Towards the next generation of dual Bcl-2/Bcl-xL inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.036

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-x _L in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the R_S versus S_S sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-x_L.

Full text of DOI:10.1016/j.bmcl.2014.05.036

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3026–3033
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Towards the next generation of dual Bcl-2/Bcl-xL inhibitors
⇑
Jeffrey G. Varnes , Thomas Gero, Shan Huang, R. Bruce Diebold, Claude Ogoe, Paul T. Grover, Mei Su,
Prasenjit Mukherjee, Jamal Carlos Saeh, Terry MacIntyre, Galina Repik, Keith Dillman, Kate Byth,
Daniel John Russell, Stephanos Ioannidis
Oncology Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural modifications of the left-hand side of compound 1 were identified which retained or improved
potent binding to Bcl-2 and Bcl-xL in in vitro biochemical assays and had strong activity in an RS4;11
apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and
comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer
(14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochem-
ical preference for the RS versus SS sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent
to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-
fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-xL.
Received 25 March 2014
Revised 10 May 2014
Accepted 12 May 2014
Available online 20 May 2014
Keywords:
Bcl-2
Bcl-xL
Navitoclax
B-cell lymphoma
Apoptosis
Ó 2014 Elsevier Ltd. All rights reserved.
The B-cell lymphoma 2 (Bcl-2) family of proteins play a central
role in regulating cell death through the outer mitochondrial path-
way of apoptosis. Antiapoptotic members, such as Bcl-2, Bcl-xL,
and Mcl-1, are comprised of four Bcl homology (BH) domains.
Three of these domains (BH1–BH3) are shared by the proapoptotic
Bcl-2 effector proteins Bax and Bak, while the proapoptotic activa-
tor proteins (e.g., Bim, Bid) only contain BH3. In a normal healthy
cell, antiapoptotic proteins sequester their apoptotic counterparts
through BH3 domain binding. In response to cellular injury, Bax
and Bak are activated, resulting in oligomerization. Oligomer inser-
tion into the outer mitochondrial membrane forms an exit pore
through which cytochrome c enters the cytosol. Subsequent initia-
tion of the caspase cascade culminates in cell death.1
inhibitor, is in Phase I/II clinical trials for solid tumors and hemato-
logic malignancies.5,6
Dose-limiting observations for Navitoclax include thrombocyto-
penia, which is a target-driven toxicity resulting from Bcl-xL inhibi-
tion in platelets.7a Bcl-xL inhibition also induces transient
thrombocytopathy, leading to increased tail-bleeding time in
mice.7b Thrombocytopenia has been managed in the clinic by start-
ing with low lead-in dosing prior to dose escalation to avoid severe
plateletnadirs,6d and blood platelet levels have been shown to revert
to pre-dosing levels after cessation of Navitoclax administration.6b
Therefore, a desirable outcome in developing second generation
dual Bcl-2/Bcl-xL inhibitors is an amelioration or at least attenuation
of the severity and duration of thrombocytopenia in patients. An
alternative approach is to develop a selective Bcl-2 inhibitor and
thereby side-step thrombocytopenia as a dose-limiting toxicity.8
Herein we discuss structural modification of the left-hand side of
known triflone 19 (Fig. 1, green box). Modifications of the right-hand
side of this compound will be discussed in a future publication. This
tool compound was selected because its synthetic accessibility
enabled the rapid profiling of scaffold changes. One goal of these
studies was to improve aqueous solubility, with the aim of increas-
ing the fraction absorbed and, ultimately, bioavailability. We were
also keen to selectively improve Bcl-2 potency to determine if this
might translate to an improved safety profile in vivo.
Pro-survival members of the Bcl-2 family of proteins are overex-
pressed in a variety of neoplastic malignancies, and this dysregula-
tion has been identified as
a critical component of tumor
development and chemotherapy resistance.2 As a result of overex-
pression, proapoptotic proteins are perpetually sequestered, and
the intrinsic apoptotic pathway is blocked. A developing treatment
to restore this process in cells that are ‘primed for death’ is the inhi-
bition of antiapoptotic Bcl-2 proteins with BH3-mimetics.3 Two
such examples are ABT-7374 and ABT-2635 (Navitoclax; Fig. 1),
which bind to Bcl-2, Bcl-xL, and Bcl-w with subnanomolar affinity.
Navitoclax, which is an orally bioavailable second generation Bcl-2
As a protein–protein interaction inhibitor that binds to a hydro-
phobic BH3 groove,10 Navitoclax is not surprisingly very lipophilic
(clogP = 12.4). There are a number of functionalities, such as the
⇑
Corresponding author. Tel.: +1 781 839 4944; fax: +1 781 839 4630.
E-mail address: Jeffrey.Varnes@Astrazeneca.com (J.G. Varnes).
http://dx.doi.org/10.1016/j.bmcl.2014.05.036
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
3027
CF3
CF3
O
O
S O
O
O
S O
NO2
H
N
H
N
H
N
S
S
S
H
N
H
N
H
N
O
O
O
S
S
S
O
O
O
O
N
O
N
O
N
N
N
N
N
N
N
Cl
Cl
Cl
ABT-737
1
ABT-263
Navitoclax
Figure 1. Structures of ABT-737, ABT-263, and 1.
biphenyl and aryl acylsulfonamide, which likely contribute to its
poor aqueous solubility. Additionally, the high acidity of the acyl-
sulfonamide (pKa 3.4) is suspected of reducing cellular permeabil-
ity.5 If this moiety could be replaced, a number of properties might
therefore be improved.
Conversion of the acylsulfonamide to the corresponding ben-
zamidine analog 211 (Table 1) resulted in a complete loss of cellular
activity and poor solubility. Similarly, sulfonamide 3 was designed
based on the ability of oxetane to act as a carbonyl isostere15 but
was inactive in both biochemical and cellular assays. A correlation
between cellular activity and acyl sulfonamide acidity has been
previously reported.5b Therefore, lack of cell potency for 2 (no cal-
culated acidic centers; ACD pKa v10, with correction library) and 3
(pKa 9.0) was attributed to reduced acidity. However, other less
quantifiable factors such as functional group binding incompat-
ability (e.g., oxetane) were not ruled out.
(clogP = 8.8; pKa 3.5) demonstrated comparable binding to Bcl-2
and Bcl-xL relative to 1. Thiazole 6 (clogP = 9.8) was less active
than analog 1, despite an increase in acidity (pKa 2.6).
As shown in Table 2 and similar to changes to the acylsulfona-
mide, heterocyclic replacements of the benzylpiperazine were well
tolerated with respect to binding, but retaining cellular activity
was a challenge (Table 2). For example, we designed piperazine
analogs 8 and 9 with the goal of leveraging the polar character of
phosphine oxides to enhance aqueous solubility. Analog 9 did
indeed have improved solubility, but this was obviated by a lack
of cell potency. Other point modifications such as thio ether 12
and sulfone 15 were equally tantalizing. For 4-substituted piperi-
dines 16–19, only the hydroxymethyl analog had appreciable
activity in cells but was still 10-fold less active than 1.16
Of the compounds in Table 2, only sulfoxide 13 met our criteria
for in vitro potency and solubility, while the corresponding sulfox-
ide diastereomer (14) was inactive in the cell. The data suggest that
the latter may be due to poor target inhibition (Bcl-2 FP IC50
0.454 lM). Docking studies (Fig. 2) were used to evaluate potential
differences stemming from changes in stereochemistry and build
Heterocyclic replacement of the phenyl ring of the aryl acylsulf-
onamide was also attempted as a means of reducing lipophilicity,
improving solubility, and retaining or enhancing cellular activity.
Pyridazine 4 (clogP = 8.7; pKa ꢀ3.6) was modestly active and
offered an improvement in solubility, while pyrimidine
5
support for stereochemical assignment of the sulfoxide itself.
Table 1
Fluorescence polarization, cellular, and solubility data for 1–6
CF3
O
O
S O
H
N
S
H
N
X
S
Ring (R)
O
N
O
A
C
B
D
Ring
N
N
N
N
N
S
N
N
Cl
a
a
b
Compd
R
X
Bcl-2 FP IC50
(l
M)
Bcl-xL FP IC50
(lM)
RS4;11 EC50
(l
M)
Solc
(lM)
19
211
3
4
5
A
A
A
B
C
D
O
NH
0.011
0.019
>1
0.270
0.018
0.119
0.016
0.063
>1
0.378
0.039
0.098
0.011
>3.19
>1.1
0.563
0.177
0.385
14
<1
<1
38
3
Oxetan-3,3-yl
O
O
O
6
2
a
b
c
Fluorescence polarization assay.12
Caspase-GloÒ cellular assay.13
Equilibrium solubility (pH 7.4).14

3028
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
Table 2
Fluorescence polarization, cellular, and solubility data for 7–19
Ring (R)
C
CF3
S O
O
A
N
B
D
N
H
N
S
H
N
O
S
P
O
N
N
O
P
O
O
N
O
N
Ring
E
N
F
G
N
H
N
X
N
NH2
OEt
OH
CN
Cl
O
Solc
(lM)
a
a
b
Compd
R
X
Bcl-2 FP IC50
(
lM)
Bcl-xL FP IC50
(l
M)
RS4;11 EC50
(l
M)
7
8
9
A
B
C
D
D
D
D
D
D
E
CH2
CH2
CH2
O
C@O
S
>0.468
>1.1
0.077
0.308
0.018
0.038
<0.010
0.031
0.011
0.054
0.021
<0.010
<0.010
0.030
<0.010
0.610
>1.1
>1.1
>0.921
>1.1
>0.516
0.090
>1.1
<1
4
0.054
0.250
0.065
0.061
0.019
0.545
0.087
<0.010
<0.010
0.017
0.059
21
35
ntd
27
26
12
27
2
10
11
12
13
14
15
16
17
18
19
S@O, isomer 1
S@O, isomer 2
SO2
CH2
CH2
CH2
CH2
>1.1
0.504
0.395
1.79
F
G
H
<1
<1.5
6
0.112
a
b
c
Fluorescence polarization assay.12
Caspase-GloÒ cellular assay.13
Equilibrium solubility (pH 7.4).14
Not tested.
d
Two surrogate ligands (Fig. 2A) were generated for 13 and 14
wherein the triflone was replaced by hydrogen and the morpholine
was changed to dimethyl amine to compensate for anticipated
induced fit movements during binding. For convenience, we refer
to these surrogate ligands as 13B (R sulfoxide) and 14B (S
sulfoxide).17
As seen in Figure 2B and C the surrogate ligands were predicted
to bind with very similar poses. The key difference between the
two ligand poses (Fig. 2D and E) was the vector of the sulfoxide
oxygen atom. For the R sulfoxide (13B) the oxygen atom points
towards the solvent. The S sulfoxide oxygen (14B) points into the
pocket in the direction of Ala104 and Phe97.18a Burial of the polar
oxygen atom of 14B into a hydrophobic region of the pocket is
expected to result in unfavorable electrostatics and contribute to
lower binding affinity.18b Based on this analysis we propose that
13 and 14 are the RS and SS sulfoxides, respectively.
Like changes made to the aryl ring of the acylsulfonamide, we
postulated that incorporation of an aromatic nitrogen into the
biphenyl moiety would decrease lipophilicity and potentially
increase solubility (Table 3). This might also offer the opportunity
to capitalize on the established plasticity5b of the P2 pocket of Bcl-
2 and Bcl-xL through perturbation of the orthogonality of the biaryl
moiety itself.10 Significant binding to both prosurvival proteins in
fluorescence polarization assays was observed for many modifica-
tions. However, pyrimidine 20 was only marginally active and
pyrazole 23 was completely inactive in the cellular assay, while a
20- to 40-fold decrease in activity was also observed for 21, 22,
and 24 relative to 1. No improvements in solubility were noted.
In contrast, appending 2-oxo dimethylacetamide and dimethyl-
aminoethoxy20 side chains at the ortho and meta positions relative
to the benzylic carbon maintained or improved binding and also
gave good activity in the RS4;11 cellular assay. Dimethylamine
27 (clogP = 10.8) was particularly potent and compared favorably
to 1.
In terms of parameterization and as shown in Figure 3, the com-
pounds described herein with clogP’s greater than nine (right of
red line) generally had improved cellular potency. Good RS4;11
activity was also secured when lowering lipophilicity compared
to Navitoclax (black line). Lipophilicity may not be the only predic-
tor of cellular activity, as compounds with PSA >170 (n = 2; Fig. 4)
were generally observed to be less likely to induce apoptosis. A
larger data set would help clarify this initial observation.
Select compounds were tested in cells overexpressing Bcl-2 and
Bcl-xL to validate the contributions of each prosurvival protein to
activity (Table 4). All compounds demonstrated good agreement
between RS4;11 cells and FDCP-1 cells overexpressing Bcl-2. This
is consistent with the assertion that activity in RS4;11 cells is dri-
ven by Bcl-2 inhibition.22 Four compounds (5, 13, 16 and 26) had
only weak activity against Bcl-xL and were largely selective for
Bcl-2. Dimethylamine 27 was the most potent compound against
all cell lines and compared favorably to both benchmark 1 and
Navitoclax in terms of binding efficiency (BEI = 8.21).
In general, compounds were prepared using a late stage
coupling (EDC, DMAP, DCM) of an acid and a known sulfonamide
such as depicted for 1 in Scheme 1.4,5a While some compounds
were derived from known acids, such as 2,11 21,19 and 22,19 the
synthesis of more esoteric moieties are described in Schemes
2–7. Both referenced methods and those detailed herein were used
to prepare any compound not explicitly discussed.
Oxetane 3 (Scheme 2) was prepared from commercially avail-
able tert-butyl sulfinamide 29. Lithium-halogen exchange of 30
with tert-butyl lithium, addition of the resulting anion to 29 and

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
3029
O
S
(A)
F3C
O
O
H
N
H
N
N
N
H
N
H
N
O
O
O
O
S
S
S
S
O
O
O
O
Modification for
docking analysis
N
S
N
S
*
*
Cl
13/14
Cl
13B/14B
(B)
(C)
(D)
(E)
Figure 2. (A) Structural modifications used to create surrogate ligands 13B/14B for rigid protein docking with Bcl-xL. (B) Docking pose of 13B (blue). (C) Docking pose of 14B
(magenta). (D) and (E) Close-up views 13B and 14B, respectively, which highlight the positioning of the sulfoxide oxygen atom relative to Phe97 and Ala104.18a
Table 3
Fluorescence polarization, cellular and solubility data for 20–28
Ring (R)
C
CF3
S O
O
O
H
N
A
B
E
D
S
H
N
O
N
N
N
S
N
N
N
H
O
N
O
G
F
N
O
O
O
O
O
N
N
N
N
H
I
N
Ring
O
X
X
Y
Solc
(lM)
a
a
b
Compd
R
X
Y
Bcl-2 FP IC50
(l
M)
Bcl-xL FP IC50
(lM)
RS4;11 EC50
(l
M)
20
A
B
C
D
E
F
G
H
I
N
Cl
Cl
Cl
H
0.021
<0.010
0.026
0.021
0.015
0.025
0.038
<0.010
<0.010
<0.010
ntd
3.98
<1
ntd
ntd
<1
<1
<1
2
2119
2219
23
CH
CH
CH
CH
CH
CH
CH
CH
0.201
0.374
>10
0.420
0.018
0.051
0.004
0.023
0.040
24
25
26
27
H
<0.010
<0.010
<0.010
<0.010
<0.010
Cl
Cl
Cl
Cl
<1
<1
28
<0.010
a
b
c
Fluorescence polarization assay.12
Caspase-GloÒ cellular assay.13
Equilibrium solubility (pH 7.4).14
nt = not tested.
d

3030
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
Table 4
RS4;11 and FDCP-1 cellular activity
a
b
c
Compd
RS4;11 EC50
M)
Bcl-2 FDCP-1 IC50
M)
Bcl-xL FDCP-1 IC50
M)
BEId
(
l
(
l
(l
Navitoclax 0.014
<0.015
0.014
0.340
0.434
0.545
0.045
0.187
0.004
0.022
0.060
0.064
2.09
1.72
2.82
0.226
2.09
0.033
0.138
8.06
8.46
7.15
7.22
6.51
7.43
6.99
8.21
7.42
1
0.011
0.177
0.090
0.504
0.018
0.051
0.004
0.023
5
13
16
25
26
27
28
a
b
c
Caspase-GloÒ cellular assay.13
Cellular assay with over-expression of Bcl-2.21
Cellular assay with over-expression of Bcl-xL.21
Figure 3. Cellular activity in RS4;11 as
a function of lipophilicity (clogP).
Compounds with Bcl-2 FP IC50 > 1.1 have been omitted. Compounds herein with a
clogP > 9 (right of red line) generally have improved cellular activity. The clogP of
Navitoclax (12.4) is shown as a black line.
d
BEI = Binding Efficiency Index = (RS4;11 pEC50 Â 1000)/MW.
deprotection with
a-chloroethyl chloroformate, and reprotection
with di-tert-butyl dicarbonate afforded bromide 39. Suzuki cou-
pling to install a vinyl moiety was followed by ozonolysis and oxi-
dation with potassium permanganate to provide the corresponding
benzoic acid. Simultaneous esterification and Boc-cleavage in the
presence of refluxing hydrochloric acid in methanol provided 40.
Reductive amination and hydrolysis gave acid 41.
Phosphine oxide 9 was prepared from dibromide 42 by first dis-
placing the benzylic bromide with diethylphosphite26 and then
using a Suzuki coupling to provide 43 (Scheme 4). Conversion of
the phosphonate to benzyl piperidine 44 in a manner analogous
to that described in Scheme 3 was followed by deprotection, addi-
tion to ethyl 4-fluorobenzoate, and hydrolysis to yield 45.
Sulfoxides 13 and 14 were generated starting with alkylation of
thiol 47 with bromide 46 (Scheme 5). Oxidation of 48 to the corre-
sponding sulfoxide and deprotection to give 49 was followed by
additon to ethyl p-fluorobenzoate. However, the only isolated
product was ethyl ester 50. The same chemistry using the sulfone
analog of 49 was successful.
Thioether 51 formed smoothly when 48 was deprotected and
added to ethyl p-fluorobenzoate. This ester was hydrolyzed and
carried on to provide 12. Oxidation of 51 with sodium periodate
under reflux conditions afforded the corresponding sulfoxide,
which was resolved using normal phase chiral HPLC. The faster
eluting sulfoxide ethyl ester provided 52a, which, upon coupling
with the triflone sulfonamide of Scheme 1, yielded 13.
Figure 4. Observed cellular (RS4;11) activity versus polar surface area (PSA).
Marked regions suggest possible preferred (blue circle) and potentially less
preferred (red box) PSA values for BH3 mimetics described herein with good cell
potency. PSA for Navitoclax = 133.4.
deprotection with dilute hydrochloric acid in methanol afforded
31. Addition to known sulfonyl chloride 325a followed by SNAr dis-
placement with chiral amine 3423 afforded the corresponding thio-
phenyl Boc carbamate. Boccleavage was accomplished by adding a
minimal amount of concentrated aqueous hydrochloric acid.
Reductive amination with commercially available aldehyde 36
then provided 3.
Phosphine oxide 8 was prepared starting from iodide 37. Palla-
dium-catalyzed coupling with diethyl phosphite in the presence of
potassium acetate24 and a subsequent three step conversion25 of
the resulting aryl phosphonate to its divinyl analog provided 38
(Scheme 3). Cyclization through the addition of benzyl amine,
Substituted piperidines 16–19 were prepared using a synthetic
strategy such as that depicted in Scheme 6. Palladium-catalyzed
coupling of 53 and 54 afforded 55 in low yield. In the case of 18
and 19, ethyl piperidine-4-carboxylate was added directly to tert-
butyl 4-fluorobenzoate in a method analogous to that described
CF3
O
O
S
O
H
N
S
H
N
O
OH
O
S
CF3
S O
O
N
O
O
O
H
N
S
N
N
H2N
N
N
S
O
N
O
1
Cl
Scheme 1. Retrosynthesis of 1.
Cl

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
3031
F
S
F
F
O
O
O
NH2
Br
N
F
H
N
SO2CF3
F
O
N
O
S
a, b
c
O
O
O
ClO2S
N
N
S
32
N
Boc
N
N
29
Boc
30
31
Boc
33
F
S
F
F
O
O
H
N
S
O
H
N
H2N
O
S
S
d,e
O
O
f
N
O
2
N
O
Cl
N
36
34
N
H
35
Scheme 2. Reagents and conditions: (a) t-butyllithium, THF, À78 °C, 52%; (b) 3 M HCl in MeOH, MeOH, 76%; (c) DIPEA, DCM/EtOAc, 49%; (d) DIPEA, DMF, 50 °C, 61%; (e) cHCl,
96%; (f) NaBH(OAc)3, DCE, 68%.
Br
O
OH
O
O
Br
Br
O
e-g
a-d
h-k
l,m
O
P
N
O
P
N
P
P
O
I
N
H
O
O
37
38
39
40
Cl
41
Scheme 3. Reagent and conditions: (a) (EtO)2HPO, Et3N, Pd(dppf)Cl2, KOAc, THF, 68 °C, 22 h, 76%; (b) TMSBr; (c) (COCl)2, DMF, CH2Cl2; (d) vinyl-MgBr, THF, À78 °C, 53% (three
steps); (e) BnNH2, H2O, 90 °C, 60%; (f) (i) -chloroethylchloroformate, toluene, reflux, (ii) MeOH, reflux; (g) Boc2O, Et3N, DCM, 35% (two steps); (h) vinyl boroxane–pyridine
a
complex, K2CO3, Pd(PPh3)4, 9:1 DME/H2O, 80 °C; (i) O3, MeOH, À78 °C, 97% (two steps); (j) KMnO4, acetone, 80%; (k) 3 M HCl in MeOH, reflux; (l) 36, NaBH(OAc)3, DCE, 50%
(two steps); (m) LiOH, THF/MeOH/H2O, 100 °C, 93%.
O
O
OH
O
N
P
P(OEt)2
Br
N
P
a,b
g-i
c-f
O
Br
Cl
Cl
42
43
44
Cl
45
Scheme 4. Reagents and conditions: (a) KN(TMS)2, (EtO)2HPO, THF, À78 °C, 96%; (b) p-Cl–C6H5B(OH)2, Pd(PPh3)4, K2CO3, 9:1 DME/H2O, 90 °C, 93%; (c) TMSBr; (d) (COCl)2,
DMF, CH2Cl2; (e) vinyl-MgBr, THF/Et2O, À78 °C, 45% (three steps); (f) BnNH2, H2O, 90 °C, 60%; (g) (i)
F–C6H4CO2Et, DIPEA, DMSO, 120 °C, 41%; (i) LiOH, THF/MeOH/H2O, 100 °C, 91%.
a-chloroethylchloroformate, toluene, reflux, (ii) MeOH, reflux, 72%; (h) 4-
in Schemes 4 and 5. Alkylation of 55 with bromide 42 was followed
by installation of the 4-chloro-phenyl moiety, and subsequent
acidic deprotection of the tert-butyl ester furnished 56.
Phenyl ethers 25–28 were prepared according to Scheme 7.
Thus reductive amination of commercially available aldehyde 57
and piperazine 58 followed by Suzuki coupling and subsequent
alkylation with 2-chloro dimethylacetamide afforded amide 59.
Acid precursors of 25 and 26 were subsequently prepared via
hydrolysis of the appropriate analog using either potassium tri-
methylsilanoate (25) or lithium hydroxide (26). Conversion of 59
to the corresponding dimethylamine was smoothly effected using
a Rh-catalyzed methodology.27 Hydrolysis then afforded 60.
In summary, we have identified several compounds with potent
activity in vitro Bcl-2/Bcl-xL binding and cellular assays. Achieving
cellular activity was challenging, and, given the high protein bind-
ing exhibited by ABT-737 and Navitoclax,5b we suspect that this

3032
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
H
because of both the lipophilic nature of this class of BH3 mimetics
and the debatable impact of small changes on the physical proper-
ties of molecules of this size. Sulfoxide 13 was intriguing because,
in addition to an improvement in solubility, docking studies
suggest that potency resides with the R sulfoxide. Lastly, of all
compounds tested, dimethylamine 27 was the most potent Bcl-2/
Bcl-xL inhibitor in RS4;11 and FDCP-1 cell assays, and both potency
and binding efficiency compared favorably to 1 and Navitoclax.
Boc
N
N
S
O
O
Boc
N
HS
Br
a,b
Cl
c,d
e
S
O
N
Br
47
46
Cl
49
48
50
O
O
O
O
OH
References and notes
1. Llambi, F.; Green, D. R. Curr. Opin. Genet. Dev. 2011, 21, 12.
2. Recent review and leading references: Kelly, P. N.; Strasser, A. Cell Death Differ.
2011, 18, 1414.
3. (a) Kang, M. H.; Reynolds, C. P. Clin. Cancer Res. 2009, 15, 1126; (b) Vogler, M.;
Dinsdale, D.; Dyer, M. J. S.; Cohen, G. M. Cell Death Differ. 2009, 16, 360.
4. Bruncko, M.; Oost, T. K.; Belli, B. A.; Ding, H.; Joseph, M. K.; Kunzer, A.;
Martineau, D.; McClellan, W. J.; Mitten, M.; Ng, S.-C.; Nimmer, P. M.; Oltersdorf,
T.; Park, C.-M.; Petros, A. M.; Shoemaker, A. R.; Song, X.; Wang, X.; Wendt, M.
D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmer, S. W. J. Med. Chem. 2007, 50,
641.
d,e
f-h
Cl
N
N
*
S
S
Cl
51
52a/52b
5. (a) Park, C.-M.; Bruncko, M.; Adickes, J.; Bauch, J.; Ding, H.; Kunzer, A.; Marsh, K.
C.; Nimmer, P.; Shoemaker, A. R.; Song, X.; Tahir, S. K.; Tse, C.; Wang, X.; Wendt,
M. D.; Yang, X.; Xhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med.
Chem. 2008, 51, 6902; (b) Wendt, M. D. Expert Opin. Drug Discov. 2008, 3, 1123.
6. (a) Gandhi, L.; Camidge, D. R.; Ribeiro de Oliveira, M.; Bonomi, P.; Gandara, D.;
Khaira, D.; Hann, C. L.; McKeegan, E. M.; Litvinovich, E.; Hemken, P. M.; Dive, C.;
Enschede, S. H.; Nolan, C.; Chiu, Y.-L.; Busman, T.; Xiong, H.; Krivoshik, A. P.;
Humerickhouse, R.; Shapiro, G. I.; Rudin, C. M. J. Clin. Oncol. 2011, 29, 909; (b)
Tse, C.; Shoemaker, A. R.; Adickes, J.; Anderson, M. G.; Chen, J.; Jin, S.; Johnson,
E. F.; Marsh, K. C.; Mitten, M. J.; Nimmer, P.; Roberts, L.; Tahir, S. K.; Xiao, Y.;
Yang, X.; Zhang, H.; Fesik, S.; Rosenberg, S. H.; Elmore, S. W. Cancer Res. 2008,
68, 2341; (c) Yecies, D.; Carlson, N. E.; Deng, J.; Letai, A. Blood 2010, 115, 3304;
(d) Wilson, W. H.; O’Connor, O. A.; Czuczman, M. S.; LaCasce, A. S.; Gerecitano, J.
F.; Leonard, J. P.; Tulpule, A.; Dunleavy, K.; Xiong, H.; Chiu, Y.-L.; Busman, T.;
Elmore, S. W.; Rosenberg, S. H.; Krivoshik, A. P.; Enschede, S. H.;
Humerickhouse, R. A. Lancet Oncol. 2010, 11, 1149.
Scheme 5. Reagents and conditions: (a) K2CO3, DMF, 85 °C, 100%; (b)
p-Cl–C6H5B(OH)2, Pd(PPh3)4, K2CO3, 9:1 DME/H2O, 90 °C, 93%; (c) m-CPBA, CH2Cl2,
91%; (d) cHCl, 83–90%; (e) DIPEA, DMSO, 120 °C, 50: 41%, 51: 81%; (f) NaIO4, 3:1
MeOH/H2O, reflux, 83%; (g) Chiral separation: AD column, 50% hexanes in (1:1
methanol/isopropanol) containing 0.1% diethylamine; (h) LiOH, THF/MeOH/H2O,
100 °C, 74–77%.
O
OH
O
O
O
O
Br
H
N
a
b-d
N
7. (a) Kaluzhny, Y.; Yu, G.; Sun, S.; Toselli, P. A.; Nieswandt, B.; Jackson, C. W.;
Ravid, K. Blood 2002, 100, 1670; (b) Schoenwaelder, S. M.; Jarman, K. E.;
Gardiner, E. E.; Hua, M.; Qiao, J.; White, M. J.; Josefsson, E. C.; Alwis, I.; Ono, A.;
Willcox, A.; Andrews, R. K.; Mason, K. D.; Salem, H. H.; Huang, D. C. S.; Kile, B.
T.; Roberts, A. W.; Jackson, S. P. Blood 2011, 118, 1663.
N
Br
Br
CN
CN
CN
8. Example references for compounds selective for Bcl-2 over Bcl-xL: (a) Petros, A.
M.; Huth, J. R.; Oost, T.; Park, C.-M.; Ding, H.; Wang, X.; Zhaing, H.; Nimmer, P.;
Mendoza, R.; Sun, C.; Mack, J.; Walter, K.; Dorwin, S.; Gramling, E.; Ladror, U.;
Rosenberg, S. H.; Elmore, S. W.; Fesik, S. W.; Hajduk, P. J. Bioorg. Med. Chem. Lett.
2010, 20, 6587; (b) Souers, A. J.; Leverson, J. D.; Boghaert, E. R.; Ackler, S. L.;
Catron, N. D.; Chen, J.; Dayton, B. D.; Ding, H.; Enschede, S. H.; Fairbrother, W. J.;
Huang, D. C. S.; Hymowitz, S. G.; Jin, S.; Khaw, S. L.; Kovar, P. J.; Lam, L. T.; Lee, J.;
Maecker, H. L.; Marsh, K. C.; Mason, K. D.; Mitten, M. J.; Nimmer, P. M.;
Oleksijew, A.; Park, C. H.; Park, C.-M. Nat. Med. 2013, 19, 202; (c) Porter, J.;
Payne, A.; de Candole, B.; Ford, D.; Hutchinson, B.; Trevitt, G.; Turner, J.;
Edwards, C.; Watkins, C.; Whitcombe, I.; Davis, J.; Stubberfield, C. Bioorg. Med.
Chem. Lett. 2009, 19, 230; (d) Bruncko, M.; Ding, H.; Doherty, G. A.; Elmore, S.
W.; Hasvold, L.; Hexamer, L.; Kunzer, A. R.; Mantei, R. A.; McClellan, W. J.; Park,
C. H.; Park, C.-M. J.; Sullivan, G. M.; Tao, Z.-F.; Wang, G. T.; Wang, L.; Wang, X.;
Wendt, M. D. WO 065,865 A2, 2010.
53
54
55
42
Cl
56
Scheme 6. Reagents and conditions: (a) Pd2dba3, Cs2CO3, tert-butyl-XPhos, DMF,
90 °C, 22%; (b) LiHMDS, TMEDA, THF, À78 °C, 45%; (c) p-Cl–C6H4B(OH)2, Na2CO3,
toluene, reflux, 60%; (d) TFA, 99%.
O
OH
O
O
O
O
9. Shah, O. J.; Shen, Y.; Lin, X.; Anderson, M.; Huang, X.; Li, J.; Leiming, L. WO
068,863 A1, 2011.
10. Lee, E. F.; Czabotar, P. E.; Smith, B. J.; Deshayes, K.; Zobel, K.; Colman, P. M.;
Fairlie, W. D. Cell Death Differ. 2007, 14, 1711.
O
OH
N
N
N
N
d,e
a-c
N
N
11. For benzamidine analogs containing a nitro group rather than a triflone, see:
Elmore, S. W.; Bruncko, M.; Park, C.-M. US 0,272,744 A1, 2005. For quinazoline
nitro and triflone variants, see: Sleebs, B. E.; Czabotar, P. E.; Fairbrother, W. J.;
Fairlie, W. D.; Flygare, J. A.; Huang, D. C. S.; Kersten, W. J. A.; Koehler, M. F. T.;
Lessene, G.; Lowes, K.; Parisot, J. P.; Smith, B. J.; Smith, M. L.; Souers, A. J.; Street,
I. P.; Yang, H.; Baell, J. B. J. Med. Chem. 2011, 54, 1914.
12. Bcl-xL and Bcl-2 FP binding affinity data were determined according to the
procedures of Wang, J.-L.; Zhang, Z.-J.; Choksi, S.; Sjam, S.; Lu, Z.; Croce, C. M.;
Alnemri, E. S.; Komgold, R.; Huang, Z. Cancer Res. 2000, 60, 1498. Procedure:
Fluorescence polarization (‘FP’) assays were developed using c-terminal 6XHIS
tagged Bcl-2 (aa 1–204) and a C-terminal 6XHIS tagged Bcl-xL (aa 1–209). The
O
O
N
Br
O
57
N
H
Cl
Cl
58
59
60
Scheme 7. Reagents and conditions: (a) NaBH(OAc)3, DCE, 98%; (b) 4-Cl–C6H4-
B(OH)2, Pd(PPh3)4, K2CO3, 9:1 DME/H2O, 80 °C, 54%; (c) ClCH2C(O)NMe2, NaH, DMF,
92%; (d) (PPh3)2(CO)RhH, Ph2SiH2, THF, 54%; (e) LiOH, THF/MeOH/H2O, 55 °C, 48%.
tracer was
a synthetic BH-3 peptide (Bim) conjugated to Fluorescein
isothiocyanate (FITC-DLRPEIRIAQELRRIGDEFNETYTRR). Dilutions of either
Bcl-2 (1.3 nM) or Bcl-xL (0.8 nM) were added to serial dilutions of antagonist
and incubated for one hour prior to the addition of 2 nM of fluorescent peptide
tracer (Anaspec, Fremont, CA) in assay buffer. Final assay buffer conditions
were 20 mM HEPES, pH 7.5, 1 mM DTT, 0.005% Tween-20 and 50 mM NaCl.
Samples were read after incubation for 20 min. Fluorescence polarization
values were plotted as a function of antagonist concentration, and IC50’s are the
average of at least two experiments. Values of 10 nM or less are reported as
was at least partly due to non-specific binding and poor membrane
permeability. Based on an analysis of the compounds of this Letter,
parameter-based design of Bcl-2/Bcl-xL inhibitors appears well
served when physical properties such as PSA and clogP are consid-
ered. Significant increases in solubility through modification of
acyl sulfonamide, biphenyl, and piperidine moieties were elusive
<0.010
lM to account for assay limitations when using the above
concentrations for very potent compounds.

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3026–3033
3033
13. Apoptosis was assessed using Promega’s Caspase-GloÒ 3/7 Assay Kit
(Catalog#G8092, Madison, WI) following the manufacturer’s directions.
Protocol: RS4;11 cells were plated in NUNC white opaque 96-well plates
(Catalog#353296, Rochester, NY) at 10,000 cells per well in Sigma’s RPMI
Media (Catalog #R7509, St. Louis, Mo) containing 10% FBS (Thermofisher
Catalog#SH30396 Waltham, MA). After 16 h, cells were treated in duplicate
with compound and incubated at 37 °C for 6 h prior to caspase detection.
DMSO was used as minimum caspase activity control, while the addition of
ABT-263 was used as a measure of maximum caspase activity. An equal
volume of Caspase-Glo 3/7 reagent was added to each well and incubated for
1 h prior to reading on a Tecan Ultra luminometer (Tecan, Durham, NC) to
quantitate luminescence. EC50’s are the average of at least two experiments.
14. Alelyunas, Y. W.; Liu, R.; Pelos-Kilby, L.; Shen, C. Eur. J. Pharm. Sci. 2009, 37, 172.
15. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
ligand–protein interaction energy for 13B is energetically more favorable
(À1053.92 kJ/mol) compared to 14B (À1002.79 kJ/mol). A similar trend was
observed for the electrostatic component of the interaction energy with 13B
being more stable (À759.05 kJ/mol) than 14B (À707.05 kJ/mol). The van der
Waals interaction energy was more comparable for 13B (À294.87 kJ/mol) and
14B (À295.74 kJ/mol).
19. ABT-737 analogs with these heteroatom modifications have been reported. In:
Bruncko, M.; Ding, H.; Elmore, S.; Kunzer, A.; Lynch, C. L.; McClellan, W.; Park,
C.-M.; Petros, A.; Song, X.; Wang, X.; Tu, N.; Wendt, M.; Shoemaker, A.; Mitten,
M. U.S. 0,072,860 A1, 2007.
20. For an example of dimethylaminoethoxy side chains applied to selective Bcl-2
inhibitors, see: Bruncko, M.; Ding, H.; Doherty, G. A.; Elmore, S. W.; Hasvold, L.;
Hexamer, L.; Kunzer, A. R.; Mantei, R. A.; McClellan, W. J.; Park, C. H.; Park, C.-
M.; Petros, A. M.; Song, X.; Souers, A. J.; Sullivan, G. M.; Tao, Z.-F.; Wang, G. T.;
Wang, L.; Wang, X.; Wendt, M. D.; Hansen, T. M. U.S. Pat. Appl. Publ. 0,298,321
A1, 2010.
16. For nitro variants of 4,4-substituted piperidines, see Ref. 4.
17. These modifications preserved the similarities and differences of these two
ligands, allowing rigid protein docking on
a
publicly available protein
21. A cell viability assay was used to determine the relative inhibition of BCL-2 and
BCL-xL test compounds. FDCP-1 cells (parental line from DSMZ) were
engineered by retroviral infection to over-express either human BCL-2 or
human BCL-xL. Both these cell lines, as well as the parental, were maintained at
37 °C in RPMI/10% FBS supplemented with 5 ng/mL IL-3. Prior to compound
treatment, cells were first washed in No IL-3 media (otherwise identical to
media above), then incubated for 48 h w/o IL3, during which time the parental
cells died out. The surviving FDCP-1 BCL-2 and FDCP-1 BCL-xL cells were then
counted, resuspended in fresh No IL-3 media, and plated at approximately 5–
7 K cells/well in 384 w microplates containing the test compounds, including
the DMSO control (untreated wells), in triplicate. The cells were then incubated
for an additional 24 h at 37 °C. After this incubation, a matching volume of Cell
Titer Glo reagent was added to each well, the plate shaken at RT for at least
15 min, and the luminescence read using a Tecan Ultra 384 (Magellan). IC50’s
structure. Method: The crystal structure of a quinazoline ligand bound to Bcl-
xL (PDB code-3QKD) was chosen for docking analysis due to the similarity of
the cognate ligand with 13 and 14. The protein complex was downloaded from
the protein data bank (www.rcsb.org) and prepared for docking using the
protein preparation workflow in Maestro (Schrodinger LLC, Portland, OR).
Ligands for docking were also prepared in Maestro using the OPLS2005/GBSA
setting for ligand minimization. Pose validation studies with the cognate
ligand were carried out in both Glide (Schrodinger LLC, Portland, OR) and Gold
(CCDC, Cambridge, UK). Default settings failed to produce desirable results and
multiple constraints were evaluated. Satisfactory results were obtained in Gold
using four hydrophobic constraints defined based on the pose of the cognate
ligand. The hydrophobic constraints were 2 A spheres defined around the
centroids of the (a) phenyl ring connected to the thioether linkage, (b) phenyl
ring with the nitro substitution, (c) piperidine ring and (d) the terminal phenyl
ring of the biphenyl moiety. The automatic GA settings were used with 200%
search efficiency and the chemscore scoring function was utilized for docking.
Energetic analysis was performed using the Embrace module of Macromodel
(Schrodinger LLC, Portland, OR) in the interaction energy mode using the
OPLS2005 potential.
were determined and reported as
a percentage of surviving cells in the
untreated control wells. Matching +IL3 controls were treated in the same
fashion, but in IL3-replete media, to verify that the compounds had minimal to
no effect under those conditions.
22. Robinson, B. W.; Behling, K. C.; Gupta, M.; Zhang, A. Y.; Moore, J. S.; Bantly, A.
D.; Willman, C. L.; Carroll, A. J.; Adamson, P. C.; Barrett, J. S.; Felix, C. A. Br. J.
Haematol. 2007, 141, 827.
23. Wendt, M. D.; Wang, S.; Kunzer, A.; McClellan, W. J.; Bruncko, M.; Oost, T. K.;
Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer, P. M.; Ng, S.-C.; Shoemaker, A. R.;
Petros, A. M.; Oleksijew, A.; Marsh, K.; Bauch, J.; Oltersdorf, T.; Belli, B. A.;
Martineau, D.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med. Chem. 2006,
49, 1165.
24. Kalek, M.; Jezowska, M.; Stawinski, J. Adv. Synth. Catal. 2009, 351, 3207.
25. Close, J.; Grimm, J.; Heidebrecht, Jr., R. W.; Kattar, S.; Miller, T. A.; Otte, K. M.;
Peterson, S.; Siliphaivanh, P.; Tempest, P.; Wilson, K. J.; Witter, D. J. WO
010,985 A2, 2008.
26. Snyder, S. A.; Sherwood, T. C.; Ross, A. G. Angew. Chem., Int. Ed. 2010, 49, 5146.
27. Kuwano, R.; Takahashi, M.; Ito, Y. Tetrahedron Lett. 1998, 39, 1017.
18. (a) The Bcl-2 residues corresponding to Bcl-xL residues Phe97 and Ala104 are
Phe101 and Asp108, respectively. Based on the observation of available Bcl-2
structures, the Asp108 residue lies in between two helices, a region which is
highly plastic, reorganizing itself in response to ligand binding. In the apo form
(PDB code-1GJH) the residue points into the pocket but shows side chain (PDB
code-1YSW) and backbone movements (PDB code-2O2F)] away from the
ligand in response to ligand binding. The acid residue in Bcl-2 may be more
rigid and therefore causes greater energetic destabilization due to lone pair
clashes between the acid and sulfoxide with the S-sulfoxide than the
R-sulfoxide, leading to the >10 fold difference in binding affinity between
Bcl-2 and Bcl-xL.
(b) Energetic analysis of the protein–ligand complex showed that the