Diversity-oriented synthesis of a cytisine-inspired pyridone library leading to the discovery of novel inhibitors of Bcl-2

Article abstract of DOI:10.1016/j.bmcl.2009.03.037

Four enantiopure cytisine-inspired scaffolds can be accessed via a versatile pyrrolidine template derived from a stereocontrolled [3+2] azomethine ylide-alkene cycloaddition. Differential ester protection allows for the selective formation of either a bridged bicyclic or tricyclic scaffold via pyridone cyclization. Solid-phase diversification of the pyridone scaffolds yielded a diverse library of 15,000 compounds enabling the discovery of a novel class of Bcl-2 inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.03.037

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2500–2503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diversity-oriented synthesis of a cytisine-inspired pyridone library leading
to the discovery of novel inhibitors of Bcl-2
à
*
Lisa A. Marcaurelle , Charles Johannes , Daniel Yohannes , Bonnie P. Tillotson, David Mann
Infinity Pharmaceuticals, Inc., 780 Memorial Drive, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Four enantiopure cytisine-inspired scaffolds can be accessed via a versatile pyrrolidine template derived
from a stereocontrolled [3+2] azomethine ylide–alkene cycloaddition. Differential ester protection allows
for the selective formation of either a bridged bicyclic or tricyclic scaffold via pyridone cyclization. Solid-
phase diversification of the pyridone scaffolds yielded a diverse library of 15,000 compounds enabling the
discovery of a novel class of Bcl-2 inhibitors.
Received 10 February 2009
Revised 10 March 2009
Accepted 10 March 2009
Available online 17 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Diversity-oriented synthesis
Pyridone
Natural product
Solid-phase
Protein–protein interaction
Apoptosis
H
N
Bn
N
In light of their structural complexity and inherent biological
activity,1 natural products can serve as useful starting points for
diversity-oriented synthesis (DOS).2 Indeed, screening of natural
product-based DOS libraries has led to the discovery of numerous
small molecules possessing biological activity unrelated to that of
the parent compound.3 In designing a DOS library for the purpose
of drug discovery, we drew inspiration from the natural product
cytisine (Fig. 1), which contains a fused pyridone ring within a
bridged bicyclic scaffold.4 Cytisine is a well-known nicotine ago-
nist, which led in part to the development of the smoking cessation
agent ChantixÒ.5 Interestingly, cytisine analogs have also been
found to possess anti-phosphatase activity.6
Previous work in the total synthesis of cytisine and related ana-
logs made use of an efficient pyridone cyclization step involving
the activation of a primary alcohol and subsequent reaction with
2-methoxypyridine.7 We chose to exploit this reaction for the syn-
thesis of a diverse compound library to be screened against multi-
ple, non-CNS related, biological targets. Below we will highlight
the strategy we used to generate an unbiased library of pyridone
compounds, which led to the discovery of several low micromolar
inhibitors of the anti-apoptotic protein Bcl-2.8
ref 7
N
MsO
N
O
OMe
(-)-cytisine
Figure 1. Retrosynthesis of the natural product (À)-cytisine.
For the design of our cytisine-based scaffolds we chose to make
use of a highly versatile pyrrolidine intermediate (1, Fig. 2) substi-
tuted with a 2-methoxypyridine unit adjacent to the pyrrolidine
nitrogen. We envisioned that both enantiomers of this densely
functionalized template could be accessed via a stereocontrolled
[3+2] cycloaddition9 between a glycine-derived imine (2) and a
chiral E-a,b-unsaturated ester (3). Cyclization to form the pyri-
done-ring could be achieved by activation of a primary alcohol
masked as an ester to form the methylene bridge. Orthogonal pro-
tection of the two ester functionalities, as an allyl or methyl ester,
would allow ring closure to occur via alternate pathways (A and B)
providing two distinct skeletons (I and II). We reasoned that the
cytisine-inspired scaffolds would serve as attractive starting points
for library synthesis given their low molecular weight (ꢀ264) and
opportunities for incorporating appendage diversity (ester and
amine).
* Corresponding author at present address: Broad Institute, Cambridge, MA
02142, USA. Tel.: +1 617 324 4373; fax: +1 617 324 9601.
E-mail address: lisa@broad.mit.edu (L.A. Marcaurelle).
As shown in Scheme 1, the
allyl (3b) esters were readily prepared starting from
a
,b-unsaturated methyl (3a) and
Present address: Forma Therapeutics, Inc., Singapore.
Present address: Targacept, Inc., Winston-Salem, NC 27101, United States.
à
D-glyceralde-
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.037

L. A. Marcaurelle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2500–2503
2501
aldehyde and the glycine allyl11 or methyl ester. Reaction of the
glycine-derived azomethine ylides with esters 3a and 3b promoted
by AgOAc/DBU yielded cycloadducts 1a and 1b in high yield with
excellent diastereoselectivity (>95:5).12
O
OP1
O
*R
O
*R
OP1
N
*R
N
B
A
O
O
N
H
N
N
H
N
H
P2O
-P1
P2O
-P2
Following protection of pyrrolidines 1a and 1b as Fmoc-carba-
mates 7 and 8, the allyl ester was removed via treatment with
1
O
OMe
Pd(PPh3)4 using N-methylaniline as the p
-allyl scavenger.13 Initial
II
I
attempts to reduce the resulting acid with BH3–DMS were unsuc-
cessful, as were attempts to reduce various mixed anhydrides.
Optimal conditions for this transformation were found to be
BOP-activation of the acid followed by reduction of the HOBt-ester
with sodium borohydride.14,15
[3+2]
OP2
O
N
+
OP1
N
OMe
*R
O
2
3
Having optimized the reduction of the acid, the key cyclization
reaction to form the pyridone ring could be carried out. This was
achieved via activation of the primary alcohol with mesyl chloride
followed by heating in toluene to afford the bridged bicyclic scaf-
fold (9) and the tricyclic core (10). Isolation of the intermediate
mesylate was not required for the success of this reaction thus pro-
viding the desired pyridone compounds in ‘one-pot’.
Figure 2. Cyclization strategy to form pyridone scaffolds I and II.
hyde acetonide (4) via a Horner–Emmons reaction with commer-
cially available phosphonates 5 and 6.10 Meanwhile, imines 2a
and 2b were prepared starting from 2-methoxypyridine-6-carbox-
O
O
O
O
O
CO2Me
O
HO
O
P
OEt
O
CO2Me
CO2Me
N
O
g
b
d-f
OMe
a
EtO
N
R
N
OMe
N
R
N
N
R
3a
O
5
O
OMe
O
O
1a: R = H
N
CO2R
O
O
c
R = Fmoc
R = Fmoc
N
7: R = Fmoc
2a: R = allyl
2b: R = Me
CHO
9
11
OMe
4
O
O
O
O
O
O
O
P
OEt
O
HO
MeO2C
O
O
g
O
N
d-f
b
O
N
N
O
EtO
N
Fmoc
MeO2C
MeO2C
N
N
Fmoc
O
3b
6
Fmoc
OMe
O
1b: R = H
10
12
c
8: R = Fmoc
Scheme 1. Reagents and conditions: (a) NaH, THF, 5 or 6; (b) 3a/2a or 3b/2b, AgOAc, DBU, toluene, À78 °C, 86–90%; (c) FmocCl, DIEA DCM, 81–90%; (d) Pd(PPh3)4, PhMeNH,
THF, 82–99%; (e) BOP, NaBH4, THF, 70–75%; (f) (i) MsCl, Et3N; (ii) toluene, reflux, 70–74%; (g) (i) HCl, THF, H2O; (ii) NaIO4; (iii) NaBH4, 76–78%.
i-Pr
i-Pr
R1
NH
i-Pr
O
Si
O
i-Pr
CO2Me
O
HO
Si
O
HO
O
O
N
c, d, h
a, b
a, b
c, d
i-Pr
i-Pr
N
Si
N
N
H
N
N
R2
N
11, ent-11
12, ent-12
OMe
PS-SynPhaseTM Lanterns
MeO2C
N
H
R1 NH
R2
d-f, h
O
O
14
=
d, g, h
13
d-f, h
HO
d, g, h
CO2H
O
O
S
O
R
O
HO
R2
O
OR1
H
N
HO
HO
N
R
N
O
HO2C
R1O
N
N
R2
R2
R2
N
OH
R
N
HN
R
*
R2
R
16: R1 = H
18: R1 = CONHR
N
20
O
S
15: R1 = H
O
17: R1 = CONHR
R
19
N
R
Scheme 2. Reagents and conditions: (a) (i) TfOH (6.0 equiv), DCM; (ii) 11, ent-11, 12 or ent-12 (1.2 equiv), 2,6-lutidine (9.0 equiv), DCM, rt, 24 h; (b) 20% piperidine, DMF, rt,
30 min; (c) AlMe3 (15 equiv), amine (20 equiv), toluene, 40 °C, 3 d; (d) RSO2Cl (20 equiv), 2,6-lutidine (25 equiv) DCM, rt, 24 h; RNCO (15 equiv), DCM, rt, 24 h; RCOCl
(10 equiv), 2,6-lutidine (15 equiv), DCM, rt, 24 h; epoxide (20 equiv), LiClO4 (20 equiv), i-propanol, 70 °C, 1–3 d; RCHO (20 equiv), Na(OAc)3BH (22 equiv), 2% AcOH, DMF, rt,
3 d; FmocNCS (5 equiv), rt, 24 h then 20% piperidine, DMF, rt, 30 min then RCOCH2Br (10 equiv), rt, 24 h; (e) LiBH4 (5 equiv), THF, rt, 1–2 d; (f) RNCO, toluene, 40 °C, 24 h; (g)
LiOH (10 equiv), THF, MeOH, H2O, rt, 24 h; (h) (i) 15% HF/pyridine, THF, rt, 2 h; (ii) TMSOMe, 10 min.

2502
L. A. Marcaurelle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2500–2503
Table 2
In order to prepare for solid-phase library synthesis, which re-
quired a primary alcohol for loading onto solid-support,16 the di-
methyl acetal was hydrolyzed, and the resulting diol was cleaved
oxidatively with sodium periodate. Reduction of the resulting alde-
hyde could be achieved by controlled reaction with sodium boro-
hydride to provide cores 11 and 12 and their corresponding
enantiomers (ent-11 and ent-12).
O
HO
R1
N
N
O
14
Having established a practical large-scale (>75 g) synthesis of
the pyridone scaffolds, the library production was undertaken.
Loading of the four scaffolds onto silicon-functionalized Lan-
terns16b was carried out via activation with TfOH to provide an
average loading level of 15 mmol of compound per Lantern
(Scheme 2). Following Fmoc removal the ester functionality was
converted into a variety of amides and pyrrolidine capping was
achieved by reaction with sulfonyl chlorides, acid chlorides, epox-
ides, isocyanates and aldehydes. 2-Aminothiazoles were formed by
capping with FmocNCS, followed by Fmoc-removal and treatment
with an assortment of bromoketones.17 Cleavage from the Lantern
was achieved via reaction with 15% HF/pyridine to afford amides
13 and 14. The ester functionality was also reduced to provide
the alcohols 15 and 16 and then reacted with isocyanates to afford
carbamates 17 and 18, while hydrolysis of the ester with LiOH pro-
vided acids 19 and 20. In total ꢀ15,000 cytisine-inspired com-
pounds were produced with purities exceeding 75% for over 75%
of the library.18
Compounds produced in the pyridone library were screened for
binding affinity against Bcl-2 and Bcl-xL in traditional solution-
based competition binding assays formatted for HTS analysis.
Competition against a fluorescently tagged BH3 peptide was mea-
sured in the presence of an inhibitor, and compounds arrayed in a
384-well format were initially screened at a single fixed concentra-
tion of approximately 10 mM. Library hits demonstrating inhibi-
tion with significant Z factors (three standard deviations above
assay error) were subsequently assayed in a multiple point dilution
series to generate Ki values in order to measure the affinity of the
compound for Bcl-2 and Bcl-xL.19 The hit rate from this library
screen was 1.1% and 0.2% against Bcl-2 and Bcl-xL respectively,
with the best inhibitors having single digit micromolar activities.
A survey of the current literature in the area of published Bcl-2
small molecule ligands suggests that the library hits identified in
this screen represent a significant accomplishment as the majority
of the reported ligands are in fact weaker in affinity that those
identified from this screen.20,21
Compound
R1
Bcl-2 Ki (
l
M)
Bcl-xL Ki (lM)
H
N
14a
4.1
>100
N
N
H
N
14b
14c
5.4
7.8
>100
>100
H
N
PhO
H
N
14d
8.4
>100
The most potent compounds derived from the bridged bicyclic
pyridone scaffold (11) were those containing a diamine at R1 and
a chloro-substituted diphenyl 2-aminothiazole at R2 (13, Table 1).
Both enantiomers in this series were equally active against Bcl-2,
perhaps indicative of non-specific binding. They also displayed
inhibitory activity against Bcl-xL except for compounds 13d and
ent-13d, which lacked the diamine at R1.
The most active compounds derived from the tricyclic pyridone
core (12) were the products of reductive alkylation and Weinreb
amidation (14). For one enantiomeric series there was a distinct
preference at R2 for cyclohexyl carboxaldehyde in combination
with primary amines containing a hydrophobic aromatic ring at
R1 (Table 2). For the enantiomeric series (ent-14) 3,4-dichloroben-
zyaldehyde as well as 4-phenoxybenzaldehyde were preferred at
R2 in combination with benzyl or phenyl substituted piperidines
at R1 (Table 3). This difference in activity for the two enantiomeric
series suggests more specific binding as compared to the 2-amino-
Table 3
O
Table 1
HO
R1
N
O
R1
HO
N
R2
Cl
=
O
S
R2
N
N
R2
ent-14
N
Compound R1
R2
Bcl-2 Ki
M)
Bcl-xL Ki
(lM)
O
(
l
13
R1
Me
Bcl-2 Ki (
lM)
Bcl-xL Ki (lM)
Cl
Compound
ent-14e
1.2
2.2
>100
>100
N
H
N
Cl
Cl
F
N
13a
ent-13a
2.0
1.3
5.7
6.6
Me
ent-14f
H
N
N
Cl
13b
ent-13b
2.5
1.5
6.0
11.0
N
ent-14g
5.2
6.8
>100
>100
N
H
N
13c
ent-13c
3.0
1.6
5.7
7.8
OPh
OPh
N
F
H
ent-14h
N
13d
ent-13d
4.3
14.0
>100
>100
MeO
N

L. A. Marcaurelle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2500–2503
2503
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series were selective for Bcl-2 over Bcl-xL.
Given the difficulty in designing inhibitors of protein–protein
interactions, the identification of low micromolar inhibitors di-
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from L-glyceraldehyde.
13. N-Methylaniline was chosen as the (-allyl scavenger given the sensitivity of
Fmoc to base: Ciommer, M.; Kunz, H. Synlett 1991, 593.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.037.
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the undesired dimethyl amide. Similar problems have been reported for
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