Synthesis and evaluation of lysine derived sulfamides as histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.075

We have recently reported on a novel class of histone deacetylase (HDAC) inhibitors bearing a sulfamide group as the zinc-binding unit. Herein, we report on the synthesis of sulfamide based inhibitors designed around a lysine scaffold and their structure-

Full text of DOI:10.1016/j.bmcl.2009.02.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1866–1870
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of lysine derived sulfamides as histone
deacetylase inhibitors
Sukhdev Manku a, Martin Allan a, Natalie Nguyen a, Alain Ajamian a, Jacques Rodrigue a, Eric Therrien a,
James Wang c, Tim Guo c, Jubrail Rahil b, Andrea J. Petschner b, Alina Nicolescu b, Sylvain Lefebvre c,
Zuomei Li c, Marielle Fournel c, Jeffrey M. Besterman c, Robert Déziel a, Amal Wahhab a,
*
a MethylGene Inc., Departments of Medicinal Chemistry, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
b Lead Discovery, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
c Pharmacology and Cell Biology, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently reported on a novel class of histone deacetylase (HDAC) inhibitors bearing a sulfamide
group as the zinc-binding unit. Herein, we report on the synthesis of sulfamide based inhibitors designed
around a lysine scaffold and their structure–activity relationships against HDAC1 and HDAC6 isotypes as
well as 293T cells. Our efforts led us to an improvement of the originally disclosed lysine-based sulfam-
ide, 2a to compound 12h which has equal potency in enzyme and cell-based assays as well as enhanced
metabolic stability and PK profile.
Received 29 January 2009
Revised 16 February 2009
Accepted 19 February 2009
Available online 23 February 2009
Keywords:
Sulfamides
HDAC inhibitors
HDAC1
Ó 2009 Elsevier Ltd. All rights reserved.
HDAC6
The acetylation status of lysine residues on the N-terminus of
core H2A/B, H3 and H4 is known to play a crucial role in chromatin
structure and hence the regulation of gene expression.1 Hyperacet-
ylation of histones is catalyzed by a family of histone acetyl-trans-
ferases (HAT) and leads to the relaxation of chromatin and, thus,
gene activation. Conversely, hypoacetylation, which is controlled
by the histone deacetylases (HDAC), causes the condensation of
chromatin resulting in transcriptional repression. The HDAC family
is classified into two categories: zinc-dependent amidohydrolases
(classes I, II and IV) and the NAD+ dependent enzymes (class III)
also known as sirtuins. Class I of the zinc-dependent HDAC family
is composed of isotypes 1, 2, 3 and 8. Class II is further subdivided
into two subclasses: class IIa (HDAC4, 5, 7 and 9) and IIb (HDAC6
and 10). HDAC11 is the only known member of class IV.1a–c
HDAC inhibitors have shown attractive anti-cancer properties
by inducing transcriptional events involved in growth arrest, cell
proliferation, and apoptosis.1,2 The HDAC inhibitor vorinostat (Zo-
linzaÒ, formerly known as SAHA)3 was approved in 2006 for the
treatment of cutaneous T-cell lymphoma (CTCL),4a and a number
of other inhibitors are in different stages of clinical development.4
Various structural classes of HDAC inhibitors have been developed,
yet most feature a common pharmacophore comprised of a zinc-
binding group, a linker (scaffold), and a surface recognition domain
(cap) which together impart potency and isotype selectivity.2
Examples of inhibitors are depicted in Figure 1 and range from
hydroxamic acids such as vorinostat to short-chained fatty acids
such as valproic acid.5 Vorinostat typically exhibits non-selective
inhibition against most of the zinc-dependent HDAC’s, while
MGCD0103,6 an aminobenzamide currently in Phase I/II clinical
trials, is more selective for class I HDAC’s over class II. Apicidin7,
N
O
H
N
OH
N
N
H
NH2
N
H
H
N
O
N
O
SAHA (vorinostat)
MGCD0103
O
O
OH
O
N
H
H
N
N
NH
O
N
OMe
valproic acid
O
S
O
O
H
apicidin
N
NH2
N
N
H
S
O2
R
1
, R = H
2a
, R = NHCbz
Figure 1. Examples of HDAC inhibitors in preclinical or various stages of clinical
development.
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.075

S. Manku et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1866–1870
1867
a, b, c
0.9
(Cbz) derived lysine scaffold was active against both HDAC1 and
HDAC6 (IC50’s 0.16 M and 0.18 M). In addition to its enzyme po-
tency, sulfamide 2a also showed whole cell HDAC inhibition in
293T cells (IC50 2.3 M) and displayed histone (H3Ac, EC50
0.7 M) and -tubulin (TubAc, EC50 0.6 M) acetylations in T24
lM) while compound 2a based on the N-carbobenzyloxy
O
and then d
S
O
H
N
NHBoc
Ph
NHR
HO
N
N
H
S
NHFmoc
3
O2
l
l
NH2
4, R = Boc
5a, R = H
j
l
either
S
O
e, f, g, h, or i
H
N
l
a
l
Ph
4
NHR
O2
N
N
H
S
cancer cells. Given the compound’s activity profile and its relative
ease of synthesis, it was chosen for further optimization.
HN
X
R = Boc
6b-d,f,h-j,
j
Diversification and replacement of the phenylthiazolylamide of
2a were achieved using the synthetic procedures described previ-
5b-d,f,h-j,
R = H
ously8b while functionalization of the
a-amino group on the lysine
Scheme 1. Reagents and conditions: (a) POCl3, pyr, 4-phenylthiazol-2-amine; (b)
4 N HCl, dioxane/DCM; (c) ClSO2NCO, tBuOH, Et3N, DCM; (d) piperidine, DCM; (e)
BOP, Et3N, DCM and 2-phenylacetic acid; (f) N,N0-disuccinimidyl carbonate, 2,6-
lutidine DCM/MeCN and alcohol; (g) Et3N, THF and 4-fluorobenzylisocyanate; (h)
PhCH2SO2Cl, pyr/DCM; (i) 2-(5-methoxy-2-methyl-1H-indol-3-yl)acetic acid, POCl3,
pyr; (j) TFA, DCM.
scaffold was performed using different orthogonally protected ly-
sine derivatives. Starting from commercially available lysine deriv-
ative 3, the protected sulfamide 4 was obtained via coupling with
4-phenylthiazol-2-amine using POCl3 to give the amide, followed
by removal of the Boc group from the
the protected sulfamide using ClSO2NCO and t-butanol, and then
deprotection of the -nitrogen from the Fmoc group (Scheme 1).
The free amine on 4 was further functionalized to the amide, car-
bamate, urea and the sulfonamide derivatives (6b–d, 6f, and 6h–
j). The removal of the Boc group from the sulfamide group was eas-
ily achieved by exposure to TFA to give the final compounds. For
the synthesis of ethyl carbamate 5e and phenyl urea 5g, lysine
derivative 7 was utilized by first installing the protected sulfamide,
e-nitrogen, installation of
O
c or d,
then e
a
O
H
N
a then b
NH2HCl
NHBoc
O2
MeO
MeO
S
NHCbz
NH2
7
8
O
H
S
O
f, g
H
N
NHBoc
Ph
HO
S
N
NH2
N
N
H
S
O2
HN
O2
X
HN
9e, g
X
5e,g
as described above, cleavage of the Cbz group from the
a-amino
Scheme 2. Reagents and conditions: (a) ClSO2NCO, tBuOH, NEt3, DCM; (b) H2, Pd/C,
MeOH; (c) ethyl chloroformate, PS-NMM, MP-isocyanate, DCM; (d) phenylisocya-
nate, DMAP, THF; (e) LiOH, THF, MeOH, H2O; (f) 4-phenylthiazol-2-amine and PS-
CDI, DMF/DCM or POCl3, pyr; (g) TFA/DCM.
group to allow for subsequent N-functionalization, followed by es-
ter hydrolysis, then amide formation and finally deprotection of
the sulfamide (Scheme 2).
Most replacements of the amide unit of 2a with various hetero-
cycles were achieved from the commercially available lysine deriv-
ative 10 and by introducing the sulfamide unit at the end of each
synthetic sequence (Scheme 3). The 1,3,4-oxadiazole 12e was ob-
tained by the coupling of 10 with benzhydrazide followed by
dehydrative cyclization. The 2-phenylthiazole containing sulfam-
ide 12a was obtained via conversion of 10 to the thioamide 13 fol-
lowed by treatment with 2-bromo-1-phenylethanone, while the 4-
phenylthiazole isomers, 12b–d, were formed via the addition and
with its ethyl ketone unit as the zinc-binding group and its cyclic
tetrapeptide as the surface recognition domain, also shows mainly
class I selectivity.2a
Previously,8 we have reported on an alternative class of HDAC
inhibitors featuring a sulfamide moiety as a novel replacement
for the more common hydroxamic acid, benzamide or ethyl ketone
zinc-binding groups. Furthermore, it was found that the nature of
the surface recognition domain that was paired with the sulfamide
led to changes in isotype selectivity. For instance, compound 1
with a linear 5-carbon linker was selective against HDAC6 (IC50
condensation of the
a-bromoketone intermediate (15) with vari-
ous thioamides. The 2-phenylimidazole analog 12j was accessible
through the reaction of benzimidamide with
a-bromoketone 15.
HO
N
NH2
O
22
N
a, b
N
O
F
i
N
NHBoc
k
Ph
HO
NHR
NHR
F
O
N
NHCbz
10
NHCbz
11, R = Boc
12e, R = SO2NH2
NHCbz
23, R = Boc
c, d
S
f
l, m, l
l, m, l
O
NHBoc
H
H2N
12f, R = SO2NH2
N
NHBoc
X
N
H
NHCbz
O
e
13
NHCbz
Br
NHBoc
j
18,
NHCbz
X = H, R = Boc
15
19, X = Me, R = Boc
X
NHCbz
S
Y
N
NH
N
N
g
or
NHR
h
Ph
N
Ph
NHR
l, m, l
X
NH2
X
NH2
S
NHCbz
X
NHR
14
, R = Boc
20, X = H, R = Boc
N
l, m, l
21,
X = Me, R = Boc
Y = S
NHCbz
12a
, R = SO2NH2
Y= NH
12j,
X = H, R = SO2NH2
16b-d, Y = S, R = Boc
12k,
X = Me, R = SO2NH2
17,
X= Ph, Y = NH, R = Boc
l, m, l
12b-d,
Y= S, R = SO2NH2
12i,
X= Ph, Y = NH, R = SO2NH2
Scheme 3. Reagents and conditions: (a) benzhydrazide, EDCI, DMAP, DCM; (b) (i) SOCl2, Pyr, THF 0 °C; (ii) toluene, reflux; (c) EDCI, HOBt, NH4OH, DCM; (d) Lawesson’s
reagent, THF, reflux; (e) 2-bromo-1-phenylethanone, MeOH; (f) (i) ethyl chloroformate, Et3N, THF; (ii) CH2N2 Et2O; (iii) 48% HBr (aq), Et2O; (g) thioamide, ethanol; (h)
benzimidamide, KHCO3, THF/water, reflux; (i) EDCI, HOBt, hydrazine or methylhydrazine, DCM; (j) methyl benzimidate hydrochloride, Et3N, THF then xylenes 155 °C; (k) 22,
DCC, DCM then pyr, reflux; (l) TFA/DCM; (m) ClSO2NCO, tBuOH, Et3N, DCM.

1868
S. Manku et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1866–1870
The 1,2,4-triazoles 12j and 12k were obtained by heating the
hydrazide and methylhydrazide analogs of lysine 10 in the pres-
ence of methyl benzimidate. Finally, coupling of 10 with hydroxy-
amidine 22 followed by cyclization gave the 1,2,4-oxadiazole 12f
after installation of the sulfamide unit. For the synthesis of benz-
imidazoles 12g–h, the lysine derivative 7 was used and the Boc-
protected sulfamide unit was attached prior to the formation of
the benzimidazole ring. Cyclization was achieved by heating benz-
amide intermediates, 26g–h in acetic acid (Scheme 4).
All synthesized compounds were screened against a panel of re-
combinant human HDAC’s 1–8.8c The IC50’s of the inhibition of
HDAC1 (representative of class I HDAC’s) and HDAC6 (representa-
tive of class IIb) are summarized in the tables below, while the re-
sults for the other HDAC’s were omitted for clarity. These
compounds were tested for their ability to inhibit the cellular
HDAC activity in 293T cells.8d
Table 1 depicts the HDAC inhibitory activities of different thia-
zole amides compared to the parent compound 2a. The unsubsti-
tuted thiazolylamide (2b) had almost negligible activity against
both HDAC1 and HDAC6, while the benzothiazole (2c) retained
its potency on HDAC6 at the dramatic cost of HDAC1 leading to
an isotype selectivity of 24 to 1. A phenyl substituent off the thia-
zole ring of 2a was more favored than the bulky t-butyl group (2d),
which lost significant activity against both enzymes. The ethyl es-
ter substitution (2e) abrogated HDAC1 activity but retained some
of it against HDAC6. The 4,5-diphenyl substituted thiazole amide
(2f) was five and threefold less active than 2a against HDAC1
and HDAC6, respectively, implicating that a mono-phenyl substitu-
ent is superior to disubstitution. Compounds 2g–j were prepared
to investigate the effect of substitution off the phenyl ring of 2a.
As can be seen from Table 1, substitution on the phenyl group
was well tolerated as demonstrated by compounds with electron
donating (2g and 2h) and electron withdrawing groups (2i), all
showing submicromolar activities. Bulkier para-aliphatic substitu-
ents on the phenyl ring such as an iso-propyl (2j) were partially tol-
erated against HDAC1 but less so against HDAC6. Overall,
monoaryl thiazoles provided the best activity, but within this ser-
ies no particular compound exhibited a significant improvement
with the exception of compound 2g which had four times the cel-
lular activity of 2a in 293T cells.
Modifications of the
a-amino substituent on 2a were also per-
formed to further explore the tolerance of substitution at this posi-
tion (see Table 2). Complete removal of the benzyl carbamate to
leave the free amine (5a) resulted in a significant reduction in
the activity against both enzymes, thus indicating the importance
of functionalization at this position. Substitution on the phenyl
unit of the benzyl carbamate of 2a, as illustrated by compounds
5b–d, gave an overall decrease in both enzyme and whole cell
HDAC activities. Aliphatic carbamates, such as the ethyl carbamate
5e, showed a noticeable loss in HDAC1 activity in both the enzyme
and cell, but remained significantly potent on HDAC6. The removal
of the oxygen from the benzyl carbamate to give the benzyl amide
analog (5f) resulted in a drop of potency against the HDAC1 en-
zyme. Likewise, the phenyl urea (5g), lost most of its activity espe-
cially on HDAC1, yet the benzyl urea derivative (5h) regained some
of it at the enzyme level thus suggesting the importance of the ex-
tra atom on the inhibitors’ side chain. Further sensitivity on HDAC1
towards amino-substitution was illustrated by the sulfonamide on
compound 5i which was poorly tolerated by the enzyme. Finally,
the use of the 2-methyl-5-methoxyindole (5j) resulted in excep-
tional HDAC1 activities (0.064 lM and 0.59 lM on HDAC1 and
whole cell, respectively). Overall, marginal improvements were ob-
served by replacing the benzyl carbamate of 2a and therefore, for
further studies we retained this group given the ease of obtaining
commercially available Cbz-protected lysine derivatives for
synthesis.
During our investigation, it was discovered that compound 2a
had a poor pharmacokinetic (PK) profile in rats (see below). Also,
preliminary evaluation of the metabolic stability of 2a indicated
that only 3% of the compound remained intact over 1 h in rat plas-
ma at 37 °C. This instability was determined to be mostly due to
cleavage of its amide bond to give the acid and 4-phenylthiazol-
2-amine. These observations lead us to explore isosteric heterocy-
NH2
25
O
O
a, b
H
N
R
NH2
NH2
HCl
NHBoc
HO
MeO
R
S
c
O2
NHCbz
R
NHCbz
O
24
7
NH
d, e
H
N
H
NHBoc
O2
N
NH2
N
N
H
S
S
O2
NHCbz
12g-h
NH2
NHCbz
26g-h
Scheme 4. Reagents and conditions: (a) ClSO2NCO, tBuOH, Et3N, DCM; (b) LiOH,
THF/MeOH/H2O; (c) dianiline, PS-CDI, HOBt, DCM; (d) AcOH, 90 °C; (e) HCl/dioxane.
Table 1
SAR on the thiazole amide capping unita
O
H
X
N
H
N
NH2
S
O2
NHCBz
R1
R2
Compound
HDAC1 IC50
(l
M)
HDAC6 IC50
(lM)
293T cells IC50 (lM)
S
X =
N
2a
2b
R
R
1 = H, R2 = Ph
1 = H, R2 = H
0.16
>10
0.18
4.1
2.3
>50
S
2c
4.5
0.19
19
X =
N
2d
2e
2f
2g
2h
2i
R
R
R
R
R
R
R
1 = H, R2 = tBu
3.6
0.84
0.58
0.45
0.20
0.42
0.25
1.4
9.1
>50
7.2
0.53
3.2
4.9
3.2
1 = H, R2 = CO2Et
>10
0.85
0.12
0.46
0.15
0.52
1 = Ph, R2 = Ph
1 = H, R2 = p-MeOC6H4
1 = H, R2 = m-MeOC6H4
1 = H, R2 = p-ClC6H4
1 = H, R2 = 4-i-PrC6H4
2j
a
Values are means of at least two experiments.

S. Manku et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1866–1870
1869
Table 2
SAR of the a
-amino substituenta
S
O
H
N
NH2
N
N
H
S
O2
NH
X
Compound
HDAC1 IC50
(l
M)
HDAC6 IC50
(l
M)
293T cells IC50 (lM)
Y
R
X =
O
2a
5a
5b
5c
5d
5e
5f
5g
5h
5i
R = PhCH2, Y = O
X = H
R = p-ClC6H4CH2, Y = O
R = m-ClC6H4CH2, Y = O
R = 3-pyridinyl-CH2, Y = O
R = Et, Y = O
R = Ph, Y = CH2
R = Ph, Y = NH
R = p-FC6H4CH2, Y = NH
X = PhCH2SO2
0.16
>10
0.57
0.35
1.5
2.5
0.70
2.3
0.18
1.1
2.3
>50
6.8
3.6
18
0.53
0.28
0.60
0.20
0.15
0.56
0.37
0.52
17
6.1
>50
29
0.35
1.4
38
X =
O
5j
0.064
1.2
0.59
O
N
H
a
Values are means of at least two experiments.
Table 3
cles as replacements for the amide unit.9 For instance; compound
12a incorporates the same phenylthiazole as 2a, yet excludes the
amide group connecting it to the rest of the lysine scaffold. Unfor-
tunately, the activity of 12a suffered significantly as a result of this
change (Table 3). However, by moving the sulfur atom on the thi-
azole next to the phenyl group to give its regioisomer 12b, the en-
SAR of amide excluded HDAC inhibitors.a
H
N
X
NH2
S
O2
NHCBz
Compound
X
HDAC1 IC50
M)
HDAC6 IC50
M)
293T cells
(l
(l
IC50
(lM)
zyme activity returned to submicromolar levels (IC50 of 0.79
and 0.40
phenyl ring of 12b improved the cellular potency from 6.7
for 12b, down to an impressive 0.12
Both oxadiazoles 12e and 12f were poor isosteres of the amide unit
and had an overall negative effect on the enzymatic activities,
while the benzimidazoles (12g–h) and the imidazole containing
compound (12i) displayed very potent profiles. Among the benz-
imidazole series, the unsubstituted benzimidazole (12g) was al-
most 30-fold more selective for HDAC6 over HDAC1, while aryl
substitution at the heterocycle’s 5-position (12h) resulted in com-
pounds with equal potency against both HDAC1 and HDAC6. Par-
ticularly noteworthy is the submicromolar activity in 293T cells
l
M
S
l
M on HDACs 1 and 6, respectively). Substitution on the
12a
12b
12c
12d
12e
12f
4.9
1.2
12
Ph
Ph
N
S
lM
l
M for the more basic 12d.
0.79
0.50
0.39
3.2
0.40
0.48
0.89
1.8
5.8
N
S
n.a.b
0.18
9.9
N
O
S
N
O
N
N
N
Ph
F
O
displayed by benzimidazole 12h at 0.62
lM which is in line with
that of vorinostat (0.6
l
M).8a The triazole analogue 12j also re-
N
O
>10
2.9
1.7
>50
9.5
vealed partial selectivity for HDAC6, whereas the N-methyltriazole
12k was almost devoid of any significant activity against both
enzymes.
N
NH
12g
0.1
Given their good combinations of enzyme and cellular activities,
the thiazole 12d and benzimidazole 12h were chosen for further
pharmacokinetic and phasma stability studies. The metabolic
stability of benzimidazole 12h was an improvement upon 2a,
being stable in both rat and human plasma (data not shown).
The PK profiles of 12d and 12h in rats revealed that both
compounds had lower clearances and increased half lives com-
pared to 2a (Table 4). However, they still suffered from poor oral
absorption as indicated by the low AUC values and dismal bioavail-
ability. Furthermore, the poor PK profile was also observed across
different species. Despite the acceptable clearance and half-life of
12h in dog (0.51 (L/h)/kg and 5.7 h, respectively), the AUC was still
N
NH
12h
0.20
0.29
0.48
0.41
0.62
2.2
S
N
HN
12i
12j
Ph
N
HN
N
2.4
4.3
0.32
2.0
6.4
Ph
Ph
N
N
N
12k
>50
poor at 0.2 lM h/(mg/kg), and the bioavailability was a mere
N
6%. The poor bioavailability displayed by this class of compounds
could be attributed to the sulfamide unit. Middleton et al.,10a and
Beaumont et al.,10b encountered a similar problem with their
a
Values are means of at least two experiments.
Not available.
b

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S. Manku et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1866–1870
6. Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Fréchette, S.; Gaudette, F.; Paquin, I.;
Table 4
Bernstein, N.; Bouchain, G.; Vaisburg, A.; Jin, Z.; Gillespie, J.; Wang, J.; Fournel,
M.; Yan, P. T.; Trachy-Bourget, M.-C.; Kalita, A.; Lu, A.; Rahil, J.; MacLeod, A. R.;
Li, Z.; Besterman, J. M.; Delorme, D. J. Med. Chem. 2008, 51, 4072.
PK profile in rat of selected compounds
Compound AUC (PO) lM h/(mg/kg) Cl (IV) (L/h)/kg Vss L/kg t1/2 (h) %F
7. Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.; Dulski, P. M.; Crumley, T. M.;
Allocco, J. J.; Cannova, C.; Meinke, P. T.; Colletti, S. L.; Bednarek, M. A.; Singh, S.
B.; Goetz, M. A.; Dombrowski, A. W.; Polishook, J. D.; Schmatz, D. M. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 13143.
2a
12d
12h
0.063
0.005
0.023
2.5
2.3
0.41
0.02
0.78
0.03
0.025
1.6
1.2
4.9
0.5
0.5
8. (a) Wahhab, A.; Smil, D.; Ajamian, A.; Allan, M.; Chantigny, Y.; Therrien, E.;
Nguyen, N.; Manku, S.; Leit, S.; Rahil, J.; Yan, T. P.; Li, Z.; Besterman, J.; Déziel, R.
Bioorg. Med. Chem. Lett. 2009, 19, 336; (b) For experimental details see: 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.; (c) The enzymatic
assay followed the fluorescent signal obtained from the HDAC catalyzed
deacetylation of coumarin-labeled lysine. The substrate used for HDAC1, 2, 3, 6,
NK2 antagonists bearing a sulfamide group, and have ascribed
their compounds’ poor oral absorption to weak intrinsic perme-
ability and transporter-mediated efflux from the GI tract.10
In conclusion, a structure–activity investigation around the
originally disclosed lysine-based sulfamide, 2a has revealed that
subtle modifications around the periphery of the molecule are tol-
erated when tested against HDAC 1 and 6 isotypes and in
293Tcells, but more significant modifications resulted in consider-
able losses in HDAC1 activity or, in some cases, both HDAC1 and
HDAC6. However, replacement of the core amide linkage on 2a
with different heterocycles led to compounds of equal potency
with improved pharmacokinetic profiles. In particular, benzimid-
azole 12h showed promising potency and metabolic stability, how-
ever, further optimization of its pharmacokinetic properties is
needed.
and 8 was Boc-Lys(
e-acetyl)-AMC (Bachem Biosciences Inc.) and Boc-Lys-(e-
trifluormethylacetyl)-AMC (synthesized in-house) for HDAC4, 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
trypsin and TSA to a final concentration of 1 mg/mL and 1
lM, respectively.
Fluorescence was measured using fluorimeter (SPECTRAMAX GeminiXS,
a
Molecular Devices). The 50% inhibitory concentrations (IC50) for inhibitors
were determined by analyzing dose–response inhibition curves with GraFit.;
(d) The whole cell assay was done in cultured Human Embryonic Kidney cells
(293T), which were treated 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
References and notes
concentration of 1 mg/mL and 1 M, respectively. The cells were lysed with
l
1% NP-40. Fluorescence was read at Ex 360 nm, Em 470 nm, using GeminiXS
fluorimeter.
1. (a) Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Discovery 2006, 5,
769; (b) De Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van
Kuilenburg, A. B. Biochem. J. 2003, 370, 737; (c) Minnucci, S.; Pelicci, G. Nat. Rev.
Cancer 2006, 6, 38; (d) Dey, P. Curr. Med. Chem. 2006, 13, 2909.
2. (a) Bieliauskas, A. V.; Pflum, M. K. H. Chem. Soc. Rev. 2008, 37, 1402; (b)
Moradei, O.; Maroun, C. R.; Paquin, I.; Vaisburg, A. Curr. Med. Chem. Anti-Cancer
Agents 2005, 5, 529.
3. 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. U.S.A. 1996, 93, 5705.
4. (a) Grant, S.; Easley, C.; Kirkpatrick, P. Nat. Rev. Drug Discovery 2007, 6, 21; (b)
Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505;
(c) Bruserud, O.; Stapnes, C.; Ersvaer, E.; Gjertsen, B. T.; Ryningen, A. Curr.
Pharm. Biotechnol 2007, 8, 388.
9. For general references on amide isosteres see: (a) Venkatesan; Kim, B. H. Curr.
Med. Chem. 2002, 9, 2243; (b) Borg, S.; Estenne-Bouhtou, G.; Luthman, K.;
Csöregh, I.; Hesselink, W.; Hacksell, U. J. Org. Chem. 1995, 61, 3112–3120; (c)
Borg, S.; Vollinga, R. C.; Labarre, M.; Payza, K.; Terenius, L.; Luthman, K. J. Med.
Chem. 1999, 42, 4331; For amide isosteres used for HDAC inhibitors see: (d)
Jones, P.; Altamura, S.; De Francesco, R.; Gonzalez Paz, O.; Kinzel, O.; Mesiti, G.;
Monteagudo, E.; Pescatore, G.; Rowley, M.; Verdirame, M.; Steinkuhler, C. A. J.
Med. Chem. 2008, 51, 2350; (e) Pescatore, G.; Kinzel, O.; Attenni, B.; Cecchetti,
O.; Foire, F.; Fonsi, M.; Rowley, M.; Schultz-Fademrecht, C.; Serafini, S.;
Steinkülher, C.; Jones, P. Bioorg. Med. Chem. Lett. 2008, 18, 5528.
10. (a) Middleton, D. S.; MacKenzie, A. R.; Newman, S. D.; Corless, M.; Warren, A.;
Marchington, A. P.; Jones, B. Bioorg. Med. Chem. Lett. 2005, 15, 3957; (b)
Beaumont, K.; Harper, A.; Smith, D. A.; Bennett, J. Eur. J. Pharm. Sci. 2000, 12, 41.
5. Chen, J. S.; Faller, D. V.; Spanjaard, R. A. Curr. Cancer Drug Targets 2003, 3, 219.