Quinazoline sulfonamides as dual binders of the proteins B-cell lymphoma 2 and B-cell lymphoma extra long with potent proapoptotic cell-based activity

Article abstract of DOI:10.1021/jm101596e

ABT-737 and ABT-263 are potent inhibitors of the BH3 antiapoptotic proteins, Bcl-x_L and Bcl-2. This class of putative anticancer agents invariantly contains an acylsulfonamide core. We have designed and synthesized a series of novel quinazoline-based inhibitors of Bcl-2 and Bcl-x_L that contain a heterocyclic alternative to the acylsulfonamide. These compounds exhibit submicromolar, mechanism-based activity in human small-cell lung carcinoma cell lines in the presence of 10% human serum. This comprises the first successful demonstration of a quinazoline sulfonamide core serving as an effective benzoylsulfonamide bioisostere. Additionally, these novel quinazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.

Full text of DOI:10.1021/jm101596e

ARTICLE
pubs.acs.org/jmc
Quinazoline Sulfonamides as Dual Binders of the Proteins B-Cell
Lymphoma 2 and B-Cell Lymphoma Extra Long with Potent
Proapoptotic Cell-Based Activity
Brad E. Sleebs,†,‡ Peter E. Czabotar,†,‡ Wayne J. Fairbrother,§ W. Douglas Fairlie,†,‡ John A. Flygare,§
David C. S. Huang,†,‡ Wilhelmus J. A. Kersten,†,‡ Michael F. T. Koehler,§ Guillaume Lessene,†,‡
Kym Lowes,†,‡ John P. Parisot,†,‡ Brian J. Smith,†,‡ Morey L. Smith,|| Andrew J. Souers,|| Ian P. Street,†,‡
Hong Yang,†,‡ and Jonathan B. Baell*,†,‡
†The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
‡Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
§Departments of Discovery Chemistry and Protein Engineering, Genentech, Inc. 1 DNA Way, South San Francisco, California 94080,
United States
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park,
Illinois 60064, United States
S Supporting Information
b
ABSTRACT: ABT-737 and ABT-263 are potent inhibitors of
the BH3 antiapoptotic proteins, Bcl-xL and Bcl-2. This class of
putative anticancer agents invariantly contains an acylsulfona-
mide core. We have designed and synthesized a series of novel
quinazoline-based inhibitors of Bcl-2 and Bcl-xL that contain a
heterocyclic alternative to the acylsulfonamide. These com-
pounds exhibit submicromolar, mechanism-based activity in
human small-cell lung carcinoma cell lines in the presence of
10% human serum. This comprises the first successful demon-
stration of a quinazoline sulfonamide core serving as an effective
benzoylsulfonamide bioisostere. Additionally, these novel qui-
nazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.
’ INTRODUCTION
interacting killer (Bik), Bcl-2 interacting mediator (Bim), Bcl-2
modifying factor (Bmf), Harakiri-Bcl-2 interacting protein
(Hrk), phorbol-12-myristate-13-acetate-induced protein 1
(Noxa), and Bcl-2 binding component 3 (Puma) and are
upregulated in response to various cellular stresses (e.g., DNA
damage, growth factor deprivation). Unlike Bax/Bak and the
prosurvival proteins that adopt a well-defined, conserved helical-
bundle three-dimensional structure, the BH3 only proteins
(which are so-called because they possess only the BH3 motif),
are intrinsically unstructured,2 with the exception of Bid.
Dysfunctional apoptosis is a hallmark of most, if not all,
cancers as it allows damaged cells that would otherwise be
removed to survive and, in some cases, proliferate.3 Various
conventional anticancer chemo- and radiotherapy treatment
regimes work by up-regulating BH3-only proteins to trigger
apoptosis in tumor cells, and their effectiveness is often blunted
by the overexpression of prosurvival proteins, a common apop-
totic defect in many cancers.4,5 In addition, their efficacy is also
diminished by upstream defects in the pathway, such as
B-cell lymphoma 2 (Bcl-2) family proteins comprise two
classes with diametrically opposed functions—those that are
prosurvival and those that are proapoptotic—and their interplay
modulates the balance between cell survival and cell death.1
Prosurvival proteins include Bcl-2, B-cell lymphoma extra long
(Bcl-xL), B-cell lymphoma w (Bcl-w), myeloid cell leukemia 1
(Mcl-1), and B-cell lymphoma 2 related protein A1 (A1), and
these lie upstream of the pro-apoptotic proteins Bcl-2 associated
protein X (Bax) and Bcl-2 antagonist/killer (Bak), which med-
iate cell death through permeabilization of the mitochondrial
outer membrane. This results in liberation of cytochrome c (and
other apoptogenic factors) into the cytosol, which activates the
caspase cascade en route to apoptosis. Despite their opposing
functions, all members of this protein family are structurally
related, possessing four Bcl-2 homology (BH) motifs: BH1,
BH2, BH3, and BH4. Prosurvival proteins bind to Bax and
Bak, thus neutralizing their ability to induce cell death. This brake
can be released when an upstream subclass of proapoptotic
proteins, called the BH3-only proteins, bind to prosurvival
proteins. The BH3-only proteins include Bcl-2-associated death
promotor (Bad), BH3 interacting death domain (Bid), Bcl-2
Received: December 15, 2010
Published: March 02, 2011
r
2011 American Chemical Society
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Figure 1. Structures of known BH3 mimetics with pro-apoptotic activity, acylsulfonamides 1 and 2, and the proposed quinazoline isosteric scaffold
(boxed).
Figure 2. Structures of 4-fluorobiaryl compound 3 and the targeted model quinazoline sulfonamide 4.
inactivating mutations of p53 (found in ∼50% of all human
cancers), which is required for transcriptional up-regulation of
prodeath proteins Puma and Noxa in response to these DNA-
damaging insults. Small-molecule mimetics of BH3-only proteins
could potentially overcome both of these issues by neutralizing
the prosurvival proteins present, even when they are produced in
excess, allowing for the release of Bax/Bak and ensuing apoptosis
in a manner that bypasses the need for activation of p53.6
Intense interest in small-molecule BH3 mimetics is reflected
in numerous recent publications. However, demonstration of
mechanism-based induction of apoptosis has generally not been
reported.6-8 An exception is the acylsulfonamide series of
inhibitors from Abbott Laboratories, exemplified by 1 (ABT-
737, Figure 1), which was reported to inhibit Bcl-xL, Bcl-2, and
Bcl-w with an IC50 of less than 1 nM, greater than 3 orders of
magnitude more potently than previously described small-mole-
cule inhibitors.9 This compound exhibited single-agent mechan-
ism-based killing of lymphoma and small cell lung carcinoma
(SCLC) cell lines as well as primary patient-derived chronic
lymphocytic leukemia cells. In murine xenograft models, 1
improved survival, caused regression of established tumors, and
produced cures in a high percentage of the animals. A related
compound, 2 (Navitoclax or ABT-263, Figure 1), was subse-
quently prepared that demonstrates oral availability across multi-
ple species, and that has now entered phase II clinical trials.10,11
A hallmark of all members of this class of drugs is the presence
of an acylsulfonamide core. This moiety represents a potential
metabolic liability and indeed has been used in prodrug ap-
proaches for sulfonamides where the acyl group is cleaved
in vivo.12 For this reason, we felt that there would be considerable
interest in the identification of an isosteric replacement of the
acylsulfonamide. We postulated that a quinazoline sulfonamide
may serve such a purpose while being topographically appro-
priate to maintain high affinity binding to Bcl-2 family proteins.
Herein, we report the synthesis of a series of quinazoline
sulfonamides, their binding affinities for Bcl-xL and Bcl-2, and
their mechanism-based cell killing activity.
’ RESULTS AND DISCUSSION
To explore the utility of the quinazoline isostere, we first
undertook the synthesis of a compound based on acylsulfona-
mide 3 (Figure 2). Compound 3 is an early generation Bcl-xL
inhibitor13 from Abbott that served as a starting point for the
generation of 1. We reasoned that the demonstration of adequate
binding to Bcl-xL by quinazoline isostere 4 would substantiate the
more intensive synthesis required for the quinazoline-based
surrogates of 1.
As shown in Scheme 1, synthesis of 4 was initiated via Suzuki
coupling of 2-amino-4-chlorobenzonitrile with 4-fluorophenyl-
boronic acid to afford intermediate 6 in an unoptimized yield of
56%. The formation of quinazoline 7 was then accomplished
through condensation with formamidine acetate at 120 °C. Acid-
mediated hydrolysis followed by chlorination afforded the
4-chloroquinazoline 9, which was then coupled with sulfonamide
1013 to afford the quinazoline sulfonamide 4 in good yield.14
The affinities of compounds 3 and 4 for Bcl-xL were then
determined via an amplified luminescent proximity homoge-
neous assay (AlphaSCREEN), based on competition between
test compound and Bim for Bcl-xL, affording IC50 values of 1.5
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Scheme 1. Synthesis of the 4-Fluorophenylquinazoline Sulfonamide 4a
a Reagents and conditions: (a) 4-Fluorophenylboronic acid, K2CO3, TBAB, Nꢀajera's catalyst, TBAB, K2CO3, reflux, 2 h [water] 56%. (b) Formamidine
acetate, MeOCH2CH2OH, 120 °C, 5 h, 75%. (c) 5 N aqueous HCl, reflux, 30 min, 92%. (d) SOCl2, CHCl3, cat. DMF, 99%. (e) DMF, K2CO3,
85 °C, 68%.
and 3 μM, respectively. This result provided initial validation of
the quinazoline design and prompted the synthesis of the more
elaborate quinazoline-bearing analogue of 1 (21, Scheme 2). As
shown in Scheme 2, regioselective nitro displacement of 2,4-
dinitrobenzonitrile with N-Boc piperazine and subsequent acid-
mediated deprotection afforded intermediate 13, which was then
alkylated with 2-bromomethylbromobenzene to furnish 14. The
4-chlorobenzene moiety was then installed via Suzuki coupling
conditions to afford intermediate 15. Iron-mediated nitro-reduction
followed by quinazoline formation as described afforded 17. Sub-
sequent hydrolysis of the 4-amino group and chlorination using
thionyl chloride furnished the desired 4-chloroquinazoline 19 in high
overall yield. Finally, base-mediated coupling with sulfonamide 2015
afforded the desired target 21 after HPLC purification.
In the AlphaSCREEN competition assay, quinazoline 21
returned an IC50 against Bcl-xL of 3 nM, as compared with that
for 1 of 1 nM (data not shown). To determine binding constants,
surface plasmon resonance (SPR) studies were undertaken, and
the results are shown in Figure 3a for 21, along with those for 1 in
Figure 3b. The binding kinetics of 21 and 1 were characterized by
extremely slow dissociation rates and returned kinetic KD values
of 4.2 and 0.37 nM, respectively, in broad agreement with the
AlphaSCREEN competition assay data. As can be observed in
Figure 3a, compound 21 exhibits a slightly decreased association
rate and increased dissociation rate as compared with 1
(Figure 3b), explaining the slight decrease in overall binding
affinity. By comparison, the 26-mer Bim BH3 peptide exhibits a
binding constant of 28 pM and is therefore 150-fold more potent
than compound 21, a result of both extremely high association
and slow dissociation rates (data not shown).
resolution (Figure 4a,b). As demonstrated in Figure 4b, 21 binds
to Bcl-xL in a manner similar to 19,16 with a high degree of overlap
between the two molecules. The chlorophenyl group of both
compounds projects identically and deeply into the P2 hydro-
phobic pocket, and the phenylthio-tethered phenylsulfonamide
similarly occupies the P4 hydrophobic pocket. Notably, however,
an electrostatic interaction between the quinazoline endocyclic nitro-
gen atom in the 1 position and the hydroxyl group of Tyr101 is evident
(Figure 4b), providing an additional specific point of contact that is not
observed in the corresponding complex of 1 and Bcl-xL.
To induce apoptosis in wild-type mouse embryonic fibroblasts
(MEFs), both Bcl-xL and Mcl-1 must be neutralized.17 Acylsul-
fonamide 1 has low affinity for Mcl-1 and thus fails to induce
apoptosis in cell lines expressing this protein. Therefore, to
fl/fl
investigate cell-based activity, wild-type (mcl-1 ) or Mcl-1-
deficient (mcl-1-/-) mouse MEFs were treated with varying
concentrations of either 1 or quinazoline 21 in the presence of
10% fetal bovine serum (Figure 5). Both 1 and quinazoline 21
potently killed MEFs lacking Mcl-1, with respective EC50 values
of 51 and 110 nM. However, they were much less potent against
wild-type MEFs, and 21 returned an EC50 of 5.8 μM, while 1 did
not show appreciable cytotoxicity even at the highest dose tested
(10 μM). This selectivity for inducing apoptosis in Mcl-1-
deficient but not wild-type MEFs is consistent with the high
affinity of these compounds for Bcl-xL but not Mcl-1. This is an
important observation because such evidence of mechanism-
based cellular activity in other reported BH3 mimetics is
generally lacking. Indeed, where tested, other reported BH3
mimetics appear to induce cell killing in a nonspecific manner.8
The quinazoline system reported here therefore represents only
the second known class of Bcl-2 family protein inhibitors shown
to induce mechanism-based cell death.
We then assessed the selectivity profile of 21 against a panel of
Bcl-2 prosurvival family proteins using an SPR-based solution
competition assay. As demonstrated in Table 1, 21 is a potent,
dual Bcl-xL/Bcl-2 competitive ligand with no measured affinity
for Mcl-1. This binding profile of 21 with respect to Bcl-2 and
Bcl-xL is similar to 1, while in contrast, the affinity of 21 for Bcl-w
is significantly less (∼10-fold higher IC50) than 1.
In the course of this work, a subsequent series of publications
by Abbott Laboratories detailed the synthesis and characteriza-
tion of a related Bcl-2/Bcl-xL inhibitor, 2 (Figure 1). These
publications reported that distinct modular changes allowed for
retention of mechanism-based cell killing via Bcl-2 family mem-
ber inhibition while conferring enhanced oral bioavailability in
preclinical species. Specifically, the modifications included the
To investigate the binding interactions of 21 with Bcl-xL, a
complex was crystallized, and the structure determined to 2.0 Å
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Figure 3. SPR study of (a) quinazoline 21 and (b) 1 with Bcl-xL. Representative sensorgrams for direct binding assays were performed on the Biacore
S51 with 1 (injections of 270, 90, 30, 10, and 3.3 nM) and 21 (injections of 500, 125, 31.25, 7.8, and 1.95 nM). The fine black line over each colored
sensorgram is the fit (one-to-one binding with mass transport limitations) provided by the Biacore S51 Evaluation Version 1.2.1 data analysis software.
Sensorgrams are characterized in both cases by extremely slow off-rates as observed in the trace from T = 100 s onward. Listed for each sensorgram are
the derived ka, kd, kt (mass transport constant: RU are response units, and MW is molecular mass in Dalton), and kinetic KD. All values are (50%.
Scheme 2. Synthesis of the Elaborated Quinazoline Sulfonamide 21a
a Reagents and conditions: (a) N-Boc-piperazine, DMSO, 25 °C, 72 h, 56%. (b) TsOH, 2 h [MeCN], 83%. (c) 2-Bromobenzyl bromide, Et3N [iPrOH],
93%. (d) 4-Chlorophenylboronic acid, PdCl2(PPh3)2, Na2CO3, [DME/EtOH/H2O], 81%. (e) Fe 60 °C, 40 min, [AcOH], 85%. (f) Formamidine
acetate, MeOCH2CH2OH, 120 °C, 6 h, 81%. (g) AcOH, concentrated HCl, 130 °C, 9 h. (h) SOCl2, CHCl3, cat DMF, 12 h, 80%. (i) DMF, K2CO3, 85 °C.
saturation of the western biphenyl group, replacement of the
nitro moiety with a trifluoromethylsulfonyl group, and variation
of the eastern basic amine tail. These reports prompted us to
target the synthesis of a discrete set of additional quinazoline-
based isosteres shown in Figure 6.
The modular synthesis of these analogues allowed for the
initial preparation of the quinazoline core, followed by the
appropriate incorporation of building blocks described pre-
viously by the Abbott group.10,15,18 To this end, 12 was reduced
and converted to the amino quinazoline 36 in high yield by
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treatment with formamidine acetate (Scheme 3). Acid hydrolysis
of the Boc protecting group followed by alkylation of the
piperazine 24 with halide synthons 22 and 23 proceeded
smoothly to afford 18 and 37, respectively.
Chlorination of these 4-hydroxyquinazolines proved trouble-
some, and purification could not be performed on silica chro-
matography without significant degradation. However, the use of
optimized reaction conditions followed by purification via alu-
mina chromatography returned good yields of target products 19
and 38. The coupling reactions of the various sulfonamide
fragments with the corresponding chloroquinazoline intermedi-
ates also proved difficult as initial attempts with various bases
were generally low yielding and produced multiple byproducts. A
number of temperature and solvent variations were also at-
tempted, and it was found that addition of a catalytic amount
of copper(I) iodide and palladium tetrakis(triphenylphosphine)
had a marked effect on the coupling. Employing this modification
in conjunction with microwave irradiation at 150 °C afforded all
eight desired quinazolines 21 and 28-34 in excellent yields
(Scheme 3).
Table 1. Selectivity Profile of 1 and Quinazoline 21 for Bcl-2
Family Members as Assessed by a Biacore 3000 Competition
Assaya
IC50 (nM)
compd
Bcl-xL
Bcl-2
Bcl-w
Mcl-1
1
<3b
7
6.1
8.7
40
>1000
>1000
21
440
a IC50 values ((50%) were determined by solution competition assays.
An 11-point 2-fold dilution series of each compound was incubated with
recombinant pro-survival proteins and percentage bound determined by
SPR using a Biacore 3000 instrument with a wild-type mBimBH3
(DLRPEIRIAQELRRIGDEFNETYTRR) peptide immobilized on the
SPR chip. b Below the lower limit of the measurement range for
this assay.
All compounds were tested for binding affinity for Bcl-2 family
fl/fl
members and cell-killing activity in wild-type (mcl-1 ) or Mcl-
1-deficient (mcl-1-/-) MEFs in both 1 and 10% serum
(Table 2).
As shown in Table 2, quinazolines 21, 29, 31, and 33 were
particularly potent Bcl-xL/Bcl-2 dual inhibitors with IC50 values
Figure 4. (a) Complex of quinazoline 21 with Bcl-xL, showing the electrostatic molecular surface with hydrophobic pockets P2 and P4 labeled as shown.
(b) Overlay of the compound 21 complex (white) with the complex of Bcl-xL and 1 (blue). A hydrogen bond between the quinazoline nitrogen and the
hydroxyl group of Tyr101 on Bcl-xL (N-O distance 2.60 Å) is apparent. This figure was created using PyMol.
Figure 5. Viability of wild-type (mcl-1 ) and Mcl-1-deficient (mcl-1-/-) MEFs 24 h after treatment with (a) quinazoline 21 or (b) 1, in the presence
fl/fl
of 10% fetal bovine serum. Means ( SEMs of a representative of three experiments.
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Figure 6. Targeted quinazoline sulfonamides and proposed convergent synthesis.
Scheme 3. Synthesis of Hydroxyquinazoline Core and Alkylation with Alkyl Halidesa
a Reagents and conditions: (a) Fe, AcOH, 60 °C, 45 min. (b) Formamidine acetate, MeOCH2CH2OH, 120 °C, 6 h. (c) 6 N HBr, 130 °C, 3 h.
(d) Compound 22 (X = Br), DIPEA, DMF, 25 °C, 20 h. (e) Compound 23 (X = Cl), DIPEA, DMF, 25 °C, 20 h. (f) POCl3, cat. DMF, DCE, 70 °C, 20 h.
(g) Cs2CO3, CuI, Pd(PPh3)4, dioxane, μW, 150 °C, 45 min, with appropriate sulfonamide coupling partner.
of less than 15 nM for both proteins. The least active quinazoline
was 34 with respective IC50 values of 29 and 84 nM, while the
activities of remaining compounds 28, 30, and 32 lay within the
range spanned by 21 and 34. All compounds exhibited weaker
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Table 2. Activity of Quinazolines against Bcl-2 Family Members and in MEFs
IC50 (nM)a
cellular EC50 (nM)b
fl/fl
fl/fl
compd
Bcl-xL
Bcl-2
Bcl-w
Mcl-1
mcl-1 (1% serum)
mcl-1 (10% serum)
mcl-1-/- (1% serum)
mcl-1-/- (10% serum)
21
28
29
30
31
32
33
34
1
7
34
6
9
21
13
81
12
21
5
440
>1000
245
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1680
5820
40 ( 11
24.8 ( 4.7
c
110 ( 13
190 ( 25
c
>10000
c
>10000
c
16
3
>1000
540
>10000
1610
>10000
4300
22.4 ( 2.5
16.0 ( 9.8
38 ( 13
149 ( 20
61 ( 11
221.6 ( 7.1
37.2 ( 3.2
328 ( 34
51 ( 11
20
3
>1000
275
>10000
2650
>10000
5460
11.6 ( 6.9
37.2 ( 6.2
2.03 ( 0.37
29
3
84
6.1
>1000
40
>10000
>10000
>10000
>10000
a IC50 values ((50%) were determined by solution competition assays. An 11-point 2-fold dilution series of each compound was incubated with
recombinant pro-survival proteins and percentage bound determined by SPR using a Biacore 3000 instrument with a wild-type mBimBH3
(DLRPEIRIAQELRRIGDEFNETYTRR) peptide immobilized on the SPR chip. b MEFs were treated with compound for 24 h, and cytotoxicity was
assessed by CellTiter Glo assay. Cellular EC50 represents the range of compound concentration in which cell viability was reduced by 50% as compared
to no treatment control. Data represents means ( SEMs for three independent experiments. c Not tested.
marginally less potent. However, 2 was substantially more potent
than the quinazolines and registered EC50 values of between 51
and 120 nM for the three different SCLC cell lines.
Table 3. Activity of Quinazolines in Cancer Cells (SCLC)
SCLC activity (EC50, μM) in 10% human seruma
compd
H146
H889
H1963
’ CONCLUSIONS
21
28
30
31
32
33
34
2
0.61
3.7
0.21
1.3
0.17
1.4
Acylsulfonamides 1 and 2 are high-affinity inhibitors of Bcl-2
and Bcl-xL and show potent cell killing in cell lines that are
dependent on these proteins for viability. Here, we have dis-
covered that the benzoylsulfonamide portion of these com-
pounds can be replaced by a quinazolinesulfonamide scaffold
to furnish a new class of Bcl-2 family inhibitors. Like the
acylsulfonamides, these quinazoline sulfonamides exhibit low
nanomolar activity against Bcl-2 and Bcl-xL but no activity toward
Mcl-1. There are some differences in the Bcl-2 family selectivity
profile between these two compound classes, however, and
unlike the acylsulfonamides, the quinazolines are significantly
weaker against Bcl-w as compared with Bcl-xL and Bcl-2. X-ray
crystallographic analysis of quinazoline 21 in complex with Bcl-xL
shows a binding mode very similar to acylsulfonamide 1, with the
exception that the nitrogen atom in the 1 position of the
quinazoline ring forms an additional electrostatic interaction
with the hydroxyl group of Tyr101 in Bcl-xL. This observation is
notable in that the corresponding interaction is not possible with
1; yet, its affinity for Bcl-xL is several times higher than the affinity
of quinazoline 21 for Bcl-xL.19
0.63
0.54
1.2
0.82
0.41
0.60
0.72
1.6
0.40
0.17
0.46
0.30
0.71
0.051
0.71
1.1
0.080
0.12
a Cells were treated for 48 h in 96-well tissue culture plates in medium
supplemented with 10% human serum. Cell viability was assessed using
an MTS proliferation assay. All values represent the average from single
experiments run in triplicate ((50%).
activity against Bcl-w and no activity against Mcl-1. Consistent
with the latter finding, the quinazoline analogues were essentially
inactive when tested against wild-type MEFs in concentrations of
up to 10 μM. Where EC50 values could be derived, these were in
the micromolar range, and we attribute this activity to nonspe-
cific cytotoxicity. In stark contrast, all quinazolines were much
more potent against Mcl-1-deficient MEFs, even in the presence
of 10% serum. Here, the weakest compound was 34, with an
EC50 for 328 nM, while 33 was considerably more potent and,
with an EC50 of 37 nM, was essentially equipotent to 1. This
correlates with the respective Bcl-xL/Bcl-2 inhibition by these
two compounds in the competition assays, and indeed, the data
in Table 2 show that this trend is remarkably consistent across
the set of quinazolines tested. As also shown in Table 2, all
compounds were more potent by 3- (21) to 9- (34) fold when
tested in the presence of only 1% serum.
Significantly, these quinazolines produce potent and mechan-
ism-based cytotoxicity in MEFs where Mcl-1 has been genetically
silenced. They also exhibit submicromolar activity against a panel
of small-cell lung carcinoma cell lines in the presence of 10%
human serum. After the acylsulfonamides, these compounds
comprise the only known class of functional BH3 mimetics that
confer mechanism-based cell killing.
’ EXPERIMENTAL SECTION
These compounds were then assessed for their ability to
induce cell killing in a panel of SCLC cell lines known to be
highly sensitive to acylsulfonamides 1 and 2. As shown in Table 3,
quinazolines 21, 30, 31, and 33 were potent even in the presence
of 10% human serum, with submicromolar EC50 values across
the three SCLC cell lines tested. Compounds 28, 32, and 34 were
Chemistry. General Chemistry Methods. All nonaqueous reac-
tions were performed in oven-dried glassware under an atmosphere of
dry nitrogen, unless otherwise specified. Tetrahydrofuran was freshly
distilled from sodium/benzophenone under N2. Dichloromethane was
freshly distilled from CaH2 under N2. All other solvents were reagent
grade. Petroleum ether describes a mixture of hexanes in the bp range
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N-(7-[4-Fluorophenyl]quinazolin-4-yl)-4-(phenylthio)ethan-2-
40-60 °C. Analytical thin-layer chromatography was performed on
Merck silica gel 60F254 aluminum-backed plates and were visualized by
fluorescence quenching under UV light. Flash chromatography was
performed with silica gel 60 (particle size 0.040-0.063 mm). NMR
spectra were recorded on a Bruker Avance DRX 300 with the solvents
indicated (1H NMR at 300 MHz). Chemical shifts are reported in
ppm on the δ scale and referenced to the appropriate solvent peak. NMR
spectra for compounds 21 and 28-34 were recorded on a Bruker 500
MHz NMR spectrometer and referenced to tetramethylsilane. The
analysis for total nitrogen, carbon, hydrogen, and sulfur was determined
by Dr. Thomas Rodemann at the Central Science Laboratory, University
of Tasmania, using a Thermo Finnigan EA 1112 Series Flash Elemental
Analyzer. HRESMS were recorded at the Australian National University
Mass Spectrometry Facility using a Waters LCT Premier XE (ESI TOF
Mass Spectrometer). Preparative HPLC was performed on a Polaris C18
5 μm column (50 mm ꢀ 21 mm). Low-resolution mass spectra were
performed on a Finnigan LCQ Advantage MAX or recorded on a Sciex
15 mass spectrometer in ESþ mode. Purity analysis of final compounds
was performed on a Berger Instruments SFC system operating at 100
bar, 35 °C, column) Berger diol, 4.6-150 mm, with a 6 min gradient of
20-60% MeOH in CO2 flowing at 2.35 mL/min (SFCd method) or a
Berger pyridine column, 4.6-150 mm, with a 6 min gradient of 5-50%
MeOH in CO2 flowing at 2.35 mL/min (SFCp). All final compounds
were analyzed using ultrahigh performance liquid chromatography/
ultraviolet/evaporative light scattering detection coupled to time-of-
flight mass spectrometry (UHPLC/UV/ELSD/TOFMS). Unless
otherwise noted, all tested compounds were found to be >95% pure
by this method.
Chemistry Experimental. 3-Amino-40-fluorobiphenyl-4-carbo-
nitrile (6). 2-Amino-4-chlorobenzonitrile (608 mg, 4 mmol), 4-fluor-
ophenylboronic acid (834 mg, 6 mmol), K2CO3 (1.10 g, 8 mmol),
TBAB (1.28 g, 4 mmol), and Nꢀajera's catalys20 (2.5 mol %) in water
(15 mL) were refluxed in air for 2 h, and the product was extracted with
warm toluene (4 ꢀ 20 mL). The organic layer was separated, dried
(MgSO4), and decanted through a silica plug, eluting product with
CH2Cl2. The crude product was recrystallized from 50% cyclohexane/
toluene to give the biaryl 6 as an off-white solid (480 mg, 56%). 1H NMR
(DMSO-d6): δ 7.64-7.59 (2H, m), 7.44 (1H, dd, J = 8.2 and 1.2 Hz),
7.32-7.26 (2H, m), 7.01 (1H, s), 6.85-6.82 (1H, m), 6.08 (2H, bs).
HRESMS found: (M þ H) 213.0833; C13H9FN2 requires (M þ H),
213.0823.
ylamino)-3-nitro benzenesulfonamide (4). A mixture of the crude
4-chloroquinazoline 9 (26 mg, 0.1 mmol), the sulfonamide 10 (35
mg, 0.1 mmol), and K2CO3 (138 mg, 1.0 mmol) in DMF (1 mL) was
heated at 60 °C for 24 h. A 10% citric acid solution (8 mL) was added,
and the solution was extracted with EtOAc (2 ꢀ 10 mL). The organic
layer was then dried (MgSO4) and concentrated in vacuo to give a
yellow solid. The solid was then triturated with hot toluene (2 ꢀ 2 mL)
and then CH3CN (2 ꢀ 2 mL) to give the sulfonamide 4 as a yellow
powder (39 mg, 68%). HRESMS found: (M þ H) 576.1170;
C28H22FN5O4S2 requires (M þ H), 576.1170. 1H NMR (DMSO-d6):
δ 8.63 (1H, m), 8.59 (1H, d, J = 2.0 Hz), 8.41, (1H, bs), 8.23 (1H, d, J =
8.4 Hz), 7.79-7.96 (5H, m), 7.07-7.40 (9H, m), 3.56-3.63 (2H, m),
3.21-3.28 (2H, m).
tert-Butyl 4-(4-Cyano-3-nitrophenyl)piperazine-1-carboxylate
(12). A mixture of 2,4-dinitrobenzonitrile (10 g, 51.8 mmol) and N-
Boc-piperazine (19.2 g, 103.6 mmol) in DMSO (60 mL) was stirred for
3 days at 25 °C. The dark brown reaction mixture was then partitioned
between EtOAc (400 mL) and water (2 ꢀ 100 mL). The layers were
separated, and the organic layer was dried (MgSO4) and concentrated
under vacuum. The resulting residue was triturated with Et2O and
filtered to give the nitrile 12 as a deep yellow powder (8 g, 47%). A
second crop was obtained from the supernatant on standing after some
evaporation had taken place (1.7 g, 10%). HRESMS found: (M þ H)
333.1536; C16H20N4O4 requires (M þ H), 333.1537. 1H NMR
(CDCl3): δ 7.69-7.04 (2H, m), 7.05 (1H, dd, J = 2.7 and 8.8 Hz),
3.65-3.61 (4H, m), 3.49-3.43 (4H, m), 1.49 (9H, s).
2-Nitro-4-(piperazin-1-yl)benzonitrile Bistosylate (13). The pipera-
zine 12 (8.64 g, 15 mmol) and p-toluenesulfonic acid (12.9 g, 75 mmol)
were dissolved in CH3CN (60 mL) and left to stand for 2 h. The product
in the form of a bis-tosylate 13 was then filtered off as course prisms (7.2
g, 83%). Mþ [ESþ] 233. 1H NMR (DMSO-d6): δ 8.80 (1H, bs), 7.88
(1H, d, J 8.82 Hz), 7.76 (1H, d, J = 2.58 Hz), 7.46 (4H, d, J = 8.07 Hz),
7.37 (1H, dd, J = 8.82 and 2.58 Hz), 7.09 (4H, d, J = 8.07 Hz), 3.6-3.7
(4H, m), 3.1-3.3 (4H, m), 2.25 (6H, s). Anal. found: C, 50.35; H, 5.12;
N, 9.22; S, 10.42; C25H28N4O8S2.H2O requires C, 50.49; H, 5.08; N,
9.42; S: 10.78%.
4-(4-(2-Bromobenzyl)piperazin-1-yl)-2-nitrobenzonitrile (14). To
the bis-tosylate 13 (2.3 g, 4.0 mmol) and 2-bromobenzylbromide
(1.49 g, 6.0 mmol) in i-PrOH (15 mL) was added Et3N (1.95 mL,
14.0 mmol), and the whole was stirred for 3 h. MeOH (20 mL) was then
added, the mixture was allowed to stand a few minutes, and the aryl
bromide product 14 was filtered off pure as an orange powder (1.5 g,
93%). Mþ [ESþ] 401, 403. 1H NMR (DMSO-d6): δ 7.80 (1H, d, J = 8.9
Hz), 7.67, d, J = 2.47 Hz), 7.58 (1H, d, J = 7.6 Hz), 7.49 (1H, d, J = 7.6
Hz), 7.36 (1H, dd, J = 7.6 and 7.6 Hz), 7.30 (1H, dd, J = 8.9 and 2.47 Hz),
7.19 (1H, dd, J = 7.6 and 7.6 Hz), 3.58 (2H, s), 3.4-3.5 (4H, m), 2.5-
2.6 (4H, m). Anal. found: C, 53.92; H, 4.50; N, 13.95; S, 10.42;
C18H17BrN4O2 requires C, 53.88; H, 4.27; N, 13.96%.
7-(4-Fluorophenyl)-4-aminoquinazoline (7). To the biaryl 6 (212
mg, 1 mmol) in ethylene glycol monomethyl ether (2 mL) was added
formamidine acetate (360 mg, 6 mmol), and the mixture was refluxed
under nitrogen for 5 h. The mixture was cooled, and the product was
filtered off to give the quinazoline 7 as colorless plates (180 mg, 75%).
1H NMR (DMSO-d6): δ 8.38 (1H, s), 8.26 (1H, d, J = 8.6 Hz), 7.74-
7.87 (6H, m), 7.31 (2H, m). Anal. found: C, 69.82; H, 3.90; N, 17.54;
C14H10FN3 requires C, 70.28; H, 4.21; N, 17.56%.
4-(4-((40-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)-2-nitrobenzo-
nitrile Tosylate (15). A mixture of the aryl bromide 14 (1.38 g, 3.43
mmol), p-chlorophenylboronic acid (703 mg, 4.46 mmol), sodium
carbonate (763 mg, 7.2 mmol), and PdCl2(PPh3)2 (5 mol %) in a
mixture of DME (6 mL), EtOH (6 mL), and water (6 mL) was heated at
90 °C for 4 h under an atmosphere of nitrogen. The reaction mixture was
partitioned between EtOAc and water and filtered through Celite, and
the organic layer was dried (MgSO4) and evaporated in vacuo. The
resulting residue was then treated with p-toluenesulfonic acid (1.72 g, 10
mmol) in CH3CN (20 mL) and Et2O (40 mL). On standing in the
freezer, the nitroarene product 15 precipitated as a yellow powder (1.68
g, 81%). Mþ [ESþ] 433, 435. 1H NMR (DMSO-d6): δ 9.57 (1H, bs),
7.85 (1H, d, J = 8.8 Hz), 7.7-7.8 (1H, m), 7.69 (1H, d, J = 2.52 Hz),
7.4-7.6 (6H, m), 7.2-7.4 (4H, m), 7.08 (2H, d, J = 7.9 Hz), 4.36 (2H,
m), 4.07 (2H, m), 3.22 (4H, m), 2.88 (2H, m), 2.25 (3H, s). Anal. found:
7-(4-Fluorophenyl)-4-hydroxyquinazoline (8). The 4-amino-quina-
zoline 7 (85 mg, 0.36 mmol) in 5 N HCl (5 mL) was heated to reflux for
30 min, and the solution was allowed to cool; on standing, a precipitate
formed, which was filtered off and washed with water to give quinazo-
linone 8 as a white solid (78 mg, 92%). 1H NMR (DMSO-d6): δ 8.20
(1H, s), 8.17 (1H, dd, J = 8.31 and 0.5 Hz), 7.89-7.80 (4H, m), 7.37-
7.31 (2H, m). Anal. found: C, 69.68; H, 4.00; N, 11.56; C14H9FN2O
requires C, 69.99; H, 3.78; N, 11.66%.
7-(4-Fluorophenyl)-4-chloroquinazoline (9). The quinazolinone 8
(103 mg, 0.43 mmol) in anhydrous CHCl3 (1 mL) and SOCl2 (1 mL)
with 1 drop of DMF was heated to reflux for 30 min. Ice water was added,
and the mixture was extracted with EtOAc (2 ꢀ 10 mL). The organic
layer was dried (MgSO4) and evaporated in vacuo to yield the
4-chloroquinazoline 9 as a creamy solid (110 mg, 99%). This sample
was on-reacted without further purification or characterization.
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C, 59.47; H, 4.99; N, 8.89; S, 4.94; C31H29ClN4O5S.H2O requires C,
59.75; H, 5.01; N, 8.99; S, 5.15%.
under vacuum to give the aniline 35 as a pale yellow powder (72%). 1H
NMR (CDCl3): δ 7.22 (1H, d, J = 8.8 Hz), 6.25 (1H, dd, J = 2.1 and 8.9
Hz), 6.09 (1H, d, J = 2.3 Hz), 4.33 (2H, bs), 3.55-3.51 (4H, m), 3.24-
3.21 (4H, m), 1.46 (9H, s). Anal. found: C, 63.49; H, 7.24; N, 18.28;
C16H22N4O2 requires C, 63.55; H, 7.33; N, 18.53%.
2-Amino-4-(4-((40-chlorobiphenyl-2-yl)methyl)piperazin-1-yl)ben-
zonitrile (16). The nitroarene tosylate 15 (1.3 g, 2.15 mmol) and iron
powder (1.1 g, 19.4 mmol) in AcOH (20 mL) were heated at 90 °C with
stirring for 10 min and partitioned between EtOAc and saturated
Na2CO3 solution, and the organic layer was separated, washed, and
evaporated to dryness to give the crude aniline as a brownish residue.
The crude residue was purified by triturating with Et2O. This gave the
aniline product 16 (518 mg, 60%); a further crop of product 16 could be
recovered from the ethereal supernatant on standing if so desired. Mþ
[ESþ] 403, 405. 1H NMR (DMSO-d6): δ 7.1-7.6 (9H, m), 6.23 (1H, d,
J = 9.2 Hz), 6.04 (1H, s), 4.23 (2H, bs), 3.40 (2H, bs), 3.19 (4H, bs),
2.34 (4H, bs). Anal. found: C, 70.87; H, 6.08; N, 13.60;
tert-Butyl 4-(4-Aminoquinazolin-7-yl)piperazine-1-carboxylate
(36). tert-Butyl 4-(3-amino-4-cyanophenyl)piperazine-1-carboxylate
35 (1.5 g, 4.97 mmol) in methoxyethanol (10 mL) at 120 °C was
treated portionwise every 1 h with formamidine acetate (4 ꢀ 12.4
mmol). The mixture was heated at that temperature for a further 6 h.
After standing for 20 h at room temperature, Et2O (40 mL) was added.
The resulting precipitate was filtered, washed with Et2O, slurried with
water (40 mL), and refiltered to afford the quinazoline 36 as a colorless
powder (1.1 g, 69%). On standing, the filtrate after evaporation of the
1
C24H23ClN4 0.25H2O requires C, 70.75; H, 5.81; N, 13.75%.
organic layer gave a further crop of crude product (22%). H NMR
3
7-(4-((40-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)quinazolin-4-
amine (17). The aniline compound 16 (216 mg, 0.54 mmol) was on-
reacted with formamidine acetate (194 mg, 3.24 mmol) in methyl glycol
(5 mL) at reflux for 3 h under nitrogen, and the product was precipitated
from the dark, cooled reaction mixture by the addition of a little water.
This was filtered and dried to give the 4-aminoquinazoline 17 as a buff
solid that could be recrystallized from aqueous DMSO after neutraliza-
tion with aqueous ammonia (198 mg, yield 81%). Mþ [ESþ] 430, 432.
1H NMR (DMSO-d6): δ 8.18, (1H, s), 7.95 (1H, d, J = 9.2 Hz), 7.47-
7.50 (1H, m), 7.45 (4H, bs), 7.29-7.36 (4H, m), 7.20-7.23 (1H, m),
7.16 (1H, dd, J = 9.2 and 2.4 Hz), 6.80 (1H, d, J = 2.3 Hz), 3.37 (2H, s),
3.23 (4H, m), 2.40 (4H, m). Anal. found: C, 68.26; H, 5.47; N, 15.54;
(DMSO-d6): δ 8.23 (1H, s), 8.02 (1H, d, J = 9.21 Hz), 7.39 (2H, bs)
7.23 (1H, dd, J = 2.6 and 9.2 Hz), 6.88 (1H, d, J = 2.5 Hz), 3.50-3.46
(4H, m), 3.35-3.30 (4H, m), 1.43 (9H, s). Anal. found: C, 61.92; H,
6.83; N, 21.25; C17H23N5O2 requires C, 61.99; H, 7.04; N, 21.26%.
7-(Piperazin-1-yl)quinazolin-4-ol dihydrobromide (24). tert-Butyl
4-(4-aminoquinazolin-7-yl)piperazine-1-carboxylate 36 (1.3 g, 4 mmol)
was then added carefully with rapid stirring (note that vigorous
effervescence occurred) to 6 N hydrobromic acid (6 mL). Further
stirring and heating at 130 °C occurred in a capped vessel for 3 h. After
the hot reaction mixture was added to hot MeOH (50 mL), the whole
was allowed to cool for 20 h. The precipitate that formed was filtered off to
afford 7-(piperazin-1-yl)quinazolin-4-ol dihydrobromide 24 as colorless
needles (1.5 g, 97%). Mþ 231. 1H NMR (DMSO-d6): δ 8.87 (2H, bs),
8.61 (1H, bs), 7.98 (1H, d, J = 9.0Hz), 7.01 (1H, d, J = 2.4 Hz), 3.71-3.61
(4H, m), 3.60-3.40 (4H, m). Anal. found: C, 34.29; H, 4.41; N, 13.24;
C25H24ClN5 0.5H2O requires C, 69.84; H, 5.63; N, 16.29%.
3
7-(4-((40-Chlorobiphenyl-2-yl)methyl)piperazin-1-yl)quinazolin-4-
ol (18). The 4-aminoquinazoline compound 17 (176 mg, 0.4 mmol)
was heated at 130 °C for 9 h in AcOH (2 mL) and 2.5 N HCl solution
(2 mL) in a small flask fitted with an air condenser. The solvent was
removed, and the residue was recrystallized from aqueous DMSO after
neutralization with minimal aqueous ammonia. This gave the hydro-
lyzed product 18 as a buff powder (153 mg, 87% yield). HRESMS
found: (M þ H) 431.1636; C25H23ClN4O requires (M þ H),
C12H16Br2N4O 1.5H2O requires C, 34.39; H, 4.57; N, 13.37%.
3
7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-4-ol (18).
DIPEA (384 μL, 2.55 mmol) was added to a stirred solution of the
7-(piperazin-1-yl)quinazolin-4-ol dihydrobromide 24 (500 mg, 1.28
mmol) in DMF (10 mL). To this solution, 2-bromomethyl-40-chloro-
biphenyl 22 (X = Br) (360 mg, 1.28 mmol) in DMF (4 mL) was added
dropwise over 30 min. The solution was allowed to stir at room
temperature for 20 h. A solution of 10% NaHCO3 (50 mL) was added
to the stirred solution. The resulting precipitate was filtered off and dried
in a vacuum oven to yield the quinazolinone 18 as a white solid (440 mg,
80%). The compound was of sufficient purity to be used in the next step
without further purification. HRESMS found: (M þ H) 431.1636;
C25H23ClN4O requires (M þ H), 431.1633. 1H NMR (DMSO-d6): δ
7.92 (1H, s), 7.86 (1H, d, J = 9.0 Hz) 7.52-7.34 (7H, m), 7.23 (1H, dd,
J = 6.9 and 1.9 Hz), 7.11 (1H, dd, J = 9.0 and 2.2 Hz), 6.88 (1H, d, J =
2.3 Hz), 3.38 (2H, s), 3.25 (4H, bs), and 2.41 (4H, bs). LCMS: Tr =
5.77 min, m/z = 431.
1
431.1633. H NMR (DMSO-d6): δ 11.8 (1H, bs), 7.90 (1H, s), 7.84
(1H, d, J = 9.0 Hz), 7.46-7.51 (1H, m), 7.44 (4H, bs), 7.30-7.38 2H,
m), 7.20-7.23 (1H, m), 7.09 (1H, dd, J = 9.0 and 2.0 Hz), 6.86 1H, d,
J = 2.0 Hz), 4.44-3.37 (2H, bs), 3.27 (4H, m), 2.38 (4H, bm).
4-Chloro-7-(4-((40-chlorobiphenyl-2-yl)methyl)piperazin-1-yl)quinazo-
line (19). The hydrolyzed product 18 (30 mg, 0.07 mmol) was treated
with dry CHCl3 (1 mL) and SOCl2 (1 mL) with a catalytic amount of
DMF (10 μL). The solution was then heated to reflux for 1 h, the
solution was poured onto ice, and the product was extracted with EtOAc
(2 ꢀ 6 mL) to give the chlorinated product 19, which was on-reacted
without characterization.
4-Chloro-7-[4-(40-chloro-biphenyl-2-ylmethyl)piperazin-1-yl]qui-
nazoline (19). A solution of POCl3 (0.5 mL) and DMF (2 drops) in
1,2-DCE (2 mL) was added dropwise over 15 min to a stirred solution
of the 7-[4-(40-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-quinazo-
lin-4-ol 37 (500 mg, 1.16 mmol) in 1,2-DCE (30 mL) at 70 °C under
an atmosphere of nitrogen. Additional POCl3 was added in increments
(1 mL) of 15 min over 1 h. The solution was allowed to stir at 70 °C for
20 h. The solution was then concentrated in vacuo to dryness and then
diluted with a solution of 10% NaHCO3 (40 mL) and CH2Cl2
(40 mL). The layers were separated, and the organic layer was dried
(MgSO4) and concentrated in vacuo. The resulting residue was then
applied to alumina column chromatography gradient eluting from
100% CH2Cl2 to 0.5% MeOH/CH2Cl2 to afford the quinazoline 19 as
a yellow foam (287 mg, 55%), which was on-reacted without
characterization.
N-{7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-
4-yl}-4-((R)-3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-
3-nitro-benzenesulfonamide (21). A portion of the chlorinated pro-
duct 19 (8 mg, 0.018 mmol), the sulfonamide 20 (8 mg, 0.018
mmol),15 and K2CO3 (40 mg, 0.288 mmol) in DMF (0.2 mL) was
heated at 85 °C for 24 h. The reaction mixture was partitioned between
EtOAc (2 mL) and water (2 mL). The organic layer was separated,
dried (MgSO4), and evaporated in vacuo to give a yellow residue. This
residue was purified by HPLC to give of the quinazoline isostere 21
(2 mg, 13%) as a yellow glass.
tert-Butyl 4-(3-Amino-4-cyanophenyl)piperazine-1-carboxylate
(35). A mixture of tert-butyl 4-(4-cyano-3-nitrophenyl)piperazine-1-
carboxylate 12 (3 g, 9.9 mmol) and iron powder (2 g, 35.8 mmol) in
AcOH (20 mL) was heated at 60 °C with rapid stirring. The reaction
mixture was diluted with EtOAc (60 mL) and filtered twice through
Celite. The filtrate was washed with a base wash using 6 N NaOH
solution. The organic layer was then dried (MgSO4) and concentrated
7-{4-[2-(4-Chloro-phenyl)-5,5-dimethyl-cyclohex-1-enylmethyl]-
piperazin-1-yl}quinazolin-4-ol (37). DIPEA (384 μL, 2.55 mmol) was
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Journal of Medicinal Chemistry
ARTICLE
added to a stirred solution of the 7-(piperazin-1-yl)quinazolin-4-ol
dihydrobromide 24 (500 mg, 1.28 mmol) in DMF (10 mL). To this
solution, 1-(2-bromomethyl-4,4-dimethyl-cyclohex-1-enyl)-4-chloro-
benzene 23 (X = Br) (401 mg, 1.28 mmol) in DMF (4 mL) was added
dropwise over 30 min. The solution was allowed to stir at room
temperature for 20 h. A solution of 10% NaHCO3 (50 mL) was added
to the stirred solution. The resulting precipitate was filtered off and dried
in a vacuum oven to yield the quinazolinone 37 as a white solid (593 mg,
84%). The compound was of sufficient purity to be used in the next step
without further purification. HRESMS found: (M þ H) 463.2281;
C27H31ClN4O requires (M þ H), 463.2259. 1H NMR (DMSO-d6): δ
7.92 (1H, s), 7.83 (1H, d, J = 9.0 Hz) 7.36 (2H, d, J = 6.5 Hz), 7.15 (2H,
d, J = 6.5 Hz), 7.06 (1H, dd, J = 9.0 and 2.4 Hz), 6.82 (1H, d, J = 2.3 Hz),
3.25 (4H, bs), 2.74 (2H, bs), 2.27-2.21 (6H, m), 1.98 (2H, s), 1.42 (2H,
t, J 6.4 Hz), and 0.96 (6H, s).
4-Chloro-7-{4-[2-(4-chloro-phenyl)-5,5-dimethyl-cyclohex-1-enyl-
methyl]piperazin-1-yl} (38). A solution of POCl3 (0.5 mL) and DMF
(2 drops) in 1,2-DCE (2 mL) was added dropwise over 15 min to a
stirred solution of 37 (537 mg, 1.16 mmol) in 1,2-DCE (30 mL) at 70 °C
under an atmosphere of nitrogen. Additional POCl3 was added in
increments (1 mL) of 15 min over 1 h. The solution was allowed to
stir at 70 °C for 20 h. The solution was then concentrated in vacuo to
dryness and then diluted with a solution of 10% NaHCO3 (40 mL) and
CH2Cl2 (40 mL). The layers were separated, and the organic layer was
dried (MgSO4) and concentrated in vacuo. The resulting residue was
then applied to alumina column chromatography gradient eluting
from 100% CH2Cl2 to 0.5% MeOH/CH2Cl2 to afford the quinazoline
38 as a yellow foam (324 mg, 58%), which was on-reacted without
characterization.
2H), 3.10-3.15 (m, 2H), 3.03-3.08 (m, 2H), 2.71 (s, 6H), 2.0-2.1 (m,
2H). LCMS: Tr = 13.46 min, m/z = 924.
N-{7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-
4-yl}-4-((R)-3-morpholin-4-yl-1-phenylsulfanylmethyl-propylamino)-
3-trifluoromethanesulfonyl-benzenesulfonamide (30). Compound 30
was prepared from 2710 and 19 following the procedure for 21. 1H NMR
(500 MHz, DMSO-d6): δ 9.65-9.75 (m, 1H), 8.18-8.32 (m, 2H), 8.02
(d, J = 2.5 Hz, 1H), 7.50-7.60 (m, 4H), 7.38-7.48 (m, 2H), 7.20-7.40
(m, 7H), 7.12-7.18 (m, 1H), 7.00-7.10 (m, 1H), 6.80-6.90 (m, 1H),
4.05-4.10 (m, 1H), 3.95-4.00 (m, 2H), 3.70 (s, 6H), 2.95-3.10 (m,
6H), 2.10-2.20 (m, 2H). LCMS: Tr = 5.57 min, m/z = 966.
N-(7-{4-[2-(4-Chloro-phenyl)-5,5-dimethyl-cyclohex-1-enylmethyl]-
piperazin-1-yl}quinazolin-4-yl)-4-((R)-3-dimethylamino-1-phenylsulfa-
nylmethyl-propylamino)-3-nitro-benzenesulfonamide (31). Com-
pound 31 was prepared from 2015 and 38 following the procedure for
1
21. H NMR (500 MHz, DMSO-d6): δ 9.32-9.42 (m, 2H), 8.50-
8.52 (m, 1H), 8.14-8.24 (m, 2H), 7.98-8.08 (m, 1H), 7.86 (d, J = 2.5
Hz, 1H), 7.34-7.84 (m, 3H), 7.20-7.30 (m, 3H), 7.05-7.15 (m,
5H), 4.10-4.20 (m, 2H), 4.00-4.10 (M, 1H), 3.60-3.65 (m, 4H),
3.02-3.12 (m, 6H), 2.75-2.85 (m, 2H), 2.70 (s, 6H), 2.25-2.30 (m,
2H), 2.13 (q, J = 7 Hz, 2H), 2.01-2.10 (M, 2H), 1.45-1.50 (m, 2H),
1.00 (s, 6H). LCMS: Tr = 5.76 min, m/z = 869.
N-(7-{4-[2-(4-Chloro-phenyl)-5,5-dimethyl-cyclohex-1-enylmethyl]-
piperazin-1-yl}quinazolin-4-yl)-4-((R)-3-morpholin-4-yl-1-phenylsulfa-
nylmethyl-propylamino)-3-nitro-benzenesulfonamide (32). Com-
pound 32 was prepared from 2515 and 38 following the procedure for
1
21. H NMR (500 MHz, DMSO-d6): δ 9.52-9.62 (m, 1H), 9.28-
9.38 (m, 1H), 8.52 (s, 1H), 8.14-8.24 (m, 2H), 7.98-8.08 (m, 1H),
7.83 (d, J = 2.5 Hz, 1H), 7.40 (d, J = 2 Hz, 2H), 7.20-7.30 (m, 3H),
7.05-7.15 (m, 6H), 4.10-4.20 (m, 2H), 3.91-4.02 (M, 4H), 3.52-
3.58 (m, 6H), 3.12-3.22 (m, 2H), 2.95-3.05 (m, 2H), 2.80-2.90 (m,
2H), 2.25-2.30 (m, 2H), 2.12-2.18 (m, 2H), 2.01-2.06 (brs, 2H),
1.45-1.50 (m, 2H), 1.00 (s, 6H). LCMS: Tr = 14.12 min, m/z = 456.
N-(7-{4-[2-(4-Chloro-phenyl)-5,5-dimethyl-cyclohex-1-enylmethyl]-
piperazin-1-yl}quinazolin-4-yl)-4-((R)-3-dimethylamino-1-phenylsul-
fanylmethyl-propylamino)-3-trifluoromethanesulfonyl-benzenesulfo-
namide (33). Compound 33 was prepared from 2610 and 38 following
the procedure for 21. 1H NMR (500 MHz, DMSO-d6): δ 9.35-9.45 (m,
2H), 8.15-8.25 (m, 2H), 7.98-8.06 (m, 2H), 7.42 (d, J = 2.5 Hz, 2H),
7.22-7.32 (m, 3H), 7.20-7.30 (m, 3H), 7.05-7.15 (m, 3H), 6.95-
7.05 (m, 1H), 6.79-6.88 (m, 1H), 4.00-4.10 (m, 2H), 3.60-3.65 (m,
2H), 3.25-3.35 (m, 4H), 3.12-3.22 (m, 2H), 2.95-3.05 (m, 2H), 2.80-
2.90 (m, 2H), 2.72 (s, 6H), 2.25-2.30 (m, 2H), 2.00-2.10 (M, 4H),
1.45-1.50 (m, 2H), 1.00 (s, 6H). LCMS: Tr = 14.93 min, m/z = 956.
N-(7-{4-[2-(4-Chloro-phenyl)-5,5-dimethyl-cyclohex-1-enylmethyl]-
piperazin-1-yl}quinazolin-4-yl)-4-((R)-3-morpholin-4-yl-1-phenyl-
sulfanylmethylpropylamino)-3-trifluoromethanesulfonyl-benzenesul-
fonamide (34). Compound 34 was prepared from 2710 and 38 following
the procedure for 21. 1H NMR (500 MHz, DMSO-d6): δ 9.65-9.75 (m,
1H), 9.32-9.42 (m, 1H), 8.15-8.25 (m, 2H), 7.98-8.04 (m, 2H), 7.40
(d, J = 2 Hz, 1H), 7.29 (d, J = 2 Hz, 2H), 7.20-7.25 (m, 2H), 7.12-7.18
(m, 3H), 7.00-7.05 (M, 1H), 6.78-6.84 (m, 1H), 3.90-4.12 (m, 4H),
3.31-3.41 (M, 6H), 3.15-3.25 (m, 2H), 2.95-3.15 (m, 4H), 2.80-2.90
(m, 2H), 2.25-2.30 (m, 2H), 2.00-2.15 (m, 4H), 1.45-1.50 (m, 2H),
1.00 (s, 6H). LCMS: Tr = 15.02 min, m/z = 998.
N-{7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-
4-yl}-4-((R)-3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-
3-nitro-benzenesulfonamide (21). A solution of the chloroquinazoline
19 (100 mg, 0.22 mmol), the sulfonamide 20 (93 mg, 0.22 mmol),
Cs2CO3 (101 mg, 0.31 mmol), Pd(PPh3)4 (2.5 mol %), and CuI
(5.7 mg, 0.03 mmol) in dioxane (4 mL) was degassed for 5 min before
being subjected to microwave irradiation (300 W, 150-180 °C, CEM
Discover Labmate) for 45 min. The mixture was filtered washing with
EtOAc and then washed with solution of 10% NaHCO3 (10 mL). The
organic layer was dried (MgSO4) and concentrated in vacuo to give a
crude residue. This residue was then subjected to preparative reverse
1
phase HPLC for purification to afford the sulfonamide 21. H NMR
(500 MHz, DMSO-d6): δ 9.3-9.4 (m, 1H), 8.52 (s, 1H), 8.15-8.25 (m,
1H), 7.98-8.08 (m, 1H), 7.85 (d, J = 2.5 Hz, 1H), 7.45-7.55 (m, 4H),
7.35-7.45 (m, 4H), 7.25-7.35 (m, 1H), 7.20-7.30 (m, 4H), 7.05-
7.15 (m, 4H), 4.1-4.2 (m, 2H), 3.05-3.15 (m, 4H), 2.72 (s, 6H), 2.11
(m, 2H). LCMS: Tr = 12.48 min, m/z = 837.
N-{7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-
4-yl}-4-((R)-3-morpholin-4-yl-1-phenylsulfanylmethyl-propylamino)-
3-nitro-benzenesulfonamide (28). Compound 28 was prepared from
1
2515 and 19 following the procedure for 21. H NMR (500 MHz,
DMSO-d6): δ 9.6-9.7 (m, 1H), 8.52 (brs, 1H), 8.15-8.25 (m, 1H),
7.99-8.09 (m, 1H), 7.87 (d, J = 2.5 Hz, 1H), 7.45-7.55 (m, 4H), 7.35-
7.45 (m, 2H), 7.28-7.38 (m, 1H), 7.20-7.30 (m, 4H), 7.05-7.15 (m,
5H), 4.1-4.15 (m, 1H), 3.85-4.00 (m, 2H), 3.35 (s, 6H), 3.12-3.28
(m, 4H), 2.98-3.08 (m, 2H), 2.10-2.20 (m, 2H). LCMS: Tr = 12.50
min, m/z = 879.
AlphaSCREEN Assay. AlphaSCREEN is a bead-based technology,
which facilitates the measurement of the interaction between molecules.
The assay consisted of two hydrogel-coated beads, which when brought
into close proximity by a binding interaction allow the transfer of singlet
oxygen from a donor bead to an acceptor bead. Upon binding and
excitation with laser light at 680 nm, a photosensitizer in the donor bead
converts ambient oxygen to a more excited singlet state. This singlet
oxygen then diffuses across to react with a chemiluminescer in the
acceptor bead. Fluorophores within the same bead are activated, resulting
N-{7-[4-(40-Chloro-biphenyl-2-ylmethyl)piperazin-1-yl]quinazolin-
4-yl}-4-((R)-3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-
3-trifluoromethanesulfonyl-benzenesulfonamide (29). Compound 29
was prepared from 2610 and 19 following the procedure for 21. 1H NMR
(500 MHz, DMSO-d6): δ 9.35-9.45 (m, 1H), 8.13-8.23 (m, 2H), 7.98
(d, J = 2.5 Hz, 2H), 7.50 (d, J = 2.0 Hz, 4H), 7.35-7.48 (m, 2H), 7.28 (d,
J = 2.0 Hz, 4H), 7.25 (d, J = 2.5 Hz, 1H), 7.21 (t, J = 2 Hz, 2H), 7.10-
7.18 (m, 1H), 6.98-7.08 (m, 1H), 6.75-6.85 (m, 1H), 4.00-4.05 (m,
1923
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Journal of Medicinal Chemistry
ARTICLE
in the emission of light at 580-620 nm. Screening of the test compounds
was performed using the AlphaSCREEN GST (glutathione S-trans-
ferase) detection kit system (Perkin-Elmer Lifesciences). Briefly, test
compounds were titrated into the assay, which consisted of GST-tagged
Bcl-xL ΔC25 protein (0.6 nM final concentration) and biotinylated Bim
BH3-26 peptide, Biotin-DLRPEIRIAQELRRIGDEFNETYTRR (5.0
nM final concentration), anti-GST-coated acceptor beads, and strepta-
vidin-coated donor beads (both bead types at a final concentration of 15
μg/mL) and a room temperature incubation time of 4 h before reading.
More specifically, (i) a 384 well-plate was prepared with 4.75 μL of buffer
and 0.25 μL ofcompound stock (20 mM inDMSO) per well;(ii) binding
partners were mixed—in one tube, Bcl-xL was added with the acceptor
beads, while in the second tube biotinylated BH3 peptide was added with
the donor beads; (iii) these two pairs of binding partners were pre-
incubated for 30 min; (iv) 10 μL of the acceptor beads/Bcl-xL protein
complex was then added to each of the 384 wells; (v) plates were sealed
and incubated at room temperature for a further 30 min; (vi) 10 μL of the
donor bead/BH3 peptide complex was then added to each of the 384
wells; (vii) plates were sealed, covered with foil, and incubated for a
further 4 h and then read. The assay buffer contained 50 mM HEPES, pH
7.4, 10 mM DTT, 100 mM NaCl, 0.05% Tween 20, and 0.1 mg/mL
casein. Bead dilution buffer contained 50 mM Tris-HCl, pH 7.5, 0.01%
Tween 20, and 0.1 mg/mL casein. The final DMSO concentration in the
assay was 1.0% (v/v). Assays were performed in 384-well white Opti-
plates (Perkin-Elmer Lifesciences) and analyzed on the PerkinElmer
Fusion Alpha plate reader (Ex680, Em520-620 nM).
one binding site model, which includes mass transport limitations. The
equilibrium dissociation constant (KD) was determined by kd/ka.
Determination of Selectivity Profile by Biacore S3000 SPR
Analysis. Recombinant Proteins. Expression and purification of the
loop-deleted form of human Bcl-xL (Δ27-82, ΔC24) for crystallization
and Bcl-2 ΔC22, Bcl-xL ΔC24, Bcl-w C29S/A128E ΔC29, and human/
mouse chimeric Mcl-1 (ΔN170,ΔC23) for Biacore studies was per-
formed as described previously.21-23
Binding Affinity Measurements—Solution Competition Assays. Solu-
tion competition assays were performed using a Biacore 3000 instrument
as described previously.24 Briefly, prosurvival proteins (10 nM) were
incubated with varying concentrations of compounds for at least 2 h in
running buffer [10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and
0.005% (v/v) Tween 20, pH 7.4] prior to injection onto a CM5 sensor
chip on which either a wild-type mBimBH3 (DLRPEIRIAQELRRI-
GDEFNETYTRR) peptide or an inert mBimBH3 (DLRPEIREAQEER-
REGDEENETYTRR) mutant peptide was immobilized. Specific binding
of the prosurvival protein to the surface in the presence and absence of
compounds was quantified by subtracting the signal from the Bim mutant
channel from that obtained on the wild-type Bim channel. The IC50 was
calculated by nonlinear curve fitting of the data with Kaleidagraph
(Synergy Software).
X-ray Structural Data. Crystallization was performed as pre-
viously described for the Bcl-xL:1 complex.16 Briefly, Bcl-xL was com-
bined with compound 21 at equimolar concentrations, the complex was
concentrated to 10 mg/mL, and crystals were grown in hanging drops at 295
K with a reservoir solution consisting of 1.1 M sodium citrate and 0.1 M Tris,
pH 8.0. Crystals were soaked for 30 s in cryoprotectant (1.1 M sodium
citrate, 0.1 M Tris, pH 8.0, 5% DMSO, and 12.5% glycerol) and flash frozen
in liquid nitrogen. X-ray data were collected at 100 K using an in-house
RAXIS-IVþþ detector with a micro-max007 rotating anode X-ray source.
Data were integrated and scaled with HKL2000.25 The R-free set from the
data used to solve the 1:Bcl-xL structure was transferred to the new data to
avoid model bias. The structure was solved by performing Rigid Body
refinement with REFMAC526 using the Bcl-xL coordinates from the Bcl-xL:1
complex (2YXJ) as a starting model. Several rounds of building in COOT27
and refinement with REFMAC5 led to the model described in the Support-
ing Information.
Determination of Binding Affinity by Biacore S51 SPR
Analysis. Immobilization of anti-GST Antibody. Anti-GST antibody
surfaces were prepared by using standard amine-coupling procedures as
instructed by the GST Capture Kit from Biacore. The running buffer was
10 mM NaH2PO4 H2O, 40 mM Na2HPO4 2H2O, 150 mM NaCl,
3
3
1.0 mM EDTA, 0.03% Tween 20, and 5% DMSO, pH 7.4. Flow cells
(spots) were activated by injecting 200 μL of (11.5 mg/mL)
NHS and (75 mg/mL) EDC. Fifty microliters of (30 μg/mL) anti-
GST antibody in 10 mM sodium acetate, pH 5.0, was injected for 10 min
at 5 μL/min, followed by the injection of 175 μL of ethanolamine
(1 M). This was followed by the injection for 36 s of Biadesorb1 solution
(0.5% SDS) and 40 s of Biadesorb2 solution (50 mM glycine, pH 9.5)
at 30 μL/min at the end of the run. These methods resulted consistently
in about 10000 RU anti-GST antibody immobilized over separate
experiments.
MEF Viability Assay. MEFs were maintained in high-glucose DME
supplemented with 250 mM L-asparagine, 50 mM 2-mercaptoethanol,
and 10% (v/v) FCS (Bovagen). Cells were exposed to compounds at the
indicated concentrations for 24 h in medium containing 1 or 10% FCS.
The cytotoxicity was assessed with CellTiter Glo (Promega).
Capture of GST-Bcl-xL. The running buffer was the same as used for
the immobilization of anti-GST antibody. GST-Bcl-xL (0.1-0.2 mg/
mL) in the running buffer was injected at 10 μL/min for 3 min across
one spot, resulting in the capture of approximately 1200 RU protein.
Kinetic Analysis of Small Molecules and GST-Bcl-xL Interactions. A
concentration series of each compound was injected at a flow rate of 90
μL/min over three spots (one spot was immobilized with GST-Bcl-xL
protein capture by anti-GST antibody, the other two spots were free) at
25 °C. The association time and dissociation time were 95 and 240 s,
respectively. The buffer blanks were also injected periodically for double
referencing. The buffer samples containing 4-6% DMSO were injected
for solvent correction. The antibody surface was regenerated between
binding cycles with 40 s injection of 10 mM glycine-HCl, pH 2.2, and 40
s injection of 0.05% SDS. Fresh protein was injected at 10 μL/min for 3
min at the beginning of each binding cycle.
SCLC Viability Assay. Cell Culture. The SCLC cell lines NCI-
H889, NCI-H1963, and NCI-H146 were purchased from the American
Type Culture Collection (Manassas, VA). Cells were cultured in RPMI
1640 (Invitrogen Corp., Grand Island, NY) supplemented with 10% fetal
bovine serum(Invitrogen), 1% sodium pyruvate, 25 mmol/L HEPES, 4.5
g/L glucose, and 1% penicillin/streptomycin (Sigma). All cell lines were
maintained in a humidified chamber at 37 °C containing 5% CO2.
Cell Treatment and Viability Assays. Cells were treated for 48 h in 96-
well tissue culture plates in a total volume of 100 μL of tissue culture
medium supplemented with 10% human serum (Invitrogen). Each
concentration was tested in duplicate at least thrice separately. Viable
cells were determined using the CellTiter 96 AQueous nonradioactive
cell proliferation MTS assay (Promega Corp., Madison, WI).
Data Analysis. All sensorgram data were processed by using double
referencing. Thus, first the response from the reference spot (no
immobilization) was subtracted from the binding response obtained
on the test spot followed by solvent correction (to correct for DMSO
bulk shift responses). Then, the response from an average of buffer
injections was also subtracted from the remaining response 1 described
above to obtain final corrected response units. To obtain kinetic rate
constants (ka and kd), corrected response data were then fit to a one-to-
’ ASSOCIATED CONTENT
S
Supporting Information. Supplementary Table 1, Crys-
b
tallographic statistics. This material is available free of charge via
the Internet at http://pubs.acs.org.
Accession Codes
The PDB ID is 3QKD.
1924
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
The latter is a topographical mimetic of the native binding epitope even
though superficially it may be hard to envisage any resemblance between
the two. The field of BH3 mimetics comprises both type II and type II
peptidomimetics. We find this distinction useful. Despite this, the
proposed classification of peptidomimetics of Ripka and Rich does
not appear to have been widely taken up.
Corresponding Author
*Tel: þ61 3 9345 2108. Fax: þ61 3 9345 2100. E-mail:
jbaell@wehi.edu.au.
(9) Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong,
R. C.; Augeri, D. J.; Belli, B. A.; Bruncko, M.; Deckwerth, T. L.; Dinges,
J.; Hajduk, P. J.; Joseph, M. K.; Kitada, S.; Korsmeyer, S. J.; Kunzer, A. R.;
Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D. G.; Ng, S.; Nimmer, P. M.;
O'Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed, J. C.; Shen, W.;
Tahir, S. K.; Thompson, C. B.; Tomaselli, K. J.; Wang, B.; Wendt, M. D.;
Zhang, H.; Fesik, S. W.; Rosenberg, S. H. An inhibitor of Bcl-2 family
proteins induces regression of solid tumours. Nature 2005,
435, 677–681.
(10) 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.; Zhang, H.; Fesik, S. W.;
Rosenberg, S. H.; Elmore, S. W. Discovery of an Orally Bioavailable
Small-molecule Inhibitor of Prosurvival B-Cell Lymphoma 2 Proteins. J.
Med. Chem. 2008, 51, 6902–6915.
(11) (a) 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. ABT-263: A potent and orally
bioavailable Bcl-2 family inhibitor. Cancer Res. 2008, 68, 3421–3428.
(b) More recent progress in phase II trials can be found at http://
clinicaltrials.gov and by searching on “Navitoclax” (or “ABT-263”) and
“phase II”.
(12) Huang, S.; Connolly, P. J.; Lin, R.; Emanuel, S.; Middleton, S. A.
Synthesis and evaluation of N-acyl sulfonamides as potential prodrugs of
cyclin-dependent kinase inhibitor JNJ-7706621. Bioorg. Med. Chem. Lett.
2006, 16, 3639–3641.
(13) Petros, A.; Dinges, J.; Augeri, D.; Baumeister, S.; Betebenner,
D.; Bures, M. G.; Elmore, S. W.; Hajduk, P. J.; Joseph, M. K.; Landis,
S. K.; Nettesheim, D. G.; Rosenberg, S. H.; Shen, W.; Thomas, S.; Wang,
X.; Zanze, I.; Zhang, H.; Fesik, S. W. Discovery of a potent inhibitor of
the antiapoptotic protein Bcl-xL from NMR and parallel synthesis. J.
Med. Chem. 2006, 49, 656–663.
(14) Alternative coupling procedures involving attempted EDC/
DMAP coupling between the hydroxyquinazoline and the sulfonamide,
or from sulfonylation of the 4-aminoquinazoline with the sulfonyl
chloride, were all unsuccessful.
(15) Wendt, M. D.; Shen, W.; 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. Discovery and Structure Activity Relationship of
Antagonists of B-Cell Lymphoma 2 Family Proteins with Chemopo-
tentiation Activity in Vitro and in Vivo. J. Med. Chem. 2006,
49, 1165–1181.
(16) Lee, E. F.; Czabotar, P. E.; Smith, B. J.; Deshayes, K.; Zobel, K.;
Colman, P. M.; Fairlie, W. D. Crystal structure of ABT-737 complexed
with Bcl-xL: implications for selectivity of antagonists of the Bcl-2 family.
Cell Death Differ. 2007, 14, 1711–1713.
’ ACKNOWLEDGMENT
This work was supported by Fellowships and Grants from the
Leukemia and Lymphoma Society (SCOR 7015-02). Crystal-
lization trials were performed at the Bio21 Collaborative Crystal-
lisation Centre, Victoria, Australia. We thank Peter Colman for
useful discussions and also the NHMRC, the Victorian State
Government, the Cancer Council of Victoria, the Australian
Cancer Research Foundation, and the Australian Research
Council for the following support: NHMRC Program Grant
461221; NHMRC IRIISS Grant #361646; Victorian State Gov-
ernment OIS grant; Cancer Council of Victoria, Fraser Fellow-
ship to P. M. Colman (2002-2006); ACRF Equipment Grant
(2002); ARC Future Fellowship; and NHMRC Project Grant
575561 to P.E.C.
’ ABBREVIATIONS USED
AlphaSCREEN, amplified luminescent proximity homogeneous as-
say; A1, B-cell lymphoma 2 related protein A1; Bad, Bcl-2-associated
death promotor; Bak, Bcl-2 antagonist/killer; Bax, Bcl-2 associated
protein X; Bcl-2, B-cell lymphoma 2; Bcl-w, B-cell lymphoma w;
Bcl-xL, B-cell lymphoma extra long; BH, Bcl homology; Bid, BH3
interacting death domain; Bik, Bcl-2 interacting killer; Bim, Bcl-2
interacting mediator; Bmf, Bcl-2 modifying factor; Hrk, Harakiri-
Bcl-2 interacting protein; Mcl-1, myeloid cell leukemia 1; MEF,
mouse embryonic fibroblast; Noxa, phorbol-12-myristate-13-acet-
ate-induced protein 1; Puma, Bcl-2 binding component 3; SCLC,
small cell lung carcinoma; SPR, surface plasmon resonance
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1925
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Journal of Medicinal Chemistry
ARTICLE
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’ NOTE ADDED AFTER ASAP PUBLICATION
Bcl-xL was defined incorrectly in the version of this paper
published on March 2, 2011. The corrected version posted to
the web on March 17, 2011.
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