Non-peptide macrocyclic histone deacetylase inhibitors derived from tricyclic ketolide skeleton

Article abstract of DOI:10.1021/jm100507q

Inhibition of histone deacetylase (HDAC) function is a validated therapeutic strategy for cancer treatment. Of the several structurally distinct small molecule histone deacetylase inhibitors (HDACi) reported, macrocyclic depsipeptides possess the most complex cap groups and have demonstrated excellent HDAC inhibition potency and isoform selectivity. Unfortunately, the development of macrocyclic depsipeptides has been hampered in part because of development problems characteristic of large peptides and the complex reaction schemes required for their synthesis. Herein we report that tricyclic ketolide TE-802 is an excellent mimetic for the peptide backbone of macrocyclic HDACi. Compounds derived from this template are particularly selective against HDACs 1 and 2 with nanomolar inhibitory activity. Interrogation of the association between a subset of these compounds and key HDAC isoforms, using AutoDock, enables a molecular description of the interaction between the HDAC enzyme's outer rim and the inhibitors' macrocyclic cap group that are responsible for compound affinity and presumably isoform selectivity.

Full text of DOI:10.1021/jm100507q

6100 J. Med. Chem. 2010, 53, 6100–6111
DOI: 10.1021/jm100507q
Non-Peptide Macrocyclic Histone Deacetylase Inhibitors Derived from Tricyclic Ketolide Skeleton
Sandra C. Mwakwari,† William Guerrant,† Vishal Patil,† Shabana I. Khan,§ Babu L. Tekwani,§ Zachary A. Gurard-Levin,
Milan Mrksich, and Adegboyega K. Oyelere*,†,‡
†School of Chemistry and Biochemistry, and ‡Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology,
Atlanta, Georgia 30332-0400, §National Center for Natural Products Research and Department of Pharmacology, School of Pharmacy,
University of Mississippi, University, Mississippi 38677-1848, and Department of Chemistry and Howard Hughes Medical Institute,
University of Chicago, Chicago, Illinois 60637-1511
Received April 26, 2010
Inhibition of histone deacetylase (HDAC) function is a validated therapeutic strategy for cancer treatment. Of
the several structurally distinct small molecule histone deacetylase inhibitors (HDACi) reported, macrocyclic
depsipeptides possess the most complex cap groups and have demonstrated excellent HDAC inhibition
potency and isoform selectivity. Unfortunately, the development of macrocyclic depsipeptides has been
hampered in part because of development problems characteristic of large peptides and the complex reaction
schemes required for their synthesis. Herein we report that tricyclic ketolide TE-802 is an excellent mimetic for
the peptide backbone of macrocyclic HDACi. Compounds derived from this template are particularly
selective against HDACs 1 and 2 with nanomolar inhibitory activity. Interrogation of the association between
a subset of these compounds and key HDAC isoforms, using AutoDock, enables a molecular description of
the interaction between the HDAC enzyme’s outer rim and the inhibitors’ macrocyclic cap group that are
responsible for compound affinity and presumably isoform selectivity.
Introduction
suberoylanilide hydroxamic acid (SAHA) (approved in 2006 by
the FDA for the treatment of cutaneous T-cell lymphoma
(CTCL)15,16), benzamides, short chain fatty acids, electophilic
ketones, and cyclic peptides such as FK-228 (romidepsin) which
was recently approved by the FDA17,18 for the treatment of
CTCL in patients who have received at least one prior systemic
therapy (Figure 1b,c).19,20 However, most of these drugs non-
specifically inhibit various HDAC isoforms. At the fore of HD-
AC drug development is the identification of isoform-selective
HDACi with the potential for enhanced potency and reduced
side effects, compared to the current pan-HDACi. However,
these efforts have been so far modestly successful, resulting in
only few HDACi that demonstrate partial selectivity.21,22
On the other hand, macrocyclic peptide HDACi have the
most complex recognition cap group moieties and present an
excellent opportunity for the selective modulation of the bio-
logical activities of HDACi. Although they possess potent
HDAC inhibition activity (nanomolar range), their progress
through clinical trials has been slow.17,18,23 The paucity of
clinically effective cyclic-peptide HDACi may be in part due
to development problems characteristic of large peptides, most
especially poor oral bioavailability. Identification of non-
peptide macrocyclic HDACi will offer a new class of macro-
cyclic HDACi with potentially more favorable druglike proper-
ties. Furthermore, this will enhance our understanding of the
roles of specific interactions between the enzyme outer rim and
inhibitor cap groups in HDACi activity and ultimately aid in
the identification of more isoform-selective HDACi.
Histone deacetylase (HDACa) and histone acetyltransfer-
ase (HAT) are two functionally opposing enzymes, many of
which tightly regulate the chromatin structure and function
via sustenance of equilibrium between the acetylated and
deacetylated states of histones. By catalyzing the removal of
acetyl groups, HDACs induce a condensed chromatin struc-
ture resulting in transcription repression, whereas acetylated
histones are associated witha moreaccessible/open chromatin
structure and activation of transcription.1-4 In addition,
many non-histone proteins such as tubulin, ERR, p53, HSP
90, NF-YA, and GATA-1 have been found in an acetylated
state and may be substrates of HDACs.5-10 Eighteen human
HDAC isoforms are known, and they are subdivided into the
classical zinc dependent HDACs comprising classes I, II, and
IV and NADþ dependent sirtuins, class III enzymes.9,11,12
HDAC inhibitors (HDACi) are an emerging class of novel
anticancer drugs with a demonstrated ability to arrest prolifera-
tion of nearly all transformed cell types, including epithelial
(melanoma, lung, breast, pancreas, ovary, prostate, colon, and
bladder) and hematological (lymphoma, leukemia, and multiple
myeloma) tumors.13 To date, several classes of small molecule
HDACi (fitting a three-motif pharmacophoric model, namely,
a zinc-binding group (ZBG), a hydrophobic linker, and a
recognition cap group14 (Figure 1a)) have been reported.
Examples include hydroxamic acids such as trichostatin A (TSA),
*To whom the correspondence should be addressed. Phone: 404-894-
4047. Fax: 404-894-2291. E-mail: aoyelere@gatech.edu.
Recently we reported that non-peptide macrocyclic skeletons
derived from 14- and 15-membered macrolides are suitable
as surrogates for the cap groups of macrocyclic HDACi
(Figure 1d). The resulting HDACi have improved enzyme
inhibition potency and isoform-selectivity.24 Herein, we report
a Abbreviations: HDAC, histone deacetylase; HAT, histone acetyl-
transferase; HDACi, histone deacetylase inhibitors; HDLP, histone
deacetylase-like protein; SAHA, suberoylanilide hydroxamic acid;
TSA, trichostatin A; ZBG, zinc-binding group; MALDI, matrix assisted
laser desorption/ionization; SAM, self-assembled monolayers.
r
pubs.acs.org/jmc
Published on Web 07/29/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6101
Figure 1. (a) Pharmacophoric model of HDACi and representative examples of (b) acyclic, (c) cyclic peptide, and (d) macrolide-based HDAC inhibitors.
that enhancement of the 14-membered macrolide ring hydro-
phobicity and rigidity facilitates specific drug interactions with
the enzyme’s outer rim residues, maximizes HDAC inhibition,
and improves drug cytotoxicity against human cancer cell lines.
Moreover, these compounds have antiparasitic activities against
causative parasites of malaria and leishmaniasis in a manner
that reveals structural attributes that confer a specific anti-
parasitic response.
macrolides clarithromycin and azithromycin, respectively,
using histone deacetylase-like protein (HDLP), revealed the
structural basis for the enhanced activity of these macrocyclic
compounds. Either ring system adopted docked orientations
that displayed molecular surface complementarities between
the macrolide skeleton and the enzyme outer rim.24 In these
docked structures, most of the hydrophobic components of the
macrolide ring optimally interact with the hydrophobic resi-
dues within HDLP hydrophobic pockets. Common to both
ring systems are the vicinal diols at C11 and C12 and at C12 and
C13 for clarithromycin and azithromycin, respectively, which
have to be accommodated within the enzyme hydrophobic
Results and Discussion
Molecular Docking Analysis. Previous molecular dock-
ing studies on HDACi derived from 14- and 15-membered

6102 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Mwakwari et al.
Figure 2. Representative examples of ketolides.
Figure 3. Docked structures of ketolide-derived HDACi at the active site of HDAC1. (a) Superposition of the low energy conformation of IIIb
(gray) and 15b (green) revealed the pocket binding preferences of inhibitors at the HDAC1 surface. (b) Relative orientation of the macrocyclic
rings of 15a (pink) and 15b (green) within the hydrophobic pocket on HDAC1 outer rim.
pocket. We postulated that refinement of the ring hydropho-
bicity through appropriate modification of the vicinal diols
could further modulate the HDAC inhibition of these non-
peptide macrocyclic HDACi. In the previous report, we ob-
served that the two macrolide skeletons contributed distinctly to
the overall HDAC binding. For 14- and 15-membered macro-
lides with C5 and C6 linkers, the 14-membered compounds are
about 2- to 5-fold more potent HDACi than their 15-membered
counterparts.24 On the basis of the foregoing, we focused on the
transformation of the 14-membered macrocyclic template.
Conversion of the 14-membered ring vicinal diols to various
five-membered carbamates is a common and synthetically trac-
table approach toward their transformation to relatively more
hydrophobic ketolide skeletons (Figure 2).25-27 Thetemplateof
ketolide 10 (TE-802),25 in which the carbamate NH group has
been further substituted with a small hydrophobic group that
results in a tricyclic ring system, is very attractive in this regard.
The rigid template of 10 minimally perturbs the ring within the
region of interest while maximizing ring hydrophobicity and
should be compatible with the enzyme outer rim hydrophobic
pocket. To verify this prospect, we proceeded to interrogate the
binding interaction between HDAC1 homology model built
from human HDAC2 X-ray structure 3MAX coordinates28
and the non-peptide macrocyclic hydroxamates 15a,b, derived
from the ketolide 10, using AutoDock as described previously.
We focused on the C5 and C6 linked compounds 15a,b because
these spacer lengths are optimal for the anti-HDAC activity of
the previous non-peptide macrocyclic HDACi.24 Molecular
docking analysis revealed that the enhanced hydrophobicity
and/or rigidity of the ketolide moiety of the C5 and C6 linker
compounds 15a and 15b, respectively, forced their rings to
adopt docked orientations that are distinct from the corre-
sponding macrolide template whose ZBG makes similar inter-
action with the active site Zn2þ ion (Figure 3a). The desosamine
0
˚
sugar 2 -OH group of both 15a and 15b is within 1.9 A of the
backbone carbonyl group of Tyr24 with which it could poten-
tially engage in a stabilizing H-bonding interaction (see Sup-
porting Information Figure S1a). Relative to that of 15a, the
ketolide ring of 15b fits optimally into a hydrophobic pocket on
HDAC1 outer rim. The aryl moiety of 15b is partly stacked on
top of His28 that forms the base of a hydrophobic cleft at the
entrance to a pocket-like active site (Figure 3b and Supporting
Information Figure S1b). The orientation of the aryl moiety
enables the C6-linker methylene group to efficiently present
the hydroxamate ZBG to the active site Zn2þ ion. To accom-
modate for the loss of one methylene group and consequently
optimally interact with the active site zinc, the ketolide ring
of 15a is slightly lifted out of the pocket while its aryl moiety
is twisted, almost perpendicular to the aryl moiety of 15b, to
make a minimal edge contact with His28 at the pocket entrance
cleft. In this orientation, 15a is more solvent exposed relative
to 15b (Figure 3b). The observed disparities in the docked
orientations of 15a and 15b may have implications on their
affinity for the enzyme.
Chemistry. To experimentally test these in silico observa-
tions, we synthesized compounds 15a-e. Our proposed
synthesis of the requisite compounds involves the intermediacy
of 4-ethynylbenzylketolide 9. We envisioned that 9 could be
accessed via two different routes (A and B) whereby the
4-ethynylbenzyl group is introduced early or late in the synthesis

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6103
Scheme 1. Synthesis of Tricyclic Ketolide 9 from Clarithromycin 1
Scheme 2. Synthesis of Tricyclic Ketolide 9 from Ketolide 10
(Schemes 1 and 2, respectively). It is gratifying to note that both
routes worked equally well in furnishing intermediate 9.
formation. Selective acetylation of the 20-OH group was accom-
plished by treating an acetone solution of 5with acetic anhydride
at 40 °C for 48 h to give compound 6 in 70% yield. Subsequent
oxidation of the 3-hydroxyl group of compound 6 to a 3-keto
functional group, under anhydrous conditions with NCS, af-
forded the ketolide 7 in a near quantitative yield. Treatment
of 7 with excess carbonyldiimidazole (CDI) and NaHMDS in a
mixture of THF/DMF afforded the 12-carbamoylimidazolide
ketolide 8 in 52% yield. Transformation of 8 into the desired
Demethylation of clarithromycin 1under standard conditions
gave 30-desmethyl-clarithromycin 2 in 70% yield.29 Subsequent
alkylation of 2 with 4-ethynylbenzyl methanesulfonate afforded
the modified 4-ethynylbenzylclarithromycin 4 which was treated
with 1 N HCl to selectively cleave the cladinose sugar and afford
compound 5. It was observed for this reaction step that time was
of essence, as longer reaction times led to extensive byproducts

6104 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Mwakwari et al.
Scheme 3. Synthesis of Triazole-Linked Tricyclic Ketolide Hydroxamates 15
Table 1. HDAC Inhibition Activity (IC50) and Isoform Selectivity of Tricyclic Ketolide-Based HDAC Inhibitors
HDAC8/nuclear
extract isoform
selectivity
HDAC6/nuclear
extract isoform
selectivity
nuclear extract
(nM)
HDAC8
(nM)
HDAC6
(nM)
compd
n
15a
15b
1
2
3
4
5
7.77
1.03
796.2
544.6
102.5
528.7
18.3
17.5
21.9
29
1180.1
728.7
151.9
707.5
16.4
11.7
15.4
1.3
15c
15d
104.2
163.6
208.2
65.0
1909.3
2859.9
4557.8
1860.0
1709.8
1916.9
3203.1
85.5
15e
SAHAa
a Cited from ref 22.
tricyclic ketolide 9 was achieved in two successive cyclization
steps adapting literature procedures.25 Reaction of imidazolide 8
with ethylenediamine followed by intramolecular Michael addi-
tion led to the formation of 11,12-cyclic carbamate. Subsequent
intramolecular dehydration completed the cyclization process,
affording the desired product 9 in 44% yield (Scheme 1).
Alternatively, intermediate 9 could be directly obtained
from 10. We synthesized 10 from clarithromycin 1 according
to a literature procedure.25,26,30,31 Subsequent N-demethyla-
tion of 10 using diethyl azodicarboxylate (DEAD) gave the
expected N-desmethyl ketolide 11 in 62% yield.31 Reductive
amination of 11 with 4-ethynylbenzaldehyde using borane-
pyridine complex (BAP) afforded the requisite intermediate
9 in 78% yield (Scheme 2).
The final transformation of 9 to 15a-e was uneventful
(Scheme 3). Copper(I) catalyzed cycloaddition reaction24 of
9 with O-trityl protected azidohydoxamate analogues 12a-e
afforded the 1,2,3-triazole linked derivatives 14a-e. Depro-
tection of the trityl group of 14a-e with TFA gave the
desired products 15a-e in good yields. A similar outcome
was obtained when trityl group deprotection was effected
Fluor de Lys assay kit (Enzo Life Sciences, Inc.).32-34 Results
show a linker-length-dependent anti-HDAC activity which
peaked with compound 15b, an analogue having six methylene
linkers separating the triazole ring from the zinc binding hydro-
xamic acid group (Table 1). Compound 15b potently inhibits the
deacetylase activity of HeLa nuclear extract with a single digit
nanomolar IC50. Although compound 15a (analogue having
five methylene linkers) also has nanomolar inhibition activity
against the nuclear extract, it is nevertheless over 7-fold worse
than 15b. The HDAC inhibition profiles of 15a and 15b
paralleled those we previously reported for the macrolide-
derived HDACi of the same linker lengths.24 Additionally, this
result is in close agreement with the in silico prediction using
HDAC1 which revealed that 15b adopted a docked orientation
with a better fit with the enzyme outer rim hydrophobic pocket
(Figure 3b). The affinity of 15a for the enzyme could be
compromised by the solvent exposure of its macrocyclic ring
and the shortness of its linker region relative to that of 15b.These
shortcomings in the docked structure of 15a may explain the
7-fold enhanced anti-HDAC activity of 15b relative to 15a.
Compounds 15c-e, analogues having longer methylene lin-
kers than those of 15b, show a progressive reduction in anti-
HDAC activity with the increase in linker length (Table 1). We
then performed further molecular docking analyses on HDAC1
with the goal of shedding more light on the chain length depen-
dence reduction in the anti-HDAC activities of these longer
chain compounds. We observed that these longer linker com-
pounds adopted various docked orientations which progres-
sively extruded the ketolide ring from the outer rim pocket into
with BF3 OEt2. Alternatively, the desired hydroxamates
3
could be obtained through direct copper(I) catalyzed cyclo-
addition between unprotected azidohydroxamates 13 and
alkyne 9, according to our published protocol.24
HDACi Activity and Selectivity of 15a-e. We first tested
compounds 15a-e for their HDAC inhibitory activity
(Table 1) against HeLa cell nuclear extract (which contains
primarily HDACs 1 and 2), HDAC6, and HDAC8 using the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6105
Figure 4. Comparison of the orientations of (a) the C6-linker compound 15b (green) and C9-linker compound 15e (yellow) at the outer rim of
HDAC1 and (b) the C5-linker compound 15a (pink) and C6-linker compound 15b (green) at the outer rim of HDAC8.
solvent exposed regions on the enzyme surface. Compound 15e,
the analogue with the longest methylene linker in the series
herein disclosed, made the least interaction with the enzyme
outer rim (Figure 4a). The observed extrusion of the macrocyclic
ring from the enzyme surface as the methylene linker length
increases could compromise the compounds’ affinity for the
enzyme. These in silico observations provide a plausible struc-
tural basis for the observed chain length dependence attenuation
of the anti-HDAC activities of these ketolide-derived HDACi.
To obtain evidence for the HDAC isoform selectivity, we
tested all triketolide HDACi herein described against HDAC6
and HDAC8 using the Fluor de Lys assay. Compared to
SAHA, compounds 15a and 15b are more selective for the
nuclear extract (HDACs 1 and 2) with selectivity indices
comparable to those of their 14-membered macrolide
congeners.24 Compounds 15c-e are less selective for either
HDAC isoform compared to 15a,b. However, 15c-e have
improved HDAC6 and comparable HDAC8 isoform selec-
tivity relative to SAHA (Table 1). The linker length dependent
dissipation of HDAC isoform selectivity could also be due to
the relief of the attractive interactions between the ketolide
macrocycle and the enzyme hydrophobic cleft, a concomitant
effect of the increase in compounds’ spacer length. HDAC8
could be particularly sensitive to changes in linker length
because it has a much shallower active site pocket relative to
HDAC1.35 To gain insight into the structural basis of the
observed isoform selectivity, we performed molecular docking
analysis on the HDAC8 structure reported by Somoza et al.35
In contrast to their docked structures on HDAC1, the orienta-
tions of 15a and 15b that enable interaction of the ZBG with the
active site Zn2þ ion are those that adopted a closed conforma-
tion (Figure 4b). In this conformation, the macrolactonic ring
of either compound is nestled atop the entrance to the enzyme
active site while the desosamine sugar, the triazolylaryl moiety,
and a portion of the methylene linker are lifted off the enzyme
surface and wrapped back under the macrolactonic ring to
enable the ZBG to interact with the Zn2þ ion at the base of a
shallow active site pocket. This closed conformation is inacces-
sible to 15e, the analogue with the longest methylene linkers in
the series herein disclosed. Instead, 15e adopted a conformation
in which its macrocyclic moiety is flanged into a distinct solvent
exposed patch on the enzyme surface to give its long linker
moiety enough room to present the ZBG to the enzyme active
site (see Supporting Information Figure S1c). Analyses of
X-ray data have revealed significant inhibitor specific changes
in the enzyme active site topology of HDAC8.35 It is therefore
possible that the observed HDAC1 isoform selectivity may be
partly due to the inability of these ketolide based HDACi to
efficiently induce active site conformational changes that
facilitate HDAC8 specific inhibitor association with the en-
zyme active site.36
Recent studies have shown that the use of fluorescently
labeled substrates can perturb enzyme activity and the effect
of inhibitors and activators on the deacetylases.37,38 In one
example, the discovery that resveratrol and various analogues
activate the deacetylase sirtuin 1 (SIRT1) was found to be an
artifact of the fluorescent assay.39 Indeed, many recommended
Fluor de Lys substrates have been shown not to be active toward
their corresponding enzymes, including HDAC8, absent the
fluorophore.40,41 In light of these data, we sought to validate that
compounds 15a-e do inhibit HDAC8 using the label-free
SAMDI mass spectrometry assay.
The SAMDI technique, wherein self-assembled mono-
layers (SAM) of alkanethiolates on gold are analyzed by
matrix assisted laser desorption/ionization (MALDI) mass
spectrometry, has been used to characterize many biochem-
ical activities, including the deacetylase enzyme family.41-43
The SAMDI assay is also amenable to analysis of homo-
geneous reactions, as the substrates are immobilized to the
SAMs after the reaction has been terminated.44 We em-
ployed the SAMDI technique to measure the inhibition
constant (Ki) of compounds 15a-e against HDAC8 using
a previously identified preferred HDAC8 substrate, Ac-
41
GRKAcFGC-NH2.
In the first example, the reactions were set up in a 96-well
plate with 1 μM HDAC8 and varying concentrations of
substrate (5, 10, 25, and 50 μM) and inhibitor 15b (0, 50, 100,
250, and 500 nM). The reactions were stopped at different
time points and applied to arrays of gold circles presenting
maleimide-terminated SAMs to immobilize the substrate via
Michael addition of the cysteine thiol with maleimide. SAMDI
spectra were then acquired for each reaction, and a shift of m/z
42 is indicative of a deacetylation reaction (Supporting Infor-
mation Figure S1). The amount of product formed was plotted
over time for each reaction condition to get the initial rates of
reaction. A Dixon plot (1/v vs [I]) shows lines corresponding
to different substrate concentrations that intersect around
200 nM, which is the Ki for compound 15b (Figure 5a). We then
measured the Ki for the remaining compounds and found that
15b has the highest affinity for HDAC8. Consistent with the in
silico data, we observed a weak affinity for compound 15a for
HDAC8. The Ki values for 15c-e are rather comparable
(Figure 5b), yet do mimic the trend observed with the fluor-
escent assay that as the length of the hydroxamic acid linker
increases, the inhibitor affinity decreases. Finally, we note that
while we observe a similar inhibition pattern with 15a-e using
the two assays, there is a disparity in the absolute magnitude of
the inhibition constants. This is anticipated because of the

6106 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Mwakwari et al.
extract HDAC. They potently inhibit the proliferation of
both the sensitive and resistant strains of P. falciparum with IC50
ranging from 0.12 to 1.5 μg/mL (Table 3). Again, 15b has the
most potent antimalarial activity, which is between 2- to 4-fold
more potent than the control compound SAHA. Moreover, 15b
is several-fold more selectively toxic to either strains of
P. falciparum compared to SAHA. This result suggests that
the parasite HDACs could be one of the compound intracellular
targets. To obtain direct evidence for the involvement of
P. falciparum HDACs as one of the potential intracellular
targets for these compounds, we investigated their effects on
the activity of P. falciparum HDAC1 (pf-HDAC1).50 All the
compounds demonstrate inhibitory activity against pf-HDAC1
with IC50 values that followed a similar pattern as their activity
against the HeLa nuclear extract HDAC (Table 4). The anti pf-
HDAC1 activity of compound 15b, the most potent in this
series, is about 3-fold enhanced relative to the previously
reported macrolide HDACi chemotypes with the same linker
length.50 The enhanced anti-pf-HDAC1 activity of 15b could be
due to a better fit to the enzyme afforded by its rigid tricyclic
ring. Overall, this result confirms that these ketolide based
HDACi partly derived their antimalarial activity through intra-
cellular inhibition of pf-HDAC1 activity.
Interestingly however, compounds 15a,b are devoid of
antileishmanial activity against the promastigote stage of
L. donovani while compounds 15c-e have moderate to good
activity that peaks with 15e, the analogue with the longest
methylene spacer in the series disclosed in this manuscript
(Table 3). This result is in contrast to the antimalarial activity
that mirrors compound anti-HDAC activity, and it may
have implications in the organization of the active sites of
the relevant P. falciparum and L. donovani HDAC isozymes.
We have observed a similar methylene spacer-length depen-
dence in the antileishmanial activity of macrocyclic HDACi
derived from 14- and 15-membered macrolides.46,50 The
foregoing observations further support the suitability of
HDAC inhibition as a viable therapeutic strategy to curb
infections caused by apicomplexan protozoans and try-
panosomatids.
Figure 5. Determination of Ki for compounds 15a-e using SAM-
DI mass spectrometry: (a) Dixon plot of compound 15b; (b) Ki
values for compounds 15a-e as determined by SAMDI.
Table 2. Antiproliferative Activity (μM) of Tricyclic Ketolide-Based
HDAC Inhibitors
Hs1.Lu normal
lung fibroblast
DU-145
(μM)
A549
(μM)
MCF-7
(μM)
compd
n
(μM)
15a
15b
15c
15d
15e
1
2
3
4
5
1.64
0.82
3.70
3.89
4.83
1.17
0.66
1.64
2.48
2.81
1.26
0.75
1.35
2.56
3.68
>10
>10
>10
>10
>10
nature of each assay (including the use of either a labeled or
unlabeled substrate) and the parameters that were measured to
compare inhibitor potency.
Encouraged by the in vitro HDAC inhibition results, we
studied the effect of 15a-e on the viability of three repre-
sentative cancer cell lines (prostate (DU-145), lung (A549),
and breast cancer (MCF-7)) and a nontransformed cell
(human lung fibroblast cell (Hs1.Lu)). We included a non-
transformed cell line in our investigation in order to obtain
evidence for compound selective toxicity. Drug concentra-
tions necessary for 50% inhibition of cell viability (EC50)
were quantitatively measured using MTS colorimetric assay
as we previously described.24,45 Table 2 shows the EC50
values of each compound. Compounds 15a-e inhibit the
proliferation of all transformed cells studied with antipro-
liferative activity that closely matched their in vitro anti-
HDAC activity. Specifically, compound 15b has the most
potent antiproliferative activity with high nanomolar effi-
cacy against all three cancer cell lines. Moreover, none of the
compounds show any discernible toxicity against normal
Hs1.Lu cells at drug concentrations in excess of 10 μM
(Table 2). These data showed that the triketolide hydroxa-
mates reported here are selectively toxic to the transformed
cells, a trait that tracks with those of many HDACi.
Rapidly growing eukaryotes including the human patho-
genic apicomplexan protozoans and trypanosomatids, such
as the causative agents of malaria and leishmaniasis, respec-
tively, have been shown to be responsive to HDACi.46-50
These earlier observations demonstrate HDACs as promis-
ing targets in the fight against the often fatal diseases caused
by these parasites. However, the structure-activity relation-
ship (SAR) of the antimalarial and antileishmanial activities
of HDACi is not entirely clear. To further profile the
biological activities of these triketolide HDACi, we investi-
gated their antiparasitic activities against Plasmodium falci-
parum and Leishmania donovani, the causative parasites of
human malaria and leishmaniasis, respectively.
Conclusion
We report that tricyclic ketolide is an excellent mimetic for
the peptide backbone of macrocyclic HDACi. The tricyclic
ketolide herein described has a rigid hydrophobic skeleton
that, as we confirmed by molecular docking analysis, favor-
ably interacts with the HDAC enzyme’s outer rim groups,
thereby resulting in enhanced HDAC inhibition profile.
Compounds derived from this template are particularly selec-
tive against HeLa nuclear extract HDACs 1 and 2 with
nanomolar inhibitory activity and have improved drug cyto-
toxicity against human cancer cell lines. Many of these
compounds have superior antiparasitic activities against cau-
sative parasites of malaria and leishmaniasis compared to
clinically useful SAHA. The pattern of the antimalarial and
antileishmanial activities of these compounds reveals struc-
tural attributes that confer a specific antiparasitic response.
Finally, ketolides, like their 14- and 15-membered macrolide
counterparts, have selective tissue/cell distribution profiles.
For example, ketolides have been reported to exceptionally
accumulate in the phagocytes, in particular, human polymor-
phonuclear neutrophils (PMNs) and other phagocytic and
epithelial cell lines.51 Therefore, these ketolide derived HDA-
Ci could find application in targeting tumor-associated
The antimalarial activities of compounds 15a-e closely
paralleled their anti-HDAC activity against HeLa nuclear

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6107
Table 3. In Vitro Antileishmanial (μg/mL) and Antimalarial (μg/mL) Activities of Tricyclic Ketolide-Based HDAC Inhibitor Derivativesa
antileishmanial activityb
IC50
antimalarial activityc
Plasmodium
falciparum (D6 clone)
IC50 (μg/mL)
Plasmodium
falciparum (W2 clone)
IC50 (μg/mL)
cytotoxicity
(VERO)c
compd
n
(μg/mL)
NA
NA
IC90(μg/mL)
IC50(μg/mL)
SI D6 (W2)
15a
15b
15c
15d
15e
1
2
3
4
5
NA
NA
0.897 ( 0.198
0.137 ( 0.027
1.317 ( 0.231
1.296 ( 0.129
1.195 ( 0.058
0.012 ( 0.002
0.0066 ( 0.0007
NT
1.789 ( 0.254
0.133 ( 0.027
1.475 ( 0.244
1.376 ( 0.079
0.897 ( 0.025
0.147 ( 0.018
0.0091 ( 0.0011
NT
NC
NC
>5.3 (>2.68)
>35.0 (>36.0)
>3.6 (>3.3)
>3.7 (>3.5)
>4.0 (>5.3)
NT
19.7 ( 2.4
14.9 ( 1.7
4.8 ( 0.5
NT
35.8 ( 4.4
32.3 ( 3.1
23.7 ( 2.4
NT
NC
NC
NC
NT
chloroquine
artemisinin
pentamidine
amphotericine B
SAHA
NT
NT
NT
NT
NT
NT
1.17 ( 0.25
0.079 ( 0.012
21.5 ( 3.51
2.45 ( 0.34
0.395 ( 0.054
51.7 ( 8.1
NT
0.279 ( 0.077
NT
0.392 ( 0.045
NT
1.375 ( 0.255
NT
4.9 (3.5)
a NA = not active up to highest concentration tested. NC = not cytotoxic up to the highest concentration tested. NT = not tested. SI = selectivity
index (IC50(Vero)/IC50). b Maximum tested concentration = 40 μg/mL. c Maximun tested concentration = 4.8 μg/mL.
(13a,b) were synthesized as we previously reported.24 Azido-O-
Table 4. In Vitro Inhibition of pf-HDAC1 by Tricyclic Ketolide-Based
HDACi
trityl alkylhydroxamate derivatives (12a-e) were synthesized by
adapting literature protocol.46
compd
n
IC50 (nM)
20-O-Acetyldescladinose-4-ethynylbenzylclarithromycin (6).
To a solution of descladinose-4-ethynylbenzylclarithromycin 5
(3.80 g, 5.50 mmol) in acetone (20 mL) was added Ac2O (0.62 g,
6.00 mmol), and the resulting mixture was stirred at 40 °C for
36 h. The reaction mixture was diluted with EtOAc (100 mL)
and washed with aqueous NaHCO3 (70 mL) and saturated brine
(70 mL). Purification on silica column (6:1 CH2Cl2/acetone)
afforded 2.8 g (70%) of the title compound as a yellowish solid.
1H NMR (CDCl3, 400 MHz) δ 0.80 (3H, t, J = 7.2 Hz), 0.90
(3H, d, J = 7.2 Hz), 1.08-1.47 (12H, m), 1.58 (6H, s), 1.63-2.05
(7H, m), 2.08 (3H, s), 2.16 (3H, s), 2.42-2.80 (3H, m), 2.92 (3H,
s), 2.94-3.00 (1H, m), 3.03-3.68 (3H, m), 3.79 (2H, s), 3.94 (1H,
s), 4.08 (1H, m), 4.54 (1H, d, J = 8.0 Hz), 4.80 (1H, m), 5.15 (1H,
dd, J = 11.6, 2.4 Hz), 7.17 (2H, d, J = 8.4 Hz), 7.38 (2H, d, J =
8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 8.0, 10.7, 12.8, 15.5,
16.4, 18.1, 19.5, 21.3, 21.5, 21.6, 31.1, 31.9, 36.0, 37.0, 37.5, 38.7,
44.3, 45.7, 50.0, 58.4, 62.5, 68.9, 69.8, 71.6, 74.4, 76.9, 77.0, 77.8,
78.1, 81.3, 83.9, 99.9, 120.7, 128.4, 132.2, 141.1, 170.1, 174.9,
221.2; HRMS (FAB, mnba) calcd for [C40H61NO11 þ H]þ
732.4323, found 732.4311.
2-Oxo-20-O-acetyldescladinose-4-ethynylbenzylclarithromy-
cin (7). Methyl sulfide (1.42 g, 22.80 mmol) was added to a
mixture of N-chlorosuccinimide (2.61 g, 19.50 mmol) and
CH2Cl2 (10 mL) while maintaining the temperature at -15 °C.
Compound 6 (10.0 g, 13.7 mmol) dissolved in CH2Cl2 (50 mL)
was added to the reaction flask, followed by Et3N (1.56 g, 15.4
mmol) and stirred at -15 °C for 4.5 h. The reaction mixture was
poured into EtOAc (350 mL) and 0.5 N aqueous NaOH (250
mL). The organic layer was separated, washed with saturated
brine (250 mL), and dried over Na2SO4. Solvent was evaporated
off and the crude was purified on silica column (2:3:0.1 EtOAc/
hexane/Et3N) to afford 9.54 g (96%) of the title compound as an
off-white solid. 1H NMR (CDCl3, 400 MHz) δ 0.80-0.86 (6H,
m), 1.09-1.57 (12H, m), 1.58 (6H, s), 1.62-2.02 (7H, m), 2.05
(3H, s), 2.15 (3H, s), 2.44-2.80 (2H, m), 2.92 (3H, s), 2.95-3.00
(1H, m), 3.05-3.66 (4H, m), 3.80 (1H, q, J = 14.0, 6.8 Hz), 3.89
(1H, s), 4.12 (1H, m), 4.38 (1H, d, J = 8.0 Hz), 4.79-4.83 (1H,
m), 5.14 (1H, dd, J = 11.2, 2.0 Hz), 7.16 (2H, d, J = 7.6 Hz), 7.38
(2H, d, J = 7.6 Hz); 13C NMR (CDCl3, 100 MHz) δ 10.8, 12.4,
14.4, 14.5, 16.5, 17.9, 19.6, 21.3, 21.5, 31.3, 36.9, 37.6, 39.2, 45.1,
46.3, 49.7, 51.1, 58.5, 62.5, 69.2, 69.6, 71.5, 74.1, 77.0, 77.1, 77.2,
78.1, 83.9, 101.1, 120.7, 128.5, 132.2, 140.9, 169.6, 170.0, 205.7,
221.1; HRMS (FAB, mnba) calcd for [C40H59NO11 þ H]þ
730.4166, found 730.4132.
15a
15b
1
2
3
4
5
36 ( 2.9
10 ( 0.5
40 ( 1.8
290 ( 16
304 ( 17
130 ( 9.2
15c
15d
15e
SAHA
macrophages and further clarify the roles of HDACi in the
inhibition of macrophage migration inhibitory factor (MIF)
gene, a phenomenon that has been suggested to be an essential
part of their antitumorigenic effects.52
Experimental Section
Materials and Methods. All commercially available starting
materials were used without further purification. Clarithromy-
cin 1 was purchased from Greenfield Chemical. 4-Ethynylben-
zyl alcohol, ethyl 6-bromohexanoate, ethyl 7-bromoheptanoate,
8-bromooctanoic acid, and methyl 10-bromodecanoate were
purchased from Sigma Aldrich. 9-Bromononanoic acid was
purchased from Karl Industries Inc. Reaction solvents were
either high performance liquid chromatography (HPLC) grade
or American Chemical Society (ACS) grade and used without
further purification. HDAC fluorimetric assay kit and recom-
binant HDACs were procured from Enzo Life Sciences, BIO-
MOL International, PA. Analtech silica gel plates (60 F254) were
used for analytical TLC, and Analtech preparative TLC plates
(UV 254, 2000 μm) were used for purification. UV light and
anisaldehyde/iodine stain were used to visualize the spots. Silica
gel (200-400 mesh) was used in column chromatography.
Nuclear magnetic resonance (NMR) spectra were recorded on
a Varian-Gemini 400 magnetic resonance spectrometer. 1H
NMR spectra were recorded in parts per million (ppm) relative
to the peak of CDCl3, (7.24 ppm). 13C spectra were recorded
relative to the central peak of the CDCl3 triplet (77.0 ppm) and
were recorded with complete heterodecoupling. High-resolution
mass spectra were recorded at the Georgia Institute of Techno-
logy mass spectrometry facility in Atlanta. Melting points were
recorded uncorrected on a MEL-TEMP II apparatus. HPLC
assays were performed on a Beckman Coulter instrument using
a Phenomenex RP C-18 column (250 mm ꢀ 4.6 mm), eluting
with solvent A (0.1% formic acid/acetonitrile) and solvent B
(0.1% formic acid/water) at a gradient of 5-50% over 30 min,
with detection at 251 nm and a flow rate of 1 mL/min. Sample
concentrations were 250 μM, injecting 5 μL. 30-Desmethylclar-
ithromycin 2, 4-ethynylbenzylclarithromycin 4, descladinose-4-
ethynylbenzylclarithromycin 5, and azidoalkylhydroxamic acid
12-Carbamoylimidazole Ketolide (8). To a suspension of 7
(1.50 g, 2.06 mmol) in a mixture of anhydrous THF (20 mL) and
anhydrous DMF (7 mL) was added CDI (1.30 g, 8.20 mmol)

6108 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Mwakwari et al.
followed by a solution of 1.0 M NaN(TMS)2 in THF (2.60 mL,
2.60 mmol) over 75 min, and the resulting mixture was stirred at
room temperature for 24 h. The reaction mixture was diluted
with EtOAc (50 mL), washed with aqueous NaHCO3 (20 mL)
and saturated brine (20 mL), and dried over Na2SO4. Solvent
was evaporated off and the crude was purified on silica column
(3:2:0.1 EtOAc/hexane/Et3N) to yield 52% (0.86 g) of the title
compound. 1H NMR (CDCl3, 400 MHz) δ 0.91 (3H, t, 7.6 Hz),
1.09 (3H, d, J = 7.2 Hz), 1.14-1.50 (9H, m), 1.80 (3H, s), 1.83
(3H, s), 1.56-1.94 (7H, m), 2.00 (3H, s), 2.05 (3H, s), 2.12 (3H,
s), 2.64-2.80 (1H, m), 3.02 (3H, s), 2.95-3.70 (4H, m), 3.73 (1H,
q, J = 14.0, 6.8 Hz), 4.10-4.06 (2H, m), 4.30 (1H, d, J = 7.6
Hz), 4.77-4.80 (1H, m), 5.68 (1H, dd, J = 11.2, 2.0 Hz), 6.77
(1H, s), 7.03 (1H, s), 7.16 (2H, d, J = 7.6 Hz), 7.36 (3H, m), 8.06
(1H, s); 13C NMR (CDCl3, 100 MHz) δ 10.7, 13.5, 14.4, 15.2,
19.1, 20.4, 21.1, 21.2, 21.5, 22.8, 31.2, 36.8, 47.6, 50.5, 51.2, 58.5,
60.6, 62.8, 69.3, 71.6, 78.7, 83.9, 84.7, 117.3, 120.7, 128.4, 131.1,
132.2, 137.3, 138.8, 140.9, 146.2, 169.1, 169.8; HRMS (FAB,
mnba) calcd for [C44H59N3O11 þ H]þ 806.4228, found 806.4210.
4-Ethynylbenzyltricyclic Ketolide (9). Protocol A. To a solu-
tion of 8 (0.60 g, 0. 74 mmol) in CH3CN (10 mL) and H2O (1 mL)
was added ethylenediamine (0.44 g, 7.40 mmol), and the mixture
was heated in a sealed tube at 50-55 °C for 24 h. Solvent was
evaporated off and the reaction mixture partitioned between
H2O (15 mL) and CH2Cl2 (20 mL) and separated. The organic
layer was washed with saturated brine (20 mL), dried over
Na2SO4, and solvent was evaporated off in vacuo. The crude
product was dissolved in anhydrous MeOH (15 mL) and heated
at 90 °C in a sealed tube for 24 h. MeOH was evaporated and the
product purified on silica column (EtOAc/Et3N 12:0.1) to give
0.27 g (48%) of the intermediate product. EtOH (6 mL) and
AcOH (0.043 g, 0.71 mmol) were added to the pure intermediate
product (0.27 g, 0.34 mmol), and the mixture was heated at 95 °C
in a sealed tube for 20 h. The reaction mixture was suspended in
dilute NH4OH (10 mL) and CH2Cl2 (15 mL) and the organic
layer separated, washed with saturated brine (10 mL), and dried
over Na2SO4. Purification on silica column (EtOAc/MeOH/
Et3N 10:0.5:0.05) afforded 0.24 g (92%), 44% overall yield of
compound 9.
Protocol B: Reductive Amination. A solution of 11 (0.20 g,
0.32 mmol) and 4-ethynylbenzaldehyde (0.21 g, 1.60 mmol) in
MeOH (4 mL) and acetic acid (37 μL, 0.64 mmol) was stirred for
30 min at room temperature. Borane-pyridine complex (80 μL,
0.64 mmol) was added and the mixture stirred for 5 h. The
mixture was diluted with EtOAc (20 mL) and washed with
saturated NaHCO3 (20 mL) and saturated brine (20 mL). The
organic layer was dried over Na2SO4, concentrated in vacuo,
and purified by preparative TLC (12:1 CH2Cl2/MeOH) to
afford 0.18 g (78%) of compound 9 as a creamish solid. 1H
NMR (CDCl3, 400 MHz) δ 0.80 (3H, t, J = 7.2 Hz), 1.00 (3H, d,
J = 6.8 Hz), 1.14-1.55 (12H, m), 1.30 (3H, s), 1.43 (3H, s),
1.60-1.96 (6H, m), 2.12 (3H, s), 2.45-2. 95 (6H, m), 3.03 (3H, s)
3.23-3.43 (4H, m), 3.64-3.78 (4H, m), 3. 94 (1H, m), 4.16 (1H,
d, J = 8.4 Hz), 4.25 (1H, d, J = 7.6 Hz), 4.90 (1H, d, J = 10.0
Hz), 7.18 (2H, d, J = 8.0 Hz), 7.39 (2H, d, J = 7.2 Hz); 13C
NMR (CDCl3, 100 MHz) δ 10.6, 11.1, 13.0, 14.6, 16.6, 19.3,
19.8, 21.4, 22.3, 29.7, 36.5, 37.1, 38.7, 42.5, 43.0, 48.3, 49.3, 49.7,
51.4, 57.6, 60.1, 65.6, 69.7, 70.5, 76.7, 78.7, 79.4, 81.7, 83.6,
104.0, 121.0, 128.8, 132.4, 140.0, 156.3, 169.8, 181.5, 204.4;
HRMS (FAB, mnba) calcd for [C41H59N3O9 þ H]þ 738.4330,
found 738.4332.
NH4OH/saturated NH4Cl (50 mL) and extracted with 10%
MeOH/CH2Cl2 (3 ꢀ 30 mL). The organic layers were combined,
dried over Na2SO4, and concentrated in vacuo. The crude pro-
duct was purified by preparative TLC (silica, 12:1:0.1 CH2Cl2/
MeOH/conc NH4OH) to give 127 mg (78%) of 14a as a yellow-
ish solid. 1H NMR (CDCl3, 400 MHz) δ 0.85 (3H, t, J = 7.6 Hz),
1.1 (3H, d, J = 6.8 Hz), 1.20-1.39 (17H, m), 1.46-2.00 (15H,
m), 2.19 (3H, s), 2.58-2.80 (6H, m), 2.84-3.00 (1H, m),
3.02-3.17 (1H, m), 3.31-3.60 (3H, m), 3.64-3.87 (5H, m),
3.96-4.0 (1H, m), 4.22 (1H, d, J = 8.4 Hz), 4.29-4.32 (3H, m),
4.93 (1H, dd, J = 10.0, 1.6 Hz), 7.23-7.45 (17H, m), 7.71 (1H,
s), 7.79 (2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 10.7,
11.1, 13.1, 14.6, 16.7, 19.3, 19.9, 21.4, 22.3, 22.8, 26.0, 29.6, 30.2,
31.0, 36.5, 37.0, 38.8, 42.5, 43.0, 48.3, 49.3, 49.7, 50.2, 51.4, 53.7,
57.7, 60.1, 65.6, 69.8, 70.5, 76.7, 78.7, 79.4, 81.8, 93.1, 104.1,
119.7, 126.0, 128.2, 128.4, 129.2, 129.4, 130.0, 138.9, 141.2,
147.6, 156.3, 169.8, 177.0, 181.5, 204.5; HRMS (ESI) calcd for
[C66H85N7O11 þ H]þ 1152.6379, found 1152.6367.
Tricyclic Ketolide-N-benzyltriazolyl-O-tritylheptahydroxa-
mate (14b). Reaction of 4-ethynylbenzyltricyclic ketolide 9 (0.10
g, 0.14 mmol) and 7-azido-O-tritylheptahydroxamate 12b (0.06
g, 0.14 mmol) within 24 h as described for the synthesis of 14a
gave 133 mg (83%) of 14b as a yellowish solid. 1H NMR
(CDCl3, 400 MHz) δ 0.85 (3H, t, J = 7.6 Hz), 1.06 (3H, d, J
= 6.8 Hz), 1.20-1.42 (19H, m), 1.45-1.98 (15H, m), 2.20 (3H,
s), 2.57-2.79 (6H, m), 2.85-3.00 (1H, m), 3.03-3.17 (1H, m),
3.30-3.57 (3H, m), 3.72-3.83 (5H, m), 3.96-4.00 (1H, m), 4.22
(1H, d, J = 8.8 Hz), 4.29-4.35 (3H, m), 4.95 (1H, dd, J = 10.4,
2.4 Hz), 7.22-7.45 (17H, m), 7.71 (1H, s), 7.78 (2H, d, J = 8.4
Hz); 13C NMR (CDCl3, 100 MHz) δ 10.7, 11.1, 13.1, 14.6, 16.6,
19.3, 19.9, 21.4, 22.3, 26.3, 29.6, 30.2, 36.5, 37.0, 38.8, 42.5, 43.0,
48.3, 49.3, 49.7, 50.4, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7, 70.5, 76.7,
78.7, 79.4, 81.8, 92.8, 104.1, 119.7, 126.0, 128.4, 129.2, 129.4,
130.0, 132.3, 138.9, 141.3, 147.6, 156.3, 169.8, 177.6, 182.5,
204.5; HRMS (ESI) calcd for [C67H87N7O11 þ H]þ 1166.6536,
found 1166.6478.
Tricyclic Ketolide-N-benzyltriazolyl-O-trityloctahydroxamate
(14c). Reaction of 4-ethynylbenzyltricyclic ketolide 9 (0.08 g,
0.10 mmol) and 8-azido-O-trityloctahydroxamate 12c (0.05 g,
0.10mmol) inanhydrous THF (5 mL) within22h asdescribed for
the synthesis of 14a gave 94 mg (79%) of 14c as a yellowish solid.
1H NMR (CDCl3, 400 MHz) δ 0.83 (3H, t, J = 7.2 Hz), 1.04 (3H,
d, J = 7.2 Hz), 1.15-1.41 (21H, m), 1.45-1.95 (15H, m), 2.18
(3H, s), 2.55-2.73 (6H, m), 2.87-2.97 (1H, m), 3.03-3.10 (1H,
m), 3.28-3.57 (3H, m), 3.70-3.82 (5H, m), 3.93-3.97 (1H, m),
4.21 (1H, d, J = 8.4 Hz), 4.27-4.34 (3H, m), 4.94 (1H, dd, J =
12.8, 2.4 Hz), 7.25-7.42 (17H, m), 7.71 (1H, s), 7.76 (2H, d, J =
8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 10.7, 11.1, 13.1, 14.6,
16.6, 19.3, 19.9, 21.4, 22.3, 26.4, 28.8, 29.6, 30.4, 36.6, 37.0, 38.8,
42.5, 42.9, 48.3, 49.3, 49.7, 50.6, 51.4, 53.7, 57.7, 60.1, 65.5, 69.8,
70.5, 76.7, 78.7, 79.4, 81.8, 93.8, 104.1, 119.6, 126.0, 127.7, 128.3,
128.8, 129.2, 129.4, 130.0, 132.7, 138.9, 141.3, 147.7, 156.3, 169.8,
177.0, 182.4, 204.4; HRMS (ESI) calcd for [C68H89N7O11 þ H]þ
1180.6692, found 1180.6637.
Tricyclic Ketolide-N-benzyltriazolyl-O-tritylnonahydroxa-
mate (14d). Reaction of 4-ethynylbenzyltricyclic ketolide 9 (0.08 g,
0.10 mmol) and 9-azido-O-tritylnonahydroxamate 12d (0.05 g,
0.10 mmol) in anhydrous THF (5 mL) within 20 h as described
for the synthesis of 14a gave 93 mg (76%) of 14d as a yellowish
solid. 1H NMR (CDCl3, 400 MHz) δ 0.85 (3H, t, J = 8.0 Hz),
1.05 (3H, d, J = 6.8 Hz), 1.16-1.44 (23H, m), 1.45-1.96 (15H,
m), 2.20 (3H, m), 2.57-2.78 (6H, m), 2.90-2.99 (1H, m),
3.02-3.12 (1H, m), 3.30-3.57 (3H, m), 3.72-3.84 (5H, m),
3.96-4.00 (1H, m), 4.29 (1H, d, J = 8.4 Hz), 4.31 (1H, d, J = 7.2
Hz), 4.37 (2H, t, J = 7.2 Hz), 4.95 (1H, dd, J = 12.8, 2.4 Hz),
Representative Procedure for the Synthesis of O-Trityl Pro-
tected Tricyclic Ketolide Hydroxamate. Tricyclic Ketolide-N-
benzyltriazolyl-O-tritylhexahydroxamate (14a). 4-Ethynylbenzyl-
tricyclic ketolide 9 (0.10 g, 0.14 mmol) and 6-azido-O-tritylhex-
ahydroxamate 12a (0.06 g, 0.14 mmol) were dissolved in anhy-
drous THF (7 mL) and stirred under argon at room temper-
ature. Copper(I) iodide (0.012 g, 0.063 mmol) and Hunig’s base
(0.6 mL) were added to the reaction mixture, and stirring
continued for 24 h. The reaction mixture was diluted with 1:4
7.19-7.45 (17H, m), 7.72 (1H, s), 7.78 (2H, d, J = 8.0 Hz); 13
C
NMR (CDCl3, 100 MHz) δ 10.7, 11.1, 13.1, 14.6, 16.6, 19.3,
19.9, 21.4, 22.3, 26.6, 28.9, 29.2, 29.6, 29.9, 30.5, 36.5, 37.0, 37.1,
38.8, 42.5, 43.0, 48.3, 49.3, 49.7, 50.6, 51.4, 57.7, 60.1, 65.5, 65.6,
69.7, 70.5, 76.7, 78.7, 79.4, 81.8, 94.3, 104.1, 119.6, 126.0, 128.3,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6109
128.8, 129.0, 129.2, 129.4, 130.0, 132.3, 138.9, 141.3, 147.6, 156.3,
169.8, 178.0, 181.5, 204.5; HRMS (ESI) calcd for [C69H91N7-
O11 þ H]þ 1194.6849, found 1194.6838.
(1 mL), thioanisole (0.3 mL), and TFA (0.3 mL), according to
method A, for 3 h afforded 56 mg (55%) of 15b as a yellow-white
solid.
Tricyclic Ketolide-N-benzyltriazolyl-O-trityldecahydroxamate
(14e). Reaction of 4-ethynylbenzyltricyclic ketolide 9 (0.08 g,
0.10 mmol) and 10-azido-O-trityldecahydroxamate 12e (0.05 g,
0.10 mmol) in anhydrous THF (5 mL) within 20 h as described for
the synthesis of 14a gave 98 mg (80%) of 14e as a yellowish solid.
1H NMR (CDCl3, 400 MHz) δ 0.83 (3H, t, J = 6.8 Hz), 1.03 (3H,
d, J = 7.2 Hz), 1.15-1.40 (25H, m), 1.44-1.96 (15H, m), 2.17 (3H,
s), 2.57-2.78 (6H, m), 2.87-2.95 (1H, m), 3.03-3.08 (1H, m),
3.28-3.53 (3H, m), 3.70-3.82 (5H, m), 3.92-3.96 (1H, m), 4.20
(1H, d, J = 8.4 Hz), 4.29 (1H, d, J = 6.8 Hz), 4.33 (2H, t, J = 7.2
Hz), 4.93 (1H, d, J = 10.0 Hz), 7.26-7.43 (17H, m), 7.73 (1H, s),
7.76 (2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 10.7,
11.1, 13.1, 14.6, 16.6, 19.3, 19.8, 21.4, 22.3, 26.6, 29.1, 29.3, 29.6,
29.9, 30.5, 36.6, 37.0, 38.8, 42.5, 43.0, 48.3, 49.3, 49.7, 50.6, 51.4,
57.7, 60.1, 65.5, 69.7, 70.5, 76.7, 78.7, 79.4, 81.8, 93.4, 104.1,
199.6, 126.0, 128.3, 129.2, 129.4, 130.0, 138,9, 147.6, 156.3, 169.8,
177.6, 182.0, 204.5; HRMS (ESI) calcd for [C70H93N7O11 þ H]þ
1208.7005, found 1208.6888.
Similarly, the reaction of 14b (0.15 g, 0.18 mmol) and
BF3 OEt2 (0.04 g, 0.26 mmol), according to method B, afforded
65 mg (55%) of 15b as a yellow-white solid.
3
Also, the reaction of 4-ethynylbenzyltricyclic ketolide 9 (0.05 g,
0.06 mmol) and 7-azidoheptahydroxamic acid 13b (0.02 g, 0.11
mmol) within 12 h, according, to method C, gave 22 mg (38%) of
15b as a brown-white solid. Mp 128-131 °C; retention time 21.233
min; 1H NMR (CDCl3, 400 MHz) δ 0.83 (3H, t, J = 7.6 Hz), 1.03
(3H, d, J = 6.4 Hz), 1.18-1.90 (32H, m), 2.10-2.13 (2H, m), 2.17
(3H, s), 2.58-2.73 (6H, m), 2.93-2.98 (1H, m), 3.03-3.07 (1H,
m), 3.28-3.60 (3H, m), 3.70-3.81 (5H, m), 3.98 (1H, m), 4.20 (1H,
d, J = 8.4 Hz), 4.29-4.38 (3H, m), 4.93 (1H, d, J = 10.4 Hz), 7.30
(2H, d, J = 7.6 Hz), 7.75 (2H, d, J = 7.6 Hz), 7.78 (1H, s); 13
C
NMR (CDCl3, 100 MHz) δ 10.6, 11.1, 13.1, 14.6, 16.5, 19.3, 19.8,
21.4, 22.3, 25.2, 26.0, 28.2, 29.7, 29.9, 30.1, 36.6, 37.0, 38.8, 42.5,
42.7, 48.2, 49.3, 49.5, 50.4, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7, 70.5,
76.7, 78.7, 79.3, 81.8, 104.0, 120.0, 126.0, 129.4, 129.8, 139.1, 147.7,
156.3, 169.8, 171.2, 182.4, 204.5; HRMS (ESI) calcd for [C48H73-
N7O11 þ H]þ 924.5440, found 924.5422.
Tricyclic Ketolide-N-benzyltriazolylhexahydroxamic Acid
(15a). Method A. To a solution of 14a (0.12 g, 0.11 mmol) in
methylene chloride (1 mL) at 0 °C was added thioanisole (0.2
mL) and TFA (0.2 mL) dropwise. Stirring was continued at 0 °C
for 2 h, after which TLC analysis indicated complete consump-
tion of the starting material. Excess TFA and solvent were
evaporated off, and the residue was immediately placed in an
ice bath followed by addition of PBS buffer (10 mL). Saturated
NaHCO3 was added dropwise until the pH was neutral. Extrac-
tion with 20% MeOH/CH2Cl2 (10 mL ꢀ 3), drying over
Na2SO4, and evaporation of solvent afforded crude product
which was then purified by preparative TLC (silica, 12:1:0.1
CH2Cl2/MeOH/conc NH4OH) to afford 47 mg (50%) of 15a as
a yellow-white solid.
Tricyclic Ketolide-N-benzyltriazolyloctahydroxamic Acid (15c).
The reaction of 14c (0.09 g, 0.08 mmol) in CH2Cl2 (1 mL),
thioanisole (0.2 mL), and TFA (0.2 mL), according to method
A, afforded 37 mg (51%) of 15c as a yellow-white solid. Mp
1
113-115 °C; retention time 22.533 min; H NMR (CDCl3, 400
MHz) δ 0.83 (3H, t, J = 6.4 Hz), 1.03 (3H, d, J = 5.6 Hz),
1.18-1.60 (21H, m), 1.66-2.1 (15H, m), 2.18 (3H, s), 2.56-2.70
(6H, m), 2.90-2.98 (1H, m), 3.02-3.07 (1H, m), 3.28-3.58 (3H,
m), 3.70-3.81 (5H, m), 3.94-3.97 (1H, m), 4.20 (1H, d, J = 7.2
Hz), 4.28 (1H, d, J = 6.0 Hz), 4.35 (2H, m), 4.93 (1H, d, J = 10.4
Hz), 7.30 (2H, d, J = 7.2 Hz), 7.73-7.76 (3H, m); 13C NMR (CD-
Cl3, 100 MHz) δ 10.6, 11.1, 13.1, 14.4, 14.6, 16.5, 19.3, 19.8, 21.3,
21.4, 22.3, 25.2, 26.1, 28.3, 29.7, 29.9, 30.2, 32.9, 36.6, 37.1, 38.8,
42.5, 42.7, 48.2, 49.4, 49.5, 50.5, 51.4, 57.7, 60.1, 60.6, 65.5, 69.7,
70.5, 76.7, 78.7, 79.3, 81.8, 104.0, 119.9, 126.0, 129.5, 129.8, 139.0,
147.7, 156.3, 169.8, 171.7, 182.4, 204.5; HRMS (ESI) calcd for
[C49H75N7O11 þ H]þ 938.5597, found 938.5556.
Method B. To a mixture of 14a (0.14 g, 0.12 mmol) in CH2Cl2/
MeOH (2 mL/2 mL) was added BF3 OEt2 (0.03 g, 0.24 mmol),
3
and the mixture was stirred at room temperature for 45 min. Dilute
NaHCO3 was added until pH 8 was obtained (approximately 20
mL), and the suspension was extracted with 10% MeOH/CH2Cl2
(10 mL ꢀx 4), dried over Na2SO4, and concentrated in vacuo. The
crude product was purified by preparative TLC (silica, 12:1:0.1
CH2Cl2/MeOH/conc NH4OH) to give 64 mg (60%) of 15a as a
yellow-white solid.
Tricyclic Ketolide-N-benzyltriazolylnonahydroxamic Acid
(15d). The reaction of 14d (0.09 g, 0.08 mmol) in CH2Cl2 (1
mL), thioanisole (0.2 mL), and TFA (0.2 mL), according to
method A, afforded 43 mg (60%) of 15d as a yellow-white solid.
Mp 112-115 °C; retention time 23.983 min; 1H NMR (CDCl3,
400 MHz) δ 0.83 (3H, t, J = 7.2 Hz), 1.03 (3H, d, J = 7.2 Hz),
1.17-1.60 (23H, m), 1.66-2.14 (15H, m), 2.17 (3H, s),
2.58-2.73 (6H, m), 2.87-2.95 (1H, m), 3.00-3.07 (1H, m),
3.27-3.55 (3H, m), 3.70-3.82 (5H, m), 3.93-3.97 (1H, m), 4.20
(1H, d, J = 8.8 Hz), 4.28 (1H, d, J = 7.2 Hz), 4.35 (2H, t, J = 6.8
Hz), 4.92 (1H, dd, J = 10.0, 1.2 Hz), 7.30 (2H, d, J = 8.0 Hz),
7.75 (3H, m); 13C NMR (CDCl3, 100 MHz) δ 10.7, 11.1, 13.1,
14.6, 16.5, 19.3, 19.8, 21.4, 22.3, 25.4, 26.3, 28.6, 28.8, 28.9, 29.6,
29.9, 30.3, 32.9, 36.6, 37.1, 38.8, 42.5, 42.8, 48.2, 49.3, 49.6, 50.6,
51.4, 57.7, 60.1, 65.5, 69.7, 70.5, 76.7, 78.7, 79.3, 81.8, 104.1,
119.8, 126.0, 129.5, 129.8, 139.0, 147.7, 156.3, 169.8, 171.3,
182.9, 204.5; HRMS (ESI) calcd for [C50H77N7O11 þ H]þ
952.5753, found 952.5728.
Tricyclic Ketolide-N-benzyltriazolyldecahydroxamic Acid
(15e). The reaction of 14e (0.10 g, 0.08 mmol) in CH2Cl2
(1 mL), thioanisole (0.2 mL), and TFA (0.2 mL), according to
method A, afforded 42 mg (55%) of 15e as a yellow-white solid.
Mp 123-126 °C; retention time 25.583 min; 1H NMR (CDCl3,
400 MHz) δ 0.83 (3H, t, J = 7.2 Hz), 1.03 (3H, d, J = 6.4 Hz),
1.18-1.61 (25H, m), 1.66-2.14 (15H, m), 2.18 (3H, s),
2.59-2.73 (6H, m), 2.89-2.94 (1H, m), 3.01-3.07 (1H, m),
3.28-3.54 (3H, m), 3.70-8.83 (5H, m), 3.93-3.97 (1H, m), 4.20
(1H, d, J = 8.4 Hz), 4.28 (1H, d, J = 6.8 Hz), 4.36 (2H, t, J = 7.2
Hz), 4.92 (1H, dd, J = 10.4, 1.6 Hz), 7.30 (2H, d, J = 8.0 Hz),
7.76 (3H, m); 13C NMR (CDCl3, 100 MHz) δ 10.7, 11.1, 13.1,
14.4, 14.6, 16.5, 19.3, 19.9, 21.4, 22.3, 25.5, 26.3, 28.7, 28.9, 29.0,
Method C. 4-Ethynylbenzyltricyclic ketolide 9 (0.05 g,
0.07 mmol) and 6-azidohexahydroxamic acid24 13a (0.03 g,
0.15 mmol) were dissolved in anhydrous THF (7 mL) with stirring
under argon at room temperature. Copper(I) iodide (0.01 g,
0.04 mmol) and Hunig’s base (0.5 mL) were added to the reaction
mixture, and stirring continued for 12 h. The reaction mixture was
diluted with 10% MeOH/CH2Cl2 (20 mL) and washed with 1:4
NH4OH/saturated NH4Cl (3 ꢀ 10 mL) and saturated NH4Cl
(10 mL). The organic layer was dried over Na2SO4 and concen-
trated in vacuo. The crude product was purified by preparative
TLC (silica, 12:1:0.1 CH2Cl2/MeOH/conc NH4OH) to give 20 mg
(34%) of 15a as a brown-white solid. Mp 127-130 °C; retention
time 20.167 min; 1H NMR (CDCl3, 400 MHz) δ 0.85 (3H, t, J =
8.0 Hz), 1.05 (3H, d, J = 7.2 Hz), 1.20-2.00 (30H, m), 2.13-2.18
(2H, m), 2.20 (3H, s), 2.58-2.72 (6H, m), 2.92-2.98 (1H, m),
3.05-3.09 (1H, m), 3.30-3.55 (3H, m), 3.70-3.84 (5H, m),
3.95-3.98 (1H, m), 4.22 (1H, d, J = 8.0 Hz), 4.31-4.38 (3H,
m), 4.95 (1H, d, J = 8.8 Hz), 7.33 (2H, d, J = 8.0 Hz), 7.77 (3H,
m); 13C NMR (CDCl3, 100 MHz) δ 10.6, 11.1, 13.1, 14.6, 16.5,
19.3, 19.8, 21.4, 22.3, 24.7, 25.9, 29.7, 29.9, 30.0, 36.6, 37.1, 38.8,
42.5, 42.8, 49.4, 49.6, 50.3, 51.4, 53.7, 57.7, 60.1, 65.5, 69.7, 70.5,
76.7, 78.7, 79.3, 81.8, 104.1, 120.1, 126.0, 129.5, 129.7, 139.1, 147.7,
156.3, 169.8, 171.1, 182.3, 204.5; HRMS (ESI) calcd for
[C47H71N7O11 þ H]þ 910.5284, found 910.5279.
Tricyclic Ketolide-N-benzyltriazolylheptahydroxamic Acid
(15b). The reaction of 14b (0.13 g, 0.11 mmol) in CH2Cl2

6110 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Mwakwari et al.
29.6, 29.9, 30.3, 33.1, 36.6, 37.1, 38.8, 42.5, 42.7, 48.2, 49.4, 49.5,
50.6, 51.4, 57.7, 60.1, 60.6, 65.5, 69.7, 70.5, 76.7, 78.7, 79.3, 81.8,
104.0, 119.8, 126.0, 129.5, 129.8, 139.0, 147.7, 156.3, 169.8,
of the effect of the compounds on growth of asynchronous
cultures of P. falciparum, determined by the assay of parasite
lactate dehydrogenase (pLDH) activity.53 The appropriate dilu-
tions of the compounds were prepared in DMSO or RPMI-1640
medium and added to the cultures of P. falciparum (2% hema-
tocrit, 2% parasitemia) set up in clear flat-bottomed 96-well
plates. The plates were placed into a humidified chamber and
flushed with a gas mixture of 90% N2, 5% CO2, and 5% O2. The
cultures were incubated at 37 °C for 72 h. Growth of the parasite
in each well was determined by pLDH assay using Malstat
reagent.53 The medium and RBC controls were also set up in
each plate. The standard antimalarial agents, chloroquine and
artemisinin, were used as the positive controls, while DMSO was
tested as the negative control. Antileishmanial activity of the
compounds was tested in vitro on a culture of Leishmania
donovani promastigotes. In a 96-well microplate assay, com-
pounds with appropriate dilution were added to the leishmania
promastigotes culture (2 ꢀ 106 cell/mL). The plates were in-
cubated at 26 °C for 72 h, and growth of leishmania promasti-
gotes was determined by Alamar blue assay.54 Pentamidine and
amphotericin B were tested as standard antileishmanial agents.
All the compounds were simultaneously tested for cytotoxicty
on VERO (monkey kidney epithilial) cells by Neutral Red
assay.55 The IC50 value for each compound was computed from
the growth inhibition curve.
171.7, 182.9, 204.5; HRMS (ESI) calcd for [C51H79N7O11
þ
H]þ 966.5910, found 966.5919.
Biological Assays. In Vitro HDAC Inhibition. (a) Fluores-
cence Assay. In vitro activity against HeLa nuclear extract was
determined as previously described.24,32,45 Briefly, 15 μL of
HeLa nuclear extract was mixed with 5 μL of 10ꢀ compound
and 5 μL of assay buffer. Fluorogenic substrate (25 μL) was
added, and mixture was allowed to proceed for 15 min at room
temperature and then stopped by addition of a developer
containing TSA. Fluorescence was monitored after 15 min at
excitation and emission wavelengths of 360 and 460 nm, respec-
tively. Assays against HDAC8 and HDAC6 were performed
according to the manufacturer’s instructions.
(b) SAMDI Assay. The maleimide-presenting SAMs and
expression of HDAC8 enzyme were prepared as previously
reported.41 The peptide substrate (Ac-GRKAcFGC-NH2) was
synthesized using standard FMOC solid phase peptide synthesis
protocols using reagents from Anaspec (San Jose, CA). The enzyme
was diluted to 1 μM in buffer (25 mM Tris-HCl (pH 8.0) containing
137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2) with varying
concentrations of inhibitor (0, 50, 100, 250, and 500 nM) for 5 min
at room temperature. The reaction was initiated upon substrate
addition (5, 10, 25, and 50 μM) to a final volume of 20 μL per
reaction, and the plate was incubated at 37 °C. At each time point
(0, 3, 6, 9, and 12 min), 3 μL aliquots were transferred to a separate
plate, and the reaction was terminated by heating to 80 °Cfor1min.
After collection of all time points, the aliquots were transferred to
the maleimide-presenting arrays and incubated at 37 °C for 1 h to
allow the immobilization of the peptide substrate. The arrays were
then washed with water, ethanol and dried under nitrogen. Matrix
(2,4,6-trihydroxyacetophenone, 0.01 μL, 20 mg/mL in acetone) was
applied, air-dried, and the arrays were analyzed by SAMDI MS.
(c) Pf-HDAC1 Inhibition Studies. These were performed as
described in ref 47.
Acknowledgment. This work was financially supported by
GeorgiaInstituteof Technology, bythe Blanchard fellowship,
and by NIH Grant R01CA131217 (A.K.O.). National Center
for Natural Products Research is partially supported by
USDA-ARS Scientific Cooperative Agreement No. 58-
6408-2-0009. W.G. is a recipient of a GAANN predoctoral
fellowship from the Georgia Tech Center for Drug Design,
Development, and Delivery. Z.A.G.-L. is supported by the
ARCS Foundation Chicago Chapter. M.M. is supported by
NIH Grant RO1 GM084188.
Mass Spectrometry and Data Analysis. Mass analysis was
performed using an Applied Biosystems 4800 MALDI TOF/
TOF mass spectrometer (Framingham, MA). A 355 nm Nd:
YAG laser was used as a desorption ionization source, and all
spectra were acquired with 20 kV accelerating voltage in positive
reflector mode. The extent of reaction was calculated as pre-
viously reported.41 Briefly, the relative amount of each com-
pound was calculated by measuring the relative peak intensities
of each molecular ion peak (Mþ) [amount of X = Ix/(Ix þ Iy]
where x refers to the deacetylated peak and y is the parent ion.
For Ki analysis, the micromoles of deacetylated peptide were
plotted over time and the slope of the linear portion was
measured to give the initial velocity of the reaction. A Dixon
plot was then created ([I] vs 1/v), and the intersecting point
corresponds to the Ki of the inhibitor.
Cell Growth Inhibition. All cell lines were maintained in a 37
°C environment containing 5% CO2 with recommended media
(ATCC). For cell growth inhibition, cells were passaged the day
before dosing into a 96-well cell culture plate and allowed to
grow for a minimum of 24 h prior to drug addition. All
compounds were diluted first to 1000ꢀ test concentration in
DMSO and then to test concentrations in cell media so that the
DMSO concentration would be constant across all wells at 0.1%
in a 100 μL volume. Cells were dosed for 72 h, and then 20 μL of
MTS reagent was added and allowed to incubate for 2 h.
Absorbance was then determined using an absorbance plate
reader set to 490 nm.
Supporting Information Available: 1H NMR and 13C NMR
spectral information, HPLC traces, and pf-HDAC1 activity
dose-response curves of 15a-e and molecular modeling out-
puts. This material is available free of charge via the Internet at
http://pubs.acs.org.
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