Structures of the small-molecule Bcl-2 inhibitor (BH3I-2) and its related simple model in protonated and deprotonated forms

Article abstract of DOI:10.1246/bcsj.77.2057

3-Bromo-5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide (BCNCPB-OH) was synthesized as a model compound of the small-molecule Bcl-2 inhibitor (BH3I-2) and characterized by ¹HNMR and IR measurements. The structures of BCNCPB-OH and its deprotonat

Full text of DOI:10.1246/bcsj.77.2057

ꢀ 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 2057–2064 (2004) 2057
Structures of the Small-Molecule Bcl-2 Inhibitor (BH3I-2) and
Its Related Simple Model in Protonated and Deprotonated Forms
Daisuke Kanamori, Taka-aki Okamura, Hitoshi Yamamoto,
Shigeomi Shimizu,1 Yoshihide Tsujimoto,1 and Norikazu Ueyama
ꢀ
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043
1Department of Post-Genomics and Diseases, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871
Received May 24, 2004; E-mail: ueyama@chem.sci.osaka-u.ac.jp
3-Bromo-5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide (BCNCPB–OH) was synthesized as a model com-
1
pound of the small-molecule Bcl-2 inhibitor (BH3I-2) and characterized by H NMR and IR measurements. The struc-
tures of BCNCPB–OH and its deprotonated form (BCNCPB–O–NEt4) were determined by X-ray analysis. BCNCPB–
OH has an intramolecular OH O=C hydrogen bond and BCNCPB–O–NEt has an intramolecular NH O(oxyanion)
ꢁꢁꢁ
ꢁꢁꢁ
4
hydrogen bond in the solid and solution states. The solution structures of protonated and deprotonated BH3I-20 (BH3I-2
1
analogue) were determined by H NMR, NOESY, and IR measurements. They have similar conformations to those of
the corresponding model compounds. A pKa value of BCNCPB–OH of 5.1 is considered a very low value for phenol
derivatives, suggesting the possibility that BH3I-2 binds to protein in the deprotonated form having an intramolecular
NH O(oxyanion) hydrogen bond.
ꢁꢁꢁ
BH3I-2 is a recently identified small-molecule inhibitor of
the Bcl-2 family, which is a regulator of apoptotic pathways
(Chart 1).1,2 A number of inhibitors which have a salicylamide
skeleton similar to BH3I-2 have been reported to inhibit vari-
ous enzymes relating to the respiratory chain,3–6 the biosynthe-
sis of melanin,7,8 oxidative phosphorylation,9–11 bacterial two
component systems,12,13 and so on. Although the precise inhib-
itory mechanisms in many cases are vague, drug development
work has indicated that electron-withdrawing substituents12,13
and hydrophobicity13 are needed for a high activity. The im-
portance of the salicylamide skeleton and hydrogen bonds on
it have been shown by the following evidence. In X-ray struc-
tures of the inhibitor–scytalone dehydratase complex7 and an-
timycin A1–cytochrome bc1 complex (the chemical structure
of the inhibitors are depicted in Chart 2),3,14 hydrogen bonds
between amino acid residues in the binding site and phenolic
OH, amide NH, and the carbonyl group on the salicylamide
moiety were found. In these structures, intramolecular
by a short distance between the phenolic oxygen and carbonyl
oxygen. The analogous inhibitor, antimycin A3, is also repre-
sented as an intramolecularly OH O=C hydrogen-bonded
ꢁꢁꢁ
conformation in the proposed structure of antimycin A3/Bcl-
xL (one of the Bcl-2 family proteins) complex.15,16 Modifica-
tions to break intra- and/or intermolecular hydrogen bonds
such as the deletion of the phenolic OH or carbonyl group,8
methylation of the phenolic17 or amide4 proton, or the insertion
of –CH2– between the aromatic ring and carbonyl group4
reduce the inhibitory activity. These results demonstrate that
the importance of the salicylamide skeleton arises from its
hydrogen bonding ability.
Suezawa et al. has reported that salicylanilide derivatives
having a halogen atom (X), a hydrogen bonding acceptor, at
the 3-position are in equilibrium between intramolecular
H
O
O
CH3
OH O=C hydrogen bonding on the inhibitor is suggested
ꢁꢁꢁ
N
H
Br
Cl
F
Inhibitor of scylatone dehydratase
O
H
O H
S
R
O
X
Br
Cl
O
H
CH2 CH2
O
n CH3
CH3
CH
O
O
CH3
N
N
H
O
O
H
N
O
O
H
O Cl
O Cl
C2H5
N
H
O
Cl
CH3
O
BCNCPB-OH (-OR = -OH)
BH3I-2 (X = Br)
BH3I-2' (X = I)
+
BCNCPB-ONEt4 (-OR = -O- NEt4
)
Antimycin A3 (n = 2), A1 (n = 4)
Chart 1. Chemical structures of BH3I-2, BH3I-20,
BCNCPB–OH, and BCNCPB–O–NEt4.
Chart 2. Other inhibitors having salicylamide moiety.
Published on the web November 11, 2004; DOI 10.1246/bcsj.77.2057

2058 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Structures of Protonated/Deprotonated BH3I-2
bic environment using the model compounds discussed below.
To realize a hydrophobic environment where BH3I-2 binds,
we use organic solvents and a micellar solution.
H
H
R
O
H
O
N
O
O
X
X
R
N
H
The determination of the precise hydrogen bonding mode
of BH3I-2 in the complex is important because it will be
valuable for understanding inhibitory mechanisms in detail.
For this purpose, 3-bromo-5-chloro-N-(2-chlorophenyl)-2-hy-
droxybenzamide (BCNCPB–OH) and its deprotonated form
(BCNCPB–O–NEt4) were synthesized and structurally ana-
lyzed as model compounds of protonated and deprotonated
BH3I-2 (Chart 1), respectively. Because the replacement of
the N-substituent does not significantly influence inhibitory ac-
tivity, the 4-chlorophenylsulfonyl group was removed to im-
prove the crystallinity of the BH3I-2 model compounds.4
The structures and hydrogen bonding modes were determined
by X-ray analysis and IR spectra in the solid state and by
1H NMR and IR spectra in solution. The structures of BH3I-
20 (Chart 1) in protonated and deprotonated forms were deter-
mined by 1H NMR and IR spectra in solution. The reason
BH3I-20 was employed is that it probably prefers a similar con-
formation to BH3I-2, which is not available commercially. To
evaluate the tendency to deprotonate the phenolic OH, the pKa
value of BCNCPB–OH was determined. The pKa value of
BH3I-2 is considered to be comparable to that of BCNCPB–
OH because introduction of a stronger electron-withdrawing
group (NO2) had a negligible influence on the pKa value.11,25
type A
type B
Chart 3. Two hydrogen bonding modes.
NH O hydrogen bonded (type A) and OH O=C hydrogen
ꢁꢁꢁ
ꢁꢁꢁ
bonded conformations (type B) (Chart 3).18 Although O-
acetylation removes the stabilization by the intramolecular
OH O=C hydrogen bond, the type B conformation is major
in solution. Salicylanilide derivatives, which can form intra-
ꢁꢁꢁ
molecular OH O=C hydrogen bonds, including BH3I-2,
ꢁꢁꢁ
probably favor a type B conformation. On the other hand, type
A is predominant in deprotonated salicylamide derivatives by
intramolecular NH O(oxyanion) hydrogen bonds.19,20
ꢁꢁꢁ
The structure of the BH3I-2/Bcl-xL complex has not been
determined by X-ray analysis but computationally proposed
using the solution structure of the BakBH3–peptide/Bcl-xL
complex.1,21,22 In this structure, BH3I-2 binds to the hydropho-
bic cleft formed by BH1, BH2, and BH3 regions and takes a
type A conformation depicted in Chart 1. Though it was not
discussed in detail whether BH3I-2 is protonated or not, we
present here the possibility of the deprotonation. The pKa val-
ues of salicylanilide derivatives have been reported below 7.5,
and further lowered by electron-withdrawing substituents.11
The pKa value of salicylamide derivatives is also reduced by
Results and Discussion
the NH O(oxyanion) hydrogen bond in its phenolate anion
form.11,19 Such intramolecular NH O hydrogen bonds are sup-
Synthesis.
The synthesis of BCNCPB–OH and
ꢁꢁꢁ
BCNCPB–O–NEt4 is shown in Scheme 1. The phenolic hy-
droxy group was protected by an acetyl group to avoid a side
reaction. The deprotonation of the phenolic OH was performed
by neutralization with NEt4OH.
Structures of Model Compound in the Protonated and
Deprotonated Forms in the Solid State. The molecular
structures of protonated (BCNCPB–OH) and deprotonated
forms (BCNCPB–O–NEt4) were determined by X-ray analy-
ꢁꢁꢁ
ported in a hydrophobic environment and efficiently lower the
pKa value, as shown previously in our report for thiophenol
(benzenethiol) and benzoic acid analogues.23,24 In the hydro-
phobic cleft of Bcl-xL, the pKa value of BH3I-2 is presumably
lowered, and its deprotonated form is stabilized.
In this paper, we determine the structure of BH3I-2 and its
deprotonated form and evaluate the pKa value in a hydropho-
NaH2PO4,
NaClO2,
OH
Cl
OH
Cl
O
O
Br
CHO
Br
COOH
Br
COOH
NEt3, CH3COCl
THF
H2NSO3H
dioxane, H2O
Cl
O
O
OH
1) NEt3, IBCF,
Br
CONH
Cl
Br
CONH
Cl
1M NaOH aq.
EtOH
2) o-chloroaniline
THF
Cl
Cl
BCNCPB-OH
NEt4
Br
O
20% NEt4OH aq.
CONH
Cl
MeOH
Cl
BCNCPB-O-NEt4
Scheme 1. Synthesis of BCNCPB–OH and BCNCPB–O–NEt4. IBCF = isobutyl chloroformate.

D. Kanamori et al.
Bull. Chem. Soc. Jpn., 77, No. 11 (2004) 2059
sis. It has been revealed in the solid state that BCNCPB–OH
prefers the type B conformation and BCNCPB–O–NEt4 pre-
fers type A.
The molecular structure of BCNCPB–OH is shown in
Fig. 1a and the selected short contacts, bond lengths, and tor-
sion angles are listed in Table 1. The presence of an intramo-
160.0(3)ꢂ (C5–C6–C7–O2) and 176.9(3)ꢂ (C7–N1–C8–C9).
The presence of an intermolecular OH O=C hydrogen bond
is suggested by the short distance between the phenolic hydro-
ꢁꢁꢁ
ꢀ
neighboring molecule (2.50(3) A) (Fig. 1b). No significant
gen atom (H1) and amide carbonyl oxygen atom (O2 ) of the
ꢀ
intermolecular interaction around the amide NH exists.
The crystal structure of the deprotonated form (BCNCPB–
O–NEt4) is shown in Fig. 1c. A different hydrogen bonding
mode from that in BCNCPB–OH was found. The short dis-
tances between the phenoxy oxygen (O1) and the amide proton
lecular OH O=C hydrogen bond is suggested by the short dis-
tances between the phenolic OH proton (H1) and the amide
ꢁꢁꢁ
ꢀ
carbonyl oxygen atom (O2) (1.91(3) A) and between the phe-
ꢀ
nolic oxygen (O1) and O2 (2.606(3) A). The chlorine atom at
the 20-position in ArB (Cl2) is located on the opposite side of
the carbonyl group to avoid electrostatic repulsion. The loca-
tion of the chlorine atom suggests the presence of the attractive
interaction between the amide NH proton (H2) and Cl2 (Cl2–
ꢀ
H2 is 2.51(2) A). The coplanarity between the amide plane and
two aromatic rings is indicated where the torsion angles are
(H1) (1.87(2) A) and between O1 and the amide nitrogen (N1)
ꢀ
ꢀ
(2.583(2) A) suggest the presence of an intramolecular
NH O(oxyanion) hydrogen bond. The relative orientation of
ꢁꢁꢁ
chlorine at the 20-position in ArB (Cl2) toward the amide
NH is similar to that in BCNCPB–OH. The coplanarity of
BCNCPB–O–NEt4 is higher than in BCNCPB–OH. The tor-
sion angle between ArA and amide plane (C5–C6–C7–O2) is
ꢃ1:2(3)ꢂ, and that between the amide plane and ArB (C7–
N1–C8–C9) is ꢃ175:6(2)ꢂ. The C1–O1 bond length in
ꢀ
BCNCPB–O–NEt4 (1.282(3) A) is shorter than that in
ꢀ
BCNCPB–OH (1.340(3) A) due to an increase in the double
bond character of the C1–O1 bond caused by the delocaliza-
tion of the negative charge of the phenoxy anion to the aryl
ring.
The most significant structural change between BCNCPB–
OH and BCNCPB–O–NEt4 is a ca. 180 degree rotation about
the C6–C7 bond.
The presence of hydrogen bonds in the solid state was con-
firmed by IR measurements in a Nujol mull (Fig. 2). In the pro-
tonated form, BCNCPB–OH, an amide NH stretching band
was observed at 3416 cmꢃ1, which is slightly lower than
non-hydrogen-bonded ꢀ(NH) (ꢀ(NH) of salicylanilide in dilute
solution20 is 3456 cmꢃ1) and comparable to NH Cl hydrogen-
ꢁꢁꢁ
bonded ꢀ(NH).26,27 The orientation of Cl toward the carbonyl
C=O and the amide NH is controlled by not only the electro-
static repulsion between the carbonyl oxygen and Cl but also
Fig. 1. ORTEP drawing (a) and packing structure (b) of
BCNCPB–OH and ORTEP drawing of anion part of
BCNCPB–O–NEt4 (c).
the NH Cl hydrogen bond. In BCNCPB–O–NEt , it is
4
ꢁꢁꢁ
Table 1. Selected Short Contacts, Bond Distance, and Torsion Angles of BCNCPB–OH
and BCNCPB–O–NEt4
BCNCPB–OH
BCNCPB–O–NEt4
ꢀ
Short contacts/A
H(2) Cl(2)
ꢁꢁꢁ
2.51(2)
1.91(3)
2.606(3)
2.50(3)
2.921(3)
H(1) Cl(2)
H(1) O(1)
O(1) N(1)
ꢁꢁꢁ
2.54(2)
1.87(2)
2.583(2)
ꢁꢁꢁ
H(1) O(2)
ꢁꢁꢁ
ꢁꢁꢁ
O(1) O(2)
ꢁꢁꢁ
ꢀ
H(1) O(2)
ꢁꢁꢁ
ꢀ
O(1) O(2)
ꢁꢁꢁ
ꢀ
Bond lengths/A
C(1)–O(1)
O(2)–C(7)
C(6)–C(7)
N(1)–C(7)
N(1)–C(8)
O(1)–H(1)
N(1)–H(2)
1.340(3)
C(1)–O(1)
O(2)–C(7)
C(6)–C(7)
N(1)–C(7)
N(1)–C(8)
N(1)–H(1)
1.282(3)
1.239(3)
1.484(3)
1.360(3)
1.400(3)
0.80(2)
1.223(4)
1.498(4)
1.353(4)
1.402(4)
0.74(3)
0.87(3)
Torsion angles/ꢂ
O(2)–C(7)–C(6)–C(5)
C(7)–N(1)–C(8)–C(9)
ꢃ160:0(3)
O(2)–C(7)–C(6)–C(5)
C(7)–N(1)–C(8)–C(9)
ꢃ1:2(3)
176.9(3)
ꢃ175:6(2)

2060 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Structures of Protonated/Deprotonated BH3I-2
type A
O
type B
Cl
Br
Cl
O H
O
H6'
BCNCPB-OH
N
O H
N
H6
H
Br
Cl
H
Cl
δ+ Cl
δ-
NOE
predominant
H6
H6'
O
N
BCNCPB-O-NEt4
Fig. 2. IR spectra in a Nujol mull of BCNCPB–OH (a) and
BCNCPB–O–NEt4 (b).
Br
O H
δ+
Cl
δ-
Chart 4. Solution structures of BCNCPB–OH and
BCNCPB–O–NEt4.
Fig. 3. 1H NMR spectra of BCNCPB–OH in CDCl3 (a)
and BCNCPB–O–NEt4 in CD3CN (b).
Fig. 4. NOESY spectrum of BCNCPB–OH in CDCl3.
0
thought that the location of Cl2 is controlled by the same inter-
actions. BCNCPB–O–NEt4 has an amide NH band at 3016
cmꢃ1, which is a very low frequency for ꢀ(NH) even if the
but not between the NH and 60-positioned aryl proton (H6 at
8.4 ppm) in the NOESY spectrum (Fig. 4). These results also
support the type B conformation. On the other hand, a minor
conformer in addition to type B was established in the IR re-
sults because of its higher time resolution than 1H NMR.
BCNCPB–OH gives NH stretching bands at 3425 and 3357
cmꢃ1 (Fig. 5a). The ratio of each of the conformers was esti-
mated to be about 90 and 10%, respectively, from the ratio of
the area of the bands. The predominant band is assigned to the
type B conformer (3416 cmꢃ1 in the solid state). The NH band
NH proton forms a hydrogen bond, (NH O(H)28 3390 cmꢃ1
,
ꢁꢁꢁ
NH O(Ac)29 approximately 3400 cmꢃ1, NH O(carboxyl-
ꢁꢁꢁ
ꢁꢁꢁ
ate)28 3024 cmꢃ1, NH O(phosphate)30 3226 cmꢃ1, etc.) and
shows that the NH O(oxyanion) hydrogen bond fomed in
BCNCPB–O–NEt4 is very strong. The ꢀ(NH) shift in
BCNCPB–O–NEt4 is comparable to that in the sodium salt
of 3,5-dibromosalicylanilide,20 which appears at 3000–2900
cmꢃ1
Solution Structures of BCNCPB–OH and BCNCPB–
ꢁꢁꢁ
ꢁꢁꢁ
.
at 3357 cmꢃ1 is assigned to the NH O hydrogen bonded con-
former (type A).18,29 The band at high frequency, 3483 cmꢃ1
ꢁꢁꢁ
,
O–NEt4.
Figure 3a shows the 1H NMR spectrum of
BCNCPB–OH in CDCl3. A phenolic OH signal was observed
at 12.0 ppm, which is shifted to a very low field compared to
non-hydrogen bonded phenolic OH (4–6 ppm), indicating the
is assigned to ꢀ(OH) in the type A conformation.20 The OH
band in the type B conformation was not observed in the spec-
trum because of line broadning.20
In the H NMR spectrum of BCNCPB–O–NEt4 (Fig. 3b),
an amide NH signal was observed at 15.2 ppm, which is down-
field compared to the non-hydrogen bonded NH, indicating the
1
formation of a OH O=C hydrogen bond (type B in Chart 4)
ꢁꢁꢁ
similar to the solid state. An amide NH signal was observed
at 8.6 ppm, which is slightly downfield compared to ꢁ(NH)
in non-hydrogen bonded benzamide (6–8 ppm), showing that
the weak NH Cl hydrogen bond established in the solid state
is maintained in solution. A NOE correlation between the NH
and the 6-positioned aryl proton (H6 at 7.6 ppm) was observed,
formation of a strong NH O(oxyanion) hydrogen bond (type
ꢁꢁꢁ
A) similar to the solid state. The NH shows no NOE correla-
tion with other protons in the NOESY spectrum (data not
shown). It also supports the type A conformation. The IR re-
sults (Fig. 5b), where an NH band was observed at 3015
ꢁꢁꢁ

D. Kanamori et al.
Bull. Chem. Soc. Jpn., 77, No. 11 (2004) 2061
O
S
I
O H
O
H6'
O
Cl
N
H6
Cl
H
δ+
Cl
δ−
NOE
H6
O
S
Cl
I
O
H6'
O
N
Cl
O H
δ+
Cl
δ−
Chart 5. Solution structures of protonated and deprotonated
forms of BH3I-20.
Fig. 5. IR spectra of 5 mM CH2Cl2 solution of BCNCPB–
OH (a), BCNCPB–O–NEt4 (b), and BH3I-20 (c).
Fig. 7. NOESY spectrum of protonated BH3I-20 in CDCl3.
NOE correlation regarding NH.
Protonated and deprotonated BH3I-20 form similar hydro-
gen bonds to the predominant conformations of BCNCPB–
OH and BCNCPB–O–NEt4, respectively. BH3I-2 probably
prefers similar conformations.
Fig. 6. 1H NMR spectra of BH3I-20 in CDCl3 (a) and de-
protonated BH3I-20 in CD3CN (b).
pKa of BCNCPB–OH in an Aqueous Micellar Solution.
The pH titration of BCNCPB–OH was performed in an aque-
ous micellar solution. Under these conditions, BCNCPB–OH
is incorporated into the hydrophobic layer where the hydrogen
bonds are supported. These conditions also realize a hydropho-
bic cleft formed in Bcl-xL where BH3I-2 binds.1 The titration
method using an aqueous micellar solution is adequate to mim-
ic the hydrophobic environments inside of protein.23,24 Be-
cause protons can move inside and outside of micelles freely,
the pKa value of BCNCPB–OH is evaluated by pH measure-
ments in the aqueous layer. BCNCPB–OH exists inside mi-
celles due to its poor solubility in water during the titration.
The pKa value of BCNCPB–OH obtained from the titration
was 5.1, which is very low compared with phenol (9.9 in wa-
ter31). In addition to the electron-withdrawing effect of Cl, Br,
and the aryl N-substituent (ArB), the existence of amide NH,
cmꢃ1, also show strong NH O(oxyanion) hydrogen bonding.
ꢁꢁꢁ
Solution Structures of BH3I-20. Figure 6a shows the
1H NMR spectrum of BH3I-20. Phenolic OH and amide NH
signals were observed at 11.7 and 8.7 ppm, respectively. These
similar chemical shifts to BCNCPB–OH show that BH3I-20
has similar hydrogen bonds (Chart 5). An observed NOE cor-
relation between the NH and the 6-positioned aryl proton (H6
at 7.5 ppm) supports the type B conformation (Fig. 7). Howev-
er, BH3I-20 gives one NH band at 3423 cmꢃ1 in the IR spec-
trum that is different from BCNCPB–OH (Fig. 5c). This is be-
cause iodine acts as a weaker hydrogen bonding accepter
(OH I) compared to bromine due to a smaller electronegativ-
ity. BH3I-2, which has bromine, probably has a minor con-
former similar to BCNCPB–OH.
ꢁꢁꢁ
In the 1H NMR spectrum of deprotonated BH3I-20 (Fig. 6b),
an amide NH signal was observed at 15.8 ppm, which is ex-
tremely downfield, similar to BCNCPB–O–NEt4. This indi-
which forms an intramolecular NH O(oxyanion) hydrogen
ꢁꢁꢁ
bond in the deprotonated form, lowers the pKa value.19,23,28
The pKa value of BH3I-2 should be equal to or slightly less
than 5.1 because an additional electron withdrawing substitu-
cates NH O(oxyanion) hydrogen bonding (type A). The type
A conformation is also supported by the disappearance of the
ꢁꢁꢁ

2062 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Structures of Protonated/Deprotonated BH3I-2
ent, the 4-chlorophenylsulfonyl group on the N-2-chlorophenyl
group, of BH3I-2 has little effect on its acidity.11
lyzed. The presence of an intramolecular OH O=C hydrogen
ꢁꢁꢁ
bond in BCNCPB–OH and an intramolecular NH O(oxyan-
ꢁꢁꢁ
Proposed Binding Structure of BH3I-2. The structure of
the BH3I-2/Bcl-xL complex has been computationally pro-
posed using the solution structure of the BakBH3–peptide/
Bcl-xL complex.1,21,22 This is based on the fact that the struc-
ture of Bcl-xL and the inhibitor–binding site are similar to
those of the BakBH3–peptide/Bcl-xL complex. In this com-
plex, BH3I-2 binds to a hydrophobic cleft formed by the
BH1, BH2, and BH3 regions. The 3-positioned bromine inter-
acts with the side chain of Tyr65 and the 5-positioned chlorine
with the side chain of Phe61 and Phe110, etc. The presence of
the interactions is demonstrated by a change in the inhibitory
activity induced by the substitution of these halogen atoms.
In the complex, the structure of BH3I-2 is represented in the
ion) hydrogen bond in BCNCPB–O–NEt4 as established
by X-ray analyses and IR spectra. The 1H NMR and IR results
of BCNCPB–O–NEt4 have revealed that the structure in the
solid state is maintained in dilute solution with similar hydro-
gen bonds. BCNCPB–OH prefers a similar conformation to
the solid state predominantly, but has a minor conformer hav-
1
ing an NH O hydrogen bond. H NMR and IR spectra show
ꢁꢁꢁ
that the protonated and deprotonated BH3I-20 form intramolec-
ular hydrogen bonds in the same manner as the corresponding
models except for no evidence of the minor conformers in
the protonated form. The deprotonation of BH3I-2 in the
BH3I-2/Bcl-xL complex is suggested by the low pKa value
of BCNCPB–OH. It is also supported by the possible OH-
protonated form and has an intramolecular NH O hydrogen
bond (type A). Our structural analysis of BH3I-2, however,
(Tyr) O(oxyanion) hydrogen bond formation between the
OH group of Tyr65 and the phenoxy oxygen atom of BH3I-2.
ꢁꢁꢁ
ꢁꢁꢁ
shows that BH3I-2 forms an intramolecular OH O=C hydro-
ꢁꢁꢁ
Experimental
gen bond (type B) in the protonated form. It is difficult to fit
BH3I-2 into the hydrophobic cleft while keeping the intramo-
Materials. BH3I-20 was purchased from CALBIOCHEM.
Other reagents were purchased from Nacalai Tesque or Tokyo
Chemical Industry and used without further purification. All
organic solvents were dried over CaH2 and distilled under an Ar
atmosphere before use.
Synthesis. 3-Bromo-5-chloro-2-hydroxybenzoic Acid (1):
This compound was prepared using a modified method reported
in the literature.32
To a solution of 3-bromo-5-chloro-2-hydroxybenzaldehyde
(278 mg, 1.3 mmol) in dioxane (15 mL) were added NaH2PO4
(800 mg, 5.1 mmol) in water (5 mL) and sulfamic acid (186
mg, 1.92 mmol). The reaction mixture was cooled to 7 ꢂC and
then NaClO2 (186 mg, 1.7 mmol) in water (0.7 mL) was added
lecular OH O=C hydrogen bond and the interactions between
ꢁꢁꢁ
bromine–Tyr65, chlorine–Phe110, and chlorine–Phe61, be-
cause the chlorine on the N-aryl ring would be too close to
the side chain of Phe61 or Ala106 under this assumption
(Fig. 8b). On the other hand, it is reasonable that BH3I-2 binds
in the deprotonated form in which an intramolecular
NH O(oxyanion) hydrogen bond is formed (Fig. 8a). This
ꢁꢁꢁ
suggestion is supported by the pKa value of BH3I-2 of about
5.1, which indicates that BH3I-2 is deprotonated under physio-
logical conditions (pH = 7.2).2 It is possible that Tyr65 does
not interact with bromine but forms an intermolecular
OH(Tyr) O(oxyanion) hydrogen bond.
ꢁꢁꢁ
ꢂ
gradually while keeping the temperature below 10 C. The reac-
ꢂ
Different from other salicylamide inhibitors noted above,
BH3I-2 binds to the protein in deprotonated form. This is
because the pKa value of BH3I-2 is lower than others due to
strong electron-withdrawing substituents (Cl (ꢂpara ¼ 0:23)
and Br (ꢂpara ¼ 0:23)) compared to others (F (ꢂpara ¼ 0:06)
or NHCHO (ꢂpara ¼ 0:00)).25
tion mixture was stirred for 30 min below 10 C. Sodium sulfite
(195 mg, 1.5 mmol) was added with stirring for 15 min below
10 ꢂC. Hydrochloric acid was added until a pH of 1 was obtained.
Volatile materials were evaporated, and the resulting solution was
cooled to deposit a white precipitate, which was collected by fil-
tration and washed with water (309 mg, 94%).
Anal. Found: C, 33.37; H, 1.63%. Calcd for C7H4BrClO3: C,
33.43; H, 1.60%. Mp: 238–239 ꢂC. 1H NMR (DMSO-d6; TMS)
ꢁ 7.91 (1H, m, Ar–H), 7.74 (1H, m, Ar–H). 13C NMR (DMSO-
d6): ꢁ 170.3, 156.7, 137.3, 128.7, 122.8, 115.3, 111.3. ESI-MS:
Conclusion
In this study, BH3I-2 models, BCNCPB–OH and
BCNCPB–O–NEt4, were synthesized and structurally ana-
a)
b)
Cl
Tyr65
Tyr65
O
O
Cl
Cl
S
OH
OH
H
H
O
N
S
O
O
O
O
Br
Br
O
N
H
Cl
H3C
H3C
O
Cl
Cl
H
H
N
N
Phe61
N
Phe61
N
O
H
H
Ala106
Ala106
Phe110
Phe110
O
O
Fig. 8. Proposed binding structure in type A conformation (a) and type B conformation (b).

D. Kanamori et al.
Bull. Chem. Soc. Jpn., 77, No. 11 (2004) 2063
ðM ꢃ HþÞꢃ, 249.1 (calcd for M ꢃ Hþ; 248.90).
removed under reduced pressure. The residue was washed with 10
mL of diethyl ether twice. The resultant powder was recrystallized
from THF/diethyl ether to give BCNCPB–O–NEt4 as hygroscop-
ic yellow crystals.
2-Acetoxy-3-bromo-5-chlorobenzoic Acid (2): 3-Bromo-5-
chloro-2-hydroxybenzoic acid (519.3 mg, 2.1 mmol) was dis-
solved in 10 mL of THF. To the solution was added acetyl chlo-
ride (0.15 mL, 2.1 mmol), followed by triethylamine (0.3 mL, 2.2
mmol). The reaction mixture was stirred overnight, and the white
precipitate was then filtered out. The filtrate was concentrated un-
der reduced pressure. The resultant white powder was used with-
out further purification (555 mg, 92%).
Anal. Found: C, 50.45; H, 5.52; N, 5.56%. Calcd for
C21H27BrꢂCl2N2O2 + (H2O)0:5: C, 50.52; H, 5.65; N, 5.61%. Mp:
1
108–111 C. H NMR (CD3CN; TMS) ꢁ 15.22 (1H, s, NH), 8.70
(1H, dd, J ¼ 8:0, 1.6 Hz, ArB–H), 7.77 (1H, dd, J ¼ 3:2, 0.8 Hz,
ArA–H), 7.40 (1H, dd, J ¼ 7:6, 1.6 Hz, ArB–H), 7.39 (1H, d, J ¼
3:2 Hz, ArA–H), 7.25 (1H, tt, J ¼ 8:0, 1.6 Hz, ArB–H), 6.96 (1H, tt,
J ¼ 7:6, 1.6 Hz, ArB–H), 3.14 (8H, q, J ¼ 7:6 Hz, 4 ꢄ CH2CH3),
1.19 (12H, tt, J ¼ 7:6, 2.0 Hz, 4 ꢄ CH2CH3). 13C NMR (CD3CN)
ꢁ 167.5, 167.2, 139.2, 134.7, 130.1, 129.4, 128.0, 123.8, 123.7,
123.1, 120.1, 119.1, 112.5, 53.1, 7.6.
Deprotonation of BH3I-20. BH3I-20 (1.5 mg, 2.8 mmol) was
dissolved in 2 mL of THF. To the solution was added one drop of
25% tetraethylammonium hydroxide aqueous solution. The solu-
tion was then concentrated in vacuo. The residue was washed with
2 mL of diethyl ether and dried in vacuo.
ꢂ
1
Mp: 153–155 C. H NMR (CDCl3; TMS) ꢁ 8.01 (1H, d, J ¼
2:4 Hz, Ar–H), 7.83 (1H, d, J ¼ 2:4 Hz, Ar–H), 2.38 (3H, s,
CH3). 13C NMR (CDCl3): ꢁ 168.0, 167.6, 147.3, 137.5, 132.1,
131.2, 125.1, 119.5, 20.7. ESI-MS: ðM ꢃ HþÞꢃ, 290.9 (calcd for
M ꢃ Hþ; 290.91).
2-(2-Chlorophenylcarbamoyl)-6-bromo-4-chlorophenyl Ace-
tate (3): 2-Acetoxy-3-bromo-5-chlorobenzoic acid (319 mg, 1.1
mmol) and triethylamine (0.20 mL, 1.4 mmol) were dissolved in
ꢂ
40 mL of THF. The solution was cooled to ꢃ20 C. To the solu-
tion was added isobutyl chloroformate (IBCF) (0.18 mL, 1.4
mmol) gradually, keeping the temperature under ꢃ15 ꢂC. After
stirring for 5 min, o-chloroaniline (0.15 mL, 1.4 mmol) in THF
(4 mL) was dropped into the reaction mixture. The reaction mix-
1H NMR Results of BH3I-20 and Deprotonated BH3I-20.
BH3I-20: 1H NMR (CDCl3; TMS) ꢁ 11.70 (1H, s, OH), 9.01
(1H, d, J ¼ 2:0 Hz, ArB–H), 8.69 (1H, s, NH), 7.96 (1H, d, J ¼
2:4 Hz, ArA–H), 7.93 (2H, d, J ¼ 8:8 Hz, ArC–H: protons on p-
chlorophenyl group), 7.73 (1H, dd, J ¼ 8:8, 2.0 Hz, ArB–H),
7.60 (1H, d, J ¼ 8:8 Hz, ArB–H), 7.56 (1H, d, J ¼ 2:4 Hz,
ArA–H), 7.52 (2H, d, J ¼ 8:8 Hz, ArC–H).
ꢂ
ture was stirred for 1 h at ꢃ15 C, and then 2 days at room tem-
perature. The solvent was removed, and the residue was extracted
with ethyl acetate and washed with 2% HCl aq., 4% NaHCO3 aq.,
and sat. NaCl aq., and then dried over anhydrous sodium sulfate.
The removal of the solvent gave a brown oily residue. The repre-
cipitation from diethyl ether/hexane gave a white powder. Recrys-
tallization from ethyl acetate/hexane gave colorless needles (169
mg, 62%).
1
Deprotonated BH3I-20: H NMR (CD3CN; TMS) ꢁ 15.85 (1H,
s, NH), 9.41 (1H, d, J ¼ 2:4 Hz, ArB–H), 7.90 (2H, d, J ¼ 9:2 Hz,
ArC–H), 7.82 (1H, d, J ¼ 2:8 Hz, ArA–H), 7.63 (1H, d, J ¼ 2:8
Hz, ArA–H), 7.58 (1H, d, J ¼ 8:4 Hz, ArB–H), 7.57 (2H, d, J ¼
9:2 Hz, ArC–H), 7.46 (1H, dd, J ¼ 8:4, 2.4 Hz, ArB–H), 3.14
(8H, q, J ¼ 7:6 Hz, 4 ꢄ CH2CH3), 1.19 (12H, tt, J ¼ 7:6, 2.0
Hz, 4 ꢄ CH2CH3).
ꢂ
1
Mp: 128–129 C. H NMR (CDCl3; TMS) ꢁ 8.54 (1H, s, NH),
8.49 (1H, d, J ¼ 7:6 Hz, ArB–H), 7.85 (1H, d, J ¼ 2:4 Hz, ArA–
H), 7.76 (1H, d, J ¼ 2:4 Hz, ArA–H: protons on phenolic moiety),
7.41 (1H, dd, J ¼ 8:4, 1.6 Hz, ArB–H: protons on N-aryl group),
7.33 (1H, t, J ¼ 8:4 Hz, ArB–H), 7.09 (1H, dt, J ¼ 7:6, 1.6 Hz,
ArB–H), 2.41 (3H, s, CH3). 13C NMR (CDCl3) ꢁ 167.6, 161.1,
144.3, 135.6, 134.1, 132.8, 131.2, 129.4, 129.1, 127.9, 125.4,
122.9, 121.8, 118.9, 21.0. ESI-MS: ðM ꢃ HþÞꢃ, 399.7 (calcd for
M ꢃ Hþ; 399.91).
Physical Measurements. 1H NMR spectra in a CDCl3 or
CD3CN solution were recorded on a JEOL GSX 400 spectrometer
and a JEOL JNM EX 270 at 30 ꢂC. NOESY spectra were recorded
ꢂ
on a Varian UNITYplus 600 MHz spectrometer at 30 C. Tetra-
methylsilane was used as a standard (0 ppm). 13C NMR spectra
in CDCl3, CD3CN, and DMSO-d6 were recorded on a JEOL
ꢂ
3-Bromo-5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide
(BCNCPB–OH): 2-(2-chlorophenylcarbamoyl)-6-bromo-4-chlo-
rophenyl acetate (169 mg, 0.42 mmol) was dissolved in a mixture
of MeOH (2 mL) and 1 M NaOH aq. (1 mL). The solution was
stirred for two days and acidified by 2% HCl aq. The precipitated
white powder was collected by filtration and recrystallized from
THF/hexane. Colorless needles were obtained (34 mg, 23%).
Anal. Found: C, 43.23; H, 2.20; N, 3.83%. Calcd for
C13H8BrCl2NO2: C, 43.25; H, 2.23; N, 3.88%. Mp: 157–160
ꢂC. 1H NMR (CDCl3; TMS) ꢁ 12.02 (1H, s, OH), 8.59 (1H, s,
NH), 8.36 (1H, dd, J ¼ 8:4, 1.6 Hz, ArB–H), 7.74 (1H, d, J ¼
2:4 Hz, ArA–H), 7.56 (1H, d, J ¼ 2:4 Hz, ArA–H), 7.46 (1H,
dd, J ¼ 7:6, 1.6 Hz, ArB–H), 7.36 (1H, dt, J ¼ 8:4, 1.6 Hz,
ArB–H), 7.16 (1H, dt, J ¼ 7:6, 1.6 Hz, ArB–H). 13C NMR
(CDCl3) ꢁ 165.9, 156.4, 137.2, 133.0, 129.2, 127.8, 126.0,
124.9, 124.2, 121.1, 122.4, 116.4, 113.4. ESI-MS: ðM ꢃ HþÞꢃ,
358.1 (calcd for M ꢃ Hþ; 357.90).
JNM EX 270 and Varian UNITYplus 600 MHz at 30 C. Signals
of the solvent were used as a standard {77.0 ppm (CDCl3), 118.2
ppm (CD3CN), and 39.7 ppm (DMSO-d6)}. ESI-MS measure-
ments were performed on a Finnigan MAT LCQ ion trap mass
spectrometer in a methanol solution. IR spectra were recorded
on a Jasco FT/IR 8300 spectrometer. Samples were prepared as
Nujol mulls and in CH2Cl2 solution (1 and 5 mM).
pH Titration. The pH of a 10 mM BCNCPB–OH solution
was determined using a Metrohm 716 DMS titrino, which is com-
bined with a Metrohm 728 stirrer and a saturated calomel LL
micro pH glass electrode. The saturated calomel micro glass elec-
trode was calibrated with a 0.05 M KHC6H4(COO)2 buffer (pH =
4.01), and a 0.025 M KH2PO4–Na2HPO4 buffer (pH = 6.86) at 30
ꢂC. BCNCPB–OH was dissolved in a small amount of THF, and
to this solution was added Triton X-100. The solution was concen-
trated to remove THF. The obtained residue was diluted with de-
gassed water to give a micellar solution. The final concentration
was a 10% Triton X-100 aqueous solution containing 10 mM of
BCNCPB–OH. The solution was titrated with 0.1 M NaOH aq.
at 30 ꢂC. The pKa value was estimated by the following equa-
tion:23,24 pKa ¼ pH ꢃ log½Naþꢅ þ logf½phenolꢅ0 ꢃ ½Naþꢅg.
Tetraethylammonium 2-Bromo-4-chloro-6-[N-(2-chloro-
phenyl)carbamoyl]phenolate (BCNCPB–O–NEt4): 3-Bromo-
5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide (44.9 mg,
0.12 mmol) was dissolved in 8 mL of methanol. To the solution
was added a 25% tetraethylammonium hydroxide aqueous solu-
tion (0.09 mL). The solution was stirred for 1 h, and solvents were
X-ray Structure Determination.
Each single crystal of
BCNCPB–OH and BCNCPB–O–NEt4 was mounted in a loop

2064 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Structures of Protonated/Deprotonated BH3I-2
with Nujol. The X-ray data were collected at 200 K on a Rigaku
Raxis-RAPID Imaging Plate diffractometer with graphite mono-
Goldschmidt, M. J. Loeloff, G. C. Webb, and J. F. Barrett,
J. Med. Chem., 41, 2939 (1998).
ꢀ
chromated Mo Kꢃ (ꢄ ¼ 0:71075 A). The basic crystallographic
13 D. J. Hlasta, J. P. Demers, B. D. Foleno, S. A. Fraga-
Spano, J. Guan, J. J. Hilliard, M. J. Macielag, K. A. Ohemeng,
C. M. Sheppard, Z. Sui, G. C. Webb, M. A. Weidner-Wells, H.
Werblood, and J. F. Barrett, Bioorg. Med. Chem. Lett., 8, 1923
(1998).
14 H. Kim, L. Esser, M. B. Hossain, D. Xia, C.-A. Yu, J. Rizo,
D. van der Helm, and J. Deisenhofer, J. Am. Chem. Soc., 121,
4902 (1999).
15 K. M. Kim, C. D. Giedt, G. Basan˜ez, J. W. O’Neil, J. J.
Hill, Y.-H. Han, S.-P. Tzung, J. Zimmerberg, D. M. Hockenbery,
and K. Y. J. Zhang, Biochemistry, 40, 4911 (2001).
16 S.-P. Tzung, K. M. Kim, G. Basan˜ez, C. D. Giedt, J.
Simon, J. Zimmerberg, K. Y. J. Zhang, and D. M. Hockenbery,
Nat. Cell Biol., 3, 183 (2001).
17 F. Mu, E. Hamel, D. J. Lee, D. E. Pryor, and M. Cushman,
J. Med. Chem., 46, 1670 (2003).
18 H. Suezawa, M. Hirota, T. Yuzuki, Y. Hamada, I.
Takeuchi, and M. Sugiura, Bull. Chem. Soc. Jpn., 73, 2335 (2000).
19 W. L. Mock and D. C. Y. Chua, J. Chem. Soc., Perkin
Trans. 2, 1995, 2069.
20 D. Welti, Spectrochim. Acta, 22, 281 (1966).
21 M. Sattler, H. Liang, D. Nettesheim, R. P. Meadows, J. E.
Harlan, M. Eberstadt, H. S. Yoon, S. B. Shuker, B. S. Chang, A. J.
Minn, C. B. Thompson, and S. W. Fesik, Science, 275, 983
(1997).
parameters are listed in Table S1 (See Supporting Information).
A symmetry-related absorption correction using the program AB-
SCOR33 was applied, which resulted in transmission factors rang-
ing from 0.43 to 0.49 for BCNCPB–OH and 0.42 to 0.53 for
BCNCPB–O–NEt4. The structure was solved by the direct meth-
od (SHELXS-9734 for BCNCPB–OH and SIR9235 for BCNCPB–
O–NEt4) and expanded using Fourier techniques using teXsan
crystallographic software36 and SHELXL-97.37 Non-hydrogen
atoms were refined anisotropically. The positions of OH and
NH were refined using fixed thermal parameters, and the other hy-
drogen atoms were placed at calculated positions.
Crystal Data.
BCNCPB–OH:
C13H8BrCl2NO2, M ¼
361:02, orthorhombic, a ¼ 23:15ð2Þ, b ¼ 15:73ð2Þ, c ¼ 7:126ð7Þ
ꢀ
ꢀ 3
A, U ¼ 2594ð10Þ A , T ¼ 200 K, space group Pccn (no. 56),
Z ¼ 8, ꢅ ¼ 35:84 cmꢃ1, 22578 reflections measured, 2836 unique
(Rint ¼ 0:096), R1 ¼ 0:036 (I > 2ꢂ), wR2 ¼ 0:070 (all data).
BCNCPB–O–NEt4: C21H27BrCl2N2O2, M ¼ 490:27, ortho-
ꢀ
rhombic, a ¼ 7:307ð4Þ, b ¼ 16:18ð1Þ, c ¼ 18:99ð1Þ A, U ¼
ꢀ 3
2245ð8Þ A , T ¼ 200 K, space group P212121 (no. 19), Z ¼ 4,
ꢅ ¼ 20:93 cmꢃ1, 21267 reflections measured, 2935 unique
(Rint ¼ 0:045), R1 ¼ 0:028 (I > 2ꢂ), wR2 ¼ 0:048 (all data).
One of the authors (DK) expresses his special thanks to the
center of excellence (21COE) program ‘‘Creation of Integrated
EcoChemistry of Osaka University’’.
22 A. Fahmy and G. Wagner, J. Am. Chem. Soc., 124, 1241
(2002).
23 N. Ueyama, M. Inohara, A. Onoda, T. Ueno, T. Okamura,
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24 A. Onoda, Y. Yamada, J. Takeda, Y. Nakayama, T.
Okamura, M. Doi, H. Yamamoto, and N. Ueyama, Bull. Chem.
Soc. Jpn., 77, 321 (2004).
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