Synthesis and Evaluation of Potent Ene-yne Inhibitors of TypeII Dehydroquinases as Tuberculosis Drug Leads

Article abstract of DOI:10.1002/cmdc.201000399

E-9: You sunk my battleship! Potent ene-yne-based inhibitors of typeII dehydroquinase are described. The majority of the compounds exhibited nanomolar inhibition of the Streptomyces coelicolor, Helicobacter pylori and Mycobacterium tuberculosis typeII deh

Full text of DOI:10.1002/cmdc.201000399

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DOI: 10.1002/cmdc.201000399
Synthesis and Evaluation of Potent Ene–yne Inhibitors of Type II
Dehydroquinases as Tuberculosis Drug Leads
Anh Thu Tran,[a] Katie M. Cergol,[a] Nicholas P. West,[b] Elizabeth J. Randall,[b] Warwick J. Britton,[b]
Syed Ali Imran Bokhari,[c] Musadiq Ibrahim,[c] Adrian J. Lapthorn,[c] and Richard J. Payne*[a]
The shikimate pathway is an essential biosynthetic route oper-
ating in plants, bacteria, fungi, and apicomplexan parasites.[1–4]
Consisting of seven enzyme-catalyzed steps, this pathway is re-
sponsible for the sequential conversion of erythrose-4-phos-
phate and phosphoenol pyruvate to chorismate, the precursor
for the biosynthesis of a range of aromatic amino acids, folates,
ubiquinone, and vitamins E and K.[1,5,6] The absence of this
pathway in mammals, alongside its essential role in the surviv-
al of plants, bacteria, fungi and apicomplexan parasites, makes
this an attractive target for the development of new herbicides
and antimicrobial agents.[1]
hydrogen (HS, Scheme 1) is facilitated by a conserved tyrosine
residue, resulting in the formation of an enol intermediate 3
(Scheme 1).[7,11,12]
Type II dehydroquinases are present in pathogenic bacteria
such as Mycobacterium tuberculosis,[13] the etiological agent of
tuberculosis (TB), and Helicobacter pylori,[14] a stomach patho-
gen that causes gastric ulcers and is linked to the development
of stomach cancer. Due to the increased resistance of these
and other microorganisms to current chemotherapies, antibac-
terial agents with novel mechanisms of action are required.
Since the 1990s, several new inhibitors of type II dehydroqui-
nases have been reported.[15] In particular, work by Gonzꢀlez-
Bello et al.[16–19] and Abell et al.[20–22] has highlighted several
specific features that contribute to the potent inhibition of this
enzyme. In particular, compounds possessing an anhydroqui-
nate core, mimicking the enol intermediate 3, attached either
directly to an aromatic moiety (e.g., 4,[18] Figure 1), or via an in-
The enzyme dehydroquinase (3-dehydroquinate dehydra-
tase, EC 4.2.1.10) catalyzes the third step in the shikimate path-
way—the reversible dehydration of 3-dehydroquinate (1) to 3-
dehydroshikimate (2). Two structurally distinct dehydroquinase
(DHQase) enzymes are known (type I and II), which catalyze
the same dehydration reaction via two mechanistically distinct
pathways.[7,8] Type I dehydroquinases are functional dimers
that catalyze the syn-elimination of water via an imine inter-
mediate with a conserved lysine residue, which enables the ab-
straction of the pro-R hydrogen from the C-2 position (HR,
Scheme 1).[9,10] In contrast, the type II dehydroquinases are do-
decamers that facilitate the anti-elimination of water via an
E1CB mechanism, whereby abstraction of the more acidic pro-S
Figure 1. Potent inhibitors of M. tuberculosis type II dehydroquinase.[17,22]
flexible linker, have proven successful. More recently, we have
reported potent inhibitors that support this design strategy, in-
corporating a rigid triazole linker between the anhydroquinate
core and a range of aromatic groups (e.g., 5, Figure 1).[23] This
design of small molecules is supported by reported co-crystal
structures of inhibitors bound to the active site of type II dehy-
droquinases from H. pylori and Streptomyces coelicolor.[17,20]
More recently, Gonzꢀlez-Bello and co-workers reported the first
co-crystal structure of M. tuberculosis type II dehydroquinase
with a substrate-based inhibitor bound in the active site.[24]
This data further highlights the role of the quinate core, which
forms several hydrogen bonds with active site residues, and
terminal aromatic substituents, which participate in favourable
p-stacking interactions with a conserved tyrosine residue
within a flexible loop.
Scheme 1. Proposed mechanism for the dehydration of 3-dehydroquinate
(1) to 3-dehydroshikimate (2) by type II dehydroquinases.
[a] A. T. Tran, Dr. K. M. Cergol, Dr. R. J. Payne
School of Chemistry F11, The University of Sydney, Sydney, 2006 (Australia)
Fax: (+61)2-9351-3329
E-mail: richard.payne@sydney.edu.au
[b] Dr. N. P. West, E. J. Randall, Prof. W. J. Britton
Mycobacterial Research Program, Centenary Institute of Cancer Medicine
and Cell Biology, NSW, AUSTRALIA and Sydney Medical School
The University of Sydney, NSW 2006 (Australia)
We have become interested in the development of type II
dehydroquinase inhibitors as potential tuberculosis drug leads,
which would operate via a mode of action that is different to
currently employed therapies. In this study, we report the de-
velopment of a range of ene–yne-based inhibitors 6–13 cen-
[c] S. A. I. Bokhari, M. Ibrahim, Dr. A. J. Lapthorn
Department of Chemistry and Division of Biochemistry and Life Science
University of Glasgow, G128QQ (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000399.
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ChemMedChem 2011, 6, 262 – 265

tred on the key inhibitory features described above
(Scheme 2). We envisioned that these inhibitors could be pre-
pared in an efficient manner by employing a palladium-cata-
lyzed Sonogashira cross-coupling reaction[25,26] between a key
Scheme 2. Retrosynthesis of ene–yne inhibitors 6–13.
ene–yne intermediate 14 and several aromatic moieties en-
compassing
a range of electronic and steric properties
(Scheme 2). In this case, the alkyne functionality serves as a
rigid, linear, hydrophobic linker between the anhydroquinate
core and the aromatic moieties, a strategy which has yet to be
exploited for type II dehydroquinase inhibition.
Figure 2. Molecular docking of 9 into the active site of a) S. coelicolor type II
dehydroquinase (PDB code: 1GU1) and b) H. pylori type II dehydroquinase
(PDB code: 2WKS).
To support our inhibitor design, the proposed ene–yne com-
pounds 6–13 were initially docked into the active site of the
type II dehydroquinases from S. coelicolor (PDB code: 1GU1)
and H. pylori (PDB code: 2WKS) using Glide (Schrçdinger
Inc.).[27] Most inhibitors possess a common predicted binding
mode whereby the anhydroquinate core formed a number of
favourable hydrogen-bonding interactions with active site resi-
dues (as has been observed in previous crystal structures of
anhydroquinate-containing inhibitors, see Supporting Informa-
tion).[17,20] In addition, the rigid alkyne linker appeared to orient
the terminal aryl and heteroaryl moieties so as to achieve a fa-
vourable p-stacking interaction with the essential tyrosine resi-
due (Tyr28 in S. coelicolor and Tyr22 in H. pylori). Representative
docking poses of 9, bearing a terminal m-trifluoromethylphen-
yl substituent, depicting this common binding mode are
shown in Figure 2. Compounds 12 and 13, containing terminal
benzothiazole and quinoline groups, appeared to dock in a
modified mode against the S. coelicolor enzyme whereby the
anhydroquinate core was oriented 180 degrees from the
normal pose (see Supporting Information). It is assumed that
this is due to the steric nature of the terminal bicyclic rings,
which, in order to partake in a favourable p-stacking interac-
tion with the conserved tyrosine residue, must present the an-
hydroquinate core in a different orientation.
from commercially available (À)-quinic acid (15) in nine steps
(Scheme 3).[23,28] With the key ene–yne 14 in hand, we directed
our attention towards the synthesis of the proposed ene–yne
inhibitors 6–13 (Scheme 3). Subjecting ene–yne 14 to palladi-
um-catalyzed Sonogashira cross-coupling reaction conditions
with a variety of aryl halides or triflates afforded the precursor
lactones that, upon saponification, resulted in the desired in-
Having established that the proposed inhibitors provided fa-
vourable binding modes in silico, we next undertook the syn-
thesis of key ene–yne 14, and the subsequent elaboration to
inhibitors 6–13. To this end, ene–yne 14 was first prepared
Scheme 3. Synthesis of ene–yne inhibitors 6–13 from (À)-quinic acid. Re-
agents and Conditions: a) R-X (X=Br, I, or OTf), Pd(PPh3)4, CuI, piperidine,
THF, 408C; b) LiOH, THF/H2O, RT, 30–85% (two steps).
ChemMedChem 2011, 6, 262 – 265
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hibitors 6–13 in moderate to good yields over the two steps
(30–85%).
terminal thiophene and benzothiazole rings also led to potent
inhibition of all three enzymes including M. tuberculosis type II
dehydroquinase.
Compounds 6–13 were next screened against type II dehy-
droquinases from three organisms, namely S. coelicolor,
H. pylori and M. tuberculosis. The inhibitors were tested using a
UV spectrophotometric assay, detecting the enone carboxylate
chromophore of the enzymatic product 3-dehydroshikimate (2)
at l=234 nm.[21] Gratifyingly, the majority of the compounds
prepared in this study proved to be potent inhibitors of all
three of the type II dehydroquinases studied, with inhibition
constants in the nanomolar range. Introduction of terminal
phenyl or 4-fluorophenyl substituents, in 6 and 7 respectively,
led to potent inhibition of all three of the type II dehydroqui-
nases (Table 1). Both compounds were low nanomolar inhibi-
tors of the S. coelicolor enzyme (Ki =12–28 nm); however, they
Having prepared a library of type II dehydroquinase inhibi-
tors with nanomolar affinity, we were interested in assessing
the antibacterial activity of these compounds. Specifically, we
chose to test the activity of 6–13 against virulent M. tubercu-
losis (strain H37Rv) to assess the potential of these compounds
as TB drug leads. The activity against replicating M. tuberculosis
was measured using the well-established microplate-based
assay with Alamar blue (resazurin) readout (MABA) for determi-
nation of bacterial growth (see Supporting Information).[29–32]
Although the inhibitors demonstrated activity, the majority of
the compounds exhibited low millimolar IC50 values against
the growth of M. tuberculosis, significantly less potent than the
in vitro activity against type II dehydroquinase from M. tubercu-
losis (see Supporting Information). The most potent inhibitor
of M. tuberculosis growth was compound 8 bearing an m-nitro-
phenyl moiety, which exhibited an IC50 value of 450 mm and a
minimal inhibition concentration (MIC) of 1.2 mm (see Support-
ing Information). A possible explanation for the reduced activi-
ty of these compounds against M. tuberculosis may be due to
the type II dehydroquinase in the shikimate pathway of M. tu-
berculosis being non-rate limiting. As such, high levels of
enzyme inhibition would be required to show significant inhib-
ition of bacterial growth. An alternative rationale for the poor
activity of these compounds is correlated with their hydrophilic
nature, which may make it difficult for the compounds to tra-
verse the waxy mycobacterial cell wall. Future work in our lab-
oratories will therefore focus on the preparation of prodrug
analogues of 6–13. It is anticipated that this will enable greater
concentrations of the inhibitors to enter the M. tuberculosis cell
for inhibition of type II dehydroquinase.
Table 1. Inhibition constants of ene–yne inhibitors 6–13 against S. coeli-
color, H. pylori, and M. tuberculosis type II dehydroquinase enzymes.[a]
Inhibitor
Type II DHQase Ki [nm]
S. coelicolor
H. pylori
M. tuberculosis
6
7
8
9
10
11
12
13
28Æ3
12Æ1
253Æ33
106Æ18
98Æ16
356Æ23
548Æ64
133Æ15
108Æ10
2194Æ404
187Æ18
126Æ9
2.2Æ0.4
6.9Æ0.9
171Æ23
7.1Æ0.8
7.5Æ0.7
168Æ20
103Æ13
712Æ137
316Æ44
94Æ11
1203Æ134
885Æ67
[a] Kinetic constants: S. coelicolor: KM =210Æ32 mm, kcat =61 sÀ1, 50 mm
Tris·HCl, pH 7.0, 258C; H. pylori: KM =75Æ10 mm, kcat =0.34 sÀ1, 50 mm
Tris·HOAc, pH 7.0, 258C; M. tuberculosis: KM =36Æ4 mm,
50 mm Tris·HCl, pH 7.0, 258C.
k ,
cat =4.6 sÀ1
In summary, we have successfully designed and synthesized
a small library of ene–yne inhibitors guided by in silico docking
studies. The compounds were synthesized in an efficient
manner using a Sonogashira cross-coupling reaction between
an alkyne-derivatized anhydroquinate core and a range of aro-
matic and heteroaromatic coupling partners. The vast majority
of the inhibitors prepared in this study exhibited nanomolar in-
hibition of type II dehydroquinases from S. coelicolor and the
medically relevant H. pylori and M. tuberculosis. The most
potent inhibitors were those possessing meta-substituted ter-
minal phenyl moieties bearing electron-withdrawing nitro- or
trifluoromethyl-substituents. The compounds proved to exhibit
only moderate antibacterial activity when screened against
M. tuberculosis, ascribed to the hydrophilic nature of the inhibi-
tors. Future work in our laboratories will involve increasing the
lipophilicity of 6–13 by utilising a prodrug approach and the
design of a second generation series of type II dehydroquinase
inhibitors with improved cell penetrating properties.
were less potent against the M. tuberculosis enzyme with inhib-
ition constants of 356 nm and 548 nm for 6 and 7, respectively.
This phenomenon has been reported for previously prepared
anhydroquinate-based inhibitors, suggesting subtle differences
in the active site structures of these two enzymes.[19,20,22] Com-
pounds 8 and 9 bearing electron withdrawing m-trifluorome-
thylphenyl and m-nitrophenyl substituents, respectively, exhib-
ited the most potent inhibition of all three of the enzymes. No-
tably, compounds 8 and 9 exhibited inhibition constants of
133 nm and 108 nm, respectively, against M. tuberculosis type II
dehydroquinase and, as such, represent two of the most
potent inhibitors ever tested against this enzyme. It is impor-
tant to note that compounds 8 and 9 were also potent inhibi-
tors of H. pylori type II dehydroquinase with inhibition con-
stants of 98 nm and 103 nm, respectively. To our knowledge,
these are the most potent inhibitors ever reported against this
enzyme. Introduction of a 3-pyridyl and 3-quinoline moiety in
10 and 13, respectively, led to a significant drop in potency Acknowledgements
against all three enzymes, suggesting that introduction of ni-
trogen heterocycles is detrimental to inhibition of the type II
We thank the Australian National Health and Medical Research
Council for Funding (632769). We would also like to acknowledge
dehydroquinases. Interestingly, compounds 11 and 12 bearing
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ChemMedChem 2011, 6, 262 – 265

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Bello, J. Med. Chem. 2010, 53, 191.
Ph.D. funding from an Australian Postgraduate Award and a
John Lamberton Research Scholarship (ATT).
[18] C. Sꢀnchez-Sixto, V. F. V. Prazeres, L. Castedo, H. Lamb, A. R. Hawkins, C.
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Chem 2008, 3, 756.
Keywords: antibiotics
chemistry · Mycobacterium tuberculosis · shikimate pathway
·
dehydroquinases
·
medicinal
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Received: September 19, 2010
Published online on November 4, 2010
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