Design, synthesis and anti-tuberculosis activity of 1-adamantyl-3- heteroaryl ureas with improved in vitro pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmc.2013.02.028

Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub μg/mL minimum inhibitory concentrations.

Full text of DOI:10.1016/j.bmc.2013.02.028

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and anti-tuberculosis activity
of 1-adamantyl-3-heteroaryl ureas with improved
in vitro pharmacokinetic properties
E. Jeffrey North a, Michael S. Scherman b, David F. Bruhn a, Jerrod S. Scarborough a, Marcus M. Maddox a,
Victoria Jones b, Anna Grzegorzewicz b, Lei Yang a, Tamara Hess b, Christophe Morisseau c, Mary Jackson b,
Michael R. McNeil b, 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 Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
c Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death world-
wide due to infectious disease. Development of new drugs that shorten the current tuberculosis treat-
ment time and have activity against drug resistant strains is of utmost importance. Towards these
goals we have focused our efforts on developing novel anti-TB compounds with the general structure
of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds
were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid trans-
port across the plasma membrane. However, these compounds suffered from poor in vitro pharmacoki-
netic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase
(sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by
three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize
PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-het-
eroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with
pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced
lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles,
Received 31 December 2012
Revised 6 February 2013
Accepted 14 February 2013
Available online xxxx
Keywords:
Tuberculosis
Antimicrobial agents
Transporter
Antibiotics
Medicinal chemistry
MmpL3
increased selectivity and good anti-TB potencies with sub
lg/mL minimum inhibitory concentrations.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and multiple M. tb epoxide hydrolases including EphB2,4 and EphE.2
However, M. tb epoxide hydrolases are individually non-essential.
In attempts to rationalize the primary target for anti-tuberculosis
activity, we used genetic approaches by generating and sequencing
resistant mutants for 1. We identified a further and essential target
for this compound series, the membrane transporter Mmpl3,
which is believed to play a major role in exporting mycolates to
mycobacterial cell surface.5 Although our first generation adaman-
tyl ureas possessed potent anti-TB activity,2 they had two principal
issues: (i) they were highly hydrophobic and consequently had
poor solubility and high human plasma protein binding (HPPB);
(ii) though while highly selective with respect to anti-TB activity
compared to cytotoxicity and activity against other bacteria, these
compounds still had potent human soluble epoxide hydrolase
(sEH) activity which may not be pharmacologically desirable
(Fig. 1).2 Thus the purpose of this study is to design and develop
analogs that maintain anti-tuberculosis activity but have improved
pharmacokinetic (PK) properties, most notably solubility and
selectivity away from human sEH inhibition. In this study we used
Mycobacterium tuberculosis (M. tb) is the pathogen responsible
for the infectious disease tuberculosis (TB) which still today is
responsible for a vast number of deaths globally.1 Increasing rates
of resistance and inadequacies of current TB chemotherapeutics
necessitate the need for new drugs which can potentially shorten
the drug treatment, increase patient compliance and have activity
against drug resistant strains including multi-drug-resistant
(MDR) and extremely drug-resistant (XDR) tuberculosis.
Previously we developed a series of 1-adamantyl-3-phenyl ur-
eas 1–6 that had potent anti-tuberculosis activity, however they
had undesirable pharmaceutical properties, particularly high lipo-
philicity and poor solubility (Fig. 1).2 The biochemical basis for
their activity is complex, as it was shown that these compounds
inhibited mammalian soluble epoxide hydrolase (sEH) inhibitors3
⇑
Corresponding author. Tel.: +1 (901) 595 6617; fax: +1 (901) 595 5715.
E-mail address: Richard.lee@stjude.org (R.E. Lee).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.028
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

2
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
F
F
H
N
H
N
F
F
O
O
F
N
H
N
H
F
N
H
N
H
O
F
F
F
1
2
3
MIC = 0.01
µg/mL
MIC = 0.4µg/mL
MIC = 0.02 µg/mL
Human sEH IC50 = 0.4 nM Human sEH IC50 = 0.4 nM Human sEH IC50 = 0.4 nM
ClogP = 5.1 ClogP = 4.0 ClogP = 5.2
Solubility < 0.1 Solubility < 0.1
HPPB = 98.8%
µ
g/mL
Solubility < 0.1
µ
g/mL
Cl
µg/mL
HPPB = 99.2%
O
H
N
H
N
O
O
O
O
N
H
N
H
N
H
N
H
OH
N
4
5
6
MIC = 0.4 µg/mL
Human sEH IC50 = 1.1 nM
MIC = 0.01 µg/mL
Human sEH IC50 = 0.4 nM
MIC = 0.4 µg/mL
Human sEH IC50 = 1.2 nM
ClogP = 4.9
ClogP = 5.8
ClogP = 4.1
Solubility < 0.1
µg/mL
Solubility < 0.1
µg/mL
Solubility = 0.51
µg/mL
HPPB = 99.0%
HPPB = 99.7%
HPPB = 96.9%
Figure 1. Previously synthesized adamantyl–phenyl ureas 1–6 with M. tb H37Rv MIC values, human sEH IC50 values, cLogP values, solubility and human protein plasma
binding (HPPB).2
rational bioisosteric replacements for the phenyl group of the first
generation urea in six miniseries (arylsulfonamides, pyridines,
isoxazoles, thiazoles, oxadiazoles and pyrazoles) in order to
increase polarity, which should help increase solubility and lower
HPPB, while maintaining good anti-tuberculosis activity and
potentially decreasing affinity to human sEH.
ities are not tolerated well by the molecular target or for tubercular
entry and are thus detrimental to anti-TB activity. This observation
was further validated with 13, which did possess anti-TB activity
(6.25 lg/mL) and contained a non-acidic sulfone rather than an
acidic aryl sulfonamide found in the other compounds in this
series.
2. Results and discussion
2.1.2. Series 2 – N-substituted heterocycles
Another common approach to increase solubility of phenyl
rings is the introduction of a heteroatoms resulting in bioisosteric
rings such as pyridyl, which may also have the benefit of being
more resistant to cytochrome mediated metabolism.11,12 Thus,
pyridine and pyridine analogs (pyrimidine and triazine) were
introduced into the adamantyl ureas according to Scheme 2. Prede-
termining the cLogP values for each molecule ensured they stayed
below our initial lead compound 1 (cLogP = 5.08) as a computa-
tional correlate of solubility. Two different methods were em-
ployed to synthesize this series. First, 1-adamantyl isocyanate
and heteroarylamine were reacted in the presence of triethylamine
in THF. If after four days of heated stirring, little to no product was
detected by TLC, method B was used to synthesize the adamantyl
heteroaryl urea (Scheme 2). Method B utilized butyl lithium to
activate the heteroarylamine followed by addition of 1-adamantyl
isocyanate.
2.1. Chemistry and SAR of adamantyl–heteroaryl ureas
2.1.1. Series 1 – Sulfonamides
One approach to boost the solubility of this series is to intro-
duce an ionizable group in the para position of the phenyl ring
which has been previously shown to be tolerated.2,6 One such func-
tion group is found in sulfonamide antibiotics, which were histor-
ically optimized by the addition of an electron deficient outer ring
to ionize the sulfonamide functionality at physiological pH
(Fig. 2).7 Though the addition of an adamantyl urea to the aniline
position of the sulfonamides will block their nascent antimicrobial
activity through dihydropteroate synthase inhibition,8 incorpora-
tion of the sulfonamide scaffold to the urea scaffold appeared to
match the preexisting SAR or our anti-TB indication.2 Thus, the
adamantyl–phenylsulfonamide ureas were rapidly synthesized
using microwave heating at 200 °C for 10 min from common sul-
fonamide antibiotics and 1-adamantyl isocyanate in the presence
of triethylamine (Scheme 1).
All pyridines, pyrimidines and triazines had relatively poor anti-
TB activity with 15 (3-pyridinyl) having the most potent MIC at
12.5
l
g/mL (Table 2). Generally, this series had solubility values
g/mL, which was not as high as the average
As desired, all the adamantyl sulfonamides did have increased
solubility (10 to 100-fold) over the first generation adamantyl phe-
nyl ureas (Table 1). Despite having improved solubility (acceptable
at approximately 7
l
solubility of the sulfonamide series, but superior to the initial ada-
mantyl–phenyl leads 1–6. Anti-TB activity for the whole series was
solubility >10
l
g/mL), all the adamantyl sulfonamides, except 13,
inferior to 1–6 with the 3-pyridinyl (15, MIC = 12.5
ing the best activity which greatly decreased with the 2-pyridinyl
substitution (16, MIC = 200 g/mL). Introduction of a second nitro-
lg/mL) show-
had a large reduction in M. tb minimum inhibitory concentration
(MIC) compared to 1 (Table 1 and Fig. 1) using microbroth dilution
MIC method.9,10 This suggests that acidic sulfonamide functional-
l
gen in the 2-pyrimidinyl analog (17) did not prove effective for
O
O
O
S
O
N
O
S
O
N
Physiological pH
N
N
H
H2N
H2N
Figure 2. Representation of sulfonamide (sulfamethoxazole) isonization at physiological pH.
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
O
O
S
O
S
Ar
Ar
a
N
H
O
N
H
O
+
NCO
H2N
N
H
N
H
7-13
Scheme 1. Synthesis of 1-(1-adamantyl)-3-(benzenesulfonamide)ureas. Reagents and conditions: (a) TEA, THF/DMF (1:1), lw, 200 °C, 10 min.
Table 1
In vitro whole cell anti-TB activity, human sEH inhibition, solubility, and cLogP of 1-(1-adamantyl)-3-(4-(N-(heteroaryl)benzenesulfonamide))ureas
Compd #
Structure
aM. tb MIC (
lg/mL)
bHuman sEH IC50 (nM)
cSolubility (
lg/mL)
dcLogP
H
N
O
N
N
S
N
N
O
O
O
O
O
O
7
25
19
20.7 0.2
3.6
O
Cl
O
N
N
H
H
H
N
O
S
8
9
O
25
7
292
13
14.1 1.2
2.0 0.2
27.1 1.4
2.5 0.1
27.3 0.9
3.4
3.4
3.4
3.5
3.1
N
H
N
H
H
N
O
N
S
O
25
O
N
H
N
H
H
N
O
O
N
S
10
11
12
>25
>25
25
O
N
N
N
H
N
H
H
N
O
S
O
17
N
H
N
H
H
N
O
O
S
N
31
O
N
H
N
H
NH2
O
S
13
6.25
0.7
0.9 0.1
3.8
O
O
N
H
N
H
a
b
c
In vitro anti-TB activity against M. tb H37Rv.
In vitro IC50 values against recombinant human sEH (1 nM).
In vitro solubility of ureas in a physiological environment at pH 7.4.
d
In silico calculation of cLogP using ChemBioDraw Ultra 12.0.
activity at 25 lg/mL. Finally, introduction of a third nitrogen to
the ring abolished anti-TB activity completely.
Method A
O
a
In an attempt to further optimize the solubility of the adaman-
tyl–heteroaryl ureas and obtain potent MIC values, oxazole and
isoxazole rings were introduced in the adamantyl–heteroaryl urea
scaffold. These compounds were synthesized according to
Scheme 2 and summarized in Table 3. The unsubstituted oxazole,
R
R
+
R
N
H
N
H
NCO
H2N
Method B
21, had a more potent MIC value, 6.25
lg/mL, than the unsubstitut-
ed isoxazole, 22 (MIC = 50 g/mL). However, the isoxazole (22) had
l
O
b
R
approximately five-times the solubility of the oxazole (21). Intro-
duction of a methyl group to the 5-position of the isoxazole ring
in 23 increased MIC potency by almost twofold compared to 22.
Increasing the hydrophobicity of the methyl group in 23 to a t-bu-
tyl group in 24 increased anti-TB activity significantly from a MIC
N
H
N
H
H2N
Scheme 2. Synthesis of 1-(1-adamantyl)-3-(heteroaryl)urea. Reagents and condi-
tions: Method A: (a) triethylamine, THF, 70 °C, 4 days; Method B: (b) (i) butyl
lithium, THF, ꢀ78 °C, 20 min; (ii) 1-adamantyl isocyanate, THF, rt, 1 h.
of 27
l
g/mL to 1.56
lg/mL. However, solubility dropped by about
half from 25.9 1.5
l
g/mL with 23 to 11.2 1.6 g/mL with
l
anti-TB activity, although the 4-pyrimidinyl (18) did have the best
24. Anti-TB activity was abolished with introduction of a bromine
solubility of this series at 15.5 0.8 lg/mL with modest anti-TB
to the ‘pseudo-ortho’ 4-position of the isoxazole ring (25,
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

4
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(1-adamantyl)-3-pyridinylurea analogs
Compd #
Structure
M. tb MIC (
l
g/mL)
Human sEH IC50 (nM)
Solubility (
7.0 0.3
lg/mL)
cLogP
O
O
O
O
O
O
N
N
14
18
174
3.2
N
N
H
H
15
16
17
18
19
12.5
200
200
25
113
1498
24,010
775
6.7 1.5
1.4 0.2
6.9 1.5
15.5 0.8
7.2 0.8
3.2
3.2
2.6
2.6
1.8
N
H
N
H
N
H
N
H
N
N
N
N
N
H
N
H
N
N
N
H
N
H
N
N
125
79
N
H
N
H
N
N
N
O
20
200
1012
6.8 0.3
2.1
N
H
N
H
Table 3
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(1-adamantyl)-3-isoxazolylurea analogs
Compd #
Structure
M. tb MIC (lg/mL)
Human sEH IC50 (nM)
Solubility (lg/mL)
cLogP
O
O
O
O
O
O
N
N
N
N
N
21
6.25
948
4.4 0.1
2.4
O
O
N
N
H
H
22
23
24
50
46
136
56
20.3 1.9
25.9 1.5
11.2 1.6
2.7
3.0
4.3
N
H
N
H
O
O
O
27
N
H
N
H
1.56
N
H
N
H
25
26
100
>50
0.8
0.1 0.0
3.0 0.1
3.3
2.1
N
H
N
H
Br
N
N
H
N
H
1551
O
N
O
O
O
27
28
12.5
0.78
117
2
5.5 0.3
3.0
3.7
N
H
N
H
N
O
CF3
0.77 0.0
N
H
N
H
N
O
O
29
100
0.5
1.5 0.5
3.2
N
H
N
H
Cl
N
O
O
N
N
30
31
100
100
25
20
1.2 0.2
5.7 2.2
2.8
2.6
H
H
N
N
O
O
N
H
N
H
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
MIC = 100
functional group by
anti-TB activity and solubility (26, MIC >50
ity = 3.0 0.1 g/mL). Switching the oxygen and nitrogen in 23 to
the reverse isoxazole in 27 gained a twofold increase in anti-TB
activity, however, there was a fivefold reduction in solubility.
Increasing the hydrophobicity of the 3-position from methyl (27,
l
g/mL). Separating the isoxazole ring from the urea
methylene was detrimental to both
g/mL, solubil-
an MIC 1.56 lg/mL. Compound 42 achieved the highest selectivity
a
for M. tb activity over human sHE inhibition among the pyrazoles.
There was an inverse relationship between M. tb activity and solu-
bility, with 43 having the most potent anti-TB activity yet the poor-
est solubility and 44 having the opposite.
l
l
It has been observed in previous studies, that, though syntheti-
cally less accessible, attachment of the adamantyl group through
the adamantyl secondary 2-position, as opposed to the tertiary 1-
position, significantly increases anti-TB activity.2 This was seen in
comparing 1 and 2, where the secondary-linked adamantyl–phenyl
urea 1 is 40-fold more potent than 2, the tertiary-linked adaman-
tyl–phenyl urea (Fig. 1). Therefore, a series of adamantyl–hetero-
aryl urea analogs, linked through the secondary 2-position on
adamantane, using the best heteroaryl ring systems generated thus
far were designed. Subsequently, these compounds were synthe-
sized by sequentially reacting a heteroarylamine and 2-adamantyl-
amine with triphosgene in the presence of diisopropylethylamine
(Scheme 3 and Table 7). All of the secondary-linked adamantyl–het-
eroaryl urea analogs had increased or equal solubility compared to
the tertiary linked adamantyl–heteroaryl precursors, except for 50
and 52 which had decreased solubility. Encouragingly, the second-
ary-linked isoxazole 46 had both a 15-fold improvement in MIC
MIC = 12.5 lg/mL) to trifluoromethyl (28, MIC = 0.78 lg/mL) of
the reverse isoxazole also significantly increased anti-TB activity.
Similarly it was noted previously, introduction of chlorine (29), cy-
ano (30) or methyl (31) into the ‘pseudo-ortho’ 4-position also
abolished anti-TB activity in the reverse isoxazole series, each with
MIC = 100 lg/mL. Of note, the oxazole 21 made a significant and
desirable improvement with regards to selectivity for anti-TB
activity over human sEH activity.
A four compound thiazole series was synthesized to evaluate
the role of oxygen over sulfur (Table 4). A twofold increase in
anti-TB activity was observed from the oxazole 21
(MIC = 6.25 lg/mL), to the thiazole 32 (MIC = 3.13 lg/mL). Unlike
the isoxazole series, anti-TB activity did not increase significantly
with the introduction of hydrophobic methyl groups to the 4-
and 5-positions of the thiazole ring (33 and 34, respectively). How-
ever, introduction of a benzathiazole (35) significantly improved
(0.10 lg/mL) and 2-fold improvement in solubility (22.7 2.2 lg/
the MIC value to 0.78
nately, none of the thiazoles had acceptable solubility with 33 hav-
ing the highest at 5.0 0.3 g/mL.
In order to help increase the solubilitiy of the isoxazole series, a
more polar oxadiazole ring was evaluated (Table 5). A similar SAR
was observed, in that increasing substituent hydrophobicity in the
5-position of the oxadiazole ring increased anti-TB activity. This
l
g/mL from 3.13
l
g/mL with 32. Unfortu-
mL) over the previous tertiary-linked version (24). The contribution
to activity of the position of adamantyl linkage on 23, which has a
poor MIC value yet good solubility, was also evaluated. As expected,
the secondary-linked compound 47 achieved a fourfold improve-
l
ment in MIC (6.25
(MIC = 27 g/mL). Of benefit, the solubility went unchanged at
ꢁ25 g/mL. A slightly different SAR was observed for the oxadiaz-
lg/mL) over the tertiary-linked compound 23
l
l
was observed with 36 (MIC = 25
in the 5-position, and 37 (MIC = 1.56
group in the 5-position. However, solubility of 36 and 37 were
g/mL. Anti-TB activity was reduced
l
g/mL), having a methyl group
oles. As seen before with the tertiary-linked adamantyl–heteroaryl
ureas, constraining the isopropyl side chain to ring systems reduces
the MIC values. This is not the case with the secondary-linked ada-
mantyl–heteroaryl ureas, however. Anti-TB activity is slightly re-
lg/mL), having an isopropyl
essentially the same at ꢁ10
l
with the restriction of the isopropyl group to cyclic groups. There
duced when the isopropyl (49, MIC = 1.56
constrained to the cyclopropyl (50, MIC = 3.13
increased when expanded to the cyclobutyl (51, MIC = 0.78 l
l
g/mL) side chain is
g/mL), yet slightly
g/
was approximately an eightfold reduction for both cyclopropyl
l
(38, MIC = 12.5
l
g/mL) and cyclobutyl (39, MIC = 12.5
lg/mL) and
64-fold reduction for thiophenyl (40, MIC = 100
l
g/mL) compared
mL). Many compounds in the secondary-linked series, particularly
oxadizoles 49 and 50 and pyrazoles 52 and 53, achieved the highest
selectivity for anti-TB activity versus sEH inhibition compared to all
other compounds studied in this series.
to 37. Interestingly, solubility was increased significantly with
the introduction of a cyclopropyl group in the 5-position. It was
also noted that the oxadiazole series had lower human sEH activity
compared to the previously evaluated series.
With the encouragement that heteroaryl rings can be modified
to modulate MIC, solubility and sHE inhibition, a small series of
pyrazoles were synthesized to further emphasize our current SAR
(Table 6). These compounds were synthesized according to
Scheme 2. The most promising compound from this series, with re-
gard to anti-TB activity, was the ethyl substituted pyrazole 43 with
2.2. ADME-toxicity properties
In order to reveal possible adverse properties of the adamantyl–
heteroaryl ureas, we determined the IC50 values for compounds
with MIC values less than 10
lg/mL against the VERO monkey kid-
ney cell line (Table 8). The majority of adamantyl–heteroaryl ureas
Table 4
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(1-adamantyl)-3-thiazolylurea analogs
Compd #
Structure
M. tb MIC (
l
g/mL)
Human sEH IC50 (nM)
Solubility (lg/mL)
cLogP
O
N
32
3.13
83
3.2 0.5
3.2
S
N
N
H
H
O
O
N
N
33
34
35
1.56
3.13
0.78
485
116
3
5.0 0.3
0.1 0.0
0.5 0.0
3.7
3.7
4.7
S
S
N
H
N
H
N
H
N
H
O
N
S
N
H
N
H
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

6
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 5
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(1-adamantyl)-3-oxadiazolylurea analogs
Compd #
Structure
M. tb MIC (
lg/mL)
Human sEH IC50 (nM)
Solubility (
l
g/mL)
cLogP
N
O
O
O
O
O
N
N
N
N
N
36
25
595
9.77 1.5
1.9
O
N
N
H
H
N
37
38
39
40
1.56
12.5
557
503
9.54 1.2
38.3 0.5
10.5 0.2
2.2 0.1
2.9
2.4
2.9
3.7
O
N
H
N
H
N
O
N
H
N
H
N
12.5
241
O
N
H
N
H
N
S
100
1575
O
N
H
N
H
Table 6
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(1-adamantyl)-3-pyrazolylurea analogs
Compd #
Structure
M. tb MIC (
l
g/mL)
Human sEH IC50 (nM)
Solubility (lg/mL)
cLogP
N
N
O
O
N
N
41
25
884
10.5 0.1
2.9
N
N
H
H
42
43
44
12.5
1.56
50
2512
751
13.2 2.8
1.43 0.0
41.1 2.2
3.2
3.4
2.4
N
H
N
H
N
N
O
O
N
N
H
N
H
N
488
N
H
N
H
H
N
H
N
O
O
a
O
b
(Cl)3C
C(Cl)3
+
R
R
C
H2N
N
R
O
O
45-53
Scheme 3. Synthesis of 1-(2-adamantyl)-3-(aromatic)ureas. Reagents and conditons: (a) DIPEA, DCM, THF, ꢀ78 °C, 20 min; (b) DIPEA, DCM, THF, rt, 1 h.
had cytotoxicity IC50 values below 50
36 g/mL. Compounds 13, 50 and 52 have IC50 values greater than
100 g/mL. Among the 18 compounds tested, 11 of them achieved
selectivity indexes greater than 10 showing good therapeutic po-
tential for the adamantyl–heteroaryl ureas. In particular, the ada-
mantyl–heteroaryl ureas linked through the secondary carbon on
adamantane.
Further evaluation of lead compounds were performed in 96-
well plate-based in vitro PK assays including human plasma pro-
tein binding (HPPB), microsomal stability and PAMPA permeabil-
l
g/mL, ranging from 6 to
human plasma protein. Traditionally it is believed that only free
drug (unbound) is able to elicit its biological response(s), therefore
low protein binding is optimal. All of the compounds had relatively
high protein binding, with compound 46 having the lowest at
95.2% bound. Incubation of the adamantyl–heteroaryl ureas with
mouse liver microsomes was used to monitor the metabolic liabil-
ity of these compounds to phase I hepatic metabolism. Compounds
24, 46, 49 and 51 had microsomal stabilities of 2.2, 1.4, 2.4 and
1.1 h half lives, respectively. Target microsomal stability is typi-
cally at least >30% of drug remaining after 90 min, which com-
pounds 24, 46, 49 and 51 all achieve. Permeability of these
compounds was determined using the parallel artificial membrane
permeability assay (PAMPA). Three out of the four lead compounds
(46, 49 and 51) all have excellent permeability, having values at
least as good as albendazole, our positive control, which has a per-
meability of approximately 441 ꢂ 10ꢀ6 cm/s.
l
l
ity. Table
9
shows the in vitro PK data for the 15 best
g/mL. One of
compounds, each with MIC values less than 3.5
l
the main focuses with the development of adamantyl heteroaryl
ureas was to boost the in vitro PK properties properties while
maintaining anti-tuberculosis activity. Rapid equilibrium dialysis
was used to determine the % binding of the adamantyl ureas to
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Table 7
In vitro whole cell anti-TB activity, human sEH inhibition, solubility and cLogP of 1-(2-adamantyl)-3-isoxazolylurea analogs
Compd #
Structure
M. tb MIC (lg/mL)
Human sEH IC50 (nM)
Solubility (
l
g/mL)
cLogP
H
H
N
N
N
45
12.5
46
14.9 2.7
3.5
O
O
O
O
O
O
O
O
N
N
S
N
N
N
H
H
N
N
46
47
48
49
50
51
52
0.10
6.25
1.56
1.56
3.13
0.78
3.13
73
5
22.7 2.2
25.5 2.8
1.5 0.2
10.1 4.0
4.8 2.6
10.3 2.2
2.9 0.3
5.3
4.0
4.7
3.9
3.4
4.0
4.2
O
H
N
H
N
O
N
H
N
H
N
710
843
2408
382
2463
H
N
H
N
O
N
H
N
H
N
O
N
H
N
H
N
O
N
H
H
N
N
N
N
N
O
O
H
N
H
N
N
53
1.56
936
2.0 0.1
4.7
Table 8
In vitro cytotoxicity and selectivity index for key adamantyl urea inhibitors with MIC <10
l
g/mL
Compd
aCytotoxicity IC50
(l
g/mL)
bSelectivity index
Compd
aCytotoxicity IC50
(
lg/mL)
aSelectivity index
13
21
24
28
32
33
34
35
37
144
22
6
6
7
0.4
23
4
4
31
7
8
7
22
7
43
46
47
48
49
50
51
52
53
26
7
>48
60
33
180 14
36
163 25
83
1
0
17
70
>8
38
21
58
46
52
53
24 15
1
5
22
13
23
17
11
5
0
4
4
1
6
1
a
In vitro human mammalian cytotoxicity against VERO cell line.
Selectivity index = cytotoxicity IC50/MIC.
b
greater nonpolarity increased anti-TB activity while the majority
of active compounds maintained solubility above 10 g/mL. A sim-
3. Conclusions
l
ilar SAR requirement was also observed with regard to substitution
of these five-membered ring heterocyles compared to substituted
phenyl groups. Substitutions in the ‘pseudo-ortho’ position was
not tolerated with regard to anti-TB activity, which has been seen
before with bulky ortho-substituted phenyl groups.6 While we
were able to achieve good anti-TB activity with increased solubil-
ity, HPPB was not significantly decreased over first generation ada-
mantyl–phenyl ureas. This is most likely due to the greasy nature
of the shared adamantyl urea core. Generally, the most active com-
pounds had acceptable microsomal stability half-lives and excel-
lent PAMPA permeability but all compounds have high levels of
protein binding. Our compounds appear to be specific to M. tb
and did not possess activity against many gram-positive and
gram-negative pathogens, including Staphylococcus aureus, Strepto-
Common approaches to optimization the pharmaceutical prop-
erties of an initial lipophilic hit, something which is common in
anti-tuberculosis drug discovery, include introducing ionizing
functional groups, heteroatoms and/or decreasing non-polar moie-
ties. In our efforts to optimize these properties for our lead series,
the adamantyl–phenyl ureas 1–6, a bioisosteric replacement strat-
egy was applied to the phenyl ring producing a new series of ada-
mantyl-heteroaromatic ureas. This strategy significantly improved
solubility over the first generation adamantyl–phenyl ureas and
was also successful in maintaining good anti-TB MIC values for
the isoxazole, thiazole, oxadiazole and pyrazole series. However,
despite increased solubility, the sulfonamide and pyridine series
failed to have substantial activity against M. tb. With regard to
the isoxazole, oxadiazole and pyrazole series, substitutions with
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

8
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 9
In vitro pharmacokinetic data for adamantyl urea inhibitors with MIC <3.5 lg/mL
Compd
aHPPB (% bound)
bMicrosomal stability (t1/2 = h)
cPermeability (ꢂ10ꢀ6 cm/s)
24
28
32
33
34
35
37
43
46
48
49
50
51
52
53
99.8 0.3
99.6 0.0
99.1 0.2
99.7 0.0
99.8 0.0
99.8 0.1
99.3 0.1
99.0 0.1
95.2 0.8
99.3 0.1
98.0 0.2
97.7 0.1
98.7 0.2
98.1 0.1
98.9 0.2
2.2 0.1
1.4 0.0
1.0 0.1
0.6 0.0
0.5 0.0
1.9 0.0
1.7 0.1
0.9 0.1
1.4 0.0
0.5 0.0
2.4 0.2
2.0 0.1
1.1 0.1
1.0 0.1
1.7 0.2
dnd
nd
543 92
766 35
30
nd
7
333 73
811 212
1157 292
696 41
484 22
860 70
843 77
989 38
398 151
a
b
c
In vitro human plasma protein binding.
In vitro half-life of adamantyl urea in mouse liver microsomes.
In vitro permeability of adamantyl ureas across an artificial membrane (PAMPA).
nd = not determined.
d
coccus pyogenes, Streptococcus pneumoniae, Bacillus subtilis, Acineto-
bacter, Klebsiella pneumoniae and Pseudomonas aeruginosa (see Sup-
plementary data Tables S1 and S2). Activity of the analogs was
confirmed as on target against MmpL3 as 46 and 51 caused the
same increase in trehalose monomycolate (TMM) and decrease in
trehalose dimycolate (TDM) and cell wall bound mycolates as the
seen by 1,5 (Supplementary data Fig. S1) and by cross resistance
testing of 46 and 51 against the M. tuberculosis strains resistant
to 1 (as described in Ref. 5).
tiator microwave was used for all microwave assisted reactions.
Melting points were determined using an Optimelt Automated
Melting Point System (Stanford Research Systems) and were
uncorrected. Compounds were purified using either a Biotage SP
silica gel or C18 column on a Biotage Isolera One. 1H NMR were re-
corded on a 400 MHz Bruker NMR and chemical shifts were re-
ported relative to solvent peak. Analytical RP-HPLC was
determined on a Waters Acquity UPLC system equipped with an
Acquity BEH C18 column (1.7 lm), flow rate of 0.5 mL/min and a
The adamantyl urea pharmacophore has been well studied in
the field of human sEH. Human sEH is involved in removing epox-
ides, a reactive oxygen species.3 Interestingly, sEH inhibition has
been shown to have an anti-inflammatory response and possess
potential benefits for many diseases, including high blood pressure
and kidney failure.3 While designing the adamantyl–heteroaryl ur-
eas, it was hoped to achieve selectivity for anti-TB activity over sEH
inhibition, and we were overall successful at reducing potency
against the human sEH by at least 10 to 100-fold. However, sEH
inhibition is not implicated in any foreseen negative side effects
and in this study we produced compounds with varying selectivity
in this regard. The sulfonamide series had poor M. tb activity, yet
they had excellent sEH IC50 values, with the majority of sulfona-
mides having <20 nM potencies. Coupled with the fact that sulfon-
amides had generally good solubility values, these compounds may
have the potential to be repurposed as human sEH inhibitors. We
were successful in being able to separate good anti-TB activity
from sEH inhibition most notably through the introduction of oxa-
diazole and pyrazole heterocylcles to the adamantyl–heteroaryl
ureas. The high therapeutic indices of the adamantyl–heteroaryl
ureas suggest they will produce good safety profiles in vivo, which
will be further evaluated as the lead compounds are progressed
forward towards development in future studies, particularly if
problems of high serum protein binding can be addressed.
gradient of solvent A (water with 0.1% formic acid) and solvent B
(acetonitrile with 0.1% formic acid): 0–0.25 min 97% A; 0.25–
3.0 min 3–100% B (linear gradient); 3.0–4.5 min 100% B; 4.5–
4.75 min 0–97% A (linear gradient); 4.75–5.0 min 97% A. Both UV
absorbance (monitored at 225–475 nm) and ELSD were used as
detection methods. All compounds were found to have >95% purity
with the described analytical methods. HRMS was determined on a
Waters XEVO QTOF LCMS. Cytotoxicity readout was performed on
a BioTek Synergy HT (BioTek, Winooski, VT). For solubility and per-
meability assays, compound quantification was performed on a
Biomek FX ADME-TOX workstation (Beckman Coulter Inc., Fuller-
ton, CA). For plasma protein binding and microsomal stability as-
says, compound quantification was performed on
Acquity UPLC system equipped with an Acquity BEH C18 column
(1.7 m) flow rate of 1.0 mL/min and a gradient of solvent A (water
a Waters
l
with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic
acid): 0–0.2 min 10–30% B (linear gradient); 0.2–1.6 min 30–95% B
(linear gradient); 1.6–1.95 min 95% B; 1.95–2 min 5–90% A (linear
gradient).
4.2. General method for adamantyl sulfonamide ureas
1-Adamantyl isocyanate (177 mg, 1.0 mmol), 4-aminosulfona-
mide (1.2 mmol) and triethylamine (502 lL, 3.6 mmol) were dis-
solved in a 1:1 mixture of THF/DMF (5 mL) in a microwave safe
tube and stirred at 200 °C for 10 min. The solvents were removed
under reduced pressure and the crude residue was purified by re-
verse-phase flash column chromatography using a 0.1% formic acid
in water to 0.1% formic acid in acetonitrile gradient.
4. Experimental
4.1. Materials and instrumentation
All chemicals and solvents were purchased from commercial
sources. All solvents were dried over desiccant and stored under
an argon atmosphere. The chemical reactions were tracked by
TLC using Silicycle Silica Gel 60F254 plates and spots were visual-
ized by UV lamp or I2 condensation. A Biotage (Charlotte, NC) Ini-
4.2.1. 1-(1-Adamantyl)-3-(4-(N-(6-chloropyridazin-3-
yl)benzenesulfonamide))urea (7)
73 mg (15.8%) as a yellow solid; MP = 190–200 °C; 1H NMR
(DMSO-d6) d = 1.63 (br, 6H), 1.85 (br, 1H), 1.93 (br, 5H), 2.03 (br,
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
3H), 3.46 (s, 1H), 6.05 (s, 1H), 7.49 (d, J = 8 Hz, 2H), 7.75 (d, J = 8 Hz,
4.3.2. Method B
Heteroarylamine (1.0 mmol) was added to THF (20 mL) and
cooled on dry ice in acetone bath. To that, butyllithium
(400 L, 1.0 mmol, 2.5 M in hexane) was added dropwise over
2H), 8.73 (s, 1H); ESI-HRMS: [MꢀH]ꢀ calcd for C21H23ClN5O3S:
460.1210, found: 460.1212.
a
l
4.2.2. 1-(1-Adamantyl)-3-(4-(N-(6-methoxypyridazin-3-yl)
benzenesulfonamide))urea (8)
20 min. The reaction mixture was removed from the dry ice in ace-
tone bath and 1-adamantyl isocyanate (177 mg, 1.0 mmol) was
added and stirred for 1 h. Methanol (4 mL) was then added to
quench any unreacted butyllithium. The solvents were then re-
moved under reduced pressure and the residue was purified by
normal phase flash column chromatography using a hexane to
ethyl acetate gradient.
70 mg (15.3%) as a yellow solid; MP = 241–246 °C; 1H NMR
(DMSO-d6) d = 1.63 (br, 6H), 1.92 (br, 6H), 2.03 (br, 3H), 3.85 (s,
3H), 5.23 (s, 1H), 6.02 (s, 1H), 7.46 (d, J = 8 Hz, 2H), 7.67 (d,
J = 8 Hz, 2H), 8.66 (s, 1H); ESI-HRMS: [MꢀH]ꢀ calcd for
C22H26N5O4S: 456.1706, found: 456.1701.
4.2.3. 1-(1-Adamantyl)-3-(4-(N-(5-methylisoxazol-3-yl)
benzenesulfonamide))urea (9)
4.3.3. 1-(1-Adamantyl)-3-(4-pyridinyl)urea (14)
Method A; 34 mg (12.4%) of white powder; MP = 200–202 °C;
1H NMR (CDCl3) d = 1.70 (br, 6H), 2.01 (br, 6H), 2.10 (br, 3H),
7.35 (d, J = 8 Hz, 2H), 8.22 (d, J = 8 Hz, 2H); ESI-HRMS: [M+H]+ calcd
for C16H22N3O: 272.1763, found: 272.1767.
69 mg (16.0%) as a white solid; MP = 235–238 °C; 1H NMR
(DMSO-d6) d = 1.63 (br, 6H), 1.93 (br, 6H), 2.03 (br, 3H), 2.29 (s,
3H), 6.07 (s, 1H), 6.11 (s, 1H), 7.51 (d, J = 8 Hz, 2H), 7.68 (d,
J = 8 Hz, 2H), 8.75 (s, 1H), 11.20 (s, 1H); ESI-HRMS: [MꢀH]ꢀ calcd
for C21H25N4O4S: 429.1597, found: 429.1595.
4.3.4. 1-(1-Adamantyl)-3-(3-pyridinyl)urea (15)
Method B; 58 mg (21.4%) of tan powder; MP = 202–204 °C; 1H
NMR (DMSO-d6) d = 1.64 (br, 6H), 1.94 (br, 6H), 2.04 (br, 3H),
6.00 (s, 1H), 7.23 (dd, J = 8 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 8.09 (d,
1H), 8.42 (s, 1H), 8.45 (s, 1H); ESI-HRMS: [M+H]+ calcd for
4.2.4. 1-(1-Adamantyl)-3-(4-(N-(4,5-dimethyloxazol-2-yl)
benzenesulfonamide))urea (10)
11 mg (5.0%) as a white solid; MP = 215–218 °C; 1H NMR
(DMSO-d6) d = 1.64 (br, 6H), 1.83–1.96 (m, 9H), 2.00–2.07 (m,
6H), 6.01 (s, 1H), 7.43 (d, J = 8 Hz, 2H), 7.67 (d, J = 8 Hz, 2H), 8.61
(s, 1H); ESI-HRMS: [M+H]+ calcd for C22H27N4O4S: 445.1910,
found: 445.1945.
C16H22N3O: 272.1763, found: 272.1767.
4.3.5. 1-(1-Adamantyl)-3-(2-pyridinyl)urea (16)
Method B; 78 mg (28.7%) of white powder; MP = 205–206 °C;
1H NMR (DMSO-d6) d = 1.65 (br, 6H), 1.98 (br, 6H), 2.04 (br, 3H),
6.88 (dd, J = 8 Hz, 1H), 7.32 (d, J = 8 Hz, 1H), 7.64 (t, J = 8 Hz, 1H),
8.09 (s, 1H), 8.14 (d, J = 8 Hz, 1H), 8.94 (s, 1H); ESI-HRMS:
[M+H]+ calcd for C16H22N3O: 272.1763, found: 272.1767.
4.2.5. 1-(1-Adamantyl)-3-(4-(N-(4-methylpyrimidin-2-yl)
benzenesulfonamide))urea (11)
20 mg (4.5%) as a white solid; MP = 130–135 °C; 1H NMR
(DMSO-d6) d = 1.63 (br, 6H), 1.92 (br, 6H), 2.03 (br, 3H), 2.74 (s,
3H), 5.23 (s, 1H), 6.04 (s, 1H), 6.88 (d, J = 8 Hz, 1H), 7.47 (d,
J = 8 Hz, 2H), 7.81 (d, J = 8 Hz, 2H), 8.30 (d, J = 8 Hz, 1H), 8.69 (s,
1H); ESI-HRMS: [MꢀH]ꢀ calcd for C22H27N5O3S: 440.1756, found:
440.1746.
4.3.6. 1-(1-Adamantyl)-3-(2-pyrimidinyl)urea (17)
Method B; 183 mg (67.2%) of white powder; MP = 267–270 °C;
1H NMR (CDCl3) d = 1.54 (br, 9H), 2.10 (br, 6H), 6.85 (t, J = 8 Hz,
1H), 7.17 (s, 1H), 8.45 (d, J = 8 Hz, 2H), 8.86 (s, 1H); ESI-HRMS:
[M+H]+ calcd for C15H21N4O: 273.1715, found: 273.1745.
4.2.6. 1-(1-Adamantyl)-3-(4-(N-(3,4-dimethylisoxazol-5-yl)
benzenesulfonamide))urea (12)
4.3.7. 1-(1-Adamantyl)-3-(4-pyrimidinyl)urea (18)
Method B; 126 mg (46.3%) of white powder; MP = 199–201 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.96 (br, 6H), 2.04 (br, 3H),
7.50 (s, 1H), 7.54 (d, J = 8 Hz, 1H), 8.43 (d, J = 8 Hz, 1H), 8.68 (s,
1H), 9.39 (s, 1H); ESI-HRMS: [M+H]+ calcd for C15H21N4O:
273.1715, found: 273.1711.
21 mg (4.7%) as a brown oil; 1H NMR (DMSO-d6) d = 1.62 (s, 3H),
1.64 (br, 6H), 1.94 (br, 6H), 2.04 (br, 3H), 2.07 (s, 3H), 6.07 (s, 1H),
7.51 (d, J = 8 Hz, 2H), 7.58 (d, J = 8 Hz, 2H), 8.76 (s, 1H), 10.79 (s,
1H); ESI-HRMS: [MꢀH]ꢀ calcd for C22H27N4O4S: 443.1753, found:
443.1747.
4.2.7. 1-(1-Adamantyl)-3-(4-((4-aminophenyl)
sulfonyl)phenyl)urea (13)
4.3.8. 1-(1-Adamantyl)-3-(4-(1,3,5-triazolyl))urea (19)
Method B; 28 mg (10.2%) of tan powder; MP = 231–233 °C; 1H
NMR (CDCl3) d = 1.72 (br, 6H), 2.09 (br, 9H), 8.58 (s, 1H), 8.82 (s,
1H); ESI-HRMS: [M+H]+ calcd for C14H20N5O: 274.1668, found:
274.1674.
12 mg (2.8%) as white solid; MP = 254–256 °C; 1H NMR (DMSO-
d6) d = 1.62 (br, 6H), 1.92 (br, 6H), 2.02 (br, 3H), 6.02 (s, 1H), 6.08 (s,
2H), 6.60 (d, J = 8 Hz, 2H), 7.46 (d, J = 8 Hz, 2H), 7.49 (d, J = 8 Hz,
2H), 7.65 (d, J = 8 Hz, 2H), 8.71 (s, 1H); 13C NMR (DMSO-d6)
d = 28.82, 35.94, 41.42, 50.03, 112.86, 116.86, 126.60, 127.75,
128.93, 134.29, 144.47, 153.16; ESI-HRMS: [M+H]+ calcd for
4.3.9. 1-(1-Adamantyl)-3-(3-(1,2,4-triazolyl))urea (20)
Method B; 63 mg (23.1%) of tan powder; MP = 201–204 °C; 1H
NMR (CDCl3) d = 1.72 (br, 6H), 2.11 (br, 9H), 7.61 (s, 1H), 8.40 (s,
1H), 8.63 (s, 1H), 8.85 (s, 1H); ESI-HRMS: [M+H]+ calcd for
C23H26N3O3S: 426.1851, found: 426.1834.
4.3. General methods for 1-(1-adamantyl)-3-(heteraryl)ureas
C14H20N5O: 274.1668, found: 274.1674.
4.3.1. Method A
4.3.10. 1-(1-Adamantyl)-3-(2-oxazolyl)urea (21)
1-Adamantyl isocyanate (177 mg, 1.0 mmol), heteroarylamine
(1.2 mmol) and triethylamine (502 lL, 3.6 mmol) were dissolved
in THF (1.5 mL) and stirred for 72 h at 70 °C. The solvent was re-
moved under reduced pressure and the residue was purified by
normal phase flash column chromatography using a hexane to
ethyl acetate gradient.
Method B; 112 mg (42.9%) of white powder; MP = 178–182 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.97 (br, 6H), 2.04 (br, 3H),
7.02 (s, 1H), 7.71 (s, 1H), 8.14 (s, 1H), 10.31 (s, 1H); 13C NMR
(DMSO-d6) d = 28.81, 35.89, 41.31, 50.32, 125.51, 133.91, 150.53,
155.72; ESI-HRMS: [M+H]+ calcd for C14H20N3O2: 262.1556, found:
262.1562.
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

10
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.3.11. 1-(1-Adamantyl)-3-(3-isoxazolyl)urea (22)
4.3.20. 1-(1-Adamantyl)-3-(3,4-dimethylisoxazol-5-yl)urea (31)
Method B; 12 mg (4.2%) of grey wax; 1H NMR (CDCl3) d = 1.68
(br, 6H), 1.87 (s, 3H), 2.02 (br, 6H), 2.09 (br, 3H), 2.18 (s, 3H),
5.69 (s, 1H), 7.40 (s, 1H); ESI-HRMS: [M+H]+ calcd for
Method B; 66 mg (25.3%) of white powder; MP = 166–167 °C;
1H NMR (CDCl3) d = 1.72 (br, 6H), 2.10 (br, 9H), 6.36 (s, 1H), 7.29
(s, 1H), 8.18 (s, 1H), 9.41 (s, 1H); ESI-HRMS: [M+H]+ calcd for
C14H20N3O2: 262.1556, found: 262.1550.
C16H24N3O2: 290.1869, found: 290.1865.
4.3.12. 1-(1-Adamantyl)-3-(5-methylisoxazol-3-yl)urea (23)
Method B; 218 mg (79.0%) of tan powder; MP = 179–180 °C; 1H
NMR (DMSO-d6) d = 1.63 (br, 6H), 1.92 (br, 6H), 2.03 (br, 3H), 2.31
(s, 3H), 6.31 (s, 1H), 6.36 (s, 1H), 8.96 (s, 1H); ESI-HRMS: [M+H]+
calcd for C15H22N3O2: 276.1712, found: 276.1721.
4.3.21. 1-(1-Adamantyl)-3-(thiazol-2-yl)urea (32)
Method A; 156 mg (56.2%) of white powder; MP = 173–174 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.93 (br, 6H), 2.04 (br, 3H),
6.33 (s, 1H), 6.99 (d, J = 4 Hz, 1H), 7.27 (d, J = 4 Hz, 1H), 10.01 (s,
1H); 13C NMR (DMSO-d6) d = 28.83, 35.87, 41.40, 50.23, 111.54,
137.20, 152.31, 159.74; ESI-HRMS: [M+H]+ calcd for C14H20N3OS:
278.1327, found: 278.1329.
4.3.13. 1-(1-Adamantyl)-3-(5-t-butylisoxazol-3-yl)urea (24)
Method B; 48 mg (15.12%) of white powder; MP = 195–196 °C;
1H NMR (CDCl3) d = 1.32 (s, 9H), 1.70 (br, 6H), 2.08 (br, 9H), 6.01
(s, 1H), 6.75 (s, 1H), 9.38 (s, 1H); 13C NMR (CDCl3) d = 28.58,
29.53, 32.76, 36.44, 41.95, 51.56, 91.90, 153.91, 159.24, 180.42;
ESI-HRMS: [M+H]+ calcd for C18H28N3O2: 318.2182, found:
318.2171.
4.3.22. 1-(1-Adamantyl)-3-(4-methylthiazol-2-yl)urea (33)
Method A; 155 mg (53.2%) of white powder; MP = 223–224 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.92 (br, 6H), 2.04 (br, 3H),
2.17 (s, 3H), 6.35 (s, 1H), 6.52 (s, 1H), 9.90 (s, 1H); 13C NMR
(DMSO-d6) d = 16.96, 28.83, 35.87, 41.40, 50.22, 105.69, 146.10,
152.31, 159.09; ESI-HRMS: [M+H]+ calcd for C15H22N3OS:
292.1484, found: 292.1473.
4.3.14. 1-(1-Adamantyl)-3-(4-bromo-5-methylisoxazol-3-
yl)urea (25)
Method B; 92 mg (26.0%) of white powder; MP = 193–197 °C;
1H NMR (CDCl3) d = 1.64 (br, 6H), 2.01 (br, 6H), 2.04 (br, 3H),
2.31 (s, 3H), 6.23 (s, 1H), 7.14 (s, 1H); ESI-HRMS: [M+H]+ calcd
for C15H22BrN3O2: 354.0817, found: 354.0812.
4.3.23. 1-(1-Adamantyl)-3-(5-methylthiazol-2-yl)urea (34)
Method A; 138 mg (47.4%) of white powder; MP = 233–235 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.92 (br, 6H), 2.04 (br, 3H),
2.27 (s, 3H), 6.32 (s, 1H), 6.93 (s, 1H), 9.80 (s, 1H); 13C NMR
(DMSO-d6) d = 11.12, 28.82, 35.88, 41.42, 50.18, 99.51, 124.08,
134.17, 152.26, 158.05; ESI-HRMS: [M+H]+ calcd for C15H22N3OS:
292.1484, found: 292.1473.
4.3.15. 1-(1-Adamantyl)-3-((5-methylisoxazol-3-yl)methyl)urea
(26)
Method A; 101 mg (34.9%) of white powder; MP = 207–209 °C;
1H NMR (DMSO-d6) d = 1.61 (br, 6H), 1.87 (br, 6H), 2.00 (br, 3H),
2.37 (s, 3H), 4.13 (d, J = 8 Hz, 2H), 5.70 (s, 1H), 6.05 (s, 1H), 6.11
(t, J = 8 Hz, 1H); ESI-HRMS: [M+H]+ calcd for C16H24N3O2:
290.1869, found: 290.1865.
4.3.24. 1-(1-Adamantyl)-3-(benzothiazol-2-yl)urea (35)
Method A; 30 mg (9.16%) of white powder; Decomposes; 1H
NMR (DMSO-d6) d = 1.66 (br, 6H), 1.97 (br, 6H), 2.06 (br, 3H),
6.57 (s, 1H), 7.20 (t, J = 8 Hz, 1H), 7.37 (t, J = 8 Hz, 1H), 7.60 (d,
J = 8 Hz, 1H), 7.86 (d, J = 8 Hz, 1H), 10.24 (s, 1H); 13C NMR (CDCl3)
d = 29.52, 36.38, 41.94, 51.87, 120.03, 121.25, 123.30, 126.06,
130.79, 149.30, 153.08, 161.81; ESI-HRMS: [M+H]+ calcd for
4.3.16. 1-(1-Adamantyl)-3-(3-methylisoxazol-5-yl)urea (27)
Method B; 125 mg (45.4%) of white powder; MP = 183–187 °C;
1H NMR (CDCl3) d = 1.65 (br, 6H), 1.97 (br, 6H), 2.05 (br, 3H),
2.23 (s, 3H), 5.82 (s, 1H), 5.92 (s, 1H), 8.84 (s, 1H); ESI-HRMS:
[M+H]+ calcd for C15H22N3O2: 276.1712, found: 276.1721.
C18H22N3OS: 328.1484, found: 328.1483.
4.3.25. 1-(1-Adamantyl)-3-(5-methyl-1,3,4-oxadiazol-2-yl)urea
(36)
4.3.17. 1-(1-Adamantyl)-3-(3-trifluoromethylisoxazol-5-yl)urea
(28)
Method B; 84 mg (30.4%) of white powder; MP = 173–176 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.96 (br, 6H), 2.05 (br, 3H),
2.38 (s, 3H), 7.46 (s, 1H), 10.46 (s, 1H); ESI-HRMS: [M+H]+ calcd
for C14H21N4O2: 277.1665, found: 277.1670.
Method B; 75 mg (22.8%) of white powder; MP = 264–268 °C;
1H NMR (CDCl3) d = 1.63 (br, 6H), 1.94 (br, 6H), 2.05 (br, 3H),
4.76 (s, 1H), 6.29 (s, 1H), 7.45 (s, 1H); 13C NMR (DMSO-d6)
d = 28.78, 35.79, 41.12, 50.49, 81.67, 119.67 (q, J = 1076 Hz),
149.83, 155.10 (q, J = 148 Hz), 164.40; ESI-HRMS: [M+H]+ calcd
for C15H19F3N3O2: 330.1429, found: 330.1476.
4.3.26. 1-(1-Adamantyl)-3-(5-isopropyl-1,3,4-oxadiazol-2-
yl)urea (37)
Method B; 76 mg (25.0%) of white powder; MP = 164–165 °C;
1H NMR (DMSO-d6) d = 1.25 (d, J = 8 Hz, 6H), 1.64 (br, 6H), 1.96
(br, 6H), 2.05 (br, 3H), 3.08 (sep, 1H), 7.47 (s, 1H), 10.44 (s, 1H);
13C NMR (DMSO-d6) d = 19.52, 25.34, 28.80, 35.85, 41.18, 50.59,
150.06, 158.74, 165.33; ESI-HRMS: [M+H]+ calcd for C16H25N4O2:
305.1978, found: 305.1986.
4.3.18. 1-(1-Adamantyl)-3-(4-chloro-3-methylisoxazol-5-
yl)urea (29)
Method B; 29 mg (9.4%) of white powder; MP = 178–183 °C; 1H
NMR (CDCl3) d = 1.63 (br, 6H), 1.97 (br, 6H), 2.04 (br, 3H), 2.17 (s,
3H), 5.80 (s, 1H); ESI-HRMS: [M+H]+ calcd for C15H21ClN3O2:
310.1322, found: 310.1317.
4.3.27. 1-(1-Adamantyl)-3-(5-cyclopropyl-1,3,4-oxadiazol-2-
yl)urea (38)
4.3.19. 1-(1-Adamantyl)-3-(4-cyano-3-methylisoxazol-5-yl)urea
(30)
Method B; 57 mg (19.0%) of white powder; MP = 191–196 °C;
1H NMR (CDCl3) d = 1.70 (br, 6H), 2.05 (br, 6H), 2.12 (br, 3H),
2.34 (s, 3H), 5.72 (s, 1H), 8.41 (s, 1H); ESI-HRMS: [M+H]+ calcd
for C16H21N4O2: 301.1665, found: 301.1677.
Method B; 53 mg (17.5%) of white powder; MP = 161–165 °C;
1H NMR (DMSO-d6) d = 0.88–0.92 (m, 2H), 1.03–1.08 (m, 2H),
1.64 (br, 6H), 1.95 (br, 6H), 2.04 (br, 3H), 2.09 (sep, 1H), 7.41 (s,
1H), 10.39 (s, 1H); ESI-HRMS: [M+H]+ calcd for C16H23N4O2:
303.1821, found: 303.1837.
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
4.3.28. 1-(1-Adamantyl)-3-(5-cyclobutyl-1,3,4-oxadiazol-2-yl)
urea (39)
1.97–2.00 (m, 4H), 4.09 (1H, d, J = 8 Hz), 8.62 (1H, d, J = 8 Hz),
9.77 (1H, s); ESI-HRMS: [M+H]+ calcd for C14H20N3O2: 262.1550,
found: 262.1583.
Method A; 173 mg (54.7%) of white powder; MP = 161–163 °C;
1H NMR (DMSO-d6) d = 1.64 (br, 6H), 1.85–1.98 (m, 7H), 2.00–2.09
(m, 4H), 2.20–2.38 (m, 4H), 3.64 (pen, 1H), 7.47 (s, 1H), 10.45 (s,
1H); ESI-HRMS: [M+H]+ calcd for C17H25N4O2: 317.1978, found:
317.1985.
4.4.2. 1-(2-Adamantyl)-3-(5-t-butylisoxazol-3-yl)urea (46)
167 mg (48.7%) of white powder; MP = 229–232 °C; 1H NMR
(DMSO-d6) d = 1.27 (s, 9H), 1.56 (s, 1H), 1.59 (s, 1H), 1.65–1.88
(m, 12H), 3.78 (d, J = 8 Hz, 1H), 6.28 (s, 1H), 6.98 (d, J = 8 Hz, 1H),
9.26 (s, 1H); 13C NMR (CDCl3) d = 27.18, 27.32, 28.63, 31.96,
32.38, 37.19, 37.63, 54.45, 91.93, 154.55, 159.14, 180.50; ESI-
HRMS: [M+H]+ calcd for C18H28N3O2: 318.2182, found: 318.2171.
4.3.29. 1-(1-Adamantyl)-3-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-
yl)urea (40)
Method A; 146 mg (42.4%) of white powder; MP = 211–213 °C;
1H NMR (DMSO-d6) d = 1.65 (br, 6H), 1.98 (br, 6H), 2.06 (br, 3H),
7.26 (t, J = 4 Hz, 1H), 7.36 (s, 1H), 7.63 (d, J = 4 Hz, 1H), 7.88 (d,
J = 4 Hz, 1H), 10.66 (s, 1H); ESI-HRMS: [M+H]+ calcd for
4.4.3. 1-(2-Adamantyl)-3-(5-methylisoxazol-3-yl)urea (47)
64 mg (24.5%) of white powder; MP = 208–212 °C; 1H NMR
(DMSO-d6) d = 1.32 (s, 1H), 1.35 (s, 1H), 1.44–1.63 (m, 12H), 3.54
(d, J = 8 Hz, 1H), 6.10 (s, 1H), 6.72 (d, J = 8 Hz, 1H), 8.95 (s, 1H);
13C NMR (CDCl3) d = 11.97, 26.54, 26.64, 31.18, 31.87, 36.62,
37.02, 53.06, 95.14, 152.81, 159.12, 168.69; ESI-HRMS: [M+H]+
calcd for C15H22N3O2: 276.1712, found: 276.1721.
C17H21N4O2S: 345.1385, found: 345.1383.
4.3.30. 1-(1-Adamantyl)-3-(1-ethylpyrazol-3-yl)urea (41)
Method A; 162 mg (56.2%) of white powder; MP = 140–146 °C;
1H NMR (DMSO-d6) d = 1.31 (t, J = 8 Hz, 3H), 1.63 (br, 6H), 1.93 (br,
6H), 2.03 (br, 3H), 3.95 (q, J = 8 Hz, 2H), 5.97 (s, 1H), 7.49 (s, 1H),
8.47 (s, 1H); ESI-HRMS: [M+H]+ calcd for C16H25N4O: 289.2028,
found: 289.2033.
4.4.4. 1-(2-Adamantyl)-3-(4-methylthiazol-2-yl)urea (48)
14 mg (9.6%) of off white powder; MP = 229–231 °C; 1H NMR
(CDCl3) d = 1.50 (1H, s), 1.53 (1H, s), 1.61 (2H, s), 1.67–1.74 (7H,
br), 1.78 (1H, s), 1.83 (2H, s), 3.88 (1H, d, J = 8 Hz), 6.17 (1H, s);
13C NMR (CDCl3) d = 17.38, 27.15, 27.44, 31.97, 32.43, 37.17,
37.64, 54.19, 104.85, 147.22, 154.22, 161.89; ESI-HRMS: [M+H]+
calcd for C15H22N3O2: 292.1478, found: 292.1473.
4.3.31. 1-(1-Adamantyl)-3-(1-ethyl-5-methylpyrazol-3-yl)urea
(42)
Method A; 158 mg (52.2%) of white powder; MP = 215–217 °C;
1H NMR (DMSO-d6) d = 1.24 (t, J = 8 Hz, 3H), 1.63 (br, 6H), 1.93 (br,
6H), 2.02 (br, 3H), 2.16 (s, 3H), 3.86 (q, J = 8 Hz, 2H), 5.75 (s, 1H),
6.91 (s, 1H), 8.37 (s, 1H); ESI-HRMS: [M+H]+ calcd for C17H27N4O:
303.2185, found: 303.2193.
4.4.5. 1-(2-Adamantyl)-3-(5-isopropyl-1,3,4-oxadiazol-2-yl)urea
(49)
96 mg (63.1%) of white powder; MP = 172–174 °C; 1H NMR
(DMSO-d6) d = 1.02 (6H, d, J = 8.0 Hz), 1.36 (1H, s), 1.39 (1H, s),
1.48 (2H, s), 1.51 (1H, s), 1.55 (2H, s), 1.58–1.61 (7H, m), 2.85
(1H, sep, J = 8 Hz), 3.62 (1H, d, J = 8 Hz), 7.97 (1H, d, J = 8 Hz),
10.23 (1H, s); 13C NMR (CDCl3) d = 19.76, 26.19, 27.12, 27.22,
31.88, 32.24, 37.10, 37.56, 54.71, 152.39, 158.75, 166.06; ESI-
HRMS: [M+H]+ calcd for C16H25N4O2: 305.1972, found: 305.1957.
4.3.32. 1-(1-Adamantyl)-3-(5-methyl-1-propylpyrazol-3-yl)urea
(43)
Method A; 160 mg (50.6%) of white powder; MP = 200–201 °C;
1H NMR (DMSO-d6) d = 0.82 (t, J = 8 Hz, 3H), 1.63 (br, 6H), 1.68
(sex, J = 8 Hz, 2H), 1.92 (br, 6H), 2.02 (br, 3H), 2.16 (s, 3H), 3.78
(q, J = 8 Hz, 2H), 5.75 (s, 1H), 6.87 (s, 1H), 8.38 (s, 1H); 13C NMR
(DMSO-d6) d = 10.59, 10.99, 22.82, 28.88, 36.04, 41.73, 48.95,
49.66, 99.51, 138.25, 147.47, 153.32; ESI-HRMS: [M+H]+ calcd for
4.4.6. 1-(2-Adamantyl)-3-(5-cyclopropyl-1,3,4-oxadiazol-2-yl)
urea (50)
C18H29N4O: 317.2341, found: 317.2349.
13 mg (8.6%) of white powder; MP = 179–181 °C; 1H NMR
(CDCl3) d = 1.12–1.14 (4H, m), 1.65 (1H, s), 1.68 (1H, s), 1.78 (2H,
s), 1.89 (6H, br), 1.96 (1H, s), 2.00 (3H, br), 2.03–2.09 (1H, m),
4.08 (1H, d, J = 8 Hz), 8.56 (1H, d, J = 8 Hz), 8.65 (1H, s); 13C NMR
(CDCl3) d = 5.95, 7.93, 27.12, 27.22, 31.89, 32.23, 37.10, 37.57,
54.76, 151.58, 157.89, 163.57; ESI-HRMS: [M+H]+ calcd for
4.3.33. 1-(1-Adamantyl)-3-(1-methylpyrazol-4-yl)urea (44)
Method A; 174 mg (63.4%) of white powder; MP = 217–218 °C;
1H NMR (DMSO-d6) d = 1.62 (br, 6H), 1.90 (br, 6H), 2.01 (br, 3H),
3.73 (s, 3H), 5.68 (s, 1H), 7.21 (s, 1H), 7.60 (s, 1H), 7.84 (s, 1H);
ESI-HRMS: [M+H]+ calcd for C15H23N4O: 275.1872, found:
275.1858.
C17H27N4O: 303.1816, found: 303.1809.
4.4. General procedure for 1-(2-adamantyl)-3-(heteroaryl)urea
4.4.7. 1-(2-Adamantyl)-3-(5-cyclobutyl-1,3,4-oxadiazol-2-
yl)urea (51)
Triphosgene (119 mg, 0.4 mmol) was dissolved in DCM (10 mL)
and cooled on an acetone and dry ice bath. A solution of heteroa-
rylamine (1 mmol) and DIPEA (393 lL, 2.2 mmol) in THF (5 mL)
was then added dropwise over 15 min. After the reaction mixture
stirred for an additional 5 min, a suspension of 2-adamantylamine
84 mg (53.1%) of white powder; MP = 193–196 °C; 1H NMR
(DMSO-d6) d = 1.61 (1H, s), 1.63 (1H, s), 1.73 (2H, s), 1.76–1.79
(2H, m), 1.83–1.85 (8H, m), 1.89–1.97 (1H, m), 1.99–2.10 (1H, s),
2.23–2.38 (4H, m), 3.65 (1H, pen, J = 8 Hz), 3.87 (1H, d, J = 8 Hz),
8.21 (1H, d, J = 8 Hz), 10.77 (1H, s); 13C NMR (CDCl3) d = 18.82,
26.78, 27.13, 27.23, 30.22, 31.89, 32.25, 37.11, 37.58, 54.73,
152.28, 158.67, 164.41; ESI-HRMS: [M+H]+ calcd for C17H25N4O2:
317.1972, found: 317.2020.
hydrogen chloride (188 mg, 1.0 mmol) and DIPEA (393 lL,
2.2 mmol) in DCM (5 mL) was added. The reaction mixture was re-
moved from the acetone and dry ice bath and stirred for 1 h. The
solvents were removed under reduced pressure and the crude res-
idue was purified by normal phase flash column chromatography
using a hexane to ethyl acetate gradient.
4.4.8. 1-(2-Adamantyl)-3-(1-ethyl-5-methylpyrazol-3-yl)urea
(52)
14 mg (9.26%) of white powder; MP = 159–163 °C; 1H NMR
(CDCl3) d = 1.32 (3H, t, J = 8.0 Hz), 1.57 (1H, s), 1.60 (1H, s), 1.69
(2H, s), 1.80 (6H, br), 1.90 (4H, br), 2.14 (3H, s), 3.87 (2H, q,
J = 8 Hz), 3.98 (1H, d, J = 8 Hz), 5.40 (1H, s), 6.55 (1H, s), 8.48 (1H,
4.4.1. 1-(2-Adamantyl)-3-(oxazol-2yl)urea (45)
53 mg (40.6%) of white powder; MP = 189–192 °C; 1H NMR
(CDCl3) d = 1.65 (1H, s), 1.68 (1H, s), 1.78 (2H, s), 1.89 (6H, br),
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

12
E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
s); 13C NMR (DMSO-d6) d = 10.40, 14.89, 26.60, 26.80, 31.36, 32.13,
36.71, 37.13, 42.50, 52.79, 93.71, 138.09, 147.58, 153.80; ESI-
HRMS: [M+H]+ calcd for C17H27N4O: 303.2179, found: 303.2170.
4.8. Solubility determination
Compounds were initially prepared by dissolving them into
DMSO at a final concentration of 10 mM. Stock drug plates were
prepared by adding 30 lL of 10 mM DMSO stocks to each well in
4.4.9. 1-(2-Adamantyl)-3-(5-methyl-1-propylpyrazol-3-yl)urea
(53)
a 96-well plate. For reference plates, compounds were diluted
600-fold in system solution buffer (SSB, pH 7.4; pION INC, Woburn,
MA) and iso-propanol (1:1, v/v). Concentrations were assessed by
UV spectrometry (230–500 nm). For sample plates, compounds
were diluted 100-fold in system solution buffer, incubated at room
temperature for 18 h to allow the compounds to be fully stable,
and then filtered through a 96-well filter plate (pION Inc., Woburn,
MA). Fractions were collected from the filtered sample plate, di-
luted with iso-propanol by 1:1 (v/v), and determined concentration
82 mg (51.8%) of white powder; MP = 206–209 °C; 1H NMR
(CDCl3) d = 0.93 (3H, t, J = 8.0 Hz), 1.65 (1H, s), 1.68 (1H, s), 1.78
(2H, s), 1.80–1.92 (8H, m), 1.99 (4H, br), 3.88 (2H, t, J = 8 Hz),
4.07 (1H, d, J = 8 Hz), 5.48 (1H, s), 6.49 (1H, s), 8.62 (1H, d,
J = 8 Hz); 13C NMR (DMSO-d6) d = 11.00, 11.21, 23.23, 27.26,
27.58, 32.13, 32.58, 37.20, 37.81, 50.08, 53.99, 93.10, 139.11,
147.64, 154.53; ESI-HRMS: [M+H]+ calcd for C18H29N4O:
317.2336, found: 317.2320.
via UV spectrometry. Calculation was carried out by
lSOL Evolu-
tion software and all compounds were tested in triplicates.
4.5. MIC determination
4.9. Human plasma protein binding determination
MIC values were determined against M. tb using microbroth
dilution method in accordance with the CLSI and to our previously
published methods.9,13 Compounds were initially dissolved in
DMSO at 10 mg/mL and stored at ꢀ80 °C until needed. Compounds
stocks were diluted in Middlebrook 7H9 media (final volume of
Compounds were initially prepared by dissolving them into
DMSO at a final concentration of 10 mM. Dulbecco’s phosphate
buffered saline (DPBS; pH 7.4) was obtained from Invitrogen
(Carlsbad, CA), single-use RED (rapid equilibrium dialysis) device
was obtained from Thermo scientific (Rockford, IL) and human
plasma was obtained from Innovative Research INC (Novi, Michi-
gan, IL).
Sample preparation for plasma protein binding was modified
from Waters’ method.17 Teflon base plates with the RED inserts
(molecular weight cut-off (MWCO) = 8000) were to used without
any pre-conditioning of the membrane inserts. Human plasma
was thawed and centrifuged at 1000 rpm for 2 min to remove
100 lL) and serially diluted in a 96-well round bottom microtitre
plate. M. tb culture was grown in Middlebrook 7H9 media with
0.05% Tween 80 at 37 °C to an OD600 of 0.003 providing approxi-
mately 105 cfu/mL. M. tb culture (100
l
L) was then added to the
L total volume and final drug concentra-
g/mL. The plates were incubated at 37 °C
drug plates giving 200
tions beginning at 200
l
l
for seven days and visual inspection was used to determine the
minimum concentration of drug that inhibited >90% of bacterial
growth.
any particulates. Each compound was prepared at 10
plasma. These samples were prepared by adding 1 L of drug stock
to 1000 L of human plasma. Spiked plasma solutions (300 L)
were placed into the sample chamber (indicated by the red ring)
and 500 L of DPBS into the adjacent chamber. The plate was
lM in human
l
4.6. Human soluble epoxide hydrolase assay
l
l
The IC50 values were determined using previously published
methods.14 The fluorescent substrate used was cyano(2-meth-
l
sealed and incubated at 37 °C on an orbital shaker (100 rpm) for
4 h. After incubation, the seal was removed from the RED plate
and the volume of the insert was inspected to ensure minimal vol-
ume change. Aliquots (50 lL) were removed from each side of the
insert and dispensed into a 96-well deep plate. An equal volume of
oxynaphthalen-6-yl)methyl
trans-(3-pheyloxyran-2-yl)methyl
carbonate (CMNPC). Inhibitor and purified recombinant human
sEH15,16 (1 nM) were incubated together for 5 min in pH 7.4 so-
dium phosphate buffer (100 mM) which contained BSA (0.1 mg/
mL) at 30 °C. CMNPC addition subsequently followed (final CMNPC
blank plasma or DPBS was added to the required wells to create
analytically identical sample matrices. To each sample, 200
concentration = 5 lM). Enzyme activity was determined by moni-
lL of
toring the appearance of 6-methoxy-2-naphthaldehyde over
10 min by fluorescence detection with excitation and emission
wavelengths of 330 and 465 nm, respectively. The IC50 values re-
ported are the average of three replicates with at least two data
points above and below the IC50. The assay as performed here
has a standard error between 10% and 20%, suggesting that differ-
ences of twofold or greater are significant.14
ACN containing the internal standard (4 g/mL warfarin) was
added. All of the plates were sealed and mixed well at 600 rpm
for 10 min and were centrifuged at 4000 rpm for 20 min. The
supernatants (120 lL) were transferred to analytical plates for
analysis by LCMS. LCMS conditions were described in the materials
and instrumentation section above. The test compound concentra-
tions were quantified in both buffer and plasma chambers via peak
areas relative to the internal standard. The percentage of the test
compound bound to plasma was calculated on following
equations:
l
4.7. Cytotoxicity determination
Vero epithelial cells (ATCC CCL-81) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) at 37 °C in a humidified incubator with 5%
CO2. Trypsinized cells were transferred to 96-well cell culture
plates (Nunc) at a seeding density of ꢁ5 ꢂ 103 cells per well. The
next day, when cells were ꢁ10% confluent, DMEM/FBS was ex-
changed for fresh media containing twofold serial dilutions of test
compounds and cells were incubated for an additional 72 h. MTT
cell proliferation assay was performed as described by manufac-
turer (Promega Corporation, Madison, WI) with absorbances read
following overnight solubilization of formazan. IC50 values were
derived from corresponding dose–response curves.
½Buffer Chamberꢃ
%Free ¼
ꢂ 100
ð1Þ
½Plasma Chamberꢃ
%Bound = 100 ꢀ %Free.
4.10. Microsomal stability determination
Compounds were initially prepared by dissolving them into
DMSO at a final concentration of 10 mM. NADPH regenerating
solutions A and B and mouse liver microsomes (CD-1) were ob-
tained from BD Gentest (Woburn, MA). Ninety-six deep well plates
were obtained from MidWest Scientific Inc. (Valley Park, MO).
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028

E. J. North et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
13
Sample preparation for microsomal stability was modified from
Acknowledgements
Di’s methods.18,19 A set of incubation times of 0, 15, 30, 60, 120,
and 240 min were used. Human or mouse liver microsomal solu-
This study was supported by the National Institutes of Health
grants RC1AI85992, AI057836, AI063054 and the American Leba-
nese Syrian Associated Charities (ALSAC), St. Jude Children’s Re-
search Hospital.
tions were prepared by adding 58
mouse liver microsomes (20 mg/mL protein concentration) to
1.756 mL of 0.1 M potassium phosphate buffer (pH 7.4) and 5
of 0.5 M EDTA to make a 0.6381 mg/mL (protein) microsomal solu-
tion. NADPH regenerating agent contained 113 L of NADPH A,
23 L of NADPH B, and 315 L of 0.1 M potassium phosphate buffer
(pH 7.4). The diluted test compound solutions (2 L) were each
added directly to a 1.79 mL of liver microsomal solution. This solu-
tion was mixed and 90 L was transferred to six time points plates
(each in triplicate wells). For the time 0 plate, 225 L of cold ace-
lL of concentrated human or
lL
l
Supplementary data
l
l
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.028.
l
l
References and notes
tonitrile with internal standard (4 mg/ml warfarin) was added to
1. Lienhardt, C.; Glaziou, P.; Uplekar, M.; Lonnroth, K.; Getahun, H.; Raviglione, M.
Nat. Rev. Microbiol. 2012, 10, 407.
each well, followed by addition of NADPH regenerating agent
(22.5
lL) and no incubation. For other five time points’ plate,
2. Brown, J. R.; North, E. J.; Hurdle, J. G.; Morisseau, C.; Scarborough, J. S.; Sun, D.;
Kordulakova, J.; Scherman, M. S.; Jones, V.; Grzegorzewicz, A.; Crew, R. M.;
Jackson, M.; McNeil, M. R.; Lee, R. E. Bioorg. Med. Chem. 2011, 19, 5585.
3. Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 311.
4. 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.
5. Grzegorzewicz, A. E.; Pham, H.; Gundi, V. A. K. B.; Sherman, M. S.; North, E. J.;
Hess, T.; Jones, V.; Gruppo, V.; Born, S. E. M.; Kordulakova, J.; Chavadi, S. S.;
Morisseau, C.; Lenaerts, A. J.; Lee, R. E.; McNeil, M. R.; Jackson, M. Nat. Chem.
Biol. 2012, 8, 334.
NADPH regenerating agent (22.5
lL) was added to each well to ini-
tiate the reaction. The plates were incubated at 37 °C for the re-
quired time, followed by quenching of the reaction by adding
225 lL of cold acetonitrile with internal standard (4 lg/ml warfa-
rin). All of the plates were sealed and mixed well at 600 rpm for
10 min followed by centrifugation at 4000 rpm for 20 min. The
supernatants (120 lL) were transferred to analytical plates for
analysis by LCMS. LCMS conditions were described in the materials
and instrumentation section above. The metabolic stability is eval-
uated via the half-life from least-squares fit of the multiple time
points based on first-order kinetics.
6. Scherman, M. S.; North, E. J.; Jones, V.; Hess, T. N.; Grzegorzewicz, A. E.;
Kasagami, T.; Kim, I.-H.; Merzlikin, O.; Lenaerts, A. J.; Lee, R. E.; Jackson, M.;
Morisseau, C.; McNeil, M. R. Bioorg. Med. Chem. 2012, 20, 3255.
7. Bordwell, F. G.; Fried, H. E.; Hughes, D. L.; Lynch, T.-Y.; Satish, A. V.; Whang, Y. E.
J. Org. Chem. 1990, 55, 3330.
8. Yun, M.-K.; Wu, Y.; Li, Z.; Zhao, Y.; Waddell, M. B.; Ferreira, A. M.; Lee, R. E.;
Bashford, D.; White, S. W. Science 2012, 335, 1110.
9. Hurdle, J. G.; Lee, R. B.; Nageshwar, R. B.; Carson, E. I.; Qi, J.; Scherman, M. S.;
Cho, S. H.; McNeil, M. R.; Lenaerts, A. J.; Franzblau, S. G.; Meibohm, B.; Lee, R. E.
J. Antimicrob. Chemother. 2008, 62, 1037.
10. Rastogi, N.; Labrousse, V.; Goh, K. S. Curr. Microbiol. 1996, 33, 167.
11. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
12. Gleeson, M. P. J. Med. Chem. 2008, 51, 817.
13. Barry, A. L.; Craig, W. A.; Nadler, H.; Reller, L. B.; Sanders, C. C.; Swenson, J. M.
Clin. Lab. Stand. Inst. 1999, 19, M26.
14. Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock, B. D.
Anal. Biochem. 2005, 343, 66.
4.11. Permeability determination
Compounds were initially prepared by dissolving them into
DMSO at a final concentration of 10 mM. Drug stock plates were
prepared by adding 6
pounds were diluted 200-fold in system solution buffer (SSB, pH
7.4; pION INC, Woburn, MA). The diluted drug stocks (180 L) were
added to donor plates (pION INC, Woburn, MA). A filter plate
(acceptor plate; pION INC, Woburn, MA) containing 200 L of
lL of DMSO solutions to each well. Com-
l
l
15. Beetham, J. K.; Tian, T.; Hammock, B. D. Arch. Biochem. Biophys. 1993, 305, 197.
16. Wixtrom, R. N.; Silva, M. H.; Hammock, B. D. Anal. Biochem. 1988, 169, 71.
17. Waters, N. J.; Jones, R.; Williams, G.; Sohal, B. J. Pharm. Sci. 2008, 97, 4586.
18. Di, L.; Kerns, E. H.; Li, S. Q.; Petusky, S. L. Int. J. Pharm. 2006, 317, 54.
19. Di, L.; Kerns, E. H.; Ma, X. J.; Huang, Y.; Carter, G. T. Comb. Chem. High
Throughput Screening 2008, 11, 469.
acceptor sink buffer (ASB, pH 7.4; pION INC, Woburn, MA) was
then placed over the donor plate. The plates were incubated and
stirred at room temperature for 0.5 h. Fractions were collected
from both the donor plate and the acceptor plate and concentra-
tions were assessed by UV spectrometry (230–500 nm). All com-
pounds were tested in triplicates.
Please cite this article in press as: North, E. J.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.028