. PK/PD Relationship of Earlier Compounds
Park et al.
Cmaxa (µM)
AUCa (µM·h)
Fa (%)
H146/10% HS EC50 (µM)
AUC/EC50
1
3
4
5
0.039
0.37
1.15
0.20
0.28
4.41
10.4
1.16
6
0.087
2.46
33.0
0.61
3.2
1.8
0.32
1.9
24
28
16
a Dosed orally at 5 mg/kg.
Table 2. SAR of Nitro Group Replacements
EC50 (nM)a
serum free
10% HS
H146
X
σm
FL5.12/Bcl-2
FL5.12/Bcl-XL
PSAb (Å2)
AUCc (µM·h)
AUC/EC50
1
3
NO2
CF3
CH3
H
OCF3
CN
C(O)CF3
SO2Me
SO2CF3
0.71
0.43
-0.07
0
0.38
0.56
0.63
0.60
0.79
7.7 ( 2.0*
42 ( 5.0
673 ( 278*
2700 ( 566
230
30 ( 8.8*
122 ( 38*
NDc
15000 ( 4200
805
225 ( 7.1
329 ( 29.0*
415 ( 20.2*
12 ( 1.6*
87 ( 3.5*
135
88
0.28
4.41
NDd
NDd
NDd
2.19
1.25
NDd
0.83
3.2
2460 ( 558*
28800 ( 3480*
78700 ( 18700*
5080 ( 624*
1930 ( 51*
1.8
25a
22d
25b
22c
22a
22b
22e
NDd
NDd
NDd
112
108
NDd
121
NDd
NDd
NDd
1.1
49 ( 13
55 ( 1.2*
170 ( 5.8*
4.7 ( 0.9*
2010 ( 300
3490 ( 92.7*
39.4 ( 10.9
0.62
NDd
21
a Values are mean ( standard deviation for two experiments run in duplicate. Asterisk indicates mean ( standard error for three or more experiments run
in duplicate. Values without error are single experiments run in duplicate. b Polar surface areas were calculated using Savol software (Tripos) from 3D
coordinates generated using Concord (Tripos). c Dosed orally at 5 mg/kg in rats. d ND ) not determined.
18). At selected time points, groups of three animals were
euthanized with carbon dioxide, exsanguinated by cardiac puncture,
and heparinized blood and/or tumor samples obtained. Plasma from
all samples was separated by centrifugation, and compounds were
separated using protein precipitation with acetonitrile.
Compound and the internal standard were separated from each
other and coextracted contaminants on a 50 mm × 3 mm Keystone
Betasil CN 5 µm column with an acetonitrile/0.1% trifluoroacetic
acid mobile phase (50:50, by volume) at a flow rate of 0.7 mL/
min. Analysis was performed on a Sciex API3000 biomolecular
mass analyzer with a heated nebulizer interface. Compound and
internal standard peak areas were determined using Sciex MacQuan
software. The plasma drug concentration of each sample was
calculated by least-squares linear regression analysis (nonweighted)
of the peak area ratio (parent/internal standard) of the spiked plasma
standards versus concentration. The plasma concentration data were
submitted to multiexponential curve fitting using WinNonlin.3. The
area under the plasma concentration-time curve was calculated
using the linear trapezoidal rule for the plasma concentration-time
profiles.
In Vivo Xenograft Modeling. Animal studies were conducted
in accordance with the guidelines established by the internal
Institutional Animal Care and Use Committee. C.B.-17 scid-bg mice
(Charles River Laboratories, Wilmington, MA) were implanted with
5 × 106 NCI-H1963 cells subcutaneously into the right flank.
Inoculation volume was 0.2 mL consisting of 50% matrigel (BD
Biosciences, Bedford, MA). When tumors reached the appropriate
tumor volume, mice were size-matched (day 0) into treatment and
control groups. Each animal was ear-tagged and followed individu-
ally throughout the experiment. Tumor volume was estimated by
twice weekly measurements of the length and width of the tumor
by electronic calipers and applying the following equation: V )

Orally BioaVailable Small Molecule Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6909
Table 3. SAR of 4-Chlorobiphenyl Group Replacements
EC50 (nM)a
serum free
10% HS
H146
R1
FL5.12/Bcl-2
FL5.12/Bcl-XL
33a
33b
33c
33d
33e
33f
A
B
C
D
E
F
51 ( 6.6*
11 ( 3.3*
<1
39 ( 8.8*
0.6
143 ( 16.5*
29 ( 3.0*
5.0
21 ( 1.4
0.9
691
25.7 ( 6.9
51.9
200
98
266
33 ( 9.9
55 ( 17*
a Values are mean ( standard deviation for two experiments run in duplicate. Asterisk indicates mean ( standard error for three or more experiments run
in duplicate. Values without error are single experiments run in duplicate.
Table 4. PK/PD Relationship of Selected Compounds
EC50 (nM)a
serum free
10% HS
R1
X
R2
NMe2
NMe2
morpholine
morpholine
FL5.12/Bcl-2
FL5.12/Bcl-XL
H146c
AUCb (µM·h)
AUC/EC50 (h)
1
A
B
B
C
NO2
NO2
SO2CF3
SO2CF3
7.7 ( 2.0*
11 ( 3.3*
1.1 ( 0.3
5.9 ( 0.7
30 ( 8.8*
29 ( 3.0*
0.7 ( 0.1
4.2 ( 1.3
87 ( 3.5*
25.7 ( 6.9
58.9 ( 5.3*
86.7 ( 8.3*
0.28
0.65
3.87
6.26
3.2
25.3
65.7
72.2
33b
34
2
a Values are mean ( standard deviation for two experiments run in duplicate. Asterisk indicates mean ( standard error for three or more experiments run
in duplicate. Values without error are single experiments run in duplicate. b Dosed orally at 5 mg/kg in rats. c H146 in the presence of 10% HS.
L(W2/2). Tumor growth inhibition (%TGI) was calculated on the
basis of the difference in mean tumor volumes for the treated group
relative to the appropriate vehicle control. Complete response
(%CR) was defined as regression of established tumor beyond the
level of detection by palpation. 2 was formulated in 10% EtOH,
30% PEG 400, and 60% Phosal 50 PG.
General Procedure I. 4-Fluoro-3-(methylsulfonyl)benzene-
sulfonamide (8b). A solution of 1-fluoro-2-(methylsulfonyl)ben-
zene40 (1.0 g, 5.7 mmol) in chlorosulfonic acid (4 mL) was heated
at 80 °C overnight and poured onto crushed ice. The mixture was
diluted with ethyl acetate, and the layers were separated. The
aqueous layer was extracted with ethyl acetate (×2), and the
combined organic layers were dried over MgSO4, filtered, and
concentrated to give a crude product (1.4 g, 93%), which was used
in the next step without further purification. To a solution of the
sulfonyl chloride (1.4 g, 5.3 mmol) in THF-MeOH (10 mL-10
mL) cooled to -50 °C was added ammonium hydroxide (28%
aqueous solution, 1.8 mL, 26.5 mmol). The reaction mixture was
allowed to warm to 0 °C, and the solvent was removed in vacuo.
The residue was purified by flash chromatography (SiO2, 10-100%
ethyl acetate in hexanes) to give 8b (945 mg, 65% over two steps).
1H NMR (500 MHz, DMSO-d6) δ 8.30 (dd, J ) 2.2, 6.2 Hz, 1H),
8.20 (ddd, J ) 2.5, 4.4, 8.4 Hz, 1H), 7.76 (dd, J ) 8.7, 9.7 Hz,
1H), 7.64 (s, 2H), 3.41 (s, 3H); MS (DCI) m/z 271.0 (M + NH4)+.
4-Fluoro-3-(2,2,2-trifluoro-1,1-dihydroxyethyl)benzenesulfon-
amide (8a). 8a was prepared from 2,2,2-trifluoro-1-(2-fluorophe-
nyl)ethanone according to general procedure I. 7a (hydrated form):
1H NMR (500 MHz, C6D6) 8.54 (dd, J ) 2.6, 6.6 Hz, 1H), 7.51
(ddd, J ) 2.6, 4.0, 8.8 Hz, 1H), 6.52 (dd, J ) 9.0, 10.4 Hz, 1H);
13C NMR (125 MHz, C6D6) 164.5 (d, J ) 266.0 Hz, CF), 131.6,

6910 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 5. Comparison of 34 and 2 Pharmacokinetics in Multiple Species
Park et al.
(R)-4-(4-(Dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-
(trifluoromethyl)benzenesulfonamide (10d). 10d was prepared
from 8d and 9 according to general procedure II with modification
(140 °C, 24 h). 1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J ) 2.0
Hz, 1H), 7.64 (dd, J ) 9.0, 2.0 Hz, 1H), 7.42-7.25 (m, 5H), 7.13
(s, 2H), 6.84 (d, J ) 7.5 Hz, 1H), 6.65 (d, J ) 8.8 Hz, 1H), 3.87
(m, 1H), 3.22 (d, J ) 6.5 Hz, 2H), 2.20 (m, 2H), 2.11 (s, 6H), 1.89
(m, 1H), 1.81 (m, 1H); MS (ESI) m/z 448 (M + H)+.
4-Bromo-N,N-bis(2,4-dimethoxybenzyl)benzenesulfonamide
(12). To a solution of 11 (1.3 g, 5.0 mmol) and bis(p-methoxy-
benzyl)ammonium tosylate (2.2 g, 5.0 mmol) in CH2Cl2 (20 mL)
was added triethylamine (5 mL) and DMAP (61 mg, 0.050 mmol).
The mixture was stirred at room temperature overnight and diluted
with ethyl acetate. The resulting solution was washed with 5% HCl,
water and brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by flash chromatography (SiO2, 20% ethyl
AUC (µM·h)
rat,
5 mg/kg
mouse,
dog,
monkey,
2.5 mg/kg
R
50 mg/kg
2.5 mg/kg
1
34
2
A
B
3.87
6.26
25.2
56.7
3.8
27.8
1.90
6.61
acetate in hexanes) to give 12 (2.1 g, 89%). H NMR (300 MHz,
CDCl3) δ 7.63 (dd, J ) 9.1, 9.1 Hz, 4H), 6.98 (d, J ) 9.0 Hz, 4H),
6.76 (d, J ) 9.0 Hz, 4H), 4.25 (s, 4H), 3.79 (s, 6H); MS (ESI) m/z
475.9 (M + H)+.
140.3, 130.5, 126.1, 123.0 (q, J ) 287.7 Hz, CF3), 118.9, 93.0 (q,
J ) 34.4 Hz, C(OH)2). 7a (keto form): 1H NMR (500 MHz, C6D6)
8.19 (dd, J ) 2.6, 6.2 Hz, 1H), 7.44 (ddd, J ) 2.9, 4.4, 9.2 Hz,
1H), 6.30 (dd, J ) 9.2, 9.2 Hz, 1H); 13C NMR (125 MHz, C6D6)
177.1 (C(O)), 164.6 (d, J ) 273.3 Hz, CF), 140.9, 135.2, 131.4,
120.5, 119.3, 116.1 (q, J ) 289.6 Hz, CF3). 8a: 1H NMR (500
MHz, DMSO-d6) δ 8.19 (dd, J ) 2.5, 7.2 Hz, 1H), 7.95 (s, 2H),
7.91 (ddd, J ) 2.8, 4.4, 8.7 Hz, 1H), 7.46 (s, 2H), 7.41 (dd, J )
8.6, 11.1 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 161.73 (d, J
) 259.0 Hz, CF), 139.82, 129.50 (d, J ) 10.1 Hz), 128.52, 125.90
(d, J ) 12.2 Hz), 123.08 (q, J ) 287.7 Hz, CF3), 117.45 (d, J )
25.0 Hz), 91.70 (dq, J ) 3.0, 33.3 Hz, C(OH)2); MS (DCI) m/z
289.0 (M + NH4)+.
3-Cyano-4-fluorobenzenesulfonamide (8c). 8c was prepared
from 3-cyano-4-fluorobenzene-1-sulfonyl chloride according to
general procedure I. 1H NMR (300 MHz, DMSO-d6) δ 8.32 (dd, J
) 2.2, 5.9 Hz, 1H), 8.18 (ddd, J ) 2.4, 5.1, 8.8 Hz, 1H), 7.76 (dd,
J ) 9.0, 9.0 Hz, 1H), 7.60 (s, 2H); MS (DCI) m/z 219.7 (M +
NH4)+.
General Procedure II. (R)-3-Cyano-4-(4-(dimethylamino)-1-
(phenylthio)butan-2-ylamino)benzenesulfonamide (10c). To a
solution of 8c (170 mg, 0.849 mmol) and 912 (191 mg, 0.849 mmol)
in THF (10 mL) was added DIPEA (297 µL, 1.698 mmol). The
solution was stirred at 50 °C overnight and concentrated. The
residue was purified by flash chromatography (SiO2, 0-10% 7 N
NH3-MeOH in CH2Cl2) to give 10c (160 mg, 47%). 1H NMR
(300 MHz, DMSO-d6) δ 7.76 (d, J ) 2.1 Hz, 1H), 7.63 (dd, J )
9.2, 2.2 Hz, 1H), 7.38-7.29 (m, 5H), 7.24 (m, 1H), 7.14 (br s,
2H), 6.66 (d, J ) 9.2, 1H), 3.90 (s, 1H), 3.26 (m, 2H), 2.44-2.20
(m, 2H), 2.14 (s, 6H), 1.91-1.79 (m, 2H); MS (ESI) m/z 405 (M
+ H)+.
General Procedure III. (R)-N,N-Bis(2,4-dimethoxybenzyl)-4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)benzene-
sulfonamide (13). To a solution of 12 (475 mg, 1.00 mmol) and 9
(280 mg, 1.25 mmol) in toluene (20 mL) was added Pd2(dba)3 (60
mg, 0.085 mmol), BINAP (120 mg, 0.20 mmol), and Cs2CO3 (1.0
g, 3.0 mmol). The mixture was heated under reflux in a nitrogen
atmosphere overnight and diluted with ethyl acetate and water. The
aqueous layer was extracted with ethyl acetate (×2), and the
combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated. The residue was purified by
flash chromatography (SiO2, 5-10% 7 N NH3-MeOH in CH2Cl2)
to give 13 (400 mg, 60%). 1H NMR (300 MHz, DMSO-d6) δ 7.55
(m, 2H), 7.40 (m, 2H), 7.40-7.24 (m, 9H), 6.98 (d, J ) 8.3 Hz,
4H), 6.77 (d, J ) 8.3 Hz, 4H), 6.39 (d, J ) 8.7 Hz, 2H), 4.18 (s,
4H), 3.77 (s, 6H), 3.18 (m, 1H), 3.03 (m, 1H), 2.45 (m, 1H), 2.35
(m, 1H), 2.23 (s, 6H), 2.00 (m, 1H), 1.89 (m, 1H); MS (ESI) m/z
620.3 (M + H)+.
(R)-4-(4-(Dimethylamino)-1-(phenylthio)butan-2-ylamino)ben-
zenesulfonamide (14). To a solution of 13 (310 mg, 0.50 mmol)
in CH2Cl2 (5 mL) was added TFA (5 mL). The solution was stirred
at room temperature overnight and concentrated. The residue was
diluted with CH2Cl2 and washed with saturated sodium bicarbonate,
brine, dried over Na2SO4 and filtered. The solution was concentrated
and the residue was purified by flash chromatography (SiO2, 5-10%
1
7N NH3-MeOH in CH2Cl2) to give 14 (176 mg, 92%). H NMR
(300 MHz, DMSO-d6) δ 7.60 (d, J ) 8.9 Hz, 2H), 7.40-7.24 (m,
5H), 6.38 (d, J ) 8.9 Hz, 2H), 5.60 (m, 1H), 4.60 (m, 2H), 3.70
(m, 1H), 3.15 (m, 1H), 2.99 (m, 1H), 2.45 (m, 1H), 2.35 (m, 1H),
2.23 (s, 6H), 2.00 (m, 1H), 1.76 (m, 1H); MS (ESI) m/z 380.0 (M
+ H)+.
(2-Fluorophenyl)(trifluoromethyl)sulfane (16). To a suspension
of methyl viologen dichloride (2.65 g, 10.3 mmol) in DMF (180
mL) saturated with trifluoromethyl iodide (81 g, 412 mmol) in a
3-neck flask equipped with a coldfinger was added 15 (22 mL, 206
mmol). The reaction mixture was treated with triethylamine (40
mL, 290 mmol) dropwise, and stirred at room temperature
overnight. The solution was diluted with water (480 mL), and the
layers were separated. The aqueous layer was extracted with ether
(×2), and the combined organic layers were washed with 1 N
aqueous NaOH, saturated NH4Cl, and brine, dried over MgSO4,
and filtered. Fractional distillation of the filtrate provided 16 (32.8
(R)-4-(4-(Dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-
(2,2,2-trifluoroacetyl)benzenesulfonamide (10a). 10a was pre-
1
pared from 8a and 9 according to general procedure II. H NMR
(500 MHz, DMSO-d6) δ 9.06 (d, J ) 9.0 Hz, 1H), 8.04 (s, 1H),
7.79 (dd, J ) 1.9, 9.4 Hz, 1H), 7.29 (m, 4H), 7.23 (t, J ) 7.6 Hz,
2H), 7.16 (t, J ) 7.3 Hz, 1H), 7.07 (d, J ) 9.4 Hz, 1H), 4.16 (m,
1H), 3.40 (dd, J ) 5.0, 14.0 Hz, 2H), 2.35 (m, 1H), 2.24 (m, 1H),
2.12 (s, 6H), 1.94 (m, 1H), 1.78 (m, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 178.60 (q, J ) 33.3 Hz, CdO), 154.09, 135.40,
133.86, 129.97, 129.45, 128.94, 128.86, 126.07, 116.64 (q, J )
291.7 Hz, CF3), 114.01, 107.62, 55.06, 50.64, 45.02, 37.20, 31.22;
MS (ESI) m/z 476.2 (M + H)+.
1
g, 81%). H NMR (400 MHz, CDCl3) δ 7.66 (m, 1H), 7.51 (m,
1H), 7.20 (m, 2H); MS (EI) m/z 196.1 (M)+.
(R)-4-(4-(Dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-
(methylsulfonyl)benzenesulfonamide (10b). 10b was prepared
from 8b and 9 according to general procedure II. H NMR (300
MHz, DMSO-d6) δ 8.04 (d, J ) 2.2 Hz, 1H), 7.73 (dd, J ) 9.0,
2.5 Hz, 1H), 7.38-7.29 (m, 4H), 7.22 (m, 1H), 7.18 (br s, 2H),
6.85 (d, J ) 9.0, 1H), 6.78 (d, J ) 8.4, 1H), 3.94 (s, 1H), 3.34 (m,
2H), 3.17 (s, 3H), 2.37 (m, 1H), 2.23 (m, 1H), 2.11 (s, 6H), 1.93
(m, 1H), 1.75 (m, 1H); MS (ESI) m/z 458 (M + H)+.
1-Fluoro-2-(trifluoromethylsulfonyl)benzene (17). To a solu-
tion of 16 (32.76 g, 167.2 mmol) in CCl4-acetonitrile-H2O (100
mL-100 mL-200 mL) cooled with a water bath was added sodium
periodate (107.3 g, 501.5 mmol) and ruthenium(III) chloride hydrate
(346 mg, 1.672 mmol). The reaction mixture was stirred at room
temperature overnight and diluted with CH2Cl2 (200 mL). The
mixture was filtered through a pad of Celite, and the filtrate was
diluted with aqueous sodium bicarbonate. The aqueous layer was
1

Orally BioaVailable Small Molecule Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6911
Figure 3. (A) Mean ((SEM, n ) 6) plasma concentrations of 2 after a 2.5 mg/kg iv or oral dose in dog. (B) Plasma and tumor concentrations of
2 following 50 mg/kg once-daily oral dosing in female scid-bg mice (mean ( SEM, n ) 3 mice per time point).
(R)-4-(4-(Dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-
(trifluoromethylsulfonyl)benzenesulfonamide (20a). 20awas pre-
1
pared from 18 and 9 according to general procedure II. H NMR
(300 MHz, DMSO-d6) δ 7.99 (d, J ) 2.4 Hz, 1H), 7.84 (dd, J )
9.2, 2.4 Hz, 1H), 7.43-7.29 (m, 7H), 7.22 (m, 1H), 6.99 (d, J )
9.2, 1H), 4.05 (s, 1H), 3.25 (m, 2H), 2.37-2.17 (m, 2H), 2.09 (s,
6H), 1.92-1.74 (m, 2H); MS (ESI) m/z 510 (M - H)-.
(R)-4-(4-Morpholino-1-(phenylthio)butan-2-ylamino)-3-(tri-
fluoromethylsulfonyl)benzenesulfonamide (20b). 20bwas prepared
1
from 18 and 1912 according to general procedure II. H NMR (500
MHz, DMSO-d6) δ 7.98 (d, J ) 1.8 Hz, 1H), 7.85 (dd, J ) 1.8, 9.2
Hz, 1H), 7.38 (s, 2H), 7.35 (d, J ) 7.3 Hz, 2H), 7.31 (t, J ) 7.6 Hz,
2H), 7.22 (t, J ) 7.2 Hz, 2H), 7.06 (d, J ) 9.5 Hz, 1H), 6.91 (d, J )
9.2 Hz, 1H), 4.10 (m, 1H), 3.50 (br, 4H), 3.37 (dd, J ) 5.2, 14.0 Hz,
1H), 3.29 (dd, J ) 7.3, 14.0 Hz, 1H), 2.29 (br, 4H), 2.18 (br, 2H),
1.95 (m, 1H), 1.74 (m, 1H); MS (ESI) m/z 554.0 (M + H)+.
Figure 4. Effect of 2 in the xenograft model of H1963 SCLC.
General Procedure IV. (R)-4-(4-((4′-Chlorobiphenyl-2-yl)m-
Compound 2 (9) was administered orally once daily at 100 mg/kg for
ethyl)piperazin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)bu-
21 days (black bar). Open squares (0) represent the vehicle. 100%
tan-2-ylamino)-3-(2,2,2-trifluoroacetyl)phenylsulfonyl)benz-
inhibition of tumor growth was observed with treatment of 2 with
amide Trifluoroacetic Acid Salt (22a). To a solution of 21 (140
complete regression of all tumors (N ) 10) by the end of the therapy
mg, 0.345 mmol) and 10a (149 mg, 0.314 mmol) in CH2Cl2 (1.6
period.
mL) were added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (121 mg, 0.628 mmol) and DMAP (38 mg, 0.314
extracted with CH2Cl2 (×2), and the combined organic layers were
mmol). The solution was stirred at room temperature for 15 h and
washed with brine, dried over MgSO4, filtered, and concentrated.
diluted with saturated NH4Cl. The layers were separated, and the
The residue was filtered through a pad of silica gel to give 17 (37.3
aqueous layer was extracted with CH2Cl2. The combined organic
1
g, 98%). H NMR (300 MHz, CDCl3) δ 8.02 (ddd, J ) 1.7, 6.7,
8.2 Hz, 1H), 7.85 (dddd, J ) 1.8, 4.9, 7.5, 12.4 Hz, 1H), 7.45 (ddd,
J ) 1.1, 7.7, 7.7 Hz, 1H), 7.35 (ddd, J ) 1.0, 8.4, 9.6 Hz, 1H);
MS (EI) m/z 228.1 (M)+.
layers were dried over MgSO4 and concentrated under vacuo. The
residue was purified by reverse phase HPLC to give 22a (178 mg,
52%). 1H NMR (500 MHz, DMSO-d6) δ 9.69 (br, 1H), 8.90 (d, J
) 9.0 Hz, 1H), 8.24 (s, 1H), 7.92 (dd, J ) 1.9, 9.4 Hz, 1H), 7.77
(m, 3H), 7.52 (m, 4H), 7.39 (d, J ) 8.4 Hz, 2H), 7.34 (m, 1H),
7.21 (d, J ) 7.2 Hz, 2H), 7.15 (d, J ) 9.4 Hz, 1H), 7.11 (t, J ) 7.3
Hz, 2H), 7.06 (t, J ) 7.3 Hz, 1H), 6.93 (d, J ) 9.0 Hz, 2H), 4.32
(br, 6H), 3.80 (m, 1H), 3.42 (dd, J ) 4.7, 14.3 Hz, 1H), 3.34 (dd,
J ) 6.6, 14.3 Hz, 1H), 3.15 (br, 4H), 2.91 (br, 2H), 2.75 (s, 3H),
2.13 (m, 2H); MS (ESI) m/z 864.4 (M + H)+. Anal. (C44H45Cl-
F3N5O4S2 ·2.75CF3CO2H·0.15H2O) C, H, N.
4-Fluoro-3-(trifluoromethylsulfonyl)benzenesulfonamide (18).
17 (37.3 g, 163.6 mmol) was dissolved in chlorosulfonic acid (32.8
mL, 490.8 mmol) and heated at 120 °C overnight. The solution
was slowly poured onto crushed ice and extracted with ethyl acetate.
The combined organic layers were washed with water (until the
pH of the aqueous layer increased to 3-4) and brine. The organic
layer was dried over MgSO4, filtered, and concentrated to give the
sulfonyl chloride (46 g, 86%).
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-(methyl-
sulfonyl)phenylsulfonyl)benzamide Trifluoroacetic Acid Salt
(22b). 22b was prepared from 10b and 21 according to general
procedure IV. 1H NMR (500 MHz, DMSO-d6) δ 8.21 (d, J ) 2.4
Hz, 1H), 7.89 (dd, J ) 2.1, 9.2 Hz, 1H), 7.76 (d, J ) 8.9 Hz, 3H),
7.53 (d, J ) 8.5 Hz, 4H), 7.40 (d, J ) 8.5 Hz, 2H), 7.34 (m, 3H),
7.28 (t, J ) 7.6 Hz, 2H), 7.19 (t, J ) 7.2 Hz, 1H), 6.96 (d, J ) 9.5
Hz, 1H), 6.93 (d, J ) 9.2 Hz, 2H), 6.67 (d, J ) 8.9 Hz, 1H),
4.50-3.77 (br, 6H), 4.00 (m, 1H), 3.38 (dd, J ) 5.2, 13.7 Hz, 1H),
3.33 (dd, J ) 7.3, 13.7 Hz, 1H), 3.21 (s, 3H), 3.12 (m, 4H), 2.89
(br, 2H), 2.75 (s, 6H), 2.08 (m, 2H); MS (ESI) m/z 846 (M + H)+.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(3-
cyano-4-(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)phe-
nylsulfonyl)benzamide Trifluoroacetic Acid Salt (22c). 22c was
To a solution of the crude sulfonyl chloride (23 g, 70.6 mmol)
in isopropanol (706 mL) cooled to -78 °C in a three-neck flask
equipped with a dropping funnel and mechanical stirrer was added
ammonium hydroxide (98 mL, 706 mmol) dropwise. The reaction
mixture was stirred at -78 °C for an hour and slowly quenched
with 6 N HCl. The solution was allowed to warm to room
temperature and concentrated. The residue was diluted with ethyl
acetate and water. The layers were separated, and the aqueous layer
was extracted with ethyl acetate (×2). The combined organic layers
were dried over MgSO4, filtered, and concentrated. The residue
was recrystallized from hot ethyl acetate and hexanes to give 18
1
(17.5 g, 81%). H NMR (500 MHz, DMSO-d6) δ 8.47 (ddd, J )
2.5, 4.4, 8.7 Hz, 1H), 8.43 (dd, J ) 2.5, 6.2 Hz, 1H), 7.97 (dd, J
1
) 8.7, 10.0 Hz, 1H), 7.78 (s, 2H); MS (EI) m/z 307.1 (M)+.
prepared from 10c and 21 according to general procedure IV. H

6912 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Park et al.
NMR (400 MHz, DMSO-d6) δ 7.93 (d, J ) 2.1 Hz, 1H), 7.78 (m,
3H), 7.72 (br s, 1H), 7.52 (m, 4H), 7.40 (d, J ) 8.6 Hz, 2H), 7.33
(m, 1H), 7.26 (m, 4H), 7.17 (m, 1H), 6.92 (d, J ) 8.6 Hz, 2H),
6.81 (dd, J ) 9.4, 2.6 Hz, 2H), 4.19-3.93 (m, 7H), 3.41 (m, 2H),
3.28 (m, 2H), 3.18-3.02 (m, 2H), 2.80 (m, 2H), 2.75 (d, 6H), 2.07
(m, 2H); MS (ESI) m/z 793 (M + H)+. Anal. (C43H45ClN6-
O3S2 ·2.3CF3CO2H·0.65H2O) C, H, N.
acetate (100 mL × 2). The combined organic layers were washed
with water and brine, dried over MgSO4, filtered, and concentrated.
The crude material was used for the next step without further
purification (2.5 g, 57%). 1H NMR (300 MHz, CDCl3) δ 7.79 (m,
1H), 7.46 (m, 1H), 7.42 (m, 1H); MS (ESI) m/z 318 (M - H)-.
N-(4-Bromo-3-methylphenylsulfonyl)-4-(4-((4′-chlorobiphenyl-
2-yl)methyl)piperazin-1-yl)benzamide (24a). 24a was prepared
from 23a and 21 according to general procedure IV, and the product
was purified by flash chromatography (0-10% MeOH in CH2Cl2).
1H NMR (300 MHz, DMSO-d6) δ 7.91 (m, 1H), 7.86 (d, J ) 8.3
Hz, 1H), 7.76 (m, 3H), 7.69 (dd, J ) 8.5, 2.2 Hz, 1H), 7.52 (m,
4H), 7.40 (m, 2H), 7.33 (m, 1H), 6.92 (d, J ) 9.1 Hz, 2H), 4.36
(br, 2H), 3.88 (br, 2H), 3.20 (br, 4H), 2.86 (m, 2H), 2.43 (s, 3H);
MS (ESI) m/z 637.9 (M + H)+.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)phenylsul-
fonyl)benzamide Trifluoroacetic Acid Salt (22d). 22d was
1
prepared from 14 and 21 according to general procedure IV. H
NMR (500 MHz, pyridine-d5) δ 8.27 (d, J ) 9.2 Hz, 2H), 8.16 (d,
J ) 8.9 Hz, 2H), 7.51 (m, 4H), 7.43 (m, 4H), 7.35 (m, 2H), 7.26
(m, 2H), 7.18 (m, 1H), 6.91 (d, J ) 9.2 Hz, 2H), 6.71 (d, J ) 8.9
Hz, 2H), 3.83 (m, 1H), 3.39 (s, 2H), 3.29 (m, 2H), 3.16 (m, 4H),
3.14 (m, 2H), 2.74 (s, 6H), 2.50 (m, 1H), 2.38 (m, 4H), 2.20 (m,
1H); MS (ESI) m/z 768.1 (M + H)+. Anal. (C42H46ClN5O3S2 ·
3.2CF3CO2H·0.3H2O) C, H, N.
N-(4-Bromo-3-(trifluoromethoxy)phenylsulfonyl)-4-(4-((4′-
chlorobiphenyl-2-yl)methyl)piperazin-1-yl)benzamide (24b). 24b
was prepared from 23d and 21 according to general procedure VI,
and the product was purified by flash chromatography (0-10%
1
MeOH in CH2Cl2). H NMR (300 MHz, CDCl3) δ 8.02 (m, 2H),
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-(trifluo-
romethylsulfonyl)phenylsulfonyl)benzamide Trifluoroacetic Acid
Salt (22e). 22e was prepared from 20a and 21 according to general
procedure IV. 1H NMR (400 MHz, DMSO-d6) δ 8.08 (d, J ) 1.8
Hz, 1H), 7.95 (dd, J ) 8.9, 1.8 Hz, 1H), 7.72 (d, J ) 8.9 Hz, 2H),
7.51 (d, J ) 7.3 Hz, 1H), 7.48 (s, 4H), 7.38 (m, 4H), 7.30 (t, J )
7.8 Hz, 2H), 7.24 (m, 1H), 7.21 (t, J ) 7.3 Hz, 1H), 6.85-6.78
(m, 4H), 3.99 (m, 1H), 3.39-3.24 (m, 6H), 3.14 (m, 6H), 2.85-2.72
(m, 2H), 2.40 (d, 6H), 1.93 (m, 2H); MS (ESI) m/z 900 (M + H)+.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-(trifluo-
romethyl)phenylsulfonyl)benzamide (3). 3 was prepared from 10d
and 21 according to general procedure IV, and the product was
purified by flash chromatography (0-10% MeOH in CH2Cl2
saturated with NH3). 1H NMR (500 MHz, DMSO-d6) δ 7.94 (d, J
) 2.0 Hz, 1H), 7.80 (dd, J ) 9.0, 2.0 Hz, 1H), 7.73 (d, J ) 9.0
Hz, 2H), 7.53 (d, J ) 6.6 Hz, 1H), 7.47 (m, 4H), 7.41-7.26 (m,
7H), 7.19 (d, J ) 7.5 Hz, 1H), 6.89 (d, J ) 9.0 Hz, 2H), 6.87 (d,
J ) 9.1 Hz, 1H), 5.94 (s, 1H), 3.94 (m, 1H), 3.42 (m, 2H), 3.26
(m, 6H), 3.10 (m, 1H), 2.96 (m, 1H), 2.67 (s, 6H), 2.41 (m, 4H),
2.13 (m, 2H); MS (ESI) m/z 836 (M + H)+.
7.65 (m, 5H), 7.25 (m, 5H), 6.69 (m, 3H), 3.25 (m, 5H), 2.57 (m,
5H); MS (ESI) m/z 710 (M + H)-.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-meth-
ylphenylsulfonyl)benzamide (25a). 25a was prepared from 24a
and 9 according to general procedure III, and the product was
purified by flash chromatography (0-10% MeOH in CH2Cl2
saturated with NH3). 1H NMR (500 MHz, DMSO-d6) δ 7.72 (d, J
) 8.7 Hz, 2H), 7.49 (m, 6H), 7.37 (m, 4H), 7.30 (t, J ) 7.6 Hz,
2H), 7.22 (m, 2H), 6.83 (d, J ) 9.1 Hz, 2H), 6.34 (d, J ) 9.1 Hz,
1H), 5.75 (s, 1H), 5.52 (d, J ) 8.4 Hz, 1H), 3.72 (m, 1H), 3.38 (m,
2H), 3.19 (m, 5H), 3.12 (dd, J ) 6.9, 13.4 Hz, 1H), 2.64 (m, 1H),
2.54 (m, 1H), 2.39 (m, 4H), 2.34 (s, 6H), 2.05 (s, 3H), 1.98 (m
1H), 1.87 (m, 1H); MS (ESI) m/z 782.2 (M + H)+.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-(trifluo-
romethoxy)phenylsulfonyl)benzamide Trifluoroacetic Acid Salt
(25b). 25b was prepared from 24b and 9 according to general
1
procedure III. H NMR (300 MHz, DMSO-d6) δ 11.95 (m, 1H),
10.74 (m, 1H), 7.74 (d, J ) 8.8 Hz, 2H), 7.50 (m, 5H), 7.27 (s,
10H), 6.92 (d, J ) 9.2 Hz, 2H), 6.78 (d, J ) 8.8 Hz, 1H), 6.55 (d,
J ) 9.2 Hz, 1H), 4.36 (m, 1H), 3.83 (m, 4H), 3.38 (m, 2H), 3.22
(m, 4H), 2.95 (m, 4H), 2.71 (s, 6H), 2.11 (m, 2H); MS (ESI) m/z
852 (M + H)+. Anal. (C43H45ClF3N5O4S2 ·2.8CF3CO2H·0.05H2O)
C, H, N.
4-Bromo-3-(trifluoromethoxy)aniline (23b). To a solution of
3-(trifluoromethoxy)aniline (3.5 g, 20 mmol) in CH2Cl2 (60 mL)
was added pyridinium bromide perbromide (7.7 g, 24 mmol). After
being stirred at 0 °C for 3 h, the mixture was diluted with ethyl
acetate (400 mL), washed with aqueous Na2S2O3, water, and brine,
dried over Na2SO4, filtered, and concentrated. The residue was
purified by flash chromatography (30% ethyl acetate in hexanes)
to give 23b (3.5 g, 68%). 1H NMR (300 MHz, CDCl3) δ 6.61 (m,
1H), 6.50 (m, 1H), 6.47 (m, 1H); MS (ESI) m/z 254 (M - H)-.
4-Bromo-3-(trifluoromethoxy)benzenesulfonamide (23d). 23b
(3.5 g, 13.7 mmol) was dissolved in concentrated HCl (10 mL)
and AcOH (3 mL) and cooled to -15 °C with a dry ice ethanol
bath. A solution of NaNO2 (1.04 g, 15 mmol) in water (3 mL) was
added slowly with the temperature not to exceed -5 °C and stirred
at -10 °C for 45 min. In a separate flask, a saturated solution of
SO2 in AcOH (30 mL) was prepared by bubbling SO2 for 45 min.
To the resulting solution was added CuCl (1.1 g, 11 mmol) in one
portion, and the solution was bubbled with SO2 for an additional
15 min. The solution was cooled to 10 °C, and the solution of
diazonium salt prepared above was added portionwise with the
temperature not to exceed 30 °C. Upon completion of the addition,
the mixture was poured onto crushed ice and stirred for 20 min.
The mixture was extracted with ether (150 mL × 3), and the
combined organic layers were washed with aqueous NaHCO3,
water, and brine, dried over Na2SO4, filtered, and concentrated. The
General Procedure V. Ethyl 4-(4-((2-Bromocyclohex-1-enyl)-
methyl)piperazin-1-yl)benzoate (29b). To a solution of DMF (20.0
g, 275 mmol) in CH2Cl2 (60 mL) cooled to 0 °C was added PBr3
(23.5 mL, 249 mmol). The mixture was allowed to warm to room
temperature and stirred for 20 min. A solution of 26b in CH2Cl2
(10 mL) was added dropwise. After being stirred at room temper-
ature for 20 min, the reaction mixture was poured onto crushed ice
and neutralized with NaHCO3. The mixture was diluted with ether,
and the layers were separated. The aqueous layer was extracted
with ether (×3), and the combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated. The residue
was purified by flash chromatography (5% ethyl acetate in hexanes)
to give 27b (7.99 g, 44%), which was used immediately for the
next step because of instability.
To a solution of 27b (7.99 g, 42.2 mmol) and 28 (10.0 g, 42.7
mmol) in EtOH (50 mL) was added sodium cyanoborohydride (2.68
g, 42.7 mmol) at room temperature. The pH of the solution was
adjusted to 5 with acetic acid, and the suspension was stirred
overnight. After filtration, the filter cake was washed with EtOH,
and the combined filtrate was concentrated in vacuo. The residue
was purified by flash chromatography (SiO2, 10-20% ethyl acetate
i
1
residue was dissolved in PrOH (100 mL) and cooled to 0 °C. A
in hexanes) to give 29b (6.59 g, 38%). 27b: H NMR (300 MHz,
solution of ammonium hydroxide (50 mL) was added dropwise,
and the resulting solution was allowed to warm to room temperature
overnight. The solvent was removed in vacuo, and the residue was
diluted with ethyl acetate (200 mL) and water (150 mL). The layers
were separated, and the aqueous layer was extracted with ethyl
CDCl3) δ 10.03 (s, 1H), 2.79-2.71 (m, 2H), 2.32-2.24 (m, 2H),
1
1.80-1.63 (m, 4H). 29b: H NMR (500 MHz, DMSO-d6) δ 7.80
(m, 2H), 6.98 (m, 2H), 4.26 (q, J ) 7.17 Hz, 2H), 3.33-3.29 (m,
4H), 3.14 (s, 2H), 2.51-2.47 (m, 6H), 2.23 (m, 2H), 1.73-1.59
(m, 4H), 1.31 (t, J ) 7.17 Hz, 3H); MS (ESI) m/z 407 (M + H)+.

Orally BioaVailable Small Molecule Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6913
Ethyl 4-(4-((2-Bromocyclopent-1-enyl)methyl)piperazin-1-yl)-
benzoate (29a). 29a was prepared from 26a via 27a according to
general procedure V. 27a: 1H NMR (300 MHz, CDCl3) δ 9.91 (s,
1H), 2.95-2.86 (m, 2H), 2.58-2.49 (m, 2H), 2.09-1.95 (m, 2H).
29a: 1H NMR (300 MHz, CDCl3) δ 7.97-7.87 (m, 2H), 6.90-6.80
(m, 2H), 4.32 (q, J ) 7.1 Hz, 2H), 3.44-3.21 (m, 4H), 3.13 (s,
2H), 2.73-2.62 (m, 2H), 2.61-2.50 (m, 4H), 2.47-2.37 (m, 2H),
2.03-1.90 (m, 2H), 1.37 (t, J ) 7.1 Hz, 3H); MS (ESI) m/z 393
(M + H)+.
(m, 2H), 1.64-1.52 (m, 4H), 1.35 (t, J ) 7.2 Hz, 3H); MS (ESI)
m/z 453 (M + H)+.
(Z)-Ethyl 4-(4-((2-(4-Chlorophenyl)cyclooct-1-enyl)methyl)pip-
erazin-1-yl)benzoate (30d). 30d was prepared from 29d according
to general procedure VI. 1H NMR (300 MHz, CDCl3) δ 7.98-7.89
(m, 2H), 7.41-7.33 (m, 2H), 7.03-6.95 (m, 2H), 6.85-6.76 (m,
2H), 4.33 (q, J ) 7.1 Hz, 2H), 3.74-3.10 (m, 12H), 2.61-2.45
(m, 6H), 1.74-1.43 (m, 4H), 1.36 (t, J ) 7.1 Hz, 3H); MS (ESI)
m/z 467 (M + H)+.
(Z)-Ethyl 4-(4-((2-Bromocyclohept-1-enyl)methyl)piperazin-
1-yl)benzoate (29c). 29c was prepared from 26c via 27c according
Ethyl 4-(4-((2-(4-Chlorophenyl)-5,5-dimethylcyclohex-1-enyl)-
methyl)piperazin-1-yl)benzoate (30e). 30e was prepared from 29e
according to general procedure VI. 1H NMR (300 MHz, CDCl3) δ
7.94-7.86 (m, 2H), 7.31-7.24 (m, 2H), 7.04-6.96 (m, 2H),
6.85-6.78 (m, 2H), 4.31 (q, J ) 7.12 Hz, 2H), 3.29-3.21 (m,
4H), 2.80 (s, 2H), 2.44-2.19 (m, 6H), 2.02 (s, 2H), 1.50-1.42
(m, 2H), 1.35 (t, J ) 7.12 Hz, 3H), 0.99 (s, 6H); MS (ESI) m/z
467 (M + H)+.
Ethyl 4-(4-((4-(4-Chlorophenyl)-5,6-dihydro-2H-pyran-3-yl)-
methyl)piperazin-1-yl)benzoate (30f). 30f was prepared from 29f
according to general procedure VI. 1H NMR (300 MHz, CDCl3) δ
7.90 (m, 2H), 7.31 (m, 2H), 7.06 (m, 2H), 6.81 (m, 2H), 4.32 (m,
4H), 3.90 (t, J ) 5.76 Hz, 2H), 3.25 (m, 4H), 2.91 (s, 2H), 2.38
(m, 6H,) 1.36 (t, J ) 7.12 Hz, 3H); MS (ESI) m/z 441 (M + H)+.
General Procedure VII. 4-(4-((2-(4-Chlorophenyl)cyclohex-
1-enyl)methyl)piperazin-1-yl)benzoic Acid (31b). To a solution
of 30a (406 mg, 0.925 mmol) in dioxane (10 mL) was added
aqueous LiOH (1 N, 2 mL, 3.00 mmol). The solution was heated
at 100 °C for 24 h. The solution was concentrated, and the pH was
adjusted to 6 with 2 N HCl. The precipitate was collected by
vacuum filtration and dried in a vacuum oven to give 31b (360
mg, 95%). 1H NMR (300 MHz, DMSO-d6) δ 12.24 (br s, 1H),
7.74 (m, 2H), 7.38 (m, 2H), 7.13 (m, 2H), 6.91 (m, 2H), 3.26-3.18
(m, 4H), 2.77 (s, 2H), 2.34-2.15 (m, 8H), 1.76-1.62 (m, 4H);
MS (ESI) m/z 411 (M + H)+.
1
to general procedure V. 27c: H NMR (300 MHz, CDCl3) δ 9.93
(s, 1H), 3.05-2.96 (m, 2H), 2.53-2.45 (m, 2H), 1.85-1.76 (m,
1
2H), 1.71-1.61 (m, 2H), 1.51-1.40 (m, 2H). 29c: H NMR (500
MHz, CDCl3) δ 7.95-7.88 (m, 2H), 6.88-6.82 (m, 2H), 4.32 (q,
J ) 7.2 Hz, 2H), 3.33-3.28 (m, 4H), 3.14 (s, 2H), 2.82-2.74 (m,
2H), 2.63-2.56 (m, 4H), 2.40-2.33 (m, 2H), 1.81-1.69 (m, 2H),
1.63-1.44 (m, 4H), 1.36 (t, J ) 7.2 Hz, 3H); MS (ESI) m/z 421
(M + H)+.
(Z)-Ethyl 4-(4-((2-Bromocyclooct-1-enyl)methyl)piperazin-1-
yl)benzoate (29d). 29d was prepared from 26d via 27d according
to general procedure V. 29d: 1H NMR (300 MHz, CDCl3) δ
7.95-7.88 (m, 2H), 6.88-6.82 (m, 2H), 4.31 (q, J ) 7.1 Hz, 2H),
3.34-3.27 (m, 4H), 3.16 (s, 2H), 2.77-2.66 (m, 4H), 2.63-2.56
(m, 4H), 2.45-2.36 (m, 4H), 1.72-1.56 (m, 4H), 1.36 (t, J ) 7.1
Hz, 3H); MS (ESI) m/z 435 (M + H)+.
Ethyl 4-(4-((2-Bromo-5,5-dimethylcyclohex-1-enyl)methyl)pip-
erazin-1-yl)benzoate (29e). 29e was prepared from 26e via 27e
according to general procedure V. 29e: 1H NMR (300 MHz,
DMSO-d6) δ 7.81-7.74 (m, 2H), 7.00-6.94 (m, 2H), 4.23 (q, J )
7.1 Hz, 2H), 3.09 (s, 2H), 2.48-2.40 (m, 4H), 2.06-1.95 (m, 4H),
1.51-1.35 (m, 4H), 1.28 (t, J ) 6.8 Hz, 2H), 0.94-0.86 (m, 9H);
MS (ESI), m/z 435 (M + H)+.
Ethyl 4-(4-((4-Bromo-5,6-dihydro-2H-pyran-3-yl)methyl)pip-
erazin-1-yl)benzoate (29f). 29f was prepared from 26f via 27f
according to general procedure V. 27f: 1H NMR (300 MHz, CDCl3)
δ 9.94 (s, 1H), 4.34 (t, J ) 2.7 Hz, 2H), 3.84 (t, J ) 5.8 Hz, 2H),
2.86-2.79 (m, 2H). 29f: 1H NMR (300 MHz, CDCl3) δ 7.99-7.86
(m, 2H), 6.91-6.79 (m, 2H), 4.37-4.26 (m, 4H), 3.86-3.79 (m,
2H), 3.35-3.26 (m, 4H), 3.21-3.15 (m, 2H), 2.69-2.53 (m, 6H),
1.36 (t, J ) 7.1 Hz, 3H); MS (ESI), m/z 409 (M + H)+.
4-(4-((2-(4-Chlorophenyl)cyclopent-1-enyl)methyl)piperazin-
1-yl)benzoic Acid (31a). 31a was prepared from 30a according to
1
general procedure VII. H NMR (500 MHz, DMSO-d6) δ 12.23
(s, 1H), 7.77-7.72 (m, 2H), 7.44-7.28 (m, 4H), 6.96-6.88 (m,
2H), 3.38-3.18 (m, 4H), 3.09 (s, 2H), 2.76-2.69 (m, 2H),
2.61-2.54 (m, 2H), 2.43-2.34 (m, 4H), 1.94-1.84 (m, 2H); MS
(ESI) m/z 397 (M + H)+.
(Z)-4-(4-((2-(4-Chlorophenyl)cyclohept-1-enyl)methyl)piper-
azin-1-yl)benzoic Acid (31c). 31c was prepared from 30c according
to general procedure VII. 1H NMR (300 MHz, DMSO-d6) δ 12.24
(br s, 1H), 7.76-7.71 (m, 2H), 7.41-7.34 (m, 2H), 7.13-7.06 (m,
2H), 6.94-6.87 (m, 2H), 3.26-3.19 (m, 4H), 2.78 (s, 2H),
2.45-2.36 (m, 4H), 2.35-2.27 (m, 4H), 1.87-1.70 (m, 2H),
1.64-1.44 (m, 4H); MS (ESI) m/z 425 (M + H)+.
(Z)-4-(4-((2-(4-Chlorophenyl)cyclooct-1-enyl)methyl)piperazin-
1-yl)benzoic Acid (31d). 31d was prepared from 30d according
to general procedure VII. 1H NMR (500 MHz, DMSO-d6) δ 12.27
(s, 1H), 7.81-7.76 (m, 2H), 7.46-7.41 (m, 2H), 7.15-7.10 (m,
2H), 6.97-6.92 (m, 2H), 3.29-3.25 (m, 4H), 2.81 (s, 2H),
2.58-2.45 (m, 4H), 2.36-2.29 (m, 4H), 1.71-1.56 (m, 6H),
1.53-1.46 (m, 2H); MS (ESI) m/z 439 (M + H)+.
4-(4-((2-(4-Chlorophenyl)-5,5-dimethylcyclohex-1-enyl)meth-
yl)piperazin-1-yl)benzoic Acid (31e). 31e was prepared from 30e
according to general procedure VII. 1H NMR (300 MHz, DMSO-
d6) δ 12.40 (s, 1H), 7.83-7.73 (m, 2H), 7.46-7.38 (m, 2H),
7.22-7.15 (m, 2H), 7.00-6.92 (m, 2H), 3.92-3.79 (m, 2H),
3.64-3.56 (m, 2H), 3.43-3.19 (m, 4H), 2.90-2.71 (m, 2H),
2.35-2.20 (m, 2H), 2.13 (s, 2H), 1.53-1.43 (m, 2H), 1.00 (s, 6H);
MS (ESI) m/z 439 (M + H)+.
4-(4-((4-(4-Chlorophenyl)-5,6-dihydro-2H-pyran-3-yl)meth-
yl)piperazin-1-yl)benzoic Acid (31f). 31f was prepared from 30f
according to general procedure VII. 1H NMR (300 MHz, DMSO-
d6) δ 12.26 (s, 1H), 7.73 (d, J ) 8.82 Hz, 2H), 7.41 (d, J ) 8.48
Hz, 2H), 7.21 (d, J ) 8.48 Hz, 2H), 6.90 (d, J ) 8.82 Hz, 2H),
4.17 (s, 2H), 3.80 (t, J ) 5.43 Hz, 2H), 3.23 (m, 4H), 2.89 (s, 2H),
2.32 (m, 6H); MS (ESI) m/z 413 (M + H)+.
General Procedure VI. Ethyl 4-(4-((2-(4-Chlorophenyl)cy-
clohex-1-enyl)methyl)piperazin-1-yl)benzoate (30b). To a suspen-
sion of 29b (1.86 g, 4.56 mmol), 4-chlorophenylboronic acid (0.80
g, 5.12 mmol), and aqueous sodium carbonate (2M, 2.5 mL, 5.0
mmol) in DME-H2O-EtOH (14 mL-6 mL-4 mL) was added
PdCl2(PPh3)2 (124 mg, 0.177 mmol). The mixture was heated at
85 °C for 3 h under nitrogen atmosphere and filtered through a
pad of Celite. The filter cake was washed with ethyl acetate, and
the combined filtrate was concentrated. The residue was purified
by flash chromatography (SiO2, 5-10% ethyl acetate in hexanes)
1
to give 30b (1.77 g, 89%). H NMR (300 MHz, CDCl3) δ 7.90
(m, 2H), 7.27 (m, 2H), 7.00 (m, 2H), 6.81 (m, 2H), 4.32 (q, J )
7.12 Hz, 2H), 3.30-3.22 (m, 4H), 2.81 (s, 2H), 2.40-2.33 (m,
4H), 2.28-2.17 (m, 4H), 1.78-1.67 (m, 4H), 1.36 (t, J ) 7.12
Hz, 3H); MS (ESI) m/z 439 (M + H)+.
Ethyl 4-(4-((2-(4-Chlorophenyl)cyclopent-1-enyl)methyl)pip-
erazin-1-yl)benzoate (30a). 30a was prepared from 29a according
to general procedure VI. 1H NMR (500 MHz, CDCl3) δ 7.97-7.91
(m, 2H), 7.41-7.34 (m, 2H), 7.10-7.05 (m, 2H), 6.86-6.78 (m,
2H), 4.33 (q, J ) 7.2 Hz, 2H), 3.88 (s, 2H), 3.79-3.60 (m, 4H),
3.48-3.31 (m, 2H), 2.82-2.74 (m, 2H), 2.73-2.64 (m, 4H),
2.09-1.99 (m, 2H), 1.36 (t, J ) 7.2 Hz, 3H); MS (ESI) m/z 425
(M + H)+.
(Z)-Ethyl 4-(4-((2-(4-Chlorophenyl)cyclohept-1-enyl)meth-
yl)piperazin-1-yl)benzoate (30c). 30c was prepared from 29c
according to general procedure VI. 1H NMR (500 MHz, CDCl3) δ
7.93-7.85 (m, 2H), 7.28-7.22 (m, 2H), 7.02-6.95 (m, 2H),
6.83-6.78 (m, 2H), 4.31 (q, J ) 7.2 Hz, 2H), 3.28-3.23 (m, 4H),
2.82 (s, 2H), 2.47-2.41 (m, 4H), 2.41-2.36 (m, 4H), 1.86-1.79

6914 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Park et al.
(R)-4-(4-((2-(4-Chlorophenyl)cyclopent-1-enyl)methyl)piper-
azin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)butan-2-ylami-
no)-3-nitrophenylsulfonyl)benzamide Trifluoroacetic Acid Salt
(33a). 33a was prepared from 31a and 3212 according to general
procedure IV. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (br s, 1H),
8.30-8.28 (m, 1H), 8.28 (d, J ) 9.2 Hz, 1H), 7.63-7.58 (m, 1H),
7.78 (d, J ) 8.9 Hz, 2H), 7.32 (d, J ) 8.6 Hz, 2H), 7.28 (d, J )
8.6 Hz, 2H), 6.99-6.95 (m, 2H), 6.94-6.83 (m, 4H), 6.97 (d, J )
8.9 Hz, 2H), 3.98-3.88 (m, 1H), 3.71-3.60 (m, 4H), 3.31-3.27
(m, 4H), 3.15-3.12 (m, 2H), 2.95-2.84 (m, 4H), 2.53-2.46 (m,
8H), 2.43-2.33 (m, 2H), 1.93-1.85 (m, 2H), 1.76-1.66 (m, 2H);
2H), 3.96-3.90 (m, 1H), 3.69 (s, 2H), 3.58 (t, J ) 5.3 Hz, 2H),
3.13 (s, 8H), 2.93-2.84 (m, 4H), 2.49 (s, 6H), 2.15-2.09 (m, 2H),
1.93-1.85 (m, 2H); MS (ESI) m/z 819 (M + H)+. Anal.
(C41H47ClN6O6S2 ·2.85CF3CO2H·0.65H2O) C, H, N.
(R)-4-(4-((2-(4-Chlorophenyl)cyclohex-1-enyl)methyl)piperazin-
1-yl)-N-(4-(4-morpholino-1-(phenylthio)butan-2-ylamino)-3-(tri-
fluoromethylsulfonyl)phenylsulfonyl)benzamide Trifluoroacetic
Acid Salt (34). 34 was prepared from 20b and 31b according to
1
general procedure IV. H NMR (300 MHz, DMSO-d6) δ 8.10 (d,
J ) 2.03 Hz, 1H), 7.93 (dd, J ) 9.15, 2.03 Hz, 1H), 7.71 (d, J )
8.81 Hz, 2H), 7.41-7.23 (m, 6H), 7.22-7.15 (m, 1H), 7.15-7.09
(m, 2H), 6.94 (d, J ) 9.15 Hz, 1H), 6.83 (d, J ) 8.81 Hz, 3H),
4.13-3.97 (m, 1H), 3.51 (br s, 4H), 3.39-3.26 (m, 2H), 3.23 (br
s, 4H), 2.83 (br s, 2H), 2.48-2.24 (m, 10H), 2.18 (br s, 4H),
2.04-1.86 (m, 1H), 1.81-1.70 (m, 1H), 1.66 (br s, 4H); MS (ESI)
m/z 946 (M + H)+.
MS (ESI) m/z 803 (M
+
H)+. Anal. (C41H47ClN6O5S2 ·
3.05CF3CO2H) C, H, N.
(R)-4-(4-((2-(4-Chlorophenyl)cyclohex-1-enyl)methyl)piperazin-
1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)butan-2-ylamino)-
3-nitrophenylsulfonyl)benzamide Trifluoroacetic Acid Salt (33b).
33b was prepared from 31b and 32 according to general procedure
IV. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (br s, 1H), 8.29 (d, J
) 2.5 Hz, 1H), 8.03 (d, J ) 9.2 Hz, 1H), 7.63-7.59 (m, 1H), 7.53
(d, J ) 8.9 Hz, 2H), 7.15 (d, J ) 8.3 Hz, 2H), 6.99-6.95 (m, 2H),
6.95-6.83 (m, 6H), 6.70 (d, J ) 9.2 Hz, 2H), 3.97-3.89 (m, 1H),
3.43-3.31 (m, 4H), 3.19-3.07 (m, 4H), 2.95-2.83 (m, 4H), 2.49
(s, 6H), 2.26-2.22 (m, 2H), 2.02-1.94 (m, 4H), 1.92-1.86 (m,
2H), 1.48-1.39 (m, 4H); MS (ESI) m/z 817 (M + H)+. Anal.
(C42H49ClN6O5S2 ·3.05CF3CO2H·0.05H2O) C, H, N.
(R)-4-(4-((2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-enyl)m-
ethyl)piperazin-1-yl)-N-(4-(4-morpholino-1-(phenylthio)butan-
2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonyl)benz-
amide (2). 2 was prepared from 20b and 31e according to general
procedure IV, and the product was purified by flash chromatography
(0-10% MeOH in dichloromethane saturated with NH3). 1H NMR
(500 MHz, DMSO-d6) δ 8.09 (d, J ) 2.1 Hz, 1H), 7.92 (dd, J )
2.1, 9.2 Hz, 1H), 7.71 (d, J ) 8.9 Hz, 2H), 7.36 (m, 4H), 7.28 (t,
J ) 7.8 Hz, 2H), 7.19 (t, J ) 7.2 Hz, 1H), 7.12 (d, J ) 8.2 Hz,
2H), 6.90 (d, J ) 9.5 Hz, 1H), 6.81 (d, J ) 8.9 Hz, 2H), 6.77 (d,
J ) 8.9 Hz, 1H), 4.03 (m, 1H), 3.51 (m, 4H), 3.33 (dd, J ) 5.5,
13.7 Hz, 1H), 3.26 (dd, J ) 6.7, 14.0 Hz, 1H), 3.17 (br, 4H), 2.78
(s, 2H), 2.32 (m, 12H), 1.99 (s, 2H), 1.95 (m, 1H), 1.73 (m, 1H),
1.43 (t, J ) 6.4 Hz, 2H), 0.97 (s, 6H); MS (ESI) m/z 974.3 (M +
H)+. Anal. (C47H55ClF3N5O6S3 ·0.15H2O) C, H, N.
(R,Z)-4-(4-((2-(4-Chlorophenyl)cyclohept-1-enyl)methyl)pip-
erazin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)butan-2-
ylamino)-3-nitrophenylsulfonyl)benzamide Trifluoroacetic Acid
Salt (33c). 33c was prepared from 31c and 32 according to general
procedure IV. 1H NMR (400 MHz, DMSO-d6) δ 12.11 (br s, 1 H),
8.56-8.53 (m, 1H), 8.29 (d, J ) 9.2 Hz, 1H), 7.88-7.84 (m, 1H),
7.78 (d, J ) 8.9 Hz, 2H), 7.40 (d, J ) 8.3 Hz, 2H), 7.26-7.06 (m,
8 H), 6.95 (d, J ) 8.9 Hz, 2H), 4.23-4.15 (m, 1H), 3.93-3.82 (m,
2H), 3.66-3.56 (m, 2H), 3.44-3.33 (m, 4H), 3.21-3.09 (m, 4H),
2.80-2.70 (m, 8H), 2.49-2.42 (m, 4H), 2.18-2.10 (m, 2H),
1.85-1.76 (m, 2H), 1.60-1.52 (m, 4H); MS (ESI) m/z 831 (M +
H)+. Anal. (C43H51ClN6O5S2 ·2.9CF3CO2H·0.05H2O) C, H, N.
(R,Z)-4-(4-((2-(4-Chlorophenyl)cyclooct-1-enyl)methyl)piper-
azin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)butan-2-ylami-
no)-3-nitrophenylsulfonyl)benzamide Trifluoroacetic Acid Salt
(33d). 33d was prepared from 31d and 32 according to general
4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(3-ni-
tro-4-(2-(phenylthio)ethylamino)phenylsulfonyl)benzamide (4).
4 was prepared from 3-nitro-4-(2-(phenylthio)ethylamino)benze-
nesulfonamide12 and 21 according to general procedure IV, and
the product was purified by flash chromatography (0-10% MeOH
1
in dichloromethane). H NMR (500 MHz, DMSO-d6) δ 8.66 (br,
1H), 8.56 (d, J ) 2.2 Hz, 1H), 7.89 (dd, J ) 2.0, 9.2 Hz, 1H), 7.73
(d, J ) 9.0 Hz, 2H), 7.51 (m, 1H), 7.47 (m, 4H), 7.37 (m, 4H),
7.28 (t, J ) 7.6 Hz, 2H), 7.25 (m, 1H), 7.18 (t, J ) 7.3 Hz, 1H),
7.11 (d, J ) 9.0 Hz, 1H), 6.86 (d, J ) 9.0 Hz, 2H), 3.64 (q, J )
6.5 Hz, 2H), 3.40 (s, 2H), 3.27 (t, J ) 6.9 Hz, 2H), 3.21 (br, 4H),
1
procedure IV. H NMR (300 MHz, DMSO-d6) δ 12.13 (s, 1H),
2.40 (br, 4H); MS (ESI) m/z 742.2 (M
(C38H36ClN5O5S2 ·0.15CH2Cl2) C, H, N.
+
H)+. Anal.
8.56-8.53 (m, 1H), 8.30 (d, J ) 9.2 Hz, 1H), 7.90-7.83 (m, 1H),
7.78 (d, J ) 9.2 Hz, 2H), 7.43 (d, J ) 8.5 Hz, 2H), 7.24-7.09 (m,
8H), 6.95 (d, J ) 9.2 Hz, 2H), 4.27-4.11 (m, 5H), 3.95-3.83 (m,
2H), 3.72-3.57 (m, 2H), 3.45-3.32 (m, 4H), 3.20-3.05 (m, 4H),
2.75 (s, 3H), 2.74 (s, 3H), 2.48-2.43 (m, 2H), 2.21-2.07 (m, 2H),
1.70-1.62 (m, 2H), 1.60-1.48 (m, 4H), 1.45-1.36 (m, 2H); MS
(ESI) m/z 439 (M + H)+. Anal. (C44H53ClN6O5S2 ·3.45CF3CO2H)
C, H, N.
(R)-4-(4-((4′-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-N-(4-
(4-morpholino-1-(phenylthio)butan-2-ylamino)-3-nitrophenyl-
sulfonyl)benzamide Trifluoroacetic Acid Salt (5). 5 was prepared
from (R)-4-(4-morpholino-1-(phenylthio)butan-2-ylamino)-3-ni-
trobenzenesulfonamide12 and 21 according to general procedure
1
IV. H NMR (500 MHz, DMSO-d6) δ 8.55 (d, J ) 2.5 Hz, 1H),
8.30 (d, J ) 9.0 Hz, 1H), 7.87 (dd, J ) 2.0, 9.2 Hz, 1H), 7.77 (d,
J ) 9.0 Hz, 2H), 7.73 (br, 1H), 7.52 (d, J ) 8.1 Hz, 4H), 7.40 (d,
J ) 8.1 Hz, 2H), 7.34 (br, 1H), 7.23 (d, J ) 7.2 Hz, 2H), 7.15 (m,
4H), 6.93 (d, J ) 9.0 Hz, 2H), 4.20 (m, 1H), 3.95 (br, 4H), 3.63
(br, 4H), 3.40 (m, 4H), 3.18 (br, 4H), 3.02 (br, 4H), 2.18 (m, 2H);
MS (ESI) m/z 855 (M + H)+. Anal. (C44H47ClN6O6S2 ·3CF3CO2H)
C, H, N.
(R)-4-(4-((2-(4-Chlorophenyl)-5,5-dimethylcyclohex-1-enyl)m-
ethyl)piperazin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)bu-
tan-2-ylamino)-3-nitrophenylsulfonyl)benzamide Trifluoroacetic
Acid Salt (33e). 33e was prepared from 31e and 32 according to
general procedure IV. 1H NMR (400 MHz, DMSO-d6) δ 12.10 (s,
1H), 8.54 (d, J ) 2.5 Hz, 1H), 8.28 (d, J ) 9.2 Hz, 1H), 7.89-7.84
(m, 1H), 7.79 (d, J ) 8.9 Hz, 2H), 7.43-7.39 (m, 2H), 7.24-7.21
(m, 2H), 7.18-7.10 (m, 6H), 6.95 (d, J ) 9.2 Hz, 2H), 4.23-4.16
(m, 1H), 3.95-3.85 (m, 1H), 3.39 (d, 2H), 3.15 (s, 6H), 2.76-2.71
(m, 8H), 2.54-2.53 (m, 3H), 2.31-2.24 (m, 2H), 2.18-2.10 (m,
2H), 2.03 (s, 2H), 1.51-1.43 (m, 2H), 0.98 (s, 6H); MS (ESI) m/z
845 (M + H)+. Anal. (C44H53ClN6O5S2 ·4.0CF3CO2H·0.7H2O) C,
H, N.
Acknowledgment. The authors thank Dr. Andrew J. Souers
for careful reading of the manuscript, and Abbott high-
throughput purification and structural chemistry groups for
compound purification and analytical services.
Supporting Information Available: Bcl-2 and Bcl-xL binding
affinity data and elemental analysis and HPLC data for all
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(R)-4-(4-((4-(4-Chlorophenyl)-5,6-dihydro-2H-pyran-3-yl)m-
ethyl)piperazin-1-yl)-N-(4-(4-(dimethylamino)-1-(phenylthio)bu-
tan-2-ylamino)-3-nitrophenylsulfonyl)benzamide Trifluoroacetic
Acid Salt (33f). 33f was prepared from 31f and 32 according to
general procedure IV. 1H NMR (500 MHz, DMSO-d6) δ 11.82 (s,
1H), 8.29 (d, J ) 2.2 Hz, 1H), 8.03 (d, J ) 9.4 Hz, 1H), 7.63-7.59
(m, 1H), 7.52 (d, J ) 9.1 Hz, 2H), 7.20-7.17 (m, 2H), 7.00-6.96
(m, 4H), 6.93-6.84 (m, 4H), 6.70 (d, J ) 9.1 Hz, 2H), 3.99 (s,
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