Non-natural macrocyclic inhibitors of histone deacetylases: Design, synthesis, and activity

Article abstract of DOI:10.1021/jm101092u

Nonpeptidic chiral macrocycles were designed on the basis of an analogue of suberoylanilide hydroxamic acid (2) (SAHA, vorinostat) and evaluated against 11 histone deacetylase (HDAC) isoforms. The identification of critical amino acid residues highly cons

Full text of DOI:10.1021/jm101092u

J. Med. Chem. 2010, 53, 8387–8399 8387
DOI: 10.1021/jm101092u
Non-Natural Macrocyclic Inhibitors of Histone Deacetylases: Design, Synthesis, and Activity†
‡
‡
‡
Luciana Auzzas,‡,§ Andreas Larsson,‡, Riccardo Matera, Annamaria Baraldi, Benoıt Deschenes-Simard,
^
Giuseppe Giannini,^ Walter Cabri,*,^ Gianfranco Battistuzzi,^ Grazia Gallo,^ Andrea Ciacci,^ Loredana Vesci,^
Claudio Pisano,^ and Stephen Hanessian*,‡
‡
§
Department of Chemistry, Universite de Montreal, P.O. Box 6128, Station Centre-ville, Montreal, QC, H3C 3J7 Canada, Istituto di Chimica
ꢀ
Biomolecolare, Consiglio Nazionale delle Ricerche, Traversa La Crucca 3, 07100 Sassari, Italy, Department of Chemistry, Umea University,
ꢀ
ꢀ
90187 Umea, Sweden, and ^Sigma-Tau Research and Development, Via Pontina Km 30.400, 00040 Pomezia, Roma, Italy
Received August 23, 2010
Nonpeptidic chiral macrocycles were designed on the basis of an analogue of suberoylanilide hydrox-
amic acid (2) (SAHA, vorinostat) and evaluated against 11 histone deacetylase (HDAC) isoforms. The
identification of critical amino acid residues highly conserved in the cap region of HDACs guided
the design of the suberoyl-based macrocycles, which were expected to bear a maximum common
substructure required to target the whole HDAC panel. A nanomolar HDAC inhibitory profile was
observed for several compounds, which was comparable, if not superior, to that of 2. A promising
cytotoxic activity was found for selected macrocycles against lung and colon cancer cell lines. Further
elaboration of selected candidates led to compounds with an improved selectivity against HDAC6 over
the other isozymes. Pair-fitting analysis was used to compare one of the best candidates with the natural
tetrapeptide apicidin, in an effort to define a general pharmacophore that might be useful in the design of
surrogates of peptidic macrocycles as potent and isoform-selective inhibitors.
Introduction
non-histone proteins whose activity is finely tuned upon lysine
modification are being identified, expanding the role and
pharmacological potential of acetylation in human cells.2,3
Among HDAC enzymes, 11 isoforms are known to belong
to the group of zinc-dependent metalloproteins4 and fall into
structurally and functionally different classes (class I, II, and
IV HDACs).5 The majority of HDAC inhibitors affect all the
isoforms with equivalent potency. To date, little is known
about the function and expression for each HDAC class and,
in particular, about the structural origin of selectivity for the
design of class- and/or isoform-selective HDAC inhibitors.6-8
Furthermore, a complete HDAC inhibitory profiling is often
not available, because the development of effective isozyme-
based assays is relatively recent and not yet a common prac-
tice.6c-e,9 Only a few isozyme and class-selective HDAC
inhibitors have been developed recently10 and are undergoing
clinical trials to assess the benefit of selectivity on potency.6a-e
Until further evidence becomes available, promiscuous HDAC
inhibitors are still considered superior and safer than class
I-selective agents, because they inhibit important class II HDAC
isoforms playing a strategic role in cancer therapy, such as
HDAC6, without exhibiting an increased toxicity.6a-e,9b In this
regard, information highlighting specific isoforms as potential
anticancer targets is continuously becoming available,6a-e
calling for the design and synthesis of new selective inhibitors.
The list of known inhibitors of class I and II HDACs covers
a wide cross section of structures,2 including simple natural
products such as trichostatin A (1, TSA),11 their unnatural
surrogates such as suberoylanilide hydroxamic acid (2) (SAHA,
vorinostat),12 and complex macrocyclic natural compounds
such as trapoxin B (3), apicidin A (4), and the recently
launched depsipeptide 6 (FK288, romidepsin) (Figure 1).13
Epigenetic therapy with agents targeting heritable changes
in gene expression that do not involve alteration in the DNA
sequences is a new and rapidly developing area in pharmacol-
ogy.1 Together with DNA methylation and RNA-associated
silencing, important post-translational modifications such as
the ones controlled by histone acetyltransferases (HATs)a and
histone deacetylases (HDACs) are among the most studied
epigenetic mechanisms. By means of a delicate balance of com-
peting activities, HATs and HDACs control the acetylation
status of highly conserved lysine residues of histone proteins,
changing the accessibility of transcription factors toDNA and
thereby affecting the chromatin remodeling process. Pertur-
bations of this balance have been linked to cancer, and
inhibition of HDACs has been shown to have an antiproli-
ferative effect on tumor cell lines, resulting in a considerable
interest in this field.2 In addition, an increasing number of
†Protein Data Bank entries 1T67, 2V5X, and 3C10.
*To whom correspondence should be addressed. S.H.: phone, (514)
343-6738; fax, (514) 343-5728; e-mail, stephen.hanessian@umontreal.
ca. W.C.: phone, þ39-06-9139-4441; fax, þ39-06-9139-3638; e-mail,
walter.cabri@sigma-tau.it.
a Abbreviations: HDAC(s), histone deacetylase(s); HATs, histone acet-
yltransferases; TSA, trichostatin A; SAHA, suberoylanilide hydroxamic
acid; DPPA, diphenyl phosphoryl azide; DEAD, diethyl azodicarboxylate;
DIAD, diisopropyl azodicarboxylate; DIBALH, diisopropylaluminum
hydride; PDC, pyridinium dichromate; EDC, N-ethyl-N0-(3-dimethylami-
nopropyl)carbodiimide; DIPEA, diisopropylethylamine; HOBt, 1-hydroxy-
benzotriazole; DEBPT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-
4(3H)-one; SRB, sulforhodamine B; DBU, 1,8-diazabicyclo[5.4.0]undec-7-
ene; Pip, D-pipecolic acid; Aoda, 2-amino-8-oxodecanoic acid; FCS, fetal
calf serum; OPLS, optimized potentials for liquid simulations; PRCG,
Polak-Ribiere Conjugate Gradient; TNCG, Truncated Newton Conjugate
Gradient; rmsd, root-mean-square deviation; PDB, Protein Data Bank.
r
2010 American Chemical Society
Published on Web 11/12/2010
pubs.acs.org/jmc

8388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Auzzas et al.
Scheme 1. Structures of Lead Compound 7, Proposed Macro-
cyclic Prototype A, and Its Assembly
comparable to 2 against HDAC1-11, and a better antipro-
liferative agent than 2 against three different human cancer
cell lines (NB4, H460, and HCT-116).20 No significant differ-
ence in inhibitory activity was found between the racemic 7
and its individual enantiomers against zinc-dependent
HDACs, and this equivalence found support in modeling of
the individual enantiomers of 7 in the crystal structure of
either HDAC87a-c or HDAC7.7d Two highly conserved Phe
residuesare presentinthe rimoutsideHDACcatalyticsites7c,h
(Phe152 and Phe208 in HDAC8), in opposite orientations,
which were found to ensure favorable π-π interactions with
one aromatic ring or the other (anilido and p-methoxybenzyl
ring) of each enantiomer of 7.21 We reasoned that this
property might be exploited for the design of constrained
analogues of 7, which could be recognized by the enzyme
regardless of their chirality.22 Considering the high level of
conservation of Phe and Asp residues among the HDAC
isoforms (Phe152, Phe208, and D101 in HDAC8),7a,c,h,23
these novel macrocycles are expected, at least in principle,
to bear pharmacophoric determinants close to a maximum
common substructural consensus required to target the whole
HDAC panel, provided that the strong zinc-binding group
hydroxamic acid is used.15,22
Figure 1. Structures of natural and unnatural HDAC inhibitors.
Typically, HDAC inhibitors are substrate mimics of the linear
acetyl lysine sidechain with a zinc-binding group replacing the
scissile acetamide bond and a “cap” motif that binds the rim
outside the active site of the enzyme.7,8 The most effective
inhibitors featurea hydroxamic acidgroupasthe zinc-binding
motif,14 although a significant cap interaction with large
peptide-based macrocycles often leads to a high activity even
in the presence of mild binding groups, resulting in an overall
potency superior to that of 2.15 Moreover, as the rim is
divergent among the different HDAC isoforms,16 cyclic
peptides have the potential to discriminate among them.13
Various analogues of natural cyclic peptides retaining the
activity of the parent natural products have been synthesized,
providing some insights into the structural requirements for
achieving high activity and, in principle, selectivity.15,17 For
instance, Ghadiri and co-workers17a,b defined the pharmaco-
phoric features required to modulate the HDAC activity of
cyclic tetrapeptides inspired by the structure of apicidin 4.
Common geometrical requirements were found within a
libraryofcyclicR3β and R2β2 peptides, which wereused forthe
design of isoform-selective inhibitors based on peptidic archi-
tectures.
Results and Discussion
Simple macrocyclic variants of 2 and 7 were first designed,
according to general structure A (Scheme 1).24 A lipophilic link-
er was chosen to force the branching point at the R-position of
the suberic acid chain in a spatially constrained template.
A small library of macrocycles that can be crafted by the com-
bination of building blocks B-D were designed to be sequen-
tially assembled by a phenolic Mitsunobu reaction or an amide
coupling, followed by the formation of the anilide bond, and a
macrocyclization using a ring closing metathesis reaction
(Scheme 1).
Toassessthe influence of macrocyclesize, we chosea simple
aliphatic linker of various lengths as in racemic compounds
(R/S)-8a-c (Figure 2). Aromatic tethers embedding a
methoxy-substituted benzyloxy motif as in 7 were hence
selected to be introduced into 14-member macrocycles
[compounds 9 and 10 (Figure 2)]. Enantiopure 9 and 10 were
also prepared to be compared to their racemic counterparts.
The synthesis commenced with the preparation of R-ally-
loxy acid building block C (Scheme 1). A common intermedi-
ate was envisaged, (S)- or (R)-11, which readily ensured the
Nonpeptidic macrocycles represent an unexplored alterna-
tive to cyclic peptides.18 By judicious design, they might offer
simplified tools for probing the cap region of HDACs, in view
of the design of analogues with increased potency and,
possibly, selectivity. However, examples of designed unnatu-
ral macrocycles with HDAC activity, such as the synthetic
compound 5 depicted in Figure 1, are somewhat scarce.19
Recently, we showed that the ω-benzyloxy-substituted
suberoyl-based hydroxamic acid 7 (Scheme 1) is an inhibitor

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8389
Scheme 3. Synthesis of Anilines 21b,c and 26
Figure 2. Macrocyclic hydroxamic acids 8-10.
Scheme 2. Synthesis of O-Allyloxy Acid (S)- and (R)-14 and
N-Allylamino Acid (S)-18
Scheme 4. Synthesis of Macrocycles (R/S)-8a-c
preparation of both enantiomers of 14 as well as the
S-configured aza analogue 18 (Scheme 2). O-Allylation of
(S)-1120a with allyl trichloroacetimidate led to (S)-12, which
was desilylated to give (S)-13. Oxidation to an aldehyde
intermediate under Swern conditions followed by Jones oxi-
dation gave carboxylic acid (S)-14 almost quantitatively. In
parallel, (R)-14 was obtained starting from the corresponding
enantiomer (R)-11.20
2-Benzyloxy aniline 26 required for macrocycles 9 and 10
(Figure 2) was prepared by reaction of o-nitrophenol 19 with
benzyl alcohol 24 in the presence of diisopropyl azodicarbox-
ylate (DIAD) and PPh3, and subsequent reduction of the
In a divergent path, intermediate (R)-11 also served for the
synthesis of amino acid (S)-18. Azidation with inversion of
configuration was performed under conventional Mitsunobu
conditions25 and led to (S)-15 in 92% yield. Hydrogenation
under Pd/C catalysis followed by N-Boc protection on the
crude resulting amine gave the protected ester intermediate,
which was smoothly N-allylated under standard conditions
(NaH, allyl iodide at 0 ꢀC) to give (S)-16. In parallel with the
same sequence described for the O-allyl ether analogue (S)-12,
the N-Boc-protected allylamino acid (S)-18 was obtained in
91% yield starting from (S)-16.
nitro derivative 25 with SnCl2 2H2O in quantitative yield
3
(Scheme 3). Intermediate 24 could be efficiently prepared by
a diisobutylaluminum hydride (DIBALH) reduction of the
corresponding methyl ester 23, in turn easily obtained from
2227 by pyridinium dichromate (PDC) oxidation to an alde-
hyde intermediate, followed by a Wittig methylenation (79%
yield for three steps). Alternatively, stryrene derivative 25
could be prepared by desymmetrization of the known benzyl
alcohol 2728 via a Mitsunobu reaction and a subsequent two-
step vinylation of 28.
Macrocycles (R/S)-8a-c were assembled as shown in
Scheme 4. A coupling reaction between racemic carboxylic
acid 14 and anilines 21a-c mediated by N-ethyl-N0-(3-di-
methylaminopropyl)carbodiimide (EDC) led to the corre-
sponding anilides (R/S)-29a-c in yields ranging from 69 to
78%. The ring closing metathesis reaction29 promoted by
2-Alkenyloxy anilines 21b,c26 required for assembling macro-
cycles (R/S)-8b,c (Figure 2) were efficiently prepared via a
phenolic Mitsunobu reaction between o-nitrophenol 19 and
the proper terminal alkenol, followed by reduction of the
resulting nitro derivatives 20b,c with SnCl2 2H2O (Scheme 3).
3

8390 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Scheme 5. Synthesis of Macrocycles 9 and 10a
Auzzas et al.
Figure 3. ORTEP view of (S)-32.
The HDAC inhibitory profile of macrocyclic hydroxamic
acids 8-10 was investigated by measuring their potency in
inhibiting 11 isolated human HDAC isozymes in the presence
of a fluorogenic peptide bound to the RHKK(Ac) fragment of
p53 (residues 379-392) as the substrate, and using 1,11 2,12
and dacinostat (NVP-LAQ824)34 as the reference compounds
(Table 1).20,24,35 Furthermore, their antiproliferative activity
on two human tumor cell lines (H460 and HCT-116) was also
evaluated (Table 2).
a Only the S-isomer is shown.
Macrocyclic compounds embedding an aliphatic linker
[compounds (R/S)-8a-c] showed an HDAC inhibitory pro-
file comparable to that of 2, albeit inferior to that of acyclic
reference compound (R/S)-7. Within this series, an improve-
ment in activity proportional with the increase in macrocycle
size and flexibility was observed. The 14-member ring macro-
cycle (R/S)-8c exhibited an inhibitory activity in the low
micromolar range against 10 HDACs (HDAC1-5 and
7-11) and nanomolar activity against HDAC6 (Table 1).
Macrocycles 9 and 10, embedding an extra aromatic ring
in the linker, displayed the highest activity, with a nanomolar
inhibitory profile against all HDAC isoforms. Only a slight
preference for the S isomer was observed for 9 against all
HDACs [IC50(S/R) = 0.08-0.6]. The same preference was less
evident between the two enantiomers of 10. Compound (S)-9
emerged as the best candidate, displaying a subnanomolar
activity on HDAC6 (0.84 nM) comparable to that of 1 and
activity 10-fold higher than that of dacinostat.
Reduction of the double bond of (S)-9 as in (S)-10 did
not appreciably affect the overall HDAC inhibitory profile
[average IC50(unsat/sat) = 0.5]. The same relationship could be
found also in the racemic and enatiomeric counterparts (R)-9
and (R)-10.
Although the HDAC profile suggested a pan-inhibitory
activity, some preference for the inhibition of HDAC6 was
observed for all the tested compounds [IC50(HDAC6/HDAC2) =
Grubbs’ second-generation catalyst30 in CH2Cl2 proceeded
smoothly, leading to the intermediate macrocyclic olefins as
a mixture of E and Z isomers in good to excellent yield
(72-99%). After reduction of the double bond under H2
and Pd/C, the resulting methyl esters (R/S)-30a-c were
quantitatively converted to the desired hydroxamic acids
(R/S)-8a-c by reaction with HONH2 in MeOH in the pres-
ence of aqueous NaOH.
In preliminary trials, coupling of (S)-14 with aniline 26 with
EDC in the presence of 1-hydroxybenzotriazole (HOBt) and
diisopropylethylamine (DIPEA) led to anilide 31, albeit in a
moderate yield [60% (Scheme 5)]. The same reaction pro-
moted by Goodman’s reagent [(3-diethoxyphosphoryloxy)-
1,2,3-benzotriazin-4(3H)-one (DEPBT)]31 in the presence of
DIPEA proved to be more efficient and was used for anilides
(S)- and (R)-31 (82% yield). Ring closing metathesis in the
presence of Grubbs’ second-generation catalyst in CH2Cl2
produced the desired macrocycles (S)- and (R)-32 in 24-48 h,
but only in a moderate yield (53%), along with homocoupling
side products and unreacted starting material. After optimi-
zation, we found that using the Hoveyda-Grubbs’ second-
generation catalyst32 in refluxing dichloroethane (DCE), the
desired macrocycle could be efficiently obtained in only 2 h in
73-78% average yield. Both conditions provided the unsatu-
rated macrocycle32 as a sole Z isomer. Thisconfiguration was
substantiated by the observation of a coupling constant of
11.2 MHz in the 1H NMR analysis and confirmed by X-ray
analysis of (S)-32 (Figure 3). With both the enantiomers of
the unsaturated macrocycle 32 in hand, the (S)-, (R)-, and
(R/S)-9 hydroxamates were prepared through the pre-
viously described protocol (HONH2, aqueous NaOH in
MeOH). Alternatively, after reduction of the double bond
(H2, Pd/C, nBuNH2)33 to racemic and enantiopure 33, macro-
cyclic hydroxamic acids (S)-, (R)-, and (R/S)-10 could be
obtained.
0.003-0.02].
A promising cytotoxic activity was found for all the macro-
cycles against lung and colon cancer cell lines [H460 and
HCT-116 human cell lines, respectively (Table 2)], which was
comparable or even superior to that of 2. No difference in
cytotoxicity was detected between the two enantiomers of 9 or
between macrocycles 9 and 10.
To rationalize the biological results and potentially reveal
how to increase the activity, we modeled the macrocycles into
the active site of HDAC8 using a flexible ligand/rigid receptor
docking method (see the Experimental Section). Docking

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8391
Table 1. In Vitro Inhibitory Activity (IC50) of Macrocycles 8-10 against Isoforms HDAC1-11a
class I class IIa
class IIb
class IV
HDAC1
HDAC2
HDAC3
HDAC8
HDAC4
HDAC5
HDAC7
HDAC9
HDCA6
HDAC10
HDAC11
1
7.12
258.00
3.23
22.95
921.00
15.70
10.32
350.00
10.50
89.53
243.00
3.84
12.07
493.00
5.82
16.48
378.00
5.58
22.46
344.00
6.11
38.12
316.00
8.24
0.42
28.60
5.93
20.10
456.00
8.41
15.15
362.00
5.58
2
dacinostat
(R/S)-7
(R/S)-8a
(R/S)-8b
(R/S)-8c
(R/S)-9
(S)-9
53.40
254.00
1930.00
1570.00
1540.00
207.00
158.00
382.00
793.00
268.00
425.00
131.00
369.00
176.00
240.00
39.60
331.00
643.00
1100.00
286.00
119.00
198.00
206.00
444.00
136.00
90.70
648.00
2580.00
1200.00
1250.00
159.00
79.40
134.00
1340.00
878.00
695.00
55.40
432.00
1720.00
738.00
764.00
72.60
247.00
1900.00
687.00
1040.00
99.60
20.10
15.60
10.10
9.38
179.00
1530.00
683.00
958.00
70.70
197.00
1210.00
564.00
546.00
57.70
949.00
560.00
439.00
30.70
1.97
0.84
31.70
78.10
55.70
89.00
62.20
27.80
60.60
62.50
22.90
97.10
(R)-9
284.00
447.00
118.00
454.00
155.00
341.00
109.00
150.00
180.00
279.00
61.20
302.00
336.00
123.00
322.00
10.20
8.44
3.43
152.00
320.00
190.00
200.00
(R/S)-10
(S)-10
(R)-10
204.00
72.20
102.00
246.00
93.60
94.50
226.00
103.00
174.00
271.00
3.92
a Values are the means of a minimum of three experiments and are given in nanomolar. For further details, see the Experimental Section.
Table 2. In Vitro Cytotoxic Activity (IC50) of Macrocycles 8-10a
H460 (lung)
HCT-116 (colon)
1
0.100
3.400
0.070
0.520
2.000
1.000
0.620
0.880
1.050
0.820
0.870
0.740
0.680
0.043
1.200
0.018
0.220
0.690
0.580
0.350
0.450
0.690
0.640
0.690
0.520
0.850
2
dacinostat
(R/S)-7
(R/S)-8a
(R/S)-8b
(R/S)-8c
(R/S)-9
(S)-9
(R)-9
(R/S)-10
(S)-10
(R)-10
a Values are the means of a minimum of three experiments and are
given in micromolar. Growth inhibition was assessed by a sulforhod-
amine B (SRB) assay. For further details, see the Experimental Section.
analysis of HDAC87a-c confirmed the adaptability of the
designed macrocycles on the cap region of this isozyme.
Among the macrocycles containing an aliphatic tether, the
best contacts were found with the 14-member cycle (R/S)-8c.
As expected, both R and S isomers were extruded from the
enzyme cavity, forming a π-π interaction with Phe152 and
Phe208, respectively, albeit an interaction weaker than those
observed with (S)- and (R)-7.20a Extra stabilization resulted
from a H-bond interaction between their anilide bond and
Asp101 (Figure SI1 of the Supporting Information).
Figure 4. Predicted mode of binding of (S)-9 in the HDAC8 crystal
structure.
Macrocycles 9 and 10 displayed comparable orientations
and interactions with HDAC8, irrespective of the presence of
the double bond (Figure 4; only (S)-9 is shown). Although
onlyweakinteractions withbotharomatic residues of9 and 10
with Phe152 and Phe208 were observed in HDAC8, favorable
contacts with Tyr100 and Lys33 further stabilized their
binding.21 Docking of R and S isomers of 9 in this isoform did
not account for any difference in their binding, consistent with
the results obtained in the enzymatic test on this isoform
(Table 1) and with our prediction (Figure SI2 of the Support-
ing Information).36
Binding mode analysis of (S)-9 and (S)-10 in HDAC8
revealed the possibility of adding an extra contact point with
the conserved Asp101 residue in the rim of this isozyme by
appropriately introducing a H-donor group within the macro-
cycle (Figure 4). Bioisosteric replacement of the macrocyclic
oxygen bridge of (S)-9 with an amino function was expected
to serve this purpose and prompted the synthesis of the aza
Figure 5. Macrocyclic hydroxamic acids 34-37.
macrocycle (S)-34 (Figure 5). Unexpectedly, a different or-
ientation was observed in docking (S)-34 in HDAC8 as
compared to (S)-9 (Figure 6), and a 30% decrease in the level
of the main interactions seen for the parental compound was
recorded. However, the complex gained stabilization through

8392 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Auzzas et al.
Scheme 7. Synthesis of Aniline 44
Figure 6. Best docking solution of (S)-34 into the HDAC8 crystal
structure.
isolated yield (37% borsm) with Hoveyda-Grubbs’ second-
generation catalyst in refluxing DCE for 48 h. An explanation
for the sluggish reaction can be found in the considerable
steric hindrance of the N-Boc-containing substrate (S)-38 in
comparison to the oxygenated counterpart 31, for which
the substantial formation of homocoupling side products and
a significant decomposition of the starting material account.40
Nevertheless, the desired (S)-34 could be obtained in a quan-
titative yield by converting the methyl ester function in (S)-39
to a hydroxamic acid intermediate and finally deblocking the
N-Boc protection with HCl in dioxane.
Scheme 6. Synthesis of Macrocycle (S)-34
The synthesis of macrocycle (S)-35required thepreparation
of the aniline building block 44 (Scheme 7). After orthogonal
protection of the commercially available aniline 40 by N-Boc
protection and esterification as a methyl ester, phenol 41 was
reacted under Mitsunobu conditions with benzyl alcohol 24,
leading to intermediate 42 in excellent yield (95%). After
reduction of the methyl ester group of 42 to benzyl alcohol
43 with DIBALH, a straightforward four-step sequence
was used to install the acetamido group, which consisted of
(i) azide formation under Thomson’s condition41 and (ii) Stau-
dinger reaction,42 followed by (iii) acetylation of the resulting
amine and (iv) final removal of the N-Boc protection. From this
sequence, aniline 44 was obtained in 67% yield overall.
a salt bridge with Asp101.37 The acetamido analogue (S)-35
was also prepared (Figure 5).38
Coupling product (S)-45, smoothly obtained by reaction of
aniline 44 and carboxylic acid (S)-14 in the presence of
DEPBT (Scheme 8), was cyclized under the same condition
described for anilide 31 (Hoveyda-Grubbs’ second-generation
catalyst in DCE at reflux temperature) and led to (S)-46 as a
sole Z isomer in moderate yield (55%). The presence of the
acetamido group in (S)-45, which may coordinate the ruthe-
nium center in the catalyst, and the difficulty in isolating
(S)-46 because of its high polarity might explain the reasons
for the low recovery after the metathesis reaction. After
Furthermore, diamido isosteres of macrocycles 9, as well
as an indole-containing macrocycle, were envisaged as prom-
ising analogues [compounds (R)- and (S)-36 and (S)-37
(Figure 5)]. We hoped these macrocycles would benefit from
enhanced interactions with the conserved Phe residues of
HDACs (Phe152 and Phe208 in HDAC8) as compared to
the parental compound 9 thanks to the additional CONH
functionality, which was thought to offer extra stabilization
by forming a H-bond with Asp101 (Figures SI4 and SI5 of the
Supporting Information).39
Nitrogen-containing macrocycle (S)-34 was synthesized
analogously to its oxygenated counterpart 9 (Scheme 6).
Unfortunately, anilide (S)-38, prepared by coupling aniline 26
with acid (S)-18 in the presence of DEPBT, underwent the
metathesis reaction to macrocyclic olefin (S)-39 in only 26%
n
reduction of the double bond (H2, Pd/C, BuNH2),33 direct
conversion of the methyl ester function in (S)-47 to hydro-
xamic acid afforded (S)-35 in quantitative yield in two steps.
In parallel with the general Scheme 1, diamide-containing
macrocycles (R)- and (S)-36 and 37 were prepared by combin-
ing allyloxy acid (R)-14 and/or (S)-14 with the appropriate
aniline building blocks 52 and 59 (Scheme 9, only the S

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8393
Scheme 10. Synthesis of Macrocycle (S)-37
Scheme 8. Synthesis of Hydroxamic Acid (S)-35
Scheme 9. Synthesis of Macrocycles (S)- and (R)-36a
(R)- and (S)-53 in 77% yield. Ring closing metathesis in the
presence of 10 mol % Hoveyda-Grubbs’ second-generation
catalyst afforded macrocycle (R)- and (S)-54 after refluxing
for 2 days in DCE (25% yield).44 The usual treatment of
methylesters (R)- and(S)-54withHONH2 in aqueous NaOH,
MeOH, and THF afforded the desired hydroxamic acids
(R)- and (S)-36.
Indole-containing macrocycle (S)-37 was prepared in an
analogous way (Scheme 10). Carboxylic acid 58 was chosen as
the precursor for the preparation of the requisite aniline 59.
Conversion of N-tosyl indole 55 to 2-allyl indole 56 was
performed following conventional methods.45 After TiCl4-
promoted formylation,46 aldehyde 57 was smoothly converted
to carboxylic acid 58 and coupled with phenylendiamine 49
(70% yield for two steps). The resulting N-Boc-protected
aniline 59 was quantitatively converted to the aniline hydro-
chloride salt intermediate, which was used without further
purification for the subsequent coupling reaction with acid
(S)-14. Diamide (S)-60 was obtained in good yield under
conventional Goodman’s conditions. After cyclization to
the olefin intermediate in the presence of Grubbs’ second-
generation catalyst, and reduction of the olefin intermediate
with H2 and Pd/C to macrocycle (S)-61, N-detosylation was
achieved in the presence of a Na/Hg alloy in THF.47 Final
conversion to the desired hydroxamic acid (S)-37 was per-
formed in the conventional way.
a Only the S isomer is shown.
isomer is shown; Scheme 10). Coupling of the orthogonally
protected aniline 4943 with the known benzoic acid 4827 in the
presence of DEPBT gave anilide 50 in 97% yield. Upon
reduction of the methyl ester group with DIBALH, vinylation
was performed as previously described in 87% yield for two
steps. Following the acid-mediated N-Boc deprotection of 52
tothe aniline intermediate, couplingwithacids(R)- and (S)-14
was performed using the usual protocol and led to diamide
The biological activity of macrocyclic hydroxamic acids
34-37 is reported in Tables 3 and 4. In general, a good
inhibitory profile was observed with close analogues of
macrocycles 9 and 10. The replacement of the oxygen at the
R-position of the suberoyl chain of (S)-9 with a nitrogen atom,
as in (S)-34, did not appreciably modify the overall activity.
The main difference was observed on HDAC8, whose level
of inhibition was 5-fold higher with (S)-34 than with (S)-9.

8394 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Auzzas et al.
Table 3. In Vitro Inhibitory Activity (IC50) of Macrocycles 34-37 against Isoforms HDAC1-11a
class I
class IIa
class IIb
class IV
HDAC1
HDAC2
HDAC3
HDAC8
HDAC4
HDAC5
HDAC7
HDAC9
HDCA6
HDAC10
HDAC11
2
258.00
31.70
27.70
921.00
158.00
171.00
128.00
3280.00
2530.00
584.00
350.00
55.70
15.80
243.00
198.00
39.30
493.00
79.40
52.10
378.00
62.20
24.70
344.00
27.80
15.90
316.00
60.60
22.60
28.60
0.84
0.75
456.00
62.50
46.50
362.00
22.90
34.80
(S)-9
(S)-34
(S)-35
(S)-36
(R)-36
(S)-37
41.30
63.10
45.40
64.80
65.20
34.30
47.70
0.40
65.10
45.90
864.00
1340.00
245.00
1114.00
885.00
331.00
534.00
682.00
297.00
5333.00
3480.00
1690.00
1660.00
1340.00
496.00
4300.00
2070.00
2230.00
2020.00
1140.00
602.00
59.00
75.00
4.40
2290.00
1880.00
627.00
948.00
1110.00
379.00
a Values are the means of a minimum of three experiments and are given in nanomolar. For further details, see the Experimental Section.
Table 4. In Vitro Cytotoxic Activity (IC50) of Macrocycles 34 and 35a
H460 (lung)
HCT-116 (colon)
2
3.400
1.050
0.510
6.800
1.200
0.690
0.400
3.740
(S)-9
(S)-34
(S)-35
a Values are the means of a minimum of three experiments and are
given in micromolar. Growth inhibition was assessed via the SRB assay.
For further details, see the Experimental Section.
The introduction of an acetamido group onto the anilide ring
of (S)-10, as in (S)-35, produced a moderate increase in
inhibitory activity. As expected, (S)-34 and (S)-35 exhibited a
pan-inhibitory activity, with a subnanomolar activity against
HDAC6 comparable to that of 1 and an activity 10-fold
higher than that of dacinostat.
Although the diamide-based macrocycles 36 and 37 exhib-
ited an overall decrease in activity, a notable selectivity toward
HDAC6 was observed (10-500-fold over the other HDACs)
(Table 3). This preference might be explained by the presence
of a hydrophobic region composed of several aromatic resi-
dues surrounding the Phe566 residue in the rim of the HDAC6
channel (Tyr100 in HDAC8), which might accommodate
the phenyl and indole rings of these relatively rigid macro-
cycles.10c,48
The cytotoxic activity of macrocycle (S)-34 against lung
and colon cancer cell lines [H460 and HCT-116 human cell
lines, respectively (Table 4)] was higher than that of 2. On the
other hand, the acetamido group-containing (S)-35 exhibited
a lower activity.
Figure 7. (A) Rigid alignment of the crystal structure of (S)-9
(yellow) and apicidin A (4) (green). (B) Rigid alignment of the
crystal structure of (R)-9 (yellow) and 4 (green). (C) Two-
dimensional (2D) model based on the structure of (S)-9 (yellow)
and 4 (green). (D) 2D model based on the structure of (R)-9 (yellow)
and 4 (green). In panels C and D, dihedral angles (φ) and distances
between key determinants (angstroms) are given. Apicidin A (4)
is shown as a cis-trans-trans-trans conformer,49 consistent with
recent findings by Ghadiri and co-workers.50 Pip denotes D-
pipecolic acid. Aoda denotes a 2-amino-8-oxodecanoic acid resi-
due. Only the first methylene was considered in the pair-fitting
analysis. Details of the calculation are available in the Experi-
mental Section.
Overall, only the aza analogue (S)-34 emerged as a candi-
date as good as its oxygenated counterpart.
Despite their rigid structural arrangement and the presence
of a stereogenic center, macrocycles 9 and 34-37 embed
critical determinants opportunely placed for the indiscrimi-
nate binding of each of the 11 HDACs through highly con-
served residues in their cap region. With the objective being
an improved understanding of the properties of these macro-
cycles, a ligand-based study considering their superimposition
to a known, selective inhibitor was performed, aiming at the
individualization of overlaid conformations displaying a maxi-
mum geometrical overlap of relevant chemical features, such
as H-bond donors and acceptors, hydrophobic groups, etc.
Hence, a pair-fitting analysis was performed by the rigid
alignment of each enantiomer of 9 with a prototypical natural
tetrapeptide, apicidin A (4), a well-known class I HDAC inhib-
itor (Figure 1).13b,49,50 We speculated that, despite the struc-
tural diversity between our synthetic macrocycles (S)- and
(R)-9 and 4, minimal but strategic contact points could be
revealed, possibly opening the way for the definition of a
simplified pan-HDAC inhibitory model.
Several observations emerged from the superimposition
of each enantiomer of 9 on the crystal structure of 4
(Figure 7).49,50 A good overlapping was revealed between
the expected key determinants in the S and R enantiomers of 9
and segments of apicidin A (4). In the rigid alignment of (S)-9
and 4 (Figure 7A,C), the suberic aliphatic chains pointed in
the same direction, albeit with a different angle. The aniline
ring overlaid on the indole five-member ring of 4, while the
methoxyphenyl ring of (S)-9 covered the region occupied by
the Leu side chain. The amide bonds between suberic acid and
anilinein(S)-9, and betweensuberic acidand Trp in4, showed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8395
good overlap as well, with both N-H bonds pointing inward.
Despite the opposite alignment, (R)-9 revealed a similar
overlay(Figure7B,D). Whilethe aromatic moieties linedupin
an opposite fashion, both the orientation of the suberic side
chain and the amide N-H bond retained a good alignment.
Interestingly, no segment of the isomers showed any overlap
with the six-membered piperidine ring of apicidin A (4). The lack
of selectivity between the enantiomers of 9 and HDACs can
be due to the virtual similarity of the relative positions of
pharmacophores (Figure 7C,D).
These pharmacophoric models highlight the role of 9 as a
prototype for probing the cap region upon judicious embodi-
ment of appropriate appendages, which might provide valu-
able structural, functional, and stereochemical insights into
the design of new inhibitors.
American Type Culture Collection. HCT-116 cells were grown
in McCoy’s 5A and NCI-H460 cells in RPMI-1640, both con-
taining L-glutamine supplemented with 10% heat-inactivated
fetal calf serum (FCS, Life Technologies) and 50 μg/mL genta-
micin.
Cell Proliferation Assay. HCT-116 and NCI-H460 tumor cell
lines were grown in a volume of 200 μL at approximately 10%
confluency in 96-well multititer plates and were allowed to
recover for an additional 24 h. Tumor cells were treated with
either varying concentrations of drugs or solvent for 24 h. After
the cells had been treated, the plates were washed to remove drug
and incubated for 48 h. The fraction of cells surviving after
compound treatment was determined using the SRB assay.51
IC50 was defined as the drug concentration causing a 50% reduc-
tion in cell number compared with that of vehicle-treated
cells and evaluated by the “ALLFIT” computer program by
analyzing dose-response inhibition curves.
Histone Deacetylase Profiling. HDAC profiling was performed
by Reaction Biology Corp. (Malvern, PA) against 11 isolated
isoforms of human HDAC (HDAC1-11) in the presence of the
fluorogenic tetrapeptide RHKKAc (p53 residues 379-382) as
the substrate (10 μM). Isolated human HDACs were obtained
by standard purification, with the exception of HDAC3, which
was isolated in complex with NCOR2 and used as such. Inhib-
itors 1, 2, and dacinostat were used as reference compounds.
Each compound was dissolved in DMSO (1 and 2 at 10 μM), and
sequentially diluted solutions were used for testing. IC50 values
were calculated from the resulting sigmoidal dose-response
inhibition slopes.
Conformational Analysis. Macrocycles were opened, and all
the single bonds were allowed to rotate (0-180ꢀ) with the
exception of hydroxamic acid and amide groups. Torsion checks
were applied to amide and double bonds to preserve the trans
and cis geometry, respectively. An implicit solvent model was
used with the OPLS-2001 force field (Optimized Potentials for
Liquid Simulations); all heavy atoms were used as a comparison,
and 10000 steps of the Monte Carlo Multiple Minimum algo-
rithm was used followed by 500 steps of Polak-Ribiere Con-
jugate Gradient (PRCG) minimization with a convergence
threshold of 0.05. A multiple minimization was conducted with
the aim of retaining mainly conformers with different macro-
cycle geometry, using 500 steps of Truncated Newton Conjugate
Gradient (TNCG) minimization with a convergence threshold of
0.001. Conformers in the range of potential energy of 20 kJ/mol
were retained, and a Boltzmann analysis was performed on the
population minima. Calculations were performed using Macro-
Conclusion
On the basis of structural data on HDACs, we have re-
ported the synthesis, biological activity, and pharmacophoric
characterization of novel non-natural macrocyclic compounds
as potential inhibitors of these enzymes. A small library of
macrocyclic hydroxamic acids based on an analogue of 2
was prepared (compound7),20 which displayedpotent activity
against 11 HDAC isoforms. A structure-based hypothesis
guided the design of the novel macrocycles, in which the
minimal functional requirements for HDAC activity were
maintained. Critical amino acid residues highly conserved in
the cap region of HDACs were taken into account in the
design. The novel compounds were crafted following a uni-
form hierarchical approach relying on the combination of
three building blocks, which were sequentially assembled by
a phenolicMitsunobureactionoranamidecoupling, followed
by the formation of an anilide bond, and a ring closing
metathesis reaction. All macrocycles were designed to contain
a single stereogenic center, which led to enantiomers with an
equivalent activity for the most promising hits. Macrocycles
embedding two simple aromatic rings in the linker displayed a
nanomolar activity profile against all isoforms [hydroxamic
acids 9, 10, (S)-34, and (S)-35]. As expected for hydroxamic
acid-containing HDAC inhibitors,15,22 a preferential activity
against HDAC6 in the nanomolar range was observed for all
macrocycles. In particular, a relevant selectivity toward this
isoform was detected within the group of diamide-based
macrocycles (R)- and (S)-36 and (S)-37 (10-500-fold over
the other HDACs). Giventhe roleof HDAC6indeacetylating
R-tubulin and Hsp90, a high activity against this isozyme
might be beneficial.6 An excellent cytotoxic activity was also
found for selected macrocycles against lung and colon cancer
cell lines (H460 and HCT-116, respectively), often compar-
able or even superior to 2.
€
model version 9.6 (Schrodinger, LLC, New York, NY).
Macrocycles 8-10 and 34-37 revealed a preferential orienta-
tion of the trans-amide bond among the minimum conformers,
which displayed the N-H bond pointing inside the cycle and
eclipsing the adjacent aromatic ring ((20ꢀ). An opposite orien-
tation could be observed when ΔE > 7 kj/mol. In the case of 9,
the first four minimum energy conformers showed the aliphatic
chain collapsed toward the macrocycle portion, interacting with
the hydroxamic acid moiety via an intramolecular H-bond. The
fifth conformer (ΔE = 4.684 kj/mol, Boltzmann population
of 6%) displayed a geometry very similar to that of the crystal
structure of (S)-32 (rmsdheavy = 0.31 A) and was chosen for
docking analysis. Conformer populations of the other cyclic
compounds were compared in an analogous way, and the
macrocycle conformation closest to the one observed in the
crystal structure of (S)-32 was chosen as a starting conformation
for docking studies.
Docking Procedures. A structure of HDAC8 cocrystallized
with 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-
benzamide (MS-344) (PDB entry 1T67) was used for docking
procedures. A conserved water molecule, H-bonded to His180
and interacting with the ligand, was retained. The protein was
prepared according to the protein preparation protocol imple-
mented in Maestro. Tyr100 dihedral χ1 and χ2 angles were
Unnatural macrocyclic hydroxamic acids (S)- and (R)-9,
together with the aza analogue (S)-34, emerged as the promis-
ing candidates from this study. Ligand-based analysis by rigid
alignment of both enantiomers of 9 with the natural HDAC
inhibitor apicidin A (4) led to the definition of general phar-
macophoric models. Further refinement of these novel non-
natural macrocyclic templates gives us hope for more potent
and selective inhibitors of HDACs.
Experimental Section
Biological Assays. The purity of all tested compounds was
assessed by HPLC and was g95%.
Cell Lines. HCT116 colon carcinoma cells and NCI-H460
non-small cell lung carcinoma cells were obtained from

8396 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Auzzas et al.
modified to increase the number of interactions with the macro-
cycles, in accord with the intrinsic mobility of this residue7a [see,
for example, HDAC8 (PDB entry 2V5X)].
A structure of HDAC7 cocrystallized with 1 (PDB entry
3C10) was used for further modeling analysis. A conserved
water molecule, H-bonded to His709 and interacting with 1,
was retained. The protein was prepared according to the protein
preparation protocol.
Flexible docking was applied on each ligand, and a constraint
was applied to allow the hydroxamic acid to correctly interact
with Zn2þ. Ten poses were recorded; the pose with the best
Emodel was used to compare score values. A further ligand
minimization was performed within the active site, kept fixed,
and out of it, and ΔE was calculated to evaluate the strain energy
of the docking pose. All docking calculations were performed
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using Glide version 5.0 (Schrodinger, LLC). The binding site,
for which the energy grid was calculated and stored, was defined
with a cubic “bounding box” centered at the centroid of the re-
spective cocrystallized ligands {4-(dimethylamino)-N-[7-(hydroxy-
amino)-7-oxoheptyl]benzamide in HDAC8 and 1 in HDAC7},
with an edge length of 12 A in both proteins, and an “enclosing
box” that would fit ligands up to lengths of 32 A (HDAC8) and
28 A (HDAC7).
Pharmacophore Model. The crystal structures of apicidin A
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CCDC 274844)49 and (S)-32 were modified by deletion of the
Aoda and suberoyl hydroxamic acid tail, respectively. A rigid
body alignment was conducted in MOE [Molecular Operating
ꢀ
Enivroment (CCG, Montreal, QC)] using the default settings.
The alignments were manually examined, and all alignments
having different trajectories of the tail region were disregarded.
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ꢀ
Dalbir Sekong, and Dr. Sylvie Bilodeau of CentreRegional de
Spectroscopie and of Service de RMN de l’Universite de
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´
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ꢀ
ꢀ
Montreal for their kind assistance. L.A. thanks the National
Research Council of Italy (CNR), Institute of Biomolecular
Chemistry, for a sabbatical leave. A postdoctoral grant from
the Wenner-Gren Foundation, Sweden, is kindly acknowl-
edged by A.L.
Supporting Information Available: Synthetic procedures;
1H and 13C NMR spectra of methyl esters (R/S)-30a-c, (S)-32,
(S)-33, (S)-39, (S)-47, (S)-54, and (S)-61 and hydroxamic acids
(R/S)-8a-c, (S)-9, (S)-10, (S)-34, (S)-35, (S)-36, and (S)-37;
further docking experiments (Figures SI1-SI5); and a CIF file
and crystallographic data for (S)-34. This material is available
free of charge via the Internet at http://pubs.acs.org.
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