Atropisomeric small molecule Bcl-2 ligands: Determination of bioactive conformation

Article abstract of DOI:10.1016/j.bmcl.2009.01.071

The separation of atropisomeric conformers of 1,2,3,4-tetrahydroisoquinoline amide Bcl-2 ligands allowed the identification of the bioactive conformer which was subsequently confirmed by X-ray crystallography.

Full text of DOI:10.1016/j.bmcl.2009.01.071

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1767–1772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Atropisomeric small molecule Bcl-2 ligands: Determination
of bioactive conformation
*
John Porter , Andrew Payne, Ian Whitcombe, Ben de Candole, Daniel Ford, Rachel Garlish, Adam Hold,
Brian Hutchinson, Graham Trevitt, James Turner, Chloe Edwards, Clare Watkins, Jeremy Davis,
Colin Stubberfield
UCB Celltech, 216 Bath Road, Slough SL1 3WE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The separation of atropisomeric conformers of 1,2,3,4-tetrahydroisoquinoline amide Bcl-2 ligands
allowed the identification of the bioactive conformer which was subsequently confirmed by X-ray
crystallography.
Received 3 December 2008
Revised 20 January 2009
Accepted 22 January 2009
Available online 27 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bcl-2
Protein–protein interaction
Apoptosis
Atropisomerism
Regulation of cell death is of fundamental importance. Bcl-2 is a
member of a family of anti- and pro-apoptotic proteins that func-
tion by binding to and sequestrating other family members.1 In
healthy cells, Bcl-2 expression is tightly controlled, but it is overex-
pressed in a wide range of human haematopoietic and solid can-
cers where it serves to prevent apoptosis induced by protective
cell-death mechanisms. This is manifested clinically as drug resis-
tance when treating with traditional cytotoxics and can also delay
apoptosis in response to radiation therapy.2 The homologous fam-
ily member, Bcl-xL, also shows significant overexpression in some
types of lung and ovarian cancers. Although the precise mechanism
of Bcl-2 family mediated apoptosis is the subject of considerable
debate,3 it is generally agreed that apoptosis is inhibited by the
binding of the pro-death proteins to their anti-apoptotic counter-
parts. Bcl-2 (and Bcl-xL) have a binding groove that accommodates
the BH3 (Bcl-2 Homology-3) domain of pro-apoptotic family mem-
bers,4 preventing their oligomerization and the initiation of the
apoptosis cascade.5 Chimaeric Bcl-2/xL NMR and X-ray crystal
structures have demonstrated that the binding groove is amenable
to small molecule intervention and studies have shown that such
molecules are able to sensitise tumour cells to apoptosis.6 Such
molecules should abrogate the anti-apoptotic effects of Bcl-2
expression and chemosensitize a significant proportion of tumours
to cell death by existing cytotoxics and new targeted therapies.
This has been most successfully demonstrated with ABT-263, a
small molecule Bcl-2/Bcl-xL inhibitor that has entered clinic trials.7
O
O
N
Cl
N
N
N
N
O
N
O
N
2
N
6
3
NH2
NH2
1
2
We have described the discovery and SAR of a series of
1,2,3,4-tetrahydroisoquinoline (THIQ) amide substituted phenyl
pyrazoles, such as 1 and 2, that display good selectivity and po-
tency against Bcl-2.8 These molecules often exhibited appreciable
line broadening in their 1H NMR spectra due to the restricted
rotation around the Ar-(C@O)-N bond. Substitution at the THIQ
3-position led to the observation of individual rotamers by
NMR. Although atropisomeric9 molecules have been identified
through drug discovery programmes,10 the presence of multiple
conformational forms adds to the complexity of the development
* Corresponding author. Tel.: +44 1753 534655.
E-mail address: john.porter@ucb-group.com (J. Porter).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.071

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J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1767–1772
process, consequently, there have been a number of reports of
attempts to overcome these issues.11 However, we postulated
that by sufficiently restricting rotation by substitution at the
phenyl 6-position, it would be possible to physically separate
and characterise the individual rotameric species. Assuming that
Bcl-2 recognises only one ligand conformation, then the protein
would select the bioactive conformation on exposure to the rota-
meric mixture. We could then determine the conformation by 1H
NMR techniques and optimise the molecule by a process of
structure based drug design. The results of this work are de-
scribed in this paper.
The phenylpyrazoles were prepared according to the method
outlined in Scheme 1. The benzoic acid 3 was converted to the
hydrazine 4 and cyclised with ethyl acetopyruvate to give the
pyrazole 5. Conversion to the amide 6 was achieved with lithiat-
ed diphenylamine followed by coupling with the THIQ to give 7.
Alternatively, the azide substituted THIQ was coupled to the acid
chloride derived from 5 followed by hydrogenolysis to give the
amine 10a. Introduction of a chlorine atom at the pyrazole 4-po-
sition was achieved by treating acid 5 with N-chlorosuccinimide,
followed by a similar sequence of reactions yielding amine 10b.
The route depicted in Scheme 2 allowed greater flexibility in the
incorporation of the group ortho to the phenyl carboxylate. Base
induced coupling of the pyrazole 12 (prepared by the reaction of
the dibutyl amino aluminate with the pyrazole ethyl ester) with
the fluoride 11 was followed by hydrolysis of the ester and pyB-
rOP mediated coupling with the azide substituted THIQ to give
15 which was reduced to give the final product 16.
O
O
N(nBu)2
N(nBu)2
N
N
F
N
N
CO2Et
X
CO2Et
X
CO2H
X
ii
i
11a, X=CF3
11b, X=NO2
13a, X=CF3
13b, X=NO2
14a, X=CF3
14b, X=NO2
O
O
N(nBu)2
N(nBu)2
N3
N
N
NH2
N
O
X
N
O
iii
iv
N
N
X
15a, X=CF3
15b, X=NO2
16a, X=CF3
16b, X=NH2
Scheme 2. Reagents and conditions: (i) 5-methyl-1H-pyrazole-3-carboxylic acid
dibutylamide 12, Cs2CO3, DMF, 110 °C, 2 h; (ii) LiOH, THF, water, 25 °C, 16 h; (iii)
(S)-3-azidomethyl-1,2,3,4-tetrahydro-isoquinoline, pyBrOP, DIEA, CH2Cl2, 18 °C,
16 h; (iv) % Pd–C, H2, EtOAc, 25 °C, 24 h.
cis or trans to the carbonyl. This analysis allowed the conformation
of the THIQ moiety system to be determined with the 3-substitu-
ent in a theoretically energetically less favoured pseudo-axial posi-
tion. This is a manifestation of a special case of the A-1,3 strain
which is commonly observed in substituted acylated cyclic
amines.12 The methyl substituted analogue, 7, (Fig. 1, R = Me) also
showed four pyrazole proton signals (Fig. 4) but this time the ratios
changed on equilibration (DMSO, 25 °C) from 1:6.4:1:2.4 at t = 0 h
to 1:1.5:0.2:1.3 at t = 48 h.
The atropisomers of 7 were sufficiently stable to enable HPLC
separation13 and isolation, the 1H NMR of the separated compo-
nents is shown in Figure 4. The separated rotamers were
screened in the Bcl-2 assay (Table 1).14 Fraction 2 is clearly
the most active, however, the other three fractions retained
some activity, particularly fraction 4. This was believed to be
due to re-equilibration of the separated rotamers during the
Slow rotation around the Ar-THIQ amide bonds give four atrop-
isomers, two orientations for the amide (s-cis and s-trans) and two
for the aryl carbonyl (axial-R and axial-S) (Fig. 1). These were evi-
dent in the 1H NMR spectrum of 1 where the 4-pyrazole proton sig-
nals proved to be diagnostic, displaying four resonances between d
5.5 and d 6.1 (in CD3OD, 25 °C) with an integral ratio of 1:2:2:8
(corresponding to the four rotamers shown in Fig. 1, R = H),
although only one peak was observed by HPLC. One other feature
of the 1H NMR spectrum were the signals associated with the THIQ
C-1 methylene diastereotopic protons which appeared as doublets
but with very large chemical shift differences between the signals
corresponding to the
a and b protons and the C-3 methine proton.
The magnitude of this effect, caused by shielding/deshielding by
the amide carbonyl, was dependent on whether the protons were
O
N
O
CO2Et
NPh2
NPh2
NH2.HCl
NH2
HN
N
N
OH
CO2H
N
CO2H
iv
N
O
v
N
i
iii
CO2H
CO2H
N
ii
3
4
5
6
7
vi
vii
CO2Et
O
X
CO2Et
Cl
X
N(nBu)2
NH2
vi
N
N3
N
N
N
O
N
8
O
N
viii
ix
CO2H
N
N
9a, X=H
9b, X=Cl
10a, X=H
10b, X=Cl
Scheme 1. Reagents and conditions: (i) NaNO2, aq HCl, 0 °C, 30 min; (ii) SnCl2, HCl, 25 °C, 3 h; (iii) ethyl acetopyruvate, gAcOH, refux, 1 h; (iv) Ph2NH, nBuLi, THF, À78 to
25 °C; (v) (S)-1-(1,2,3,4-tetrahydro-isoquinolin-3-yl)-methanol, HOAT, EDCI, NMM, CH2Cl2, 25 °C, 16 h; (vi) (S)-3-azidomethyl-1,2,3,4-tetrahydro-isoquinoline, (COCl)2,
CH2Cl2, 25 °C, 16 h; (vii) NCS, CCl4, 25 °C, 16 h; (viii) nBu2NH, EDCI, HOBT, Et3N, 25 °C, 16 h; (ix) 10% Pd–C, H2, EtOH, 25 °C, 1 h.

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1767–1772
1769
N
R
O
H
N
Ar-C=O
bond rotation
N
O
N
N
s
Me
H3
H
N
N
R
O
N
slow
N
H
HO
H2
w
HO
HO
cis, a-R
H1
cis, a-S
H
H
s
O=C-N
bond
rotation
O=C-N
bond
rotation
Me
fast
m
R
Figure 3. The cis, a-R (bioactive) atropisomer of compound 7 showing observed
nOe interactions (s = strong, m = medium, w = weak). The diastereotopic protons on
the THIQ C-1 and C-3 atoms are labelled. The pyrazole ring has been simplified for
clarity.
N
O
N
N
O
N
N
Ar-C=O
bond rotation
R
N
H
OH
HO
H
experimentally by monitoring the change in integrated signal
area of the pyrazole 4-H 1H NMR signals with time, the exam-
ples for fractions 2 and 4 are shown in Figure 2. The curves
show that the rate of equilibration of fraction 2 (Fig. 2A) is rel-
atively slow with approximately 90% of fraction 2 remaining
after 90 min (the approximate time course of the LANCE assay).
However, inter-conversion from fraction 4 to fraction 2 is fast
(Fig. 2B) with approximately 40% of fraction 2 present after
90 min. The apparent activity of fraction 4 may therefore be ex-
plained as a result of the rapid conversion to the active rotamer,
fraction 2. Attempts to measure the energy of the barrier to rota-
tion by observation of the coalescence of the NMR signals of the
diastereotopic protons with increasing temperature were unsuc-
cessful because of the high temperatures required (no coales-
cence of peaks in the 600 MHz 1H spectrum of the equilibrium
mixture in DMSO-d6 was observed at 50 °C). The barrier to rota-
tion around the amide bond of 2,6-disubstituted aryl amides has
trans, a-R
trans, a-S
Figure 1. Inter-conversions between the four isomers of compound 7: cis and trans
are used to denote the relative configuration between the carbonyl oxygen and the
3-THIQ substituent in the amide moiety; R and S denote the axial chirality arising
from the restricted rotation around the Ar–C@O bond. The pyrazole substituent has
been simplified in the interests of clarity.
been reported to be in excess of 100 kJ molÀ1 15
lineshape analysis has been made at this time.
.
No attempt at
The 1D selective pulse 1H NMR nOe spectra of the four sep-
arated atropisomers were measured and, as expected, found to
give a unique pattern of cross-peaks indicative of their confor-
mational differences, an example of the nOe interactions is
shown in Figure 3. A simple molecular mechanics model of 7
was generated using Sybyl16 and low energy conformations
determined for each of the four rotameric forms. It then proved
possible to uniquely assign the nOe information for each of the
physically isolated rotameric forms to the appropriate geometry
of the models, and thus assign the rotameric conformations of
the models to the physically separated compounds. As the
activity against Bcl-2 of the separated atropisomers was known,
it was possible to assign the conformation of the active rotamer
as cis, a-R (Table 1). From the assignment of the above con-
formers, it was possible to predict the equilibria between each
peak through single, stepwise bond rotations, summarised in
Figure 1. These equilibria correlate with those observed experi-
mentally in the 1H NMR time-course equilibrations described
above.
A Bcl-2 affinity assay gave further evidence for the identity of
the atropisomeric eutomer of 7.17 Bcl-2 protein was added to the
equilibrium mixture of rotamers, resulting in only the active con-
former binding to Bcl-2. The compound/protein complex was sep-
arated
from
unbound
compound
by
size
exclusion
Figure 2. (A) Re-equilibration of compound 7 fraction 2 in DMSO solution at RT, as
determined by integration of the pyrazole 4-H 1H NMR signal. (B) Re-equilibration
of compound 7 fraction 4 in DMSO solution at RT, as determined by integration of
the pyrazole 4-H 1H NMR signal.
chromatography, the complex denatured with acetonitrile to re-
lease the compound which was isolated and characterised by LC–
MS (Fig. 3). The results are in agreement with the Bcl-2 assay find-
ings: the rotamer corresponding to HPLC fraction 2 was the major
component of the isolated fraction (Fig. 5). (The presence of other
rotamers observed is due to flow-through.)
time of the experiment. To further investigate this, the re-equil-
ibration of the isolated rotamers in DMSO solution was followed

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J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1767–1772
Figure 4. The 1H NMR spectra (600 MHz) of 7 showing the diagnostic region between d 3.6 and d 6.3. (a) The equilibrium mixture after 48 h, (b) fraction 1, (c) fraction 4, (d)
fraction 3, (e) fraction 2.
Table 1
Bcl-2 activity, 600 MHz NMR data and assignment of the atropisomeric forms of compound 7
Compound
HPLC
Assignment
Pyrazole 4-H d
H1, H2
–
4.92, 4.24
4.40, 4.14
4.33, 3.98
5.18, 4.00
d
H1, H2 J (Hz)
H3
–
3.67
4.70
4.63
3.68
d
Bcl-2 IC50 (nM)
7
7
7
7
7
Mixture
–
–
–
600
1100
210
6100
390
Fraction 1
Fraction 2
Fraction 3
Fraction 4
trans, a-S
cis, a-R
cis, a-S
trans, a-R
5.51
6.11
5.96
5.74
18.0
16.0
16.8
17.8
the equivalent conformation at the THIQ amide as compound 7.
The apparent activity of fraction 2 is due to the presence of approx-
imately 20% of fraction 1 as an impurity. The chloro analogue, 10b
was not separated but the equilibrium mixture was even more po-
tent than 10a. We surmise that the chloro group locks the di-n-
butylamide in a favourable position. The equilibrium mixture of
16a exists as two major conformers and was readily separable,
but both conformers were inactive. The amino analogue 16b was
also less active, the conformation of these species was not deter-
mined. Following the conclusion of this work, an X-ray crystal
structure of a close analogue, 1 complexed with a soluble mutant
of the Bcl-2 protein was solved.8,18 This structure confirmed that
the bound conformation had the same rotameric conformation as
the active fraction (fraction 2) of 7 (see Fig. 6). This work and our
SAR observations8 shows that the conformation of the THIQ group
plays a significant role in determining the potency of these
molecules.
Figure 5. Plot of the HPLC peak area for the 4 rotamers of 7 following incubation
with Bcl-2, size exclusion chromatography to remove unbound ligand and protein
denaturation to release the bioactive conformation. The plot should be compared
with the standard equilibrium mixture shown on the left to see the clear
enrichment of fraction 2.
Armed with this information we set about designing mole-
cules that would be locked into the bioactive conformation to
afford enhanced potency and remove the issues associated with
the development of atropisomeric drugs. In conclusion, we have
utilised the atropisomeric properties of some potent inhibitors
of Bcl-2 to identify the bioactive conformation of these
compounds.
A limited SAR study was conducted on analogues of 7 (Table 2).
Replacement of the pyrazole diphenyl amide with dibutyl groups
has been shown to give improved potency against Bcl-2.8 Com-
pound 10a, which, at equilibrium, exists as two major conformers
was separated and analysed by NMR in a similar fashion to that de-
scribed above to show that the bioactive conformer, fraction 1, had

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1767–1772
1771
Table 2
Bcl-2 activity and 600 MHz NMR data for the atropisomeric forms of analogues of compound 7
O
X
N(nBu)2
NH2
N
N
O
N
R
Compound
R
X
HPLC
Pyrazole 4-H d
H1, H2
–
4.44, 4.29
4.27, 4.15
4.84, 4.19
5.21, 3.85
–
d
H1, H2 J (Hz)
H3
–
4.61
4.54
3.76
3.69
–
d
Bcl-2 IC50 (nM)
10a
Me
H
Mixture
–
–
175
55
358
nd
nd
42
Fraction 1
Fraction 2
Fraction 3
Fraction 4
Mixture
Fraction 1
Fraction 2
Mixture
6.33
6.46
6.36
6.43
n/a
6.35
6.43
–
16.6
16.6
18.0
18.0
–
16.2
16.7
–
10b
16a
Me
CF3
Cl
H
4.35, 4.18
4.24, 4.07
–
4.50
4.46
–
16% at 30
14,400
1070
lM
16b
NH2
H
Compound 10a fractions 3 and 4 were not physically separated, their NMR data was determined from the spectrum of the mixture, nd = not done.
Figure 6. The X-ray conformation of 1 (white) and the nOe-derived solution-phase conformation model of the cis, a-R bioactive atropisomer of 7 (cyan). Comparison of the
structures shows that they share a common rotameric conformation about the THIQ amide. The absence of nOe information relating to the diphenyl amide prevents
assignment of a preferred orientation of this group, however it is likely that this part of the molecule rotates freely in solution.
C.; Wang, X.; Wendt, M. D.; Yang, X.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.;
Elmore, S. W. J. Med. Chem. 2008, 51, 6902.
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13. HPLC separation of compound 7 was performed using a Waters HPLC system
Wang, B.; Wendt, M. D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H. Nature 2005,
controlled by Fractionlynx software fitted with a Phenomenex Gemini 10
lm
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C18 110 Å 150 Â 21 mm HPLC column (Phenomenex, Macclesfield, Cheshire,
UK). Mobile phase composition of 0.1% formic acid in water + 10% acetonitrile
(phase A) and 0.1% formic acid in acetonitrile + 10% water (phase B). 0–0.5 min,
95% A 5% B; 0.5–9.5 min, 50% A 50% B; 9.5–10 min, 40% A 60% B; 10–11.5 min,
5% A 95% B; 11.5–12 min, 95% A 5% B, at a constant flow rate of 20 mL/min. The
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.;
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2008, 68, 3421; (b) Park, C.-M.; Bruncko, M.; Adickes, J.; Bauch, J.; Ding, H.;
Kunzer, A.; Marsh, K. C.; Nimmer, P.; Shoemaker, A. R.; Song, X.; Tahir, S. K.; Tse,
compound was dissolved in DMSO (10 mg/mL, 250 lL) and loaded onto the

1772
column through a 500
J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1767–1772
l
L injection loop. Fractions were collected automatically
16. Tripos Software, Inc., St. Louis, MO, USA.
into test tubes that were combined as necessary. Solvent was immediately
removed by rotary evaporation followed by freeze drying.
14. HTRF-LANCE assays detected interactions of Bcl-2 proteins expressed as GST-
fusion proteins and biotinylated 16-mer BH3 peptides. Interactions were
detected using europium-conjugated anti-GST antibody and APC-labelled
streptavidin (Perkin-Elmer) using a Wallac Victor 2 plate reader.
17. Recombinant Bcl-2 protein was diluted to 40 lM in Assay Buffer (25 mM
Tris–Cl, pH 7.4, 150 mM NaCl, 0.1% ß-lactoglobulin). An equal volume of
compound was added in 2% DMSO in Assay Buffer and the mixture
incubated for 5 min at 4 °C. Protein–compound complexes were separated
from unbound compound by passing through
a Protein Desalting Spin
Column. Complexes were denatured in an equal volume of acetonitrile and
15. Ahmed, A.; Bragg, A.; Clayden, J.; Lai, L. W.; McCarthy, C.; Pink, J. H.; Westlund,
N.; Yasin, S. A. Tetrahedron 1998, 54, 13277.
characterised by LC–MS.
18. PDB code 2W3L.