An E/Z conformational behaviour study on the trypanocidal action of lipophilic spiro carbocyclic 2,6-diketopiperazine-1-acetohydroxamic acids

Article abstract of DOI:10.1016/j.tetlet.2013.03.128

An explanation for the vast difference observed in the trypanocidal activity between the new secondary (N-methylated) hydroxamic acids 5 and 6, and their primary (nonmethylated) congeners 1a and 2, based on their E/Z conformational behaviour in DMSO, is p

Full text of DOI:10.1016/j.tetlet.2013.03.128

Tetrahedron Letters 54 (2013) 3238–3240
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Tetrahedron Letters
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An E/Z conformational behaviour study on the trypanocidal action
of lipophilic spiro carbocyclic 2,6-diketopiperazine-1-acetohydroxamic acids
Alexandra Tsatsaroni a, Grigoris Zoidis a, Panagiotis Zoumpoulakis b, Andrew Tsotinis a, Martin C. Taylor c,
John M. Kelly c, George Fytas a,
⇑
a Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimioupoli-Zografou, GR-15771 Athens, Greece
b Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 11635 Athens, Greece
c Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
An explanation for the vast difference observed in the trypanocidal activity between the new secondary
(N-methylated) hydroxamic acids 5 and 6, and their primary (nonmethylated) congeners 1a and 2, based
on their E/Z conformational behaviour in DMSO, is presented.
Received 8 January 2013
Revised 15 March 2013
Accepted 28 March 2013
Available online 8 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Lipophilic hydroxamic acid derivatives
E/Z conformational behaviour
Trypanocidal action
NMR
Molecular modelling studies
Primary and secondary hydroxamic acids [R0CON(R)OH, R = H or
alkyl] constitute a class of metal ion chelating agents with great
therapeutic potential.1,2a A large number of naturally occurring
hydroxamate-based siderophores (Fe3+ carriers) have been isolated
and much effort has been devoted to the synthesis of bioactive
hydroxamic acids.1b,2,3a,3b Due to their metal ion binding ability,
hydroxamic acids often behave as inhibitors of metalloenzymes
(e.g., Fe3+-containing lipoxygenase, Zn2+-containing MMPs and
HDACs and Ni2+-containing urease), which are implicated in the
pathophysiology of human diseases.1a,2a,3
The structures of hydroxamic acid analogues have been studied
extensively using NMR and molecular modelling techniques.4,5 For
particular hydroxamic acid structures, the existence of different
possible conformations in solution has been found to depend on
the sample concentration, the temperature and the solvent.6 More
specifically, the hydroxamate group may adopt E and Z conforma-
tions which are separated by a high energy barrier.7 Furthermore,
tautomerism between the amide and the imide forms is possible,
but the imide forms are found to be absent in solution.7 Various
researchers have concluded that the Z conformation of the amide
structure prevails since it becomes stabilized via hydrogen bond
formation, either intramolecularly or intermolecularly to a polar
solvent.7 Interestingly, in some cases, the proton NMR spectra of
hydroxamic acid analogues at room temperature (ꢀ25 °C) clearly
show a double set of characteristic peaks, arising from the two dif-
ferent isomers (E/Z), while in others, a single set of characteristic
peaks is observed, which is attributed to one of the two isomers.
Thus far, the most extensive conformational study reported refers
to benzohydroxamic acid (BHA) in acetone.7 Moreover, it is well-
known that hydroxamic acids, R0CON(R)OH (R = H or alkyl), chelate
with a variety of metal ions via the oxygen atoms, the carbonyl
oxygen and the deprotonated OH (O,O-co-ordination). In this well
documented O,O-type co-ordination mode, the amide structure of
the hydroxamic group must adopt the Z(cis) conformation (ion-
binding conformation) for effective ion complexation.8
In a previous publication, we reported on acetohydroxamic acid
analogues, derived from conformationally constrained lipophilic
spiro carbocyclic 2,6-diketopiperazine scaffolds by attaching the
acetohydroxamic acid group to their imide nitrogen, as a potential
metal ion complexing group. The primary hydroxamic acid deriva-
tives 1a–e, 2, 3a–d, 4a and 4b (Fig. 1) exhibited excellent trypano-
cidal properties.9a We have also demonstrated that the hydroxamic
acid group (CONHOH) is a requirement for activity. On this basis,
we assumed that this class of compounds acts by inhibiting a vital
parasite metalloenzyme, through metal ion binding at this group.
As part of our ongoing search to probe the stereoelectronic
requirements for optimal trypanocidal activity, we present herein
the synthesis and biology of the new secondary hydroxamic acids
5 and 6 (Fig. 1). Surprisingly, the trypanocidal activity results ob-
⇑
Corresponding author. Tel.: +30 210 727 4810; fax: +30 210 727 4747.
E-mail address: gfytas@pharm.uoa.gr (G. Fytas).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.03.128

A. Tsatsaroni et al. / Tetrahedron Letters 54 (2013) 3238–3240
CH2COOH
3239
CH2CONHOH
CH2CONHOH
CH2CON(CH3)OBn
O
N
O
O
O
O
N
O
N
(i) CDI, THF, 28 oC, 1 h, Ar
(ii) HN(CH3)OBn, 28 oC,
R
25 h, then 1 h, 45 oC, Ar
O
N
O
(CH2)n
N
N
H
N
R
R
N
H
R
R'
R'
n=7 : 3a-d
n=6 : 4a, 4b
7
80% for 9
69% for 10
1a-e, 2
: R=H
9: R=H
8: R=CH2Ph
CH2CON(CH3)OH
10: R=CH2Ph
O
N
O
a:R=R'=H
CH2CON(CH3)OH
b:R=H, R'=Me
O
N
O
c:R=Me, R'=H, S-enantiomer
N
H
d
:R=CH2Ph, R'=H, S-enantiomer
:R=CH2Ph, R'=H, R-enantiomer
R
e
H2/Pd-10%, EtOH, 50 psi, rt, 3 h
2:R=CH2Ph, R'=H, racemic
N
H
: R=H
R
5
86% for 5
94% for 6
: R=CH Ph, racemic
6
2
5: R=H
6
: R=CH2Ph
Figure 1. Structures of the hydroxamic acid derivatives 1a–e, 2, 3a–d, 4a and 4b
and the structures of the new N-methyl hydroxamate analogues 5 and 6.
Scheme 1. Synthesis of the new N-methyl hydroxamate analogues 5 and 6.
tained for these compounds indicated a dramatic decrease in po-
tency (ꢀ2000-fold), compared to their nonmethylated counter-
parts 1a and 2, as shown in Table 1.
This vast difference in the trypanocidal action of the new ana-
logues can probably be attributed to the predominance of different
conformer(s). In order to verify this argument, we conducted a ser-
ies of NMR experiments on hydroxamates 1a, 2, 5 and 6, accompa-
nied by theoretical calculations.
The 1H NMR spectra of compounds 2 and 6 are depicted in Fig-
ure 2 (downfield region), and their full 1H NMR spectra in Supple-
mentary data. Full assignment tables of the 1H and 13C signals for
the four analogues studied are also provided in Supplementary
data.
For compound 2, the integration of the four signals from 8.83 to
10.52 ppm indicated that the two outermost peaks at 8.83 and
10.52 ppm are assigned to one isomer (isomer A), while the two in-
ner ones, at 9.25 and 10.12 ppm, belong to isomer B, with an A:B
ratio equal to 75:25. Furthermore, C8 of the hydroxamate carbonyl
resonated at 164.1 ppm for the A isomer and at 169.5 ppm for the B
isomer.
In order to determine the E/Z conformation, a 2D NOESY exper-
iment was conducted. Figure 3 presents the expansion of the
NOESY spectrum of compound 2. Off diagonal signals due to the
H7 methylene at 4.15 ppm are observed with two resonances for
the major isomer (10.52 and 8.83 ppm) and one signal at
10.12 ppm for the minor isomer.
From molecular modelling studies of the E and Z isomers of
compound 2, the distances between H9, H10 and H7 in both iso-
mers have been calculated, and only H10 (OH) of the Z conforma-
tion is a long distance from H7 (ꢀ5 Å), which may explain the
absence of an NOE signal; thus H10 of the Z isomer is assigned to
the signal at 9.25 ppm, and consequently H9 is assigned to that
at 10.12 ppm.
The synthesis of the new methylated hydroxamic acids 5 and 6
is shown in Scheme 1. Reaction of the carboxylic acids 7 and 8, pre-
pared as previously reported,9 with O-benzyl-N-methyl hydroxyl-
amine in the presence of CDI in dry THF, led to the formation of
the respective O-benzyl-N-methyl hydroxamates 9 and 10. Subse-
quent O-benzyl deprotection, by hydrogenolysis, gave the corre-
sponding methyl hydroxamic acid analogues 5 and 6 in high yields.
The NMR experiments were performed in DMSO-d6 at ambient
temperature. DMSO was the solvent of choice since it simulates the
biological environment. A previous NMR study on simple mono-
alkylhydroxamic acids using DMSO-d6 as the solvent4 concluded
that the Z isomer predominated.
Structure elucidation was performed for compounds 1a, 2, 5
and 6 using routine 1D 1H, 13C and 2D gCOSY, gHSQC, gHMBC
and NOESY NMR techniques. Interestingly, compounds 1a and 2
exhibited four distinct peaks for the NH and OH protons of the
hydroxamate group, which correspond to the E and Z isomers.
Moreover, two distinct 13C NMR resonances appeared for the ter-
tiary carbon atoms of the hydroxamate carbonyl group attributed
to each of the two isomers. On the other hand, compounds 5 and
6, which bear a CON(CH3)OH moiety instead of the CONHOH group
of 1a and 2, respectively, showed only one resonance for the OH
proton and a single NCH3 signal. Furthermore, the 13C NMR spectra
of compounds 5 and 6 showed a single resonance for each of the
hydroxamate carbonyl and NCH3 carbons.
As a result, the minor isomer is Z and the major is E. This is also
in accordance with theoretical energy calculations of the E and Z
isomers showing 66.3 kcal molꢁ1 for the Z and 65.4 kcal molꢁ1 for
Table 1
Activity of acetohydroxamic acids 1a, 2, 5 and 6 tested against cultured bloodstream-
form T. brucei (pH = 7.4) (see Supplementary data)
Compd
T. brucei
a,b
a,b
IC50 (nM)
IC90 (nM)
1a
2
5
90 16 (79 6)
155 7 (148 8)
26 3 (24 1)
17 1 (18 1)
246 ꢂ 103 (106 ꢂ 103)
523 ꢂ 103 (198 ꢂ 103)
6
37 ꢂ 103 (35 ꢂ 103)
47 ꢂ 103 (45 ꢂ 103)
a
Concentrations required to inhibit growth of T. brucei by 50% and 90%,
respectively. For the active compounds 1a and 2, IC50 and IC90 data are the mean of
triplicate experiments SEM (standard error of the mean).
b
IC50 and IC90 data for the respective hydrochlorides are shown in parentheses.
Figure 2. Downfield region of compounds 2 and 6 including proton assignments.

3240
A. Tsatsaroni et al. / Tetrahedron Letters 54 (2013) 3238–3240
NCH3 peak at 3.06 ppm and the methylene H7 protons, provided
evidence for the E configuration of the molecule.
Systematic probing of the C7-C8-N-H9 dihedral angle of com-
pound 2 produced two energy minima, one for the E conformation
(58.4 kcal molꢁ1
)
and the other for the
Z
conformation
(59.8 kcal molꢁ1, Fig. 4). Similarly, compound 6 provides one en-
ergy minimum for the E (58.8 kcal molꢁ1) and one for the Z
(62.4 kcal molꢁ1). The energy barrier for the E/Z interconversion
for compound 6 is higher than that of 2, with an energy difference
of ꢀ5 kcal molꢁ1 (Fig. 5).
In conclusion, NMR studies indicate that the primary hydroxa-
mic acid derivatives 1a and 2, in their amide form, adopt preferen-
tially the E conformation (E/Z = 75/25) in DMSO at ꢀ25 °C, whereas
in their respective secondary N-methylated congeners 5 and 6, the
E conformer is the only one present in solution. These results are in
accordance with in silico theoretical calculations.
Given that the Z conformation is a prerequisite for complexa-
tion with a metal ion, the failure of the methylated hydroxamic
acid analogues 5 and 6 to produce a similar biological effect to their
congeners 1a and 2 might be attributed to the absence of the
respective Z conformer in the binding site of the metalloenzyme.
In the case of the active molecules 1a and 2, as the minor Z con-
former is complexed to the metal ion in the catalytic site of the en-
zyme, the E/Z equilibrium is shifted to the active conformer
Figure 3. Expansion of the 2D NOESY spectrum of compound 2.
(
)
inducing metalloenzyme activity
inhibition.
Acknowledgments
J.M.K. acknowledges the support of the Wellcome Trust (Grant
no. 092573).
Supplementary data
Figure 4. E (left) and Z (right) low energy conformations of bioactive compound 2.
Supplementary data (experimental procedures and spectral
data) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.tetlet.2013.03.128.
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the E isomer. Finally, H9 and H10 of the E isomer are assigned to
the signals at 10.52 and 8.83 ppm, respectively. The above assign-
ments are in agreement with the 2D heteronuclear gHMBC spec-
trum, where a correlation between C8 and H9 is observed.
The same methodology was used for the assignment of the sig-
nals obtained for compound 6 (Supplementary data). Once again,
an NOE signal was observed between H10 (OH) at 10.05 ppm and
the H7 methylene, while the absence of an NOE between the