Analysis of β-amino alcohols as inhibitors of the potential anti-tubercular target N-acetyltransferase

Article abstract of DOI:10.1016/j.bmcl.2010.12.099

The synthesis and inhibitory potencies of a novel series of β-amino alcohols, based on the hit-compound 3-[3′-(4″-cyclopent-2?- en-1?-ylphenoxy)-2′-hydroxypropyl]-5,5 dimethylimidazolidine-2,4- dione as specific inhibitors of mycobacterial N-acetyltransferase (NAT) enzymes are reported. Effects of synthesised compounds on growth of Mycobacterium tuberculosis have been determined.

Full text of DOI:10.1016/j.bmcl.2010.12.099

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1185–1190
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Analysis of b-amino alcohols as inhibitors of the potential anti-tubercular target
N-acetyltransferase
Elizabeth Fullam a, Areej Abuhammad a, David L. Wilson b, Matthew C. Anderton a, Steve G. Davies b,
Angela J. Russell a,b, Edith Sim a,
⇑
a Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
b Department of Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and inhibitory potencies of a novel series of b-amino alcohols, based on the hit-compound
3-[30-(400-cyclopent-2000-en-1000-ylphenoxy)-20-hydroxypropyl]-5,5 dimethylimidazolidine-2,4-dione as
specific inhibitors of mycobacterial N-acetyltransferase (NAT) enzymes are reported. Effects of synthes-
ised compounds on growth of Mycobacterium tuberculosis have been determined.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 17 November 2010
Revised 14 December 2010
Accepted 18 December 2010
Available online 24 December 2010
Keywords:
Tuberculosis
Arylamine N-acetyltransferase
b-Amino alcohols
Enzyme inhibition
Drug discovery
Tuberculosis (TB) remains one of the leading causes of death
from a single infectious disease worldwide each year. In 2009 the
World Health Organisation (WHO) reported 1.7 million people
died from TB, equal to 4700 deaths a day, (http://www.who.int/
tb/publications/global_report/2010/) and also reported 9.4 million
new cases of TB in the same year. The increase in prevalence of HIV
and the emergence of multi-drug resistant (MDR) and extreme
drug resistant strains (XDR) means that new drugs are urgently re-
quired to prevent a potential pandemic.1,2
The arylamine N-acetyltransferase (NAT) enzyme has been
identified in a number of eukaryotic and prokaryotic species
including Mycobacterium tuberculosis, the causative agent of TB
and has been identified as a potential new target for the treatment
of tuberculosis. When the gene was deleted from Mycobacterium
hydroxyamines and aryl hydrazines4 and acyl hydrazides including
isoniazid,5 which is one of the front-line treatments for tuberculo-
sis. The crystal structures of NAT enzymes from a number of spe-
cies have been solved, including Salmonella typhimurium,6
Pseudomonas aeruginosa,7 Nocardia farcinica,8 human NAT29 and
the mycobacterial species Mycobacterium smegmatis10 and Myco-
bacterium marinum.11 All NAT enzymes are found to have very sim-
ilar 3-dimensional structures and consist of three distinct domains
with the active site containing a catalytic triad formed from a cys-
teine, a histidine and an aspartate residue. Recently the cofactor
binding site has been identified in human NAT29 and M. marinum11
and found to differ between mammalian and bacterial species.11
In order to further understand and investigate the role of NAT as
an essential target within mycobacteria, small molecule inhibitors
of NAT enzymes are required so that a chemical genomic approach
can be undertaken to complement the genetic studies which had
bovis BCG (
have low levels of mycolic acids and an alteration in the cell wall
architecture.3 The
nat mutant strain was found to have increased
Dnat) the resulting knockout organism was found to
D
been previously carried out on D
nat strains of M. bovis BCG3 and
sensitivity to the antibiotics hygromycin and gentamycin that have
previously been shown to have little effect on wild-type M. bovis
M. smegmatis.12 Therefore, an in-house library of 5000 selected
commercial compounds were screened for in vitro enzymic inhibi-
tion against a panel of prokaryotic and eukaryotic NAT en-
zymes.13,14 A smaller manual screen had been previously carried
out and been successful in identifying inhibitors of prokaryotic
NAT enzymes.15 From the larger, automated high-throughput
screen, six compounds were identified as specific inhibitors of
the prokaryotic NAT enzymes with these compounds displaying
no inhibitory effect on the eukaryotic enzymes also screened.13
One of the compounds identified from the more extensive screen
BCG and M. tuberculosis. Importantly these
Dnat mutant strains
were found to be more susceptible to intracellular killing within
mouse macrophages.3
NAT enzymes utilise the donor cofactor acetyl coenzyme A to
acetylate a broad range of substrates including arylamines, N-aryl-
⇑
Corresponding author. Tel.: +44 1865 271884; fax: +44 1865 271853.
E-mail address: edith.sim@pharm.ox.ac.uk (E. Sim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.099

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E. Fullam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1185–1190
was the b-amino alcohol 3-[30-(400-cyclopent-2000-en-1000-ylphen-
oxy)-20-hydroxypropyl]-5,5-dimethylimidazolidine-2,4-dione
1.
The novel class of compound, that inhibited prokaryotic NATs with
greater than 80% inhibition at a concentration of 30 M, had not
l
previously been reported to have antibacterial activity. However,
it was interesting to note that a b-amino-alcohol motif has been
incorporated into ethambutol 2, Figure 1,16 a front-line drug cur-
rently used for the treatment of tuberculosis. Also, phenanthracene
derivatives 3, Figure 1, have been reported to have anti-tubercular
activity at a minimum inhibitor concentration (MIC) as low as
Scheme 1. Synthesis of ortho- and para-cyclopentenylphenol. Reagents and con-
ditions: (a) H3PO4, toluene, rt, 2 h then cyclopentadiene, toluene, rt, 2 h.
3.12 l
g/mL,17 Figure 1. We report here on the synthesis of a series
of b-amino alcohols which have diversification around the aryl and
hydantoin moieties of the purported hit compound 1, the in vitro
evaluation of this series of compounds as inhibitors of NAT en-
zymes and growth of mycobacteria species and the identification
of preliminary structure–activity relationships.
O
O
N
H
OH
OH
R2
R3
O
A series of b-amino alcohols were synthesized with derivatisa-
tion occurring either on the aryl ring and/or on the hydantoin moi-
ety of the molecule, Schemes 1 and 2.18,19 In order to resynthesise
the hit compound 1, ortho- and para-substituted cyclopentenyl-
phenol 4 and 5, respectively, were prepared via the reaction of
freshly prepared cyclopentadiene with phenol, Scheme 1.18 The
reaction proceeded in a non-regioselective manner. Subsequently
the product mixture containing ortho-4 and para-5 isomers was
separated using column chromatography purification. Other
substituted phenols at the ortho-, meta- and para-positions were
commercially available and purchased. The corresponding b-amino
alcohols were synthesized in two steps starting from the respective
substituted phenols, via an epoxide intermediate that was then re-
acted with commercially available hydantoins,19 affording resyn-
thesised hit compound 1, the ortho-analogue of the hit
compound 620 and analogues 7–17, Scheme 2. The hit compound
1 contained approximately 30% impurity, as determined by 1H
NMR. This impurity is believed to be another para-substituted
compound with isomerisation occurring of the double bond on
the cyclopentenyl ring. We were unable to separate this impurity
from compound 1 and therefore the results obtained from com-
pound 1 described in this study are from this mixture of para-
cyclopentenyl compounds. This product showed poor inhibition,
Table 1.
The series of b-amino alcohols were tested for their in vitro
activity against three bacterial NAT enzymes:15 NAT from M.
smegmatis (MSNAT) and NAT from P. aeruginosa (PANAT) which
were two of the prokaryotic enzymes used in the high-throughput
screen13 and the NAT from M. marinum (MMNAT) which is the
closest recombinant homologue of the NAT enzyme from M. tuber-
culosis which was available at the time.11
Upon testing of the resynthesised hit compound 1 using DTNB
to measure the rate of hydrolysis of acetyl CoA with both isoniazid
(INH) and 5-aminosalicylate (5AS) as substrates, no inhibitory ef-
fect upon the activity of PANAT, MSNAT or MMNAT at a concentra-
a
b
O
N
O
R1
R1
R1
Scheme 2. Synthesis of b-amino alcohols. Reagents and conditions: (a) epichloro-
hydrin, K2CO3, acetone, reflux 12 h; (b) requisite hydantoin, cat. pyridine, ethanol,
reflux, 4 h.
the ortho-analogue 6 of the hit compound 1 had inhibitory activity
against each of these prokaryotic NAT enzymes, with greatest po-
tency shown against the PANAT version of the NAT enzymes. The
IC50 values for compound 6 with PANAT were 9
(INH) was used as a substrate and 17 M when 5-aminosalicylate
(5AS) was used as a substrate, Table 1. For MMNAT IC50 values for
compound 6 of 37 and 33 M were observed for INH and 5AS,
respectively, Table 1. When lower concentrations of hydralazine
(HLZ) are used as a substrate21 the IC50 values are even lower for
compound 6, Table 1.
The sub-library of synthesized derivatives of compound 1 were
tested for their inhibitory effects on MMNAT enzymic activity and
the results displaying the percentage inhibition are shown in Ta-
lM when isoniazid
l
l
ble 2. The compounds were all tested at 30 lM and the most po-
tent compound was the ortho-cyclopentenyl analogue 6 (58%
inhibition) followed by the 1-naphthyl derivative 14 (28% inhibi-
tion) and then the ortho-phenyl 11 substituted compound (24%
inhibition) and ortho-bromo substitution 8 (24% inhibition). Com-
pounds that had no substitution of the phenyl ring (15–17) were
found to have no inhibitory effect upon the activity of MMNAT.
The replacement of the ortho-hydrogen by a fluorine atom 7 also
resulted in no increase in inhibitory potency of the compound.
When the halogen at the ortho-position is a bromine atom 8 rather
than fluorine 7, then an increase in the percentage inhibition of
MMNAT from 0% to 24% is observed.
As the crystal structure of MMNAT is available11 in silico dock-
ing studies were carried out for the compounds 1, 6, 11 and 14 in
tion of 50 lM was found. However, interestingly it was found that
Figure 1. b-Amino alcohols. Compound 1 is the hit compound identified from the high-throughput screen, compound 2 is ethambutol currently used as a front-line drug for
the treatment of tuberculosis and analogues of compound 3 have been reported to inhibit the growth of Mycobacterium tuberculosis.16,17,24

E. Fullam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1185–1190
1187
Table 1
Comparison of compound 1 and 6 as inhibitors of NAT enzymes
Enzyme
PANAT
MSNAT
MMNAT
MMNAT
Substrate
INH
5AS
INH
5AS
INH
5AS
HLZ
IC50
IC50
(
l
M)
M)
1 para
6 ortho
>50
9.4 1.4
>50
17.7 1.5
>50
37.0 1.2
>50
16.2 0.9
>50
37.0 1.8
>50
33.1 1.5
ND
1.5 0.09
(l
The IC50
protein in the presence of five concentrations of compound 1 and 6 with the substrates isoniazid (INH) or 5AS (500
150 M. Each inhibitor concentration was carried out in triplicate. The concentration at which there was 50% inhibition of activity was determined graphically. ND is not
(l
M) values for compounds 1 and 6. The NAT activity assay14 was used to determine the specific activities of the pure recombinant MMNAT, MSNAT or PANAT
M). Hydralazine (HLZ) was used at a concentration of
l
l
determined.
Table 2
Effect of analogues of 1 on MMNAT enzymic activity
O
R4
R5
O
N
R1
R2
OH
NH
O
R3
Compound
R1
R2
R3
H
R4
R5
% inhibition
6
1
7
8
9
10
11
12
13
14
15
16
17
Cyclopentenyl
H
F
Br
Me
NO2
Ph
H
H
H
H
H
H
H
H
Ph
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
58
0
0
24
0.5
4
24
6
8
28
0
0
0
Cyclopentenyl
H
H
H
H
H
H
Ph
H
H
H
H
H
Naphthyl
Naphthyl
H
H
H
H
H
H
H
H
The substitution of the aryl ring R1–R3 and substituted hydantoin R4–R5 are shown. The percentage inhibition of MMNAT enzymic activity at 30
l
M compound is calculated
by comparison to a 5% DMSO control. The NAT activity assay14 was used to determine the activity with isoniazid as substrate. Each measurement was carried out in triplicate.
an effort to rationalise the preliminary structure–activity relation-
ships for this series of b-amino alcohols. Compounds were docked
into the MMNAT structure (PDB code 2VFB) using the GOLD suite of
programs.22 Ten docking solutions for each ligand were outputted
by the program and the docking solution with the best score, was
selected and analysed. The docking solutions indicated that both
the ortho- and para-compounds 6 and 1 are able to enter a cleft
of MMNAT that leads up to the active-site cysteine, although re-
mote from the cofactor acetyl-CoA binding site, Figure 2.11 A num-
ber of hydrophobic binding interactions are predicted for each.
Both compounds (1 and 6) are positioned in a similar location in
the protein with the main difference between the two predicted
docking solutions of compounds 1 and 6 being the orientation of
the cyclopentenyl group. In the ortho-isomer 6 the cyclopentenyl
MMNAT enzymic activity by 24% whilst the para-biphenyl ana-
logue 13 shows only 8% inhibition and the meta-biphenyl com-
pound 12 inhibits MMNAT activity by just 6%. These results for
the biphenyl series of compounds 11-13 show the same trend as
for the cyclopentenyl derivatives 1 and 6 in which the ortho-substi-
tuted compounds are the most potent inhibitors of MMNAT enzy-
mic activity. It should be noted that the ortho-biphenyl substituted
derivate 11 (24% inhibition) has less inhibitory potency than the
ortho-cyclopentenyl counterpart 6 (58% inhibition).
To explain these experimental results in structural terms,
docking studies were carried out (Fig. 3). The docking results sug-
gest that each inhibitor binds within the same position of the
MMNAT protein with each ligand being within a distance of 4 Å
from the active-site Cys70. These docking results suggest that
the ortho-phenyl substituent 11 is orientated away from the ac-
tive site cysteine, in a manner that stabilises the binding interac-
group is orientated towards the Sc of active site cysteine. In the
case of the para-isomer 1 the cyclopentenyl group is orientated
away from the active site cysteine. This is consistent with the
experimental data that ortho-compound 6 is an inhibitor of pro-
karyotic NAT enzymes, whereas the para-compound 1 is a poor
inhibitor of NAT enzymic activity.
In order to understand the effects of ortho-, meta- and para-sub-
stitution the appropriate biphenyl compounds ortho-biphenyl 11,
meta-biphenyl 12 and para-biphenyl 13 were synthesised and
tested for activity on the MMNAT enzyme and the results are
shown in Table 2. The ortho-biphenyl derivative 11 inhibits
tion between the protein and ligand through
p-stacking
interactions between the phenyl-substituent group of the ligand
and with residue Phe130 (Fig. 3D). This may explain the experi-
mental result in which compound 11 is a less potent inhibitor
than compound 6. Furthermore, compound 6 may result in great-
er inhibition of MMNAT enzymic activity than compound 11 as a
result of ortho-cyclopentenyl compound 6 having fewer confor-
mational restrictions than the ortho-biphenyl compound 11. The
cyclopentenyl-ring of compound 6 may adopt an envelope con-

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E. Fullam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1185–1190
Figure 2. Comparison of the ortho- and para-cyclopentenyl substitution on interaction with MMNAT. (A) Compounds 1 and 6 were docked into the GOLD docking program. The
residues which are predicted to interact with the ligands are shown in stick format. The ortho isomer 6 carbon atom’s are shown in magenta and the para isomer 1 carbon
atoms are coloured blue. (B) A surface representation of MMNAT (PDB code 2VFB), indicating the predicted cleft for binding of the compounds 1 and 6. The CoA binding site is
indicated.
Figure 3. Comparison of the ortho-substitution of the b-amino alcohol inhibitors interaction with MMNAT. Compounds 6 (magenta, A), 8 (orange, B), 14 (yellow, C), 11 (grey,
D) were docked into the MMNAT protein (PDB code 2VFB) using the GOLD docking program. The residues which are predicted to interact with the ligands are shown in stick
format.
formation thus enabling close contacts between the inhibitor and
the active-site of the cleft of MMNAT to occur. The rigid aromatic
biphenyl compound 11 is locked in its orientation, is less flexible,
and therefore may not fit the MMNAT pocket as well as com-
pound 6 resulting in less inhibitory potency of the ortho-biphenyl
compound 11. ortho-Naphthyl 14 also contains rigid aromatic
rings and has similar potency to 11. Docking studies of the naph-
thyl analogue 14 show that this compound is also predicted to
bind in the same orientation as the cyclopentenyl substituted
compound 6, with the naphthyl group orientated towards the ac-
tive-site cysteine (Fig. 3C). The bromo-compound 8 is also pre-
dicted to bind in a similar position in the MMNAT protein,
Figure 3B. Bromo-compound 8 is likely to be a better inhibitor
than fluoro-compound 7, which does not have any inhibitory ef-
fect upon the activity of MMNAT, as a result of the increased size
of the bromine atom, compared to fluorine.
Recently the structure of MMNAT has been solved with the sub-
strate hydralazine (HLZ) (PDB code 3LTW,23). When the docking re-
sults of the ortho-cyclopentenyl compound 6 are overlaid upon the
solved HDZ-MMNAT structure it is observed that the ortho-cyclo-
pentenyl moiety of compound 6 is in the same position of the
HDZ substrate, Figure 4. Therefore it is proposed that the mecha-
nism of inhibition of these ortho-b-amino alcohol compounds is
through their ability to prevent substrates of the MMNAT protein
being able to access and undergo acetylation at the active-site
Cys70.

E. Fullam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1185–1190
1189
Figure 4. Comparison of docked ortho-isomer 6 with the binding site of hydralazine in MMNAT. (A) The binding site of hydralazine (dark blue) in the active-site of MMNAT is
shown (PDB code 3LTW). (B) The docking result of the ortho-isomer 6 is overlaid upon the hydralazine-MMNAT structure. The ortho-isomer 6 carbon atoms are shown in
magenta.
The predicted conformations of the inhibitors of MMNAT indi-
cate that they each bind in a similar orientation and position in
the MMNAT protein, Figure 5A. However, the ortho-cyclopentenyl
compound 6 docking result predicts that the hydroxyl moiety of
the b-amino alcohol functionality points in an opposing direction
with compounds 8, 11 and 14. The orientation of this hydroxyl-
moiety of 6 is positioned ideally to form a hydrogen bond with
Thr109 and therefore this extra ligand-protein interaction formed
may go some way to explaining why compound 6 is a better inhib-
itor than 8, 11 and 14, Figure 5. Previous studies of compounds that
have been identified as substrates and inhibitors of NAT proteins
have suggested that more lipophilic compounds are preferred.15
In this study the orientation, conformation, size and steric factors
also appear to play an important role in the inhibitory effect of this
series of compounds studied.
Substituted hydantoin compounds 15, 16 and 17, had no effect
on the activity of the protein, suggesting that this moiety of the po-
tential inhibitor has little impact on enzymic activity.
Having shown that the ortho-compound 6 is able to inhibit the
activity of MMNAT, the effects of this compound and also the ‘hit’
para-compound 1 on the growth of M. marinum were investi-
gated.24 Compound 6 displayed moderate inhibition of growth
Figure 5. The predicted orientation of the docked ortho-substituted inhibitors in MMNAT. (A) An overlay of docked solutions of the ortho-cyclopentenyl compound 6
(magenta), the bromo-compound 8 (orange), the naphthyl-compound 14 (yellow) and the ortho-biphenyl compound 11 (grey). (B) The docked solutions in the active site of
MMNAT (PDB code 2VFB). (C) The ortho-cyclopentenyl compound 6 (magenta) is shown indicating the H-bond with Thr109. The Oxygen atoms are indicated in red.

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E. Fullam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1185–1190
against M. marinum and at a concentration of 80 lg/mL no growth
Supplementary data
of M. marinum could be observed. The compound in the library
purported to be the para-analogue 1 had no inhibitory effect on
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.099.
the growth of M. marinum at concentrations as high as 100
mL. Compound 6 moderately inhibited the growth of M. tuberculo-
sis at 50 g/mL by 20% compared to the DMSO only control. The
lg/
l
References and notes
remaining synthesised analogues of 1 were not tested for their ef-
fect on the inhibition of mycobacterial growth given that they were
poor inhibitors of MMNAT at 30 lM and only weak anti-mycobac-
terial activity was observed for the ortho-isomer 6, which was
found to be the most potent MMNAT inhibitor of this series of
compound.
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the ortho-compound 6 reported in this study.17,25 Panda et al. sug-
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thracene compounds synthesized by Panda et al. are more hydro-
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In conclusion, a series of b-amino alcohols were synthesized
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ring in this series of compounds plays an essential role in the
inhibitory properties of this class of compound in NAT enzymes.
Designing future compounds which have a substituent at the
ortho-position of the phenyl-ring that is primed to interact with
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the Sc of Cys70 of MMNAT, may result in a compound with en-
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ortho-6 and para-1 compounds as NAT inhibitors is also reflected
in their relative potencies as inhibitors of growth of M. marinum
and M. tuberculosis although they are each very weak inhibitors
of mycobacterial growth.
Furthermore, we have also demonstrated the absolute impor-
tance of verifying a hit compound that has been identified as a
result of undertaking a high-throughput screen through both
resynthesis and biological testing of the compound. These
studies have shown that b-amino alcohol compounds represent
an interesting class of compound that are worth further
investigation in order for novel anti-tubercular drugs to be
identified.
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R.; Srivastava, B. S. Bioorg. Med. Chem. 2004, 12, 5269.
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20. Spectroscopic data for 6: 1H NMR (CDCl3) 400 MHz d, p. H., s), 1.41 (3H, s), 1.62–
1.68 (1H, m), 2.12 (1H, s), 2.32-2.52 (4H, m), 3.73–3.81 (2H, m), 3.93–4.01 (2H,
m), 4.25 (1H, br s), 5.78–5.82 (1H, m), 5.91–5.95 (1H, m), 6.78–8.82 (1H, m),
6.88–7.92 (1H, m), 7.11–7.19 (2H, m).
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Acknowledgements
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The authors wish to thank Dr. Isaac Westwood, Dr. Akane
Kawamura and Dr. Adam Le Gresley for helpful discussions.
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