Parallel medicinal chemistry approaches to selective HDAC1/HDAC2 inhibitor (SHI-1:2) optimization

Article abstract of DOI:10.1016/j.bmcl.2008.12.083

The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by

Full text of DOI:10.1016/j.bmcl.2008.12.083

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1168–1172
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Parallel medicinal chemistry approaches to selective HDAC1/HDAC2
inhibitor (SHI-1:2) optimization
a
a
a
a
Solomon D. Kattar a, , Laura M. Surdi , Anna Zabierek , Joey L. Methot , Richard E. Middleton ,
Bethany Hughes a, Alexander A. Szewczak a, William K. Dahlberg a, Astrid M. Kral b, Nicole Ozerova b,
Judith C. Fleming b, Hongmei Wang b, Paul Secrist b, Andreas Harsch a, Julie E. Hamill a, Jonathan C. Cruz c,
Candia M. Kenific c, Melissa Chenard c, Thomas A. Miller a, Scott C. Berk a, Paul Tempest a
*
a Department of Drug Design and Optimization, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Department of Cancer Biology and Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
c Department of Oncology Pharmacology, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The successful application of both solid and solution phase library synthesis, combined with tight inte-
gration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural ser-
ies of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An
initial lead from a small parallel library was found to be potent and selective in biochemical assays.
Advanced compounds were the culmination of iterative library design and possess excellent biochemical
and cellular potency, as well as acceptable PK and efficacy in animal models.
Received 3 November 2008
Revised 17 December 2008
Accepted 19 December 2008
Available online 25 December 2008
Keywords:
Histone deacetylases
Parallel synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
ZolinzaTM
Histone deacetylases (HDACs) are a family of enzymes that cat-
alyze the deacetylation of lysine residues located on the N-termi-
nal tails of histone proteins.1 HDACs are divided into four distinct
classes.2 The chosen targets, HDAC1 and HDAC2, are class 1 en-
zymes involved in chromatin-modifying activities, that are thought
to play a role in carcinogenesis.3,5–9 As shown inFigure 1, the struc-
tural characteristics of HDAC inhibitors contain three pharmaco-
phores: a surface recognition domain, a linker region, and metal
binding region.4,5,7,10–13
O
H
N
N
H
OH
O
Surface
Recognition
Linker
Metal
Binding
Figure 1. Common pharmacophore of HDAC inhibitors, as exemplified by ZolinzaTM
.
ZolinzaTM, is Merck’s first-in-class HDAC 1, 2, 3, and 6 inhibitor
for the treatment of cutaneous manifestations of T-cell lym-
phoma.5 Following the success of ZolinzaTM, efforts were initiated
to improve the efficacy and tolerability of this clinical agent. 16,17,21
The Parallel Medicinal Chemistry Group at MRL-Boston is
charged with assisting project teams using parallel/technology en-
abled chemistry wherever applicable in order to solve issues of po-
tency, selectivity, pharmacokinetics, etc. Towards this end,
members of the group are ‘embedded’ within a project team with
a mission of identifying areas where library synthesis is both appli-
cable and useful. Use of automated systems and databases allows
us to generate SAR quickly in order to push projects forward in a
useful and rapid manner. Embedded within the HDAC1/HDAC2
project team, we made small focused libraries to quickly generate
information in order to advance the project.
At the start of our efforts in the HDAC1/HDAC2 program, the
team’s goal was to identify new lead series, which would show im-
proved tolerability and selectivity over HDAC3 as compared to
ZolinzaTM.
Keeping the metal binding domain constant, we initially var-
ied both the surface recognition and linker domains in a library
format to identify novel active lead series and establish SAR for
these regions. Bi-functional linker scaffolds were coupled with a
diverse amine surface recognition set to generate 16 libraries
totaling approximately 500 compounds. Nine of these diverse
linker scaffolds exhibited an IC50 of <1
lM in the HDAC1 assay
(see Fig. 2).16
Due to its low molecular weight, chemical tractability, and
activity in the HDAC1 biochemical assay (see Table 1), we chose
* Corresponding author. Tel.: +1 617 992 2099; fax: +1 617 992 2043.
E-mail address: sam_kattar@merck.com (S.D. Kattar).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.083

S. D. Kattar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1168–1172
1169
O
O
OH
OH
N
H
O
n
O
O
OH
R1
OH
OH
N
H
H
N
N
H
N
H
H
N
R1
R1
O
O
O
H
N
R1
OH
O
N
HN
H
N
N
H
H
N
H
N
R1
O
R1
O
O
O
Scaffold 1
H
N
R1
H
N
R1
N
H
N
H
N
S
R1
H
NH
OH
O
OH
OH
O
O
O
Figure 2. Summary of various linkers used in the initial library synthesis.
Table 1
Table 2
HDAC1 inhibition from Library A
Aminophenol SAR
R1
O
OH
O
N
H
H
N
R1
R1-NH2
Cl
N
H
H
N
OH
R2
Compound
HDAC1 inhibition IC50 (nM)
Compound
R1
R2-NH2
HDAC1 inhibition
IC50 (nM)
HCT-116-96
h (nM)
1
348
6
Ph
Ph
Ph
Ph
Ph
Me
CI
972
n/a
O
2
3
4
477
7
112
n/a
Cl
Cl
1006
1620
8
32
1357
1825
1735
n/a
N
9
13
N
10
11
12
13
14
15
16
10
5
24,060
N
N
N
N
N
N
N
20,570
5032
2013
5223
1568
7964
n/a
to follow-up on Scaffold 1.18 Our first follow-up library of 48 com-
pounds around Scaffold 1 was synthesized in two steps using com-
mercially available methyl 4-formylbenzoate as the linker scaffold
(see Fig. 3).
SAR for this initial series indicated that benzyl amines were fa-
vored over their phenethyl or aniline counterparts. It was also
shown that aromatically substituted amines were more potent
than allylic building blocks. See Table 2.
F
n/a
Br
n/a
H
n/a
In addition to gaining potency, replacement of the hydroxamate
moiety became a priority. Based on an HTS hit, 2-aminophenols
were chosen as a potential replacement for the hydroxamate
groups used in the previous library (see Fig. 4). For our next itera-
tion, we devised a rapid solid phase synthetic route to incorporate
this new Zn binding motif.
OMe
n/a
compounds 22–26 (see Table 4). Based on the hypothesis that
the phenol group limited cellular activity, our next library replaced
the hydroxyl group of the aminophenol moiety with the primary
Preliminary SAR suggested that having an aromatic group at R1
is optimal for binding (this theory was further substantiated by
O
R1
H
R1
H
R1NH2
a
N
N
H
H
OH
N
O
OH
b
O
O
O
Figure 3. Synthetic scheme for Library A derived from Scaffold 1. (a) PS-NaBH(OAc)3, DMF; (b) NH2OH (aq), DMF.

1170
S. D. Kattar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1168–1172
O
Cl
Cl
Br
O2N
H2N
a
b
c
O
O
NH2
N
O
O
N
H
H
N
N
H
OH
d
Cl
O
N
e
Figure 4. Synthetic scheme of 2-aminophenol derived libraries. (a) Cs2CO3, DMF, microwave at 40 °C; (b) SnCl2, DMF; (c) DIPEA, DMAP, DCM; (d) NaI, proton sponge, DMF; (e)
1:1 TFA:DCM.
H
O
N
Cl
O
O
Cl
H2N
Cl
N
O
O
O
HN
H
O
N
H
N
O
HN
O
HN
a
b
C
O
O
O
N
H
d
N
N
NH2
H
21
Figure 5. Synthetic scheme of libraries derived from diamino-based zinc binders. (a) NaBH(OAc)3, 5% AcOH/DCE; (b) DIPEA, DCM; (c) NaI, proton sponge, DMF; (d) TFA:DCM.
Table 3
Table 4
Biphenyl diamine SAR
Biphenyl diamine SAR
R1
R1
O
O
N
H
N
H
R2
NH2
R2
NH2
Compound R1
R2-NH2
HDAC1
HDAC3
HCT-
hERG
Compound
R1
R2-NH2
HDAC1
inhibition
IC50 (nM)
HDAC3
inhibition
IC50 (nM)
HCT-116-72
ha (nM)
inhibition inhibition 116-72 h activity
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
N
H
N
S
NH2
22
4
8
9617
8706
82
73
1597
1492
17
18
Ph
Ph
10
10
6390
5384
1745
3609
N
N
H
N
S
N
H
N
23
N
N
19
20
Ph
Ph
13
33
19,320
6928
670
309
NH2
N
N
N
H
N
24
16
47
17,390
5727
470
183
167
HN
F
H
N
N
21
Ph
8
16,480
118
F
H
N
25
1067
a
HCT-116-96 cellular assay was changed to a 72 h assay.
F
N
H
N
amine (Fig. 5).14,15 It was only after transitioning to this group that
we began to see smaller shifts in the biochemical to cell assay po-
tency (Table 3).19
26
13
4803
276
237

S. D. Kattar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1168–1172
1171
SAR in the R2 portion of the scaffold was consistent from the
aminophenol to the diamine based libraries (see Table 3). Amino
spirocyclic building blocks like those in compounds 20–21 were
identified as potent inhibitors of HDAC1. Inhibitor 21 showed
excellent enzymatic and cellular potency, with approximately
2000-fold selectivity against HDAC3 (Table 3). Pharmacokinetic
properties of compound 21 in rat plasma include excellent bio-
availability (70%), a half life of 8 h, but a moderately high clearance
and volume of distribution of 45 mL/min/kg and 22 L/kg,
respectively.
Introduction of various other Zn binding elements such as 4-(2-
thienyl)benzene-1,2-diamine and 4-(3-thienyl)benzene-1,2-dia-
mine) improved selectivity against HDAC3, however, these com-
pounds exhibited IC50’s of 1.6 lM and 1.5 lM, respectively, in the
biochemical hERG binding assays, indicating the potential for car-
Incorporation of terephthalamide linkers (Table 5) proved to be
a successful way of mitigating hERG liabilities. A library of 26
members was synthesized using the synthetic route outlined in
Figure 6.
Enzymatic and cellular potencies, as well as the low serum shift
of 3 and selectivity were maintained in this set of compounds.
Compound 30 was identified as a potent and selective inhibitor
of HDAC1. Ancillary pharmacology showed it to be inactive, at
<10 l
M, against hERG and Ca+ ion channels as well as the Cyto-
chrome P450 isozymes (CYP’s). Pharmacokinetic studies in rat
and dog revealed good drug-like properties, but with low oral bio-
availability (Table 6).
In spite of having low bioavailability values of 4% and 17% in
dog and rat, respectively, the molecule was advanced into
in vivo pharmacodynamic (PD) studies. Compound 30 proved
efficacious, as the 25 mg/kg dose IP gave a threefold increase
in Ac H2B/Total H2B in the acute PD HCT-116 xenograft model
study.20
diovascular toxicity.
Figure 7 shows how linear library synthesis with embedded par-
allel chemists rapidly improved chemical properties and aided in
the development of an HDAC1 inhibitor.
Table 5
Terephthalamide SAR
Through tight integration with the HDAC1/HDAC2 project
chemists and the synthesis of parallel libraries,
a Parallel
R1
Medicinal Chemistry group was able to identify and explore
a novel class of HDAC-selective inhibitors. Compound 30 pos-
sessed excellent biochemical and cellular potency, acceptable
PK profile and off-target activity profiles that exhibit selectivity
distinct from that of ZolinzaTM, which has been shown to be a
broad spectrum HDAC inhibitor; equipotent against HDAC’s 1,
2, 3 and 6. Compound 30 also proved to be efficacious in
the acute pharmacodynamic (PD) HCT-116 xenograft model
study.
O
NH
N
H
N
NH2
O
Compound
R1
HDAC1
inhibition
IC50 (nM)
HDAC3
inhibition
IC50 (nM)
HCT-116-72 h
IC50 (nM)
hERG activity
IC50 (nM)
S
27
28
29
30
11
11
12
8
4330
70
25,090
—
Table 6
>50,000
3202
290
138
103
F
Phamacokinetic properties of Compound 30
Rat
2.0
26
14
7.0
4.0
Dog
F
IV dose (mg/kg)
Cl (ml/min/kg)
Vdss (L/kg)
t1/2 (h)
PO dose (mg/kg)
0.5
8.2
5.1
8.5
1.0
0.17
4
—
5102
12,390
AUCN (
l
M h kg/mg)
0.25
17
F (%)
H
N
O
H2N
N
O
O
O
HN
O
N
H
O
OH
O
O
O
HN
O
O
a
b
a
O
O
O
N
H
N
H
N
N
N
NH2
H
c
O
HN
N
O
O
O
O
O
30
Figure 6. Synthetic scheme of terephthalamide based library. (a) PS-CDI, HOBt, DMF; (b) NaOH, MeOH; (c) 1:1 TFA:DCM.

1172
S. D. Kattar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1168–1172
ZolinzaTM
H
O
Medicinal Chemistry Optimization Starting Point
Approved by the FDA for the treatment of CTCL
N
N
H
OH
O
Library Synthesis
H
O
OH
N
H
Most Potent Compound: 348nM
Number of Compounds: 26
Liabilities: Biochemical Potency, Selectivity
N
R1
Library Synthesis
O
O
N
H
H
Most Potent Compound: 10nM
Number of Compounds: 81
Liabilities: Selectivity, Cell Potency
N
OH
R1
R1
Library Synthesis
Most Potent Compound: 7 nM
Number of Compounds: 12
Liabilities: hERG
N
H
H
N
NH2
R1
R2
Library Synthesis
HN
O
Most Potent Compound: 7 nM
Number of Compounds: 38
Liabilities: hERG, PK
N
H
n
NH2
N
Library Synthesis
HN
Most Potent Compound: 6 nM
Number of Compounds: 26
Liabilities: PK
O
N
H
n
NH2
N
O
Figure 7. Schematic of the progression of the parallel medicinal chemistry library series.
(c) Acharya, M.; Sparreboom, A.; Sausville, E. A.; Conley, B. A.; Doroshow, J. H.;
References and notes
Venitz, J.; Figg, W. D. Cancer Chemother. Pharmacol. 2006, 57, 275.
15. Moradei, O.; Leit, S.; Zhou, N.; Frechette, S.; Paquin, I.; Raeppel, S.; Gaudette, F.;
Bouchain, G.; Woo, S. H.; Vaisburg, A.; Fournel, M.; Kalita, A.; Lu, A.; Trachy-
Bourget, M.-C.; Yan, P. T.; Liu, J.; Li, Z.; Rahil, J.; MacLeod, A. R.; Besterman, J. M.;
Delorme, D. Bioorg. Med. Chem. Lett. 2006, 16(.), 4048.
16. Siliphaivanh, P.; Harrington, P.; Witter, D.; Otte, K.; Tempest, P.; Kattar, S.; Kral,
A.; Fleming, J.; Deshmukh, S.; Harsch, A.; Secrist, P.; Miller, T. Bioorg. Med.
Chem. Lett. 2007, 17, 4619.
17. Hamblett, C. L.; Methot, J. L.; Mampreian, D. M.; Sloman, D. L.; Stanton, M. G.;
Kral, A. M.; Fleming, J. C.; Cruz, J. C.; Chenard, M.; Ozerova, N.; Hitz, A. M.;
Wang, H.; Deshmukh, S. V.; Nazef, N.; Harsch, A.; Hughes, B.; Dahlberg, W. K.;
Szewczak, A. A.; Middleton, R. E.; Mosley, R. T.; Secrist, J. P.; Miller, T. A. Bioorg.
Med. Chem. Lett. 2007, 17, 5300.
1. Verdin, E.; Dequiedt, F.; Kasler, H. Trends Genet. 2003, 19, 286.
2. Yang, X.; Seto, E. Nat. Rev. Mol. Cell Biol. 2008, 9, 206.
3. Hake, S.; Allis, C. Br. J. Cancer 2004, 90, 761.
4. Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46, 5097.
5. Grant, S.; Easley, C.; Kirkpatrick, P. Nat. Rev. Drug Discov. 2007, 6, 21.
6. Huang, L. J. Cell. Physiol. 2006, 209, 611.
7. Liu, T.; Kuljaca, S.; Tee, A.; Marshall, G. M. Cancer Treat. Rev. 2006, 32, 157.
8. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Discov. 2006, 5,
769.
9. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
10. Richon, V. M.; Webb, Y.; Merger, R.; Sheppard, T.; Jursic, B.; Ngo, L.; Civoli, F.;
Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. 1996, 93, 5705.
11. Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.; Brunetti, M.; Renzoni, D.;
Chakravarty, P.; Paolini, C.; De Francesco, R.; Gallinari, P.; Steinkuhler, C.; Di
Marco, S. Proc. Natl. Acad. Sci. USA 2004, 101, 15064.
12. Yoshida, M.; Horinouchi, S.; Beppu, T. Bioessays 1995, 17, 423.
13. Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9, 3.
14. (a) Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.; Tsuchiya, K.; Ando, T.;
Suzuki, T.; Tsuruo, T.; Nakanishi, O. Proc. Natl. Acad. Sci. 1999, 96, 4592; (b)
Suzuki, T.; Mariko, Y.; Tsuchiya, K.; Fukazawa, N. J. Med. Chem. 1999, 42, 3001;
18. For enzyme assay procedure, please see Ref. 18.
19. For cell viability assay procedure, please see Ref. 18.
20.
A
solution of the inhibitor in 10%DMSO/45%PEG-400/45% water was
administered once-daily ip for up to 21 days.
21. Methot, J. L.; Hamblett, C. L.; Mampreian, D. M.; Jung, J.; Harsch, A.; Szewczak,
A. A.; Dahlberg, W. K.; Middleton, R. E.; Hughes, B.; Fleming, J. C.; Wang, H.;
Kral, K. M.; Ozerova, N.; Cruz, J. C.; Haines, B.; Chenard, M.; Kenific, C. M.;
Secrist, J. P.; Miller, T. A. Bioorg. Med. Chem. Lett. 2008, 18, 6104.