Sulfamides as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.081

The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted in potent HDAC1 and HDAC6 inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.11.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 336–340
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sulfamides as novel histone deacetylase inhibitors
a
a
a
a
a
Amal Wahhab a, , David Smil , Alain Ajamian , Martin Allan , Yves Chantigny , Eric Therrien , Natalie
Nguyen a, Sukhdev Manku a, Silvana Leit a, Jubrail Rahil b, Andrea J. Petschner b, Ai-Hua Lu b,
Alina Nicolescu b, Sylvain Lefebvre c, Samuel Montcalm c, Marielle Fournel c, Theresa P. Yan c, Zuomei Li c,
Jeffrey M. Besterman c, Robert Déziel a
*
a Department of Medicinal Chemistry, MethylGene Inc., 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
b Department of Lead Discovery, MethylGene Inc., 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
c Department of Molecular Biology, MethylGene Inc., 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of
these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-
chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted
in potent HDAC1 and HDAC6 inhibitors.
Received 4 November 2008
Revised 20 November 2008
Accepted 21 November 2008
Available online 27 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Sulfamide
HDAC inhibitors
HDAC1
HDAC6
Histone deacetylases (HDACs) are a family of 18 enzymes that
play an important role in the regulation of gene expression, cell
and hydroxyl groups of the hydroxamic acid chelate the zinc ion
in the active site in a bidentate fashion.
growth, and proliferation by regulating the deacetylation of
e
-N-
SAHA3a is reported to be a pan-HDACi while MGCD0103, an o-
aminobenzanilide currently in Phase I/II cancer clinical trials, is
an isotype-selective inhibitor.4f
acetyl groups on the -lysine residues at the N-terminal tails of core
L
histones, tubulin, and other proteins.1a These enzymes are divided
into two categories: zinc-dependent (HDAC1-11) and NAD+-
dependent enzymes (known as sirtuins, Sirt1-7, or HDAC class
III). The zinc-dependent enzymes are divided into class I (HDAC1,
2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and
10), and class IV (HDAC11), which exhibits properties of both class
I and class II HDACs.1b,c
The nature of the ZBG seems to confer both the activity and
selectivity of these inhibitors. Search for ZBGs that could serve as
a suitable pharmacophore in the design of novel HDACis is the sub-
ject of our current research. In this letter, we detail the identifica-
tion of a new class of HDACis bearing the sulfamide moiety as the
ZBG. Sulfamides are well-documented inhibitors of carbonic anhy-
drase, among other zinc-dependent enzymes.7a–c
To test our hypothesis that the sulfamide moiety could act as an
efficient ZBG for HDACis, we fixed the ‘cap region’ using biphenyla-
mide and varied both the sulfamide parent structure and the
length of the linker.
Sulfamides 4a–c were prepared according to Scheme 1 starting
from the Boc-protected amino acids utilizing standard coupling
conditions. The one-step procedure for the synthesis of sulfamide
4a from amine 3a utilizing sulfuric diamide8 resulted in low yields
and required high temperatures. We therefore opted for the two-
step procedure, making the benzyl carbamates9a,b 4b and 4c, fol-
lowed by hydrogenation to furnish sulfamides 5a and 5b. The reac-
tion of 3b with ClSO2NCO and t-BuOH gave the t-butyl carbamate
intermediate which was methylated with MeI to produce 6. Depro-
tection with TFA furnished the mono-methylated sulfamide 7a.
The majority of the known HDAC inhibitors (HDACis) are
hydroxamic acids.2a,2b SAHA (ZolinzaÒ, Vorinostat) was approved
in 2006 to treat cutaneous T-cell lymphoma (CTCL),3a and a num-
ber of other HDACis are in different stages of clinical develop-
ment.3b–d The quest for HDACis resulted in a number of diverse
structures,4a,b such as aliphatic acids,4c hydroxamic acids,2a,b,4d,e
o-aminoanilides,4f cyclic peptides,4g electrophilic ketones,4h and
thiols4i–k (Fig. 1). These structures all share a common pharmaco-
phore composed of a zinc-binding group (ZBG), a linker (scaffold),
and a surface recognition domain (cap).5 The crystal structures of
both the histone deacetylase-like protein (HDLP) complexed with
SAHA and TSA6a, and that of HDAC86b,c suggest that the carbonyl
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.081

A. Wahhab et al. / Bioorg. Med. Chem. Lett. 19 (2009) 336–340
337
O
O
OH
H
N
OH
N
H
O
valproic acid
SAHA (Zolinza(R),Vorinostat)
N
O
H
N
NH
HO
N
N
H
NH2
H
N
N
HN
O
LBH-589
MGCD0103
O
O
S
O
O
N
H
H
N
N
NH
N
N
N
H
CF3
6
OMe
O
Trifluoromethylketone
O
O
O
Apicidin
SH
6
Ph
N
H
thiol
Figure 1. HDAC inhibitors.
O
O
O
H
N
H
a,b
c
NHBoc
n
N
NH2
n
HO
or
S
R
Ph
N
H
Ph
N
n
d
H
O
O
n=4, 5, or 6
3a, n=4
3b, n=5
3c, n=6
h
4a, n=4, R= H
4b, n=5, R= CO2Bn
4c, n=6, R= CO2Bn
e
f
O
H
N
NMe2
Ph
N
H
S
O
H
5
O
O
N
NH2
O
R1
N
7b
S
Ph
N
H
H
N
n
O
O
Ph
N
H
S
O
R2
5
5a, n=5
5b, n=6
O
i
6
g
R1= Me, R2= CO2t-Bu
O
8
O
H
N
H
N
H
N
H
N
O
S
Ph
N
H
Ph
N
H
S
Me
5
5
O
O
O
O
7a
Scheme 1. Reagents and conditions: (a) 3-phenylaniline, BOP, Et3N, DMF; (b) TFA, DCM, or 4 N HCl, dioxane; (c) 3a, H2NSO2NH2, Et3N, toluene, 120 °C; (d) 3b or 3c, ClSO2NCO,
benzyl alcohol, DCM, Et3N; (e) H2/10%Pd–C, MeOH; (f) i—3b, ClSO2NCO, t-BuOH, DCM, Et3N, ii—MeI, DBU, CH3CN, 0 °C; (g) TFA, DCM; (h) 3b, ClSO2NMe2, Et3N, THF, rt, 18 h;
(i) 5a, Ac2O, DBU, DMF.
O
O
O
Compound 10 (Scheme 2), was obtained starting from 6-meth-
oxy-6-oxohexanoic acid by an amide coupling reaction with 3-
phenylaniline followed by reduction of the intermediate ester
(not shown in Scheme 2) with LiAlH4 to furnish alcohol 9. Oxida-
tion of 9 with Dess–Martin periodinane to form the corresponding
aldehyde (not shown in Scheme 2) followed by reductive amina-
tion with methylamine yielded the N-methylamino-intermediate
which was then converted into sulfamide 10 using sulfuric dia-
mide. To obtain sulfamate 11, alcohol 9 was reacted with ClSO2N-
CO and formic acid in acetonitrile.10
a, b
OH
Ph
N
H
HO
Ph
O
2
3
9
f
c, d, e
O
O
O
NH2
N
NH2
N
H
S
S
Ph
N
H
5
5
O
O
O
O
11
10
Scheme 2. Reagents and conditions: (a) 3-phenylaniline, BOP, Et3N, DMF; (b)
LiAlH4, THF; (c) Dess–Martin periodinane, DCM; (d) MeNH2, NaBH4, MeOH; (e)
H2NSO2NH2, Et3N, toluene, 120 °C; (f) ClSO2NCO/formic acid/CH3CN.
Sulfamoyl 12 was obtained by the reaction of 7-ethoxy-7-oxo-
heptanoic acid with 3-phenylaniline followed by hydrolysis and
standard CDI coupling with sulfuric diamide (Scheme 3).
Analogs 13a–f with diverse capping groups were prepared
according to Scheme 4.
The dimethylated sulfamide 7b was obtained from the reaction of
3b with a preformed solution of ClSO2NMe2 (Scheme 1). Sulfamoyl
8 was obtained by the reaction of 4b with acetic anhydride and
DBU in DMF.
The synthesized inhibitors 4a, 5a, 5b, 7a, 7b, 8, 10, 11, and
1211a were screened against a panel of recombinant human HDACs

338
A. Wahhab et al. / Bioorg. Med. Chem. Lett. 19 (2009) 336–340
in Table 1, while the results for the other HDACs were omitted
for clarity.
Unlike hydroxamic acid 1,11c which is a low-nanomolar inhibi-
O
O
O
H
N
a, b, c
Et
NH2
HO
O
Ph
N
H
S
3
4
O
O
O
tor of both HDAC1 and HDAC6, or o-aminobenzamide 2, a sub-
micromolar inhibitor of HDAC1,11dnone of the compounds 4a, 5a,
5b, 7a, 7b, 8, 10, 11, or 12 showed activity against HDAC1.11e
The same compounds were also inactive against HDAC6, with the
exception of sulfamide 5a, which showed a low micromolar activ-
ity. Similar to other HDACis, the inhibitory activity of the sulfamide
‘ZBG’ was directly affected by the length of the linker tying it to the
capping region. Optimal activity was observed with the 5-methy-
lene linker, 5a, while 4a and 5b were devoid of activity. In addition,
methyl or acetyl substitution at the proximal or distal nitrogen of
sulfamide 5a was deleterious to the activity (7a, 7b, 8, 10, and
12), as was the replacement of the proximal sulfamide nitrogen
by an oxygen (compound 11). To study the SAR of the capping re-
gion, compounds 13a–f were synthesized using standard coupling
methodology replacing the 3-aminobiphenyl with other aryl and
heteroaryl amines (Scheme 4). Compounds 13b–f turned out to
be more potent against HDAC6 than both 13a and 5a (Table 2).
However, these linear sulfamides 13b–f (Table 2), were 40- to
50-fold less active than the corresponding hydroxamic acids and
12
Scheme 3. Reagents and conditions: (a) 3-phenylaniline, BOP, Et3N, DMF; (b) LiOH,
THF, H2O; (c) CDI, DBU, NH2SO2NH2, DMF.
Table 1
Effect of zinc-binding group and chain length on HDAC1 and 6 inhibitory activitya
O
R
Ph
N
H
n
Compound
SAHAb
n
R
HDAC1 IC50
0.1
(l
M)
HDAC6 IC50
0.2
(lM)
H
N
1b
2b
4a
6
0.009
0.009
OH
O
O
NH
6
4
0.49
>50
NH2
O
O
H
N
a, b, c
NH2
O
R'
NHBoc
N
H
S
HO
3
3
O
O
O
13a-f
S
>10
>10
N
H
NH2
Scheme 4. Reagents and conditions: (a) R0-NH2, BOP, Et3N, DMF; or R0-NH2, PS-CDI,
HOBt, DCM; or Vilsmeier reagent, R0-NH2, Et3N, DCM; (b) TFA, DCM, or 4 N HCl in
dioxane; (c) H2NSO2NH2, Et3N, toluene, 120 °C; or ClSO2NCO, benzyl alcohol, DCM,
Et3N, followed by H2/10%Pd–C, MeOH; or ClSO2NCO, t-BuOH, DCM, Et3N, followed
by TFA, DCM.
O
O
S
S
S
S
5a
5b
7a
7b
10
11
12
5
6
5
5
5
5
5
5
>10
>10
>10
>10
>10
>10
>10
>10
1.1
>10
>10
>10
>10
>10
>10
>10
N
H
NH2
O
O
N
H
NH2
Table 2
Effect of cap on the HDAC activitya
O
O
O
H
NH2
R'
N
N
N
H
N
S
H
H
3
O
O
O
O
N
Compound
R0
HDAC1 IC50
>10
(l
M)
HDAC6 IC50
1.5
(lM)
N
H
13a
O
N
O
S
NH2
N
13b
13c
13d
13e
>10
>10
>10
>10
>10
0.39
0.49
0.71
0.44
0.9
O
O
O
S
NH2
O
O
O
N
S
N
H
NH2
H
N
H
N
O
N
8
S
O
O
N
a
Values are means of at least two experiments.
HDAC inhibitory activities for SAHA and for compounds 1 and 2 were deter-
mined in our laboratories.
b
MeO
S
13f
N
1-8.11b The IC50s of the inhibition of HDAC1 (representative of class
I HDACs) and HDAC6 (representative of class IIb) are summarized
a
Values are means of at least two experiments.

A. Wahhab et al. / Bioorg. Med. Chem. Lett. 19 (2009) 336–340
339
O
O
H
a, b, c, d
NH2
HCl
Ar
N
NH2
MeO
N
H
S
O
NHCbz
O
NHCbz
N
S
14a
14b
Ar=
N
MeO
Scheme 5. Reagents and conditions: (a) ClSO2NCO tBuOH, Et3N, DCM: (b) LiOH/THF MeOH/H2O; (c) Ar-NH2, BOP, Et3N, DMF; or Ar-NH2, PS–CDI, HOBt, DCM; or Vilsmeier
reagent, Et3N, DCM, Ar-NH2; or POCl3/Py, Ar-NH2; or iso-BuOCOCl, Et3N, DCM, Ar-NH2; (d) TFA, DCM, or 4 N HCl in dioxane.
Table 3
HDAC enzyme and cellular activitiesa
O
H
N
Ar
NH2
N
H
S
O
O
R"
HDAC-6 IC50
Compound
SAHA
Ar
R00
HDAC-1 IC50
0.1
(l
M)
(
l
M)
293TV cells IC50
(lM)
H3Acb EC50
0.12
(lM)
TubAcc EC50
0.25
(lM)
0.2
0.6
S
S
13f
H
>10
0.9
>50
2.3
20
3.5
0.6
N
N
14a
NH-Cbz
0.16
0.18
0.7
N
13e
H
>10
0.44
0.06
>50
1.8
>25
1
MeO
MeO
N
14b
NH-Cbz
0.46
0.35
0.2
a
Values are means of at least two experiments.
HAc, histone H3 acetylation.
Tub Ac, a-tubulin acetylation.
b
c
they were still not active against the other HDACs (data not
shown). Attempts to improve the HDAC inhibitory activity by using
a more rigid scaffold or by introducing a phenyl ring in the chain
resulted in loss of all activity (data not shown).
and 14b, which inhibited HDAC1, demonstrated a clear ability to
abrogate HDAC activity in the cell with IC50 = 2.3 and 1.8 lM,
respectively, in line with the observed enzymatic profile.14d By
comparison, SAHA, brought down cellular HDAC activity with
Jones et al., reported potent HDACis bearing a methylketone
IC50 = 0.6 lM. These results suggest that the contribution of HDAC6
ZBG utilizing
fied apicidin scaffold.12 We investigated whether we could achieve
similar success utilizing -lysine bearing the sulfamide moiety at
the -nitrogen.
Scheme 5 depicts the synthesis of these sulfamides from the
commercially available Z-Lys-OMe. Interestingly, we observed a
new activity profile when the linear scaffold (as in 5a) was changed
L
-Aoda (
L
-2-amino-8-oxodecanoic acid) as a simpli-
to total cellular HDAC activity is minimal.
The observed enzymatic profile and cellular activities were fur-
ther corroborated in functional cellular end points, namely, histone
L
e
(H3Ac) and
a-tubulin acetylation (TubAc) in T24 bladder cancer
cells.15
As expected, compounds 13e and 13f had no effect on H3Ac,
nevertheless caused a measurable induction of TubAc with EC50
to a lysine scaffold (14a and 14b). Branching at the
a-amino-posi-
of 1.0 and 3.5 lM, respectively, in line with their observed enzy-
tion resulted in beneficial interactions and conferred activity to
these sulfamides against HDAC1 in addition to HDAC613 (Table 3).
Compounds 13e, 13f, 14a, and 14b were further profiled in cel-
lular assays measuring their ability to inhibit total HDAC class I
activity in the whole cell (293TV).14a–c Compounds 13f and 13e,
which are specific inhibitors of HDAC6, showed no measurable de-
crease in HDAC cellular activity (Table 3), whereas sulfamides 14a
matic and cellular potencies. This is in agreement with literature
reports implicating HDAC6 in tubulin deacetylation.16 On the other
hand, 14a and 14b, which were potent inhibitors of both HDAC1
and HDAC6 displayed increased levels of both H3Ac and TubAc
acetylation. Compound 14b showed H3Ac and TubAc EC50s of
0.35 and 0.2
lM, respectively, comparable to the observed poten-
cies of SAHA (Table 3).

340
A. Wahhab et al. / Bioorg. Med. Chem. Lett. 19 (2009) 336–340
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 7024; (c) Winum, J.-Y.;
In conclusion, we have identified the sulfamide moiety for the
Scozzafava, A.; Montero, J.-L.; Supuran, C. T. Med. Res. Rev. 2006, 26, 767.
8. Alker, D.; Campbell, S. F.; Cross, P. E.; Burges, R. A.; Carter, A. J.; Gardiner, D. G. J.
Med. Chem. 1990, 33, 585.
9. (a) Krueger, A. C.; Madigan, D. L.; Jiang, W. W.; Kati, W. M.; Liu, D.; Maring, C. J.;
Masse, S.; McDaniel, K. F.; Middleton, T.; Mo, H.; Molla, A.; Montgomery, D.;
Pratt, J. K.; Rockway, T. W.; Zhang, R.; Kempf, D. J. Bioorg. Med. Chem. Lett. 2006,
16, 3367; (b) Casini, A.; Winum, J.-Y.; Montero, J.-L.; Scozzafava, A.; Supuran, C.
T. Bioorg. Med. Chem. Lett. 2003, 13, 837.
10. Espino, C. G.; Wehn, P. M.; Chow, J.; Du Bois, J. J. Am. Chem. Soc. 2001, 123, 6935.
11. a All experimental details can be found in MethylGene patent application:
Smil, D.; Leit, S.; Ajamian, A.; Allan, M.; Chantigny, Y. A.; Déziel, R.; Therrien, E.;
Wahhab, A.; Manku, S. International Patent WO 07/143822, 2007.; b The
enzymatic assay followed the fluorescent signal obtained from the HDAC
catalyzed deacetylation of coumarin-labeled lysine. The substrate used for
design of novel HDACis with bona fide HDAC cellular activities in
accordance with their observed enzymatic potencies. In addition,
we were able to manipulate the selectivity of these inhibitors.
The lysine-based inhibitors were HDAC1 and HDAC6 active, while
the long-chain compounds were selective toward HDAC6 and did
not show activity against the other HDACs tested. These com-
pounds are novel HDACis worthy of further investigation and
optimization.
Acknowledgments
HDAC1, 2, 3, 6, and 8 was Boc-Lys(
e-acetyl)-AMC (Bachem Biosciences Inc.)
The authors are grateful to Dr. Arkadii Vaisburg for valuable
suggestions and corrections and to Dr. A. Lori Martell for proof
reading this manuscript.
and Boc-Lys-( -trifluormethylacetyl)-AMC (synthesized in-house) for HDAC4,
e
5, and 7. Recombinant enzymes expressed in baculovirus were used. HDAC1, 2,
and 3 were C-terminal FLAG-tagged and HDAC4 (612–1034), HDAC5 (620–
1122), HDAC6, HDAC7 (438–915), and HDAC8 are N-terminal His-tagged. The
enzymes were incubated with the compounds in assay buffer (25 mM Hepes,
pH 8.0, 137 mM NaCl, 1 mM MgCl2 and 2.7 mM KCl) for 10 min at ambient
temperature in black 96-well plates. The substrate was added into enzyme–
compound mixture and incubated at 37° C. Reaction was quenched by adding
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compounds also showed activity against HDAC2 and HDAC3 but not against
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lM). For
example 14a IC50 is 0.24 and 0.11 M on HDAC2 and HDAC3, respectively.
l
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with inhibitors for 16 h and then incubated with Boc-Ac-Lys-AMC,
a
membrane permeable HDAC substrate. After 90 min at 37° C, the reaction
was quenched with trypsin and TSA by to a final concentration of 1 mg/mL and
1 lM, respectively. The cells were lysed with 1% NP-40. Fluorescence was read
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sulfamide inhibitors was comparable to MGCD0103, which was used as
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decrease of total HDAC activity in intact cells in comparison to SAHA.
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treatment in 96-well plates. The following day, the cells were incubated for 3 h
with the HDAC inhibitors at different concentrations. The cells were fixed on
the plate and the appropriated acetylated histone H3 and tubulin antibody was
added. The reaction was developed by Amplex Red. The cell number was
normalized by Alamar blue.
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