The structure-activity relationship of urea derivatives as anti-tuberculosis agents

Article abstract of DOI:10.1016/j.bmc.2011.07.034

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure-activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.

Full text of DOI:10.1016/j.bmc.2011.07.034

Bioorganic & Medicinal Chemistry 19 (2011) 5585–5595
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The structure–activity relationship of urea derivatives
as anti-tuberculosis agents
Joshua R. Brown a, , Elton J. North a, , Julian G. Hurdle a, Christophe Morisseau b, Jerrod S. Scarborough a,
Dianqing Sun a, Jana Korduláková c, Michael S. Scherman d, Victoria Jones d, Anna Grzegorzewicz d,
Rebecca M. Crew d, Mary Jackson d, Michael R. McNeil d, Richard E. Lee a,
⇑
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 1000, Memphis, TN 38105, USA
b Department of Entomology and Cancer Center, University of California, Davis, CA 95616, USA
c Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84215 Bratislava, Slovak Republic
d Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug
resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action,
be discovered and developed. The direct anti-tubercular testing of a small compound library led to dis-
covery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis
of an optimization library. This library was generated by systematically replacing each section of the mol-
ecule with a similar moiety until a clear structure–activity relationship was obtained with respect to anti-
tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and
had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted
that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide
hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative
epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the
optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity
is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibi-
tion of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must
be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis
enzymes.
Received 12 June 2011
Revised 12 July 2011
Accepted 19 July 2011
Available online 24 July 2011
Keywords:
Urea
Tuberculosis
Epoxide hydrolase
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
discovery programs.2 On the contrary, direct phenotypic based
MIC screening of commercial and proprietary libraries has recently
Increasing drug resistance and poor activity of existing thera-
pies towards the latent stage of Mycobacterium tuberculosis infec-
tion has produced a clear need to develop novel therapeutics to
treat tuberculosis.1 Thus fast-acting drugs with novel mechanisms
of action that are not cross resistant to existing drugs are being
sought actively. To tackle this problem two primary screening
strategies are being applied in tuberculosis drug discovery—target
based high throughput screening and phenotypic minimum inhib-
itory concentration (MIC) based screening of whole cell bacteria.
Although target/enzyme based high throughput screening for
new tuberculosis therapeutics has been widely adopted, this strat-
egy has not produced many notable successes, an experience that
mirrors the success of this approach in other antibacterial drug
produced a number of interesting clinical candidates including the
diaryl quinolone, TMC207, and benzothiazinone, BTZ043,3,4 for
which the ultimate enzymatic target and antitubercular mode of
action for these compounds was derived after potent inhibitors
were identified.
We have previously adopted an analogous approach to screen
various available chemical libraries directly for anti-tuberculosis
activity. After the identification of selective hits, mode of action
studies are performed producing novel validated anti-tubercular
drug candidates.5 In this study a ꢀ12,000 compound library from
LeadScreen (Tripos) was screened for anti-tuberculosis activity by
microbroth dilution in Middlebrook 7H9 media. Three hundred
and eight compounds initially showed activity at 10 lM. One of
the best of these hits was the urea hit 1, which upon resynthesis
and retesting displayed a confirmed anti-tuberculosis MIC of
⇑
Corresponding author. Tel.: +1 901 595 6617; fax: +1 901 595 5715.
E-mail address: Richard.lee@stjude.org (R.E. Lee).
0.03
lM (0.01 lg/ml) (Fig. 1). Interestingly, the structure of the
urea compound 1 was found to be very similar to inhibitors of
These authors contributed equally.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.034

5586
J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
F
F
tubercular structure–activity relationships (SAR) for potency and
O
selectivity. These positions are shown as R0 (aryl) and R (adaman-
tyl) in Scheme 1. The synthesis of these derivatives was carried
out by reacting the desired amine with the corresponding
isocyanate in dichloromethane in the presence of triethylamine
(Scheme 1). Using this facile chemistry, an array of compounds
(1–30) was rapidly synthesized using parallel synthesis.
N
H
N
H
F
1
Figure 1. Lead compound 1.
The second series of compounds was synthesized focusing on
modifications of the urea moiety. The first compound targeted
was the thiourea derivative 31 to explore the structural similarity
to already known thiourea anti-tubercular drugs such as thiocar-
lide and thiacetazone.12,13 As shown in Scheme 2A, this synthesis
was identical to the urea synthesis with the exception that the iso-
thiocyanate was used in place of the isocyanate to give 31 in good
yield.
Scheme 2B shows the synthesis of the carbamate derivative 32.
This synthesis coupled the adamantyl alcohol with the phenyl iso-
cyanate in order to form the desired product again in high yield.
Further modification of the urea moiety is shown by the synthesis
of the mono- and dimethylated derivatives 33 and 34 shown in
Scheme 2C. The synthesis of the 33 started from 1, which was
selectively deprotonated at the more acidic urea nitrogen adjacent
to the phenyl ring with n-butyl lithium at low temperature. The
anion was then reacted with iodomethane to form compound 33
in good yield. Di-N-methylated urea 34 was obtained in an analo-
gous fashion using increased equivalents of n-butyl lithium and
iodomethane.
A
O
R
R'
N
H
N
H
R and R' = Alkyl (linear and cyclic) and Aryl (substituted and non-substituted).
B
O
O
O
F3C
O
N
H
N
H
N
H
N
H
EphB = 19 nM
sEH = 390 nM
EphB = 31,600 nM
sEH = 12 nM
Cl
Cl
Cl
O
O
N
H
N
H
N
H
N
H
EphB = 220 nM
sEH = 13 nM
EphB = 130 nM
sEH = 2 nM
The final series of compounds synthesized aimed to decrease
the lipophilicity by the introduction of oxygen containing substit-
uents, as solubility issues were noted for some of the most prom-
ising compounds in preliminary testing of the first two series. The
first compound targeted was a methyl para-amino salicylic ester
substituted urea 36, a known inhibitor of human sEH and EphB,6
Figure 2. (A) Summary of current urea-based sEH and M. tuberculosis EphB
inhibitors.6,7 (B) Specific examples of urea-based sEH M. tuberculosis EphB inhibitors
with IC50 values.6,7
mammalian soluble epoxide hydrolase (sEH6,7 and the recently re-
ported inhibitors of the M. tuberculosis epoxide hydrolase (EH) en-
zyme B (EphB).6 Figure 2 shows the general structure and specific
examples for the urea-based sEH and M. tuberculosis EphB inhibi-
tors. This lead us to hypothesize that hit 1 targets the Eph enzymes
of M. tuberculosis. Initial analysis of the tuberculosis genome
showed it contained at least six putative EH enzymes.8 This unusu-
ally large number of EHs compared to other bacteria, suggests
these enzymes play important roles in the physiology of M. tuber-
culosis; notably, lipid metabolism and detoxification of reactive
oxygen species derived from the host’s immune system. In this re-
gard, Eph enzymes represent promising targets for anti-tubercular
drug discovery. Moreover, they represent potentially druggable
targets with an active site suitable for small molecule therapeutic
intervention.6,9 Since M. tuberculosis contains several EH enzymes
with similar active sites and perhaps redundancy in function this
presents a considerable challenge to obtain compounds with
anti-tubercular activity through target based discovery. We there-
fore adopted a direct MIC approach for rapidly determining the ini-
tial therapeutic potential of these inhibitors. Currently, there are
no reports on the whole cell anti-tubercular activity of EH inhibi-
tors, though molecules with similar structures have recently been
described with good inhibition of M. tuberculosis EphB6 or antitu-
bercular MICs.10,11 In this study, the three sections—the aryl ring,
the alkyl ring and urea—of hit 1 were systematically modified to
develop a detailed anti-tubercular structure–activity relationship
for this series of compounds and the results were rationalized with
respect to the binding site of EphB.
O
NH2
OCN
a
R'
R
+
R
R'
N
H
N
H
Scheme 1. Reagents: (a) triethylamine, dry dichloromethane, room temperature,
overnight.
F
F
F
S
a
a
+
+
A
B
NH2
OH
N
H
N
H
F
SCN
OCN
F
F
F
31 92%
F
F
F
F
O
O
N
H
F
32 97%
F
F
O
N
H
N
b
F
F
F
O
33 92%
C
N
H
N
H
F
c
O
F
1
N
N
F
F
34 44%
2. Chemistry
Scheme 2. Reagents and conditions: (a) TEA, dry DCM, room temperature,
overnight; (b) (i) n-BuLi, dry THF, ꢁ78 °C; (ii) iodomethane, reflux, 24 h; (c) (i) n-
BuLi, dry THF, ꢁ78 °C; (ii) iodomethane, reflux, 24 h.
The optimization of compound 1 began by modifying each side
of the urea moiety with a selection of substituents to probe anti-

J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
5587
O
O
O
N
N
H
OH
c
H
O
O
a
b
42 33%
d
NO2
O
O
NH2
NO2
S
N
N
H
H
43 50%
Scheme 3. Reagents and conditions: (a) 1-bromo-4-methylpentane, NaOH, DMF, 65 °C, overnight; (b) Pd/C, H2, EtOH, overnight; (c) triphosgene, TEA, DCM, room
temperature, overnight; (d) thiophosgene, TEA, DCM, room temperature, overnight.
which has been shown to be orally bioavailable. Compound 36 was
synthesized according to the published synthesis of Kasagami
et al.14,15
positions (Compounds 10–14) were more tolerated with those
substitutions not having a major impact on the anti-tuberculosis
activity. In the group of compounds which contain the trifluor-
ophenyl moiety (Compounds 1–9), the adamantyl compounds 1
Other oxygenated and potentially more soluble urea analogs
37–41 were synthesized according to Scheme 1. To complete the
series we were interested in generating and testing compound
42, the urea derivative and metabolite of the diphenylurea tuber-
culosis drug isoxyl. Compound 42 was synthesized by alkylating
4-nitrophenol with 1-bromo-4-methylpentane to produce the O-
alkylated intermediate (Scheme 3). This nitro intermediate was
then reduced by hydrogenolysis to produce the aniline intermedi-
ate. This compound was then reacted with triphosgene or thio-
phosgene to produce the 42 and 43 (isoxyl) in acceptable yields.
and 3 exhibit the best activity with potent MIC values of 0.01 lg/
mL. With the exception of compounds 3 and 6, all compounds 1–
9 inhibited EphB greater than 50%. Within this series the bulky
hydrophobic adamantyl group is preferred over a straight-chain al-
kyl group 4. With regard to compounds that only contain aliphatic
groups (Compounds 15–21), the best MIC value is displayed by the
compound with the n-hexyl alkyl chain 20. Interestingly,
compound 20 has a relatively poor EphB inhibition percentage at
32% but potent EphE inhibition at 100%. When comparing the
compounds with the 3-chloro-4-methylphenyl substitution
(Compounds 22–26), compounds with a bulky alkyl ring on the
opposite side (adamantylmethylene and cyclooctyl) result in
the most active chemicals (22 and 24). This trend continues in
the compounds in the 2-fluoro-3-(trifluoromethyl)phenyl arylurea
series 27–30 further suggesting a reproducible SAR that favors
bulky alkyl and aryl substituted ureas for maximal antituberculosis
activity.
Table 2 shows the MIC data for the second series of compounds
in which the central urea moiety was modified. In this series, it is
clearly seen that the unmodified urea moiety is preferred for best
anti-tuberculosis activity, human sEH and Eph inhibition. Intro-
duction of a thiourea 31 produces a 80-fold decrease in MIC activ-
ity. Replacing the urea with a carbamate 32 produces an additional
10-fold larger decrease in anti-M. tuberculosis potency. Mono N-
methylation of the urea 33 shows a similar decrease in activity
and di-N-methylation 34 produces an even greater decrease in
MIC activity. In this small library there is a clear correlation be-
tween anti-tuberculosis activity and Eph inhibition, strongly sup-
porting our hypothesis that Eph enzymes are the anti-tubercular
targets of these inhibitors.
3. Results and discussion
Table 1 shows the M. tuberculosis MIC activity for the first series
of compounds. In addition to whole cell phenotypic MIC screening
we tested these compounds against pure recombinant EphB
(Rv1938) and EphE (Rv3670). For the EphB and EphE enzyme inhi-
bition assays, urea inhibitors were tested in duplicates at 10 nM
against the fluorescent EphB and EphE substrate cyano(2-meth-
oxynaphthalen-6-yl)methyl trans-(3-phenyl-oxyran-2-yl) methyl
carbonate (Epoxy Fluor 7, Cayman Chemical, Ann Arbor, MI), at a
substrate final concentration of 5 lM. The tables below show the
results expressed as percent inhibition of the control reaction (con-
taining solely EphB and substrate or EphE and substrate). IC50 val-
ues were determined against the mammalian sEH also using
Cyano(2-methoxynaphthalen-6-yl)methyl
trans-(3-phenyl-oxy-
ran-2-yl) methyl carbonate as the fluorescent substrate.16
Overall, these compounds show good activity primarily against
M. tuberculosis (H37Rv). In this series, compounds 1, 3, 11, 20, 24
showed the best activity with an MIC value of 0.01
comparable to the anti-tuberculosis drug isoniazid (MIC 0.01
l
g/mL that is
g/
l
Table 3 shows the M. tuberculosis MIC and Eph data for the third
series of oxygenated ureas designed in part to increase the solubil-
ity of this series and to expand the SAR. Compounds 35 and 36
showed good antitubercular activity while the remaining com-
pounds 37–40 had much poorer activity. It is of interest to note
that 35 and 36 were the only two in this series which contained
the preferred SAR arrangement of having a bulky aliphatic ring
on one side of the molecule and having an aromatic ring on the
other. When comparing compounds 37 and 38 to aliphatic analog
19, placing the oxygen in the alkyl chain dramatically decreases
antitubercular activity. Similar trends were observed for EphB
and EphE inhibition. Compound 41, the urea analog and metabolite
of thiourea tuberculosis drug thiocarlide 42, was found to be inac-
tive. This is in contrast to the comparison of compounds 1 and 31,
where replacing the urea moiety with a thiourea decreased but did
mL), which was used as a control. Interestingly, of these five com-
pounds, only 1 and 24 inhibited EphB greater than 50% but all (1, 3,
11, 20, 24) showed >70% inhibition of EphE. This suggests that the
potent M. tuberculosis MIC values are more closely correlated to
EphE inhibition rather than EphB inhibition and that inhibition of
multiple Eph enzyme isoforms is likely a contributing factor to
the potent anti-tubercular activity of compounds in this series.
Some significant SAR emerged from this data. The substituent at
the one position has a strong preference for the bulky aliphatic ring
system such as adamantyl with the other urea substituent at the
three position favoring an aryl ring. Substituting adamantyl of 1
for a cyclohexyl 8 or cyclopentyl 9 considerably decreased the
activity, while a cyclooctyl 7 ring system only minimally decreased
the activity to
a level comparable to the drug ethambutol
(0.8 g/mL). Substitution in the aryl ring at the meta and/or para
l

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J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
Table 1
No.
Structure
M. tb MIC (
l
g/mL)a
Cytotox (
l
g/mL)b
% EphB inhibition
at 10 nM
% EphE inhibition
at 10 nM
Human sEH
IC50 (nM)
F
O
O
O
O
1
0.01
219
72.8
89.7
44.2
87.5
71.4
92.2
90.8
92.8
0.4
0.4
0.4
0.4
F
F
F
N
H
N
H
2
3
4
0.4
0.01
6
725.8
1.18
F
F
F
N
H
N
H
F
F
F
N
H
N
H
129.7
F
N
H
N
H
F
F
O
O
5
0.02
16
76.3
86.1
0.4
F
F
F
N
H
N
H
6
7
8
9
0.2
0.8
12.5
50
15.5
63.4
15.9
ndc
38.6
95.7
96.4
86.4
102.9
91.6
65.6
36.0
0.4
F
N
H
N
H
F
F
O
O
O
0.4
F
F
F
N
H
N
H
1.2
56.2
F
F
F
N
H
N
H
F
N
H
N
H
F
N
O
10
11
12
0.4
7.1
9.0
72.7
87.9
31.4
1.1
4.5
0.5
N
H
N
H
O
0.01
0.1
30.2
9.7
34.1
80.6
N
N
H
H
O
N
H
N
H
O
O
13
3.1
4.2
nid
34.1
14.8
N
N
H
H
O
14
15
16
1.6
50
26.4
23.4
371.9
9.8
58.3
57.3
55.4
4.5
Cl
N
N
H
H
O
27.2
20.7
84.4
24.2
N
H
N
H
O
12.5
N
H
N
H

J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
5589
Table 1 (continued)
Structure
No.
17
18
19
20
21
M. tb MIC (
l
g/mL)a
Cytotox (
170.9
25
l
g/mL)b
% EphB inhibition
at 10 nM
% EphE inhibition
at 10 nM
Human sEH
IC50 (nM)
O
O
12.5
51.9
11.6
58.6
32.3
37.1
73.6
70.2
90.5
100.4
99.9
61.4
N
H
N
H
10
0.4
6.4
1.0
0.5
N
H
N
H
O
0.39
11.6
N
H
N
H
O
0.01
174.8
20
N
H
N
H
O
0.2
N
H
N
H
O
22
23
24
0.02
3.1
4.34
33.9
91.0
83.6
94.1
99.3
98.2
0.4
1.6
0.4
Cl
Cl
N
N
H
H
O
186.4
>400
N
H
N
H
O
O
0.01
Cl
Cl
N
H
N
H
F
25
26
6
6
342.3
>400
18.6
81.9
19.0
97.4
16.6
15.0
N
H
N
H
O
Cl
N
H
N
H
O
27
28
3
nd
5.5
46.8
56.3
0.4
0.4
F3C
N
N
H
H
F
O
0.8
25.3
17.0
F3C
F3C
F3C
N
N
H
H
F
F
F
O
O
29
30
1.6
1.6
38.5
ni
63.9
15.5
1.9
3.7
N
H
N
H
Cl
193.2
16.6
N
H
N
H
a
b
c
In vitro antituberculosis activity of urea compounds against M. tuberculosis H37Rv.
Cytotoxicity was determined against mammalian Vero cell line.
nd = not determined.
d
ni = no inhibition.
not eliminate all antitubercular activity demonstrating the contri-
bution of the adamantyl and phenyl groups to the overall activity.
This also suggests that thiocarlide and its metabolites act on a dif-
ferent target than the compounds selected here, in agreement with
prior mode of action studies for thiocarlide.12
In this study, clear structure–activity relationships (SAR) for
anti-tubercular activity of urea-based inhibitors has emerged with
the best compounds containing a 1-cycloalkyl-3-phenyl disubsti-
tuted urea structure. The 1–3 disubstituted urea moiety was
shown to be most active over other urea substitution patterns

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J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
Table 2
In vitro antitubercular activity of compounds bearing extra substitutions to the urea core
F
Y
X
Z
F
No.
M. tb MIC (lg/mL)
% EphB inhibition at 10 nM
% EphE inhibition at 10 nM
Human sEH IC50 (nM)
F
X
Y
Z
1
31
32
33
34
NH
NH
O
NH
NMe
O
S
O
O
O
NH
NH
NH
NMe
NMe
0.01
0.8
6
6
12
72.8
21.3
10.2
6.7
71.4
35.5
21.4
22.8
25.2
0.4
4.8
463.8
38.1
<1
1838.1
Table 3
In vitro antituberculosis activity and cytotoxicities of oxygenated urea compounds
No.
Structure
M. tb MIC (
mL)
lg/
Cytotox (
mL)
lg/
% EphB inh. at
10 nM
% EphE inh. at
10 nM
Human sEH IC50
(nM)
O
O
35
0.4
0.4
13.4
30.3
95.1
83.1
79.5
14.3
1.2
N
H
N
H
O
O
O
36
26.9
HO
N
H
N
H
O
O
O
O
37
38
39
40
50
nda
18.7
23.7
11.9
16.6
17.6
35.9
14.7
35.0
524.8
386.1
260.4
242.6
N
H
N
H
100
25
20.4
6.4
O
N
H
N
H
O
N
H
N
H
O
O
25
264.8
N
H
N
H
H
H
N
N
41
200
2.5
nd
nd
14.6
88.8
22
6.5
nd
O
S
O
O
O
O
42*
(Isoxyl)
H
N
H
N
nib
*
EphB and EphE inhibition data are IC50 values (nM).
a
nd = not determined.
b
ni = no inhibition.
and urea bioisosteres. For the cycloalkyl ring substituent, a strong
preference for bulky groups such as adamantyl was preferred for
anti-tubercular activity. This is consistent with previous findings
by Biswal et al.,6 which indicates the importance of the adamantyl
substituent in inhibiting M. tuberculosis EphB. Therefore, bulky
groups such as adamantyl appear relevant for both enzyme and
whole cell activities. This is noteworthy as other adamantyl ana-
logs have been shown to have good anti-tubercular activity; most
notably the clinical candidate adamantyl diamine SQ109 and
adamantyl amides discovered through high throughput screening
by the NIH sponsored TAACF program.10,17,18 Substitutions to the
phenyl ring were well tolerated; suggesting further analogs of this
position may be worthwhile.
Most compounds had some activity against EphB and EphE with
some being so potent that they are close to the accurate limit of
detection for our assay, further supporting our hypothesis that
these urea-based compounds are readily recognized by EH en-
zymes. Fourteen compounds inhibited EphB >50% and 28 com-
pounds inhibited EphE >50% at 10 nM. In general, compounds
containing adamantane and the 2,3,4-trifluorophenyl were more
active against EphB and EphE than the other compounds. Com-
pounds 3, 10, 11, 20, 22 have good MIC values good EphE activity

J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
5591
but relatively poor EphB inhibition suggesting that EphE inhibition
is the major contributor to MIC activity. However, there are many
putative EH enzymes encoded in the tuberculosis genome, sug-
gesting that the highly potent phenotypic MIC values may be
attributed to these compounds hitting multiple EH targets. Com-
pounds in this series displayed varying cytotoxicity, which did
not correlate with either antitubercular MIC or Eph inhibition.
However, the most potent antitubercular compounds all had
acceptable therapeutic indexes (Cytotoxicity/MIC >100). Twenty-
three of the forty-two compounds tested had significant inhibition
of human sEH (<10 nM), suggesting the need for the identification
of new urea-based inhibitors possessing selectivity for M. tubercu-
losis EH enzymes over sEH enzymes in future studies.
compounds 32 and 42 mass spectra were recorded on a Bruker Es-
quire LCMS using ESI. Purity of the products was confirmed before
testing by analytical RP-HPLC on a Shimadzu HPLC system, and all
final compounds had a purity of 95% or greater as determined by
RP-HPLC. Gradient Conditions M1: solvent A (0.1% formic acid in
water) and solvent B (0.1% formic acid in MeOH): 0–1.00 min
95% A, 1.00–6.00 min 0–95% B (linear gradient), 6.00–9.50 min
100% B, 9.50–9.75 min 0–95% A, 9.75–10.0 min 95% A, detection
by UV at 254 nm and by ELSD. Gradient Conditions M2: same as
M1 except solvent B is (0.1% formic acid in acetonitrile). Melting
points were obtained on the OptiMelt MPA100 Automated Melting
Point System.
The SAR and Eph inhibition data provide support that
a/b-
4.2. Synthesis of urea compounds. General method
hydrolase fold EHs may be the actual targets of these ureas in M.
tuberculosis due to the structural similarities of these compounds
to human sEH inhibitors.19 It therefore appears that urea hit 1
and derivatives competitively bind to the active site of EH enzymes
to cause inhibition. By interacting with this conserved catalytic do-
main it is highly likely that these compounds inhibit multiple EH
targets by binding in similar ways, which may explain their potent
MIC activity. Future ligand design using the active site of model EH
EphB may therefore act as a platform for future discoveries against
the multiple EHs found in M. tuberculosis. This may lead to com-
pounds with potent MIC activity that likely results from binding
In a round bottom flask equipped with a stir bar, 1.2 mmol of
the appropriate amine was added to 10 mL of anhydrous dichloro-
methane. To this solution, 1.0 mmol of the appropriate isocyanate
followed by 3.6 mmol of triethylamine was added. The reaction
was stirred at room temperature overnight. The solvent was re-
moved by rotary evaporation. The resulting residue was purified
by flash chromatography using a petroleum ether to ethyl acetate
gradient to elute the final compound.
4.2.1. 1-(2-Adamantyl)-3-(2,3,4-trifluorophenyl)urea (1)
Yield: 303 mg (93%); Mp: 229–230 °C; 1H NMR (DMSO-d6): d
1.56 (1H, s), 1.59 (1H, s), 1.69–1.90 (12H, m), 3.78 (1H, d,
J = 8.0 Hz), 6.96 (1H, d, J = 8.0 Hz), 7.16–7.24 (1H, m), 7.96 (1H,
m), 8.51 (1H, s); ESI-HRMS m/z: [M+H]+ calcd: 325.1528, found:
325.1533.
of the inhibitors to multiple related members of
a/b-hydrolase fold
family producing an additive lethal effect.20
Evidence for a targeted mode of action is bolstered by a lack of a
pure correlation between lipophilicity and MIC against M. tubercu-
losis, suggesting that activity was not due to disruption of the
lipophilic tubercular cell wall, though it does appear that the com-
pounds do need to have adequate lipophilicity to enter the bacte-
ria. Specificity of this series is also supported by the most active
compounds also exhibiting good activity against Mycobacterium
smegmatis and the general lack of activity of most analogs against
other gram-positive and -negative bacterial pathogens including
Staphylococcus aureus, Bacillus anthracis, Escherichia coli, Pseudomo-
nas aeruginosa and Klebsiella pneumoniae (see Supplementary data).
In general all urea-based compounds with potent MICs that were
4.2.2. 1-(1-Adamantyl)-3-(2,3,4-trifluorophenyl)urea (2)
Yield: 326 mg (81%); Mp: 216–219 °C; 1H NMR (DMSO-d6): d
1.63 (6H, s), 1.93 (6H, s), 2.03 (3H, s), 6.44 (1H, s), 7.13–7.21 (1H,
m), 7.84–7.92 (1H, m), 8.26 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
325.1528, found: 325.1533.
4.2.3. 1-(1-(1-Adamantyl)methyl)-3-(2,3,4-trifluorophenyl)urea
(3)
comparable to isoniazid and ethambutol (0.01–1.6
lg/mL), showed
Yield: 297 mg (88%); Mp: 171–173 °C; 1H NMR (DMSO-d6): d
1.55–1.74 (12H, m), 1.90–1.95 (3H, m), 2.81 (2H, d, J = 6.0 Hz),
6.60 (1H, t, J = 5.5 Hz), 7.15–7.23 (1H, m), 7.88–7.96 (1H, m), 8.44
(1H, s); ESI-HRMS m/z: [M+H]+ calcd: 339.1685, found: 339.1670.
good therapeutic selectivity for M. tuberculosis with selectivity
indices of 16.5–>21,900. Importantly, this indicates that phenotyp-
ically selective antitubercular ureas may be obtained. Increasing
solubility, metabolic stability and selectivity away from inhibition
of human sEH are currently the principal design drivers that are
being used to further optimize this series.
4.2.4. 1-Heptyl-3-(2,3,4-trifluorophenyl)urea (4)
Yield: 350 mg (98%); Mp: 96–98 °C; 1H NMR (DMSO-d6): d 0.87
(3H, t, J = 6.5 Hz), 1.24 (8H, s), 1.36–1.46 (2H, m), 3.08 (2H, q,
J = 6.5 Hz), 6.57 (1H, t, J = 5.0 Hz), 7.15–7.23 (1H, m), 7.81–7.88
(1H, m), 8.40 (1H, s); ESI-HRMS m/z: [M+H]+ calcd: 289.1528,
found: 289.1545.
4. Experimental
4.1. Reagents and instrumentation
4.2.5. 1-(2,3,4-Trifluorophenyl)-3-(2,6,6-trimethylbicyclo[3.1.1]-
heptan-3-yl)urea (5)
All anhydrous solvents and starting materials were purchased
from Aldrich Chemical Co. (Milwaukee, WI). All reagent grade sol-
vents used from chromatography were purchased from Fisher Sci-
entific (Suwanee, GA) and flash column chromatography silica
cartridges were obtained from Biotage Inc. (Lake Forest, VA). The
reactions were monitored by thin-layer chromatography (TLC) on
pre-coated Merch 60 F254 silica gel plates and visualized using
UV light (254 nm). A Biotage FLASH column chromatography sys-
tem was used to purify mixtures. All 1H NMR spectra were re-
corded on a Varian INOVA-500 spectrometer. Chemical shifts (d)
are reported in parts per million relative to the residual solvent
peak or internal standard (tetramethylsilane), and coupling con-
stants (J) are reported in hertz (Hz). High resolution mass spectra
were recorded on a Waters Xevo G2 QTOF LCMS using ESI. For
Yield: 399 mg (99%); Mp: 156–159 °C; 1H NMR (DMSO-d6): d
0.90 (1H, d, J = 9.5 Hz), 1.00 (3H, s), 1.06 (3H, d, J = 7.0 Hz), 1.20
(3H, s), 1.49–1.56 (1H, m), 1.75–1.82 (2H, m), 1.88–1.94 (1H, m),
2.34–2.49 (2H, m), 3.97 (1H, quintuplet, J = 8.0 Hz), 6.70 (1H, d,
J = 8.0 Hz), 7.14–7.22 (1H, m), 7.84–7.92 (1H, m), 8.26 (1H, s);
ESI-HRMS m/z: [M+H]+ calcd: 327.1685, found: 327.1694.
4.2.6. 1-((6,6-Dimethylbicyclo[3.1.1]heptan-2-yl)methyl)-3-
(2,3,4-trifluorophenyl)urea (6)
Yield: 336 mg (83%); Mp: 105–107 °C; 1H NMR (DMSO-d6): d
0.86 (1H, d, J = 9.5 Hz), 1.00 (3H, s), 1.17 (3H, s), 1.39–1.49 (1H,
m), 1.78–1.94 (5H, m), 2.06–2.15 (1H, m), 2.29–2.36 (1H, m),
3.02–3.14 (2H, m), 6.62 (1H, t, J = 5.5 Hz), 7.14–7.22 (1H, m),

5592
J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
7.82–7.90 (1H, m), 8.38 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
327.1685, found: 327.1694.
4.2.16. 1-(2-Adamantyl)-3-tert-butylurea (16)
Yield: 185 mg (74%); Mp: >300 °C; 1H NMR (DMSO-d6): d 1.22
(9H, s), 1.49 (1H, s), 1.52 (1H, s), 1.65–1.86 (12H, m), 3.64 (1H, d,
J = 8.0 Hz), 5.70 (1H, s), 5.95 (1H, d, J = 8.5 Hz); ESI-HRMS m/z:
[M+H]+ calcd: 251.2124, found: 251.2121.
4.2.7. 1-Cyclooctyl-3-(2,3,4-trifluorophenyl)urea (7)
Yield: 371 mg (99%); Mp: 160–163 °C; 1H NMR (DMSO-d6): d
1.42–1.66 (13H, m), 1.76 (2H, t, J = 9.5 Hz), 6.63 (1H, d,
J = 8.0 Hz), 7.14–7.22 (1H, m), 7.84–7.93 (1H, m), 8.31 (1H, s);
ESI-HRMS m/z: [M+H]+ calcd: 301.1528, found: 301.1541.
4.2.17. 1-(2-Adamantyl)-3-propylurea (17)
Yield: 199 mg (84%); Mp: 194–196 °C; 1H NMR (DMSO-d6): d
0.84 (3H, t, J = 7.0 Hz), 1.37 (2H, sextuplet, J = 7.5 Hz), 1.49 (1H,
s), 1.52 (1H, s), 1.66–1.86 (12H, m), 2.94 (2H, q, J = 7.0 Hz), 3.66
(1H, d, J = 8.0 Hz), 5.81 (1H, t, J = 5.5 Hz), 6.02 (1H, d, J = 8.0 Hz);
ESI-HRMS m/z: [M+H]+ calcd: 237.1968, found: 237.1964.
4.2.8. 1-Cyclohexyl-3-(2,3,4-trifluorophenyl)urea (8)
Yield: 247 mg (63%); Mp: 188–191 °C; 1H NMR (DMSO-d6): d
1.30–1.28 (3H, m), 1.28–1.37 (2H, m), 1.48–1.57 (1H, m), 1.59–
1.70 (2H, m), 1.74–1.84 (2H, m), 3.42–3.52 (1H, m), 6.60 (1H, d,
J = 9.0 Hz), 7.15–7.23 (1H, m), 7.85–7.93 (1H, m), 8.33 (1H, s);
ESI-HRMS m/z: [M+H]+ calcd: 273.1215, found: 273.1203.
4.2.18. 1-(2-Adamantyl)-3-cyclohexylurea (18)
Yield: 234 mg (85%); Mp: >300 °C; 1H NMR (DMSO-d6): d 1.01–
1.32 (5H, m), 1.49 (1H, s), 1.52 (1H, s), 1.58–1.86 (17H, m), 3.65
(1H, d, J = 7.5 Hz), 5.75 (1H, d, J = 8.0 Hz), 5.98 (1H, d, J = 8.5 Hz);
ESI-HRMS m/z: [M+H]+ calcd: 277.2281, found: 277.2284.
4.2.9. 1-Cyclopentyl-3-(2,3,4-trifluorophenyl)urea (9)
Yield: 265 mg (71%); Mp: 184–186 °C; 1H NMR (DMSO-d6): d
1.30–1.43 (2H, m), 1.47–1.68 (4H, m), 1.77–1.89 (2H, m), 3.94
(1H, q, J = 6.5 Hz), 6.66 (1H, d, J = 7.5 Hz), 7.14–7.23 (1H, m),
7.84–7.94 (1H, m), 8.27 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
259.1059, found: 259.1046.
4.2.19. 1-(2-Adamantyl)-3-pentylurea (19)
Yield: 246 mg (93%); Mp: 151–153 °C; 1H NMR (CDCl3): d 0.93
(3H, t, J = 6.5 Hz), 1.30–1.39 (4H, m), 1.53 (2H, quintet, J = 7.5 Hz),
1.62 (2H, s), 1.76 (2H, s), 1.80 (1H, s), 1.86 (7H, t, J = 11.0 Hz),
1.93 (2H, s), 3.19 (2H, q, J = 7.0 Hz), 3.81 (1H, s), 4.26 (1H, s), 4.64
(1H, s); ESI-HRMS m/z: [M+H]+ calcd: 265.2281, found: 265.2297.
4.2.10. 1-(2-Adamantyl)-3-(4-cyanophenyl)urea (10)
Yield: 265 mg (90%); Mp: 217–219 °C; 1H NMR (DMSO-d6): d
1.57 (1H, s), 1.60 (1H, s), 1.68–1.89 (12H, m), 3.78 (1H, d,
J = 7.5 Hz), 6.67 (1H, d, J = 7.5 Hz), 7.55 (2H, d, J = 8.5 Hz), 7.67
(2H, d, J = 8.5 Hz), 8.92 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
296.1764, found: 296.1769.
4.2.20. 1-(2-Adamantyl)-3-hexylurea (20)
Yield: 200 mg (71%); Mp: 95–98 °C; 1H NMR (DMSO-d6): d 0.87
(3H, t, J = 6.5 Hz), 1.21–1.39 (8H, m), 1.49 (1H, s), 1.52 (1H, s), 1.66–
1.86 (12H, m), 2.97 (2H, q, J = 6.5 Hz), 3.65 (1H, d, J = 8.0 Hz), 5.78
(1H, t, J = 5.5 Hz), 6.01 (1H, d, J = 8.5 Hz); ESI-HRMS m/z: [M+H]+
calcd: 279.2437, found: 279.2443.
4.2.11. 1-(2-Adamantyl)-3-phenethylurea (11)
Yield: 229 mg (77%); Mp: 115–117 °C; 1H NMR (DMSO-d6): d
1.49 (1H, s), 1.51 (1H, s), 1.65–1.86 (12H, m), 2.68 (2H, t,
J = 7.0 Hz), 3.24 (2H, q, J = 7.0 Hz), 3.67 (1 H, d, J = 8.0 Hz), 5.82
(1H, t, J = 5.5 Hz), 6.11 (1H, d, J = 8.5 Hz), 7.21 (3H, d, J = 6.0 Hz),
7.30 (2H, t, J = 7.5 Hz); ESI-HRMS m/z: [M+H]+ calcd: 299.2124,
found: 299.2128.
4.2.21. 1-(2-Adamantyl)-3-heptylurea (21)
Yield: 292 mg (85%); Mp: 83–86 °C; 1H NMR (CDCl3): d 0.90
(3H, t, J = 6.0 Hz), 1.24–1.38 (8H, m), 1.52 (2H, t, J = 6.5 Hz), 1.48–
1.56 (2H, m), 1.75 (2H, s), 1.77 (1H, s), 1.86 (7H, t, J = 11.0 Hz),
1.93 (2H, s), 3.18 (2H, q, J = 6.5 Hz), 3.81 (1H, s), 4.34 (1H, s),
4.70 (1H, s); ESI-HRMS m/z: [M+H]+ calcd: 293.2594, found:
293.2582.
4.2.12. 1-(3-Acetylphenyl)-3-(2-adamantyl)urea (12)
Yield: 212 mg (72%); Mp: 213–215 °C; 1H NMR (DMSO-d6): d
1.57 (1H, s), 1.60 (1H, s), 1.69–1.91 (13H, m), 2.55 (3H, s), 3.79
(1H, d, J = 7.5 Hz), 6.50 (1H, d, J = 7.5 Hz), 7.38 (1H, t, J = 8.0 Hz),
7.50 (1H, d, J = 8.0 Hz), 7.58 (1H, d, J = 8.0 Hz), 8.00 (1H, s); ESI-
HRMS m/z: [M+H]+ calcd: 313.1917, found: 313.1909.
4.2.22. 1-(1-(1-Adamantyl)methyl)-3-(3-chloro-4-methyl-
phenyl)urea (22)
Yield: 301 mg (91%); Mp: 183–185 °C; 1H NMR (DMSO-d6): d
1.45 (5H, s), 1.56–1.76 (7H, m), 1.95 (3H, s), 2.23 (3H, s), 2.79
(2H, d, J = 6.0 Hz), 6.30–6.38 (1H, m), 7.04–7.10 (1H, m), 7.17
(1H, d, J = 8.0 Hz), 7.65–7.71 (1H, m), 8.46 (1H, s); ESI-HRMS m/z:
[M+H]+ calcd: 333.1734, found: 333.1730.
4.2.13. 1-(2-Adamantyl)-3-benzylurea (13)
Yield: 234 mg (81%); Mp: 205–207 °C; 1H NMR (DMSO-d6): d
1.51 (1H, s), 1.53 (1H, s), 1.66–1.88 (12H, m), 3.70 (1H, d,
J = 8.0 Hz), 4.21 (2H, d, J = 6.0 Hz), 6.18 (1H, d, J = 8.0 Hz), 6.27
(1H, t, J = 6.0 Hz), 7.24 (3H, q, J = 7.5 Hz), 7.33 (2H, t, J = 7.5 Hz);
ESI-HRMS m/z: [M+H]+ calcd: 285.1968, found: 285.1974.
4.2.23. 1-(3-Chloro-4-methylphenyl)-3-heptylurea (23)
Yield: 266 mg (90%); Mp: 99–101 °C; 1H NMR (CD3OD): d 0.93
(3H, t, J = 6.5 Hz), 1.29–1.42 (8H, m), 1.50–1.58 (2H, m), 2.30 (3H,
s), 3.19 (2H, t, J = 6.5 Hz), 7.09–7.17 (2H, m), 7.53 (1H, s); ESI-HRMS
m/z: [M+H]+ calcd: 283.1578, found: 283.1566.
4.2.14. 1-(2-Adamantyl)-3-(3-chloro-2-methylphenyl)urea (14)
Yield: 261 mg (82%); Mp: 254–257 °C; 1H NMR (DMSO-d6): d
1.57 (1H, s), 1.59 (1H, s), 1.70–1.86 (10H, m), 1.91 (2H, d,
J = 12.5 Hz), 2.26 (3H, s), 3.79 (1H, d, J = 7.5 Hz), 6.91 (1H, d,
J = 8.0 Hz), 7.03 (1H, d, J = 7.5 Hz), 7.10 (1H, t, J = 8.0 Hz), 7.84–
7.91 (2H, m); ESI-HRMS m/z: [M+H]+ calcd: 319.1578, found:
319.1569.
4.2.24. 1-(3-Chloro-4-methylphenyl)-3-cyclooctylurea (24)
Yield: 277 mg (94%); Mp: 179–182 °C; 1H NMR (DMSO-d6): d
1.42–1.68 (14H, m), 2.23 (3H, s), 3.65–3.73 (1H, m), 6.13 (1H, d,
J = 8.0 Hz), 7.05 (1H, d, J = 8.0 Hz), 7.16 (1H, d, J = 8.0 Hz), 7.65
(1H, s), 8.35 (1H, s); ESI-HRMS m/z: [M+H]+ calcd: 295.1578, found:
295.1555.
4.2.15. 1-(2-Adamantyl)-3-isopropylurea (15)
Yield: 162 mg (69%); Mp: 240–242 °C; 1H NMR (DMSO-d6): d
1.02 (6H, d, J = 6.5 Hz), 1.49 (1H, s), 1.52 (1H, s), 1.66–1.85 (12H,
m), 3.59–3.68 (2H, m), 5.66 (1H, d, J = 7.5 Hz), 5.95 (1H, d,
J = 8.0 Hz); ESI-HRMS m/z: [M+H]+ calcd: 237.1968, found:
237.1964.
4.2.25. 1-(3-Chloro-4-methylphenyl)-3-(3-fluorobenzyl)urea
(25)
Yield: 235 mg (80%); Mp: 146–148 °C; 1H NMR (CD3OD): d 2.30
(3H, s), 4.40 (2H, s), 7.00 (1H, t, J = 8.5 Hz), 7.08 (1H, d, J = 10.0 Hz),

J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
5593
7.12–7.19 (3H, m), 7.35 (1H, q, J = 6.5 Hz), 7.54 (1H, s); ESI-HRMS
m/z: [M+H]+ calcd: 293.0858, found: 293.0864.
4.2.35. 1-(1-Adamantyl)-3-(2-isopropoxyethyl)urea (39)
Yield: 244 mg (87%); Mp: 92–95 °C; 1H NMR (CDCl3): d 1.18
(6H, d, J = 6.0 Hz), 1.68 (6H, s), 1.98 (6H, s), 2.08 (3H, s), 3.30 (2H,
q, J = 5.5 Hz), 3.50 (2H, t, J = 5 Hz), 3.61 (1H, m), 4.50 (1H, s), 4.61
(1H, s); ESI-HRMS m/z: [M+H]+ calcd: 281.2230, found: 281.2228.
4.2.26. 1-(3-Chloro-4-methylphenyl)-3-(4-phenylbutan-2-yl)-
urea (26)
Yield: 245 mg (78%); Mp: 111–113 °C; 1H NMR (CD3OD): d 1.20
(3H, d, J = 6.5 Hz), 1.77 (2H, q, J = 7.0 Hz), 2.29 (3H, s), 2.62–2.76
(2H, m), 3.78–3.86 (1H, m), 7.08–7.31 (7H, m), 7.54 (1H, s); ESI-
HRMS m/z: [M+H]+ calcd: 317.1421, found: 317.1401.
4.2.36. 1-(1-Adamantyl)-3-(2-propoxyethyl)urea (40)
Yield: 271 mg (97%); Mp: 87–90 °C; 1H NMR (CDCl3): d 0.95
(3H, t, J = 7.5 Hz), 1.58–1.66 (2H, m), 1.69 (6H, s), 1.98 (6H, s),
2.09 (3H, s), 3.33 (2H, q, J = 5.0 Hz), 3.42 (2H, t, J = 6.5 Hz), 3.50
(2H, t, J = 5.0 Hz), 4.39 (1H, s), 4.54 (1H, s); ESI-HRMS m/z:
[M+H]+ calcd: 281.2230, found: 281.2228.
4.2.27. 1-(2-Fluoro-3-(trifluoromethyl)phenyl)-3-heptylurea
(27)
Yield: 385 mg (87%); Mp: 77–79 °C; 1H NMR (DMSO-d6): d 0.86
(3H, t, J = 6.5 Hz), 1.20–1.33 (8H, m), 1.43 (2H, t, J = 6.5 Hz), 3.10
(2H, q, J = 6.5 Hz), 6.67 (1H, t, J = 5.5 Hz), 7.23–7.32 (2H, m), 8.42
(1H, t, J = 7.0 Hz), 8.55 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
321.1591, found: 321.1581.
4.3. 1-(2-Adamantyl)-3-(2,3,4-trifluorophenyl)thiourea (31)
In a round bottom flask equipped with a stir bar, 2-adamantan-
amine (0.24 g, 1.3 mmol) was dissolved in 10 mL of anhydrous
dichloromethane. To this solution, 2,3,4-trifluorophenyl isothiocy-
anate (0.14 mL, 1.1 mmol) followed by triethylamine (0.53 mL,
3.8 mmol) was added. The reaction was stirred at room tempera-
ture overnight. The solvent was removed by rotary evaporation.
The resulting residue was purified by flash chromatography using
a petroleum ether to ethyl acetate gradient to elute the final com-
pound (330 mg, 92%). Mp: 176–179 °C; 1H NMR (DMSO-d6): d 1.61
(1H, s), 1.64 (1H, s), 1.67–1.83 (10H, m), 2.01 (2H, s), 4.33 (1H, s),
7.23–7.31 (1H, m), 7.66 (1H, s), 8.19 (1H, s), 9.30 (1H, s); ESI-HRMS
m/z: [M+H]+ calcd: 341.1300, found: 341.1314.
4.2.28. 1-Cyclooctyl-3-(2-fluoro-3-(trifluoromethyl)phenyl)urea
(28)
Yield: 442 mg (96%); Mp: 127–129 °C; 1H NMR (CDCl3): d 1.54–
1.79 (14H, m), 1.85–1.94 (2H, m), 3.82–3.90 (1H, m), 7.25 (2H, d),
8.33–8.40 (1H, m); ESI-HRMS m/z: [M+H]+ calcd: 333.1591, found:
333.1580.
4.2.29. 1-(2-Fluoro-3-(trifluoromethyl)phenyl)-3-(4-phenyl-
butan-2-yl)urea (29)
Yield: 463 mg (95%); Mp: 145–148 °C; 1H NMR (DMSO-d6): d
1.13 (3H, d, J = 6.5 Hz), 1.71 (2H, d, J = 7.0 Hz), 2.62 (2H, d,
J = 7.5 Hz), 3.64–3.74 (1H, m), 6.68 (1H, d, J = 7.0 Hz), 7.13–7.32
(7H, m), 8.40–8.52 (2H, m); ESI-HRMS m/z: [M+H]+ calcd:
355.1434, found: 355.1419.
4.4. 2-Adamantyl 2,3,4-trifluorophenylcarbamate (32)
In a round bottom flask equipped with a stir bar, 2-adamantanol
(0.42 g, 2.8 mmol) was dissolved in 10 mL of anhydrous dichloro-
methane. To this solution, 2,3,4-trifluorophenyl isocyanate
(2.8 mL, 2.3 mmol) followed by triethylamine (1.2 mL, 8.3 mmol)
was added. The reaction was stirred at room temperature over-
night. The solvent was removed by rotary evaporation. The result-
4.2.30. 1-(3-Chlorobenzyl)-3-(2-fluoro-3-(trifluoromethyl)-
phenyl)urea (30)
Yield: 451 mg (94%); Mp: 152–153 °C; 1H NMR (DMSO-d6): d
4.35 (2H, d, J = 6.0 Hz), 7.22 (1H, t, J = 5.5 Hz), 7.26–7.35 (4H, m),
7.36–7.41 (2H, m), 8.40–8.46 (1H, m), 8.76 (1H, s); ESI-HRMS m/
z: [M+H]+ calcd: 347.0575, found: 347.0589.
ing residue was purified by flash chromatography using
a
petroleum ether to ethyl acetate gradient to elute the final com-
pound (727 mg, 97%). Mp: 114–116 °C; 1H NMR (DMSO-d6): d
1.52 (1H, s), 1.56 (1H, s), 1.67–1.90 (10H, m), 2.00 (1H, s), 4.78
(1H, s), 7.22–7.35 (1H, m), 7.38–7.51 (1H, m), 9.46 (1H, s); ESI-
MS m/z: 348.1 [M+Na]+.
4.2.31. 1-(2-Adamantyl)-3-(4-methoxyphenyl)urea (35)
Yield: 250 mg (83%); Mp: 125–128 °C; 1H NMR (DMSO-d6): d
1.56 (1H, s), 1.58 (1H, s), 1.68–1.90 (12H, m), 3.70 (3H, s), 3.76
(1H, d, J = 8.0 Hz), 6.34 (1H, d, J = 8.0 Hz), 6.82 (2H, d, J = 8.5 Hz),
7.28 (2H, d, J = 9.0 Hz), 8.20 (1H, s); ESI-HRMS m/z: [M+H]+ calcd:
301.1917, found: 301.1932.
4.5. 1-(2-Adamantyl)-3-methyl-3-(2,3,4-trifluorophenyl)urea
(33)
To a round bottom flask containing 1 (0.20 g, 0.62 mmol), anhy-
drous THF (10 mL) was added under argon. The solution was then
cooled to ꢁ78 °C. To the cold solution, n-butyl lithium (0.39 mL,
0.62 mmol) was added drop wise and allowed to stir at ꢁ78 °C
for 10 min. To this solution, iodomethane (0.038 mL, 0.62 mmol)
was added drop wise at ꢁ78 °C. The solution was then allowed
to warm to room temperature and then refluxed for 24 h. The sol-
vent was then removed by rotary evaporation. The resulting resi-
due was purified by flash chromatography using a petroleum
ether to ethyl acetate gradient to elute the final compound as an
oil (192 mg, 92%). 1H NMR (DMSO-d6): d 1.44 (1H, s), 1.47 (1H,
s), 1.65–1.84 (10H, m), 1.86 (2H, s), 3.14 (3H, s), 370 (1H, s), 5.62
(1H, d, J = 5.5 Hz), 7.26–7.31 (1H, m), 7.31–7.38 (1H, m); ESI-HRMS
m/z: [M+H]+ calcd: 339.1685, found: 339.1670.
4.2.32. Methyl 4-(3-(1-adamantyl)ureido)-2-hydroxybenzoate
(36)
Yield: 850 mg (41%); Mp: 207–210 °C; 1H NMR (CDCl3): d 1.68
(6H, s), 2.02–2.16 (9H, m), 3.90 (3H, s), 4.73 (1H, s), 6.62 (1H, s),
6.83 (1H, s), 6.98 (1H, d, J = 9 Hz), 7.70 (1H, d, J = 8.5 Hz),
10.83 (1H, s); ESI-HRMS m/z: [M+H]+ calcd: 345.1815, found:
345.1801.
4.2.33. 1-(1-Adamantyl)-3-(2-ethoxyethyl)urea (37)
Yield: 249 mg (94%); Mp: 116–118 °C; 1H NMR (CDCl3): d 1.23
(3H, t, J = 7.0 Hz), 1.69 (6H, s), 1.98 (6H, s), 2.09 (3H, s), 3.33 (2H,
q, J = 5.0 Hz), 3.49–3.56 (4H, m), 4.41 (1H, s), 4.56 (1H, s); ESI-
HRMS m/z: [M+H]+ calcd: 267.2073, found: 267.2083.
4.2.34. 1-(1-Adamantyl)-3-(3-methoxypropyl)urea (38)
Yield: 251 mg (94%); Mp: 135–137 °C; 1H NMR (CDCl3): d 1.69
(6H, s), 1.77 (2H, quintet, J = 6.0 Hz), 1.98 (6H, s), 2.09 (3H, s),
3.25 (2H, q, J = 6.0 Hz), 3.36 (3H, s), 3.49 (2H, t, J = 5.5 Hz); ESI-
HRMS m/z: [M+H]+ calcd: 267.2073, found: 267.2083.
4.6. 1-(2-Adamantyl)-1,3-dimethyl-3-(2,3,4-trifluorophenyl)-
urea (34)
To a round bottom flask containing 1 (0.40 g, 1.2 mmol), anhy-
drous THF (25 mL) was added under argon. The solution was then

5594
J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
cooled to ꢁ78 °C. To the cold solution, n-butyl lithium (1.5 mL,
2.5 mmol) was added drop wise and allowed to stir at ꢁ78 °C for
10 min. To this solution, iodomethane (0.15 mL, 2.5 mmol) was
added drop wise at ꢁ78 °C. The solution was then allowed to warm
to room temperature and then refluxed for 24 h. The solvent was
then removed by rotary evaporation. The resulting residue was
purified by flash chromatography using a petroleum ether to ethyl
acetate gradient to elute the final compound (190 mg, 44%). Mp:
71–74 °C; 1H NMR (DMSO-d6): d 1.50 (1H, s), 1.52 (1H, s), 1.66–
1.84 (10 H, m), 2.18 (2H, s), 2.66 (3H, s), 3.11 (3H, s), 3.61 (1H,
s), 7.20–7.26 (1H, m), 7.34–7.41 (1H, m); ESI-HRMS m/z: [M+H]+
calcd: 353.1841, found: 353.1837.
of the aniline was slowly added into the solution of thiophosgene
under argon and allowed to stir overnight at room temperature.
The product (683 mg, 50%) crashed out of solution and was filtered
off and washed with dichloromethane. Mp: 139–141 °C; 1H NMR
(DMSO-d6): d 0.94 (12H, d, J = 7.0 Hz), 1.61 (4H, q, J = 7.0 Hz),
1.74–1.84 (2H, m), 3.98 (4H, t, J = 7.0 Hz), 6.90 (4H, d, J = 9.0 Hz),
7.10 (4H, d, J = 9.0 Hz), 9.45 (2H, s); ESI-MS m/z: 423.2 [M+Na]+.
4.9. MIC determination
MIC values were determined against M. tuberculosis (H37Rv)
and other bacteria by the microbroth dilution method.21 A broth
culture of M. tuberculosis or M. smegmatis mc2155 was grown in
Middlebrook 7H9 medium with 10% ADC supplement to an
OD600 of 0.4–0.6. The culture was diluted with 7H9 medium to
4.7. 1,3-Bis(4-(isopentyloxy)phenyl)urea (41)
To a round-bottom flask, 4-nitrophenol (2.0 g, 14 mmol) and
sodium hydroxide (1.7 g, 43 mmol), was dissolved in N,N-dimeth-
ylformamide (50 mL). The solution was stirred at room tempera-
ture for 15 min. After stirring, 1-bromo-4-methylpentane (5.4 mL,
43 mmol) was added to the solution and refluxed for 3 h. The
resulting solution was washed with 0.7 N HCl (3 ꢂ 20 mL) then ex-
tracted with diethyl ether (3 ꢂ 20 mL). The combined organic frac-
tions were then washed with brine (3 ꢂ 20 mL) and dried over
magnesium sulfate. The solution was then filtered and the solvent
by using rotary evaporation. The crude mixture was then purified
by flash chromatography using a petroleum ether to ethyl acetate
gradient to obtain pure intermediate (2.69 g, 90%). The intermedi-
ate nitro compound was then converted to an aniline by hydroge-
nation using Pd/C catalyst overnight. The aniline was used in the
next step without further purification (1.85 g, 80%). In a round bot-
tom flask, the aniline (1.27 g, 7.08 mmol) was dissolved in dichlo-
romethane and then triethylamine (2.0 mL, 14 mmol) was added
and the solution was allowed to stir at room temperature for
15 min. In a separate round bottom flask, triphosgene (0.70 g,
2.4 mmol) was dissolved in dichloromethane. Then the solution
of the aniline was slowly added into the solution of thiophosgene
under argon and allowed to stir overnight at room temperature.
The product (301 mg, 33%) crashed out of solution and was filtered
off and washed with dichloromethane. Mp: 173–175 °C; 1H NMR
(DMSO-d6): d 0.93 (12H, d, J = 6.5 Hz), 1.59 (4H, q, J = 6.5 Hz),
1.72–1.82 (2H, m), 3.94 (4H, t, J = 6.5 Hz), 6.85 (4H, d, J = 9.0 Hz),
7.31 (4H, d, J = 9.0 Hz), 8.35 (2H, s); ESI-HRMS m/z: [M+H]+ calcd:
385.2492, found: 385.2501.
an OD600 of 0.01, and 100
taining twofold serial dilutions of the tested compounds for a final
volume of 200 L. The plates were incubated at 37 C for 7 days. The
MIC90 was determined by visual inspection and defined as the con-
centration that inhibited 90% of growth. MICs against other bacte-
ria were performed according to the CLSI method and also read by
visual inspection.21,22
lL was added to a microtiter plate con-
l
4.10. Cytotoxicity study
Cytotoxicity assays were performed using the Vero monkey kid-
ney cell line (CCL-81) obtained from the American Type Culture
Collection (ATCC, Manassas, VA). Vero cells were propagated in
Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal
bovine serum and maintained in a humidified incubator (37 °C, 5%
CO2). After dislodging cells with a cell scraper, they were collected
by centrifugation, resuspended in fresh medium at ꢀ106 cells/mL,
dispensed into 96-well microtiter plates (100 ll/well) and incu-
bated for 18 h at 37 °C before being used for cytotoxicity assays.
Test compounds were subsequently added at concentrations rang-
ing from 400 to 0.2 lg/mL and incubation continued for another
72 h before the cytopathic effects of compounds was determined
using the MTT Cell Proliferation Assay (ATCC). The cytotoxic IC50
defined as the concentration causing 50% reduction in Vero cell
viability, was obtained from a dose–response curve plotted from
percentage activity versus log10 concentration. Therapeutic selec-
tivity was then determined as the Cytotoxic IC50 divided by the
MIC against M. tuberculosis.
4.8. 1,3-Bis(4-(isopentyloxy)phenyl)thiourea (isoxyl) (42)
4.11. EphB and EphE inhibition determination
To a round-bottom flask, 4-nitrophenol (2.0 g, 14 mmol) and
sodium hydroxide (1.7 g, 43 mmol), was dissolved in N,N-dimeth-
ylformamide (50 mL). The solution was stirred at room tempera-
ture for 15 min. After stirring, 1-bromo-4-methylpentane (5.4 mL,
43 mmol) was added to the solution and refluxed for 3 h. The
resulting solution was washed with 0.7 N HCl (3 ꢂ 20 mL) then ex-
tracted with diethyl ether (3 ꢂ 20 mL). The combined organic frac-
tions were then washed with brine (3 ꢂ 20 mL) and dried over
magnesium sulfate. The solution was then filtered and the solvent
by using rotary evaporation. The crude mixture was then purified
by flash chromatography using a petroleum ether to ethyl acetate
gradient to obtain pure intermediate (2.69 g, 90%). The intermedi-
ate nitro compound was then converted to an aniline by hydroge-
nation using Pd/C catalyst overnight. The aniline was used in the
next step without further purification (1.85 g, 80%). In a round bot-
tom flask, the aniline (1.9 g, 11 mmol) was dissolved in dichloro-
methane and then triethylamine (2.9 mL, 21 mmol) was added
and the solution was allowed to stir at room temperature for
15 min. In a separate round bottom flask, thiophosgene (0.26 mL,
3.4 mmol) was dissolved in dichloromethane. Then the solution
The fluorescent assay used to measure the inhibition of EphB
(Rv1938) and EphE (Rv3670) by urea derivatives was similar to
that described by Biswal and colleagues and used the fluorescent
substrate cyano(6-methoxy-2-napthalenyl)methyl[(2,3)-3-pheny-
loxiranyl]methyl ester carbonate (Epoxy Fluor 7 from Cayman
Chemical, Ann Arbor, MI).6 Briefly, the purified EphB protein
(0.8 l
g per well; [E] = 106 nM)6 or crude extracts prepared from
E. coli BL21AI/pGEFII-EphE expressing ephE (a kind gift from Drs.
M. Arand and A. Cronin, University of Zurich, Switzerland)
(10 lg; estimated [E] = 10–15 nM) were pre-incubated with inhib-
itors ([I] = 10 nM in 1% DMSO final concentration) for 10 min in
Bis–Tris–HCl buffer (25 mM, pH 7.0, containing 0.1 mg/ml of bo-
vine serum albumin) at 30 °C prior to substrate introduction
([S] = 5 lM). All assays were run for 30 min at 30 °C and performed
in duplicate. Hydrolysis of the substrate epoxide was monitored at
excitation and emission wavelengths of 320 and 460 nm, respec-
tively. Results are expressed as percentage inhibition of the control
reaction containing no inhibitor. E. coli BL21AI extracts not
expressing EphE showed no activity under the assay conditions
used here.

J. R. Brown et al. / Bioorg. Med. Chem. 19 (2011) 5585–5595
5595
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4. Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova, O.; Saint-Joanis,
B.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli, A. P.; Milano, A.; De Rossi, E.;
Belanova, M.; Bobovska, A.; Dianiskova, P.; Kordulakova, J.; Sala, C.; Fullam, E.;
Schneider, P.; McKinney, J. D.; Brodin, P.; Christophe, T.; Waddell, S.; Butcher,
P.; Albrethsen, J.; Rosenkrands, I.; Brosch, R.; Nandi, V.; Bharath, S.; Gaonkar, S.;
Shandil, R. K.; Balasubramanian, V.; Balganesh, T.; Tyagi, S.; Grosset, J.; Riccardi,
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Lenaerts, A. J.; Slayden, R. A.; McNeil, M. R.; Lee, R. E. Bioorg. Med. Chem. 2009,
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IC50 values were determined using a sensitive fluorescent based
assay.16 Cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phe-
nyl-oxyran-2-yl) methyl carbonate (CMNPC) was used as the fluo-
rescent substrate. Purified recombinant Human sEH (1 nM)23 was
incubated with the inhibitor for 5 min in pH 7.4 sodium phosphate
buffer (100 mM) containing 0.1 mg/mL of BSA at 30 °C prior to sub-
strate introduction ([S] = 5 lM). Activity was determined by mon-
itoring the appearance of 6-methoxy-2-naphthaldehyde over
10 min by fluorescence detection with an excitation wavelength
of 330 nm and an emission wavelength of 465 nm. Reported IC50
values are the average of three replicates with at least two datum
points above and at least two below the IC50. The fluorescent assay
as performed here has a standard error between 10% and 20%, sug-
gesting that differences of twofold or greater are significant.
6. Biswal, B. K.; Morisseau, C.; Garen, G.; Cherney, M. M.; Garen, C.; Niu, C.;
Hammock, B. D.; James, M. N. J. Mol. Biol. 2008, 381, 897.
7. Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 311.
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Sect. F Struct. Biol. Crystallogr. Commun. 2006, 62, 136.
9. Silver, L. L. Nat. Rev. Drug Disc. 2007, 6, 41.
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Laughon, B. E.; Maddry, J. A.; Mehta, A.; Rasmussen, L.; Reynolds, R. C.; Secrist, J.
A., 3rd; Shindo, N.; Showe, D. N.; Sosa, M. I.; Suling, W. J.; White, E. L.
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Acknowledgments
12. Phetsuksiri, B.; Jackson, M.; Scherman, H.; McNeil, M.; Besra, G. S.; Baulard, A.
R.; Slayden, R. A.; DeBarber, A. E.; Barry, C. E., 3rd; Baird, M. S.; Crick, D. C.;
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Reynolds, R. C.; Coxon, G. D.; Kremer, L. PLoS One 2007, 2, e1343.
14. Knölker, H.-J.; Wolpert, M. Tetrahedron 2003, 59, 5317.
We thank National Institutes of Health grants AI057836,
AI063054, RC1AI85992-01 and the American Lebanese Syrian
Associated Charities (ALSAC) for financial support.
15. Kasagami, T.; Kim, I.-H.; Tsai, H.-J.; Nishi, K.; Hammock, B. D.; Morisseau, C.
Bioorg. Med. Chem. Lett. 2009, 19, 1784.
Supplementary data
16. Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock, B. D.
Anal. Biochem. 2005, 343, 66.
17. Lee, R. E.; Protopopova, M.; Crooks, E.; Slayden, R. A.; Terrot, M.; Barry, C. E., 3rd
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Supplementary data (RP-HPLC purity and retention times and
MIC data for non-tubercular antibacterial testing) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.07.034.
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