Exploring alternative Zn-binding groups in the design of HDAC inhibitors: Squaric acid, N-hydroxyurea, and oxazoline analogues of SAHA

Article abstract of DOI:10.1016/j.bmcl.2006.06.090

Analogues of suberoylanilide hydroxamic acid (SAHA) were prepared by replacing the Zn-binding group with squaric acid, N-hydroxyurea, and 4-hydroxymethyl oxazoline units, also varying the length of the aliphatic chain. No inhibitory activity on HDAC was observed below 1.0 μM and no cytotoxic activity on different tumor cell lines was seen below 20.0 μM.

Full text of DOI:10.1016/j.bmcl.2006.06.090

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4784–4787
Exploring alternative Zn-binding groups in the design
of HDAC inhibitors: Squaric acid, N-hydroxyurea,
and oxazoline analogues of SAHA
Stephen Hanessian,a,* Valerio Vinci,a Luciana Auzzas,a,b Mauro Marzic
and Giuseppe Gianninic
aDepartment of Chemistry, Universite´ de Montre´al, PO Box 6128, Station Centre-ville, Montre´al, Que., Canada H3C 3J7
bIstituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Traversa La Crucca 3, I-07040 Sassari, Italy
cSigma-Tau Research & Development, Via Pontina Km 30.400, 00040 Pomezia, Roma, Italy
Received 21 June 2006; accepted 26 June 2006
Available online 25 July 2006
Abstract—Analogues of suberoylanilide hydroxamic acid (SAHA) were prepared by replacing the Zn-binding group with squaric
acid, N-hydroxyurea, and 4-hydroxymethyl oxazoline units, also varying the length of the aliphatic chain. No inhibitory activity
on HDAC was observed below 1.0 lM and no cytotoxic activity on different tumor cell lines was seen below 20.0 lM.
Ó 2006 Elsevier Ltd. All rights reserved.
HDACs are nuclear enzymes that play a major role in
regulating gene expression.1 They catalyze the deacetyla-
tion of the N-acetyl lysine residues thereby changing the
accessibility of transcription factors to DNA, thus
affecting the chromatin remodeling process. Cell-specific
patterns of gene expression depending on histone acety-
lation result from a balance of the competing activities
of two classes of enzymes, the histone acetyl transferases
(HATs) and the histone deacetylases (HDACs). Pertur-
bation of this balance has been linked to cancer, and
inhibition of HDAC has been shown to have antiprolif-
erative effects on tumor cell lines, resulting in consider-
able interest in this field.2
zinc-binding groups (ZBGs) that can be incorporated in
the structures of metalloproteases inhibitors in general.5
O
O
OH
OH
N
H
N
Trichostatin A (TSA)
O
NH
N
H
O
SAHA
The list of known inhibitors of HDACs covers a wide
cross-section of structures including natural products
such as Trichostatin A (TSA)3 and unnatural surrogates
such as suberoylanilide hydroxamic acid (SAHA)4
(Fig. 1). A large body of work has been devoted to the
synthesis of hydroxamic acid analogues of acyclic and
heterocyclic compounds.
O
O
X
H
H
N
N
O
Y
n
n
O
O
N
n = 2, 3, 4
n = 4
HO
X = -O, -SNa, -SMe, -N(Me)OH
Y = -N-, -S-
In spite of the promising results, the potential toxicity of
hydroxamic acids has also instigated the search for other
O
H
N
OH
N
N
H
n
H
O
n = 3, 4, 5
Figure 1. Structures of known inhibitors and proposed prototypes.
Keywords: Metalloprotease inhibitor.
*
Corresponding author. E-mail: stephen.hanessian@umontreal.ca
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.090

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4784–4787
4785
We wish to report on our efforts to prepare analogues of
SAHA in which the hydroxamic acid residue has been
replaced by squaric acid, N-hydroxyurea, and hydroxy-
methyl oxazoline units individually (Fig. 1). To the best
of our knowledge, these well-known motifs have not
been synthesized and tested as non-hydroxamate HDAC
inhibitors.6
Alternatively, treatment of the esters 5a–c with aqueous
sodium hydrogen sulfide in ethanol afforded the corre-
sponding thiol monoacids as their sodium salts 7a–c.13
The methylthioesters 8a–c were obtained upon treat-
ment with methyl iodide (Scheme 1).
Alternatively, intermediate 4 was converted into the cor-
responding monosodium thiolate by treatment with
sodium hydrogen sulfide in methanol and then coupled
with the readily available bromide 10 (prepared in two
steps from d-valerolactone)6,15 in DMF, to afford 11
(Scheme 2). Treatment with N-methyl hydroxyl-
amine gave the thio squaric N-methyl hydroxamate
analogue 12.
Being cognizant that the zinc-binding geometry of
hydroxamic acids7 may be different compared to squaric
acids or the N-hydroxyurea units, we also varied the
length of the aliphatic chain to allow for greater flexibil-
ity and accommodation within the active site domain.8
Squaric acid derivatives. Since the first synthesis of squa-
ric acid in 1959,9 a number of derivatives have been
reported and their chemistry has been well studied in dif-
ferent contexts over the years.10,11 Squaric acid-based
inhibitors of matrix metalloproteases were recently
reported.12 Due to its different resonance forms, squaric
acid can mimic acidic functions, which was the main de-
sign element in this study, although it was not clear
whether the spatial requirements could be satisfied in
the active site of HDAC enzymes.8
O
O
a
b
4
OC4H9
NaS
73%
(2 cycles)
9
O
O
H
c
N
OC4H9
S
2
2
52%
We therefore focused first on the synthesis of SAHA-
type analogues of different chain lengths and harboring
a squaric acid unit that contains exocyclic nitrogen or
sulfur groups to simulate vinylogous hydroxamic acids
and thioether counterparts as potential zinc binders. A
series of N-Boc x-amino carboxylic acids 1a–c was con-
verted to the corresponding phenylamides 2a–c, and
then cleaved to the amines 3a–c (Scheme 1). Treatment
with the readily available di-n-butyl squarate 4 led to
the corresponding monoamides 5a–c in good yields.13
Mineral acid hydrolysis gave the corresponding squaric
acid SAHA analogues 6a–c in excellent yields.14
O
O
11
O
O
H
N
OH
S
N
12
H
N
Br
2
O
10
Scheme 2. Reagents: (a) NaHSÆH2O, MeOH; (b) 10, DMF; (c)
MeNHOHÆHCl, NaOH, MeOH.
H
H
a
b
c
HO
N
N
NH2. TFA
NHBoc
NHBoc
n
n
n
O
O
O
1a n = 2
1b n = 3
1c n = 4
2a (80%)
2b (83%)
2c (97%)
3a-c
O
O
O
O
O
O
e
f
H
N
H
N
H
N
SNa
S
N
OC4H9
N
N
n
n
n
H
H
H
O
O
O
5a (67% for two steps)
5b (77% for two steps)
5c (71% for two steps)
7a (80%)
7b (83%)
7c (97%)
8a (94%)
8b (88%)
8c (96%)
d
O
O
H
N
N
OH
O
O
n
H
O
6a (80%)
6b (84%)
6c (79%)
C4H9O
OC4H9
4
Scheme 1. Reagents and conditions: (a) PhNH2, PyBop, Et3N, CH2Cl2; (b) TFA, CH2Cl2, 0 °C to rt; (c) 4, Et3N, EtOH; (d) 4 N HCl/dioxane, H2O;
(e) aq NaHS, EtOH; (f) MeI.

4786
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4784–4787
N-Hydroxyurea derivatives. The replacement of
a
A series of resulting hydroxy acids 16–18 were converted
to the corresponding anilides 19–21, which were
O-alkylated to give O-PMB ethers 22–24. Intermediate
23 (n = 5) was converted into the corresponding
N-OTHP N-hydroxyurea 25 and the latter subjected to
mild acid hydrolysis to afford 26.
hydroxamic acid moiety with an N-hydroxyurea coun-
terpart has received only scant attention in the search
for HDAC inhibitors. In principle, hydroxamic acids
should be more acidic compared to an N-hydroxyurea,
although the requisite terminal functionality to bind
the zinc atom is present in both.
Hydroxymethyl oxazolines. Cook and coworkers18 have
explored the core of 4-hydroxymethyl oxazolines as
potential MMP inhibitors. We therefore prepared
SAHA analogues in which the hydroxamic acid group
was replaced with this moiety as shown in Scheme 5.
Suzuki and coworkers6 recently reported that a SAHA
N-hydroxyurea derivative was weakly active as an inhib-
itor of HDAC. Our intention was to probe the influence
of the hydrocarbon chain length, knowing that the ac-
tive site domain of SAHA and TSA can accommodate
a limited number of carbon atoms in an acyclic
chain.5a,8
Amide 29 was prepared in very good yields starting from
monomethyl suberate 27 and the chiron 28 (readily
available from L-serine). Subsequent treatment of 29
Treatment of the readily available amines 3b–d with
hydroxylamine in the presence of carbonyl diimidazole
led to the intended analogues 13b–d in very good overall
yields (Scheme 3).
OBn
O
O
a
OTBDPS
HCl . H2N
+
O
OH
82%
6
27
28
Ether and amide analogues of SAHA have been recently
disclosed in a patent.16 We therefore prepared an ether
prototype in the N-hydroxyurea series. Briefly, ( )-
aminopimelic acid 14 was selectively protected as the
lactone acetal 15, diazotized, and the resulting alcohol
then chain-extended via an Arndt-Eistert reaction
(Scheme 4).17
OBn
OBn
OH
O
O
O
O
b
OTBDPS
O
c
N
H
O
N
84%
6
6
H
29
30
O
O
O
O
OBn
OR
d
N
N
N
O
60%
66%
6
H
6
H
H
H
31
32 R = Bn
H
a
. TFA
NH2
2
N
N
N
N
e
OH
2
55%
O
O
O
33 R = H
3b n = 4
3c n = 5
3d n = 6
13b (88%)
13c (87%)
13d (74%)
Scheme 5. Reagents and conditions: (a) HATU, DIEA, DMAP,
CH2Cl2. (b) TBAF, THF. (c) DAST, K2CO3, CH2Cl2, À78 °C. (d) (i)
LiOHÆH2O, MeOH, H2O, THF; (ii) PhNH2, PyBOP, Et3N, DMAP,
DMF. (e) BCl3, CH2Cl2, À40 °C.
Scheme 3. Reagents: (a) CDI, Et3N, NH2OHÆHCl, CH2Cl2.
OH
OH
NH2
O
H
a
b
c
d
O
N
CO2Me
CO2Me
CO2H
CO2H
HO2C
HO2C
72%
84-96%
30-39%
75-84%
n
n
4
4
O
O
14
15
16 n = 4
17 n = 5
18 n = 6
19 n = 4
20 n = 5
21 n = 6
O
O
O
O
O
O
O
e
f
H
N
H
H
H
N
H
H
H
CO2Me
N
N
N
N
N
OTHP
OH
78%
75%
n
5
5
O
O
O
O
22 n = 4
23 n = 5
24 n = 6
25
26
Scheme 4. Reagents and conditions: (a) (i) NaNO2, 2 N HCl, 0 °C to rt; (ii) 2,2-dimethoxypropane, PTSA. (b) For n = 4: (i) CH2N2ÆEt2O; (ii) 70%
AcOH, 60 °C, 84% for two steps; for n = 5: (i) SOCl2, cat. DMF, CH2Cl2, À10 °C to rt, then À5 °C, CH2N2, Et2O, rt, 86% for two steps; (ii) AgOBz,
Et3N, MeOH, À25 °C to rt, 99%; for n = 6: (i) BH3ÆDMS, THF, 0 °C to rt; (ii) (COCl)2, DMSO, CH2Cl2, À78 °C, then Et3N, rt; (iii)
Ph3P@CHCO2Me, CH2Cl2, 82% for three steps; (iv) NaBH4, NiCl2Æ6H2O, MeOH, 0 °C to rt; (v) 70% AcOH, 65 °C, 92% for two steps. (c)
C6H5N@S@O, 1,2,4-triazole, CH2Cl2, 0 °C to rt; (d) PMBO(C@NH)CCl3, cat. BF3ÆEt2O, CH2Cl2–Et2O. (e) (i) NaOH–THF–MeOH; (ii) diphenyl
phosphoryl azide, Et3N, toluene, reflux, then THPONH2, reflux; (f) HCl–MeOH, THF.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4784–4787
4787
6. Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.;
Maruyama, S.; Kurotaki, M.; Nakagawa, H.; Miyata, N.
J. Med. Chem. 2005, 48, 1019.
with TBAF afforded 30 which underwent DAST-medi-
ated ring-closure to give 31 in very good yields.
7. (a) Levin, J. I. Curr. Top. Med. Chem. 2004, 4, 1289; (b)
Lim, N. C.; Morton, M. D.; Jenkins, H. A.; Bruckner, C.
J. Org. Chem. 2003, 68, 9233; (c) Muri, E. M. F.; Nieto,
M. J.; Sindelar, R. D.; Williamson, J. S. Curr. Med. Chem.
2002, 9, 1631.
Saponification of the methyl ester and introduction of
the anilide moiety led to 32 which finally underwent
debenzylation by BCl3 in CH2Cl2 to afford the oxazoli-
dine analogue 33.
8. (a) Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho,
J. D.; Jennings, A. J.; Luong, C.; Arvai, A.; Buggy, J. J.;
Chi, E.; Jie Tang, J.; Sang, B.-C.; Verner, E.; Wynands,
R.; Leahy, E. M.; Dougan, D. R.; Snell, G.; Navre, M.;
Knuth, M. W.; Swanson, R. V.; McRee, D. E.; Tari, L. V.
Structure 2004, 12, 1325; (b) Finnin, M. S.; Donigian, J.
R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P.
A.; Breslow, R.; Pavletich, N. P. Nature 1999, 401, 188.
9. Cohen, S.; Lacher, J. R.; Park, J. D. J. Am. Chem. Soc.
1959, 81, 3480.
Unfortunately, none of the new analogues in the series
exhibited HDAC inhibitory activity below 1.0 lM. Fur-
thermore, no cytotoxic activity on different tumor cell
lines was seen below 20.0 lM.
Acknowledgments
We thank Consiglio Nazionale delle Ricerche, Istituto di
Chimica Biomolecolare, Italy, for a sabbatical leave to
L.A. and Sigma-Tau Industrie Farmaceutiche Riunite
S.p.A. for biological testing.
10. Schmidt, A. H. Synthesis 1980, 961.
11. For biomedical applications, see: (a) Butera, J. A.;
Antane, M. M.; Antane, S. A.; Argentieri, T. M.;
Freeden, C.; Graceffa, R. F.; Hirth, B. H.; Jenkins, D.;
Lennox, J. R.; Matelan, E.; Norton, N. W.; Quagliato,
D.; Sheldon, J. H.; Spinelli, W.; Warga, D.; Wojdan, A.;
Woods, M. J. Med. Chem. 2000, 43, 1187; (b) Bang-
Andersen, B.; Ahmadian, H.; Lenz, S. M.; Stensbøl, T.
B.; Madsen, U.; Bøgesø, K. P.; Krogsgaard-Larsen, P.
J. Med. Chem. 2000, 43, 4910; (c) Gilbert, A. M.;
Antane, M. M.; Argentieri, T. M.; Butera, J. A.;
Francisco, G. D.; Freeden, C.; Gundersen, E. G.;
Graceffa, R. F.; Herbst, D.; Hirth, B. H.; Lennox, J.
R.; McFarlane, G.; Norton, N. W.; Quagliato, D.;
Sheldon, J. H.; Warga, D.; Wojdan, A.; Woods, M.
J. Med. Chem. 2000, 43, 1203.
12. Onaran, M. B.; Comeau, A. B.; Seto, C. T. J. Org. Chem.
2005, 70, 10792.
13. See for example: Zhou, H.-B.; Zhang, J.; Lu, S.-M.; Xie,
¨
R.-G.; Zhou, Z.-Y.; Choi, M. C. K.; Chan, A. S. C.; Yang,
T.-K. Tetrahedron 2001, 57, 9325.
14. See for example: Chan, P. C. M.; Roon, R. J.; Koerner, J.
F.; Taylor, N. J.; Honek, J. F. M. J. Med. Chem. 1995, 38,
4433.
References and notes
1. Wu, J.; Grunstein, M. Trends Biochem. Sci. 2005, 25, 619.
2. For recent reviews, see: (a) Minucci, S.; Pelicci, P. G. Nat.
Rev. Cancer 2006, 6, 38; (b) Iizuka, M.; Smith, M. M.
Curr. Opin. Genet. Dev. 2003, 13, 154; (c) Roth, S. Y.;
Denu, J. M.; Allis, C. D. Annu. Rev. Biochem. 2001, 70, 81;
(d) Khochbin, S.; Verdel, A.; Lemercier, C.; Seigneurin-
Berny, D. Curr. Opin. Genet. Dev. 2001, 11, 162; (e)
Archer, S. Y.; Hodin, R. A. Curr. Opin. Genet. Dev. 1999,
9, 171; see also, Muri, E. M. F.; Nieto, M. I.; Sindelar, R.
D.; Williamson, J. D. Curr. Med. Chem. 2002, 9, 1631;
Yang, K.; Lou, B. Mini-Rev. Med. Chem. 2003, 3, 349.
3. Isolation and activity: (a) Tsuji, N.; Kobayashi, M.;
Nagashima, K.; Wakisaka, Y.; Koizumi, K. J. Antibiot.
1976, 29, 1; (b) Yoshida, M.; Kijima, M.; Akita, M.; Beppu,
T. J. Biol. Chem. 1990, 265, 17174. Synthesis: Mori, K.;
Koseki, K. Tetrahedron 1998, 44, 6013.
4. (a) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.;
Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 3003; (b) Butler, L. M.; Agus,
D. B.; Scher, H. I.; Higgins, B.; Rose, A.; Cordon-Cardo,
C.; Thaler, H. T.; Rifkind, R. A.; Marks, P.; Richon, V.
M. Cancer Res. 2000, 60, 5165; (c) Huang, L.; Pardee, A.
B. Mol. Med. 2000, 6, 849; (d) Gediya, L. K.; Pankaj
Chopra, P.; Purushottamachar, P.; Maheshwari, N.; Njar,
V. C. O. J. Med. Chem. 2005, 48, 5047.
5. For recent reviews, see: (a) Moradei, O.; Maroun, C. R.;
Paquin, I.; Vaisburg, A. Curr. Med. Chem. Anti-Canc.
Agents 2005, 5, 529; (b) Suzuki, T.; Miyata, N. Curr. Med.
Chem. 2005, 12, 2867, and references therein.
15. Bigg, D. C. H.; Lesimple, P. Synthesis 1992, 277.
16. Breslow, R.; Belvedere, S.; Gershell, L.; Miller, T. A.;
Marks, P. A.; Richon, V. M.; Rifkind, R. US Patent
6,511,990-B1, 2003.
17. Toyooka, N.; Okomura, M.; Nemoto, H. J. Org.
Chem. 2002, 67, 6078; (a) Leading references: Bach-
man, W. E.; Struve, W. S. Org. React.; John Wiley
and Sons, Inc., New York, NY, 1942; Vol. 1, p 38;
(b) Newman, M. S.; Beal, P. F., III. J. Am. Chem.
Soc. 1950, 72, 5163. .
18. Cook, G. R.; Manivannan, E.; Underdahl, T.; Lukacova,
V.; Zhang, Y.; Balaz, S. Bioorg. Med. Chem. Lett. 2004,
14, 4935.