Tetrahydroisoquinoline amide substituted phenyl pyrazoles as selective Bcl-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.113

Anti-apoptotic Bcl-2 protects cells from apoptosis by binding to pro-apoptotic members of the Bcl-2 family thereby playing a role in tumour survival in response to chemo- or radiation therapy. We describe a series of phenyl pyrazoles that have high affini

Full text of DOI:10.1016/j.bmcl.2008.10.113

Bioorganic & Medicinal Chemistry Letters 19 (2009) 230–233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydroisoquinoline amide substituted phenyl pyrazoles
as selective Bcl-2 inhibitors
*
John Porter , Andrew Payne, Ben de Candole, Daniel Ford, Brian Hutchinson, Graham Trevitt, James Turner,
Chloe Edwards, Clare Watkins, Ian Whitcombe, Jeremy Davis, Colin Stubberfield
UCB Celltech, 216 Bath Road, Slough SL1 3WE, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-apoptotic Bcl-2 protects cells from apoptosis by binding to pro-apoptotic members of the Bcl-2 fam-
ily thereby playing a role in tumour survival in response to chemo- or radiation therapy. We describe a
series of phenyl pyrazoles that have high affinity for Bcl-2 and rationalise the observed SAR by means of
an X-ray crystal structure.
Received 2 October 2008
Revised 22 October 2008
Accepted 25 October 2008
Available online 31 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Bcl-2
Bcl-xL
Apoptosis
Protein–protein interaction
In normal cells Bcl-2, an anti-apoptotic 26 kDa protein, regulates
the activity of pro-apoptotic proteins by direct binding and sequestra-
tion.1 Bcl-2isoverexpressedinawiderangeofhumanhaematopoietic
and solid cancers and serves to prevent apoptosis induced by protec-
tive 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 highly homologous
familymember, Bcl-xL, also shows significant overexpression insome
types of lung and ovarian cancers. The precise mechanism of Bcl-2
mediated apoptosis is the subject of considerable debate.3
The Bcl-2 family of proteins can be divided into three groups
based on the presence of Bcl-2 homology domains (BH1–BH4).
Anti-apoptotic members such as Bcl-2 and Bcl-xL have a binding
groove that accommodates the BH3 domain of pro-apoptotic fam-
ily members,4 preventing their oligomerization and the initiation
of the apoptosis cascade.5 Chimaeric Bcl-2/xL NMR and X-ray crys-
tal structures have demonstrated that the binding groove is ame-
nable to small-molecule intervention6 and further studies have
shown that such molecules are able to sensitise tumour cells to
apoptosis. It is envisaged that a small-molecule inhibitor of Bcl-2
function would prevent binding and sequestration of pro-apoptotic
Bcl-2 partner proteins. Such a molecule would abrogate the anti-
apoptotic effects of Bcl-2 expression and chemosensitize a signifi-
cant proportion of tumours to cell-death by existing cytotoxics and
new targeted therapies. This has been most successfully demon-
strated with ABT-737, 1, a small molecule Bcl-2/Bcl-xL inhibitor,7
an analogue of which has now entered the clinic.8
Cl
N
N
H
O
N
N
O
S
O
Thephenylpyrazole 2 was identifiedby a HTS campaignas a mod-
O
O
O
N
eratelypotentand selectiveBcl-2 inhibitor(IC50 Bcl-2 1.5 lM; Bcl-xL
N
NH
16.6 l
M). 15N–1H HSQC NMR experiments showed that phenylpyr-
azole 2 binds to a similar region of Bcl-2 as ABT-737.6 We now de-
scribe some of our attempts to develop 2 and rationalise the SAR
observed with reference to an X-ray crystal structure.
N
O
S
N
N
The target molecules were prepared by two methods. Initially
the pyrazole ring was constructed from the 2-hydrazinebenzoic
acid9 and ethylacetopyruvate, Scheme 1, to give a 4:1 mixture of
regioisomers. Amide formation with lithiated amine gave 4, fol-
lowed by coupling with the second amine gave the desired product
5. This method was limiting for the more highly functionalised tar-
1
2
* Corresponding author. Tel.: +44 1753 534655.
E-mail address: john.porter@ucb-group.com (J. Porter).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.113

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 230–233
231
(16-mer). DMPK studies showed that both THIQ groups were
extensively metabolised so initial efforts focused on modifying
these groups. Initial SAR studies indicated that while the phenyl
THIQ amide appeared essential for activity; the requirement for
the pyrazole THIQ amide was less critical. Consequently, we em-
barked on the systematic replacement of the pyrazole THIQ amide,
with the results for some representative examples shown in Table
1. A preference for tertiary over secondary amides (compare 10
with 9) presumably reflects a conformational preference for the
pyrazole amide substituent. The benzyl analogue, 11, was inactive
and para-substituents on the aromatic ring were disfavoured
(compare 12 with 13 or 14). Larger amide N-substituents were tol-
erated, culminating in the diphenyl analogue 18 being amongst the
most potent. Interestingly, this compound showed good levels of
selectivity for Bcl-2 over Bcl-xL. Reversal of the amide by switching
the nitrogen and carbonyl positions; replacement with sulfon-
amide, carbamate, ester or carbonyl functionality; or directly
attaching a phenyl group to the pyrazole completely abolished
activity.
O
N
O
N
NPh2
OEt
H2N
NH
O
i
ii
N
O
N
O
OH
OH
OH
O
N
4
3
NPh2
N
O
iii
N
OH
5
Scheme 1. Reagents and conditions: (i) ethyl acetopyruvate, gAcOH, reflux 2 h,
59%; (ii) nBuLi
2
equiv, Ph2NH, THF, À78 to 25 °C, 98%; (iii) (S)-1-(1,2,3,4-
tetrahydroisoquinolin-3-yl)-methanol, DIEA, EDC, HOBT, DCM, 25 °C, 16 h, 30%.
Attempts to replace the phenyl THIQ substituent with a wide
range of isosteres were unsuccessful. However, the addition of a
methanol group at the 3-position, Table 2, gave a marked improve-
ment in potency. Although the direct attachment of carboxylate
functionality, for example 23, at this position was detrimental,
(presumably an unfavourable interaction with the amide carbonyl)
a range of homologated substituents were active. In fact, it became
clear that size, hydrogen bonding potential or charge of these
groups were not critical, and that the observed activity must come
from a conformational lock exerted by the 3-substituent. This was
confirmed by NMR studies showing the 3-substituted THIQ in an
theoretically energetically unfavoured pseudo-axial conformation.
This is a manifestation of a special case of the A-1,3 strain which is
commonly observed in substituted acylated cyclic amines.13 All of
these compounds were prepared from homochiral starting materi-
als, with the exception of the 3-methyl analogue, HPLC separation
of the two enantiomers was accomplished to give the eutomer 27
and the distomer 28. There is a clear preference for the S-enantio-
mer at this position, again suggesting a specific conformational
requirement for this part of the molecule. Interestingly, the NMR
spectra of these 3-substituted THIQ compounds typically show a
mixture of rotamers (as determined by integration of the pyrazole
proton), with one rotamer conformation predominating. Although
these rotamers can make analysis difficult, their presence sug-
get molecules so an alternative method using the base mediated
coupling of a pyrazole 6 to an activated phenyl, was employed,
Scheme 2.
Confirmation that the desired regioisomer 7 had been prepared
was obtained by nOe NMR. Hydrolysis of the ester, amide forma-
tion and reduction gave the desired product 8.
3-Substituted tetrahydroisoquinolines (THIQ) were prepared
from the commercially available chiral material by standard func-
tional group interconversions.10 For example, preparation of the
aminomethyl analogue was achieved through the azidation of
the alcohol using DPPA. The azide THIQ was then coupled to the
benzoic acid and subsequently reduced to give the amine. The 3-
methyl analogue was prepared as the racemate by reduction of
3-methyl isoquinoline using NaBH4 and NiBr2.11
Screening for binding affinity against Bcl-2 was done by means
of a LANCE based assay using a 16-residue Bak peptide.12 As bind-
ing affinity improved the peptide substrate was switched to the
higher affinity 26-residue analogue, which typically gave an
approximate 10-fold drop-off in activity compared to the 16-mer
peptide. A similar system was used to screen against Bcl-xL.
Phenylpyrazole 2 was a moderately potent inhibitor with an
IC50 of 1.5
l
M against Bcl-2 (16-mer) and 16.6
l
M against Bcl-xL
O
Cl
NBu2
O
O
N
OEt
Cl
i, ii
NBu2
iii
N
O
N
N
N
OEt
N
H
H
6
NO2
7
O
O
Cl
NBu2
Cl
NBu2
N
iv, v
vi
N
O
N
N
O
N
N
N3
NO2
NH2
NH2
8
Scheme 2. Reagents and conditions: (i) N-Chlorosuccinimide, CCl4, benzoyl peroxide, reflux, 88%; (ii) nBuLi, nBu2NH, THF, À78 to 25 °C, 88%; (iii) ethyl 2-fluoro-5-nitro-
benzoate, K2CO3, DMF, 100 °C, 91%; (iv) NaOH, EtOH, water, 25 °C, 98%; (v) (S)-3-azidomethyl-1,2,3,4-tetrahydroisoquinoline HCl, DIEA, EDC, HOBT, DCM, 25 °C, 20%; (vi) H2,
10% Pd/C, EtOH, 25 °C.

232
J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 230–233
Table 1
gested a way of determining the bioactive conformation. Our at-
tempts to achieve this will be described elsewhere.
Modifications to pyrazole amide substituent.
R1
A significant advance came from the observation that bromina-
tion at the pyrazole 4-position led to a considerable improvement
in potency, Table 3, 29. Chloro-substitution was equipotent, for
example 30, but other functionality such as phenyl and nitro were,
at best, only equipotent with the non-substituted pyrazole. With
these modifications in hand we returned to the pyrazole amide
substituent. Replacement of the diphenylamide with dialkylamides
was relatively successful, Table 3, with the di-n-butyl group
appearing to be optimum, for example 36. The dialkylamides also
appear to have greater Bcl-xL activity than the phenylamides, com-
pare for example 36 with 31.
O
N
R2
N
N
O
N
Compound
R1
R2
Bcl-2 (16mer)
Bcl-xL (16mer)
IC50 (lM)
IC50
(
lM)
9
H
Ph
Ph
50
2.5
Inactive
10.7
2.3
3.0
1.9
7.7
1.4
10
11
12
13
14
15
16
17
18
Me
Me
Me
Me
Me
Et
i-Pr
c-Hex
Ph
The X-ray crystal structure of 31 complexed to a chimaeric Bcl-2/
Bcl-xL protein was solved, Figure 1.14 Comparison with the pub-
lished structures shows that 31 binds to part of the same cleft as that
occupied by the Bak, Bad, and Bim peptides and small molecules4,15
and is consistent with the observed SAR. Peptide binding to Bcl-2/
Bcl-xL is mediated via hydrophobic interactions of the side-chains
(i, i + 4, i + 7, and i + 11) in discrete sub-pockets in the cleft. 31 occu-
pies some of these sites (i, i + 4), but also makes a unique interaction
through the THIQ group to a site that is not seen by the peptides or,
the Abbott ligand.15 31 exhibits the predicted conformation deter-
mined from the 1H NMR derived molecular models of the isolated
rotamers of conformationally locked analogues. The key hydropho-
bicpyrazoleamide substituent prefersto be cisto thepyrazolewhich
may have implications in the design of suitable amide replacements.
There is clearly a hydrophobic ‘second-site’ binding pocket that is
occupied by the peptides and the Abbott compounds. Accessing this
pocket would be expected to improve potency by maximising ligand
interaction with Bcl-2. The crystal structure suggests that this may
be achieved by substituting in the 4-position of the central phenyl
ring. Initial studies were performed on the non-substituted THIQ
scaffold, Table 4. The most potent examples contained carbonyl
functionalityand ahydrophobicgroupalso appearedtobe preferred.
Although these molecules had poor solubility, results were suffi-
ciently encouraging to prepare the 3-aminomethyl THIQ analogues,
the most potent being the ether, 42. Interestingly, the thioether, 45
has much improved Bcl-xL activity compared to the ether analogue
46, suggesting a key binding interaction with the protein in this
binding pocket.
CH2Ph
4-F-Ph
3-F-Ph
2-F-Ph
Ph
Ph
Ph
Ph
4.11
12.4
0.8
Table 2
Modifications to the THIQ-3-substituent.
O
NPh2
N
N
N
O
R
Compound
R
Bcl-2 (16mer)
IC50
Bcl-xL
(16mer) IC50 (lM)
(
lM)
5
(S)-CH2OH
0.19
0.11
0.16
0.73
1.05
5.61
0.28
0.15
0.14
0.11
19
20
21
22
23
24
25
26
27
28
(S)-CH2NH2
(S)-CH2OMe
(S)-CH2NHAc
11.36
5.57
9.69
(S)-CH2NHSO2Me
(S)-CO2Et
40% @ 33
18.0
lM
(S)-CH2CO2Me
(S)-CH2NMe2
(S)-CH2NHMe
(R)-Me
5.17
6.79
12.63
5.24
4% @ 30 lM
In conclusion we have identified a series of phenylpyrazoles
with high binding affinity to Bcl-2 and Bcl-xL. These small mole-
cules have a novel mode of binding and with the appropriate func-
tionality display selectivity for Bcl-2 over Bcl-xL. Access to a small
molecule that specifically blocks Bcl-2 function would be of value
(S)-Me
27% @ 30
lM
Table 3
Pyrazole modifications.
R2
R1
N
N
O
N
R3
M)
Compound
R1
R2
R3
Bcl-2 16mer IC50
(
l
Bcl-2 26mer IC50
(lM)
Bcl-xL 16mer IC50
(
lM)
Bcl-xL 26mer IC50
9% @ 30
(lM)
29
30
31
32
33
34
35
36
CONPh2
CONPh2
CONPh2
CONEt2
CON(CH2CF3)2
CON(nBu)2
CON(nBu)2
CON(nBu)2
Br
Cl
Cl
Br
Cl
H
CH2OH
CH2OH
CH2NH2
CH2OH
CH2OH
H
0.04
0.05
0.03
3.67
0.21
0.48
0.10
17.3
2.41
2.28
1.61
lM
29% @ 30
28% @ 30
14% @ 10
l
l
l
M
M
M
6% @ 10
2.01
l
M
13% @ 30
0.25
lM
4% @ 30
25.20
6.94
lM
0.23
0.10
0.02
Cl
Cl
CH2OH
CH2NH2
0.78
0.16
5.05

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 230–233
233
Table 4
Phenyl substituents.
O
R1
NBu2
N
N
O
N
R2
R3
Compound
R1
R2
R3
Bcl-2 16mer IC50
(l
M)
Bcl-2 26mer IC50
(l
M)
Bcl-xL 26mer IC50
7.99
(lM)
37
38
39
40
41
43
44
45
46
8
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
–NH2
–NHCOPh
0.09
0.81
0.04
0.04
0.250
0.02
0.01
0.70
5.66
0.27
0.13
3.15
0.33
0.13
0.60
0.51
0.38
0.03
0.65
7% @ 30
2.37
lM
–NHCOCH2Ph
–NHCOCH2OPh
–NHSO2CH2Ph
–OCH2COPh
–OCH2CO2H
NHCOCH2SPh
NHCOCH2OPh
–NH2
2.38
19% @ 10
3.24
lM
5.64
0.40
3.05
6.16
H
Cl
Cl
Cl
CH2NH2
CH2NH2
CH2NH2
42
47
–NHCOCH2OPh
NHCH2(4-pyridyl)
0.808
17% @ 30
lM
98, 3012; (b) 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.-
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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.-C.; Nimmer, P. M.; O’Connor, J. M.;
Oleksijew, A.; Petros, A. M.; Reed, J. C.; Shen, W.; Tahir, S. K.; Thompson, C.
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2007, 14, 1711.
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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.
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Figure 1. X-ray crystal structure of 31 (magenta) in Bcl-2/Bcl-xL. The BH3 binding
cleft of the protein is represented as a cyan Connolly surface, with the ‘second-site’
(Ile85) binding pocket on the right The 16-mer Bak peptide (PDB ID: 1BXL) shown
as a yellow helix is aligned to the structure. Bak residues involved in binding to the
hydrophobic cleft (l to r: i, i + 4, i + 7, and i + 11) are also shown in yellow.
9. Stephenson, E. F. M. Org. Syn. Coll. Vol. 1966, 3, 475.
10. Page, D.; Naismith, A.; Schmidt, R.; Coupal, M.; Labarre, M.; Gosselin, M.;
Bellemare, D.; Payza, K.; Brown, W. J. Med. Chem. 2001, 44, 2387.
11. Nose, A.; Kudo, T. Chem. Pharm. Bull. 1988, 36, 1529.
12. HTRF-LANCE assays detected interactions of Bcl-2 proteins expressed as
in revealing the individual roles of this family of anti-apoptotic
proteins, particularly as blockade of Bcl-xL has been associated
with an apoptosis-like response in platelets that is distinct from
platelet activation and results in enhanced clearance of platelets
in vivo by the reticuloendothelial system.16 Solution of the X-ray
crystal structure of chimaeric Bcl-2/Bcl-xL with a phenylpyrazole
ligand has identified key areas for further optimisation. Inhibitor
profiling in cell assays either by themselves or in combination with
cytotoxics will be reported elsewhere.17
GST-fusion proteins and a biotinylated 16- or 26-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.
13. Malhotra, S. K.; Johnson, F. Chem. Commun. 1968, 19, 1149.
14. Crystallisation and solution of structure performed by Sareum Ltd, 2 Iconix
Park, Pampisford, Cambridge, CB22 3EG, United Kingdom, http://
www.sareum.co.uk. Crystallographic data for the structure in this paper have
been deposited with the PDB.
15. Bruncko, M.; Oost, T. K.; Belli, B.; Ding, H.; Joseph, M. K.; Kunzer, A. R.;
Martineau, D.; McClellan, W. J.; Mitten, M. J.; Ng, S.-C.; Nimmer, P. M.;
Oltersdorf, T.; Park, C.-M.; Petros, A. M.; Shoemaker, A. R.; Song, X.; Wang, X.;
Wendt, M. D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med.
Chem. 2007, 50, 641.
16. Zhang, H.; Nimmer, P. M.; Tahir, S. K.; Chen, J.; Fryer, R. M.; Hahn, K. R.; Iciek, L.
A.; Morgan, S. J.; Nasarre, M. C.; Nelson, R.; Preusser, L. C.; Reinhart, G. A.;
Smith, M. L.; Rosenberg, S. H.; Elmore, S. W.; Tse, C. Cell Death Differ. 2007, 14,
943.
17. Payne, A.; Porter, J.; Watkins, C.; Edwards, C; Ford, D.; de Candole, B.;
Hutchinson, B.; Turner, J.; Lemmens, I.; Tavernier, J.; Davis, J.; Stubberfield, C.;
Abstracts of Papers, Molecular Targets and Cancer Therapeutics EORTC-AACR-
NCI, San Francisco, CA, October 22–26, 2007; B297.
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