Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo

J. Med. Chem. 2006, 49, 1165-1181

1165

Discovery and Structure-Activity Relationship of Antagonists of B-Cell Lymphoma 2 Family

Proteins with Chemopotentiation Activity in Vitro and in Vivo

Michael D. Wendt,*^,†Wang Shen,^†Aaron Kunzer,^†William J. McClellan,^†Milan Bruncko,^†Thorsten K. Oost,^†Hong Ding,^†

Mary K. Joseph,^†Haichao Zhang,^†Paul M. Nimmer,^†Shi-Chung Ng,^†Alexander R. Shoemaker,^†Andrew M. Petros,^†

Anatol Oleksijew,^†Kennan Marsh,^†Joy Bauch,^†Tilman Oltersdorf,^‡Barbara A. Belli,^‡Darlene Martineau,^‡Stephen W. Fesik,^†

Saul H. Rosenberg,^†and Steven W. Elmore^†

Cancer Research, Global Pharmaceutical R & D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, and

Idun Pharmaceuticals, 9380 Judicial DriVe, San Diego, California 92121

ReceiVed August 1, 2005

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X_Lfunction,

is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-

µM binding but were strongly deactivated in the presence of serum. The dominant component of serum

deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of

inhibitors bound to both Bcl-X_Land HSA domain III indicated two potential optimization sites for separation

of affinities. Modifications at both sites resulted in compounds with improved Bcl-X_Lbinding and greatly

increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X_Lwith a K_i

of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X_Loverexpression against cytokine

deprivation in FL5.12 cells with an EC₅₀of 0.47 µM. 73R showed little effect on the viability of the human

non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated

the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in

a mouse xenograft model.

Introduction

their fully elaborated pro-apoptotic counterparts Bax and Bak.¹³

In response to cellular stress, some BH3-only proteins (Bim,

Bid) directly activate Bax and Bak, a process which is inhibited

by antiapoptotic Bcl-2 family members.^13,14Other BH3-only

proteins, such as Bad, cannot directly activate Bax and Bak but

instead bind to antiapoptotic Bcl-2 family members, freeing the

BH3-only proteins that are capable of activating Bax and Bak.

Thus, members of this subgroup behave as sensitizers rather

than direct activators. A small molecule that preferentially

interacts with the BH3-binding groove of Bcl-2 or Bcl-X_Lwould

then be expected to mimic the activity of a Bad-like protein.

Such a compound would be capable of restoring the inherent

cellular potential for apoptotic response, thereby potentiating

the effects of existing therapies, improving sensitivity, and

overcoming resistance. Cellular data using various BH3-derived

peptides are in support of this model.^15,16

Programmed cell death, or apoptosis, is a highly regulated

process used to eliminate defective and unnecessary cells.¹

Disregulation of this process is strongly associated with cancer.²

Impaired apoptosis has been shown to be a key contributor to

various stages of neoplastic progression^3,4and also provides an

innate defense to cytotoxic chemotherapy.^5,6A group of

important players in the apoptotic process is the Bcl-2 (B-cell

lymphoma) family of proteins. Members of this family share

up to four Bcl-2 homology (BH) domains, and the family is

composed of both prosurvival (Bcl-2, Bcl-X_L, Bcl-w, Mcl-1,

A1) and proapoptotic members. Proapoptotic proteins are further

subdivided into two groups, the Bax subfamily (Bax, Bak, Bok)

and the larger group of BH3-only proteins (Bad, Bid, Bim, Bik,

Puma, Noxa, and others).⁷

The proapoptotic Bax and Bak are direct mediators of

apoptosis, respectively localized in the cytosol and mitochondria

under normal conditions. Following multiple death stimuli, both

Bax and Bak form aggregates within the mitochondrial outer

membrane, releasing cytochrome c and triggering the mito-

chondrial apoptosis pathway.⁸Antiapoptotic Bcl-2 family

proteins (e.g. Bcl-2 and Bcl-X_L) inhibit cytochrome c release

by blocking Bax/Bak activation.⁹The exact mechanism of action

of Bcl-2 and Bcl-X_Lhas not been unambiguously determined.

It is known they can form heterodimers with pro-apoptotic Bcl-2

family proteins and that the ratio of pro- to anti-apoptotic

proteins is associated with cell survival.^10,11Bcl-2 and Bcl-X_L

do act via sequestration of pro-apoptotic BH3-only proteins.¹²

The BH3-only proteins appear to perform different roles than

There is a wealth of evidence that overexpression of anti-

apoptotic Bcl-2 family proteins, especially Bcl-2 and Bcl-X_L,

are associated with tumor progression, poor prognosis, and drug

resistance. In particular, Bcl-X_Loverexpression shows a more

consistent correlation with intractability of cancer cell lines than

does Bcl-2. As an illustration of this, an informatics study on

the NCI 60 tumor cell line panel demonstrated that Bcl-X_L

expression exhibited a strong negative correlation with sensitiv-

ity to both 122 standard chemotherapeutic agents and a larger

set of 1200 cytotoxic agents.¹⁷This association was p53

independent, was not observed for Bcl-2 or Bax, and was more

significant than the correlation between cytotoxicity and p53

mutational status. Our goal was then to specifically target Bcl-

X_Lactivity as a strategy for developing a small molecule that

would act primarily as a potentiator in conjunction with standard

cancer chemotherapies. We recently disclosed the discovery of

ABT-737, which represents the culmination of efforts that began

from compound 1 (Figure 1a).¹⁸Here we show detailed SAR

of a portion of that work, which delineates our efforts to

* To whom correspondence should be addressed: Abbott Labora-

tories, Department R4N6, Bldg. AP10-3, 100 Abbott Park Road, Abbott

Park, IL 60064-6101. Tel: (847) 937-9305. Fax: (847) 935-5165. E-mail:

mike.d.wendt@abbott.com.

^†Abbott Laboratories.

^‡Idun Pharmaceuticals.

Published on Web 01/11/2006

1166 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

Figure 1. (a) Structure of 1 with substructure nomenclature. (b) Diagram of 1 bound to Bcl-X_L, showing bent-back conformation. Arrows indicate

locations of proposed structure modifications to 1. (c) Diagram of 2 bound in extended conformation to HSA-III, with arrows pointing to structure

modification sites.

Table 1. Binding of 1

(HSA) present in 1% human serum by itself showed a similar

µM^a

deactivation, while the quantity of R₁-acid glycoprotein in 1%

serum displayed little effect.²¹We further refined our search

by focusing on domain III of HSA (HSA-III). This domain

contains the primary binding site within albumin for medium-

and long-chain fatty acids and displays high affinity for small

anionic aromatic compounds; thus HSA-III was a likely

candidate to bind the aryl acylsulfonamide 1.²²In the event,

the 68-fold deactivation exhibited by the 1% serum equivalent

of HSA-III indicated its likely identity as the main driver of

serum deactivation of 1.

We were able to obtain an NMR-derived structure of 2, a

closely related dimethyl analogue of 1, bound to HSA-III.²⁰The

protein is populated by 2 at a single site within subdomain IIIA.

This subdomain was previously reported to be the primary

binding region for the biphenyl carboxylic acid diflunisal²³and

is also the site occupied by two myristic acid molecules in an

HSA cocrystal complex.²⁴As further confirmation of the

importance of this interaction, results of a direct, competitive

binding assay measuring affinity to this particular binding site

of HSA-III by displacement of a dansyl sarcosine probe provided

a K_dof <100 nM for 1.²⁵

assay

Bcl-2 K_i

Bcl-X_LK_i

Bcl-X_LK_i1% HS

Bcl-X_LK_i10% HS

0.433 ( 0.020*

0.036 ( 0.009*

2.50 ( 0.58*

1% inhib @ 10 µM

^aValues with standard error and asterisk if three or more experiments.

Table 2. Deactivation of 1 by Serum Components

added protein

none

IC₅₀(µM)^a

deactivation

0.093

1% human serum

>10*

>100

68

HSA from 1% HS^b

HSA-III from 1% HS^b

R₁-AG from 1% HS^b

>10

6.30

0.122 ( 0.006

1.3

^aValues with standard deviation if two experiments were performed;

with standard error and asterisk if three or more experiments. ^bSee

Experimental Section for concentrations.

uncouple Bcl-X_Land serum affinity, resulting in a phenylpip-

eridine analogue acting via Bcl-X_Linhibition and constituting

the first compound to show efficacy in vitro and in vivo.

We next attempted to apply structure-based design to de-

couple affinity for Bcl-X_Lfrom that of HSA-III. The differences

in the binding modes of 1 and 2 bound to Bcl-X_Land HSA-III,

respectively, suggested two ways in which albumin binding

might be reduced (Figure 1).²⁰First, in contrast to the bent-

back conformation of 1 bound to Bcl-X_L, 2 displays an extended

structure within domain III, with the Site 3 phenylthioethyl tail

buried by nonpolar residues. This suggested to us the possibility

of adding to the structure of 1 by building off of the ethylene

group. These substitutions should not be easily accommodated

by HSA-III, while Bcl-X_Lshould more readily tolerate such

groups, allowing them to extend into solvent. Moreover,

appended groups should optimally be polar, considering the

aqueous and hydrophobic environments to be encountered by

binding to Bcl-X_Land HSA-III, respectively. A recent chemo-

metric analysis of ligand binding to HSA-III found that various

amines, and to a lesser extent polar, uncharged groups such as

carbamates, amides, and sulfones, most effectively decreased

binding to HSA-III.²⁶A second opportunity for modifications

was located at the terminal biphenyl end of 1/2. The Bcl-X_L-

bound structure is presented with additional space at the fluoro

end of the biphenyl and is partially solvent-exposed, while the

fluorophenyl in HSA-III is more thoroughly surrounded by

nonpolar residues. We thus felt it might be possible to maintain

or improve the affinities of our compounds toward Bcl-X_Land

reduce affinities toward HSA-III by either increasing the polarity

of the Site 1 fragment or by extending the end of the fragment

by again appending a polar group. One caveat that should be

Rationale

Three-dimensional structures of complexes of Bcl-X_Lwith

Bax-¹⁹and Bad-derived²⁰BH3 peptides have been described.

The amphipathic R-helical BH3 domains bind to the long

hydrophobic groove formed by the combination of BH1, BH2

and BH3 domains of Bcl-X_L. Compound 1, generated from a

combination of NMR-based screening and directed parallel

synthesis, spans most of the length of the BH3-binding groove

of Bcl-X_Land interestingly exhibits a bent-back π-stacked

structure within the groove, with its phenylthio group tucked

under its nitroaryl ring. Data in Table 1 show 1 to bind to Bcl-

X_Lwith a K_iof 36 nM and with moderate activity against Bcl-

2. However, Bcl-X_LK_idetermination in the presence of 1%

human serum provided evidence of tight binding by one or more

serum components, resulting in a 69-fold deactivation, and

binding in the presence of 10% human serum was almost

completely abolished. The aim of the present study was to

increase Bcl-X_Laffinity in the presence of serum to provide a

compound which we could more thoroughly examine in vitro

and in vivo.

Given the extreme serum effect on binding, an immediate

goal was to determine the exact component or components in

serum that were responsible for the deactivation. We began by

determining Bcl-X_L/1 binding affinity in the presence of human

serum, and separate serum components known to bind small

molecules (Table 2). While 1% human serum deactivated 1 by

a factor of over 100, the quantity of human serum albumin

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1167

Chart 1. General Coupling Protocol

Scheme 1^a

Scheme 3^a

^aReagents and conditions: (a) 4-chloro-3-nitrobenzenesulfonamide,

EDCI, DMAP, CH₂Cl₂; (b) 2-aminoethanol, dioxane, 80 °C; (c) Bu₃P,

ADDP, 2,4-dimethylthiophenol, THF.

Scheme 2^a

^aReagents and conditions: (a) 3,3-dimethylglutaric anhydride, AcCl,

ClCH₂CH₂Cl; (b) 2-methoxyethyl ether, BF₃‚Et₂O, NaBH₄; (c) LiOH, THF,

H₂O.

2/3 sulfonamide unit 7 in near quantitative yield. The acid 9

was produced via S_NAr reaction on ethyl 4-fluorobenzoic acid

to give the ester 8, followed by LiOH-mediated hydrolysis. The

acid and sulfonamide were coupled using EDCI to give the

BOC-protected 10 in good yield. The free secondary amine was

deprotected with aqueous HCl to give 11, and 12 and 13 were

produced by reaction with the appropriate carbonyl chloride.

The EDCI coupling to form the acylsulfonamide was a key

step in the syntheses of our compounds and, with the exceptions

of the cases just discussed, constituted the final step of all

syntheses presented here (Chart 1). In rare cases a deprotection

step was incorporated into the workup of the coupling reaction

(see Experimental Section). Thus, synthetic plans were reduced

to targeting the appropriate acids and sulfonamides.

^aReagents and conditions: (a) chlorosulfonic acid, 80 °C, then NH₄OH,

-78 °C; (b) 2-(phenylsulfanyl)ethanamine, DMSO; (c) BOC-piperazine,

K₂CO₃, DMSO, 120 °C; (d) LiOH, THF; (e) EDCI, DMAP, CH₂Cl₂; (f) 4

M HCl, dioxane; (g) AcCl or Me₂NCOCl, pyridine, Et₃N.

The dimethylpiperidine-containing acid 16 was made in three

steps from ethyl 4-aminobenzoic acid (Scheme 3). Condensation

with 3,3-dimethylglutaric anhydride gave 14 in 95% yield. The

imide was reduced with NaBH₄and BF₃‚OEt₂to give the ester

15,²⁹which was saponified with LiOH to give 16.

applied to such an analysis is that HSA is notoriously elastic in

its binding conformations, and protein-protein binding sites in

general involve flexible topology,²⁷but the above analysis

nevertheless suggested most likely venues for structure modi-

fication.

Schemes 4 and 5 illustrate syntheses of compounds wherein

the fluoro group of 1 was replaced by polar tails of varying

lengths. The synthetic precursor 17, the triflate ester of vanillin,

is easily made and has been reported.³⁰Suzuki coupling of 17

to 4-carbomethoxybenzeneboronic acid cleanly gave 18. The

aldehyde was oxidized to the acid using the Sharpless protocol,³¹

and the subsequent acid chloride was reacted with dimethy-

lamine to give 19. The amide was reduced with borane to the

amine 21, and both 19 and 21 were saponified to the respective

acids 20 and 22. For the one-carbon homologous compounds,

18 was subjected to the two-step aldehyde-to-amide homolo-

gation described previously.³²The aldehyde was cleanly trans-

formed into the dibromoolefin 23, and hydrolysis with dime-

thylamine and water in DMF produced 24 in excellent yield.

Compounds 25-27 were then produced in a fashion analogous

to 19-21 above.

For the two-carbon homologated acids, 18 again served as

starting material (Scheme 5). Reaction with the commercially

available acetate-derived phosphorane gave 28. The resulting

olefin was hydrogenated with Wilkinson’s catalyst to give 29,

and the tert-butyl group was removed with TFA/Et₃SiH to

quantitatively provide the acid 30. The amidoester 31 and

aminoester 33 and corresponding acids 32 and 34 were then

Synthesis

Compound 2 was synthesized as part of an SAR study on

thiophenol substitution (Scheme 1). The commercially available

acid 3 was coupled to 4-chloro-3-nitrobenzenesulfonamide with

EDCI to give the acylsulfonamide 4. Aromatic substitution of

the activated chloride with aminoethanol cleanly gave 5, which

subsequently was subjected to modified Mitsunobu conditions

to give 2.²⁸

The new Site 1 compounds featuring replacement of the

fluorophenyl ring with a piperazine moiety, were synthesized

by a route allowing for late exchange of the terminal nitrogen

substituents (Scheme 2). Thus, 2-fluoronitrobenzene was chlo-

rosulfonylated, the deactivated ring requiring neat ClSO₃H at

80 °C, and the resulting sulfonyl chloride was carefully subjected

to ammonolysis to produce 6, with low temperature required

in order to not displace the activated fluoro group. Subsequent

desired displacement of the fluorine produced the complete Site

1168 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

Scheme 4^a

Scheme 5^a

^aReagents and conditions: (a) 4-(CO₂Me)PhB(OH)₂, PdCl₂(dppf), CsF,

dioxane, 90 °C; (b) NaIO₄, RuCl₃, CCl₄, CH₃CN, H₂O, then (COCl)₂,

Me₂NH, CH₂Cl₂; (c) BH₃, THF; (d) LiOH, THF, H₂O; (e) CBr₄, PPh₃,

CH₂Cl₂; (f) Me₂NH, DMF, H₂O, 80 °C.

^aReagents and conditions: (a) Ph₃PdCHCO₂tBu, THF; (b) RhCl(PPh₃)₃,

H₂, toluene, 60 °C; (c) TFA, Et₃SiH, 50 °C; (d) (COCl)₂, CH₂Cl₂, amine;

(e) BH₃, THF; (f) LiOH, THF, H₂O; (g) TBSOTf, 2,6-lutidine, CH₂Cl₂.

we chose to reduce 54 to the amine before deprotection and

reaction with 6, though these steps could likely have been

performed in the reverse order, as with the other series of

compounds.

Finally, the four-carbon tail synthesis (Scheme 8) began with

reduction of the acid functionality of Fmoc-D-Lys(BOC)-OH,

again using the chloroformate/NaBH₄method, giving 57 in

excellent yield. The PhS group 58 was installed using the

standard disulfide method, though here heating to 80 °C was

required.³⁴The BOC group was removed and the tertiary

dimethylamine 60 formed via reductive amination. The sul-

fonamides 59 and 61 were produced from 58 and 60 using the

one-pot Fmoc-deprotection/addition protocol.

synthesized as before. The corresponding morpholine-derived

compounds 35-38 were also produced similarly. Finally, 30

was employed in the synthesis of the hydroxy acid 41, via

borane reduction to 39, TBS-protection of the resulting alcohol,

and saponification.

Sulfonamides with a two-carbon side chain were synthesized

as shown in Scheme 6. We began with either antipode of Fmoc-

Asp(OtBu)-OH, with the D form leading to compounds with

the R configuration throughout, and the L form producing

S-sense compounds. Reduction of the acid side chains with

NaBH₄via the mixed anhydride gave 42R,S, typically in

excellent yield.³³This method is known to maintain chirality

of N-protected amino acids, and is also mild enough not to

remove the Fmoc group. Next, the modified Mitsunobu proce-

dure was again used to install the phenylthio group of 43R,S.

The Fmoc group was removed and the resulting amine displaced

the highly activated fluoro group of 6 in one pot at room

temperature, resulting in 44R,S. The tert-butyl ester was

removed quantitatively with aqueous HCl to give 45R,S, which

were transformed into the amides 46R,S and 48R,S using EDCI.

The amides were again selectively reduced with borane to

provide the amines 47R,S and 49R,S, typically in 70-80%

yield.

Three-carbon side chain compounds were synthesized as

shown in Scheme 7. The hydroxy group of N-BOC-L-serine

methyl ester was transformed into the thioether under neutral

conditions via conversion to the mesylate and immediate in situ

displacement by thiophenol. The resulting ester 50 was reduced

to the aldehyde 51 with DIBAL in moderate yield, with

separation of small amounts of starting ester and alcohol

required. 51 was homologated using the Wittig phosphorane to

give the olefin 52, which was quantitatively both hydrogenated

with Wilkinson’s catalyst, and saponified to give the saturated

acid 53. The dimethyl amide of 54 was again installed via EDCI

coupling, and the combination of BOC-deprotection and S_NAr

displacement on 6 was again employed to provide 55. For 56

Results and Discussion

Structure-Activity Relationships. Compounds were evalu-

ated in a fluorescence polarization assay (FPA) for their ability

to displace a Bad-derived peptide from Bcl-X_L, and a Bax-

derived peptide from Bcl-2.³⁵We tracked Bcl-2 binding, because

while we were primarily concerned with activity against Bcl-

X_L, the high structural homology of the BH3 binding grooves

of Bcl-2 and Bcl-X_Lsuggested that compounds would possess

significant Bcl-2 affinity.³⁶We note that the relative importance

of these related proteins in a clinical setting is not yet known.

More importantly, to track deactivation by HSA-III and other

serum components, compounds were also evaluated against Bcl-

X_Lin the presence of human serum. We initially used a

concentration of 1% serum for these assessments.

We began our work by assessing Site 1 modification as an

approach to decreasing serum deactivation. In the first part of

our two-pronged study, a series of polar tails of various lengths,

capped primarily by amides and amines, were appended to the

end of a methoxy-substituted Site 1 biphenyl (Table 3). The

methoxy group came out of a small investigation of ring-

substitution effects on binding of biphenyl-containing com-

pounds; the methoxy group increased potency by a small amount

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1169

Scheme 6^a

i

^aReagents and conditions: (a) BuOCOCl, THF, then NaBH₄, MeOH; (b) ADDP, Bu₃P, PhSH, THF; (c) 6, DIPEA, DMF; (d) 4 M HCl, dioxane; (e)

amine, EDCI, DMAP, DMF; (f) BH₃, THF.

Scheme 7^a

^aReagents and conditions: (a) MsCl, DIPEA, CH₂Cl₂, then PhSH; (b) DIBAL, CH₂Cl₂, -78 °C; (c) Ph₃PdCHCO₂tBu, THF; (d) RhCl(PPh₃)₃, H₂,

toluene, 50 °C, then LiOH, THF, H₂O; (e) Me₂NH, EDCI, DMAP, DMF; (f) 4 M HCl, dioxane, then 6, DIPEA, DMF; (g) BH₃, THF.

Scheme 8^a

in particular, fully protonated under physiological conditions,

were very effective in this regard, showing no dependence on

chain length and reducing deactivation in this assay from 69-

fold to from 1.5- to 7-fold, resulting in improved binding over

1 in the 1% HS assay for even 62 and 64. Nevertheless,

combined with intrinsic affinities, the three-carbon compounds,

and in particular the amines 66 and 68, appeared to stand out

among this group of compounds. Furthermore, in an FPA with

10% serum present, only 66 and 68 displayed Bcl-X_Lactivity

of less than 10 µM, with K_ivalues of 5.66 and 4.3 µM,

respectively. While the serum K_idata for 68 was clearly driven

less by a reduced serum effect than was the data for 66, the

superior intrinsic Bcl-X_Lbinding led us going forward to focus

primarily on the morpholino moiety of 68 from this group. It is

also of note that 68 and 70 appeared to have gained some

separation between Bcl-X_Land Bcl-2 affinity, driven solely by

gains toward Bcl-X_L.

The second aspect of our approach to Site 1 modification

emphasized an increase in polarity of the existing framework,

as opposed to an explicit lengthening of the Site 1 fragment.

Key compounds deriving from this approach are included in

Table 4. The piperazines 10, 12, and 13 appear similar to 62-

65 in Table 3, with short, polar appendages leading to significant

drops in Bcl-X_Laffinity. Compounds 11 and 71, presenting their

polar NH and O groups still closer to the center of the

hydrophobic BH3-binding groove, are unsurprisingly further

deactivated. While the relative serum deactivation of the polar

compounds improved as a group, only the relatively nonpolar

72 showed an affinity comparable to 1, an indication that any

significant increase in polarity in the vicinity of the fluorophenyl

^aReagents and conditions: (a) BuOCOCl, DME, then NaBH₄, H₂O;

(b) PhSSPh, Bu₃P, toluene, 80 °C; (c) 6, DIPEA, DMF; (d) TFA, CH₂Cl₂,

then CH₂O, NaBH₃CN, THF.

i

compared to parent (data not shown). A clear length dependence

on affinity is immediately evident. One- and two-carbon

compounds 62-65 proved severely detrimental to Bcl-X_L(and

Bcl-2) binding, while three-carbon compounds displayed Bcl-

X_Laffinity roughly equal to, or in the cases of 68 and 70, slightly

greater than that of 1. We surmise that the chain lengths of 62-

65 are too short to deliver the polar groups beyond the end of

the flexible hydrophobic BH3-binding site and into more

solvent-accessible space. Equally important is that all of the

amines and the three-carbon amides 67 and 69 produced a

suppression of deactivation by 1% serum. The dimethylamines

1170 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Table 3. Site 1 Biphenyl Tail SAR

Wendt et al.

Table 4. Site 1 Fluorophenyl Replacement SAR

^aValues with standard deviation if two experiments were performed;

with standard error and asterisk if three or more experiments.

^aValues with standard deviation if two experiments were performed;

with standard error and asterisk if three or more experiments.

ring-binding region would almost certainly have too detrimental

an effect on Bcl-X_Lbinding to be of interest. 72 also showed a

decrease in serum deactivation from 1 of from 69-fold to 34-

fold.

(Table 5). FL5.12 is an IL-3 dependent murine pro-B lymphoid

cell line characterized by low-moderate Bcl-X_Lexpression

levels. Transfection with human Bcl-X_Lproduces cells that

express roughly 10-fold higher protein levels as quantitated by

Western blot analysis, thus allowing cells to survive in the

absence of IL-3.

In general, then, the hypothesis that an increase in the polarity

of compounds at Site 1 would lead to decreased serum

deactivation was validated. However, affinity to Bcl-X_Lwas in

most cases also reduced by an unacceptable amount, underscor-

ing a need for either a minimal polarity increase to the Site 1

core or moving the source of polarity sufficiently near the end

of the peptide-binding region. On the basis of these results, we

narrowed our choices of modifications to Site 1 as we turned

to Site 3 modification, choosing to go forward with the Site 1

fragments from 68 and 72. We felt, based on the obtained

structural information,²⁰that the most straightforward point of

attachment for Site 3 augmentation with polar groups was at

the carbon atom adjacent to the aniline nitrogen. This neces-

sitated creation of a chiral center; however, routes to a variety

of compounds arising from a number of commercially available

D- and L-amino acid derivatives were readily apparent. Thus

we synthesized both enantiomers of selected inhibitors. We again

focused primarily on amides and amines, and also focused on

a two-carbon chain length because it appeared from the structure

that a two-carbon linker would provide a length sufficient to

situate the polar groups at the edge of the Bcl-X_Lsurface.

Furthermore, we felt that increasing the chain length of the Site

3 amines might well be neutral toward Bcl-X_Lbinding but would

be more likely to influence binding to HSA-III. Thus, we also

synthesized some compounds with chain lengths of three and

four carbon atoms.

Table 5 collects results from key compounds of both the 68-

and 72-derived series. It was immediately evident from the

amine pairs 73R,S, 77R,S, and 79R,S that R-chirality was

preferred. This result fit well with structural information, which

suggested that the carbon chains of the R-tails would be more

likely to maintain close contact with Bcl-X_L, while the S-tails

would immediately enter solvent. All of the R-chiral compounds

displayed extremely high, roughly 1 nM affinity to Bcl-X_L.

Clearly, the Site 3 modifications as a group markedly improved

Bcl-X_Laffinity. It is not clear from observation of the structure

of complexed inhibitors that the increased affinity comes from

a single source; rather, it seems likely that both the carbon chains

and the amines contribute to binding. With regard to serum

deactivation, the 68- and 72-derived groups showed similar

patterns. Both the pair of amides 78 and 84 and the pair of

morpholines 77R and 83 were much less effective than the rest

of the amines in reducing serum deactivation in the binding

assay; thus, it appears that at least at this site, a charged species

is particularly effective at reducing serum binding.

Interestingly, all of the dimethylamines as well as the primary

amines 76 and 82 showed similar levels of serum deactivation,

indicating that variation in chain length not only did little to

influence Bcl-X_Lbinding but also had little effect on serum

binding. However, cellular EC₅₀data separated compounds

further. The two-carbon dimethylamines 73R and 79R were

more active both under serum-free conditions and in the

presence of 3% FBS than their three- and four-carbon coun-

terparts; this chain-length variation is unexplained. The mor-

Anticipating some measure of additivity within this study

toward reducing serum binding, we evaluated serum deactivation

with 10% rather than 1% serum in our binding assay. Cellular

EC₅₀data were obtained using Bcl-X_L-transfected FL5.12 cells

in both the absence and presence of 3% fetal bovine serum

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1171

Table 5. Combination Substitution

^aValues with standard deviation if two experiments were performed; with standard error and asterisk if three or more experiments.

pholines and amides, unsurprisingly, were also less potent than

their comparators 73R and 79R in the presence of 3% FBS,

though both groups showed less deactivation in this cell line

than the binding data would have predicted. Ultimately, the

dimethylamines 73R and 79R emerged from this final group

of compounds. We verified that this pair of compounds indeed

derived much of their improved binding in the presence of serum

to greatly decreased affinity for HSA-III, with K_dvalues for

affinity to the key HSA-III binding site of 13.6 and 94 µM,

respectively. We also verified the dominant affinity of 73R

against Bcl-X_Lcompared to other Bcl-2 family members;

affinity to Bcl-w and Mcl-1 were determined to be 0.459 µM

and >10 µM, respectively. On the basis of ease of synthesis,

prospects for future modifications, and pharmacokinetic profile,

we chose to examine 73R in a further series of in vitro and in

vivo experiments designed to test its ability to cooperate with

standard chemotherapies in combating tumor growth.

Initially, treatment with UV-C radiation in 10% FBS was

chosen in order to eliminate the possibility that potentiation was

due to a change in cellular uptake of the apoptotic stimulus. As

illustrated in Figure 2a, 73R induced a leftward shift of the

UV-C dose-response curve, driving a two- to 3-fold dose-

dependent potentiation of UV-C induced cytotoxicity, as

measured by shifts in EC₅₀values. It was also observed that in

the absence of 73R, a plateau was reached in the ability of UV-C

irradiation to block cell growth; at a UV-C dose of 16 mJ/cm²,

the fraction of viable cells (35 ( 6%) was not significantly

different than at 32 mJ/cm²(29 ( 3%). In combination,

however, 73R also induces a concentration-dependent increase

in extent of cell kill at high UV-C doses, which was observed

at concentrations as low as 2.5 µM. In comparison, the

enantiomer 73S, employed as a mechanistic control compound,

shows no effect under the same conditions (Figure 2b).

The ability of 73R to enhance the effects of a chemothera-

peutic agent was evaluated next. Figure 2c shows the result of

an experiment run with paclitaxel under serum-free conditions.

As was observed with UV-C irradiation, 73R in a concentration-

dependent fashion decreased the fraction of viable cells at the

highest paclitaxel dose. Furthermore, the addition of 73R 48 h

after the initiation of paclitaxel treatment resulted in a 21-fold

potentiation of paclitaxel cytotoxicity. Again, the enantiomer,

73S, had no effect on cell viability in similar experiments (data

not shown).

Chemo- and Radiopotentiation in Human Tumor Cells.

Results from the experiments with IL-3 deprived FL5.12 cells,

combined with the currently accepted hypothesis for the role

of Bcl-X_Lin the apoptotic process, indicated that 73R would

be most effective in the presence of an apoptotic stimulus. We

used A549 cells to evaluate the effect of combination treatment.

A549 is a human nonsmall cell lung carcinoma line,³⁷whose

cells express large amounts of Bcl-X_L.^17,38Cells were treated

with serial dilutions of 73R plus serial dilutions of the

costimulus. At 48 h posttreatment, cell viability was measured

by the MTS assay and cell viability was normalized to untreated

cells. Treatment with 73R alone at up to 20 µM had little effect

on cell viability.

In Vivo Evaluation. We next evaluated 73R in an A549

xenograft tumor model. A549 tumors grow relatively slowly in

immunocompromised mice, requiring 35-40 days to reach 1

cm³on a Scid background. Xenograft studies from numerous

1172 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

Figure 3. Kaplan-Meier analysis of 73R plus paclitaxel in the A549

NSCLC model. Treatment with paclitaxel at 30 mg/kg/day (dark gray),

paclitaxel at 30 mg/kg/day plus 73R at 75 mg/kg/day (light gray) or

vehicle (black). The %ILS, measured as the median time to 1 cm³tumor

volume, was 95 for paclitaxel and for the 73R/paclitaxel combination

was 182. See Experimental Section for more details.

Figure 4. Paclitaxel plasma concentrations after ip dosing (15 mg/

kg) alone or in combination with 73R (100 mg/kg) in Scid mouse.

Figure 2. (a) Effect of 73R in combination with 16 mJ/cm²UV-C

radiation on A549 human NSCLC cells in the presence of 10% FBS,

showing potentiation of radiation. (b) Corresponding graph of 73S in

combination with UV-C radiation, showing no effect potentiation with

73S. (c) Effect of 73R in combination with paclitaxel on A549 cells.

Experiment run under serum-free conditions. Cells were treated with

paclitaxel for 96 h and with 73R from the 48 to 96 h time points.

Viability was compared to untreated samples.

the effect of 73R monotherapy was always statistically the same

as vehicle.

To demonstrate that the effects of combination therapy were

due to the interaction of 73R with Bcl-X_Land not due to

enhancement of paclitaxel exposure, we assessed potential

pharmacokinetic interactions between the two agents. Studies

involving coadministration of 73R and paclitaxel in Scid mice

demonstrated that neither the C_maxnor the AUC of paclitaxel

was significantly altered by the presence of 73R (Figure 4).

laboratories have demonstrated that this tumor line is resistant

to most commonly used cytotoxic agents; however, administra-

tion of paclitaxel at the maximum tolerated dose (MTD) can

effect a reduction of tumor growth rate of 60-70%.³⁹In an

established, staged tumor model experiment, 73R enhanced the

antitumor activity of paclitaxel with no overt evidence of

increased toxicity (Figure 3). A549 cells were inoculated

subcutaneously and allowed to grow to approximately 240 mm³

(day 15), at which point mice were assigned to treatment groups

and therapy was initiated. Paclitaxel was given at its MTD of

30 mg/kg/day on days 1, 5, and 9 with and without cotreatment

with 73R at 75 mg/kg/day for 21 days (see Experimental Section

for details). Treatment with the MTD of paclitaxel alone resulted

in tumor growth inhibition for two weeks after treatment. In

contrast, the combination of paclitaxel plus 73R caused regres-

sion of established A549 tumors during the treatment period,

resulting in 75% tumor growth inhibition and significantly

enhanced tumor growth delay with a time to 1 cm³tumor

volume of 182%. We also note that although treatment with

73R alone was not included in this particular experiment, we

analyzed its effect in numerous studies similar to this one, and

Conclusions

Rationally designed modifications to an inhibitor of Bcl-X_L

function have been described that directly addressed deleterious

binding to serum components, specifically to domain III of HSA.

Polarity-driven modification of the core structure at Site 1

maintained affinity to Bcl-X_Land modestly decreased serum

deactivation, while combination with modifications to Site 3

produced affinity improvements of at least an order of magni-

tude, resulting in compounds 73R and 79R, with roughly 1 nM

affinity for Bcl-X_L. These compounds exhibited 67- and 230-

fold lower affinity for Bcl-2, respectively, and 73R was also

shown to have much lower affinity for Bcl-w and Mcl-1.

Compounds also displayed cellular efficacy in cells stressed with

an apoptotic stimulus. Furthermore, deactivation from serum

binding was greatly reduced; in particular, the targeted HSA-

III affinity was reduced by over 2 orders of magnitude. 73R

demonstrated the ability to potentiate the activity of UV radiation

in vitro and paclitaxel in both in vitro and in vivo models of

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1173

The solid was filtered, rinsed with 1 M HCl (1L) and water (2 L)

and dried at 50 °C to provide 162.4 g (74%) of 6. H NMR (400

MHz, DMSO-d₆) δ 8.52 (dd, J ) 7.0, 2.5 Hz, 2H), 8.19 (ddd, J )

8.8, 4.1, 2.4 Hz, 2H), 7.78 (dd, J ) 11.1, 8.7 Hz, 1H), 7.70 (br s,

2H). MS (ESI) m/z 219 (M - H)^-.

human tumor growth, thus verifying the potential utility of a

small-molecule BH3-mimetic as an anticancer agent.

1

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

3-Nitro-4-(2-phenylsulfanylethylamino)benzenesulfonamide

(7). A solution of 6 (314 mg, 1.33 mmol), 2-(phenylsulfanyl)-

ethanamine (204 mg, 1.33 mmol), and DIPEA (0.5 mL) in DMSO

(5 mL) was stirred for 16 h, diluted with EtOAc (100 mL), washed

sequentially with 3 M HCl, water, and brine, dried (Na₂SO₄),

filtered, and concentrated. The residue was chromatographed on

silica gel with 25% EtOAc/hexane to give 450 mg (97%) of 7. ¹H

NMR (300 MHz, CDCl₃) δ 8.74 (d, J ) 2.4 Hz, 1H), 8.61 (br s,

1H), 7.83 (dd, J ) 8.5, 2.4 Hz, 1H), 7.41 (m, 2H), 7.30 (m, 3H),

6.81 (d, J ) 9.2 Hz, 1H), 4.76 (br s, 2H), 3.58 (dt, J ) 6.6, 5.6 Hz,

1H), 3.22 (t, J ) 6.6 Hz, 1H). MS (DCI) m/z 354 (M + H)⁺.

4-(4-Ethoxycarbonylphenyl)piperazine-1-carboxylic Acid tert-

Butyl Ester (8). A suspension of 4-fluorobenzoic acid ethyl ester

(16.8 g, 100 mmol), piperazine-1-carboxylic acid tert-butyl ester

(18.6 g, 100 mmol) and K₂CO₃(20.7 g, 150 mmol) in DMSO (100

mL) was stirred at 120 °C for 10 h. The reaction mixture was cooled

to room temperature and poured into water (1 L). The solid

precipitate was filtered, washed with water and dried in a vacuum

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 ammonia, ESI indicates

electron spray ionization, APCI indicates atmospheric pressure

chemical ionization with ammonia. Elemental analyses were

performed by Quantitative Technologies, Inc., Whitehouse, NJ.

Column chromatography 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 Waters Symmetry

C8 column (25 × 100 mm, 7 µm particle size) using a gradient of

10-100% CH₃CN:0.1% TFA over 8 min (10 min run time) at a

flow rate of 40 mL/min.

1

oven at 40 °C for 24 h to provide 13.38 g (40%) of 8. H NMR

(300 MHz, DMSO-d₆) δ 7.79 (d, J ) 8.8 Hz, 2H), 6.98 (d, J ) 9.2

Hz, 2H), 4.24 (q, J ) 7.1 Hz, 2H), 3.45 (m, 4H), 3.29 (m, 4H),

1.42 (s, 9H), 1.29 (t, J ) 7.1 Hz, 3H). MS (ESI) m/z 335 (M +

H)⁺.

4-Chloro-N-(4′-fluorobiphenyl-4-carbonyl)-3-nitrobenzene-

sulfonamide (4). A solution of 3 (4.54 g, 21.0 mmol), 4-chloro-

3-nitrobenzenesulfonamide (4.74 g, 20.0 mmol), EDCI (4.80 g, 25.0

mmol), and DMAP (1.23 mg, 10.0 mmol) in CH₂Cl₂(60 mL) was

stirred for 16 h, diluted with EtOAc (200 mL), washed sequentially

with 1 M HCl (50 mL), water (50 mL), and brine (20 mL), dried

(MgSO₄), filtered, and concentrated. The concentrate was flash

chromatographed on silica gel with 50% EtOAc/hexanes to provide

4-(4-Carboxyphenyl)piperazine-1-carboxylic Acid tert-Butyl

Ester (9). 9 was prepared from 8 using the procedure for the

preparation of 27. ¹H NMR (300 MHz, DMSO- d₆) δ 12.29 (br s,

1H), 7.78 (d, J ) 9.2 Hz, 2H), 6.98 (d, J ) 9.2 Hz, 2H), 3.43 (m,

4H), 3.29 (m, 4H), 1.42 (m, 9H). MS (ESI) m/z 305 (M - H)^-.

General Coupling Procedure. A suspension of acid (1 equiv),

sulfonamide (1 equiv), EDCI (2-4 equiv) and DMAP (0.5-1

equiv) in CH₂Cl₂(20 mL/mmol substrate) was stirred for 24 h.

The reaction mixture was diluted with 5-10 volumes CH₂Cl₂and

washed with water. The organic phase was separated, dried

(MgSO₄), filtered, and condensed. The crude reaction mixture was

purified either by HPLC or by silica gel chromatography.

4-{4-[3-Nitro-4-(2-phenylsulfanylethylamino)benzenesulfonyl-

aminocarbonyl]phenyl}piperazine-1-carboxylic Acid tert-Butyl

Ester (10). A suspension of 9 (246 mg, 0.8 mmol), 7 (284 mg, 0.8

mmol), EDCI (308 mg, 1.6 mmol) and DMAP (98 mg, 0.8 mmol)

in CH₂Cl₂(10 mL) was stirred overnight. The reaction mixture was

diluted with CH₂Cl₂(20 mL) and washed with water (10 mL). The

organic phase was separated, dried (MgSO₄), filtered, and con-

densed. The crude reaction mixture was chromatographed on silica

gel using 5% MeOH/CH₂Cl₂to yield 333 mg (65%) of 10. 1H

NMR (300 MHz, DMSO-d₆) δ 8.77 (t, 1H), 8.60 (d, 1H), 7.91

(dd, 1H), 7.76 (d, 2H), 7.36 (d, 2H), 7.14-7.29 (m, 4H), 6.94 (d,

2H), 3.66 (q, 2H), 3.37-3.45 (m, 4H), 3.23-3.30 (m, 6H), 1.41

(s, 9H). MS (ESI) m/z 640 (M - H)^-. Anal. (C₃₀H₃₅N₅O₇S₂) C, H,

N.

1

6.5 g (75%) of 4. H NMR (300 MHz, DMSO-d₆) δ 8.62 (d, J )

2.2 Hz, 1H), 8.25 (dd, J ) 8.8, 2.2 Hz, 1H), 8.05 (d, J ) 8.4 Hz,

1H), 7.98 (d, J ) 8.5 Hz, 2H), 7.79 (m, 4H), 7.33 (dd, J ) 8.9, 8.8

Hz, 2H). MS (ESI) m/z 433 (M - H)^-.

N-(4′-Fluorobiphenyl-4-carbonyl)-4-(2-hydroxyethylamino)-

3-nitrobenzenesulfonamide (5). A solution of 4 (2.5 g, 5.75 mmol)

and 2-aminoethanol (10 mL) in dioxane (10 mL) was stirred at 80

°C for 20 min, taken up in 1 M HCl (100 mL), extracted with

EtOAc (3 × 100 mL), washed with 2 × 1 M HCl, water and brine,

dried (Na₂SO₄), filtered and concentrated to provide a yellow oil

which was flash chromatographed on silica gel eluting with 5%

MeOH/EtOAc to provide 2.57 g (97%) of 5. 1H NMR (300 MHz,

DMSO-d₆) δ 8.72 (t, J ) 6 Hz, 1H), 8.67 (d, J ) 2 Hz, 1H), 7.95

(m, 3H), 7.80 (m, 4H), 7.31 (m, 3H), 5.00 (br m, 1H), 3.65 (t, J )

6 Hz, 2H), 3.51 (dt, J ) 6, 6 Hz, 2H). MS (ESI) m/z 458 (M -

H)^-.

4-[2-(2,4-Dimethylphenylsulfanyl)ethylamino]-N-(4′-fluorobi-

phenyl-4-carbonyl)-3-nitrobenzenesulfonamide (2). A 0 °C solu-

tion of Bu₃P (155 µL, 0.62 mmol) and 1,1′-(azodicarbonyl)-

dipiperidine (157 mg, 062 mmol) in THF (4 mL) was treated with

5 (149 mg, 0.32 mmol) and 2,4-dimethylthiophenol (50 µL, 0.37

mmol), stirred for 48 h, and concentrated. The concentrate was flash

chromatographed on silica gel with 70% EtOAc/hexanes to provide

3-Nitro-4-(2-phenylsulfanylethylamino)-N-(4-piperazin-1-yl-

benzoyl)benzenesulfonamide (11). A solution of 10 (4.49 g, 7.0

mmol) in CH₂Cl₂(20 mL) and dioxane (20 mL) was treated with

4 M HCl in dioxane (50 mL) for 3 h. The reaction mixture was

condensed and purified by reverse phase chromatography (Sep-

pak C18, 0-40% MeCN/water/0.1% HCl) to give 3.31 g (95%) of

1

78 mg (41%) of 2. H NMR (300 MHz, DMSO-d₆) δ 8.51 (m,

2H), 7.95 (br d, J ) 8 Hz, 2H), 7.87 (dd, J ) 2, 9 Hz, 1H), 7.73

(dd, J ) 6, 9 Hz, 2H), 7.60 (br d, J ) 8 Hz, 2H), 7.29 (m, 3H),

6.98 (m, 3H), 3.54 (dt, J ) 7, 7 Hz, 2H), 3.18 (t, J ) 7 Hz, 2H),

2.27 (s, 3H), 2.22 (s, 3H). MS (ESI) m/z 578 (M - H)^-.

1

11. H NMR (300 MHz, DMSO-d₆) δ 8.50 (t, J ) 5.8 Hz, 1H),

8.47 (d, J ) 2.0 Hz, 1H), 7.86 (dd, J ) 8.8, 2.0 Hz, 1H), 7.77 (d,

J ) 8.8 Hz, 2H), 7.40 (m, 2H), 7.31 (m, 2H), 7.20 (m, 1H), 6.96

(d, J ) 9.2, 1H), 6.88 (d, J ) 8.8 Hz, 2H), 3.59 (q, J ) 6.3 Hz,

2H), 3.38 (m, 4H), 3.26 (m, 2H), 3.20 (m, 4H). MS (ESI) m/z 540

(M - H)^-. Anal. (C₂₅H₂₇N₅O₅S₂‚HCl‚1.5H₂O) C, H, N.

4-Fluoro-3-nitrobenzenesulfonamide (6). A mixture of 2-fluo-

ronitrobenzene (141.2 g, 1.0 mol) and chlorosulfonic acid (300 mL)

was heated to 95 °C for 18 h, cooled to room temperature, and

slowly added over 1 h to a mixture of 2-propanol (3.2 L) and

concentrated NH₄OH (800 mL) maintained between -35 and -20

°C. The mixture was stirred an additional 30 min at -35 °C and

concentrated HCl was added until the solution was acidic. The

resulting slurry was partially concentrated in vacuo, water was

added, and the process was repeated to give an aqueous slurry (3L).

N-[4-(4-Acetylpiperazin-1-yl)benzoyl]-3-nitro-4-(2-phenylsulf-

anylethylamino)benzenesulfonamide (12). To a solution of 11

(54.1 mg, 0.1 mmol) in pyridine (2 mL) and Et₃N (1 mL) was

added acetyl chloride (14.3 µL, 0.2 mmol), and the reaction mixture

was stirred for 24 h. The reaction mixture was condensed, and the

1174 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

product was purified by HPLC. ¹H NMR (300 MHz, DMSO-d₆) δ

12.04 (s, 1 H), 8.77 (t, J ) 5.4 Hz, 1H), 8.60 (d, J ) 2.0 Hz, 1H),

7.91 (dd, J ) 9.2, 2.1 Hz, 1H), 7.76 (d, J ) 9.2 Hz, 2H), 7.36 (m,

2H), 7.14-7.28 (m, 4H), 6.95 (d, J ) 9.2 Hz, 2H), 3.67 (q, J )

6.5 Hz, 2H), 3.55 (m, 4H), 3.38 (m, 2H), 3.27 (m, 4H), 2.03 (s,

3H). MS (ESI) m/z 582 (M - H)^-. Anal. (C₂₇H₂₉N₅O₆S₂‚0.5H₂O)

C, H, N.

for 1 h and condensed. The crude acid chloride was taken up in

THF (100 mL) and 2 M Me₂NH in THF (7 mL) was added. The

reaction was stirred for 30 min and then poured into 1 M HCl (100

mL). The mixture was extracted with ether (2 × 100 mL), and the

combined extracts were rinsed with saturated Na₂CO₃and brine,

dried (Na₂SO₄), filtered, and concentrated. The concentrate was

flash chromatographed on silica gel with 30% EtOAc/hexanes to

1

provide 626 mg (49%) of 19. H NMR (300 MHz, DMSO-d₆) δ

4-{4-[3-Nitro-4-(2-phenylsulfanylethylamino)benzenesulfonyl-

aminocarbonyl]phenyl}piperazine-1-carboxylic Acid Dimeth-

ylamide (13). 13 was prepared from 11 and dimethylcarbamoyl

8.01 (d, J ) 8.5 Hz, 2H), 7.66 (d, J ) 8.5 Hz, 2H), 7.40 (d, J )

7.5 Hz, 1H), 7.14 (d, J ) 2 Hz, 1H), 7.07 (dd, J ) 7.5, 2 Hz, 1H),

3.88 (s, 3H), 3.81 (s, 3H), 3.01 (s, 3H), 2.98 (s, 3H). MS (ESI) m/z

314 (M + H)⁺.

1

chloride using the procedure for the preparation of 12. H NMR

(300 MHz, DMSO-d₆) δ 8.64 (m, 1H), 8.54 (d, J ) 2.4 Hz, 1H),

7.88 (dd, J ) 9.2, 2.0 Hz, 1H), 7.75 (d, J ) 8.8 Hz, 2H), 7.38 (m,

2H), 7.29 (m, 2H), 7.18 (m, 1H), 7.08 (d, J ) 9.2 Hz, 1H), 6.89

(d, J ) 9.2 Hz, 2H), 3.63 (m, 2H), 3.18-3.34 (m, 10H), 2.77 (s,

6H). MS (ESI) m/z 611 (M - H)^-. Anal. (C₂₈H₃₂N₆O₆S₂‚0.5H₂O)

C, H, N.

4′-Dimethylaminocarbamoyl-2′-methoxybiphenyl-4-carbox-

ylic Acid (20). A solution of 19 (622 mg, 2.0 mmol), LiOH^.H₂O

(336 mg, 8.0 mmol), THF (50 mL), water (15 mL), and MeOH

(15 mL) was stirred for 24 h. The mixture was poured into 1 M

HCl (100 mL), and the resulting mixture extracted with EtOAc (3

× 100 mL). The extracts were washed with brine, dried (Na₂SO₄),

filtered, and concentrated to provide 585 mg (98%) of 20. ¹H NMR

(300 MHz, DMSO-d₆) δ 12.62 (br s, 1H), 7.99 (d, J ) 8.8 Hz,

2H), 7.63 (d, J ) 8.8 Hz, 2H), 7.39 (d, J ) 8.1 Hz, 1H), 7.14 (d,

J ) 1.4 Hz, 1H), 7.07 (dd, J ) 8.1, 1.4 Hz, 1H), 3.81 (s, 3H), 3.00

(s, 3H), 2.98 (s, 3H). MS (ESI) m/z 300 (M + H)⁺.

Ethyl 4-(4,4-Dimethyl-2,6-dioxopiperidin-1-yl)benzoate (14).

A solution of ethyl-4-aminobenzoate (2.2 g, 13.1 mmol), 3,3-

dimethylglutaric anhydride (2.0 g, 13.1 mmol), and 1,2-dichloro-

ethane (33 mL) was refluxed for 4 h. After cooling to room

temperature, AcCl (1.9 mL, 27 mmol) was added dropwise, and

the reaction was refluxed for 1 h and then cooled to room

temperature. The solution was diluted with CH₂Cl₂(150 mL),

washed with water, saturated aqueous NaHCO₃, and brine, dried

4′-Dimethylaminomethyl-2′-methoxybiphenyl-4-carboxylic Acid

Methyl Ester (21). 21 was prepared from 20 using the procedure

1

(MgSO₄), and condensed to afford 3.64 g (95%) of 14. H NMR

1

for the preparation of 33. H NMR (300 MHz, CDCl₃) δ 8.06 (d,

(300 MHz, CDCl₃) δ 8.14 (d, J ) 8.5 Hz, 2H), 7.16 (d, J ) 8.8

Hz, 2H), 4.39 (q, J ) 7.1 Hz, 2H), 2.69 (s, 4H), 1.39 (t, J ) 7.1

Hz, 3H), 1.22 (s, 6H). MS (DCI) m/z 290 (M + H)⁺.

J ) 8.5 Hz, 2H), 7.60 (d, J ) 8.5 Hz, 2H), 7.28 (d, J ) 7.5 Hz,

1H), 7.01 (s, 1H), 6.96 (d, J ) 7.5 Hz, 1H), 3.93 (s, 3H), 3.84 (s,

3H), 3.51 (s, 2H), 2.33 (s, 6H). MS (ESI) m/z 300 (M + H)⁺.

Ethyl 4-(4,4-Dimethylpiperidin-1-yl)benzoate (15). 15 was

synthesized according a literature procedure.²⁹A solution of 14

(900 mg, 3.06 mmol) and 2-methoxyethyl ether (10 mL) was stirred

at 0 °C as BF₃‚Et₂O (0.88 mL, 6.8 mmol) was added dropwise. A

suspension of NaBH₄(248 mg, 6.48 mmol) in 2-methoxyethyl ether

(8.3 mL) was then added slowly. The reaction was stirred for 15

min at 0 °C and for 4 h at room temperature. After cooling to 0

°C, the reaction was quenched by the cautious addition of ice,

followed by excess water, and the mixture was stirred at room

temperature for 1 h. The solid was filtered, washed with water,

and dried to afford 0.61 g (76%) of 15. ¹H NMR (300 MHz, CDCl₃)

δ 7.90 (d, J ) 9 Hz, 2H), 6.86 (d, J ) 9 Hz, 2H), 4.32 (q, J ) 7.1

Hz, 2H), 3.32 (t, J ) 6.1 Hz, 4H), 1.49 (t, J ) 6.1 Hz, 4H), 1.36

(t, J ) 7.1 Hz, 3H), 0.99 (s, 6H). MS (DCI) m/z 262 (M + H)⁺.

4-(4,4-Dimethylpiperidin-1-yl)benzoic Acid (16). A solution

of 15 (9.5 g, 36.3 mmol), LiOH^.H₂O (1.52 g, 36.3 mmol), THF

(500 mL), water (125 mL), and MeOH (125 mL) was stirred for

18 h. Solvent was evaporated, and 1 M HCl was added. The

resultant solid was filtered, washed with water, and dried to afford

8.1 g (96%) of 16. ¹H NMR (300 MHz, DMSO-d₆) δ 7.74 (d, J )

8.8 Hz, 2H), 6.93 (d, J ) 9.1 Hz, 2H), 3.32 (m, 4H), 1.40 (t, J )

5.8 Hz, 4H), 0.95 (s, 6H). MS(DCI) m/z 234 (M + H)⁺.

4′-Formyl-2′-methoxybiphenyl-4-carboxylic Acid Methyl Es-

ter (18). A mixture of 3-methoxy-4-trifluoromethanesulfonyloxy-

benzaldehyde (36.0 g, 127 mmol), 4-methoxycarbonylphenylboronic

acid (27.4 g, 152 mmol), PdCl₂(dppf)‚CH₂Cl₂(2.93 g, 4.0 mmol),

and CsF (39.5 g, 260 mmol) in dioxane (400 mL) was heated to

70 °C, stirred for 16 h, cooled, filtered through a pad of silica gel,

and rinsed with ether (250 mL) and concentrated. The concentrate

was triturated with EtOAc/hexanes to provide 30.0 g (87%) of 18.

¹H NMR (300 MHz, DMSO-d₆) δ 10.06 (s, 1H), 8.02 (d, J ) 8

Hz, 2H), 7.70 (d, J ) 8 Hz, 2H), 7.63 (m, 3H), 3.90 (s, 3H), 3.89

(s, 3H). MS (DCI) m/z 271 (M + H)⁺.

4′-Dimethylaminomethyl-2′-methoxybiphenyl-4-carboxylic Acid

(22). 22 was prepared from 21 using the procedure for the

preparation of 27. ¹H NMR (300 MHz, DMSO-d₆) δ 7.98 (d, J )

8.5 Hz, 2H), 7.62 (d, J ) 8.5 Hz, 2H), 7.39 (d, J ) 7.5 Hz, 1H),

7.37 (s, 1H), 7.15 (d, J ) 7.5 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 2H),

2.59 (s, 6H). MS (ESI) m/z 286 (M + H)⁺.

4′-(2,2-Dibromovinyl)-2′-methoxybiphenyl-4-carboxylic Acid

Methyl Ester (23). A solution of 18 (1.35 g, 5.0 mmol) in CH₂Cl₂

(30 mL) was treated with CBr₄(1.82 g, 5.5 mmol) and PPh₃(2.88

g, 11 mmol), stirred for 1 h, treated with hexanes (50 mL), and

filtered through silica gel (50 g). The solution was rinsed with 1:1

water/CH₂Cl₂(100 mL), the layers were separated, and the organic

phase was concentrated. The concentrate was flash chromatographed

on silica gel with 2-10% EtOAc/hexanes to provide 2.07 g (97%)

1

of 23. H NMR (300 MHz, CDCl₃) δ 8.08 (d, J ) 8.5 Hz, 2H),

7.62 (d, J ) 8.5 Hz, 2H), 7.52 (s, 1H), 7.34 (d, J ) 8 Hz, 1H),

7.22 (d, J ) 8 Hz, 1H), 3.93 (s, 3H), 3.83 (s, 3H). MS (DCI) m/z

427 (M + H)⁺.

4′-Dimethylcarbamoylmethyl-2′-methoxybiphenyl-4-carbox-

ylic Acid Methyl Ester (24). A mixture of 23 (213 mg, 0.5 mmol)

and 2 M Me₂NH in THF (1 mL), DMF (1.5 mL), and water (0.25

mL) was heated to 80 °C for 8 h, diluted with EtOAc (100 mL),

washed with water (45 mL) and brine (10 mL), dried (MgSO₄),

filtered, and concentrated. The concentrate was flash chromato-

graphed on silica gel with 2-10% MeOH/CH₂Cl₂to provide 140

1

mg (86%) of 24. H NMR (300 MHz, CDCl₃) δ 8.05 (d, J ) 6.5

Hz, 2H), 7.59 (d, J ) 6.5 Hz, 2H), 7.27 (d, J ) 7.5 Hz, 1H), 6.93

(s, 1H), 6.91 (d, J ) 7.5 Hz, 1H), 3.93 (s, 3H), 3.81 (s, 3H), 3.76

(s, 2H), 3.06 (s, 3H), 3.00 (s, 3H). MS (ESI) m/z 328 (M + H)⁺.

4′-Dimethylcarbamoylmethyl-2′-methoxybiphenyl-4-carbox-

ylic Acid (25). 25 was prepared from 24 using the procedure for

the preparation of 20. ¹H NMR (300 MHz, DMSO-d₆) δ 12.87 (br

s, 1H), 7.96 (d, J ) 7.5 Hz, 2H), 7.59 (d, J ) 7.5 Hz, 2H), 7.27 (d,

J ) 7.5 Hz, 1H), 7.01 (d, J ) 1 Hz, 1H), 6.91 (dd, J ) 7.5, 1 Hz,

1H), 3.76 (s, 3H), 3.74 (s, 2H), 3.04 (s, 3H), 2.85 (s, 3H). MS

(ESI) m/z 314 (M + H)⁺.

4′-(2-Dimethylaminoethyl)-2′-methoxybiphenyl-4-carbox-

ylic Acid Methyl Ester (26). 26 was prepared from 24 using the

procedure for the preparation of 33. ¹H NMR (300 MHz, DMSO-

d₆) δ 7.97 (d, J ) 8.4 Hz, 2H), 7.62 (d, J ) 8.4 Hz, 2H), 7.24 (d,

J ) 8.0 Hz, 1H), 7.02 (s, 1H), 6.92 (d, J ) 8.0 Hz, 1H), 3.87 (s,

4′-Dimethylcarbamoyl-2′-methoxybiphenyl-4-carboxylic Acid

Methyl Ester (19). A mixture of 18 (1.10 g, 4.07 mmol), NaIO₄

(1.75 g, 8.15 mmol), and RuCl₃(20 mg) in CCl₄(10 mL), CH₃CN

(10 mL), and water (15 mL) was stirred for 1 h. The mixture was

poured into water (50 mL) and extracted with CH₂Cl₂(3 × 70

mL), and the extracts were washed with brine, dried (Na₂SO₄),

filtered, and concentrated. The crude acid (600 mg, 2.1 mmol) was

taken up in CH₂Cl₂(30 mL), and to the solution was added (COCl)₂

(220 µL, 2.5 mmol) and a drop of DMF. The reaction was stirred

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1175

3H), 3.78 (s, 3H), 2.76 (t, J ) 8.2 Hz, 2H), 2.50 (t, J ) 8.2 Hz,

2H), 2.22 (s, 6H). MS (ESI) m/z 314 (M + H)⁺.

mixture was poured into 6 M HCl (200 mL) and stirred for 2 h.

The solution was adjusted to pH >10 with solid KOH and water,

and the resulting solution was extracted with EtOAc (2 × 400 mL),

washed with water and brine, dried (Na₂SO₄), filtered, and

concentrated to provide a yellow oil which was flash chromato-

graphed on silica gel eluting with 50% EtOAc/hexanes to provide

4′-(2-Dimethylaminoethyl)-2′-methoxybiphenyl-4-carbox-

ylic Acid (27). A solution of 26 (105 mg, 0.335 mmol), LiOH^.H₂O

(564 mg, 1.35 mmol), THF (20 mL), water (7 mL), and MeOH (7

mL) was stirred for 24 h. 1 M HCl (2 mL) was added, the mixture

was poured into saturated NaH₂PO₄(50 mL), and the resulting

mixture was extracted with EtOAc (3 × 50 mL). The extracts were

washed with brine, dried (Na₂SO₄), filtered, and concentrated to

1

3.90 g (82%) of 33. H NMR (300 MHz, DMSO-d₆) δ 7.97 (d, J

) 8 Hz, 2H), 7.62 (d, J ) 8 Hz, 2H), 7.25 (d, J ) 7.5 Hz, 1H),

6.98 (s, 1H), 6.90 (d, J ) 7.5 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 3H),

2.63 (t, J ) 7.8 Hz, 2H), 2.25 (t, J ) 7.3 Hz, 2H), 2.15 (s, 6H),

1.74 (tt, J ) 7.5, 7.3 Hz, 2H). MS (ESI) m/z 328 (M + H)⁺.

4′-(3-Dimethylaminopropyl)-2′-methoxybiphenyl-4-carbox-

ylic Acid Methyl Ester. 34 was prepared from 33 using the

procedure for the preparation of 27. ¹H NMR (300 MHz, DMSO-

d₆) δ 7.96 (d, J ) 8.5 Hz, 2H), 7.59 (d, J ) 8.5 Hz, 2H), 7.27 (d,

J ) 8 Hz, 1H), 7.02 (s, 1H), 6.93 (d, J ) 7.5 Hz, 1H), 3.79 (s,

3H), 2.88 (m, 1H), 2.75 (m, 1H), 2.62 (dt, J ) 8, 2 Hz, 2H), 2.56

(s, 6H), 2.01 (m, 2H). MS (ESI) m/z 314 (M + H)⁺.

1

provide 96 mg (96%) of 27. H NMR (300 MHz, DMSO-d₆) δ

7.94 (d, J ) 8.5 Hz, 2H), 7.56 (d, J ) 8.5 Hz, 2H), 7.23 (d, J )

8 Hz, 1H), 7.01 (d, J ) 1 Hz, 1H), 6.93 (dd, J ) 8, 1 Hz, 1H),

3.77 (s, 3H), 2.76 (t, J ) 8 Hz, 2H), 2.53 (t, J ) 8 Hz, 2H), 2.22

(s, 6H). MS (ESI) m/z 300 (M + H)⁺.

4′-((E)-2-tert-Butoxycarbonylvinyl)-2′-methoxybiphenyl-4-car-

boxylic Acid Methyl Ester (28). A mixture of (tert-butoxycarbo-

nylmethylene)triphenylphosphorane (2.25 g, 5.5 mmol) and 18 (1.35

g, 5.0 mmol) in THF (20 mL) was stirred for 3 h, diluted with

hexanes (30 mL), and filtered through silica gel (50 g). The silica

gel was rinsed with 50% CH₂Cl₂/ether, and the combined solutions

2′-Methoxy-4′-(3-morpholin-4-yl-3-oxopropyl)biphenyl-4-car-

boxylic Acid Methyl Ester (35). 35 was prepared from 30 and

morpholine using the procedure for the preparation of 31. ¹H NMR

(300 MHz, DMSO-d₆) δ 7.97 (d, J ) 8 Hz, 2H), 7.60 (d, J ) 8

Hz, 2H), 7.24 (d, J ) 8 Hz, 1H), 7.03, (d, J ) 1 Hz, 1H), 6.93 (dd,

J ) 8, 1 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.51 (m, 4H), 3.44

(m, 4H), 2.78 (t, J ) 7.5 Hz, 2H), 2.69 (t, J ) 7.5 Hz, 2H). MS

(ESI) m/z 384 (M + H)⁺.

1

were concentrated to provide 1.66 g (90%) of 28. H NMR (300

MHz, CDCl₃) δ 8.07 (d, J ) 8.5 Hz, 2H), 7.61 (d, J ) 8.5 Hz,

2H), 7.59 (d, J ) 16 Hz, 1H), 7.34 (d, J ) 8 Hz, 1H), 7.20 (d, J

) 8 Hz, 1H), 7.11, (s, 1H), 6.41 (d, J ) 16 Hz, 1H), 3.94 (s, 3H),

3.85 (s, 3H), 1.55 (s, 9H). MS (ESI) m/z 367 (M - H)^-.

4′-(2-tert-Butoxycarbonylethyl)-2′-methoxybiphenyl-4-car-

boxylic Acid Methyl Ester (29). A mixture of 28 (20.0 g, 54.0

mmol) and RhCl(PPh₃)₃(2.5 g) in toluene (300 mL) was stirred

under a hydrogen atmosphere at 60 °C for 24 h, cooled, and

concentrated. The residue was taken up in EtOAc and filtered

through a pad of Celite (100 g) and silica gel (100 g), and the pad

was rinsed with EtOAc (200 mL) and concentrated to provide 20.0

g (100%) of 29. ¹H NMR (300 MHz, DMSO-d₆) δ 7.97 (d, J ) 8

Hz, 2H), 7.61 (d, J ) 8 Hz, 2H), 7.24 (d, J ) 8 Hz, 1H), 7.02, (d,

J ) 1 Hz, 1H), 6.91 (dd, J ) 8, 1 Hz, 1H), 3.88 (s, 3H), 3.77 (s,

3H), 2.78 (t, J ) 7.5 Hz, 2H), 2.58 (t, J ) 7.5 Hz, 2H), 1.40 (s,

9H). MS (ESI) m/z 371 (M + H)⁺.

2′-Methoxy-4′-(3-morpholin-4-yl-3-oxopropyl)biphenyl-4-car-

boxylic Acid (36). 36 was prepared from 35 using the procedure

1

for the preparation of 20. H NMR (300 MHz, DMSO-d₆) δ 7.94

(d, J ) 8 Hz, 2H), 7.57 (d, J ) 8 Hz, 2H), 7.24 (d, J ) 8 Hz, 1H),

7.02, (d, J ) 1 Hz, 1H), 6.93 (dd, J ) 8, 1 Hz, 1H), 3.78 (s, 3H),

3.51 (m, 4H), 3.44 (m, 4H), 2.87 (t, J ) 7.5 Hz, 2H), 2.69 (t, J )

7.5 Hz, 2H), 0.86 (s, 9H), 0.03 (s, 6H). MS (ESI) m/z 370 (M +

H)⁺.

2′-Methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbox-

ylic Acid Methyl Ester (37). 37 was prepared from 35 using the

procedure for the preparation of 33. ¹H NMR (300 MHz, DMSO-

d₆) δ 7.97 (d, J ) 8 Hz, 2H), 7.61 (d, J ) 8 Hz, 2H), 7.25 (d, J )

7.5 Hz, 1H), 6.99 (s, 1H), 6.89 (d, J ) 8 Hz, 1H), 3.87 (s, 3H),

3.78 (s, 3H), 3.58 (m, 4H), 3.38 (m, 4H), 2.33 (m, 4H), 1.78 (t, J

) 7 Hz, 2H). MS (ESI) m/z 370 (M + H)⁺.

4′-(2-Carboxyethyl)-2′-methoxybiphenyl-4-carboxylic Acid

Methyl Ester (30). A solution of 29 (1.75 g, 4.73 mmol) in TFA

(20 mL) and Et₃SiH (5 mL) was stirred at 50 °C for 24 h. The

solution was condensed and then condensed from heptane (2×) to

1

provide 1.53 g (99%) of 30. H NMR (300 MHz, DMSO-d₆) δ

2′-Methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbox-

ylic Acid (38). 38 was prepared from 37 using the procedure for

12.16 (br s, 1H), 7.97 (d, J ) 9 Hz, 2H), 7.61 (d, J ) 9 Hz, 2H),

7.25 (d, J ) 8 Hz, 1H), 7.03, (d, J ) 1 Hz, 1H), 6.92 (dd, J ) 8,

1 Hz, 1H), 3.86 (s, 3H), 3.77 (s, 3H), 2.88 (t, J ) 7.5 Hz, 2H),

2.60 (t, J ) 7.5 Hz, 2H). MS (ESI) m/z 313 (M - H)^-.

1

the preparation of 27. H NMR (300 MHz, DMSO-d₆) δ 12.4 (br

s, 1H), 7.96 (d, J ) 9 Hz, 2H), 7.59 (d, J ) 9 Hz, 2H), 7.28 (d, J

) 7.5 Hz, 1H), 7.03 (d, J ) 1.5 Hz, 1H), 6.94 (dd, J ) 7.5, 1.5

Hz, 1H), 3.88 (m, 2H), 3.79 (s, 3H), 3.69 (dt, J ) 13, 4 Hz, 2H),

2.83 (m, 4H), 2.64 (m, 2H), 2.09 (m, 2H). MS (ESI) m/z 356 (M

+ H)⁺.

4′-(2-Dimethylcarbamoylethyl)-2′-methoxybiphenyl-4-car-

boxylic Acid Methyl Ester (31). A solution of 30 (500 mg, 1.59

mmol) in CH₂Cl₂(5 mL) was treated with 2 M oxalyl chloride in

CH₂Cl₂(1 mL) and a drop of DMF, stirred for 1 h, concentrated

under vacuum, and dissolved in CH₂Cl₂(5 mL). The mixture was

treated with 2 M Me₂NH in THF (1.0 mL), and the resulting slurry

was filtered through silica gel (10 g). The silica gel was rinsed

with EtOAc and concentrated. The concentrate was flash chro-

matographed on silica gel with 30% EtOAc/hexanes to provide 490

4′-(3-Hydroxypropyl)-2′-methoxybiphenyl-4-carboxylic Acid

Methyl Ester (39). A solution of 30 (2.92 g, 9.3 mmol) in THF

(10 mL) was treated with 1 M BH₃in THF (18.6 mL), stirred for

24 h, and slowly quenched at 0 °C with MeOH (5 mL). The mixture

was poured into 4 M HCl (200 mL) and stirred for 1 h. The solution

was extracted with EtOAc (3 × 150 mL), washed with water and

brine, dried (Na₂SO₄), filtered, and concentrated. The crude product

was flash chromatographed on silica gel eluting with 50% EtOAc/

hexanes to provide 1.51 g (57%) of 39. ¹H NMR (300 MHz,

DMSO-d₆) δ 7.97 (d, J ) 7 Hz, 2H), 7.62 (d, J ) 7 Hz, 2H), 7.24

(d, J ) 8 Hz, 1H), 6.98, (d, J ) 1 Hz, 1H), 6.89 (dd, J ) 8, 1 Hz,

1H), 4.50 (t, J ) 5 Hz, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.46 (dt, J

) 7, 5 Hz, 1H), 2.67 (t, J ) 8 Hz, 2H), 1.77 (tt, J ) 8, 7 Hz, 2H).

MS (ESI) m/z 301 (M + H)⁺.

1

mg (91%) of 31. H NMR (300 MHz, CDCl₃) δ 8.05 (d, J ) 8.5

Hz, 2H), 7.59 (d, J ) 8.5 Hz, 2H), 7.25 (d, J ) 7.5 Hz, 1H), 6.90

(d, J ) 7.5 Hz, 1H), 6.88 (s, 1H), 3.93 (s, 3H), 3.81 (s, 3H), 3.02

(t, J ) 8 Hz, 2H), 2.98 (s, 6H), 2.67 (t, J ) 8 Hz, 2H). MS (ESI)

m/z 342 (M + H)⁺.

4′-(2-Dimethylcarbamoylethyl)-2′-methoxybiphenyl-4-car-

boxylic Acid (32). 32 was prepared from 31 using the procedure

for the preparation of 20. ¹H NMR (300 MHz, DMSO-d₆) δ 12.72

(br s, 1H), 7.95 (d, J ) 8 Hz, 2H), 7.59 (d, J ) 8 Hz, 2H), 7.23 (d,

J ) 8 Hz, 1H), 7.02, (d, J ) 1 Hz, 1H), 6.92 (dd, J ) 8, 1 Hz,

1H), 3.78 (s, 3H), 2.96 (s, 3H), 2.75 (t, J ) 7.5 Hz, 2H), 2.72 (s,

3H), 2.65 (t, J ) 7.5 Hz, 2H). MS (ESI) m/z 328 (M + H)⁺.

4′-(3-Dimethylaminopropyl)-2′-methoxybiphenyl-4-carbox-

ylic Acid Methyl Ester (33). A solution of 31 (4.90 g, 12.8 mmol)

in THF (30 mL) was treated with 1 M BH₃in THF (51 mL), stirred

for 24 h and slowly quenched at 0 °C with MeOH (8 mL). The

4′-[3-(tert-Butyldimethylsilanyloxy)propyl]-2′-methoxybiphen-

yl-4-carboxylic Acid Methyl Ester (40). A solution of 39 (234

mg, 0.66 mmol), TBDMSOTf (165 µL, 0.72 mmol), and 2,6-

lutidine (92 µL, 0.79 mmol) in CH₂Cl₂(10 mL) at 0 °C was stirred

for 30 min, and flash chromatographed on silica gel with 50%

1

EtOAc/hexanes to provide 300 mg (97%) of 40. H NMR (300

MHz, DMSO-d₆) δ 7.92 (d, J ) 8 Hz, 2H), 7.57 (d, J ) 8 Hz,

2H), 7.20 (d, J ) 8 Hz, 1H), 6.91, (d, J ) 1 Hz, 1H), 6.84 (dd, J

1176 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

) 8, 1 Hz, 1H), 3.82 (s, 3H), 3.72 (s, 3H), 3.59 (t, J ) 7.5 Hz,

2H), 2.62 (t, J ) 8 Hz, 2H), 1.76 (tt, J ) 7.5, 8 Hz, 2H), 0.84 (s,

9H), 0.00 (s, 6H). MS (ESI) m/z 415 (M + H)⁺.

(S)-3-(2-Nitro-4-sulfamoylphenylamino)-4-phenylsulfanylbu-

tyric acid (45S). 45S was prepared from 44S using the procedure

for the preparation of 45R. MS (ESI) m/z 410 (M - H)^-.

(R)-N,N-Dimethyl-3-(2-nitro-4-sulfamoylphenylamino)-4-phen-

ylsulfanylbutyramide (46R). A solution of 45R (411 mg, 1 mmol),

2 M Me₂NH in THF (1 mL), EDCI (296 mg, 1.5 mmol), and DMAP

(10 mg) in DMF (10 mL) was stirred for 16 h, diluted with EtOAc

(200 mL), washed sequentially with 1 M HCl (50 mL), water (50

mL), and brine (20 mL), dried (MgSO₄), filtered, and concentrated.

The concentrate was flash chromatographed on silica gel with 100%

4′-[3-(tert-Butyldimethylsilanyloxy)propyl]-2′-methoxybiphen-

yl-4-carboxylic acid (41). 41 was prepared from 40 using the

procedure for the preparation of 27. ¹H NMR (300 MHz, DMSO-

d₆) δ 7.93 (d, J ) 8 Hz, 2H), 7.57 (d, J ) 8 Hz, 2H), 7.23 (d, J )

8 Hz, 1H), 6.94, (d, J ) 1 Hz, 1H), 6.86 (dd, J ) 8, 1 Hz, 1H),

3.75 (s, 3H), 3.62 (t, J ) 7 Hz, 2H), 2.64 (t, J ) 8 Hz, 2H), 1.80

(tt, J ) 7, 8 Hz, 2H), 0.86 (s, 9H), 0.03 (s, 6H). MS (ESI) m/z 399

(M - H)^-.

1

EtOAc to provide 245 mg (56%) of 47R. H NMR (300 MHz,

DMSO-d₆) δ 8.77 (d, J ) 9.5 Hz, 1H), 8.39 (s, 1H), 7.72 (dd, J )

9.2, 2.3 Hz, 1H), 7.24-7.35 (m, 6H), 7.21 (m, 1H), 7.09 (d, J )

9.5 Hz, 1H), 4.40 (m, 1H), 3.41 (d, J ) 6.4 Hz, 2H), 2.94 (m, 1H),

2.91 (s, 3H), 2.79 (s, 3H), 2.72 (m, 1H). MS (ESI) m/z 439 (M +

H)⁺.

(S)-N,N-Dimethyl-3-(2-nitro-4-sulfamoylphenylamino)-4-phen-

ylsulfanylbutyramide (46S). 46S was prepared from 45S using

the procedure for the preparation of 46R. MS (ESI) m/z 439 (M +

H)⁺.

(R)-3-(9H-Fluoren-9-yloxycarbonylamino)-4-hydroxybutyr-

ic Acid tert-Butyl Ester (42R). A solution of Fmoc-D-Asp(OtBu)-

OH (9.0 g, 21.8 mmol) and DIPEA (4.6 mL) in THF (100 mL) at

-40 °C was treated with isobutyl chloroformate (3.1 mL, 24.1

mmol), warmed to 0 °C over 30 min, cooled to -20 °C, and treated

slowly with NaBH₄(1.64 g, 43.6 mmol) and MeOH (10 mL). The

reaction was gradually warmed to room temperature over 2 h,

diluted with EtOAc (200 mL), washed with water (100 mL) and

brine (50 mL), dried (MgSO₄), filtered, and concentrated to provide

8.22 g (95%) of 42R. ¹H NMR (300 MHz, CDCl₃) δ 7.77 (d, J )

8 Hz, 2H), 7.59 (d, J ) 7.5 Hz, 2H), 7.40 (dd, J ) 7.5, 7.5 Hz,

2H), 7.31 (dd, J ) 8, 7.5 Hz, 2H), 5.45 (m, 1H), 4.41 (br d, J )

6.8 Hz, 2H), 4.22 (t, J ) 6.8 Hz, 1H), 4.03 (m, 1H), 3.72 (m, 2H),

2.56 (m, 2H), 2.34 (m, 1H), 1.45 (m, 9H). MS (ESI) m/z 398 (M

+ H)⁺.

4-((R)-3-Morpholin-4-yl-3-oxo-1-phenylsulfanylmethylpropyl-

amino)-3-nitrobenzenesulfonamide (48R). 48R was prepared from

45R and morpholine using the procedure described for the

1

preparation of 46R. H NMR (300 MHz, DMSO-d₆) δ 8.70 (d, J

) 9.5 Hz, 1H), 8.39 (d, J ) 2 Hz, 1H), 7.73 (dd, J ) 9.5, 2 Hz,

1H), 7.22-7.36 (m, 6H), 7.18 (t, J ) 7 Hz, 1H), 7.09 (d, J ) 9.5

Hz, 1H), 4.42 (m, 1H), 3.42-3.56 (m, 4H), 3.39 (m, 4H), 3.30 (d,

J ) 7 Hz, 2H), 3.00 (dd, J ) 16, 7 Hz, 1H), 2.78 (dd, J ) 16, 5.5

Hz, 1H). MS (ESI) m/z 481 (M + H)⁺.

(S)-3-(9H-Fluoren-9-yloxycarbonylamino)-4-hydroxybutyr-

ic Acid tert-Butyl Ester (42S). 42S was prepared from Fmoc-L-

Asp(OtBu)-OH using the procedure for the preparation of 42R.

MS (ESI) m/z 398 (M + H)⁺.

4-((S)-3-Morpholin-4-yl-3-oxo-1-phenylsulfanylmethylpropyl-

amino)-3-nitrobenzenesulfonamide (48S). 48S was prepared from

45S and morpholine using the procedure described for the prepara-

tion of 46R. MS (ESI) m/z 481 (M + H)⁺.

(R)-3-(9H-Fluoren-9-yloxycarbonylamino)-4-phenylsulfanyl-

butyric Acid tert-Butyl Ester (43R). A solution of Bu₃P (7.3 mL,

29.2 mmol) and 1,1′-(azodicarbonyl)dipiperidine (7.35 g, 29.2

mmol) in CH₂Cl₂(100 mL) was treated with 42R (9.65 g, 24.3

mmol) and thiophenol (5.0 mL, 48.6 mmol), stirred for 24 h, and

concentrated. The concentrate was flash chromatographed on silica

4-((R)-3-Dimethylamino-1-phenylsulfanylmethylpropylamino)-

3-nitrobenzenesulfonamide (47R). A mixture of 46R (4.06 g, 9.25

mmol) and 1 M BH₃in THF (20 mL) was stirred for 16 h, treated

with MeOH (5.0 mL) and concentrated HCl (2 mL), stirred at 80

°C for 3 h, cooled to room temperature, adjusted to pH 10 with 4

M Na₂CO₃, diluted with EtOAc (150 mL), washed with water (50

mL) and brine (10 mL), dried (MgSO₄), filtered, and concentrated.

The concentrate was flash chromatographed on silica gel with 20%

1

gel with 50% EtOAc/hexanes to provide 8.5 g (72%) of 43R. H

NMR (300 MHz, CDCl₃) δ 7.76 (d, J ) 7.4 Hz, 2H), 7.57 (d, J )

7.1 Hz, 2H), 7.40 (m, 4H), 7.30 (m, 4H), 7.19 (t, J ) 7.5 Hz, 1H),

5.48 (m, 1H), 4.35 (br d, J ) 7.4 Hz, 2H), 4.19 (t, J ) 7.1 Hz,

1H), 4.13 (m, 1H), 3.26 (m, 1H), 3.11 (m, 1H), 2.64 (m, 2H), 1.43

(m, 9H). MS (DCI) m/z 490 (M + H)⁺.

(S)-3-(9H-Fluoren-9-yloxycarbonylamino)-4-phenylsulfanyl-

butyric Acid tert-Butyl Ester (43S). 43S was prepared from 42S

using the procedure for the preparation of 43R. MS (DCI) m/z 490

(M + H)⁺.

1

MeOH/CH₂Cl₂to provide 3.88 g (99%) of 47R. H NMR (300

MHz, DMSO-d₆) δ 8.64 (br d, J ) 9 Hz, 1H), 8.39 (d, J ) 2 Hz,

1H), 7.71 (dd, J ) 9, 2 Hz, 1H), 7.31 (m, 4H), 7.27 (m, 2H), 7.19

(m, 1H), 7.08 (br d, J ) 9 Hz, 1H), 4.12 (m, 1H), 3.37 (m, 2H),

2.40 (m, 1H), 2.20 (m, 1H), 2.11 (s, 6H), 1.94 (m, 1H), 1.82 (m,

23.4

1H). MS (ESI) m/z 425 (M + H)⁺. [R]_D

acetone).

) -342° (c 0.51,

(R)-3-(2-Nitro-4-sulfamoylphenylamino)-4-phenylsulfanylbu-

tyric Acid tert-Butyl Ester (44R). A mixture of 43R (600 mg,

1.23 mmol), 6 (298 mg, 1.34 mmol), and DIPEA (3 mL) in DMF

(3 mL) was stirred for 12 h, diluted with EtOAc (100 mL), washed

with water (45 mL) and brine (10 mL), dried (MgSO₄), filtered,

and concentrated. The concentrate was flash chromatographed on

silica gel with 30% EtOAc/CH₂Cl₂to provide 390 mg (68%) of

4-((S)-3-Dimethylamino-1-phenylsulfanylmethylpropylamino)-

3-nitrobenzenesulfonamide (47S). 47S was prepared from 46S

using the procedure for the preparation of 47R. MS (ESI) m/z 425

23.1

(M + H)⁺. [R]_D) +334° (c 0.42, acetone).

4-((R)-3-Morpholin-4-yl-1-phenylsulfanylmethylpropylamino)-

3-nitrobenzenesulfonamide (49R). 49R was prepared from 48R

using the procedure described for the preparation of 47R. ¹H NMR

(300 MHz, DMSO-d₆) δ 8.41 (d, J ) 9.2 Hz, 1H), 8.40 (d, J ) 2

Hz, 1H), 7.73 (dd, J ) 9.5, 2 Hz, 1H), 7.22-7.35 (m, 6H), 7.19 (t,

J ) 7 Hz, 1H), 7.11 (d, J ) 10 Hz, 1H), 4.18 (m, 1H), 3.54 (m,

4H), 3.38 (m, 2H), 2.28-2.40 (m, 4H), 2.21 (m, 2H), 2.00 (m,

1H), 1.92 (m, 1H). MS (ESI) m/z 467 (M + H)⁺.

1

44R. H NMR (300 MHz, CDCl₃) δ 8.68 (s, 1H), 8.66 (d, J ) 7

Hz, 1H), 7.75 (d, J ) 7 Hz, 1H), 7.55 (m, 1H), 7.37 (m, 2H), 7.27

(m, 4H), 6.67 (d, J ) 9.1 Hz, 1H), 4.83 (s, 2H), 4.17 (m, 1H), 3.20

(d, J ) 6.4 Hz, 2H), 2.77 (ddd, J ) 10.5, 9, 6.4 Hz, 1H), 1.42 (m,

9H). MS (ESI) m/z 468 (M + H)⁺.

(S)-3-(2-Nitro-4-sulfamoylphenylamino)-4-phenylsulfanylbu-

tyric Acid tert-Butyl Ester (44S). 44S was prepared from 43S using

the procedure for the preparation of 44R. MS (ESI) m/z 468 (M +

H)⁺.

4-((S)-3-Morpholin-4-yl-1-phenylsulfanylmethylpropylamino)-

3-nitrobenzenesulfonamide (49S). 49S was prepared from 48S

using the procedure described for the preparation of 47R. MS (ESI)

m/z 467 (M + H)⁺.

(R)-3-(2-Nitro-4-sulfamoylphenylamino)-4-phenylsulfanylbu-

tyric acid (45R). A mixture of 44R (2.8 g, 6 mmol) and 4 M HCl

(50 mL) in 1,4-dioxane (50 mL) was stirred for 6 h. The solution

was concentrated and then concentrated from toluene to provide

(R)-2-tert-Butoxycarbonylamino-3-phenylsulfanylpropionic Acid

Methyl Ester (50). A 0 °C solution of N-(tert-butoxycarbonyl)-L-

i

serine methyl ester (30 g, 137 mmol) and Pr₂NH (58 mL, 330

1

2.47 g (99%) of 45R. H NMR (300 MHz, CDCl₃) δ 8.67 (m,

mmol) in CH₂Cl₂(250 mL) was treated with methanesulfonyl

chloride (11.65 mL, 151 mmol), stirred for 20 min, treated with

thiophenol (15.5 mL, 151 mmol), warmed to room temperature,

stirred for 30 min, and concentrated. The crude material was flash

2H), 8.03 (s, 2H), 7.74 (d, J ) 9.1 Hz, 1H), 7.37 (m, 2H), 7.16 (m,

3H), 6.67 (d, J ) 9.2 Hz, 1H), 4.22 (m, 1H), 3.777 (m, 2H), 3.22

(d, J ) 6.1 Hz, 2H). MS (ESI) m/z 410 (M - H)^-.

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1177

chromatographed on silica gel with 10% EtOAc/hexanes to provide

25.0 g (59%) of 50. H NMR (300 MHz, DMSO-d₆) δ 7.35 (m,

5H), 7.24 (m, 1H), 4.10 (m, 1H), 3.60 (s, 3H), 3.35 (dd, J ) 14, 6

Hz, 1H), 3.14 (dd, J ) 14, 10 Hz, 1H), 1.38 (s, 9H). MS (ES) m/z

310 (M - H)^-.

mmol) and 1 M BH₃in THF (94 mL) was stirred for 24 h, treated

with MeOH (10 mL) and 4 M HCl (100 mL), and stirred for 24 h.

The mixture was adjusted to pH > 12 with KOH, extracted with

EtOAc (3 × 200 mL), and the extracts were washed with water

(200 mL) and brine (200 mL), dried (Na₂SO₄), filtered, and

concentrated. The amine was taken up in DMF (100 mL), 6 (5.9 g,

26.8 mmol) and DIPEA (5 mL) were added, and the reaction was

stirred for 4 h. The reaction was poured into water (400 mL) and

extracted with EtOAc (3 × 300 mL). The extracts were washed

with water (3 × 200 mL), dried (Na₂SO₄), filtered, concentrated,

and flash chromatographed on silica gel with 1/10/89 TEA/MeOH/

EtOAc to provide 9.8 g (88%) of 56. ¹H NMR (300 MHz, DMSO-

d₆) δ 8.39 (d, J ) 2 Hz, 1H), 8.27 (br d, J ) 9 Hz, 1H), 7.74 (dd,

J ) 9, 2 Hz, 1H), 7.31 (m, 4H), 7.14-7.25 (m, 4H), 4.09 (m, 1H),

3.33 (m, 2H), 2.15 (t, J ) 7 Hz, 2H), 2.05 (s, 6H), 1.74 (m, 2H),

1

((R)-1-Formyl-2-phenylsulfanylethyl)carbamic Acid tert-Butyl

Ester (51). A solution of 50 (8.1 g, 26.0 mmol) in CH₂Cl₂at -78

°C was treated with 1 M DIBAL in CH₂Cl₂(52 mL), stirred for 3

h, quenched with MeOH (20 mL), and poured into saturated NaH₂-

PO₄. The mixture was extracted with EtOAc (3 × 300 mL), and

the combined extracts were dried (Na₂SO₄), filtered, concentrated,

and flash chromatographed on silica gel with 30% EtOAc/hexanes

1

to provide 5.0 g (68%) of 51. H NMR (300 MHz, DMSO-d₆) δ

9.47 (s, 1H), 7.34 (m, 5H), 7.23 (m, 1H), 4.00 (m, 1H), 3.40 (dd,

J ) 14, 6 Hz, 1H), 3.07 (dd, J ) 14, 10 Hz, 1H), 1.39 (s, 9H). MS

(ESI) m/z 280 (M - H)^-.

1.47 (m, 2H). MS (ESI) m/z 439 (M + H)⁺. [R]_D) -321° (c

23.7

(E,R)-4-tert-Butoxycarbonylamino-5-phenylsulfanylpent-2-

enoic Acid tert-Butyl Ester (52). 51 (8.4 g, 30 mmol) in THF (25

mL) at 0 °C was treated with a solution of Ph₃PdCHCO₂tBu, (13.6

g, 36 mmol) in THF (150 mL), warmed to room temperature, stirred

for 24 h, treated with hexanes (100 mL), filtered through a pad of

silica gel, and concentrated. The concentrate was flash chromato-

graphed on silica gel with 10% EtOAc/hexanes to provide 9.5 g

0.33, acetone).

((R)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-hydroxy-

hexyl)carbamic Acid tert-Butyl Ester (57). A solution of Fmoc-

D-Lys(BOC)-OH (2.102 g, 4.5 mmol) in DME (5 mL) at -15 °C

was treated successively with N-methylmorpholine (0.56 mL, 5.0

mmol) and isobutyl chloroformate (0.7 mL, 5 mmol), stirred for 2

min, and filtered. The filter cake was washed with DME (3 × 5

mL), and the combined filtrate and washings were cooled to -5

°C and treated with NaBH₄(0.3 g, 7.5 mmol) and water (5 mL)

and additional water (250 mL) immediately afterward. The mixture

was stirred for 15 min and filtered. The filter cake was washed

with water and dried to provide 1.94 g (95%) of 57. ¹H NMR (300

MHz, CDCl₃) δ 7.76 (d, J ) 8 Hz, 2H), 7.59 (d, J ) 7.5 Hz, 2H),

7.39 (dd, J ) 7.5, 7.3 Hz, 2H), 7.31 (dd, J ) 8, 7.3 Hz, 2H), 5.07

(m, 1H), 4.58 (m, 1H), 4.41 (d, J ) 5.8 Hz, 2H), 4.21 (t, J ) 6.8

Hz, 1H), 3.62 (m, 4H), 3.09 (m, 2H), 1.26-1.54 (m, 6H), 1.43 (s,

9H). MS (APCI) m/z 455 (M + H)⁺.

((R)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-phenyl-

sulfanylhexyl)carbamic Acid tert-Butyl Ester (58). A mixture of

57 (2.0 g, 4.4 mmol), PhSSPh (1.44 g, 6.6 mmol), and PBu₃(1.65

mL, 6.6 mmol) in toluene (50 mL) at 80 °C was stirred for 18 h

and concentrated. The concentrate was flash chromatographed on

silica gel with 25% EtOAc/hexanes to provide 1.83 g (76%) of 58.

¹H NMR (300 MHz, CDCl₃) δ 7.76 (d, J ) 8 Hz, 2H), 7.52 (m,

4H), 7.25-7.42 (m, 6H), 7.21 (t, J ) 7 Hz, 1H), 5.46 (m, 2H),

4.50 (m, 2H), 4.36 (m, 1H), 4.06 (m, 2H), 1.18-1.66 (m, 6H),

1.43 (s, 9H). MS (APCI) m/z 547 (M + H)⁺.

((R)-5-Dimethylamino-1-phenylsulfanylmethylpentylcar-

bamic Acid 9H-Fluoren-9-ylmethyl Ester (59). A solution of 58

(1.36 g, 2.4 mmol) in CH₂Cl₂(5 mL) and TFA (5 mL) was stirred

at room temperature for 30 min, and concentrated. The concentrate

was dissolved in AcOH (1 mL) and 37% aqueous formaldehyde

(5 mL), treated with 1 M NaCNBH₃in THF (10 mL), stirred for

30 min, adjusted to pH 7 with saturated NaHCO₃solution, and

extracted with EtOAc (3 × 100 mL). The combined extracts were

washed with water and brine, dried (Na₂SO₄), filtered, and

concentrated. The concentrate was flash chromatographed on silica

gel with 48:48:4 EtOAc/CH₂Cl₂/MeOH to provide to provide 638

mg (56%) of 59. ¹H NMR (300 MHz, DMSO-d₆, TFA salt) δ 7.89

(d, J ) 7.5 Hz, 2H), 7.69 (m, 2H), 7.42 (dd, J ) 7.5, 7 Hz, 2H),

7.32 (m, 7H), 7.15 (m, 1H), 6.04 (m, 2H), 3.55 (m, 2H), 3.01 (m,

2H), 1.15-1.60 (m, 6H). MS (ESI) m/z 475 (M + H)⁺.

1

(83%) of 52. H NMR (300 MHz, DMSO-d₆) δ 7.38-7.49 (m,

4H), 7.21 (m, 2H), 6.76 (dd, J ) 16, 6 Hz, 1H), 5.78 (dd, J ) 16,

2 Hz, 1H), 4.14 (m, 1H), 3.19 (m, 1H), 3.01 (m, 1H), 1.41 (s, 9H).

MS (ESI) m/z 378 (M - H)^-.

(R)-4-tert-Butoxycarbonylamino-5-phenylsulfanylpentanoic

Acid (53). A mixture of 52 (5.0 g, 13.2 mmol) and RhCl(PPh₃)₃(1

g) in toluene (125 mL) was stirred under H₂at 50 °C for 24 h,

cooled, filtered through silica gel, and concentrated. The concentrate

was dissolved in THF (90 mL), water (30 mL), and MeOH (30

mL), treated with LiOH (2.77 g, 66 mmol), and stirred for 24 h.

The mixture was poured into saturated NaH₂PO₄solution (200 mL)

and extracted with EtOAc (3 × 200 mL). The combined extracts

were washed with brine, dried (Na₂SO₄), filtered, and concentrated

to provide 4.12 g (96%) of 53. ¹H NMR (300 MHz, DMSO-d₆) δ

7.33 (m, 4H), 7.18 (m, 1H), 6.72 (br d, J ) 9.5 Hz, 1H), 3.55 (m,

1H), 2.99 (d, J ) 7 Hz, 2H), 2.21 (m, 2H), 1.85 (m, 1H), 1.58 (m,

1H), 1.38 (s, 9H). MS (ESI) m/z 326 (M + H)⁺.

((R)-3-Dimethylcarbamoyl-1-phenylsulfanylmethylpropyl)-

carbamic Acid tert-Butyl Ester (54). A solution of 53 (10.6 g,

32.6 mmol), 2 M Me₂NH in THF (32.6 mL), EDCI (12.5 g, 65

mmol), and DMAP (4.0 g, 32.6 mmol) in CH₂Cl₂(10 mL) was

stirred for 24 h, diluted with EtOAc (200 mL), washed sequentially

with 1 M HCl (50 mL), water (50 mL), and brine (20 mL), dried

(Na₂SO₄), filtered, and concentrated. The concentrate was flash

chromatographed on silica gel with EtOAc to provide 9.0 g (78%)

1

of 54. H NMR (300 MHz, DMSO-d₆) δ 7.32 (m, 4H), 7.18 (m,

1H), 6.82 (br d, J ) 9.5 Hz, 1H), 3.55 (m, 1H), 3.01 (d, J ) 7 Hz,

2H), 2.91 (s, 3H), 2.79 (s, 3H), 2.27 (m, 2H), 1.83 (m, 1H), 1.59

(m, 1H), 1.38 (s, 9H). MS (ESI) m/z 353 (M + H)⁺.

(R)-4-(2-Nitro-4-sulfamoylphenylamino)-5-phenylsulfanylpen-

tanoic Acid Dimethyl Amide (55). A mixture of 54 (1.13 g, 3.22

mmol) in dioxane (50 mL) and 4 M HCl (50 mL) was stirred for

2 h, poured into saturated Na₂CO₃(400 mL), and extracted with

EtOAc (3 × 300 mL). The combined extracts were dried (Na₂-

SO₄), filtered, and concentrated to provide the pure primary amine.

The amine was taken up in DMF (10 mL), 6 (880 mg, 4 mmol),

and DIPEA (1 mL) were added, and the reaction was stirred for 4

h. The reaction was poured into water (200 mL) and extracted with

EtOAc (3 × 100 mL). The extracts were washed with water (3 ×

100 mL) and brine (100 mL), dried (Na₂SO₄), filtered, concentrated,

and flash chromatographed on silica gel with 50% EtOAc/hexanes

((R)-5-(2-Nitro-4-sulfamoylphenylamino)-6-phenylsulfanyl-

hexyl)carbamic Acid tert-Butyl Ester (60). 60 was prepared from

1

59 using the procedure for the preparation of 44R. H NMR (300

MHz, CDCl₃) δ 8.69 (s, 1H), 8.38 (d, J ) 9 Hz, 1H), 7.74 (d, J )

9 Hz, 1H), 7.36 (d, J ) 8 Hz, 2H), 7.14-7.36 (m, 6H), 6.65 (d, J

) 9 Hz, 1H), 4.80 (m, 2H), 4.49 (m, 1H), 3.55 (m, 2H), 1.93 (m,

2H), 1.73 (m, 4H), 1.43 (s, 9H). MS (ESI) m/z 523 (M - H)^-.

1

to provide 890 mg (61%) of 55. H NMR (300 MHz, DMSO-d₆)

δ 8.40 (d, J ) 2 Hz, 1H), 8.28 (br d, J ) 9 Hz, 1H), 7.72 (dd, J

) 9, 2 Hz, 1H), 7.14-7.39 (m, 8H), 4.12 (m, 1H), 3.38 (m, 2H),

2.88 (s, 3H), 2.78 (s, 3H), 2.41 (m, 2H), 1.97 (m, 2H). MS (ESI)

m/z 453 (M + H)⁺.

4-((R)-4-Dimethylamino-1-phenylsulfanylmethylbutylamino)-

3-nitrobenzenesulfonamide (56). A mixture of 54 (9.0 g, 25.5

4-((R)-5-Dimethylamino-1-phenylsulfanylmethylpentylamino-

3-nitrobenzenesulfonamide (61). 61 was prepared from 59 using

1

the procedure for the preparation of 44R. H NMR (300 MHz,

DMSO-d₆) δ 8.39 (d, J ) 2 Hz, 1H), 8.26 (d, J ) 9 Hz, 1H), 7.74

(dd, J ) 9, 2 Hz, 1H), 7.13-7.37 (m, 8H), 4.08 (m, 1H), 3.16 (m,

1178 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

2H), 2.21 (m, 2H), 2.14 (s, 6H), 1.75 (m, 2H), 1.47 (m, 4H). MS

7.7 Hz, 1H), 3.99 (m, 2H), 3.78 (s, 3H), 3.67 (m, 2H), 3.51 (m,

4H), 3.29 (t, J ) 6.8 Hz, 2H), 3.11 (m, 4H), 2.68 (t, J ) 8.4 Hz,

2H), 2.02 (m, 2H). MS (ESI) m/z 691 (M - H)^-. Anal.

(C₃₅H₃₈N₄O₇S₂‚1.5C₂HF₃O₂) C, H, N.

23.8

(ESI) m/z 453 (M + H)⁺. [R]_D) -327° (c 0.21, acetone).

N-(4′-Dimethylaminomethyl-2′-methoxybiphenyl-4-carbonyl)-

3-nitro-4-(2-phenylsulfanylethylamino)benzenesulfonamide (62).

62 was prepared from 22 and 7 using the general coupling

procedure. ¹H NMR (300 MHz, DMSO-d₆, TFA salt) δ 9.45 (br s,

1H), 8.80 (t, J ) 7.5 Hz, 1H), 8.63 (d, J ) 2.4 Hz, 1H), 7.94 (dd,

J ) 8.8, 2.4 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 2H), 7.61 (d, J ) 8.5

Hz, 2H), 7.38 (m, 3H), 7.27 (m, 4H), 7.17 (m, 2H), 4.31 (d, J )

4.4 Hz, 2H), 3.80 (s, 3H), 3.68 (d, J ) 6.8 Hz, 2H), 3.29 (d, J )

6.8 Hz, 2H), 2.78 (s, 6H). MS (ESI) m/z 621 (M + H)⁺. Anal.

(C₃₁H₃₂N₄O₆S₂‚4.5C₂HF₃O₂) C, H, N.

N-[2′-Methoxy-4′-(3-morpholin-4-yl-3-oxopropyl)biphenyl-4-car-

bonyl]-3-nitro-4-(2-phenylsulfanylethylamino)benzenesulfon-

amide (69). 69 was prepared from 36 and 7 using the general

1

coupling procedure. H NMR (300 MHz, DMSO-d₆) δ 12.50 (br

s, 1H), 8.79 (t, J ) 7.4 Hz, 1H), 8.62 (d, J ) 2.4 Hz, 1H), 7.93

(dd, J ) 9.1, 2.4 Hz, 1H), 7.88 (d, J ) 8.8 Hz, 2H), 7.56 (d, J )

8.4 Hz, 2H), 7.37 (d, J ) 8.1 Hz, 2H), 7.24 (m, 5H), 7.02 (s, 1H),

6.92 (dd, J ) 7.8, 1.4 Hz, 1H), 3.76 (s, 3H), 3.67 (m, 2H), 3.52

(m, 4H), 3.45 (m, 4H), 3.27 (m, 2H), 2.86 (t, J ) 8.5 Hz, 2H),

2.67 (t, J ) 8.5 Hz, 2H). MS (ESI) m/z 703 (M - H)^-. Anal.

(C₃₅H₃₆N₄O₈S₂‚H₂O) C, H, N.

2-Methoxy-4′-[3-nitro-4-(2-phenylsulfanylethylamino)ben-

zenesulfonylaminocarbonyl]biphenyl-4-carboxylic Acid Dimeth-

ylamide (63). 63 was prepared from 20 and 7 using the general

1

coupling procedure. H NMR (500 MHz, DMSO-d₆) δ 12.45 (br

N-[4′-(3-Hydroxypropyl)-2′-methoxybiphenyl-4-carbonyl]-3-

nitro-4-(2-(phenylsulfanylethylamino)benzenesulfonamide (70).

70 was prepared from 41 and 7 using the general coupling

procedure. The crude product was stirred with 4 M HCl (10 mL)

s, 1H), 8.78 (t, J ) 7.5 Hz, 1H), 8.63 (d, J ) 2.5 Hz, 1H), 7.94

(dd, J ) 9.1, 2.5 Hz, 1H), 7.91 (d, J ) 8.7 Hz, 2H), 7.62 (d, J )

8.7 Hz, 2H), 7.38 (m, 3H), 7.27 (dd, J ) 8.1, 7.2 Hz, 2H), 7.21 (d,

J ) 9.4 Hz, 1H), 7.18 (t, J ) 7.5 Hz, 1H), 7.13 (s, 1H), 7.06 (d,

J ) 7.8 Hz, 1H), 3.79 (s, 3H), 3.68 (d, J ) 6.9 Hz, 2H), 3.29 (d,

J ) 6.9 Hz, 2H), 3.00 (s, 3H), 2.96 (s, 3H). MS (ESI) m/z 633 (M

- H)^-. Anal. (C₃₁H₃₀N₄O₇S₂‚0.4H₂O) C, H, N.

1

and dioxane (10 mL) for 1 h prior to purification. H NMR (300

MHz, DMSO-d₆) δ 12.45 (br s, 1H), 8.80 (t, J ) 7.5 Hz, 1H), 8.63

(d, J ) 2.4 Hz, 1H), 7.94 (dd, J ) 9.1, 2.1 Hz, 1H), 7.88 (d, J )

8.8 Hz, 2H), 7.58 (d, J ) 8.4 Hz, 2H), 7.37 (d, J ) 7.1 Hz, 2H),

7.24 (m, 5H), 6.96 (s, 1H), 6.88 (d, J ) 7.8 Hz, 1H), 4.50 (br m,

1H), 3.76 (s, 3H), 3.68 (d, J ) 7 Hz, 2H), 3.44 (t, J ) 6.4 Hz, 2H),

3.27 (m, 2H), 2.65 (t, J ) 7.6 Hz, 2H), 1.76 (tt, J ) 7.6, 6.4 Hz,

2H). MS (ESI) m/z 620 (M - H)^-. Anal. (C₃₁H₃₁N₃O₇S₂‚0.25C₂-

HF₃O₂) C, H, N.

N-[4′-(2-Dimethylaminoethyl)-2′-methoxybiphenyl-4-carbonyl]-

3-nitro-4-(2-phenylsulfanylethylamino)benzenesulfonamide (64).

64 was prepared from 27 and 7 using the general coupling

1

procedure. H NMR (300 MHz, DMSO-d₆) δ 12.45 (br s, 1H),

8.79 (t, J ) 7.5 Hz, 1H), 8.62 (d, J ) 2.5 Hz, 1H), 7.94 (dd, J )

9.2, 1.2 Hz, 1H), 7.91 (d, J ) 8.4 Hz, 2H), 7.57 (d, J ) 8.7 Hz,

2H), 7.14-7.40 (m, 7H), 7.07 (s, 1H), 6.96 (d, J ) 7.8 Hz, 1H),

3.78 (s, 3H), 3.68 (t, J ) 6.5 Hz, 2H), 3.42 (m, 2H), 3.19 (m, 2H),

3.02 (m, 2H), 2.85 (s, 6H). MS (ESI) m/z 635 (M + H)⁺.

N-(4-Morpholin-4-ylbenzoyl)-3-nitro-4-(2-phenylsulfanylethyl-

amino)benzenesulfonamide (71). 71 was prepared from 4-(4-

morpholinyl)benzoic acid and 7 using the general coupling proce-

1

dure. H NMR (300 MHz, DMSO-d₆) δ 12.05 (br s, 1H), 8.75 (t,

J ) 5.9 Hz, 1H), 8.59 (d, J ) 2.0 Hz, 1H), 7.90 (dd, J ) 9.2, 2.0

Hz, 1H), 7.77 (d, J ) 9.2 Hz, 2H), 7.37 (m, 2H), 7.27 (m, 2H),

7.17 (d, J ) 8.8 Hz, 2H), 6.95 (d, J ) 9.2 Hz, 1H), 3.63-3.74 (m,

6H), 3.22-3.32 (m, 6H). MS (ESI) m/z 542 (M + H)⁺. Anal.

(C₂₅H₂₆N₄O₆S₂) C, H, N.

N-[4-(4,4-Dimethylpiperidin-1-yl)benzoyl]-3-nitro-4-(2-phen-

ylsulfanylethylamino)benzenesulfonamide (72). 72 was prepared

from 16 and 7 using the general coupling procedure. ¹H NMR (300

MHz, DMSO-d₆) δ 11.98 (br s, 1H), 8.77 (t, J ) 6.1 Hz, 1H), 8.59

(d, J ) 2.3 Hz, 1H), 7.91 (dd, J ) 9.1, 2.0 Hz, 1H), 7.71 (d, J )

9.1 Hz, 2H), 7.36 (d, J ) 8.8 Hz, 2H), 7.17-7.27 (m, 4H), 6.91

(d, J ) 9.2 Hz, 2H), 3.67 (dt, J ) 6.6, 6.4 Hz, 2H), 3.25-3.37 (m,

6H), 1.37 (m, 4H), 0.95 (s, 6H). MS (ESI) m/z 569 (M + H)⁺.

Anal. (C₂₈H₃₂N₄O₅S₂) C, H, N.

2-{2-Methoxy-4′-[3-nitro-4-(2-phenylsulfanylethylamino)ben-

zenesulfonylaminocarbonyl]biphenyl-4-yl}-N,N-dimethylacet-

amide (65). 65 was prepared from 25 and 7 using the general

1

coupling procedure. H NMR (300 MHz, DMSO-d₆) δ 12.45 (br

s, 1H), 8.78 (t, J ) 7.5 Hz, 1H), 8.62 (d, J ) 2.3 Hz, 1H), 7.93

(dd, J ) 9.2, 2.4 Hz, 1H), 7.89 (d, J ) 8.5 Hz, 2H), 7.58 (d, J )

8.1 Hz, 2H), 7.37 (d, J ) 7.1 Hz, 2H), 7.20-7.28 (m, 4H), 7.19 (t,

J ) 7.5 Hz, 1H), 7.00 (s, 1H), 6.89 (d, J ) 7.8 Hz, 1H), 3.74 (s,

3H), 3.73 (s, 2H), 3.68 (d, J ) 6.5 Hz, 2H), 3.28 (d, J ) 6.5 Hz,

2H), 3.03 (s, 3H), 2.84 (s, 3H). MS (ESI) m/z 647 (M - H)^-.

N-[4′-(3-Dimethylaminopropyl)-2′-methoxybiphenyl-4-car-

bonyl]-3-nitro-4-(2-phenylsulfanylethylamino)benzenesulfon-

amide (66). 66 was prepared from 34 and 7 using the general

coupling procedure. ¹H NMR (500 MHz, DMSO-d₆) δ 9.50 (br s,

1H), 8.78 (t, J ) 7.5 Hz, 1H), 8.63 (d, J ) 2.5 Hz, 1H), 7.94 (dd,

J ) 9.3, 2.5 Hz, 1H), 7.89 (d, J ) 8.8 Hz, 2H), 7.58 (d, J ) 8.7

Hz, 2H), 7.38 (d, J ) 7.2 Hz, 2H), 7.24 (m, 4H), 7.19 (t, J ) 7.2

Hz, 1H), 7.01 (s, 1H), 6.92 (d, J ) 6.6 Hz, 1H), 3.78 (s, 3H), 3.68

(m, 2H), 3.29 (t, J ) 6.9 Hz, 2H), 3.09 (m, 2H), 2.79 (s, 6H), 2.67

(t, J ) 7.1 Hz, 2H), 2.00 (m, 2H). MS (ESI) m/z 647 (M - H)^-.

Anal. (C₃₃H₃₆N₄O₆S₂‚0.5C₂HF₃O₂) C, H, N.

4-((R)-3-Dimethylamino-1-phenylsulfanylmethylpropylami-

no)-N-[4-(4,4-dimethylpiperidin-1-yl)benzoyl]-3-nitrobenzenesul-

fonamide (73R). 73R was prepared from 16 and 47R using the

1

general coupling procedure. H NMR (300 MHz, DMSO-d₆) δ

12.45 (br s, 1H), 8.46 (d, J ) 2.0 Hz, 1H), 8.24 (d, J ) 8.8 Hz,

1H), 7.81 (dd, J ) 8.8, 2.0 Hz, 1H), 7.72 (d, J ) 9.2 Hz, 2H), 7.32

(d, J ) 7.2 Hz, 2H), 7.25 (dd, J ) 7.2, 7.1 Hz, 2H), 7.17 (t, J )

7.1 Hz, 1H), 6.90 (d, J ) 9.5 Hz, 1H), 6.81 (d, J ) 9.1 Hz, 2H),

4.07 (m, 1H), 3.37 (m, 2H), 3.21 (t, J ) 5.6 Hz, 4H), 2.87 (m,

2H), 2.50 (s, 6H), 2.04 (m, 2H), 1.39 (t, J ) 5.6 Hz, 4H), 0.94 (s,

6H). MS (ESI) m/z 640 (M + H)⁺. [R]_D^23.0) -232° (c 0.42, DMF).

M.p. 217 °C. Anal. (C₃₂H₄₁N₅O₅S₂) C, H, N.

3-{2-Methoxy-4′-[3-nitro-4-(2-phenylsulfanylethylamino)ben-

zenesulfonylaminocarbonyl]biphenyl-4-yl}-N,N-dimethylpropi-

onamide (67). 67 was prepared from 32 and 7 using the procedure

1

for the general coupling procedure. H NMR (300 MHz, DMSO-

d₆) δ 12.50 (br s, 1H), 8.79 (t, J ) 7.5 Hz, 1H), 8.63 (d, J ) 2.3

Hz, 1H), 7.94 (dd, J ) 9.2, 2.4 Hz, 1H), 7.88 (d, J ) 8.5 Hz, 2H),

7.57 (d, J ) 8.5 Hz, 2H), 7.37 (d, J ) 7.4 Hz, 2H), 7.24 (m, 5H),

7.02 (s, 1H), 6.92 (d, J ) 7.8 Hz, 1H), 3.76 (s, 3H), 3.68 (m, 2H),

3.29 (m, 2H), 2.95 (s, 3H), 2.84 (m, 2H), 2.83 (s, 3H), 2.64 (t, J )

8.1 Hz, 2H). MS (ESI) m/z 661 (M - H)^-. Anal. (C₃₃H₃₄N₄O₇S₂‚

0.5C₂HF₃O₂) C, H, N.

4-((S)-3-Dimethylamino-1-phenylsulfanylmethylpropylami-

no)-N-[4-(4,4-dimethylpiperidin-1-yl)benzoyl]-3-nitrobenzenesulfon-

amide (73S). 73S was prepared from 16 and 47S using the general

1

coupling procedure. H NMR (500 MHz, DMSO-d₆, TFA salt) δ

11.97 (br s, 1H), 9.48 (br s, 1H), 8.56 (d, J ) 2.5 Hz, 1H), 8.29 (d,

J ) 9.4 Hz, 1H), 7.88 (dd, J ) 9.0, 2.1 Hz, 1H), 7.74 (d, J ) 9.1

Hz, 2H), 7.14-7.25 (m, 6H), 6.92 (d, J ) 9.4 Hz, 2H), 4.18 (m,

1H), 3.32-3.41 (m, 6H), 3.14 (m, 2H), 2.75 (s, 6H), 2.14 (m, 2H),

N-[2′-Methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-car-

bonyl]-3-nitro-4-(2-phenylsulfanylethylamino)benzenesulfon-

amide (68). 68 was prepared from 38 and 7 using the general

1.37 (t, J ) 5.8 Hz, 4H), 0.95 (s, 6H). MS (ESI) m/z 640 (M +

23.8

H)⁺. [R]_D

H, N.

) +226° (c 0.33, DMF). Anal. (C₃₂H₄₁N₅O₅S₂) C,

1

coupling procedure. H NMR (300 MHz, DMSO-d₆, TFA salt) δ

9.68 (br s, 1H), 8.80 (t, J ) 7.4 Hz, 1H), 8.62 (d, J ) 2.4 Hz, 1H),

7.93 (m, 1H), 7.88 (d, J ) 8.4 Hz, 2H), 7.58 (d, J ) 8.4 Hz, 2H),

7.37 (d, J ) 8.1 Hz, 2H), 7.24 (m, 5H), 7.01 (s, 1H), 6.93 (d, J )

4-((R)-4-Dimethylamino-1-phenylsulfanylmethylbutylami-

no)-N-[4-(4,4-dimethylpiperidin-1-yl)benzoyl]-3-nitrobenzenesul-

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1179

fonamide (74). 74 was prepared from 16 and 56 using the general

coupling procedure. ¹H NMR (300 MHz, DMSO-d₆) δ 9.95 (br s,

1H), 8.48 (d, J ) 2.0 Hz, 1H), 8.18 (d, J ) 9.0 Hz, 1H), 7.81 (dd,

J ) 9.0, 2.0 Hz, 1H), 7.72 (d, J ) 8.8 Hz, 2H), 7.29 (d, J ) 7.7

Hz, 2H), 7.21 (dd, J ) 7.4, 7.1 Hz, 2H), 7.15 (t, J ) 7.1 Hz, 1H),

7.03 (d, J ) 9.1 Hz, 1H), 6.83 (d, J ) 9.1 Hz, 2H), 4.06 (m, 1H),

3.38 (m, 2H), 3.24 (m, 4H), 2.94 (m, 2H), 2.72 (s, 6H), 1.72 (m,

4H), 1.39 (t, J ) 5.6 Hz, 4H), 0.94 (s, 6H). MS (ESI) m/z 654 (M

+ H)⁺. Anal. (C₃₃H₄₃N₅O₅S₂‚0.5H₂O) C, H, N.

J ) 7.1 Hz, 1H), 7.04 (d, J ) 9.1 Hz, 1H), 7.00 (s, 1H), 6.89 (d,

J ) 7.8 Hz, 1H), 4.08 (m, 1H), 3.77 (s, 3H), 3.25-3.42 (m, 6H),

2.95-3.11 (m, 8H), 2.72 (s, 6H), 2.06 (m, 2H), 1.76 (m, 4H). MS

(ESI) m/z 760 (M - H)^-. Anal. (C₃₉H₄₇N₅O₇S₂‚2C₂HF₃O₂‚H₂O)

C, H, N.

4-((S)-3-Dimethylamino-1-phenylsulfanylmethylpropylamino)-

N-[2′-methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbonyl]-

3-nitrobenzenesulfonamide (79S). 79S was prepared from 38 and

1

47S using the general coupling procedure. H NMR (300 MHz,

4-((R)-5-Dimethylamino-1-phenylsulfanylmethylpentylami-

no)-N-[4-(4,4-dimethylpiperidin-1-yl)benzoyl]-3-nitrobenzenesul-

fonamide (75). 75 was prepared from 16 and 61 using the general

DMSO-d₆, TFA salt) δ 9.70 (br s, 1H), 9.40 (br s, 1H), 8.58 (d, J

) 2.3 Hz, 1H), 8.32 (d, J ) 9.1 Hz, 1H), 7.92 (d, J ) 8.5 Hz, 1H),

7.89 (dd, J ) 9.2, 2.4 Hz, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.12-

7.28 (m, 7H), 7.01 (s, 1H), 6.93 (d, J ) 7.8 Hz, 1H), 4.19 (m, 1H),

3.96 (m, 2H), 3.77 (s, 3H), 3.64 (m, 2H), 3.40 (m, 4H), 3.12 (m,

6H), 2.75 (s, 6H), 2.68 (t, J ) 7.6 Hz, 2H), 2.14 (m, 2H), 2.01 (m,

2H). MS (ESI) m/z 762 (M + H)⁺. Anal. (C₃₉H₄₇N₅O₇S₂‚2.5C₂-

HF₃O₂) C, H, N.

4-((R)-4-Dimethylamino-1-phenylsulfanylmethylbutylamino)-

N-[2′-methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbonyl]-

3-nitrobenzenesulfonamide (80). 80 was prepared from 38 and

56 using the general coupling procedure. ¹H NMR (500 MHz,

DMSO-d₆) δ 10.95 (br s, 1H), 8.56 (d, J ) 2.4 Hz, 1H), 8.33 (d,

J ) 9.2 Hz, 1H), 7.91 (d, J ) 8.2 Hz, 1H), 7.90 (dd, J ) 9.2, 2.1

Hz, 1H), 7.59 (d, J ) 8.2 Hz, 2H), 7.26 (m, 4H), 7.13 (m, 3H),

7.01 (s, 1H), 6.93 (d, J ) 7.6 Hz, 1H), 4.15 (m, 1H), 3.97 (m, 2H),

3.78 (s, 3H), 3.65 (m, 2H), 3.34 (m, 2H), 3.14 (m, 2H), 3.08 (m,

2H), 2.99 (m, 2H), 2.73 (s, 6H), 2.68 (m, 2H), 2.02 (m, 2H), 1.79

(m, 2H), 1.60 (m, 2H), 1.36 (m, 2H). MS (ESI) m/z 774 (M -

H)^-.

4-((R)-5-Dimethylamino-1-phenylsulfanylmethylpentylamino)-

N-[2′-methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbonyl]-

3-nitrobenzenesulfonamide (81). 81 was prepared from 38 and

61 using the general coupling procedure. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.56 (d, J ) 2.4 Hz, 1H), 8.33 (d, J ) 9.2 Hz, 1H),

7.91 (d, J ) 8.2 Hz, 1H), 7.89 (dd, J ) 8.8, 2.1 Hz, 1H), 7.59 (d,

J ) 8.2 Hz, 2H), 7.25 (m, 4H), 7.15 (m, 3H), 7.01 (s, 1H), 6.93 (d,

J ) 7.6 Hz, 1H), 3.97 (m, 1H), 3.74 (s, 3H), 3.65 (m, 2H), 3.34

(m, 2H), 3.14 (m, 2H), 3.08 (m, 2H), 2.99 (m, 2H), 2.73 (s, 6H),

2.68 (m, 2H), 2.02 (m, 2H), 1.79 (m, 2H), 1.60 (m, 2H), 1.36 (m,

2H). MS (ESI) m/z 788 (M - H)^-. Anal. (C₄₁H₅₁N₅O₇S₂‚2.5C₂-

HF₃O₂) C, H, N.

1

coupling procedure. H NMR (300 MHz, DMSO-d₆) δ 8.46 (d, J

) 2.4 Hz, 1H), 8.18 (d, J ) 9.1 Hz, 1H), 7.81 (dd, J ) 9.2, 2.1

Hz, 1H), 7.72 (d, J ) 8.9 Hz, 2H), 7.32 (d, J ) 7.1 Hz, 2H), 7.25

(dd, J ) 7.2, 7.1 Hz, 2H), 7.16 (t, J ) 7.2 Hz, 1H), 6.99 (d, J )

9.5 Hz, 1H), 6.81 (d, J ) 9.2 Hz, 2H), 4.03 (m, 1H), 3.37 (m, 2H),

3.25 (m, 4H), 2.95 (m, 2H), 2.69 (s, 6H), 1.77 (m, 2H), 1.57 (m,

2H), 1.39 (m, 4H), 1.35 (m, 2H), 0.94 (s, 6H). MS (ESI) m/z 668

(M + H)⁺. Anal. (C₃₄H₄₅N₅O₅S₂‚2.25C₂HF₃O₂) C, H, N.

4-((R)-5-Amino-1-phenylsulfanylmethylpentylamino)-N-[4-

(4,4-dimethylpiperidin-1-yl)benzoyl]-3-nitrobenzenesulfonam-

ide (76). 76 was prepared from 16 and 60 using the general coupling

procedure. The crude product was stirred with 4 M HCl (10 mL)

1

and dioxane (10 mL) for 3 h prior to purification. H NMR (300

MHz, DMSO-d₆) δ 12.00 (s, 1H), 8.53 (d, J ) 2.0 Hz, 1H), 8.31

(d, J ) 9.2 Hz, 1H), 7.86 (dd, J ) 9.2, 2.1 Hz, 1H), 7.74 (d, J )

9.2 Hz, 2H), 7.69 (br s, 2H), 7.09-7.25 (m, 6H), 6.93 (d, J ) 9.2

Hz, 2H), 4.08 (m, 1H), 3.57 (s, 2H), 3.29 (m, 4H), 2.72 (m, 2H),

1.77 (m, 2H), 1.77 (m, 2H), 1.52 (m, 2H), 1.37 (m, 6H), 0.95 (s,

6H). MS (ESI) m/z 640 (M + H)⁺. Anal. (C₃₂H₄₁N₅O₅S₂‚

0.25CH₂O₂) C, H, N.

N-[4-(4,4-Dimethylpiperidin-1-yl)benzoyl]-4-((R)-3-morpho-

lin-4-yl-1-phenylsulfanylmethylpropylamino)-3-nitrobenzene-

sulfonamide (77R). 77R was prepared from 16 and 49R using the

general coupling procedure. ¹H NMR (300 MHz, DMSO-d₆) δ 8.48

(d, J ) 2.0 Hz, 1H), 8.35 (d, J ) 9.2 Hz, 1H), 7.79 (dd, J ) 9.2,

2.0 Hz, 1H), 7.72 (d, J ) 8.8 Hz, 2H), 7.30 (d, J ) 6.8 Hz, 2H),

7.23 (dd, J ) 7.5, 7.1 Hz, 2H), 7.15 (t, J ) 7.1 Hz, 1H), 6.98 (d,

J ) 9.5 Hz, 1H), 6.85 (d, J ) 9.2 Hz, 2H), 4.13 (m, 1H), 3.51 (m,

4H), 3.37 (m, 2H), 3.26 (m, 4H), 2.37 (m, 4H), 2.28 (m, 2H), 2.00

(m, 1H), 1.83 (m, 1H), 1.39 (t, J ) 5.5 Hz, 4H), 0.94 (s, 6H). MS

(ESI) m/z 682 (M + H)⁺. Anal. (C₃₄H₄₃N₅O₆S₂‚0.5CH₂O₂) C, H,

N.

N-[4-(4,4-Dimethylpiperidin-1-yl)benzoyl]-4-((S)-3-morpho-

lin-4-yl-1-phenylsulfanylmethylpropylamino)-3-nitrobenzene-

sulfonamide (77S). 77S was prepared from 16 and 49S using the

general coupling procedure. ¹H NMR (500 MHz, DMSO-d₆) δ 8.50

(d, J ) 2.1 Hz, 1H), 8.25 (d, J ) 9.1 Hz, 1H), 7.80 (dd, J ) 9.1,

2.0 Hz, 1H), 7.71 (d, J ) 9.0 Hz, 2H), 7.21 (m, 4H), 7.10 (m, 2H),

6.94 (d, J ) 8.7 Hz, 2H), 4.21 (m, 1H), 3.87 (m, 4H), 3.31 (m,

4H), 3.12 (m, 2H), 2.95 (m, 4H), 2.28 (m, 2H), 2.20 (m, 2H), 1.71

(m, 2H), 1.36 (t, J ) 5.3 Hz, 4H), 0.94 (s, 6H). MS (ESI) m/z 682

(M + H)⁺. Anal. (C₃₄H₄₃N₅O₆S₂‚0.5CH₂O₂) C, H, N.

4-((R)-5-Amino-1-phenylsulfanylmethylpentylamino)-N-[2′-

methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbonyl]-3-ni-

trobenzenesulfonamide (82). 82 was prepared from 38 and 60

using the general coupling procedure. The crude product was stirred

with 4 M HCl (10 mL) and dioxane (10 mL) for 3 h prior to

1

purification. H NMR (300 MHz, DMSO-d₆) δ 8.47 (d, J ) 2.4

Hz, 1H), 8.14 (d, J ) 9.1 Hz, 1H), 7.87 (d, J ) 8.5 Hz, 1H), 7.82

(dd, J ) 8.8, 2.0 Hz, 1H), 7.39 (d, J ) 8.8 Hz, 2H), 7.31 (d, J )

6.7 Hz, 2H), 7.25 (dd, J ) 7.5, 7.1 Hz, 2H), 7.18 (d, J ) 7.5 Hz,

2H), 6.94 (s, 1H), 6.91 (d, J ) 9.5 Hz, 1H), 6.85 (d, J ) 7.8 Hz,

1H), 3.97 (m, 1H), 3.74 (s, 3H), 3.58 (m, 4H), 3.34 (m, 2H), 2.73

(t, J ) 7.1 Hz, 2H), 2.62 (t, J ) 7.5 Hz, 2H), 2.35 (m, 4H), 2.32

(m, 2H), 1.77 (m, 2H), 1.49 (m, 2H), 1.37 (m, 2H). MS (ESI) m/z

760 (M - H)^-. Anal. (C₃₉H₄₇N₅O₇S₂‚3HCl) C, H, N.

(R)-3-{4-[4-(4,4-Dimethylpiperidin-1-yl)benzoylsulfamoyl]-2-ni-

trophenylamino}-N,N-dimethyl-4-phenylsulfanylbutyramide (78).

78 was prepared from 16 and 46R using the general coupling

N-[2′-Methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbo-

nyl]-4-((R)-3-morpholin-4-yl-1-phenylsulfanylmethylpropylamino)-

3-nitrobenzenesulfonamide (83). 83 was prepared from 38 and

1

procedure. H NMR (300 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.85

1

(d, J ) 9.5 Hz, 1H), 8.54 (d, J ) 2.4 Hz, 1H), 7.83 (dd, J ) 9.5,

2.4 Hz, 1H), 7.72 (d, J ) 9.1 Hz, 2H), 7.25 (d, J ) 9.1 Hz, 2H),

7.10-7.19 (m, 4H), 6.92 (d, J ) 9.2 Hz, 2H), 4.43 (m, 1H), 3.41

(m, 2H), 3.27 (m, 4H), 2.98 (m, 1H), 2.90 (s, 3H), 2.78 (s, 3H),

2.75 (m, 1H), 1.37 (t, J ) 5.6 Hz, 4H), 0.95 (s, 6H). MS (ESI) m/z

654 (M + H)⁺. Anal. (C₃₂H₃₉N₅O₆S₂) C, H, N.

49R using the general coupling procedure. H NMR (500 MHz,

MeOH-d₄) δ 8.71 (d, J ) 2.2 Hz, 1H), 7.94 (d, J ) 9.0 Hz, 1H),

7.91 (d, J ) 8.5 Hz, 2H), 7.52 (d, J ) 8.1 Hz, 2H), 7.29 (dd, J )

8.1, 1.6 Hz, 2H), 7.23 (d, J ) 7.5 Hz, 2H), 7.13 (m, 3H), 6.96 (d,

J ) 9.5 Hz, 1H), 6.95 (s, 1H), 6.89 (d, J ) 7.8 Hz, 1H), 4.14 (m,

1H), 3.97 (m, 2H), 3.78 (s, 3H), 3.65 (m, 2H), 3.34 (m, 2H), 3.14

(m, 2H), 3.08 (m, 2H), 2.99 (m, 2H), 2.73 (s, 6H), 2.68 (m, 2H),

2.02 (m, 2H), 1.79 (m, 2H), 1.60 (m, 2H), 1.36 (m, 2H). MS (ESI)

m/z 802 (M - H)^-. Anal. (C₄₁H₄₉N₅O₈S₂‚0.5H₂O) C, H, N.

(R)-3-(4-{[2′-Methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-

4-carbonyl]sulfamoyl}-2-nitrophenylamino)-N,N-dimethyl-4-

phenylsulfanylbutyramide (84). 84 was prepared from 38 and 46R

using the general coupling procedure. ¹H NMR (500 MHz, MeOH-

4-((R)-3-Dimethylamino-1-phenylsulfanylmethylpropylamino)-

N-[2′-methoxy-4′-(3-morpholin-4-ylpropyl)biphenyl-4-carbonyl]-

3-nitrobenzenesulfonamide (79R). 79R was prepared from 38 and

1

47R using the general coupling procedure. H NMR (500 MHz,

DMSO-d₆) δ 8.49 (d, J ) 2.2 Hz, 1H), 8.12 (d, J ) 9.0 Hz, 1H),

7.90 (d, J ) 8.4 Hz, 1H), 7.86 (dd, J ) 9.1, 2.0 Hz, 1H), 7.42 (d,

J ) 8.1 Hz, 2H), 7.30 (d, J ) 7.5 Hz, 2H), 7.22 (m, 4H), 7.14 (t,

1180 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3

Wendt et al.

d₄) δ 8.72 (s, 1H), 8.69 (d, J ) 9.1 Hz, 1H), 7.94 (d, J ) 8.5 Hz,

2H), 7.52 (dd, J ) 9.0, 2.3 Hz, 1H), 7.46 (d, J ) 8.3 Hz, 2H), 7.34

(d, J ) 7.3 Hz, 2H), 7.14-7.21 (m, 4H), 6.92 (s, 1H), 6.87 (d, J

) 9.5 Hz, 2H), 4.40 (m, 1H), 3.77 (s, 3H), 3.74 (m, 2H), 3.34 (m,

4H), 2.85 (m, 4H), 2.98 (s, 3H), 2.87 (s, 3H), 2.71 (m, 4H), 1.96

(m, 2H), 1.29 (m, 2H). MS (ESI) m/z 774 (M - H)^-. Anal.

(C₃₉H₄₅N₅O₈S₂‚H₂O) C, H, N.

Protein Preparation. A previously described loop-deleted

version of Bcl-X_Lwhich lacked the putative transmembrane helix

was employed for NMR studies and biological assays.^18,19The Bcl-2

protein used was a chimera based on isoform 2 (A96T and G110R)

in which residues 35-91 were replaced with residues 35-50 from

Bcl-X_L, and the C-terminal end (residues 208-219) was excised.⁴⁰

For both Bcl-X_Land Bcl-2, uniformly ¹⁵N-labeled and ¹⁵N-, ¹³C-

labeled protein was expressed in E. coli containing the appropriate

plasmid, on minimal media containing ¹⁵N-labeled ammonium

chloride as the sole nitrogen source with or without ¹³C-labeled

glucose as the sole carbon source. Proteins were purified by affinity

chromatography on a Nickel-ProBond column (Invitrogen), con-

centrated, and exchanged into 40 mM disodium phosphate buffer,

pH 7.0, containing either 10% or 100% D₂O plus 5 mM deuterated

dithiothreitol. Protein samples for NMR were 0.5-1.0 mM in

microcells. Ligands were added to the protein from concentrated

(100 mM) stock solutions prepared in DMSO-d₆).

NMR-Based Structural Studies. NMR spectra for structural

studies were recorded on Bruker DRX600 and DRX800 spectrom-

eters at 303 K. Resonance assignments for ligand-bound Bcl-X_L

were extrapolated from those of the apo protein by comparing two-

dimensional ¹³C- and ¹⁵N-HSQC spectra and three-dimensional ¹³C-

edited and ¹⁵N-edited NOESY spectra of the liganded to the

unliganded protein. Intraprotein NOEs for those residues of Bcl-

X_Lwhich line the binding groove were extracted from three-

dimensional ¹³C-edited and ¹⁵N-edited NOESY spectra recorded

with a mixing time of 80 ms. Protein-ligand NOEs were extracted

from three-dimensional ¹³C-edited, ¹²C-filtered NOESY spectra

recorded with mixing times ranging from 150 to 250 ms.

Fluorescence Polarization Assay. K_iand IC₅₀values were

determined using a competitive fluorescence polarization assay as

described previously.^20,35A series dilution of compounds were used

to compete the binding of 1 nM f-Bad peptide (NLWAAQRYGREL-

RRMSDK(FITC)FVD) and 6 nM Bcl-X_Lor 1 nM f-Bax peptide

(FITC-QDASTKKLSECLKRIGDELDS) and 10 nM Bcl-2. The

effects of 1% and 10% human serum, and of separate serum

proteins, were detected using 30 nM f-Bad peptide and 60 nM Bcl-

X_L. Serum and serum components were added to assay buffer.

Concentrations for serum components were as follows: HSA, 0.42

mg/mL; HSA-III, 0.146 mg/mL; R₁-acid glycoprotein, 0.0095 mg/

mL. Individual determinations were the result of duplicate values.

K_iand IC₅₀values were calculated using Microsoft Excel.

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 per liter of â-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 per liter of â-mercaptoethanol, 1% penicillin-streptomycin, 3%

FBS). Compounds in series dilutions were added, and the cells were

cultured for 24 h. Cell viability was assayed by MTS or cell titerGlo

from Premega. Individual determinations were the result of duplicate

values.

treating with 73R (in DMEM containing 10% FBS). Cell viability

was measured by MTS readout at 48 h postexposure and percent

viability determined by comparison to untreated samples. For

paclitaxel, cells in the same media were pretreated with dilutions

of paclitaxel (starting dose 20 nM, 3-fold dilutions) for 48 h prior

to a 48 h coincubation with paclitaxel and 73R (in DMEM

containing 0.34% gelatin). The total incubation volume was 100

µL. Cell viability was measured by the MTS (Promega) assay.

In Vivo Tumor Efficacy Model. Animal studies were conducted

following the guidelines of the Institutional Animal Care and Use

Committee. Immunocompromised male Scid mice (C.B-17-Prkdc-

^scid) were obtained from Charles River Laboratories (Wilmington,

MA) and trials were initiated when mice were 7-10 weeks of age.

A-549 NSCLC cells were obtained from the American Type Culture

Collection (Manassa, VA). 5 × 10⁶cells in 50% Matrigel (BD

Biosciences, Bedford, MA) were inoculated subcutaneously into

the flank. Tumors were allowed to reach an average volume of

250 mm³(day 15 post inoculation), and mice were assigned to

treatment groups of equal tumor size (N )10 mice per group).

Therapy was initiated the following day. Tumor size was evaluated

by twice weekly measurements with digital calipers. Tumor volume

was estimated using the formula: V ) L × W². 73R was

administered ip in a vehicle of 5% Tween 80, 20% poly(ethylene

glycol), 75% 0.1 M NaPO₄, pH ∼ 4.0 at 75 mg/kg/day from day

16-36 Paclitaxel was obtained from the Bristol-Myers Squibb

Company (Princeton, NJ) and prepared and administered ip at 30

mg/kg/day on days 16, 20, and 24 according to the manufacturers

formulation guidelines.

Paclitaxel/73R Pharmacokinetic Study. 73R and paclitaxel

were administered at doses of 100 and 15 mg/kg, respectively, in

the same manner as described in the previous section, to Scid mice.

A single method was developed for the simultaneous quantitation

of 73R and paclitaxel in plasma samples. The initial analytical

method used a protein precipitation with CH₃CN to separate both

compounds from the plasma. A plasma aliquot (200 µL, sample or

spiked standard) was combined with 50 µL of internal standard

and 1 mL of acetonitrile. Following vortexing and centrifugation,

the supernatant was transferred to a clean tube. An aliquot (10 µL)

of DMSO was added to each tube. Supernatant was evaporated to

near dryness with dry nitrogen over low heat, and samples were

reconstituted by vortexing with 0.2 mL of mobile phase. Samples

were analyzed simultaneously with spiked plasma standards. 73R,

paclitaxel, and internal standard were separated on a 50 × 3 mm

Keystone Aquasil 5µm C18 column with a CH₃CN: 0.1% TFA

mobile phase (85:15, by volume) at a flow rate of 0.35 mL/min.

Analysis was performed on a Sciex API365 Biomolecular Mass

Analyzer with a turbo-ionspray interface. Analytes were ionized

in the positive ion mode. Detection was in multiple reaction

monitoring (MRM) mode at m/z 640.5 f 216.4 for 73R and m/z

854.4 f 286.1 for paclitaxel. 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 method for quantitation for 73R and

paclitaxel, evaluated over the concentration range 0-8 µg/mL, was

linear (correlation coefficient > 0.99), with mean accuracy values

from 91.7 to 108.5% and 85.5-109.4% of theory, respectively, for

the analysis of triplicate standards at seven separate concentrations.

The limit of quantitation was estimated to be ∼10 ng/mL from a

0.2 mL plasma sample. The three mice with highest concentrations

were averaged to provide the peak plasma concentration (C_max) and

the time to peak plasma concentration (T_max). The mean plasma

concentration data were submitted to multiexponential curve fitting

using WinNonlin. The area under the mean plasma concentration-

time curve from 0 to t hours (time of the last measurable plasma

concentration) after dosing (AUC_0-t) was calculated using the linear

trapezoidal rule for the plasma concentration-time profiles. The

residual area extrapolated to infinity, determined as the final

measured plasma concentration (C_t) divided by the terminal

In Vitro Chemopotentiation. Using a 96-well plate format,

A549 cells were seeded in DMEM containing 5% FBS at 5 × 10³

cells/well the day prior to the experiment. The following day,

medium was removed and a dose range of UV-C (starting energy

32 mJ/cm², with 50% reductions each step) was applied prior to

Antagonists of B-Cell Lymphoma 2 Family Proteins

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1181

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Supporting Information Available: A table of combustion

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JM050754U

Article Doi

Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo

DOI: 10.1021/jm050754u

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Article abstract of DOI:10.1021/jm050754u

Full text of DOI:10.1021/jm050754u

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