Synthesis and biochemical evaluation of highly enantiomerically pure (R,R)- and (S,S)-alexidine

Article abstract of DOI:10.1016/j.bmc.2013.09.057

Alexidine is in everyday human use as oral disinfectant and contact lens disinfectant. It is used as a mixture of stereoisomers. Since all of alexidine's known biological targets are chiral, the biological activity of any of its chiral stereoisomers could be significantly higher than that of the mixture of stereoisomers. This makes a synthetic methodology for obtaining the individual enantiomers of the chiral diastereoisomer highly desirable. Here, we describe the first synthesis of both enantiomers of alexidine in high enantiomeric purity, and demonstrate their activity against the protein-protein interaction between the anti-apoptotic protein Bcl-x_L and the pro-apoptotic protein Bak.

Full text of DOI:10.1016/j.bmc.2013.09.057

Bioorganic & Medicinal Chemistry 21 (2013) 7357–7363
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biochemical evaluation of highly enantiomerically
pure (R,R)- and (S,S)-alexidine
a
a
b
a,
⇑
Corinna Gröst , Martin Gräber , Michael Hell , Thorsten Berg
a
University of Leipzig, Institute of Organic Chemistry, Johannisallee 29, 04103 Leipzig, Germany
Max Planck Institute of Biochemistry, Department of Molecular Biology, Am Klopferspitz 18, 82152 Martinsried, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2013
Accepted 20 September 2013
Available online 2 October 2013
Alexidine is in everyday human use as oral disinfectant and contact lens disinfectant. It is used as a mix-
ture of stereoisomers. Since all of alexidine’s known biological targets are chiral, the biological activity of
any of its chiral stereoisomers could be significantly higher than that of the mixture of stereoisomers. This
makes a synthetic methodology for obtaining the individual enantiomers of the chiral diastereoisomer
highly desirable. Here, we describe the first synthesis of both enantiomers of alexidine in high enantio-
meric purity, and demonstrate their activity against the protein–protein interaction between the anti-
Keywords:
Protein–protein interactions
Anti-apoptotic proteins
Inhibitors
L
apoptotic protein Bcl-x and the pro-apoptotic protein Bak.
Ó 2013 Elsevier Ltd. All rights reserved.
Drug design
Chirality
1
. Introduction
Alexidine (1) contains two stereogenic centers, but because of
its symmetry, only three isomers of alexidine exist: two enantio-
mers (R,R)-1 and (S,S)-1, and the achiral meso diastereoisomer
(R,S)-1 (Fig. 1).
1
Alexidine is a bisbiguanide which is currently in widespread hu-
Ò
man use as the active ingredient of the oral disinfectant Esemdent ,
and the contact lens disinfectant Complete RevitaLens . The anti-
bacterial activity of alexidine is thought to be based on interactions
between the positively charged biguanide moieties and the nega-
tively charged phospholipids on the bacterial membrane. Recently,
the mitochondrial dual specificity phosphatase PTPMT1 has been
identified as another target of alexidine. Other activities of alexi-
dine reported in the literature include those against tumor cells de-
rived from oral tissues and multiple myeloma.
Ò
One of the quintessential requirements for chiral drugs admit-
ted for human use today is a solid understanding of the biological
effects of the individual enantiomers. In striking contrast to this
principle, no studies have been published which analyze the bio-
logical effects of the individual chiral isomers of alexidine. All sam-
ples of alexidine used either as disinfecting agents or in basic
research studies to date consist of the mixture of the two chiral
enantiomers (R,R)-1 and (S,S)-1, and the achiral isomer (R,S)-1.
Assuming a statistical distribution of the stereogenic centers in
the mixture of alexidine isomers, each chiral isomer contributes
2
3
4
5
We recently discovered that alexidine inhibits protein–protein
interactions mediated by the anti-apoptotic Bcl-2-family protein
Bcl-x
and has been established as a target for cancer drug discovery.
Via a hydrophobic groove comprised of its Bcl-2 homology (BH) do-
6
L
. Bcl-x
L
is overexpressed in a large number of human tumors
7
–9
NH NH
H
N
H
N
H
N
N
H
N
H
N
H
L
mains BH1, BH2, and BH3, Bcl-x binds to the BH3 domains of pro-
apoptotic Bcl-2 proteins.10 This leads to sequestration and thereby
functional inhibition of pro-apoptotic Bcl-2 proteins and prevents
the induction of apoptosis in tumor cells. Alexidine was found to
NH NH
(R,R)-Alexidine (1)
NH NH
H
N
H
N
H
N
bind to the BH3 domain binding pocket of Bcl-x
interfere with protein–protein interactions between Bcl-x
the pro-apoptotic proteins Bad and Bak. As a consequence, alexidine
induces apoptosis in a number of tumor cell lines derived from the
tongue and the pharynx via the intrinsic apoptosis pathway.
L
, and to thereby
and
N
H
N
H
N
H
NH NH
L
(S,S)-Alexidine (1)
NH NH
6
H
N
H
N
H
N
N
H
N
H
N
H
NH NH
(
R,S)-Alexidine (1)
⇑
Corresponding author. Tel.: +49 341 97 36527.
E-mail address: tberg@uni-leipzig.de (T. Berg).
Figure 1. Isomers of alexidine 1.
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.057

7
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C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
2-ethyl-1-hexylamines (R)-2 and (S)-2,11 respectively, and dicyano
R2
NH NH
H
N
H
N
H
N
R1
R1
compound 3 as the key step of the synthesis. Both 2 and 3 can
be obtained from readily available starting materials, and are
subsequently combined to yield the target molecules (R,R)-1 and
(S,S)-1 in a convergent manner.
N
H
N
H
N
H
R2
NH NH
9,R2 = C
R1 = n-C
4
H
2 5
H
: (R,R)-Alexidine 1
1
2
R
= C H5,R = n-C H : (S,S)-Alexidine 1
2
4
9
The synthetic route towards (2S)-ethyl-1-hexylamine (S)-2
started with the published procedure towards the enantiomeri-
cally enriched (2S)-ethyl-1-hexanol (S)-8, and is based on pseudo-
R2
NH
H
N
H
N
N
NH
2
+
12
R1
N
H
N
ephedrine (R,R)-4 (Fig. 3). Synthesis of (2R)-ethyl-1-hexanol (R)-8
N
H
NH
was carried out in an analogous fashion using the enantiomeric
pseudoephedrine (S,S)-4. To this end, pseudoephedrines (S,S)-4
and (R,R)-4 were acylated with butyric acid anhydride. After depro-
tonation of the pseudoephedrine amides (S,S)-5 and (R,R)-5, they
were alkylated with butyl iodide to obtain (S,S,R)-6 and (R,R,S)-6,
respectively (Fig. 3). Acid hydrolysis of the chiral auxiliary and lith-
ium aluminium hydride reduction of the liberated acids (R)-7 and
(S)-7 provided the alcohols (R)-8 and (S)-8. The assignment of the
absolute configuration of the newly generated stereogenic centers
is based on the positive value of the optical rotation of alcohol (S)-8
(
R
R)-2
= n-C
3
1
9,R2 = C
4
H
2
4
H
5
(
R
S)-2
= C
1
5,R2 = n-C
2
H
H
9
Figure 2. Retrosynthetic approach to alexidine 1.
2
5% of the overall mixture, and the achiral isomer contributes 50%.
Since all of alexidine’s molecular targets reported so far are chiral,
it is conceivable that the biological activity of any of the chiral iso-
mers is up to four times higher than the biological activity of the
mixture of isomers. This makes it highly desirable to have access
to the individual chiral enantiomers of alexidine for biological test-
ing. However, despite alexidine’s biological relevance, no synthesis
of its enantiomers has been published to date. We therefore sought
to develop a synthetic strategy leading to both enantiomers of
alexidine in high enantiomeric purity.
Here, we report the first synthesis of highly enantiomerically
enriched (R,R)- and (S,S)-alexidine 1. The synthetic strategy can
be adapted to the synthesis of enantiomerically enriched alexidine
analogs, allowing for the analysis of their structure–activity rela-
tionships in biological assays.
2
D
2
ð½a
ꢀ
þ 2:88; c 0:1; CHCl Þ, which is consistent with the assignment
3
of the (S)-configuration to the respective 2-ethyl-1-hexanol enan-
tiomer throughout the literature.
1
1–14
Alcohols (R)-8 and (S)-8 were converted to N-alkylated phthal-
imides (R)-9 and (S)-9 under Mitsunobu conditions. Hydrazinolysis
of 9 furnished the desired enantiomerically enriched 2-ethylhexyl-
1-amines (R)-2 and (S)-2 as their hydrochloride salts. The determi-
nation of the optical purities of amines 2 was accomplished by
conversion into their corresponding Mosher amides. HPLC analysis
of the Mosher amides revealed an enantiomeric excess (ee) of
95.6% for (R)-2, and 94.8% for (S)-2. The high enantiomeric purities
of amines 2 are consistent with the high enantiomeric excess of
9
6% reported for generation of the alcohol (S)-8 in the literature,12
2
2
. Results and discussion
demonstrating that the procedure can also be applied to the gener-
ation of the enantiomeric alcohol (R)-8.
.1. Synthesis
Our synthetic approach to highly enantiomerically pure
2
-ethyl-1-hexylamines 2 requires only six synthetic steps and is
11
The retrosynthetic analysis (Fig. 2) towards alexidine
enantiomers (R,R)-1 and (S,S)-1 identifies the reaction between
thus two steps shorter than the previously reported route. In
addition, while the reported procedure involves a reductive step
a) LDA, LiCl
O
O
R3 R4
NH
R3 R4
N
O
THF, -78 °C
R3 R4
N
O
O
b) n-BuI, 0 °C
R1 R2
R1 R2
R1 R2
R5 R6
THF, r.t.
(
S,S)-4
(S,S)-5: 76%
(S,S,R)-6: 71%
1
= OH, R2 = H, R3 = H, R4 = Me)
R,R)-4
= H, R2 = OH, R3 = Me, R4 = H)
(R = OH, R2 = H, R3 = H, R4 = Me)
1
(R = OH, R = H, R3 = H,
1
2
(R
R
= Me, R5 = H, R6 = n-Bu)
4
(
(R,R)-5: 97%
1
(R = H, R2 = OH, R3 = Me, R4 = H)
1
(R
(R,R,S)-6: 70%
1
= H, R = OH, R3 = Me,
2
(
R
R
4
= H, R5 = n-Bu, R6 = H)
1
2 4
8N H SO /
dioxane
reflux
O
NH
LiAlH , Et O
4
2
O
O
0
°C
reflux
O
N
HO
HO
R5 R6
O
R5 R6
R5 R6
PPh
3
, DEAD
THF, r.t.
R)-9 (R = H, R6 = n-Bu): 86%
5
(R)-8 (R5 = H, R6 = n-Bu): 91%
(S)-8 (R5 = n-Bu, R6 = H): 89%
(R)-7 (R5 = H, R6 = n-Bu): 88%
(S)-7 (R5 = n-Bu, R6 = H): 79%
(
(
5
6
S)-9 (R = n-Bu, R = H): 89%
a) H
EtOH, reflux
b) HCl
2 2 2
NNH x H O
2
H N
R5 R6
x HCl
R)-2: (R = H, R6 = n-Bu): 39%, ee = 96%
5
(
(
S)-2 (R = n-Bu, R6 = H): 28%, ee = 95%
5
Figure 3. Synthesis of (R)- and (S)-2-ethyl-1-hexylamines 2.

C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
7359
-
+
Cl H
3
N
NH3+Cl-
NaN(CN)
0 %
2
8
H
N
H
N
NH
CN
N
H
N
NC
NH
H
3
(
R)-2 or (S)-2
R5 R6
NH NH
H
N
H
N
H
N
N
H
N
H
N
NH NH
H
R6 R5
x 2 HCl
(
(
R,R)-1 (R5 = H, R6 = Et): 48%, ee > 99%, de = 91%
S,S)-1 (R = Et, R6 = H): 60%, ee > 99%, de = 90%
5
Figure 4. Synthesis of (R,R)- and (S,S)-alexidines 1.
L
Figure 6. Binding of chiral alexidine isomers to Bcl-x . (A) Overlay of the binding
mode of ABT-737 (carbon atoms shown in green) to Bcl-x
L
according to the co-
crystal structure21 (PDB entry: 2YXJ) and the docking pose of (R,R)-alexidine 1
(
carbon atoms shown in yellow). Amino acids previously shown to display strong,
dose-dependent shifts in the H, N-HSQC-NMR are marked in red.6 (B) Overlay of
1
15
the docking pose of (R,R)-alexidine 1 (carbon atoms shown in yellow) and (S,S)-
L
alexidine 1 (carbon atoms shown in green) bound to Bcl-x . The Figure was
generated using PyMol.
Figure 5. Alexidine inhibits binding between Bcl-x
assay. A peptide derived from the Bak BH3 domain (shown in red) is labeled with a
L
and Bak. (A) Principle of the
18
L
fluorophore (shown in green). When bound to Bcl-x (shown in light gray), the
was calculated as exceeding 99.8% (see Supplementary data for cal-
culation of enantiomeric excesses). The increased enantiomeric ex-
cesses of the alexidines 1 as compared to the precursor amines 2 is
caused by generation of the achiral diastereoisomer (S,R)-1, in the
event that one molecule of the respective major enantiomer, and
one molecule of the respective minor enantiomer of 2 is incorpo-
rated into 1. Thus, incorporation of one molecule of the respective
minor enantiomer of 2 into 1 does not impact the enantiomeric
purity, but instead the diastereomeric purity of 1. This effect has
emitted fluorescence has a high polarization owing to the large molecular weight of
the peptide/protein complex. An inhibitor of protein/peptide binding liberates the
fluorophore-labeled peptide, leading to a reduction of fluorescence polarization.
1
7
The structures are based on PDB entry 1BXL. The Figure was generated using
PyMol.18 (B) Activities of (R,R)- and (S,S)-alexidine and the commercially available
mixture of all three isomers against the interaction between Bcl-x
domain-derived peptide (5-carboxyfluorescein)-GQVGRQLAIIGDDINR-NH
analyzed in fluorescence polarization assays. The mutant Bcl-x 45–84, in which
the flexible loop previously shown to be dispensable for the anti-apoptotic activity
L
and the Bak BH3
19
2
as
L
D
1
7,20
L
of Bcl-x is deleted, was used in these assays.
been described in the literature for other compounds which can
using Na–Hg amalgam,11 the approach described here avoids the
use of toxic heavy metals.
15,16
form achiral meso-diastereoisomers.
Nevertheless, both (S,S)-1
and (R,R)-1 were still calculated to display high diastereomeric
excesses of 90% and 91%, respectively (see Supplementary data
for calculation of diastereomeric excesses). Physical separation of
the meso diastereoisomer by HPLC was found to be not feasible.
The optical rotation of the alexidine enantiomers was determined
Dicyano compound 3 was prepared from 1,6-diaminohexane
1
(
used as the dihydrochloride) and sodium dicyanamide. Next,
the amines (R)-2 and (S)-2 were reacted separately with 3 follow-
1
ing the published procedure to afford the enantiomers of alexi-
2
D
2
22
D
dine (Fig. 4). Assuming an unbiased incorporation of the
individual enantiomers of 2-ethyl-1-hexylamine 2 into the target
compounds 1, the enantiomeric excess of both (S,S)-1 and (R,R)-1
as ½
a
ꢀ
þ 3:6 (c 0.1 in methanol) for (R,R)-1, and ½
aꢀ
ꢁ 4:3 (c 0.1
in methanol) for (S,S)-1, thus confirming the enantiomeric nature
of (R,R)-1 and (S,S)-1.

7
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C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
2
.2. Biochemical analysis
3. Experimental
In order to investigate whether Bcl-x
L
can discriminate between
3.1. Synthesis and spectroscopic characterization of compounds
the two alexidine enantiomers, we carried out a fluorescence
polarization assay. This assay analyzes binding of a fluorophore-la-
beled peptide derived from the BH3 domain of the pro-apoptotic
1-3, 5–9
3.1.1. (1S,2S)-Pseudoephedrine butyramide 5
Bcl-2 protein Bak to the Bcl-x
compounds (Fig. 5A). Surprisingly, we found that the IC50-values
for (R,R)-alexidine (IC50 = 21.3 ± 0.8 M) and (S,S)-alexidine
IC50 = 25.7 ± 0.5 M) are not substantially different from one
L
protein in the presence of the test
l
H
O
(
l
N
another. They are also very similar to the data obtained with the
HO
H
commercially available mixture of all three alexidine isomers
(
S,S)-5
(
IC50 = 18.3 ± 0.7
.3. Molecular docking
To rationalize the results of the fluorescence polarization as-
l
M) (Fig. 5B).6
The synthesis was carried out according to the published proce-
dure. Yield: 76%. Melting point: 84–86 °C (Ref.: 82.5–84 °C).
3
): d = 0.94 (m, 3H), 1.11 (d, J(H,H) = 7 Hz,
H), 1.63 (m, 2H), 2.24 (m, 2H), 2.80 (s, 3H), 3.72 (s, 1H), 4.43
m, 1H), 4.56 (m, 1H), 7.34 (m, 5H) ppm; C NMR (75 MHz, CDCl ):
3
d = 14.0, 14.6, 18.5, 36.4, 58.5, 75.6, 76.7, 126.5, 127.7, 128.4,
2
4
25 1
H
2
3
NMR (300 MHz, CDCl
3
(
1
3
say, we carried out molecular docking experiments of the individ-
ual alexidine enantiomers against the BH3-domain binding
2
1
22
e
1
(
1
1
(
42.6,175.6 ppm; IR (KBr):
m
= 3289 (s), 3058 (w), 3027 (w), 2993
pocket of Bcl-x
ing pocket of Bcl-x
subpocket includes residues Leu108, Val126, Glu129, and
Phe146. In the crystal structure of Bcl-x bound to the high-affin-
L
using AutoDock Vina (Fig. 6A). The BH3 bind-
w), 2964 (s), 2930 (m), 2876 (m), 1624 (s), 1604 (s), 1454 (s),
427 (m), 1408 (m), 1378 (w), 1362 (w), 1348 (w), 1320 (m),
265 (m), 1238 (w), 1203 (w), 1126 (m), 1096 (m), 1051 (s), 921
w), 906 (w), 790 (w), 767 (s), 706 (s), 607 (m), 516 cm (m);
ESI-MS C14H21NO calcd: 236.2 [M+H ], found: 236.2.
L
consists of two subpockets. The narrower
L
ꢁ1
8
ity inhibitor ABT-737, this subpocket is occupied by the 4-chlo-
robiphenyl moiety of the inhibitor.21 The wider subpocket
comprising Gly94 and Asn136 is filled with the acylsulfonamide
+
2
2
1
3.1.2. (1R,2R)-Pseudoephedrine butyramide 5
part of ABT-737. All of these amino acids have previously been
1
15
shown to display strong shifts in the H, N-HSQC-NMR in the
presence of the mixture of alexidine isomers, suggesting that
6
alexidine binds to the BH3 binding pocket of Bcl-x
L
. Consistent
with the NMR shifts, molecular docking placed (R,R)-alexidine
into the same pocket as ABT-737 (Fig. 6). However, (S,S)-alexi-
dine 1 fitted equally well into the BH3-domain binding pocket
Fig. 6B). This indicated that both hydrophobic cavities of Bcl-x
O
H
6
N
1
H
OH
(
R,R)-5
(
L
The synthesis was carried out according to the published proce-
dure. Yield: 97%. Melting point: 84–86 °C (Ref.: 85–86.5 °C).
): d = 0.93 (m, 3H), 1.08 (d, J(H,H) = 7 Hz,
H), 1.63 (m, 2H), 2.24 (m, 2H), 2.79 (s, 3H), 3.83 (s, 1H), 4.43 (m,
H), 4.57 (m, 1H), 7.33 (m, 5H) ppm; C NMR (100 MHz, CDCl ):
3
d = 14.0, 14.6, 18.5, 36.4, 58.5, 75.6, 76.7, 126.5, 127.7, 128.4,
m = 3290 (s), 3058 (w), 3027 (w), 2993
w), 2965 (s), 2930 (m), 2876 (m), 1626 (s), 1604 (s), 1454 (s),
can be occupied by the individual alexidine enantiomers to the
same extent owing to the conformational flexibility of the alkyl
chains. Therefore, the configuration of the chiral center does not
appear to affect the degree of hydrophobic interactions mediated
by the 2-ethyl hexyl substituents of (S,S)- and (R,R)-alexidine
substantially.
2
4
25 1
H
3
NMR (300 MHz, CDCl
3
3
1
1
3
1
(
1
1
(
42.6, 175.6 ppm; IR (KBr): e
2
.4. Conclusions
427 (m), 1408 (m), 1379 (w), 1362 (w), 1348 (w), 1320 (m),
265 (m), 1238 (w), 1203 (w), 1126 (m), 1096 (m), 1051 (s), 921
w), 906 (w), 790 (w), 767 (s), 706 (s), 607 (m), 516 cm (m);
In summary, we have presented the first synthesis of (S,S)- and
ꢁ1
(R,R)-alexidine in high enantiomeric purity. Our approach includes
+
ESI-MS C14
H21NO
2
calcd: 236.2 [M+H ], found: 236.2.
a shorter synthetic route to the building blocks (S)- and (R)-2-
ethyl-1-hexylamine 2 than previously reported,11 which also does
not require the use of toxic heavy metals. The versatility of the
3
.1.3. (1S,2S)-Pseudoephedrine-(2R)-ethylhexanamide 6
pseudoephedrine-based approach to yield
a
-alkylated alcohols in
excellent enantiomeric excesses,2
3,24
in combination with their
racemization-free conversion under Mitsunobu conditions, will
facilitate the generation of diverse chiral primary amines that are
suitable for the synthesis of various chiral bisbiguanides. Therefore,
the new methodology presented here will allow biological research
studies aimed at analyzing the mode of action of chiral bisbigua-
nides such as alexidine and its analogs.
H
O
N
HO
(
H
S,S,R)-6
The synthesis was carried out analogous to the published
2
4
1
Even though Bcl-x
individual alexidine enantiomers, it is conceivable that other chiral
molecular targets of Bcl-x , such as the phospholipids in the bacte-
L
does not appear to discriminate between the
procedure.
3
Yield: 71%. H NMR (300 MHz, CDCl ): d = 0.86
3
(t, J(H,H) = 7 Hz, 6H), 1.06–1.71 (m, 11H), 2.48 (m, 1H), 2.84 (s, 3H),
3.87 (s, 1H), 4.4 (m, 1H), 4.6 (m, 1H), 7.33 (m, 5H) ppm; 13C NMR
L
3
rial membrane, the mitochondrial phosphatase PTPMT1, or other
targets of alexidine to be discovered in the future, may interact
substantially different with the individual enantiomers. The syn-
thetic route to highly enantiomerically enriched enantiomers of
alexidine presented here will therefore enable future research
aimed at understanding the biological activities of alexidine on
the molecular level.
(75 MHz, CDCl
3
): d = 12.1, 14.1, 14.7, 23.0, 26.1, 29.8, 32.6, 44.1,
53.5, 58.3, 75.5, 76.5, 126.4, 127.6, 128.4, 142.8,178.9 ppm; IR
m = 3376 (m), 3062 (w), 3030 (w), 2960 (s), 2930 (s), 2872
(m), 2359 (w), 1617 (s), 1482 (m), 1455 (m), 1411 (w), 1377 (w), 1307
(w), 1258 (w), 1197 (w), 1150 (w), 1112 (m), 1087 (w), 1051
(m), 1028 (w), 763 (w), 737 (w), 701 (m), 608 (w), 518 cm (m);
2
ESI-MS C18H29NO calcd: 292.2 [M+H ], found: 292.3.
(Nujol): e
ꢁ1
+

C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
7361
3
.1.4. (1R,2R)-Pseudoephedrine-(2S)-ethylhexanamide 6
e
m
1
= 3443 (w), 2961 (m), 2925 (m), 2870 (w), 2854 (w), 1634 (m),
457 (w), 1261 (m), 1096 (m), 1021 (m), 800 (m), 702 (w), 668
ꢁ
1
(
w), 608 (w), 470 cm (w). For MS analysis, the alcohol was reacted
with (S)-(+)-
a-methoxy-a-trifluoromethylphenylacetyl chloride to
H
O
afford the corresponding Mosher ester. HR-ESI-MS of the Mosher es-
N
+
H
OH
ter: C18
25 3 3
H F O calcd: 369.1648 [M+Na ], found: 369.1649.
(
R,R,S)-6
3
.1.8. (2S)-Ethylhexanol 8
The synthesis was carried out according to the published proce-
NMR (300 MHz, CDCl ): d = 0.86
3
t, J(H,H) = 7 Hz, 6H), 1.08–1.71 (m, 11H), 2.48 (m, 1H), 2.84 (s, 3H),
.99 (s, 1H), 4.39 (m, 1H), 4.60 (m, 1H), 7.33 (m, 5H) ppm;
): d = 12.1, 14.1, 14.7, 23.0, 26.1, 29.8, 32.6,
4.0, 58.2, 60.5, 75.5, 76.5, 126.4, 127.6, 128.4, 142.8,178.8 ppm;
2
5
1
dure.
(
3
Yield: 70%.
H
3
1
3
HO
C
NMR (100 MHz, CDCl
3
4
(S)-8
IR (Nujol): e
m
= 3375 (m), 3062 (w), 3029 (w), 2960 (s), 2930 (s),
The synthesis was carried out according to the published proce-
2
4
22
D
1
3
2
(
872 (m), 2360 (w), 1616 (s), 1481 (m), 1456 (m), 1411 (w), 1378
w), 1309 (w), 1258 (w), 1197 (w), 1151 (w), 1113 (m), 1087 (w),
dure. Yield: 89%. ½
a
ꢀ
þ 2:88 (c 0.1 g/100 mL, CHCl ). H NMR
3
(300 MHz, CDCl ): d = 0.9 (m, 6H), 1.3–1.4 (m, 8H), 1.7 (m, 1H), 3.5
3
13
1
5
051 (m), 1028 (w), 838 (w), 763 (w), 737 (w), 701 (m), 609 (w),
18 cm (m); ESI-MS C18
3
(d, J(H,H) = 5 Hz, 2H) ppm; C NMR (75 MHz, CDCl ): d = 11.2,
ꢁ1
+
H29NO
2
calcd: 292.2 [M+H ], found: 292.2.
14.2, 23.2, 23.5, 29.3, 30.3, 42.1, 65.4 ppm; IR (Nujol): e
m = 3288 (w),
2
(
6
959 (s), 2924 (s), 2869 (m), 2853 (m), 1738 (w), 1461 (m), 1377
3
.1.5. (2R)-Ethylhexanoic acid 7
m), 1261 (s), 1095 (s), 1021 (s), 974 (w), 863 (w), 801 (s), 702 (w),
ꢁ1
62 (w), 606 (w), 491 (w), 456 cm (w). For MS analysis, the alcohol
-methoxy- -trifluoromethylphenylacetyl
chloride to afford the corresponding Mosher ester. HR-ESI-MS of the
was reacted with (S)-(+)-
a
a
O
+
HO
Mosher ester: C18
25 3 3
H F O calcd: 369.1648 [M+Na ], found: 369.1649.
3
.1.9. Synthesis of 2-(2-ethylhexyl)isoindolin-1,3-diones 9
(
R)-7
The synthesis was carried out analogous to the published proce-
A solution of 521 mg (4 mmol, 1 equiv) 2-ethylhexanol, 589 mg
4 mmol, 1 equiv) phthalimide and 1.05 g (4 mmol, 1 equiv) tri-
2
4
1
dure.
Yield: 88%.
H
3
NMR (400 MHz, CDCl ): d = 0.89
(
3
3
(
1
(
1
1
1
t, J(H,H) = 7 Hz, 3H), 0.91 (d, J(H,H) = 7 Hz, 3H), 1.31 (m, 4H),
.57 (m, 4H), 2.28 (m, 1H), 10.45 (s, 1H) ppm;
phenylphosphine in 4 ml THF is cooled to 0 °C. To this solution, a
solution of 679 mg (4 mmol, 1 equiv) DEAD in 4 ml THF is added
and the mixture is stirred at room temperature for 22 h. The sol-
vent is removed in vacuo, the residue suspended in diethylether
and filtrated. After evaporation of the solvent in vacuo, the crude
product is purified by flash column chromatography (dichloro-
1
3
C NMR
100 MHz, CDCl
3
): d = 11.9, 14.1, 22.8, 25.3, 29.7, 31.6, 47.3,
83.2 ppm; IR (Nujol): e
m = 2963 (s), 2935 (s), 2875 (m), 2863 (m),
707 (s), 1462 (w), 1417 (w), 1384 (w), 1289 (w), 1272 (w),
ꢁ1
229 (w), 1205 (w), 945 (w), 783 (w), 605 (w), 458 cm (w);
+
ESI-MS C H
8 16
O
2
calcd: 143.1 [MꢁH ], found: 143.0.
26
methane). Racemic 9 has been reported.
3
.1.6. (2S)-Ethylhexanoic acid 7
3
.1.9.1. 2-((2R)-Ethylhexyl)isoindoline-1,3-dione 9
O
O
N
HO
O
(
S)-7
The synthesis was carried out according to the published proce-
(R)-9
2
4
1
1
dure.
Yield: 79%.
H
NMR (400 MHz, CDCl
3
): d = 0.89
Yield: 893 mg (86%). H NMR (400 MHz, CDCl ): d = 0.91 (m, 6H),
1.28 (m, 8H), 1.83 (m, 1H), 3.56 (s, 1H), 3.58 (s, 1H), 7.71 (m, 2H),
7.82 (m, 2H) ppm; C NMR (100 MHz, CDCl ): d = 10.6, 14.2, 23.1,
3
3
3
(
(
t, J(H,H) = 7 Hz, 3H), 0.94 (d, J(H,H) = 7 Hz, 3H), 1.31 (m, 4H), 1.64
m, 4H), 2.28 (m, 1H), 10.09 (s, 1H) ppm; 13C NMR (75 MHz, CDCl
):
13
3
3
d = 11.9, 14.1, 22.8, 25.3, 29.7, 31.6, 47.2, 183.1 ppm; IR (Nujol):
24.0, 28.7, 30.7, 38.5, 42.0, 123.3, 132.3, 134.0, 168.9 ppm; IR (Nu-
e
m
= 2963 (s), 2935 (s), 2875 (m), 2862 (m), 1706 (s), 1460 (m),
417 (w), 1383 (w), 1289 (w), 1271 (w), 1229 (m), 1205 (w),
150 (w), 1098 (w), 945 (w), 782 (w), 730 (w), 637 (w),
e
jol):
m
= 2959 (s), 2931 (s), 2872 (m), 2860 (m), 1773 (m), 1714
1
1
4
(s), 1614 (w), 1467 (m), 1436 (m), 1397 (s), 1364 (m), 1334 (w),
1188 (w), 1173 (w), 1065 (m), 944 (w), 902 (m), 800 (m), 793 (w),
ꢁ1
+
ꢁ1
54 cm (w); ESI-MS C
H
8 16
O
2
calcd: 143.1 [M+H ], found: 143.1.
722 (s), 713 (m), 694 (w), 624 (w), 530 cm
H21NO calcd: 260.1645 [M+H ], found: 260.1646.
16 2
(w); HR-ESI-MS
+
C
3
.1.7. (2R)-Ethylhexanol 8
3
.1.9.2. 2-((2S)-Ethylhexyl)isoindolin-1,3-dione 9
HO
O
N
(
R)-8
The synthesis was carried out analogous to the published proce-
O
2
4
22
1
dure. Yield: 91%. [
400 MHz, CDCl ): d = 0.89 (m, 6H), 1.28–1.38 (m, 8H), 1.70 (m,
H), 3.53 (s, 1H), 3.53 (s, 1H) ppm; C NMR (100 MHz, CDCl
d = 11.2, 14.2, 23.2, 23.5, 29.3, 30.3, 42.1, 65.4 ppm; IR (Nujol):
D 3
a] = -2.40 (c 0.1 g/100 mL, CHCl ). H NMR
(
S)-9
(
1
3
13
Yield: 925 mg (89%). 1H NMR (400 MHz, CDCl ): d = 0.91 (m,
6H), 1.28 (m, 8H), 1.83 (m, 1H), 3.57 (s, 1H), 3.58 (s, 1H), 7.7 (m,
3
):
3

7
362
C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
2
2
H), 7.8 (m, 2H) ppm; 13C NMR (100 MHz, CDCl
3.1, 24.0, 28.7, 30.7, 38.5, 42.1, 123.3, 132.3, 134.0, 168.9 ppm.
3
): d = 10.6, 14.2,
27.9, 29.5, 36.9, 41.6, ppm; IR (Nujol): e
m = 3443 (s), 3022 (s),
2960 (s), 2929 (s), 2874 (s), 2360 (w), 1648 (m), 1636 (m), 1603
IR (Nujol): e
m = 2953 (s), 2922 (s), 2850 (s), 1718 (m), 1699 (w),
(m), 1558 (w), 1496 (m), 1465 (m), 1458 (m), 1380 (w), 1331
ꢁ
1
1
1
683 (w), 1646 (w), 1507 (w), 1457 (m), 1419 (w), 1396 (w),
376 (w), 1365 (w), 1113 (w), 779 (w), 721 (w), 640 (w), 601
(w), 1082 (w), 792 (w), 683 (w), 628 (w), 563 cm (w); ESI-MS
+
C H
8
19N calcd: 130.2 [M+H ], found: 130.3.
ꢁ1
(
w), 456 cm
(w); HR-ESI-MS
C
16
H
21NO
2
calcd: 260.1645
Determination of the enantiomeric excess of (2S)-ethylhexyl-
+
[
M+H ], found: 260.1647.
amine hydrochloride 2, was carried out analogous to its enantio-
mer (R)-2 by conversion of the amine to the Mosher amide. This
was analyzed using HPLC (ChiralcelÒ OJ column, 4.6 mm ꢂ 25 cm).
Ee = 95%; Retention time: 16.3 min (corresponding to the Mosher
amide of (S)-2 as the major isomer) and 19.1 min (corresponding
to the Mosher amide of (R)-2 as the minor isomer).
3
.1.10. Synthesis of 2-ethylhexylamine hydrochlorides 2
A solution of 778 mg (3.0 mmol, 1 equiv) 2-(2-ethylhexyl)isoin-
dolin-1,3-dione 5 and 150 mg (3.0 mmol, 1 equiv) hydrazine
monohydrate in 12 ml ethanol is heated to reflux for 15 min. The
resulting suspension is filtrated, the filtrate acidified with 5 mL of
3
1
3.1.11. 1,6-Di-(N -cyano-N -guanidino)hexane 3
6
M hydrochloric acid, and washed with diethylether. The aqueous
phase is made alkaline by addition of 2 M NaOH and extracted with
diethylether. The organic phases are combined, dried with magne-
sium sulfate and filtrated. Four milliliter of 6 M hydrochloric acid is
added and the solvent removed in vacuo to afford the pure amine
as the hydrochloric salt.
NH
H
N
H
N
CN
N
H
N
NC
NH
H
3
3
.1.10.1. (2R)-Ethylhexylamine hydrochloride 2
Compound 3 was synthesized according to the published proce-
dure. In brief, a solution of 3.78 g (20.0 mmol, 1 equiv) 1,6-hexam-
1
ethylenediamine dihydrochloride and 3.56 g (40.0 mmol, 2 equiv)
sodium dicyanamide in 28 mL n-butanol was heated to reflux for
8 h. After cooling to room temperature, the solid was filtered off
and washed with butanol and cold water. Recrystallization from
water afforded pure compound 3. Yield: 80%. Melting point:
H N
2
x HCl
(
R)-2
2
D
2
Yield: 194.0 mg (39%, 95% ee). Melting point: 80 °C; ½
a
ꢀ
þ 1:5
1
1
1
95 °C (Ref.: 202–203 °C).
6
H NMR (300 MHz, [D ]DMSO):
1
(
(
c 0.1 g/100 mL, CH
3
OH). H NMR (300 MHz, [D
6
]DMSO): d = 0.82
d = 1.23 (m, 4H), 1.39 (m, 4H), 3.02 (m, 4H), 6.63–6.83 (m, 6H)
m, 6H), 1.25 (m, 8H), 1.57 (m, 1H), 2.65 (m, 2H), 8.11 (s, 3H)
ppm; C NMR (75 MHz, [D ]DMSO): d = 10.2, 13.9, 22.3, 22.8,
6
1
3
ppm; C NMR (100 MHz, [D
6
]DMSO): d = 25.9, 28.9, 40.8, 118.4,
1
3
1
2
61.2 ppm; IR (KBr): e
m = 3372 (s), 3159 (s), 2940 (s), 2940 (m),
857 (m), 2172 (s), 1650 (s), 1625 (s), 1475 (m), 1439 (s), 1383
2
2
(
(
7
8.0, 29.5, 36.9, 41.5, ppm; IR (Nujol): e
m = 3442 (s), 3019 (s),
961 (s), 2929 (s), 2874 (s), 2863 (s), 2665 (w), 1717 (m), 1661
(
m), 1322 (m), 1128 (m), 928 (w), 733 (w), 689 (w), 561 (m),
m), 1601 (w), 1495 (m), 1466 (m), 1396 (w), 1380 (w), 1349
w), 1330 (w), 1306 (w), 1263 (w), 1171 (w), 1082 (w), 1023 (w),
ꢁ
1
+
5
41 cm (m); ESI-MS C10
H
18
N
8
calcd: 249.2 [MꢁH ], found: 249.3.
ꢁ1
91 (w), 782 (w), 725 (w), 628 (w), 494 (w), 477 cm (w); ESI-MS
3
.1.12. Synthesis of 1,6-bis[5-(2-ethylhexyl)biguanido]hexane
+
C
8
H
19N calcd: 130.2 [M+H ], found: 130.2.
In order to determine the enantiomeric excess of (2R)-ethylhex-
ylamine hydrochloride 2, 10 mg (0.08 mmol) were dissolved in
ml dichloromethane and cooled to 0 °C. Triethylamine (18 mg,
.18 mmol) and (S)-(+)- -methoxy- -trifluoromethylphenylacetyl
chloride (22 mg, 0.09 mmol) were added, and the reaction mixture
was stirred at room temperature for 1 h. After completion of the
reaction, water and diethyl ether were added. The aqueous phase
was extracted three times with diethyl ether. Subsequently, the or-
ganic phase was dried over sodium sulfate and evaporated under
reduced pressure. The Mosher amide was analyzed using HPLC to
dihydrochlorides 1
3
1
6
3.0 mg (0.25 mmol, 1 equiv) 1,6-di-(N -cyano-N -guanidino)-
1
0
hexane 3 and 82.9 mg (0.5 mmol, 2 equiv) (S)-2-ethylhexylamine
hydrochloride 2 (for synthesis of (S,S)-1) or (R)-2-ethylhexylamine
hydrochloride (R)-2 (for synthesis of (R,R)-1) were thoroughly
mixed and heated to 155 °C for 2 h. After completion of the
reaction, the crude product was recrystallized from ethanol–ether.
a
a
3
.1.12.1. (R,R)-Alexidine 1
Ò
determine the enantiomeric excess of the amine (Chiralcel OJ col-
umn, 4.6 mm ꢂ 25 cm). Ee = 95%; retention time: 16.6 min (corre-
sponding to the Mosher amide of (S)-2 as the minor isomer) and
NH NH
H
N
H
N
H
N
1
9.5 min (corresponding to the Mosher amide of (R)-2 as the major
N
H
N
H
N
H
isomer).
NH NH
(R,R)-1)
3
.1.10.2. (2S)-Ethylhexylamine hydrochloride 2
Yield: 70.0 mg (48%). Melting point: 208 °C (Ref.:
2
7
22
D
2
CH
19–221 °C);
3
Ee >99%; de = 91%; ½
OH). H NMR (300 MHz, CD
(m, 18H), 1.60 (m, 6H), 3.25 (m, 8H) ppm; C NMR (75 MHz,
CD OD): d = 11.3, 14.5, 24.1, 25.2, 27.7, 30.0, 30.4, 32.0, 40.9,
a
ꢀ
þ 3:6 (c 0.1 g/100 mL,
H N
1
2
3
OD): d = 0.94 (m, 12H), 1.34–1.42
1
3
x HCl
(
S)-2
3
4
2.5, 45.4, 160.4, 160.5 ppm; IR (KBr): e
m = 3424 (s), 2925 (m),
2
D
2
Yield: 141.0 mg (28%, 95% ee). Melting point: 83 °C; ½
a
ꢀ
ꢁ 4:4
2855 (m), 2340 (w), 2134 (w), 2025 (w), 1632 (m), 1548
(m), 1462 (m), 1384 (m), 1165 (m), 1112 (m), 593 (w), 551 (m),
1
(
(
c 0.1 g/100 mL, CH
3 6
OH). H NMR (300 MHz, [D ]DMSO): d = 0.82
m, 6H), 1.24 (m, 8H), 1.57 (m, 1H), 2.66 (m, 2H), 8.05 (s, 3H)
ppm; C NMR (75 MHz, [D ]DMSO): d = 10.2, 14.0, 22.4, 22.8,
6
ꢁ
1
+
473 cm
56
(m); HR-ESI-MS C26H N10 calcd: 509.4762 [M+H ],
1
3
found: 509.4765.

C. Gröst et al. / Bioorg. Med. Chem. 21 (2013) 7357–7363
7363
3
.1.12.2. (S,S)-Alexidine 1
extend our thanks to the core facility of the Max Planck Institute
of Biochemistry, Martinsried, for experimental support, and to An-
gela Hollis for critical reading of the manuscript. This study was
supported by the University of Leipzig.
NH NH
H
N
H
N
H
N
N
H
N
H
N
H
NH NH
(S,S)-1
Supplementary data
Yield: 87.0 mg (60%). Melting point: 221 °C (Ref.: 219–
1
2
7
22
D
Supplementary data associated ( H and 13C NMR spectra of the
new compounds, calculation of enantiomeric and diastereomeric
excesses of alexidines) with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.09.057.
2
21 °C); Ee >99%; de = 90%; ½
a
ꢀ
ꢁ 4:3 (c 0.1 g/100 mL, CH
3
OH).
1
H NMR (400 MHz, CD
8H), 1.58 (m, 6H), 3.19 (m, 4H), 3.23 (m, 4H) ppm; C NMR
100 MHz, CD OD): d = 11.3, 14.5, 24.1, 25.2, 27.7, 30.1, 30.6,
3
OD): d = 0.92 (m, 12H), 1.32–1.40 (m,
1
3
1
(
3
3
2
1
2.0, 40.8, 42.5, 45.4, 160.1, 160.2 ppm; IR (KBr): e
m = 3313 (m),
959 (m), 2928 (m), 2871 (m), 2341 (w), 2130 (w), 2025 (w),
632 (s), 1547 (s), 1462 (m), 1384 (m), 1166 (m), 1032 (m), 668
References and notes
ꢁ1
1. Rose, F. L.; Swain, G. J. Chem. Soc. 1956, 4422.
(
56
w), 591 (w), 550 (w), 411 cm (m); HR-ESI-MS C26H N10 calcd:
2
3
.
.
Dyall, S. D.; Brown, M. T.; Johnson, P. J. Science 2004, 304, 253.
Doughty-Shenton, D.; Joseph, J. D.; Zhang, J.; Pagliarini, D. J.; Kim, Y.; Lu, D.;
Dixon, J. E.; Casey, P. J. J. Pharmacol. Exp. Ther. 2010, 333, 584.
Yip, K. W.; Ito, E.; Mao, X.; Au, P. Y.; Hedley, D. W.; Mocanu, J. D.; Bastianutto,
C.; Schimmer, A.; Liu, F. F. Mol. Cancer Ther. 2006, 5, 2234.
Feng, R.; Rios, J. A.; Onishi, T.; Lokshin, A.; Gorelik, E.; Lentzsch, S. Leuk. Res.
2010, 34, 917.
Gräber, M.; Hell, M.; Gröst, C.; Friberg, A.; Sperl, B.; Sattler, M.; Berg, T. Angew.
Chem., Int. Ed. 2013, 52, 4487.
+
5
09.4762 [M+H ], found: 509.4767.
4
5
6
7
.
.
.
.
3
.2. Fluorescence polarization assay
The effect of the test compounds on binding between Bcl-x
a 5-carboxyfluorescein-labeled Bak-derived peptide was essen-
tially carried out as described.6 In brief, Bcl-x
(amino acids
–209, 45–84) cloned into pET29, a kind gift from Professor Ho
L
and
L
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,
1
D
17
Sup Yoon (Nanyang Technical University, Singapore), was ex-
6
8, 3421.
pressed from Escherichia coli Rosetta (Novagen) as previously
8.
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. Nature 2005, 435, 677.
2
8
described, purified by affinity chromatography, and extensively
dialyzed against buffer (50 mM Hepes, 100 mM NaCl, 10% (v/v)
glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet-40 substitute, pH
7
.5). Binding of this protein to the Bak BH3-derived peptide (5-car-
19
boxyfluorescein)-GQVGRQLAIIGDDINR-NH
2
was analyzed by
9. Lessene, G.; Czabotar, P. E.; Colman, P. M. Nat. Rev. Drug Disc. 2008, 7,
89.
0. Petros, A. M.; Olejniczak, E. T.; Fesik, S. W. Biochim. Biophys. Acta 2004, 1644,
3.
11. Miura, M.; Toriyama, M.; Motohashi, S. Synth. Commun. 2006, 36, 259.
9
fluorescence polarization. Peptide and protein stocks were diluted
in 10 mM Tris, 50 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 10%
DMSO, pH 8.0. Final concentrations in the assay buffer: 5-carboxy-
1
8
12. Grenier, C. R.; George, S. J.; Joncheray, T. J.; Meijer, E. W.; Reynolds, J. R. J. Am.
Chem. Soc. 2007, 129, 10694.
fluorescein-labeled peptide: 10 nM; Bcl-x
45–84): 40 nM. Fluorescence polarization was read in an Infinite
00 plate reader (Tecan). Assays were carried out in black 384-well
L
(amino acids 1–209,
D
13. Kondakov, D. Y.; Negishi, E. J. Am. Chem. Soc. 1996, 118, 1577.
5
14. Huang, Y.; Zhang, F.; Gong, Y. Tetrahedron Lett. 2005, 46, 7217.
1
1
1
5. Vigneron, J. P.; Dhaenens, M.; Horeau, A. Tetrahedron 1973, 29, 1055.
6. Soai, K.; Hori, H.; Kawahara, M. J. Chem. Soc., Chem. Commun. 1992, 106.
7. Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.; Eberstadt,
M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S.
W. Science 1997, 275, 983.
plates (Corning no. 3573). Inhibition was calculated based on curve
fits using SigmaPlot (SPSS Science Software). The mixture of alexi-
dine isomers (as dihydrochloride, CAS# 1715-30-6) used in the
fluorescence polarization assay was purchased from Toronto Re-
search Chemicals and purified by reversed phase chromatography
at the Core Facility of the Max-Planck Institute of Biochemistry.
18. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: Palo
Alto, CA, USA, 2002.
19. Wang, J. L.; Liu, D.; Zhang, Z. J.; Shan, S.; Han, X.; Srinivasula, S. M.; Croce, C. M.;
Alnemri, E. S.; Huang, Z. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7124.
2
0. Muchmore, S. W.; Sattler, M.; Liang, H.; Meadows, R. P.; Harlan, J. E.; Yoon, H.
S.; Nettesheim, D.; Chang, B. S.; Thompson, C. B.; Wong, S. L.; Ng, S. L.; Fesik, S.
W. Nature 1996, 381, 335.
3
.3. Molecular docking
Docking experiments were performed with AutoDock Vina.22
21. Lee, E. F.; Czabotar, P. E.; Smith, B. J.; Deshayes, K.; Zobel, K.; Colman, P. M.;
Fairlie, W. D. Cell Death Differ. 2007, 14, 1711.
Default settings were used with the exception of the exhaustive-
ness, which was set to 50. Docking experiments were run using
the Bcl-x structure from PDB entry 2YXJ after removal of the li-
L
2
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Acknowledgments
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We would like to thank Prof. Ho Sup Yoon (Nanyang Technical
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A.; Berg, T. ACS Chem. Biol. 2011, 6, 1008.
University, Singapore) for the Bcl-x
L
expression plasmid. We