Elucidation of Mycobacterium tuberculosis TypeII Dehydroquinase Inhibitors using a Fragment Elaboration Strategy

Article abstract of DOI:10.1002/cmdc.201100606

A library of novel Mycobacterium tuberculosis typeII dehydroquinase (DHQase) inhibitors were discovered through the use of a fragment elaboration approach. Putative active site binding fragments were initially assessed insilico which led to the selection of two small aromatic fragments for further investigation. Synthetic elaboration of the fragments provided a library of 34 inhibitors that exhibited low-micromolar inhibition of typeII DHQase. A number of these inhibitors also showed antibacterial activity in the low-micromolar range in screens against M. tuberculosis invitro; these now serve as lead compounds for further development of therapeutics for the treatment of tuberculosis.

Full text of DOI:10.1002/cmdc.201100606

DOI: 10.1002/cmdc.201100606
Elucidation of Mycobacterium tuberculosis Type II
Dehydroquinase Inhibitors using a Fragment Elaboration
Strategy
Anh Thu Tran,[a] Nicholas P. West,[b] Warwick J. Britton,[b] and Richard J. Payne*[a]
A library of novel Mycobacterium tuberculosis type II dehydro-
quinase (DHQase) inhibitors were discovered through the use
of a fragment elaboration approach. Putative active site bind-
ing fragments were initially assessed in silico which led to the
selection of two small aromatic fragments for further investiga-
tion. Synthetic elaboration of the fragments provided a library
of 34 inhibitors that exhibited low-micromolar inhibition of
type II DHQase. A number of these inhibitors also showed anti-
bacterial activity in the low-micromolar range in screens
against M. tuberculosis in vitro; these now serve as lead com-
pounds for further development of therapeutics for the treat-
ment of tuberculosis.
Introduction
Since its initial identification in 1882, Mycobacterium tuberculo-
sis, the causative agent of tuberculosis (TB), has remained
a major global health risk.[1] According to the World Health Or-
ganization, there were 9.4 million new cases of TB in 2009 and
1.7 million deaths, with these figures expected to rise consider-
ably over the next decade.[1] The current situation has been at-
tributed to poor public health management, particularly in de-
veloping countries, where there is a high TB burden and rapid
spread of TB infection.[1] A further challenge for TB treatment is
the ability of M. tuberculosis to remain dormant in macrophag-
es in very low numbers for decades after initial infection, re-
sulting in a lifelong risk of disease reactivation, especially in
immune-compromised individuals such as those with human
immunodeficiency virus (HIV) co-infection.[1,2]
genome are enzymes in the shikimate pathway.[7] This biosyn-
thetic pathway operates in plants, bacteria, fungi and apicom-
plexan parasites, but is absent in mammals and is therefore an
attractive target for the development of small-molecule inhibi-
tors to serve as herbicides, antibacterials, fungicides, and anti-
parasitic agents.[8] The shikimate pathway consists of seven
enzyme-catalyzed steps and is responsible for the conversion
of erythrose-4-phosphate 1 and phosphoenol pyruvate 2 into
chorismate 3 and is essential for M. tuberculosis growth in vitro
(Scheme 1).[9] Chorismate serves as a precursor for the biosyn-
thesis of a range of aromatic amino acids, folate, ubiquinone,
and vitamins E and K.[10] The presence of the shikimate path-
way in M. tuberculosis has led to significant interest in the de-
velopment of inhibitors of enzymes in this pathway to serve as
TB drug leads.[11]
In recent years, the emergence of multidrug-resistant (MDR)
and extensively drug-resistant (XDR) strains of M. tuberculosis
has threatened global control of TB. MDR M. tuberculosis strains
are defined as being resistant to the major first-line antituber-
cular agents isoniazid and rifampicin and, as such, require the
use of second-line treatments such as fluoroquinolones and in-
jectable agents (e.g., aminoglycosides and polypeptides).[3] The
use of second-line drugs inevitably increases the exposure of
M. tuberculosis to a wider range of antibacterials which has led
to the emergence of XDR M. tuberculosis strains, defined as
those resistant to isoniazid, rifampicin, and any fluoroquino-
lone and one of the three injectable drugs.[4] The treatment of
individuals infected with XDR M. tuberculosis is confined to
a small number of highly toxic and less effective antibiotics; as
a result, XDR-TB is often untreatable, highlighted by a 98%
mortality rate in a recent outbreak in South Africa.[5]
The third step of the shikimate pathway, the reversible dehy-
dration of 3-dehydroquinate 4 to 3-dehydroshikimate 5, is cat-
alyzed by the enzyme dehydroquinase (DHQase, 3-dehydroqui-
nate dehydratase, EC 4.2.1.10; Scheme 1). Interestingly, two
structurally and mechanistically distinct enzymes (type I and
type II DHQases) have evolved to catalyze the same dehydra-
tion reaction.[12] Type I DHQases are homodimeric proteins that
catalyze the syn elimination of water via formation of a Schiff
base with a conserved lysine residue, which enables abstrac-
tion of the pro-R hydrogen atom from the C2 position.[13] In
contrast, type II enzymes are homododecamers that catalyze
[a] A. T. Tran, Dr. R. J. Payne
School of Chemistry, Building F11
The University of Sydney, Sydney, NSW 2006 (Australia)
E-mail: richard.payne@sydney.edu.au
Given the alarming rise in the number of MDR and XDR
M. tuberculosis infections combined with the high rate of HIV
co-infection, there is an urgent need for new TB drugs that op-
erate via novel modes of action to combat dwindling treat-
ment options.[6] Among some of the promising drug targets
identified by the complete analysis of the M. tuberculosis
[b] Dr. N. P. West, Prof. W. J. Britton
Mycobacterial Research Program
Centenary Institute of Cancer Medicine and Cell Biology, and
Sydney Medical School, The University of Sydney, NSW 2006 (Australia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100606.
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In particular, compounds that
possess an anhydroquinate core
as a mimic of the enol enzyme
intermediate of type II DHQase,
either attached directly or by
a rigid linker to a terminal aro-
matic moiety, have provided
compounds with inhibition con-
stants in the nanomolar range
against a range of type II DHQas-
es, including the M. tuberculosis
enzyme
(compounds
6–9,
Figure 1). The binding modes of
these inhibitors have been pre-
dicted by molecular modeling
and confirmed by enzyme–inhib-
itor co-crystal structures.[16c,19]
These studies have established
the importance of the anhydro-
quinate core, which forms a hy-
Scheme 1. The shikimate pathway.
the dehydration reaction via an E1CB mechanism.[14] The reac-
tion is initiated by abstraction of the more acidic pro-S hydro-
gen atom by a conserved tyrosine residue, resulting in the for-
mation of an enol intermediate (Scheme 2). Anti elimination of
water then occurs, facilitated by a conserved water molecule
and asparagine and histidine residues (Scheme 2).[15]
Figure 1. Previously synthesized inhibitors and inhibition constants against
M. tuberculosis type II DHQase.[16c,17a,18]
drogen bonding network with active site residues, and the ter-
minal aromatic moieties, which participate in a p–p stacking
interaction with the essential Tyr residue present on a flexible
loop (Figure 2).
Scheme 2. Proposed mechanism for the dehydration of 3-dehydroquinate
(4) to 3-dehydroshikimate (5) by type II DHQase.
The essentiality of type II DHQase in M. tuberculosis and
other pathogenic bacteria such as Helicobacter pylori (the etio-
logical agent of gastric ulcers) has fueled research efforts into
the design and synthesis of type II DHQase inhibitors as anti-
bacterial leads. Since the 1990s, several inhibitors of this
enzyme have been reported based on the substrate and
enzyme-bound intermediate. In particular, work by Abell and
co-workers,[16] Gonzꢁlez-Bello and colleagues,[17] as well as from
our research group[18] has identified several key features that
contribute to the potent inhibition of type II DHQases from
a variety of organisms.
Figure 2. Crystal structure of S. coelicolor type II DHQase (PDB code: 2CJF)
with thioether 8 bound.[16c]
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M. tuberculosis Type II DHQase Inhibitors
Results and Discussion
We recently reported a number of potent type II DHQase in-
hibitors that support the aforementioned design strategy
through the incorporation of a rigid triazole or alkyne linker
between the anhydroquinate core and a range of aromatic
moieties (e.g., 7 and 9, Figure 1).[18] Despite potent inhibition
(in the nanomolar range) against M. tuberculosis type II
DHQase, these compounds exhibited poor antibacterial activity
when tested against the virulent H37Rv strain of M. tuberculosis
in vitro.[18b] The poor antibacterial activity was attributed to the
hydrophilic nature of the triol carboxylate motif present in the
anhydroquinate core of these compounds. Indeed, improved in
vitro antibacterial activity of anhydroquinate-based inhibitors
has recently been observed by the use of a prodrug strategy,
whereby the carboxylic acid of a similar class of inhibitors was
derivatized as the corresponding n-propyl esters.[17c] Continu-
ing our efforts to develop type II DHQase inhibitors as TB drug
leads, in this study we sought to identify alternate, simplified
scaffolds to replace the highly polar anhydroquinate core tradi-
tionally employed in type II DHQase inhibition studies.
In silico fragment selection
We used an in silico screening approach to identify small, func-
tionally simple molecules capable of establishing favorable in-
teractions with the active site of M. tuberculosis type II DHQase.
Specifically, we selected a total of 13 putative active site bind-
ing fragments that were present as part of a 42-compound li-
brary of proposed type II DHQase inhibitors recently reported
by Kumar et al. in a virtual screening campaign (see Support-
ing Information).[20] The 13 aryl-based fragments with ClogP
values ranging from ꢀ0.3 to 2.4 were docked into the active
site of M. tuberculosis type II DHQase using Glide (Schrçdinger
Inc.)[21] to evaluate the suitability of these compounds as more
hydrophobic replacements of the anhydroquinate core (see
Supporting Information). Two fragments, namely 3,4-dihydrox-
yacetophenone 11 and the corresponding acetonide analogue
12, were subsequently selected for synthetic elaboration.
These two fragments were chosen based on favorable ClogP
values and Glide XP scores, and because these scaffolds are
amenable to synthetic elaboration to generate inhibitors.
Figure 3. Molecular docking of A) 2,3-dehydroquinate 10, B) 3,4-dihydroxya-
cetophenone 11, and C) acetonide analogue 12 into the active site of M. tu-
berculosis type II DHQase (PDB code: 2XB8).[19a]
The results of molecular docking studies of the two frag-
ments are shown in Figure 3 along with the predicted binding
mode of 2,3-dehydroquinate 10. The carbonyl moiety of 3,4-di-
hydroxyacetophenone 11 was predicted to make hydrogen
bonding interactions with the amide backbone between
His101 and Ile102 as well as the side chain of Asn75 in a simi-
lar manner to the C1 carboxylate and C1 hydroxy group of 2,3-
anhydroquinate 10. Importantly, 2,3-anhydroquinic acid is pre-
dicted to form other hydrogen bonding interactions that are
absent in the molecular docking of 11. These include hydrogen
bonding interactions between the C1 carboxylate of 10 with
the amide backbone between Ile102 and Ser103 and the hy-
droxy side chain of Ser103. Furthermore, the C1 hydroxy group
of 10 was predicted to form a hydrogen bond with the side
chain of His101. Acetonide-derived acetophenone 12 was pre-
dicted to bind to M. tuberculosis type II DHQase in exclusively
one binding mode, different from that predicted for 11. Specif-
ically, only the carbonyl group of the acetophenone moiety
was predicted to form a hydrogen bonding interaction with
the amide backbone between His101 and Ile102, whereas the
acetonide moiety is oriented into a subsidiary binding pocket.
Extra binding affinity in this pose can be attributed to a p-
stacking interaction with Tyr24. We chose to pursue inhibitors
based on 12 irrespective of this altered binding mode, as in-
corporation of aryl groups onto the acetophenone moiety of
12 was predicted to reorient the acetonide core and provide
a similar binding pose to 11 (see below). In addition, inhibitors
based on this acetonide core would be synthetic intermediates
en route to inhibitors based on 11.
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The suitability of these two fragments as mimics of the an-
hydroquinate core and as inhibitors of type II DHQase was fur-
ther verified by screening for their inhibitory activity against
M. tuberculosis type II DHQase using a continuous kinetic UV
spectrophotometric assay[18a] that detects the formation of the
enone–carboxylate chromophore of 3-dehydroshikimate 5 at
l=234 nm (Table 1). We were pleased to find that compounds
11 and 12 exhibited micromolar inhibition against the enzyme
residue (Tyr24) present on the flexible loop, which was predict-
ed to enhance potency. Ether and thioether linkages to 11 and
12 were proposed by implementing a broad range of aryl and
heteroaryl alcohols and thiols to provide a series of inhibitors
with variation in the steric and electronic nature of the termi-
nal aryl and heteroaryl moieties (e.g., general structures 13
and 14, Figure 4).
Table 1. ClogP values and activity of compounds 10–12 against M. tuber-
culosis type II DHQase (Ki) and M. tuberculosis strain H37Ra (MIC50).
Compound
Ki [mm]
MIC50 [mm][c]
ClogP
Figure 4. Generalized inhibitor structures bearing an acetonide (compound
13) or catechol (compound 14) core linked to aryl or heteroaryl rings. X=O
or S; R=variety of aryl and heteroaryl groups.
10
11
200ꢁ20[a]
>1000
ꢀ2.7
A number of proposed inhibitors were assessed for suitabili-
ty by the use of in silico molecular docking into the active site
of M. tuberculosis type II DHQase studies using Glide (Support-
ing Information). These docking studies suggested that the
majority of the inhibitors are capable of binding with the cate-
chol and aryl acetonide moieties that occupy the active site of
the enzyme, thus enabling the formation of hydrogen bonding
interactions with key active site residues. Notably, the elaborat-
ed inhibitors of the aryl acetonide core (see general structure
13, Figure 4) led to a reorientation in binding mode relative to
that predicted for fragment 12 alone, such that the pose was
similar to that observed for 11 and the elaborated inhibitors of
this fragment. The docking studies also suggested that the ter-
minal aromatic moieties of all inhibitors are capable of occupy-
ing the subsidiary binding site to interact with Tyr24 (located
on the flexible loop) via an offset face-to-face or edge-to-face
p-stacking interaction (see the Supporting Information for
binding poses).
173ꢁ34[b]
275ꢁ49
330ꢁ14
1.0
2.4
12
120ꢁ23[b]
[a] Inhibition constant for 10 was determined by Frederickson et al.[22] Ki-
netic constants for this study: KM =64ꢁ6 mm; 50 mm Tris·HCl, pH 7.0,
258C. [b] Kinetic constants: KM =25ꢁ3 mm, kcat =4.5 sꢀ1; 50 mm Tris·HCl,
pH 7.0, 258C. [c] Isoniazid MIC50 =176ꢁ14 nm; rifampicin MIC50 =1.7ꢁ
0.4 nm; Glide XP scores: 10 ꢀ10, 11 ꢀ7.7, 12 ꢀ7.2. Data represent the
mean ꢁSD from two replicate assays for M. tuberculosis type II DHQase
and three replicate assays for M. tuberculosis H37Ra.
(Ki: 173 and 120 mm, respectively). Importantly, these small mol-
ecules showed inhibition constants similar to that of 2,3-dehy-
droquinate 10 (Ki =200 mm),[22] yet possessed significantly more
favorable ClogP values (Table 1). Both fragments were also
tested against replicating M. tuberculosis (H37Ra strain) using
an Alamar blue (resazurin) bacterial growth assay.[23] Both 11
and 12 exhibited significant antibacterial activity (MIC50: 275
and 330 mm, respectively) and, as such, were further confirmed
as suitable fragments for chemical elaboration to generate
a novel library of type II DHQase inhibitors with antitubercular
activity.
Synthesis
Having designed a library of target inhibitors in silico, we next
undertook the synthesis of the proposed O- and S-linked inhib-
itors 15a–q (containing the acetonide core of 12) from cate-
chol 17 (Scheme 3). Friedel–Crafts acylation using acetyl chlo-
ride in the presence of excess aluminum chloride provided 3,4-
dihydroxyacetophenone 11 in 95% yield. Subsequent protec-
tion of the diol by treatment with acetone in the presence of
excess phosphorus pentoxide furnished the acetonide-protect-
ed 3,4-dihydroxyacetophenone 12 in 40% yield. From here,
bromination of 12 using copper(II) bromide in ethyl acetate
and dichloromethane at reflux afforded the acetonide-protect-
ed a-bromoketone 18 in 57% yield which served as a key in-
termediate for the preparation of the proposed inhibitor li-
brary. Reaction of 18 with a range of aryl- and heteroaryl-de-
rived alcohols and thiols facilitated nucleophilic substitution of
the bromide and provided the 17 acetonide-based O- and S-
linked inhibitors 15a–q in moderate to excellent yields (59–
91%). These compounds were subsequently deprotected using
Fragment elaboration
Having demonstrated the suitability of 11 and 12 as small scaf-
folds with considerable inhibitory activity against M. tuberculo-
sis type II DHQase, we next focused on elaborating these frag-
ments to provide more potent inhibitors. Specifically, we chose
to investigate conjugates of 11 and 12 in which a range of aryl
and heteroaryl moieties were appended. These groups were
designed to occupy the subsidiary binding pocket to form a fa-
vorable p–p stacking interaction with the conserved tyrosine
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M. tuberculosis Type II DHQase Inhibitors
Scheme 3. Synthesis of O- and S-linked inhibitors 15a–q and 16a–q. Reagents and conditions: a) 1. AcCl, DCE, AlCl3, 108C!RT, 2. HCl(aq), 108C, 95%;
b) (CH3)2CO, P2O5, PhCH3, 758C, 40%; c) CuBr2, EtOAc/CH2Cl2 (1:1 v/v), reflux, 57%; d) Ar-OH or Ar-SH, K2CO3, acetone, reflux, 4–5 h, 59–91%; e) 90% TFA, RT,
1.5 h, 57–100%.
90% aqueous trifluoroacetic acid to afford 17 catechol-based
in prior inhibitor studies.[16c,18a] Inspection of docking results of
the inhibitors and comparison with the fragments suggests
weaker hydrogen bonding interactions between the carbonyl
moiety of the acetophenone core and the amide backbone be-
tween Ile102 and Ser103 in the inhibitors as a result of the
newly formed p-stacking interaction. In contrast, the hydrogen
bonding interactions in the active site of the anhydroquinate-
based inhibitors are not reorganized if a terminal aromatic
moiety is appended to facilitate a p-stacking interaction,
owing to the pre-organized nature of the hydrogen bonding
functionalities. This may reflect a limitation of using planar
scaffolds in place of the chiral anhydroquinate core for potent
inhibition of type II dehydroquinases.
O- and S-linked inhibitors 16a–q (Scheme 3).
Enzyme inhibition assays
Having successfully prepared a library of 34 inhibitors, we next
evaluated these compounds for their enzyme inhibitory activi-
ty. To this end, compounds 15a–q and 16a–q were screened
against M. tuberculosis type II DHQase using a previously de-
scribed kinetic assay.[18a] The majority of the catechol-based in-
hibitors 16a–q possessed considerably enhanced inhibitory ac-
tivities (Ki: 5–39 mm) against M. tuberculosis type II DHQase over
compounds 11 and 12 alone (Ki: 173 and 120 mm, respectively;
Table 2). Although the majority of the compounds clearly fitted
a competitive, reversible model of inhibition, compounds 16g,
16i, 16j, and 16p fitted a mixed inhibition model tending
toward a competitive mode of inhibition (alpha=1.3–2.2, see
Experimental Section and Supporting Information). This could
be explained by the presence of a bulky terminal aromatic
moiety in each of these compounds, which may bias interac-
tions with the subsidiary binding pocket and enable substrate
binding to the active site. Inhibitors 15a–q, containing the
acetonide core, exhibited similar inhibitory activity (Ki: 12–
48 mm), albeit slightly less potent than 16a–q against M. tuber-
culosis type II DHQase (Table 3). In a similar manner to the cate-
chol-based inhibitors, the incorporation of bulky aromatic moi-
eties generally resulted in better fitting to a mixed model of
enzyme inhibition (see Experimental Section and Supporting
Information). Notably, whereas fragments 11 and 12 are capa-
ble of inhibiting type II DHQase with effectiveness similar to
that of 2,3-anhydroquinate 10, the incorporation of aryl and
heteroaryl groups into the acetophenone moiety resulted in—
on average—a tenfold increase in inhibitory potency com-
pared with an increase of three orders of magnitude for similar
aromatic moieties appended to C3 of the anhydroquinate core
For both series of compounds (15a–q and 16a–q) altering
the electronic or steric properties of the aromatic moieties did
not result in dramatic differences in the inhibition of the
enzyme (see Tables 2 and 3). The flat structure–activity data
from this study suggest that the subsidiary binding pocket of
the M. tuberculosis enzyme is capable of accommodating a di-
verse range of aryl and heteroaryl rings for favorable p-stack-
ing interactions.[16c,18a] While large variations in inhibition activi-
ty were not observed between the compounds, inhibitors 16 f,
16g, and 16m, bearing 2-naphthyl, 1-naphthyl, and 4-chloro-
phenyl moieties, respectively, exhibited the most potent inhibi-
tion of type II DHQase, with respective inhibition constants of
5, 8, and 6 mm.
Inhibition of Mycobacterium tuberculosis
Having demonstrated that the fragment-derived inhibitors are
capable of exhibiting low-micromolar inhibition of M. tubercu-
losis type II DHQase in vitro, we were next interested in evalu-
ating the in vitro activity against M. tuberculosis to evaluate the
potential of these compounds to serve as new TB drug leads.
We were pleased to observe that most inhibitors exhibited sig-
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Table 2. Activity of acetophenone-based inhibitors 16a–q against M. tu-
berculosis type II DHQase (Ki) and M. tuberculosis strain H37Ra (MIC50).
Table 3. Activity of acetonide-based inhibitors 15a–q against M. tubercu-
losis type II DHQase (Ki) and M. tuberculosis strain H37Ra (MIC50).
Compd
16a
R
X
O
S
Ki [mm][a]
18ꢁ2
MIC50 [mm][b]
330ꢁ14
Compd
15a
R
X
O
S
Ki [mm][a]
29ꢁ5
MIC50 [mm][b]
115ꢁ28
90ꢁ7
16b
12ꢁ2
370ꢁ84
15b
42ꢁ16[c]
16c
16d
16e
16 f
O
O
O
O
20ꢁ3
14ꢁ2
13ꢁ3
5ꢁ1
505ꢁ134
375ꢁ35
360ꢁ14
370ꢁ14
15c
15d
15e
15 f
O
O
O
O
22ꢁ5[c]
41ꢁ5
15ꢁ2
13ꢁ3
690ꢁ56
140ꢁ1
690ꢁ14
40ꢁ7
16g
16h
O
O
8ꢁ2[c]
12ꢁ2
455ꢁ7
15g
15h
O
O
18ꢁ3
28ꢁ3
55ꢁ7
570ꢁ42
835ꢁ35
16i
16j
16k
O
O
S
39ꢁ9[c]
8ꢁ3[c]
36ꢁ5
195ꢁ49
350ꢁ14
>1000
15i
15j
15k
O
O
S
48ꢁ16[c]
30ꢁ6[c]
>1000
363ꢁ10
>1000
27ꢁ10[c]
16l
S
S
14ꢁ3
6ꢁ1
485ꢁ134
295ꢁ106
15l
S
S
35ꢁ7[c]
19ꢁ2
73ꢁ18
70ꢁ7
16m
15m
16n
S
14ꢁ2
270ꢁ1
15n
S
12ꢁ5[c]
33ꢁ4
16o
16p
S
S
17ꢁ2
>1000
15o
15p
S
S
18ꢁ2
10ꢁ1
10ꢁ2[c]
350ꢁ28
19ꢁ4[c]
575ꢁ120
16q
S
13ꢁ2
690ꢁ70
15q
S
19ꢁ2
850ꢁ14
[a] Kinetic constants: KM =25ꢁ3 mm, kcat =4.5 sꢀ1; 50 mm Tris·HCl, pH 7.0,
258C. [b] Isoniazid MIC50 =176ꢁ14 nm; rifampicin MIC50 =1.7ꢁ0.4 nm.
[c] Mixed model inhibition. Data represent the mean ꢁSD from two repli-
cate assays for M. tuberculosis type II DHQase and three replicate assays
for M. tuberculosis H37Ra.
[a] Kinetic constants: KM =22ꢁ3 mm, kcat =4.4 sꢀ1; 50 mm Tris·HCl, pH 7.0,
258C. [b] Isoniazid MIC50 =176ꢁ14 nm; rifampicin MIC50 =1.7ꢁ0.4 nm.
[c] Mixed model inhibition. Data represent the mean ꢁSD from two repli-
cate assays for M. tuberculosis type II DHQase and three replicate assays
for M. tuberculosis H37Ra.
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M. tuberculosis Type II DHQase Inhibitors
corded on a Finnigan LCQ Deca ion trap mass spectrometer (ESI).
High-resolution mass spectra were recorded on a Bruker 7T Fourier
Transform Ion Cyclotron Resonance (FTICR) mass spectrometer.
Melting points were recorded using a Stanford Research Systems
OptiMelt Automated Melting Point System. Infrared (IR) absorption
spectra were recorded on a Bruker Alpha Spectrometer with atte-
nuated total reflection (ATR) capability and were processed with
OPUS 6.5 software.
nificant inhibition of M. tuberculosis growth in vitro (MIC50: 10–
835 mm). Although there was no clear correlation between the
inhibition of M. tuberculosis type II DHQase and the MIC50
values against M. tuberculosis growth, some interesting SAR
trends emerged. Firstly, the majority of acetonide-based com-
pounds were at least twofold more potent than the corre-
sponding catechol-based inhibitors against M. tuberculosis
growth in vitro. This finding may reflect the increased hydro-
phobicity of the acetonide-based compounds, a factor that has
been shown to be important in the penetration of the waxy
cell wall of M. tuberculosis.[24] It is feasible that the acetonide
moiety of these compounds is hydrolyzed after entry into the
mycobacterial cell. However, it is entirely possible, given the
variation in activity against M. tuberculosis, that the compounds
prepared in this study inhibit alternate or multiple pathways in
vitro. Nonetheless, the incorporation of bicyclic moieties such
as benzothiazole and 2-naphthalene groups in 15o and 15n
resulted in the most potent inhibitors of M. tuberculosis
growth, with MIC50 values of 10 and 33 mm, respectively. Com-
pound 15o was also shown to possess significant activity
against the virulent H37Rv strain of M. tuberculosis (MIC50 =
33ꢁ5 mm) and, although significantly less potent than the
frontline TB drugs isoniazid and rifampicin, now serves as
a lead for further investigation.
Analytical reversed-phase HPLC was performed on
a Waters
System 2695 separations module with an Alliance series column
heater at 308C and 2996 photodiode array detector. Inhibitors 11
and 16a–q were analyzed using a Waters Sunfire BEH300 5 mm,
2.1ꢂ150 mm column (C18) at a flow rate of 0.2 mLminꢀ1 using
a mobile phase of 0.1% TFA in H2O (Solvent A) and 0.1% TFA in
CH3CN (Solvent B) and a linear gradient of 0 to 100% B over
40 min. Inhibitors 19 and 15a–q were analyzed with a Waters Sun-
fire 300 5 mm, 2.1ꢂ150 mm column (C4) at
a flow rate of
0.2 mLminꢀ1 using a mobile phase of 0.1% TFA in H2O (Solvent A)
and 0.1% TFA in CH3CN (Solvent B) and a linear gradient of 20 to
100% B over 40 min. Results were analyzed with Waters Empower
software. The purity of the final inhibitors was shown to be >97%
by analytical HPLC.
Materials: Analytical thin-layer chromatography (TLC) was per-
formed on commercially prepared silica plates (Merck Kieselgel 60
0.25 mm F254). Flash column chromatography was performed using
230–400 mesh Kieselgel 60 silica eluting with distilled solvents as
described. Ratios of solvents used for TLC and column chromatog-
raphy are expressed in v/v as specified. Compounds were visual-
ized by UV light at l 254 nm or by using vanillin or cerium molyb-
date stain. Commercial materials were used as received, unless oth-
erwise noted. Dichloromethane and methanol were distilled from
calcium hydride, and THF and diethyl ether were distilled over
sodium/benzophenone. Anhydrous DMF was purchased from
Sigma–Aldrich.
Conclusions
In summary, we successfully employed an in silico fragment
screening approach to identify 3,4-dihydroxyacetophenone
and acetonide cores as replacements for the 2,3-anhydroqui-
nate core previously employed in type II DHQase inhibitors.
Elaboration of these fragments to incorporate a range of aryl
and heteroaryl moieties led to a library of inhibitors that pos-
sess low-micromolar inhibition of type II DHQase. Importantly,
this study has revealed compounds with promising activity
against M. tuberculosis growth in vitro. Future work in our re-
search groups will involve co-crystallization of acetophenone
and acetonide-based inhibitors with M. tuberculosis type II
DHQase; this will aid in the rational design of more potent
type II DHQase inhibitors. In addition, work is underway to
study the molecular mechanism of the antitubercular activity
exhibited by these compounds, the results of which will be re-
ported in due course.
Molecular modeling
Protein preparation: The crystal structures of M. tuberculosis
type II DHQase were downloaded from the Research Collaboratory
for Structural Bioinformatics (RCSB) Protein Data Bank (PDB code:
2XB8).[19a] The structures were subjected to further modification to
ensure suitability for molecular docking using Glide (version 5.0).[21]
The ligand–protein co-crystal structures were imported into Accel-
rys DS visualizer 2.0 (Accelrys Software Inc.) where the dodecamers
were simplified into a dimer (Subunits A and B). (2R)-2-Methoxy-
benzyl-3-dehydroquinic acid was maintained in subunit A. Structur-
al water molecules were also removed. The simplified type II
DHQase protein structure was then imported into Maestro (ver-
sion 9.1) and prepared using the Protein Preparation Wizard tool, in
which bond orders were assigned to the ligands. Hydrogen atoms
were added to both ligands and the protein in a manner consis-
tent with physiological pH (pH 7.0) using an all-atom force field.
Restrained minimization of the protein structure was then conduct-
ed using impref using an OPLS-AA force field until the root mean
square deviation (RMSD) of non-hydrogen atoms reached 0.3.
Experimental Section
General synthesis procedures
1H NMR spectra were recorded at 300 K using Bruker Avance
DRX200, DRX300, DPX400, or III-600 NMR spectrometers at a fre-
1
quency of 200.1, 300.2, 400.2, and 600.0 MHz, respectively. H NMR
chemical shifts are reported in parts per million (ppm) and are ref-
Ligand preparation: Ligands were built in Maestro (version 9.1)
and used as maegz files. The ligands were then subjected to treat-
ment with Ligprep (version 2.3), which generates low-energy 3D
structures with tautomers and possible states at pH 7.
erenced to solvent residual signals: CDCl3 (d=7.26 ppm), MeOD
1
(d=3.31 ppm), (CD3)2CO (d=2.05 ppm). H NMR data are reported
as chemical shift (dH), relative integral, multiplicity (s=singlet, d=
doublet, t=triplet, q=quartet, dd=doublet of doublets, ddd=
doublet of doublet of doublets), coupling constant (J in Hz), and
assignment where possible. Low-resolution mass spectra were re-
Glide docking: A receptor grid file was generated using the Recep-
tor Grid Generation utility in Glide (version 5.0). The ligand chosen
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R. J. Payne et al.
MED
was (2R)-2-methoxybenzyl-3-dehydroquinic acid as the center of
receptor grid generation; van der Waals radius scaling and partial
charge cutoff default values were used. Per-atom scale factors,
which soften receptor potential, were not used. After the receptor
grids were generated, ligands were docked into the active site
using extra precision (XP) mode.
added
a
suitable aryl or heteroaryl alcohol or thiol (0.22–
0.32 mmol), and the reaction mixture was stirred at room tempera-
ture for 15 min. Bromide 18 (0.16–0.21 mmol) in acetone (0.4 mL)
was added dropwise, and the reaction was stirred at reflux for 4–
5 h. The reaction was diluted with Et2O (10 mL) and washed with
H2O (2ꢂ10 mL). The organic layer was dried over anhydrous
MgSO4 and the solvent removed in vacuo to yield a crude residue
that was purified by column chromatography to afford the corre-
sponding ether or thioether.
Synthesis
1-(3,4-Dihydroxyphenyl)ethanone (11): Anhydrous AlCl3 (0.98 g,
7.4 mmol) was dissolved in 1,2-dichloroethane (DCE), and the reac-
tion was cooled to 108C for 30 min. Catechol 17 (0.32 g, 2.9 mmol)
was added in three portions over 5 min, and the reaction was
stirred at 108C for a further 30 min. Acetyl chloride (0.26 mL,
3.2 mmol) was added dropwise to the reaction mixture, which was
allowed to warm to room temperature and stirred for 20 h. The re-
action was subsequently cooled to 108C before quenching with
1m HCl (10 mL), and the reaction mixture was allowed to stir for
a further 2 h. The reaction mixture was diluted with CH2Cl2 (30 mL),
and the organic layer was separated. The aqueous layer was ex-
tracted with EtOAc (2ꢂ30 mL), and the combined organic layers
were washed with brine (40 mL) and dried over anhydrous Na2SO4.
The solvent was removed in vacuo to afford acetophenone 11 as
a red solid, which was used without further purification (0.43 g,
95%). Rf (8:1 v/v CH2Cl2/Et2O)=0.30; mp: 120–1218C; 1H NMR
(400 MHz, CDCl3): d=7.47 (1H, dd, J=4.0, 8.0 Hz, Ar-H, C-6), 7.42
(1H, d, J=4.0 Hz, Ar-H, C-2), 6.88 ppm (1H, d, J=8.0 Hz, Ar-H, C-5);
13C NMR (100 MHz, [D6]acetone): d=195.9, 150.2, 145.3, 130.1,
122.2, 115.6, 114.8, 25.5 ppm; IR (ATR): n˜ =3000, 1796 cmꢀ1. These
data are in agreement with those previously reported by Xiao
et al.[25]
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-phenoxyethanone
(15a): Phenol (21 mg, 0.22 mmol) was reacted with bromide 18
(57 mg, 0.21 mmol) using the general procedure described above
and purified by column chromatography (3:2 v/v CH2Cl2/hexane) to
afford 15a as a pale-yellow oil (40 mg, 67%). Rf (3:2 v/v CH2Cl2/
1
hexane)=0.45; H NMR (300 MHz, CDCl3): d=7.58 (1H, dd, J=1.5,
8.1 Hz, Ar-H), 7.40 (1H, d, J=1.5 Hz, Ar-H), 7.27 (2H, t, J=8.4 Hz,
2ꢂAr-H), 6.97–6.92 (3H, m, 3ꢂAr-H), 6.78 (1H, d, J=8.1 Hz, Ar-H),
5.17 (2H, s, CH2), 1.64 (6H, s, 2ꢂCH3); 13C NMR (100 MHz, CDCl3):
d=192.7, 158.4, 152.9, 148.2, 129.8, 129.1, 124.4, 121.8, 120.1,
115.0, 108.4, 108.2, 70.6, 26.1 ppm; IR (ATR): n˜ =1693 cmꢀ1; HRMS
calcd for C17H16O4Na [M+Na]+ 307.0941, found 307.0944.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(phenylthio)ethanone
(15b): Thiophenol (33 mL, 0.32 mmol) was reacted with bromide 18
(57 mg, 0.21 mmol) in acetone (0.4 mL) using the general proce-
dure described above and purified by column chromatography
(3:1 v/v hexane: CH2Cl2!3:2 v/v CH2Cl2/hexane) to afford 15b as
a pale-yellow oil (28 mg, 45%). Rf (3:2 v/v CH2Cl2/hexane)=0.43;
1H NMR (400 MHz, CDCl3): d=7.50 (1H, dd, J=1.5, 8.0 Hz, Ar-H),
7.39 (2H, dd, J=1.4, 7.7 Hz, 2ꢂAr-H), 7.34 (1H, d, J=1.5 Hz, Ar-H),
7.28 (2H, t, J=7.7 Hz, 2ꢂAr-H), 7.23–7.20 (1H, m, Ar-H), 6.74 (1H,
d, J=8.0 Hz, Ar-H), 4.20 (2H, s, CH2), 1.70 (6H, s, 2ꢂCH3); 13C NMR
(100 MHz, CDCl3): d=192.5, 152.1, 148.3, 135.1, 131.7, 129.6, 129.1,
127.0, 124.8, 119.7, 108.3, 107.9, 41.9, 22.8 ppm; IR (ATR): n˜ =
1671 cmꢀ1; HRMS calcd for C17H16O3SNa [M+Na]+ 323.0712, found
323.0708.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)ethanone (12): A mixture
of 3,4-dihydroxyacetophenone 11 (3.87 g, 25 mmol) and P2O5
(220 mg) in toluene (13 mL) was heated at 758C. Acetone (7.4 mL,
100 mmol) was added dropwise to the suspension over 3 h. After
the addition, four portions of P2O5 (4ꢂ220 mg) were added to the
reaction mixture every 30 min. The reaction was allowed to stir at
758C for 5 h. The reaction was quenched with 25% NaOH(aq)
(15 mL), and the solvent was removed in vacuo to yield a crude
residue that was purified by column chromatography (2:1 v/v
CH2Cl2/hexane) to afford acetonide 12 as a pale-yellow oil (1.96 g,
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(3-(trifluoromethyl)-
phenoxy)ethanone (15c): 3-(Trifluoromethyl)phenol (39 mL,
0.32 mmol) was reacted with bromide 18 (57 mg, 0.21 mmol) using
the general procedure described above and purified by column
chromatography (3:2 v/v CH2Cl2/hexane) to afford 15c as a white
solid (67 mg, 90%). Rf (1:1 v/v CH2Cl2/hexane)=0.24; mp: 115–
116 8C; 1H NMR (282 MHz, CDCl3): d=7.54 (1H, dd, J=1.8, 8.1 Hz,
Ar-H), 7.42–7.33 (2H, m, 2ꢂAr-H), 7.26–7.20 (1H, m, Ar-H), 7.19–
7.14 (1H, m, Ar-H), 7.13–7.04 (1H, m, Ar-H), 6.79 (1H, d, J=8.1 Hz,
Ar-H), 5.23 (2H, s, CH2), 1.71 ppm (6H, s, 2ꢂCH3); 13C NMR
(100 MHz, CDCl3): d=191.9, 158.2, 152.7, 148.3, 131.9 (q, J=33 Hz),
130.2, 128.4, 125.3 (q, J=270 Hz, CF3), 124.2, 119.9, 118.3 (q, J=
3.1 Hz), 118.1, 111.9 (q, J=3.2 Hz), 108.1, 107.8, 70.4, 26.0 ppm; IR
1
40%). Rf (2:1 v/v CH2Cl2/hexane)=0.29; H NMR (400 MHz, CDCl3):
d=7.53 (1H, dd, J=1.6, 8.0 Hz, Ar-H, H-6), 7.35 (1H, s, Ar-H, H-2),
6.75 (1H, d, J=8.0 Hz, Ar-H, H-5), 2.52 (3H, s, CH3), 1.69 (6H, s, 2ꢂ
CH3); 13C NMR (100 MHz, CDCl3): d=196.5, 151.7, 147.9, 131.6,
124.4, 119.4, 107.9, 107.7, 26.5, 25.9 ppm; IR (ATR): n˜ =1675 cmꢀ1
MS (ESI): m/z 193 [M+H]+.
;
2-Bromo-1-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)ethanone (18):
A solution of acetonide 12 (380 mg, 1.9 mmol) was added to a solu-
tion of CuBr2 (830 mg, 3.7 mmol) in EtOAc (2.5 mL) and CH2Cl2
(2.5 mL). The reaction mixture was held at reflux with vigorous stir-
ring for 9 h. The reaction mixture was then filtered, the solvent re-
moved in vacuo, and the crude residue purified by column chro-
matography (1:1 v/v CH2Cl2/hexane!2:1 v/v CH2Cl2/hexane) to
afford a-bromoketone 18 as a pale-yellow oil (290 mg, 57%). Rf
(1:1 v/v CH2Cl2/hexane)=0.26; 1H NMR (300 MHz, CDCl3): d=7.55
(1H, dd, J=1.8, 8.4 Hz, Ar-H, H-6), 7.36 (1H, d, J=1.8 Hz, Ar-H, H-2),
6.77 (1H, d, J=8.4 Hz, Ar-H, H-5), 4.36 (2H, s, CH2), 1.70 ppm (6H,
s, 2ꢂCH3); 13C NMR (75 MHz, CDCl3): d=190.0, 152.9, 148.6, 128.5,
(ATR): n˜ =1694 cmꢀ1
;
HRMS calcd for C18H15F3O4Na [M+Na]+
375.0815, found 375.0813.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(2-fluorophenoxy)-
ethanone (15d): 2-Fluorophenol (28 mL, 0.32 mmol) was reacted
with bromide 18 (57 mg, 0.21 mmol) using the general procedure
described above and purified by column chromatography (2:1 v/v
CH2Cl2/hexane) to afford 15d as a white solid (62 mg, 97%). Rf (2:1
v/v CH2Cl2/hexane)=0.43; mp: 113–1148C; 1H NMR (282 MHz,
CDCl3): d=7.57 (1H, dd, J=1.8, 8.4 Hz, Ar-H), 7.38 (1H, d, J=
1.8 Hz, Ar-H), 7.08 (1H, dt, J=1.8, 8.1 Hz, Ar-H), 7.00 (1H, dd, J=
1.8, 8.1 Hz, Ar-H), 6.95–6.89 (2H, m, 2ꢂAr-H), 6.78 (1H, d, J=8.4 Hz,
Ar-H), 5.25 (2H, s, CH2), 1.70 ppm (6H, s, 2ꢂCH3); 13C NMR
(100 MHz, CDCl3): d=192.2, 153.3 (d, J=258 Hz, ipso-C), 152.5,
125.6, 120.2, 108.8, 108.3, 30.9, 26.3 ppm; IR (ATR): n˜ =1668 cmꢀ1
.
General procedure for nucleophilic substitution of bromide 18:
To a suspension of K2CO3 (0.18–0.23 mmol) in acetone (0.4 mL) was
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M. tuberculosis Type II DHQase Inhibitors
148.2, 146.1 (d, J=9.5 Hz, ortho-C), 128.5, 124.3 (d, J=4.8 Hz, para-
C), 124.2, 122.2 (d, J=7.9 Hz, meta-C), 119.8, 116.6 (d, J=19 Hz,
ortho-C), 116.0, 108.1, 107.8, 71.9, 26.0 ppm; IR (ATR): n˜ =
1692 cmꢀ1; HRMS calcd for C17H15FO4Na [M+Na]+ 325.0845, found
325.0842.
25.6 ppm; IR (ATR): n˜ =1697 cmꢀ1; HRMS calcd for C20H17NO5Na
[M+Na]+ 374.0998, found 374.1002.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(4-(naphthalen-2-yl)-
phenoxy)ethanone (15i): 4-(Naphthalen-2-yl)phenol (40 mg,
0.18 mmol) was reacted with bromide 18 (45 mg, 0.16 mmol) using
the general procedure described above and purified by column
chromatography (3:2 v/v CH2Cl2/hexane) to afford 15i as a white
solid (40 mg, 62%). Rf (2:1 v/v CH2Cl2/hexane)=0.40; mp: 119–
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(3-nitrophenoxy)etha-
none (15e): 3-Nitrophenol (32 mg, 0.23 mmol) was reacted with
bromide 18 (57 mg, 0.21 mmol) using the general procedure de-
scribed above and purified by column chromatography (3:1 v/v
CH2Cl2/hexane) to afford 15e as an off-white solid (41 mg, 59%). Rf
1
1208C (dec.) H NMR (400 MHz, CDCl3): d=7.99 (1H, s, Ar-H), 7.90–
7.84 (3H, m, 3ꢂAr-H), 7.70 (1H, d, J=8.3 Hz, Ar-H), 7.66–7.61 (3H,
m, 3ꢂAr-H), 7.51–7.44 (3H, m, 3ꢂAr-H), 7.05 (2H, d, J=8.4 Hz, 2ꢂ
Ar-H), 6.81 (1H, d, J=8.1 Hz, Ar-H), 5.24 (2H, s, CH2), 1.72 ppm (6H,
s, 2ꢂCH3); 13C NMR (100 MHz, CDCl3): d=192.5, 157.8, 152.3, 148.2,
138.0, 134.4, 132.5, 128.8, 128.5, 128.4, 128.0, 127.6, 126.2, 125.7,
125.4, 125.1, 124.2, 119.7, 115.2, 108.0, 107.9, 70.6, 25.7 ppm; IR
(ATR): n˜ =1691 cmꢀ1; HRMS calcd for C27H23O4 [M+H]+ 411.1596,
found 411.1591.
1
(3:2 v/v CH2Cl2/hexane)=0.28; mp: 171–1728C; H NMR (300 MHz,
1:1 v/v CDCl3/[D6]acetone): d=7.87 (1H, dd, J=1.6, 8.1 Hz, Ar-H),
7.79 (1H, d, J=1.6 Hz, Ar-H), 7.64 (1H, dd, J=1.6, 8.1 Hz, Ar-H),
7.50 (1H, t, J=8.1 Hz, Ar-H), 7.36–7.33 (2H, m, 2ꢂAr-H), 6.85 (1H,
d, J=8.1 Hz, Ar-H), 5.46 (2H, s, CH2), 1.74 (6H, s, 2ꢂCH3); 13C NMR
(100 MHz, 1:1 v/v CDCl3/[D6]acetone): d=196.5, 164.0, 157.7, 154.3,
153.4, 135.4, 133.5, 129.2, 127.0, 125.0, 121.4, 114.5, 113.3, 112.7,
75.6, 30.9 ppm; IR (ATR): n˜ =1653 cmꢀ1
;
HRMS calcd for
C17H15NO6Na [M+Na]+ 352.0792, found 352.0793.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(3-(naphthalen-2-yl)-
phenoxy)ethanone (15j): 3-(Naphthalen-2-yl)phenol (40 mg,
0.18 mmol) was reacted with bromide 18 (45 mg, 0.16 mmol) using
the general procedure described above and purified by column
chromatography (3:2 v/v CH2Cl2/hexane) to afford 15j as a foam
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(naphthalen-2-yl-
oxy)ethanone (15 f): 2-Naphthol (31 mg, 0.22 mmol) was reacted
with bromide 18 (57 mg, 0.21 mmol) using the general procedure
described above and purified by column chromatography (3:2 v/v
CH2Cl2/hexane) to afford 15 f as a white solid (70 mg, 91%). Rf (3:2
v/v CH2Cl2/hexane)=0.45; mp: 129–1308C (dec.); 1H NMR
(300 MHz, CDCl3): d=7.77–7.75 (2H, m, 2ꢂAr-H), 7.71 (1H, d, J=
8.4 Hz, Ar-H), 7.63 (1H, dd, J=1.6, 8.0 Hz, Ar-H), 7.45–7.43 (2H, m,
2ꢂAr-H), 7.34 (1H, t, J=7.2 Hz, Ar-H), 7.26 (1H, dd, J=2.4, 8.8 Hz,
Ar-H), 7.11 (1H, d, J=2.4 Hz, Ar-H), 6.80 (1H, d, J=8.0 Hz, Ar-H),
5.30 (2H, s, CH2), 1.72 ppm (6H, s, 2ꢂCH3); 13C NMR (75 MHz,
CDCl3): d=192.9, 156.5, 152.8, 148.6, 134.7, 130.1, 129.8, 129.3,
128.1, 127.3, 126.9, 124.6, 124.4, 120.0, 119.1, 108.4, 108.3, 107.7,
71.1, 26.3 ppm; IR (ATR): n˜ =1689 cmꢀ1; HRMS calcd for C21H18O4Na
[M+Na]+ 357.1097, found 357.1091.
1
(38 mg, 58%). Rf (2:1 v/v CH2Cl2/hexane)=0.50; H NMR (400 MHz,
CDCl3): d=8.02 (1H, d, J=1.4 Hz, Ar-H), 7.92–7.85 (3H, m, 3ꢂAr-H),
7.72 (1H, dd, J=1.8, 8.5 Hz, Ar-H), 7.62 (1H, dd, J=1.8, 8.2 Hz, Ar-
H), 7.51–7.48 (2H, m, 2ꢂAr-H), 7.44 (1H, d, J=1.8 Hz, Ar-H), 7.40
(1H, app.t, J=7.8 Hz, Ar-H), 7.36 (1H, app.t, J=1.4 Hz, Ar-H), 7.34–
7.32 (1H, m, Ar-H), 6.94 (1H, ddd, J=1.4, 2.6, 7.8 Hz, Ar-H), 6.80
(1H, d, J=8.2 Hz, Ar-H), 5.26 (2H, s, CH2), 1.73 ppm (6H, s, 2ꢂCH3);
13C NMR (100 MHz, CDCl3): d=192.6, 158.9, 152.2, 147.9, 142.7,
137.9, 133.6, 132.7, 129.9, 128.8, 128.4, 128.2, 127.6, 126.3, 126.0,
125.9, 125.6, 124.7, 120.8, 119.7, 114.3, 113.3, 108.0, 107.9, 70.7,
26.3 ppm; IR (ATR): n˜ =1706 cmꢀ1; HRMS calcd for C27H23O4 [M+
H]+ 411.1596, found 411.1591.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(naphthalen-1-yl-
oxy)ethanone (15g): 1-Naphthol (31 mg, 0.22 mmol) was reacted
with bromide 18 (57 mg, 0.21 mmol) using the general procedure
described above and purified by column chromatography (3:2 v/v
CH2Cl2/hexane) to afford 15g as a colorless oil (54 mg, 77%). Rf
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(2-(trifluoromethyl)-
phenylthio)ethanone (15k): 2-Trifluoromethyl benzenethiol (43 mL,
0.32 mmol) was reacted with bromide 18 (57 mg, 0.21 mmol) using
the general procedure described above and purified by column
chromatography (3:2 v/v CH2Cl2/hexane) to afford 15k as a pale-
1
1
(3:2 v/v CH2Cl2/hexane)=0.51; H NMR (300 MHz, CDCl3): d=8.40–
yellow oil (38 mg, 49%). Rf (3:2 v/v CH2Cl2/hexane)=0.79; H NMR
8.37 (1H, m, Ar-H), 7.83–7.79 (1H, m, Ar-H), 7.65 (1H, dd, J=1.5,
8.1 Hz, Ar-H), 7.52–7.46 (4H, m, 4ꢂAr-H), 7.32 (1H, t, J=8.1 Hz, Ar-
H), 6.80–6.75 (2H, m, 2ꢂAr-H), 5.33 (2H, s, CH2), 1.71 (6H, s, 2ꢂ
CH3); 13C NMR (75 MHz, CDCl3): d=193.1, 154.3, 152.7, 148.6, 135.0,
129.3, 127.8, 126.9, 126.1, 126.0, 125.9, 124.8, 122.6, 121.6, 120.0,
108.3, 105.7, 71.6, 26.3 ppm; IR (ATR): n˜ =1698 cmꢀ1; HRMS calcd
for C21H19O4 [M+H]+ 335.1278, found 335.1280.
(400 MHz, CDCl3): d=7.65 (1H, m, Ar-H), 7.59–7.50 (2H, m, 2ꢂAr-
H), 7.46 (1H, t, J=7.6 Hz, Ar-H), 7.35–7.31 (2H, m, 2ꢂAr-H), 6.76
(1H, d, J=8.0 Hz, Ar-H), 4.24 (2H, s, CH2), 1.07 (6H, s, 2ꢂCH3);
13C NMR (100 MHz, CDCl3): d=192.2, 152.6, 148.4, 134.7, 133.4,
132.6, 130.8 (q, J=30 Hz), 129.8, 127.4, 127.2 (q, J=5.5 Hz), 125.4
(q, J=272 Hz, CF3), 125.2, 120.0, 108.6, 108.2, 41.4, 26.0 ppm; IR
(ATR): n˜ =1672 cmꢀ1; HRMS calcd for C18H15F3O3SNa [M+Na]+
391.0586, found 391.0588.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(2-(isoxazol-5-yl)phe-
noxy)ethanone (15h): 2-(5-Isoxazolyl)phenol (38 mg, 0.23 mmol)
was reacted with bromide 18 (57 mg, 0.21 mmol) using the general
procedure described above and purified by column chromatogra-
phy (4:1 v/v CH2Cl2/hexane) to afford 15h as an off-white solid
(38 mg, 52%). Rf (4:1 v/v CH2Cl2/hexane)=0.32; mp: 138–1398C
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-((4-fluorophenyl)-
thio)ethanone (15l): 4-Fluorothiophenol (34 mL, 0.32 mmol) was
reacted with bromide 18 (57 mg, 0.21 mmol) using the general
procedure described above and purified by column chromatogra-
phy (3:2 v/v CH2Cl2/hexane) to afford 15l as a pale-yellow oil
1
1
(dec.); H NMR (300 MHz, 1:1 v/v CDCl3/[D6]acetone): d=8.38 (1H,
(42 mg, 63%). Rf (3:2 v/v CH2Cl2/hexane)=0.28; H NMR (300 MHz,
s, Ar-H), 7.98 (1H, d, J=8.0 Hz, Ar-H), 7.68–7.65 (1H, m, Ar-H), 7.48
(1H, s, Ar-H), 7.42–7.38 (2H, m, 2ꢂAr-H), 7.15 (1H, d, J=8.0 Hz, Ar-
H), 7.10 (1H, t, J=8.0 Hz, Ar-H), 6.82 (1H, d, J=8.0 Hz, Ar-H), 5.52
(2H, s, CH2), 1.71 (6H, s, 2ꢂCH3); 13C NMR (100 MHz, 1:1 v/v CDCl3/
[D6]acetone): d=192.0, 154.6, 152.3, 151.2, 148.2, 131.1, 128.6,
127.4, 123.9, 121.4, 119.7, 116.7, 112.6, 108.0, 107.3, 103.9, 70.4,
CDCl3): d=7.47 (1H, dd, J=1.8, 8.1 Hz, Ar-H), 7.45–7.36 (2H, m, 2ꢂ
Ar-H), 7.31 (1H, d, J=1.8 Hz, Ar-H), 7.00–6.94 (2H, m, 2ꢂAr-H), 6.75
(1H, d, J=8.1 Hz, Ar-H), 4.08 (2H, s, CH2), 1.72 ppm (6H, s, 2ꢂCH3);
13C NMR (100 MHz, CDCl3): d=192.6, 162.0 (d, J=210 Hz, ipso-C),
152.4, 148.6, 134.1 (d, J=8.1 Hz, meta-C), 130.0 (d, J=3.1 Hz, para-
C), 129.8, 125.1, 120.0, 116.5 (d, J=22 Hz, ortho-C), 108.6, 108.2,
ChemMedChem 0000, 00, 1 – 14
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R. J. Payne et al.
MED
42.0, 26.0 ppm; IR (ATR): n˜ =1649 cmꢀ1
;
HRMS calcd for
solid which was used without further purification (55 mg, 71%). Rf
(9:1 v/v CH2Cl2/acetone)=0.54; mp: 219–2208C (dec.); 1H NMR
(400 MHz, [D6]DMSO): d=8.04 (1H, dd, J=1.2, 7.9 Hz, Ar-H), 7.76
(1H, dd, J=1.7, 8.2 Hz, Ar-H), 7.72 (1H, dt, J=1.5, 8.5 Hz, Ar-H),
7.50 (1H, d, J=1.7 Hz, Ar-H), 7.42 (1H, dt, J=1.2, 7.9 Hz), 7.27 (1H,
app.d, J=8.5 Hz, Ar-H), 7.05 (1H, d, J=8.2 Hz, Ar-H), 4.82 (2H, s,
CH2), 1.73 ppm (6H, s, 2ꢂCH3); 13C NMR (100 MHz, [D6]DMSO): d=
192.0, 161.6, 155.6, 151.9, 148.4, 147.4, 134.9, 130.7, 126.6, 126.1,
125.0, 120.3, 120.1, 108.5, 108.1, 37.8, 26.0 ppm; IR (ATR): n˜ =
1661 cmꢀ1; HRMS calcd for C19H17N2O4S [M+H]+ 369.0904, found
369.0903.
C17H15FO3SNa [M+Na]+ 341.0618, found 341.0615.
2-((4-Chlorophenyl)thio)-1-(2,2-dimethylbenzo[d][1,3]dioxol-5-
yl)ethanone (15m): 4-Chlorothiophenol (45 mg, 0.32 mmol) was
reacted with bromide 18 (57 mg, 0.21 mmol) using the general
procedure described above and purified by column chromatogra-
phy (3:2 v/v CH2Cl2/hexane) to afford 15m as a pale-yellow oil
1
(31 mg, 44%). Rf (3:2 v/v CH2Cl2/hexane)=0.59; H NMR (300 MHz,
CDCl3): d=7.50 (1H, dd, J=1.5, 8.1 Hz, Ar-H), 7.33 (2H, d, J=
8.8 Hz, 2ꢂAr-H), 7.26–7.23 (3H, m, 3ꢂAr-H), 6.75 (1H, d, J=8.1 Hz,
Ar-H), 4.17 (2H, s, CH2), 1.70 ppm (6H, s, 2ꢂCH3); 13C NMR
(100 MHz, CDCl3): d=192.4, 152.6, 148.5, 133.9, 133.5, 132.2, 129.9,
129.6, 125.1, 120.0, 108.6, 108.2, 41.4, 26.3 ppm; IR (ATR): n˜ =
1672 cmꢀ1; HRMS calcd for C17H16ClO3S [M+H]+ 335.0503, found
335.0502.
General procedure for the deprotection of 15a–q: Aqueous tri-
fluoroacetic acid (90%, 0.3–0.4 mL) was added dropwise to aceto-
nide-based inhibitor 15a–q (0.017–0.12 mmol), and the reaction
was stirred at room temperature or at 508C for 1.5–5 h. At this
point, the solvent was removed in vacuo to yield a crude residue
that was purified by column chromatography.
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-(naphthalen-2-yl-
thio)ethanone (15n): 2-Naphthalenethiol (51 mg, 0.31 mmol) was
reacted with bromide 18 (57 mg, 0.21 mmol) using the general
procedure described above and purified by column chromatogra-
phy (3:2 v/v CH2Cl2/hexane) to afford 15n as a pale-yellow oil
1-(3,4-Dihydroxyphenyl)-2-phenoxyethanone (16a): Acetonide-
based inhibitor 15a (27 mg, 0.095 mmol) was deprotected using
the general procedure described above at room temperature for
1.5 h and purified by column chromatography (95:5 v/v CH2Cl2/
CH3OH) to afford inhibitor 16a as an off-white solid (20 mg, 87%).
Rf (95:5 v/v CH2Cl2/CH3OH)=0.26; mp: 157–1588C (dec.); 1H NMR
(400 MHz, [D6]acetone): d=7.55–7.57 (2H, m, 2ꢂAr-H), 7.25–7.29
(2H, m, 2ꢂAr-H), 6.91–6.96 (4H, m, 4ꢂAr-H), 5.38 ppm (2H, s, CH2);
13C NMR (100 MHz, [D6]acetone): d=192.7, 159.0, 150.9, 145.5,
129.7, 128.0, 122.0, 121.2, 115.3, 115.1, 115.0, 70.4 ppm; IR (ATR):
n˜ =3404 (br. OH str.), 3367 (br. OH str.), 1676 cmꢀ1; HRMS calcd for
C14H12O4Na [M+Na]+ 267.0628, found 267.0624.
1
(39 mg, 53%). Rf (2:1 v/v CH2Cl2/hexane)=0.53; H NMR (300 MHz,
CDCl3): d=7.83–7.73 (4H, m, 4ꢂAr-H), 7.55 (1H, dd, J=1.8, 8.1 Hz,
Ar-H), 7.48 (1H, d, J=1.8 Hz, Ar-H), 7.47–7.43 (2H, m, 2ꢂAr-H), 7.36
(1H, d, J=1.5 Hz, Ar-H), 6.75 (1H, d, J=8.1 Hz, Ar-H), 4.30 (2H, s,
CH2), 1.69 ppm (6H, s, 2ꢂCH3); 13C NMR (75 MHz, CDCl3): d=192.7,
152.5, 148.5, 134.1, 132.9, 132.6, 130.1, 129.0, 128.3, 128.1, 127.7,
126.9, 126.4, 125.1, 119.9, 108.7, 108.2, 41.3, 26.3 ppm; IR (ATR): n˜ =
1668 cmꢀ1; HRMS calcd for C21H18O3SNa [M+Na]+ 373.0869, found
373.0863.
2-(Benzo[d]thiazol-2-ylthio)-1-(2,2-dimethylbenzo[d][1,3]dioxol-
5-yl)ethanone (15o): 2-Mercaptobenzothiazole (66 mg, 0.36 mmol)
was reacted with bromide 18 (50 mg, 0.18 mmol) using the general
procedure described above and purified by column chromatogra-
phy (49:1 v/v CH2Cl2: acetone) to afford 15o as a pale-yellow oil
1-(3,4-Dihydroxyphenyl)-2-(phenylthio)ethanone (16b): Aceto-
nide-based inhibitor 15b (25 mg, 0.083 mmol) was deprotected
using the general procedure described above at room temperature
for 1.5 h and purified by column chromatography (98:2 v/v CH2Cl2/
CH3OH) to afford inhibitor 16b as a pale-yellow oil (23 mg, 93%).
Rf (9:1 v/v CH2Cl2/CH3OH)=0.43; 1H NMR (300 MHz, [D6]acetone):
d=7.53–7.50 (2H, m, 2ꢂAr-H), 7.41–7.39 (2H, dd, J=1.2, 7.5 Hz,
2ꢂAr-H), 7.29 (2H, t, J=7.5 Hz, 2ꢂAr-H), 7.19 (1H, t, J=7.5 Hz, Ar-
H), 6.92 (1H, d, J=8.4 Hz, Ar-H), 4.41 ppm (2H, s, CH2); 13C NMR
(100 MHz, [D6]acetone): d=192.5, 150.9, 145.4, 136.6, 129.4, 129.3,
128.7, 126.5, 122.8, 115.7, 115.2, 40.3 ppm; IR (ATR): n˜ =3474 (br.
OH str.), 3270 (br. OH str.), 1657 cmꢀ1; HRMS calcd for C14H12SO3Na
[M+Na]+ 283.0399, found 283.0395.
1
(39 mg, 61%). Rf (2:1 v/v CH2Cl2/hexane)=0.20; H NMR (400 MHz,
CDCl3): d=7.83 (1H, app.d, J=8.0 Hz, Ar-H), 7.74 (1H, app.d, J=
7.8 Hz, Ar-H), 7.67 (1H, app.d, J=7.5 Hz, Ar-H), 7.44–7.38 (2H, m,
2ꢂAr-H), 7.31–7.26 (1H, m, Ar-H), 6.80 (1H, d, J=8.0 Hz, Ar-H), 4.87
(2H, s, CH2), 1.71 ppm (6H, s, 2ꢂCH3); 13C NMR (100 MHz, CDCl3):
d=190.7, 164.8, 152.5, 151.9, 147.4, 135.0, 129.2, 125.6, 124.8,
123.8, 121.1, 120.8, 119.2, 107.7, 107.4, 40.3, 25.5 ppm; IR (ATR): n˜ =
1673 cmꢀ1; HRMS calcd for C18H16NO3S2 [M+H]+ 358.0566, found
358.0569.
1-(3,4-Dihydroxyphenyl)-2-(3-(trifluoromethyl)phenoxy)ethanone
(16c): Acetonide-based inhibitor 15c (20 mg, 0.057 mmol) was de-
protected using the general procedure described above at room
temperature for 1.5 h and purified by column chromatography
(95:5 v/v CH2Cl2/CH3OH) to afford inhibitor 16c as a white solid
(14 mg, 78%). Rf (95:5 v/v CH2Cl2/CH3OH)=0.32; mp: 192–1938C
1-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-((4,5-diphenyloxazol-
2-yl)thio)ethanone (15p): 4,5-Diphenyloxazole-2-thiol (61 mg,
0.24 mmol) was reacted with bromide 18 (50 mg, 0.18 mmol) using
the general procedure described above and purified by column
chromatography (2:1 v/v CH2Cl2/hexane) to afford 15p as a color-
less oil (52 mg, 65%). Rf (2:1 v/v CH2Cl2/hexane)=0.29; 1H NMR
(400 MHz, CDCl3): d=7.65 (1H, dd, J=1.6, 8.0 Hz, Ar-H), 7.62 (2H,
dd, J=1.6, 8.0 Hz, 2ꢂAr-H), 7.54 (2H, dd, J=1.6, 8.0 Hz, 2ꢂAr-H),
7.44 (1H, d, J=1.6 Hz, Ar-H), 7.39–7.31 (6H, m, Ar-H), 6.79 (1H, d,
J=8.0 Hz, Ar-H), 4.77 (2H, s, CH2), 1.71 ppm (6H, s, 2ꢂCH3);
13C NMR (100 MHz, CDCl3): d=191.1, 158.3, 152.5, 148.2, 147.4,
136.4, 129.6, 128.7, 128.6, 128.5, 126.4, 124.8, 119.7, 108.1, 107.8,
40.6, 25.6 ppm; IR (ATR): n˜ =1675 cmꢀ1; HRMS calcd for C26H22NO4S
[M+H]+ 444.1264, found 444.1264.
1
(dec.); H NMR (282 MHz, [D6]acetone): d=7.56–7.49 (3H, m, Ar-H),
7.29–7.24 (3H, m, Ar-H), 6.95 (2H, d, J=8.0 Hz, 2ꢂAr-H), 5.56 ppm
(2H, s, CH2); 13C NMR (100 MHz, [D6]acetone): d=191.1, 158.6,
150.4, 144.6, 130.6 (q, J=32 Hz), 129.9, 126.9, 122.4 (q, J=262 Hz,
CF3), 121.1, 118.1, 117.5 (app.d, J=3.7 Hz), 114.5, 114.2, 111.0
(app.d, J=3.4 Hz), 69.6 ppm; IR (ATR): n˜ =3508 (br. OH str.), 3349
(br. OH str.), 1678 cmꢀ1; HRMS calcd for C15H11F3O4Na [M+Na]+
335.0502, found 335.0501.
1-(3,4-Dihydroxyphenyl)-2-(2-fluorophenoxy)ethanone
(16d):
2-((2-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)-2-oxoethyl)thio)qui-
nazolin-4(3H)-one (15q): 2-Mercaptoquinazolin-4(3H)-one (56 mg,
0.31 mmol) was reacted with bromide 18 (57 mg, 0.21 mmol) using
the general procedure described above to yield 15q as a white
Acetonide-based inhibitor 15d (17 mg, 0.056 mmol) was depro-
tected using the general procedure described above at room tem-
perature for 1.5 h and purified by column chromatography (98:2 v/
v CH2Cl2/CH3OH) to afford inhibitor 16d as a white solid (14 mg,
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ChemMedChem 0000, 00, 1 – 14
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M. tuberculosis Type II DHQase Inhibitors
98%). Rf (98:2 v/v CH2Cl2/CH3OH)=0.20; mp: 190–1918C (dec.);
1H NMR (282 MHz, [D6]acetone): d=7.54–7.51 (2H, m, Ar-H), 7.13
(1H, t, J=7.8 Hz, Ar-H), 7.06–7.03 (2H, m, 2ꢂAr-H), 6.94–6.92 (2H,
m, 2ꢂAr-H), 5.46 ppm (2H, s, CH2); 13C NMR (75 Hz, [D6]acetone):
d=193.1, 153.7 (d, J=244 Hz, ipso-C), 152.1, 147.9 (d, J=11 Hz,
ortho-C), 146.4, 128.6, 125.6 (d, J=3.8 Hz, para-C), 122.9, 122.6 (d,
J=6.8 Hz, meta-C), 117.2 (d, J=18 Hz, ortho-C), 116.5, 116.2, 115.7,
71.9 ppm; IR (ATR): n˜ =3320 (br. OH str.), 1691 cmꢀ1; HRMS calcd
for C14H11FO4Na [M+Na]+ 285.0534, found 285.0537.
J=8.4 Hz, Ar-H), 7.19 (1H, t, J=7.8 Hz), 7.00 (1H, d, J=8.7 Hz, Ar-
H), 5.69 ppm (2H, s, CH2); 13C NMR (100 MHz, [D6]acetone): d=
191.3, 164.8, 154.9, 151.2, 150.9, 145.3, 131.2, 127.2, 127.1, 121.6,
121.2, 116.5, 115.1, 114.6, 112.9, 104.3, 70.4 ppm; IR (ATR): n˜ =3352
(br. OH str.), 3234 (br. OH str.), 1683 cmꢀ1; HRMS calcd for
C17H13NO5Na [M+Na]+ 334.0686, found 334.0689.
1-(3,4-Dihydroxyphenyl)-2-(4-(naphthalen-2-yl)phenoxy)etha-
none (16i): Acetonide-based inhibitor 15i (10 mg, 0.024 mmol)
was deprotected using the general procedure described above at
room temperature for 1.5 h and purified by column chromatogra-
phy (9:1 v/v CH2Cl2/CH3OH) to afford inhibitor 16i as a foam
1-(3,4-Dihydroxyphenyl)-2-(3-nitrophenoxy)ethanone (16e): Ace-
tonide-based inhibitor 15e (22 mg, 0.067 mmol) was deprotected
using the general procedure described above at room temperature
for 1.5 h and purified by column chromatography (95:5 v/v CH2Cl2/
CH3OH) to afford inhibitor 16e as an off-white solid (16 mg, 85%).
Rf (95:5 v/v CH2Cl2/CH3OH)=0.21; mp: 206–2078C (dec.); 1H NMR
(300 MHz, [D6]acetone): d=7.83 (1H, dd, J=1.2, 8.1 Hz, Ar-H), 7.81
(1H, d, J=2.7 Hz, Ar-H), 7.60–7.54 (3H, m, 3ꢂAr-H), 7.41 (1H, dd,
J=1.6, 8.0 Hz, Ar-H), 6.95 (1H, d, J=8.0 Hz, Ar-H), 5.62 ppm (2H, s,
CH2); 13C NMR (75 MHz, [D6]acetone): d=192.6, 160.6, 152.2, 150.5,
146.5, 131.6, 128.5, 123.0, 122.9, 116.9, 116.3, 115.9, 110.5,
71.6 ppm; IR (ATR): n˜ =3491 (br. OH str.), 3398 (br. OH str.),
1679 cmꢀ1; HRMS calcd for C14H11NO6Na [M+Na]+ 312.0479, found
312.0476.
1
(7.5 mg, 84%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.56; H NMR (400 MHz,
[D6]acetone): d=8.13 (1H, s, Ar-H), 7.98–7.96 (2H, m, 2ꢂAr-H), 7.91
(1H, d, J=8.4 Hz, Ar-H), 7.81 (1H, dd, J=1.8, 8.6 Hz, Ar-H), 7.75
(2H, d, J=8.8 Hz, 2ꢂAr-H), 7.60–7.58 (2H, m, 2ꢂAr-H), 7.54–7.47
(2H, m, 2ꢂAr-H), 7.11 (2H, d, J=8.8 Hz, 2ꢂAr-H), 6.97 (1H, d, J=
8.0 Hz, Ar-H), 5.46 ppm (2H, s, CH2); 13C NMR (100 MHz,
[D6]acetone): d=192.2, 158.3, 150.9, 145.2, 137.9, 133.9, 133.4,
132.4, 128.3, 128.1, 128.0, 127.5, 126.2, 125.6, 125.1, 124.7, 121.6,
115.1, 114.9, 114.7, 70.0 ppm; IR (ATR): n˜ =3300 (br. OH str.),
1726 cmꢀ1; HRMS calcd for C24H19O4 [M+H]+ 371.1278, found
371.1278.
1-(3,4-Dihydroxyphenyl)-2-(3-(naphthalen-2-yl)phenoxy)etha-
none (16j): Acetonide-based inhibitor 15j (21 mg, 0.051 mmol)
was deprotected using the general procedure described above at
room temperature for 1.5 h and purified by column chromatogra-
phy (9:1 v/v CH2Cl2/CH3OH) to afford inhibitor 16j as a white foam
1-(3,4-Dihydroxyphenyl)-2-(naphthalen-2-yloxy)ethanone (16 f):
Acetonide-based inhibitor 15 f (39 mg, 0.12 mmol) was deprotect-
ed using the general procedure described above at room tempera-
ture for 1.5 h and purified by column chromatography (95:5 v/v
CH2Cl2/CH3OH) to afford inhibitor 16 f as an off-white solid (31 mg,
88%). Rf (95:5 v/v CH2Cl2/CH3OH)=0.36; mp: 174–1758C (dec.);
1H NMR (300 MHz, [D6]acetone): d=7.86–7.84 (2H, m, 2ꢂAr-H),
7.75 (1H, d, Ar-H, J=8.1 Hz), 7.62–7.60 (2H, m, 2ꢂAr-H), 7.43 (1H,
dt, J=1.5, 8.1 Hz, Ar-H), 7.36–7.32 (2H, m, 2ꢂAr-H), 7.25 (1H, dd,
J=2.4, 9.0 Hz, Ar-H), 6.98 (1H, d, J=8.7 Hz, Ar-H), 5.51 (2H, s, CH2);
13C NMR (100 MHz, [D6]acetone): d=191.6, 156.1, 150.2, 144.6,
134.2, 128.9, 128.8, 127.2, 127.1, 126.3, 125.8, 123.2, 121.2, 118.2,
114.5, 114.4, 106.8, 69.7 ppm); IR (ATR): n˜ =3423 (br. OH str.), 3283
(br. OH str.), 1682 cmꢀ1; HRMS calcd for C18H14O4Na [M+Na]+
317.0784, found 317.0787.
1
(16 mg, 84%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.50; H NMR (400 MHz,
[D6]acetone): d=8.17 (1H, s, Ar-H), 7.99–7.96 (2H, m, 2ꢂAr-H), 7.92
(1H, dd, J=2.1 Hz, 8.8 Hz, Ar-H), 7.81 (1H, dd, J=1.8, 8.5 Hz, Ar-H),
7.66–7.56 (2H, m, 2ꢂAr-H), 7.54–7.50 (2H, m, 2ꢂAr-H), 7.41–7.39
(3H, m, 3ꢂAr-H), 7.00 (1H, ddd, J=1.7, 2.4, 7.6 Hz, Ar-H), 6.95 (1H,
d, J=8.2 Hz), 5.49 ppm (2H, s, 2ꢂCH2); 13C NMR (100 MHz,
[D6]acetone): d=192.0, 159.2, 150.9, 145.1, 142.1, 138.0, 133.7,
132.9, 129.9, 128.4, 128.2, 127.5 (ꢂ2) 126.2, 126.0, 125.6, 125.2,
121.7, 119.9, 114.9, 114.7, 113.7, 113.5, 69.8 ppm; IR (ATR): n˜ =3339
(br. OH), 1689 cmꢀ1; HRMS calcd for C24H19O4 [M+H]+ 371.1278,
found 371.1273.
1-(3,4-Dihydroxyphenyl)-2-(naphthalen-1-yloxy)ethanone (16g):
Acetonide-based inhibitor 15g (39 mg, 0.12 mmol) was deprotect-
ed using the general procedure described above at room tempera-
ture for 1.5 h and purified by column chromatography (98:2 v/v
CH2Cl2/CH3OH) to afford inhibitor 16g as a colorless oil (31 mg,
88%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.51; 1H NMR (300 MHz,
[D6]acetone): d=8.40–8.37 (1H, m, Ar-H), 7.88–7.85 (1H, m, Ar-H),
7.63–7.61 (2H, m, 2ꢂAr-H), 7.55–7.47 (3H, m, 3ꢂAr-H), 7.38 (1H, t,
J=7.8 Hz, Ar-H), 6.99–6.94 (2H, m, 2ꢂAr-H), 5.57 ppm (2H, s, CH2);
13C NMR (75 MHz, [D6]acetone): d=192.4, 154.5, 151.2, 145.6, 135.1,
127.9, 127.8, 126.7, 126.2, 126.1, 125.5, 122.6, 122.5, 120.8, 115.5,
115.3, 105.8, 70.8 ppm; IR (ATR): n˜ =3518 (br. OH str.), 3430 (br. OH
str.), 1705 cmꢀ1; HRMS calcd for C18H14O4Na [M+Na]+ 317.0784,
found 317.0787.
1-(3,4-Dihydroxyphenyl)-2-((2-(trifluoromethyl)phenyl)thio)etha-
none (16k): Acetonide-based inhibitor 15k (18 mg, 0.049 mmol)
was deprotected using the general procedure described above at
room temperature for 1.5 h and purified by column chromatogra-
phy (98:2 v/v CH2Cl2/CH3OH) to afford inhibitor 16k as an off-white
solid (13 mg, 86%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.45; mp: 169–
1
1708C (dec.); H NMR (300 MHz, CD3OD): d=7.68 (1H, dd, J=1.2,
7.8 Hz, Ar-H), 7.62 (1H, dd, J=1.2, 7.8 Hz, Ar-H), 7.53 (1H, dt, J=
1.5, 7.8 Hz, Ar-H), 7.48–7.43 (2H, m, 2ꢂAr-H), 7.35 (1H, dt, J=1.2,
7.5 Hz, Ar-H), 6.82 (1H, d, J=8.5 Hz, Ar-H), 4.87 ppm (2H, s, CH2);
13C NMR (100 MHz, [D6]acetone): d=191.9, 151.1, 145.4, 136.4,
132.9, 131.2, 128.9, 128.6, 127.0 (q, J=28 Hz), 126.3, 125.9 (q, J=
271 Hz, CF3), 122.9, 115.6, 115.3, 40.7 ppm; IR (ATR): n˜ =3485 (br.
OH str.), 3367 (br. OH str.), 1657 cmꢀ1; HRMS calcd for C15H12F3O3S
[M+H]+ 329.0454, found 329.0454.
1-(3,4-Dihydroxyphenyl)-2-(2-(isoxazol-5-yl)phenoxy)ethanone
(16h): Acetonide-based inhibitor 15h (29 mg, 0.082 mmol) was de-
protected using the general procedure described above at room
temperature for 1.5 h and purified by column chromatography (9:1
v/v CH2Cl2/CH3OH) to afford inhibitor 16h as an off-white solid
(23 mg, 92%). Rf (9:1 CH2Cl2/CH3OH)=0.50; mp: 198–1998C (dec.);
1H NMR (300 MHz, [D6]acetone): d=8.53 (1H, d, J=1.8 Hz, Ar-H),
8.03 (1H, dd, J=1.5, 7.8 Hz, Ar-H), 7.66–7.64 (2H, m, 2ꢂAr-H), 7.56
(1H, d, J=1.8 Hz, Ar-H), 7.50 (1H, t, J=8.4 Hz, Ar-H), 7.33 (1H, d,
1-(3,4-Dihydroxyphenyl)-2-((4-fluorophenyl)thio)ethanone (16l):
Acetonide-based inhibitor 15l (29 mg, 0.091 mmol) was deprotect-
ed using the general procedure described above at room tempera-
ture for 1.5 h and purified by column chromatography (98:2 v/v
CH2Cl2/CH3OH) to afford inhibitor 16l as a pale-yellow oil (18 mg,
73%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.42; 1H NMR (300 MHz,
[D6]acetone): d=7.58–7.42 (4H, m, 4ꢂAr-H), 7.09–7.04 (2H, m, 2ꢂ
Ar-H), 6.78 (1H, d, J=7.8 Hz, Ar-H), 4.35 ppm (2H, s, CH2); 13C NMR
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MED
(100 MHz, [D6]acetone): d=192.1, 162.0 (d, J=240 Hz, ipso-C),
150.6, 145.0, 132.4 (d, J=8.0 Hz, meta-C), 131.3 (d, J=3.1 Hz, para-
C), 128.2, 122.5, 116.0 (d, J=20 Hz, ortho-C), 115.3, 114.7, 40.9 ppm;
IR (ATR): n˜ =3527 (br. OH str.), 3329 (br. OH str.), 1654 cmꢀ1; HRMS
calcd for C14H11FO3SNa [M+Na]+ 301.0305, found 301.0301.
508C for 5 h and purified by column chromatography (9:1 v/v
CH2Cl2/CH3OH) to afford inhibitor 16q as a white solid (16 mg,
83%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.48; mp: 214–2158C; 1H NMR
(400 MHz, [D6]DMSO): d=9.99 (1H, s, OH), 9.43 (1H, s, OH), 8.01
(1H, d, J=8.2 Hz, Ar-H), 7.70 (1H, dt, J=1.1, 8.0 Hz, Ar-H), 7.53 (1H,
dd, J=1.7, 8.5 Hz, Ar-H), 7.43 (1H, d, J=1.7 Hz, Ar-H), 7.39 (1H,
app.t, J=8.0 Hz, Ar-H), 7.27 (1H, app.d, J=8.2 Hz, Ar-H), 6.88 (1H,
d, J=8.2 Hz, Ar-H), 4.76 ppm (2H, s, CH2); 13C NMR (100 MHz,
[D6]DMSO): d=191.7, 161.5, 155.5, 151.5, 148.7, 145.6, 135.1, 128.2,
126.5, 126.2, 126.1, 122.3, 120.3, 115.6 (ꢂ2), 37.5 ppm; IR (ATR): n˜ =
3164 (br. OH str.), 1667 cmꢀ1; HRMS calcd for C16H13N2O4S [M+H]+
329.0591, found 329.0590.
2-((4-Chlorophenyl)thio)-1-(3,4-dihydroxyphenyl)ethanone
(16m): Acetonide-based inhibitor 15m (20 mg, 0.060 mmol) was
deprotected using the general procedure described above at room
temperature for 1.5 h and purified by column chromatography
(98:2 v/v CH2Cl2/CH3OH) to afford inhibitor 16m as a pale-yellow
oil (17 mg, 98%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.45; 1H NMR
(300 MHz, [D6]acetone): d=7.52–7.49 (2H, m, 2ꢂAr-H), 7.38 (2H, d,
J=8.7 Hz, 2ꢂAr-H), 7.30 (2H, d, J=8.7 Hz, 2ꢂAr-H), 6.92 (1H, d,
J=8.8 Hz, Ar-H), 4.45 ppm (2H, s, CH2); 13C NMR (75 MHz,
[D6]acetone): d=193.1, 151.9, 146.3, 136.5, 132.8, 131.9, 130.1,
129.5, 123.8, 116.6, 116.1, 41.2 ppm; IR (ATR): n˜ =3545 (br. OH str.),
3442 (br. OH str.), 1709 cmꢀ1; HRMS calcd for C14H11ClO3SNa [M+
Na]+ 317.0010, found 317.0015.
Biological assays
M. tuberculosis type II DHQase assay: Enzyme assays for type II
DHQase from M. tuberculosis (overexpressed and purified as de-
scribed previously)[15,23,24] were carried out using a Shimadzu UV/
Vis 1800 spectrometer with a 6ꢂ6 Peltier cell holder using 1 cm
path length quartz cuvettes at l 234 nm to monitor the formation
of the product, 3-dehydroshikimate. Initial reaction rates were mea-
sured by the increase in absorbance at 234 nm from formation of
the enone–carboxylate chromophore of 3-dehydroshikimate (e=
1.2ꢂ104 mꢀ1 cmꢀ1). The assays were performed at 258C in Tris·HCl
buffer (0.05m, pH 7.0) for M. tuberculosis type II DHQase. The
assays contained 3.80 nm enzyme, and were performed in dupli-
cate. The assay mixtures were prepared in 1 mL quartz cuvettes,
and the assays were initiated by the addition of the substrate (3-
dehydroquinate, synthesized by a published procedure)[26] to the
mixture after incubating the buffer, inhibitor in 0.5% DMSO, and
enzyme at 258C for 3 min. The kinetic data for inhibition studies
were obtained by measuring the initial rates of reaction over
a range of inhibitor concentrations (3–4 different concentrations)
at five different substrate concentrations (0.9KM–4KM). The inhibi-
tion constants (Ki) and the standard deviations were determined
using nonlinear regression fitting to the competitive model by
GraphPad Prism (version 5.03 for Windows). Kinetic data that were
poorly fitted to a competitive model (R2 <0.95) were fitted to
a mixed inhibition model in GraphPad Prism using nonlinear re-
gression to derive the inhibition constants (Ki) and the standard de-
viations.
1-(3,4-Dihydroxyphenyl)-2-(naphthalen-2-ylthio)ethanone (16n):
Acetonide-based inhibitor 15n (30 mg, 0.086 mmol) was depro-
tected using the general procedure described above at room tem-
perature for 1.5 h and purified by column chromatography (98:2 v/
v CH2Cl2/CH3OH) to afford inhibitor 16n as a white solid (20 mg,
75%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.71; mp: 125–1268C (dec.);
1H NMR (400 MHz, [D6]acetone): d=7.95–7.84 (4H, m, 4ꢂAr-H),
7.63–7.60 (2H, m, 2ꢂAr-H), 7.57–7.48 (3H, m, 3ꢂAr-H), 6.98 (1H, d,
J=8.0 Hz, Ar-H), 4.62 ppm (2H, s, CH2); 13C NMR (100 MHz,
[D6]acetone): d=192.1, 150.7, 145.1, 134.0, 133.9, 131.9, 128.4,
128.3, 127.7, 127.1, 127.0, 126.7, 126.5, 125.8, 122.6, 115.3, 114.9,
39.8 ppm; IR (ATR): n˜ =3470 (br. OH str.), 3384 (br. OH str.),
1667 cmꢀ1; HRMS calcd for C18H14O3SNa [M+Na]+ 333.0556, found
333.0554.
2-(Benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanone
(16o): Acetonide-based inhibitor 15o (6 mg, 0.017 mmol) was de-
protected using the general procedure described above at room
temperature for 1.5 h and purified by column chromatography
(98:2 v/v CH2Cl2/CH3OH) to afford inhibitor 16o as a yellow oil
1
(4.4 mg, 83%). Rf (9:1 v/v CH2Cl2/CH3OH)=0.37; H NMR (400 MHz,
[D6]acetone): d=7.95 (1H, d, J=8.0 Hz, Ar-H), 7.81 (1H, d, J=
8.0 Hz, Ar-H), 7.63 (1H, dd, J=1.8, 8.0 Hz, Ar-H), 7.60 (1H, d, J=
1.8 Hz, Ar-H), 7.46 (1H, app.t, J=8.0 Hz, Ar-H), 7.36 (1H, app.t, J=
8.0 Hz, Ar-H), 6.99 (1H, d, J=8.0 Hz, Ar-H), 5.03 ppm (2H, s, CH2);
13C NMR (100 MHz, [D6]acetone): d=190.5, 167.0, 153.2, 151.0,
145.2, 135.3, 128.2, 126.2, 124.4, 122.4, 121.4, 121.3, 115.2, 115.0,
40.3 ppm; IR (ATR): n˜ =3287 (br. OH str.), 1671 cmꢀ1; MS (ESI): m/z
318 [M+H]+.
Microplate-based assay with Alamar blue (resazurin) readout
for M. tuberculosis
M. tuberculosis H37Ra (ATCC 25177) was grown in Middlebrook
7H9 broth medium supplemented with OADC (Difco Laboratories,
Detroit, MI, USA), 0.5% glycerol, and 0.05% Tween-80. Freshly
seeded cultures were grown at 378C, for approximately 14 days, to
mid-exponential phase (OD600 0.4–0.8) for use in the inhibition
assays. The effect of the type II DHQase inhibitors against M. tuber-
culosis growth were measured by a resazurin reduction microplate
assay, using the procedure previously described by Taneja and
Tyagi.[23a] M. tuberculosis grown to mid-exponential phase
(OD600 0.4–0.8) was diluted to OD600 0.002 in 7H9S media (Middle-
brook 7H9 with OADC, 0.5% glycerol, 0.02% tyloxapol, 1% tryp-
tone) containing 0.5% DMSO; 96-well microtiter plates were set up
with 100 mL inhibitors, serially diluted into 7H9S. Diluted M. tuber-
culosis (100 mL, representing ~2ꢂ104 CFUmLꢀ1) was added to each
well. Plates were incubated for 5 days at 378C in a humidified incu-
bator prior to the addition of a 0.02% resazurin solution (30 mL)
and 20% Tween-80 (12.5 mL) to each well. Sample fluorescence
was measured after 48 h on a BMG Labtech Polarstar Omega in-
1-(3,4-Dihydroxyphenyl)-2-((4,5-diphenyloxazol-2-yl)thio)etha-
none (16p): Acetonide-based inhibitor 15p (40 mg, 0.090 mmol)
was deprotected using the general procedure described above at
room temperature for 1.5 h and purified by column chromatogra-
phy (9:1 v/v CH2Cl2/CH3OH) to afford inhibitor 16p as a white solid
(30 mg, 83%). Rf (95:5 v/v CH2Cl2/CH3OH)=0.35; mp: 190–1918C
(dec.); 1H NMR (400 MHz, [D6]DMSO): d=7.51–7.38 (12H, m, 12ꢂ
Ar-H), 6.85 (1H, d, J=8.5 Hz, Ar-H), 4.96 ppm (2H, s, CH2); 13C NMR
(100 MHz, [D6]DMSO): d=191.4, 158.8, 151.5, 146.7, 145.8, 136.2,
132.1, 129.4, 129.2, 127.9, 126.6, 122.6, 115.7, 115.6, 40.2 ppm; IR
(ATR): n˜ =3257 (br. OH str.), 1659 cmꢀ1; HRMS calcd for C23H18NO4S
[M+H]+ 404.0951, found 404.0951.
2-((2-(3,4-Dihydroxyphenyl)-2-oxoethyl)thio)quinazolin-4(3H)-
one (16q): Acetonide-based inhibitor 15q (18 mg, 0.049 mmol)
was deprotected using the general procedure described above at
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M. tuberculosis Type II DHQase Inhibitors
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Acknowledgements
We thank the Australian National Health and Medical Research
Council for funding (Project Grant number 632769) and PhD sup-
port from a John Lamberton Research Scholarship and an Aus-
tralian Postgraduate Award (ATT). We also thank Dr. Adrian Lap-
thorn (Department of Chemistry and Division of Biochemistry
and Life Science, University of Glasgow) for providing the plasmid
for M. tuberculosis type II DHQase and Ms. Angel Pang for tech-
nical assistance.
Keywords: antibiotics
·
dehydroquinases
·
inhibitors
·
medicinal chemistry · Mycobacterium tuberculosis
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Revised: March 7, 2012
Published online on && &&, 0000
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These are not the final page numbers!

FULL PAPERS
A. T. Tran, N. P. West, W. J. Britton,
R. J. Payne*
Extend to improve: Novel inhibitors of
Mycobacterium tuberculosis type II dehy-
droquinase were discovered through
a fragment elaboration approach. A
number of low-micromolar inhibitors of
the enzyme were elucidated which pos-
sess significant activity against M. tuber-
culosis in vitro.
&& – &&
Elucidation of Mycobacterium
tuberculosis Type II Dehydroquinase
Inhibitors using a Fragment
Elaboration Strategy
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ChemMedChem 0000, 00, 1 – 14
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These are not the final page numbers!