Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.04.106

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic pro

Full text of DOI:10.1016/j.bmcl.2012.04.106

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3951–3956
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL
antagonists
Gretchen M. Schroeder a, , Donna Wei a, , Patrizia Banfi b, Zhen-Wei Cai a,à, Jonathan Lippy a,
⇑
,
Maria Menichincheri b, Michele Modugno b, Joseph Naglich a, Becky Penhallow a, Heidi L. Perez a, John Sack a,
Robert J. Schmidt a, Andrew Tebben a, Chunhong Yan a, Liping Zhang a, Arturo Galvani b, Louis J. Lombardo a,§
Robert M. Borzilleri a
,
a Bristol-Myers Squibb Research, PO Box 4000, Princeton, NJ 08543-4000, USA
b Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 30 April 2012
5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as
potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92,
I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure con-
firmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in
MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar
antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.
Keywords:
Bcl-2
Bcl-xL
Isolated mitochondria
Protein–protein interaction
Cytochrome c
Ó 2012 Elsevier Ltd. All rights reserved.
The B-cell lymphoma 2 (Bcl-2) family of proteins are key medi-
ators of apoptosis or programmed cell death.1 Anti-apoptotic fam-
ily members including Bcl-2, Bcl-xL, and Mcl-1 bind and sequester
pro-apoptotic members such as Bim, Puma, Noxa, Bad, and Bax,
thereby preventing mitochondria-mediated cell death. Dysregula-
tion of this critical process has been implicated in cancer. For
example, up-regulation of anti-apoptotic family members provides
a mechanism for cells to evade apoptosis, allowing for tumorigen-
esis and resistance to chemotherapy and radiation.
Protein–protein interactions have proven challenging targets in
drug discovery as they are frequently characterized as large, diffuse,
and solvent-exposed.2 Despite these challenges, small molecule
protein mimetics have made considerable progress in recent
totic proteins Bcl-2 and Bcl-xL.4,5 The most advanced Bcl-2 inhibi-
tor currently in human clinical trials, ABT-263 (1), demonstrates
single agent activity in xenograft models of small-cell lung cancer
and acute lymphoblastic leukemia as well as the ability to potenti-
ate the effects of cytotoxic agents in B-cell lymphoma and multiple
myeloma where single agent activity is modest or absent (Fig. 1).6,7
While ABT-263 (1) has generated considerable interest, albumin
binding has been shown to greatly diminish sensitivity of chronic
lymphocytic leukemia (CLL) cells to ABT-263 (1) and may limit
the effectiveness of this promising agent in vivo.8
In the preceding paper, we described a novel series of pyrazole-
derived phenylacylsulfonamides 2 as potent dual Bcl-2/Bcl-xL
antagonists (Fig. 1).9 We have further developed this initial lead
series to generate additional potent dual Bcl-2/Bcl-xL antagonists
from two distinct chemotypes with improved cellular activity.
Herein we describe the synthesis and SAR of regiosiomeric
pyrazoles 3 and 2-aminopyrimidines 4.
A versatile synthetic route to the target regioisomeric pyrazoles
was developed to allow for substitution of the pyrazole, tetrahy-
droisoquinoline (THIQ), and acyl sulfonamide moieties (Scheme 1).
Commercially available 3-(tert-butoxycarbonyl)benzoic acid (5)
was first selectively iodinated in the ortho position to give 2-iodo
acid 6.10 Following amide formation with THIQ 7, the iodide was then
carbonylated to give aldehyde 9. Use of trioctylsilane in this reaction
was critical as the more typical (and more reactive) triethylsilane
prematurely reduced the aryl-halide bond before CO insertion
could occur. Aldehyde 9 was then converted to nitroalcohol 10
years. In particular,
number of groups to disrupt protein–protein interactions.3 Bcl-2
family proteins interact through binding of -helical pro-apoptotic
a-helix mimetics have been described by a
a
Bcl-2 family members to a conserved hydrophobic groove present
on anti-apoptotic family members. Small molecule inhibitors of
this heterodimerization event have been described for anti-apop-
⇑
Corresponding author. Tel.: +1 609 252 3965.
E-mail address: gretchen.schroeder@bms.com (G.M. Schroeder).
Authors contributed equally to the composition of this manuscript.
Current address: PharmaResources (Shanghai) Co., Ltd, Building 1B, No. 528
à
RuiQing Rd., Pudong, Shanghai 201201, PR China.
§
Current address: Hoffman-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110,
USA.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.106

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G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3951–3956
O
O
CF3
S
S
O
H
O
S
O
NH
N
O
N
N
1
Cl
navitoclax (ABT-263)
O
R
O
N
N
Cl
N
N
N
N
N
R =
N
Cl
N
HN
O
OH
S
O
O
2
3
4
Figure 1. Bcl-2 family antagonists.
which was itself dehydrated to give nitroolefin 11 in good yield
over two steps. The pyrazole ring was then constructed with
complete regiocontrol by reaction of the appropriately substituted
hydrazone 12 in the presence of potassium tert-butoxide followed
by a TFA quench.11 Finally, deprotection of the tert-butyl ester
followed by acylsulfonamide formation gave the desired compounds
3.
improvement against both proteins (0.66
lM versus Bcl-2 and
0.042 M versus Bcl-xL). Alteration of the ethyl ester at the pyrazole
l
C-3 position also significantly impacted Bcl-2 and Bcl-xL activity.
For example, acid 3c saw further improvements in both Bcl-2
and Bcl-xL activity (0.17 lM and 0.030 lM, respectively). Alcohol
3d was optimum giving an IC50 of 61 nM for Bcl-2 and 11 nM for
Bcl-xL. Substitution of the pyrazole N-1 aryl group was studied
extensively. Small alkoxy substituents in the para position of the
pyrazole N-phenyl ring were generally tolerated (i.e., methoxy
compound 3e). Addition of a larger, more lipophilic phenoxy group
in the meta (3f) and para (3g) positions was examined. While both
analogs gave enhanced activity versus Bcl-2, only the para substi-
tution was tolerated for Bcl-xL.
Encouraged by the improved dual inhibition demonstrated by
phenyl ether 3g, we examined this lead further. As observed be-
fore, reduction of the ethyl ester to give alcohol 3h was well toler-
ated. Addition of a chloride to the phenoxy ring gave an additional
increase in activity. The 3-chlorophenyl derivative 3j was superior
to the 4-chlorophenyl analog 3i inhibiting Bcl-2 with an IC50 of
8 nM and Bcl-xL with an IC50 of 18 nM. Replacement of the phen-
oxy group with butoxy (3k) gave a modest reduction in Bcl-2
and Bcl-xL activity. Elimination of the ether linker to give biphenyl
3l was well tolerated and inhibited Bcl-2 and Bcl-xL with IC50’s of 7
and 13 nM, respectively. Recognizing that inhibitors of protein–
protein interactions tend to be rather lipophilic with high clogP’s,
we examined appending alcohols to the pyrazole N-phenyl ring
to improve aqueous solubility and create more drug-like leads.
To that end, para alcohols 3m and 3n were generated and, gratify-
ingly, the appended alcohols were also well accommodated by
both Bcl-2 and Bcl-xL. A meta substituted alcohol 3o was slightly
less active than its para counterparts.
The ethyl ester present in pyrazole 3b could be further deriva-
tized to give the corresponding acid 3c by hydrolysis with aqueous
sodium hydroxide. Careful reduction of the ethyl ester with lithium
borohydride could also be achieved to give the alcohol 3d.
Preparation of 2-aminopyrimidine phenylacylsulfonamides 4 is
shown in Scheme 2. Aryl boronate 16 was prepared according to
the method reported by Miura and coworkers.12 Suzuki coupling
of boronate 16 with 2,4-dichloropyrimidine (R2 = H), 2,4-di-
chloro-5-methylpyrimidine (R2 = methyl), or 2,4,5-trichloropyrim-
idine (R2 = Cl) chemoselectively afforded the corresponding biaryl
17. Displacement of the 2-chloride on the pyrimidine ring with
different dialkylamines provided 2-aminopyrimidine intermediate
18. After removing the Boc protecting group, tetrahydroisoquino-
lines were introduced by HATU-mediated coupling. Hydrolysis of
methyl ester 19 under basic conditions afforded the corresponding
acids which were coupled with sulfonamides 15 to form analogs
4a–e. The synthetic sequence was flexible in that the order of steps
for the acylsulfonamide and THIQ amide formation could be re-
versed to give 4f–g, allowing for more efficient survey of THIQ sub-
stituents or replacements. Finally, (S) 3-aminomethyl THIQ 4h was
prepared from the corresponding azide by palladium catalyzed
hydrogenation.
We began by exploring SAR of the regioisomeric pyrazoles 3
with initial efforts directed at exploring the pyrazole ring itself (Ta-
ble 1). The alkyl group at C-5 was examined and it was determined
that small alkyl groups gave inferior results as compared to larger
alkyl groups. For example, methyl substituted pyrazole 3a was
Next we turned our attention to SAR of the tetrahydroisoquino-
line ring (Table 2). Borrowing from our previous experience with
substituted THIQs,9 we prepared alcohol 3p. As expected, this
compound demonstrated a marked improvement in Bcl-2 potency
relative to the unsubstituted THIQ 3d, generating a potent dual
only 4.19 and 0.64
lM versus Bcl-2 and Bcl-xL, respectively
whereas, butyl substituted pyrazole 3b showed nearly 10-fold

G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3951–3956
3953
NO2
R2
HO
O
O
O
O
O
O
I
O
I
N
N
HO
HO
N
a
b
c
d
R1
R1
R1
O
O
NH
R1
O
O
5
O
O
6
O
O
O
10
R3
8
9
7
R3
3
R
N
N
R2
O
CO2Et
NO2
R2
N
N
N
N
N
R2
O
2
R
CO2Et
O
CO2Et
O
h
R1
g
e
f
N
N
N
R4
HN
S
O
R1
O
R1
S
O
NH2
15
R1
O
N
CO2Et
R4
O
N
H
R3
O
O
O
O
HO
14
O
12
11
13
3
N
N
N
N
N
N
OH
OH
O
O
N
O
O
O
O
i
j
N
N
HN
S
O
HN
S
O
HN
S
O
O
O
O
O
O
O
3d
3c
3b
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, PhI(OAc)2, I2, Bu4NI, DCE, 80 °C, 6 h, 62%; (b) HATU, 2,6-lutidine, THF/DMF, rt, 1 h, 54–65%; (c) Pd(dppf)Cl2, Oct3SiH, DIPEA,
CO, DMF, 70 °C, 16 h, 40–50%; (d) R2CH2NO2, KOtBu, THF, rt, 3 h; (e) Ac2O, cat. DMAP, THF, rt, 30 min, then DMAP, DCM, rt, 16 h, 66–80% (2 steps); (f) KOtBu, THF, À78 °C, then
TFA, À78 °C, 2 h, 17–68%; (g) TFA, DCM, rt, 1 h; (h) EDC, DMAP, DMF, rt, 24–90% (2 steps); (i) 1 N aqueous NaOH, THF, rt, 16 h, 100%; (j) LiBH4, THF, rt, 2 h, 64–55%.
R2
O
R2
O
O
O
O
S
O
N
N
N
N
N
HN
Ar
N
OMe e, f
c, d
R3
R3
R3
R3
N
N
R2
R2
O
O
O
O
O
O
19
N
N
B
N
4a-e
a
b
OMe
Me
OMe
N
N
R2
Cl
3
O
R
O
Cl
R2
R2
O
N
O-t-Bu
R3
O-t-Bu
O-t-Bu
O
O
O
17
18
O
S
16
Cl
O
N
N
N
N
c, d
e, f
N
HN
S
Ar
N
HN
Ar
R3
N
O
R3
O
O
3
R3
R1
R
O-t-Bu
20
4f-g
Cl
Cl
Cl
Cl
O
O
O
N
N
N
HN
S
O
HN
S
O
g
N
n-Bu
N
O
n-Bu
N
O
n-Bu
H2N
N
n-Bu
N
N3
4h
20g
Scheme 2. Reagents and conditions: (a) PdCl2dppf2, Na2CO3, 1,4-dioxane, 80 °C, 15 h, 78%; (b) NH(R3)2, K2CO3, DMF, 60 °C, 5–12 h, >80%; (c) TFA/DCM, 100%; (d) THIQ, HATU,
DIPEA, rt, 2 h, 52–80%; (e) 2 N aqueous NaOH, THF/MeOH, rt, 2–3 h, 90–100%; (f) sulfonamide 15, EDC, DMAP, rt, 57–70%, (g) Pd/C, H2, MeOH, rt, 2 h, 57%.
inhibitor of Bcl-2 and Bcl-xL. Introduction of amine substituted
THIQ 3q was also well tolerated giving IC50’s against Bcl-2 and
Bcl-xL of 24 and 7 nM, respectively.
Lastly, SAR of the acylsulfonamide was explored (Table 2).
Replacement of the aromatic naphthyl ring with a trimethylsilyl-
ethyl group (3r) was found to be detrimental for Bcl-2 activity
(3-fold less active than 3d), but inconsequential with regards to
Bcl-xL activity. The simple ethyl acylsulfonamide (3s) was deter-
mined to be less potent for both enzymes. Extending the alkyl
group from ethyl to pentyl as in compound 3t restored some Bcl-
activity (IC50 = 65 nM) but had no effect on Bcl-xL
2
(IC50 = 120 nM). Substitution of the naphthyl ring with a chloride
in the 8-position (3u) gave a slight improvement in both Bcl-2
and Bcl-xL activity as compared to unsubstituted naphthyl 3j.
An X-ray crystal structure of compound 3r complexed to Bcl-xL
demonstrated that these pyrazole-based inhibitors bind in the con-
served hydrophobic groove normally occupied by pro-apoptotic
family members such as Bim (Fig. 2).13 As designed, the THIQ moi-

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G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3951–3956
Table 1
Table 2
Regioisomeric pyrazoles: pyrazole ring SARa
Regioisomeric pyrazoles: THIQ and acylsulfonamide SARa
R3
1
R
N
N
N
N
R1
O
OH
R2
R2
O
N
N
HN
S
O
HN
O
O
O
S
O
O
R3
Compd
R1
R2
H
CH2OH
CH2NH2
H
H
H
H
R3
IC50
Bcl-2
(l
M)
Compd
R1
R2
R3
IC50
Bcl-2
(l
M)
Bcl-xL
Bcl-xL
3d
3p
3q
3r
3s
3t
H
H
H
H
Naphthyl
Naphthyl
Naphthyl
(CH2)2TMS
CH2CH3
(CH2)4CH3
Naphthyl,
8-Cl
0.061
0.011
0.024
0.19
0.19
0.065
0.006
0.011
0.008
0.007
0.013
0.14
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
Me
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
Bu
CO2Et
CO2Et
CO2H
CH2OH
CO2Et
H
H
H
H
4.19
0.66
0.17
0.061
1.79
0.14
0.10
0.13
0.022
0.008
0.045
0.007
0.020
0.014
0.071
0.64
0.042
0.030
0.011
0.048
0.47
0.055
0.035
0.035
0.018
0.026
0.013
0.006
0.007
0.015
O(3-Cl)Ph
O(3-Cl)Ph
O(3-Cl)Ph
4-OMe
3-OPh
4-OPh
4-OPh
4-O(Ph, 4-Cl)
4-O(Ph, 3-Cl)
4-OButyl
0.12
0.015
CO2Et
CO2Et
3u
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
a
For assay conditions see Ref. 9.
4-Ph
4-(CH2)2OH
4-(CH2)3OH
3-CH2OH
a
For assay conditions see Ref. 9.
ety resides in the I95 pocket and interacts with Phe97, Tyr101, and
Phe105 via a combination of -stacking and edge-face interactions.
p
The pyrazole C-5 butyl group extends into the L92 pocket. The loss
in potency in shortening this butyl group to a methyl as in 3a can
now be rationalized as the methyl group is not long enough to fill
this hydrophobic space normally occupied by the amino acid side
chain of leucine. The pyrazole N-1 phenyl ring points to the I88
pocket and biaryls such as 3j likely extend into this space. The alco-
hol at C-3 of the pyrazole ring makes a hydrogen bond with
Leu130. Finally, the acyl sulfonamide forms several critical interac-
tions with the Bcl-xL protein. For example, the acyl sulfonamide
carbonyl accepts a hydrogen bond from the Tyr101 hydroxyl
group. The trimethylsilylethyl group projects into a rather large
hydrophobic pocket defined by Ala93, Phe97, Arg100, Val141,
and Tyr195. Normally occupied by Phe99 of Bim, it is clear that
groups like naphthyl would be well accomodated in such an
environment.
Next, we applied lessons learned from the pyrazole series to the
design of 2-aminopyrimidine based antagonists (Table 3). The par-
ent compound in this series, pyrimidine 4a, gave modest potency
against Bcl-2 (IC50 = 700 nM) and Bcl-xL (IC50 = 500 nM). Addition
of a halide (4b,c) to the naphthyl ring gave approximately five-fold
improvement in Bcl-2 activity, but had little effect on Bcl-xL. Like-
wise, replacement of the naphthyl ring by 1-(3,4-dichloroben-
zyl)indoline to give 4d had a similar effect. As before, addition of
amino- or hydroxy- methyl groups at the THIQ 3-position gave
improved results (4e,f). A significant discovery came from the
observation that substitution of the pyrimidine ring at C-5 with
Figure 2. Co-crystal structure of compound 3r in Bcl-xL (green) superimposed with
Bim from the Bim/Bcl-xL complex 1PQ1 (orange). The Bim residues mimicked by 3r
(I88, A89, L92, I95, and F99) are labeled in orange. Key interactions between 3r and
Bcl-xL are labeled in green. The hydrogen bonds to the side chain hydroxyl from
Y101 and the backbone carbonyl of L130 are denoted with dashed lines.
chloro or methyl groups led to improvement in potency (4g,h).
Finally, replacement of the dibutylamino group on the pyrimidine
ring with dipropylamino (4i) was optimum as the ethyl and pentyl
analogs showed diminished activity (data not shown). Compounds
from this series are expected to bind Bcl-2 and Bcl-xL analogously
to pyrazole 3r. Molecular modeling suggests the dipropylamine

G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3951–3956
3955
Table 3
2-Aminopyrimidinesa
R2
O
O
S
O
N
N
HN
Ar
N
O
R3
R3
R1
Compd
Ar
R1
R2
R3
IC50 (lM)
Bcl-2
0.70
Bcl-xL
N
N
4a
4b
4c
H
H
H
n-Butyl
n-Butyl
n-Butyl
0.50
0.48
0.97
Cl
0.12
0.14
I
N
N
Cl
4d
H
n-Butyl
0.12
0.71
N
Cl
N
N
4e
4f
H
H
n-Butyl
n-Butyl
0.43
0.16
0.16
0.13
H2N
Cl
HO
N
4g
4h
4i
Cl
n-Butyl
n-Butyl
n-Propyl
0.16
0.11
0.15
0.23
0.12
0.25
N
CH3
H
N
Cl
Cl
N
N
4j
Cl
Cl
n-Propyl
n-Propyl
0.015
0.009
0.014
0.009
H2N
HO
4k
a
For assay conditions see Ref. 9.
simultaneously fills both the I88 and L92 pockets which explains
why the length of the alkyl chain is so critical.
strating submicromolar activity in the MV-4-11 AML cell line. In
addition, pyrimidine 4j maintained submicromolar activity in
MV-4-11 cells in the presence of 10% fetal calf serum
Optimized acylsulfonamide, THIQ, and pyrimidine substituents
were combined to give pyrimidines 4j and 4k. These compounds
demonstrated potent dual Bcl-2/Bcl-xL activity with greater
than 50-fold improvement in activity compared to the parent
compound 4a.
Several compounds from the regioisomeric pyrazole and 2-ami-
nopyrimidine series were examined for their effects on cell prolif-
eration in Bcl-2/Bcl-xL dependent cell lines (Table 4).14 Cellular
antiproliferative activity paralleled that observed in the biochemi-
cal assays with potent dual antagonists from the pyrazole series
(3j, 3u) performing better than less potent analogs (3k, 3o). The
most potent compounds in the cell proliferation assay came from
the aminopyrimidine series with compounds 4j and 4k demon-
(IC50 = 0.73 lM).
With compounds in hand demonstrating excellent biochemical
potency against Bcl-2 and Bcl-xL, select analogs were examined in
isolated mitochondria from MV-4-11 AML cells for cytochrome c
release and BAK oligomerization (Fig. 3).15 Pyrimidines 4j and 4k
were tested at concentrations of 0.1 and 1 lM and were shown
to induce cytochrome c release and BAK oligomerization in a
dose-dependent manner. These results demonstrate 4j and 4k
antagonize Bcl-2 and Bcl-xL function on the surface of mitochon-
dria, resulting in initiation of the intrinsic apoptotic pathway.
Pyrazoles such as 3j and 3o also demonstrated cytochrome c release
and BAK oligomerization at low concentrations, however, they

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G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3951–3956
Table 4
In summary, potent pyrazole and pyrimidine acylsulfonamide
Cellular antiproliferative activity (2% FCS)a
inhibitors with dual activity against Bcl-2 and Bcl-xL were
identified. Compounds were optimized for binding to essential
pockets such as I88, L92, I95, and F99 which are normally occupied
by pro-apoptotic family members. An X-ray co-crystal structure
with Bcl-xL confirmed the proposed binding mode. Compounds
demonstrated on-target mechanistic inhibition of Bcl-2 and Bcl-xL
through cytochrome c release and BAK oligomerization in isolated
mitochondria. Further evidence of target inhibition was demon-
strated by whole cell mitochondria depolarization in MV-4-11
cells. Improved cellular antiproliferative activity was achieved
relative to the original lead pyrazole typified by 2.
Compd
SUDHL-4 IC50
(
lM)
MV-4-11 IC50 (lM)
3j
2.35
2.49
>10
2.02
6.64
3.79
0.70
1.43
5.06
0.58
0.33
0.33
3k
3o
3u
4j
4k
a
For assay conditions see Ref. 9.
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X.; Wendt, M. D.; Yang, X.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S.
W. J. Med. Chem. 2008, 51, 6902.
7. (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. Cancer Res.
2008, 68, 3421; (b) Ackler, S.; Mitten, M. J.; Foster, K.; Oleksijew, A.; Refici, M.;
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W.; Shoemaker, A. R. Cancer Chemother. Phamacol. 2010, 66, 869.
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Figure 3. Cytochrome c release and BAK oligomerization from isolated mitochon-
dria of MV-4-11 cells.
9. Perez, H. L.; Banfi, P.; Bertrand, J.; Cai, Z.-W.; Grebinski, J.; Kim, K.; Lippy, J.;
Modugno, M.; Naglich, J.; Schmidt, R. J.; Tebben, A.; Vianello, P.; Wei, D. D.;
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M.; Tsukamoto, S.-I. Synth. Commun. 2006, 36, 3809.
13. The structure was deposited in the PDB as 4EHR.
14. Deng, J.; Carlson, N.; Takeyama, K.; Dal Cin, P.; Shipp, M.; Letai, A. Cancer Cell
2007, 12, 171.
15. For assay conditions see Ref. 9.
16. Representative characterization data: (a) 3j; 1H NMR (CD3OD) d 8.73 (s, 1H),
8.12–7.98 (m, 6H), 7.73–7.66 (m, 2H), 7.63–6.94 (m, 13H), 4.62–4.18 (m, 4H),
3.65–3.17 (m, 2H), 2.79–2.45 (m, 4H), 1.31–0.91 (m, 4H), 0.65–0.58 (m, 3H);
MS(ESI+) m/z 825.1 (M+H)+; (b) 4j, 1H NMR (CDCl3, 1:1 mixture of amide
rotamers) d 9 .16 (s, 1H), 8.42–8.14 (m, 4 H), 8.02 (d, J = 8.1 Hz, 1H), 7.82 (d,
J = 8.6 Hz, 1H), 7.76–7.47 (m, 5H), 7.15–7.04 (m, 2 H), 6.94 (d, J = 6.0 Hz, 0.5H),
6.85 (d, J = 6.0 Hz, 0.5H), 5.43 (br s, 1H), 4.48 (d, J = 17.6 Hz, 1H), 4.28 (d,
J = 17.6 Hz, 1H), 3.45–2.98 (m, 5H), 2.88–2.48 (m, 5H), 1.65–1.23 (m, 4H), 1.01–
0.51 (m, 6H); MS(ESI+) m/z 745.1 (M+H)+; (c) 4k, 1H NMR (CD3OD, 1:1 mixture
of amide rotamers) d 9.05 (s, 1H), 8.96 (br s, 1H), 8.24 (br s, 1H), 8.14–8.04 (m,
2H), 7.85–7.49 (m, 5H), 7.16–6.89 (m, 3H), 6.82 (br s, 1H), 5.22–5.05 (m, 1H),
4.53 (br s, 1H), 4.24 (br s, 1H), 3.45–3.05 (m, 5H), 2.80–2.34 (m, 3H), 1.65–1.50
(m, 4H), 0.99–0.52 (m, 6H); MS(ESI+) m/z 746.1 (M+H)+.
17. Whole cell depolarization assay: The human acute myeloid leukemia (AML)
cell line, MV-4-11 (CRL-9591), was purchased from the American Type Culture
Collection (Manassas, VA) and routinely maintained in RPMI 1640 medium
(GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37 °C in
humidified atmosphere of 5% CO2. Logarithmically growing cells were
concentrated by low-speed centrifugation, suspended in serum-free RPMI-
Figure 4. Average percent population of depolarized mitochondria in MV-4-11
cells.
failed to show induction of BAK oligomerization at higher concen-
trations (data not shown). This may be due to poor aqueous solu-
bility at higher concentrations.
1640 medium and stained using the cationic dye, MitoProbe JC-1 (2 lM, 0.5%
DMSO, 30 min, 37 °C), following the manufacturer’s instructions (Molecular
Probes, Eugene, OR). Stained cells were concentrated by low-speed
centrifugation, suspended in RPMI-1640 medium supplemented with 2%
fetal bovine serum and subjected to compound treatment (5 Â 105 per well
Finally, pyrazole 3j and pyrimidines 4j and 4k were tested for
their effects on mitochondria depolarization in whole cells
(Fig. 4).16 All three compounds induced ꢀ25% depolarized
mitochondria in MV-4-11 AML cells after treatment for 7 h at a
of
a 96-well plate, 7 h, 37 °C). Cellular mitochondria depolarization was
measured by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA) and
the data analyzed using FlowJo software (Tree Star Inc., Ashland, OR).
concentration of 3
evidence of Bcl-2 and Bcl-xL inhibition.
l
M.17 This activity provides further mechanistic