Selective and potent urea inhibitors of cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

Article abstract of DOI:10.1021/jm3007917

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC₅₀ < 2 nM) against CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.

Full text of DOI:10.1021/jm3007917

Article
pubs.acs.org/jmc
Selective and Potent Urea Inhibitors of Cryptosporidium parvum
Inosine 5′-Monophosphate Dehydrogenase
Suresh Kumar Gorla,† Mandapati Kavitha,† Minjia Zhang,† Xiaoping Liu,‡ Lisa Sharling,‡
Deviprasad R. Gollapalli,† Boris Striepen,‡ Lizbeth Hedstrom,†,∥ and Gregory D. Cuny*,§,⊥
∥
†Departments of Biology and Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States
‡Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens,
Georgia 30602, United States
§Laboratory for Drug Discovery in Neurodegeneration, Brigham & Women’s Hospital, Harvard Medical School,
65 Landsdowne Street, Cambridge, Massachusetts 02139, United States
⊥Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston,
549A Science and Research Building 2, Houston, Texas 77204, United States
S
* Supporting Information
ABSTRACT: Cryptosporidium parvum and related species are
zoonotic intracellular parasites of the intestine. Cryptosporidium
is a leading cause of diarrhea in small children around the
world. Infection can cause severe pathology in children and
immunocompromised patients. This waterborne parasite is
resistant to common methods of water treatment and therefore
a prominent threat to drinking and recreation water even in
countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis
revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine
nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput
screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against
CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset
of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.
C. parvum and C. hominis).8 Remarkably, these parasites appear
INTRODUCTION
■
to have obtained their IMPDH gene from an ε-proteobacterium
Cryptosporidium parasites such as C. parvum and C. hominis are
by lateral gene transfer.9 The bacterial origin of CpIMPDH and
major causes of the “vicious cycle of diarrhea and malnutrition”
the different kinetic properties of these enzymes compared with
that afflicts the developing world.1 This infection is self-limiting
their human counterparts suggested CpIMPDH-selective inhib-
in healthy adult individuals, but can be chronic and fatal in
itors could be obtained.10 A high-throughput screen for selective
immunocompromised patients and children under 5 years of
inhibitors yielded an urea-based inhibitor with an IC50 value of
age.2 Parasite oocysts are resistant to commonly used methods of
340 nM against CpIMPDH (Compound 1, Table 1). Herein, we
water treatment, and contaminated drinking and recreational
report a SAR study of 1 with respect to CpIMPDH inhibition and
water are major sources of host-to-host transmission.3 While
antiparasitic activity and optimization of mouse microsomal
cryptosporidiosis is more prevalent in developing countries,4 the
stability.
developed world also has a significant disease burden. Fifteen
documented Cryptosporidium waterborne outbreaks were
RESULTS AND DISCUSSION
■
reported during 2000 in the USA, resulting in enormous medical
expenses.5 Since the oocysts can be obtained with relative ease and
High-Throughput Screen. Following our previously
described assay protocol,10 we performed a high-throughput
the water supply is readily accessed, C. parvum has the potential to
screen of 85 000 compounds to identify inhibitors of CpIMPDH.
This screen targeted the NAD site, which is the most highly
diverged region of the active site relative to the human enzymes.
This screen yielded 50 compounds that inhibited CpIMPDH
but not human IMPDH type 2 (IC50 > 5 μM). Six of these
compounds were urea-based, of which the most potent was
be used for bioterrorism. Consequently, it is considered a class B
biowarfare agent.6 Neither vaccines nor the approved drug
nitazoxanide are effective treatments for cryptosporidiosis in
immunocompromised patients, so there is an urgent need for new
chemotherapeutic agents.
Genomic analysis revealed that C. parvum and C. hominis rely on
the enzyme inosine 5′-monophosphate dehydrogenase (IMPDH)
for the production of guanine nucleotides2b,7 (Scheme 1; CpIMPDH
will be used to denote the parasite enzyme, which is identical in
Received: June 6, 2012
Published: September 5, 2012
© 2012 American Chemical Society
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a
Scheme 1. IMPDH Catalyzed Conversion of IMP to XMP
a
R5P = ribose-5′-phosphate.
a
ureas 5 and 6 were obtained. The acetyl group on 6 was converted
to the corresponding oxime 7 and methyl oxime 8 in good yields
by heating with a slight excess of hydroxylamine hydrochloride
and methoxyamine hydrochloride, respectively, in pyridine.12
Oxime 7 was alkylated in the presence of sodium hydride with
2-chloroethylamine hydrochloride to give amine derivative 9.
Lithium aluminum hydride (LAH) reduction of acetyl derivative
6 gave alcohol 10. Hydrogenation of isoprenyl derivative 5 with
10% palladium on carbon yielded the saturated urea derivative 11
in good yield.
Scheme 3 outlines the reaction sequence used for the synthesis of
a monomethyl urea derivative. 3-(1-Cyanoethyl)benzoic acid, 12,
was converted into acetyl derivative 13 using a stepwise process
that involved the conversion of the acid to the corresponding
acid chloride followed by treatment with Meldrum’s acid in
the presence of base and then hydrolytic decarboxylation.13
Subsequent hydrolysis of nitrile derivative 13 gave substituted
hydratropic acid 14, which was treated with thionyl chloride
followed by sodium azide in water to generate acyl azide 15 that
was used without isolation. Acyl azide 15 was heated to reflux in
benzene under a nitrogen atmosphere to generate isocyanate 16.14
Finally, the isocyanate was treated with 4-chloroaniline to give urea
derivative 17.
Table 1. SAR of the Anilide
IC50 (nM)
ID
R
(-) BSA
(+) BSA
410 20
5a
5b
5c
5d
5e
5f
Ph
250 20
4-Cl-Ph
4-Br-Ph
3-Cl-Ph
2-Cl-Ph
20
10
7
4
37
7
20 10
330 30
n.d.
70 10
b
>5000
>5000
4-OMe-Ph
9
b
1
21
7
5g
5h
5i
4-tBu-Ph
n.d.
3,4-di-ClPh
6
4
1
1
50 20
3-CF3-4-Cl-Ph
3-OMe,4-Cl-Ph
3-CONH2,4-Cl-Ph
3-CONHCH3,4-Cl-Ph
3-CON(Me)2,4-Cl-Ph
3,4-(OCH2CH2O)-Ph
2-Naphthyl
20
5
6
1
1
3
5j
1.3 0.2
2.3 0.8
5k
5l
3
7
8
2
1
14
5m
5n
5o
5p
5q
5r
5s
5t
11.7 0.5
110 30
40 20
n.d.
7.2 0.6
2.1 0.8
b
1-Naphthyl
>5000
An analogue of 1 that lacks both methyl groups on the benzylic
position was prepared according to the procedure outlined in
Scheme 4. 3-Acetylbenzonitrile, 18, was treated with ethylene
glycol15 to give the ketal protected benzonitrile derivative 19.
LAH reduction generated benzylamine derivative 20, which was
subsequently converted to urea 22 in two steps via the inter-
mediate p-nitrophenyl carbamate 21.16 Removal of the ketal was
carried out under acidic conditions to give 23.
6-Quinolyl
1.8 0.4
0.8 0.1
70 10
4
6
1
2
8
7-Quinolyl
3-Quinoline
308
2-Quinoline
250 20
1100 300
a
Assays as described in methods. No significant inhibition of human
IMPDH type 2 was observed (IC50 ≥ 5 μM). n.d., not determined.
b
One determination.
A series of carbamate and amide derivatives were prepared
using the methods outlined in Scheme 5. Isocyanate 3 was
refluxed with 8N HCl to give benzylamine derivative 24 in good
yield. This material was converted to carbamate 25 by treatment
with 4-chlorophenyl chloroformate in the presence of base.
Amine 24 was also converted to amide 26 in good yield. In
addition, 24 was treated with chlorosulfonic acid, followed by
exposure to aqueous potassium nitrite and subsequent decom-
position with sulfate buffer (pH = 2.8) to give the substituted
benzyl alcohol 27.17 Upon treatment with 4-chlorophenyl
isocyanate, this material was converted to carbamate 28.
Several biphenyl derivatives were prepared using the methods
outlined in Scheme 6. 3-Phenylcyanobenzene, 29, was allowed to
react with 3 equiv of methyl magnesium bromide in the presence
of the Lewis acid catalyst titanium(IV) isopropoxide to give
tert-alkyamine 30.18 Biphenylcyclopropylamine 31 was similarly
prepared from 29 by treatment with 2 equiv of ethyl magnesium
bromide and titanium(IV) isopropoxide.19 Amines 30, 31, and
32 were converted to corresponding urea derivatives 36, 37, and
Figure 1. CpIMPDH inhibitor identified by HTS.
compound 1 (Figure 1) with IC50 of 340 nM. Interestingly,
two urea-based inhibitors of human IMPDHs, merimepodib and
AVN944, have entered clinical trials,11 which further encouraged
development of this series.
Chemistry. Various derivatives of 1 were prepared following
the synthetic method outlined in Scheme 2. Ozonolysis followed
by reductive workup with dimethyl sulfide transformed
3-isopropenyl-α,α-dimethyl benzyl isocyanate 2 into 3-acetyl-
α,α-dimethyl benzyl isocyanate 3 in moderate yield. Upon
treating 2 and 3 with substituted anilines 4, the corresponding
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a
Scheme 2. General Procedure for Preparation of Urea Derivatives.
a
Reagents and conditions: (a) (i) O3, DCM, −78 °C; (ii) Me2S, 10 h, 58%; (b) DCM, R-PhNH2 (4), 2−4 h, 70−85%; (c) THF, R-PhNH2 (4), 6 h,
70 °C, 70−75%; (d) NH2OH·HCl, pyridine, 2 h, 90 °C, 80−85%; (e) NH2OMe·HCl, pyridine, 2 h, 90 °C, 60−85%; (f) (i) NaH, THF, 0 °C,
30 min; (ii) Cl(CH2)2NH2•HCl, rt, 10 h, 53%; (g) LAH, THF, 1 h, 0 °C, 74%; (h) H2/Pd−C, MeOH, 1 h, 90%.
a
Scheme 3. Preparation of Mono-Methyl Derivative 17
a
Reagents and conditions (a) (i) SOCl2, reflux, 2 h; (ii) Meldrum’s acid, pyridine, DCM, 0−5 °C, 2 h; (iii) AcOH, H2O, reflux, 4 h, 28%; (b) Conc.
HCl, 1,4-dioxane, reflux, 2 h, 78%; (c) (i) SOCl2, reflux; (ii) NaN3, DCM; (d) benzene, reflux, 1.5 h, 92%; (e) 4-Cl-PhNH2, THF, reflux, 6 h, 70%.
a
Scheme 4. Preparation of Benzyl Derivative 23
a
Reagents and conditions: (a) (CH2OH)2, p-TSA, benzene, 4 h, 110 °C, Dean−Stark, 76%; (b) LAH, ether, 0 °C, 1.5 h, 60%; (c) DIPEA,
4-NO2-PhOCOCl, DCM/THF (1:1), rt, 24 h, 82%; (d) 4-Cl-PhNH2, DCM, rt, 10 h, 71%; (e) 2N HCl, THF, rt, 2 h, 91%.
38, respectively, using a two-step procedure via intermediates 33,
34, and 35.
Evaluation of Enzyme Inhibition. IMPDH activity was
assayed by monitoring the production of NADH. The IC50 values
reported in the tables are the average of three independent
experiments unless otherwise noted. Nonspecific protein binding
was assessed by measuring IC50 values in the presence of 0.05%
fatty acid free bovine serum albumin (BSA).20 Our previous
experience indicates that the IC50 in the presence of BSA is the
better predictor of antiparasitic activity.21
For the SAR study, initial attempts were made to optimize the
anilide substituent. Removal of the cyclic urea moiety in 1 yielded
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a
Scheme 5. Preparation of Carbamate and Amide Derivatives.
a
Reagents and conditions: (a) 8N HCl, 70 °C, 3 h, 55%; (b) 4-ClPhOCOCl, DIPEA, DCM/THF, rt, 24 h, 81%; (c) 4-Cl-PhCH2COCl, DCM, 0 °C
to rt, 80%; (d) (i) ClSO3H, CHCl3, 0 °C; (ii) KNO2; (iii) Water, pH = 2.8, 30%; (e) TEA, benzene, 4-Cl-Ph-NCO, 70 °C, 3 h, 62%.
a
Scheme 6. Preparation of Biphenyl Derivatives 36, 37, and Unsubstituted Benzylderivative 38
a
Reagents and conditions: (a) (i) 3 equiv. CH3MgBr, ether, rt, 30 min; (ii) Ti(i-PrO)4, 10 h, 57%; (b) (i) 2 equiv EtMgBr, Ti(i-PrO)4, −78 °C to rt,
1 h; (ii) 2 equiv BF3·OEt2, 1 h, 52%; (c) 4-NO2PhOCOCl, DIPEA, DCM/THF, 10 h; (d) 4-Cl-PhNH2, TEA, DCM, rt, 10 h, 71%.
compound 5a, which demonstrated a slight increase in CpIMPDH
inhibitory activity with an IC50 of 250 nM (Table 1). Replacing the
imidazolone with a 4-ClPh or 4-BrPh increased potency by 16-fold
(5b) and 32-fold (5c), respectively. A similar trend has also been
observed with other CpIMPDH inhibitor series.22 However,
translocation of the chlorine atom to the 3-position (5d) resulted
in a 3-fold loss of activity, while the 2-chloro derivative (5e) was
completely inactive. The electronic nature of the substituent in the
4-position had little effect on inhibitory activity. For example, an
electron-donating methoxy (5f) retained activity comparable to
the bromide (5c) with IC50 of 9 nM. However, sterics did have an
effect as illustrated by the 4-tert-butyl (5g) derivative lacking
activity. Introduction of substituents in the 3,4-positions increased
activity. For example, 3,4-dichloro (5h), 4-chloro-3-trifluoro-
methyl (5i), and a variety of 3-amido-4-chloro (5k, 5l, and 5m)
substituents increased potency by about 2.5 to 10-fold as
compared to 5b. Again, the electronic nature of the 3,4-substitents
had little effect as exemplified by 4-chloro-3-methoxy (5j) and
the 3,4-methylenedioxy (5n) analogues, although the latter
compound demonstrated significantly weaker activity in the
presence of BSA. Given these results, several fused carbo- and
heteroaromatic derivatives were examined. The 2-naphthyl
derivative (5o) retained potent activity, while the regioisomer
1-naphthyl was devoid of activity. However, the inhibitory activity
of 5o decreased in presence of BSA. Replacement of the
2-naphthyl with 6- and 7-quinolyl (5q and 5r) resulted in highly
potent compounds that were only modestly reduced in the
presence of BSA. The 2- and 3-quinolyl analogues (5t and 5s)
were less potent.
Next, the isoprenyl group was examined (Table 2). Saturation
of the alkene to the resulting isopropyl (11) or removal (38)
resulted in complete loss of inhibitory activity. Quite surprisingly,
replacement of the alkene with an alcohol (10) resulted in
moderate activity suggesting that polar substituents could be
tolerated at this position. Incorporation of the alkene into a
phenyl (36a) retained potent activity. However, transposing
the phenyl (36b) to the para-position resulted in complete
loss of inhibitory activity. Given the relative importance of the
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c
Table 2. SAR of the Isopropenyl Substituent
a
b
c
One determination. Two determinations. n.d., not determined.
sp2-hybridized substituent at the 3-position and moderate
tolerance of a hydrophilic alcohol in 10, replacement of the
alkene with a ketone was explored. In three cases examined
(6a−c), good to moderate inhibitory activity was observed.
The SAR of the central portion of 1 was interrogated next
(Table 3). First, the importance of the gem-dimethyl was
confirmed. For example, the monomethyl derivative 17 was less
active compared to 6a, and elimination of the methyl groups (23)
resulted in complete loss of activity. Incorporation of the two
methyl groups into a cyclopropane (37) was also detrimental.
Several changes to the urea were examined. For example,
N-methylation (5u) of the aniline portion of the urea and replace-
ment of the nitrogen with oxygen (25) or carbon (26) were
deleterious. Replacement of the benzylamine nitrogen with
oxygen (28) was better tolerated, demonstrating only a 2-fold
reduction in activity.
the O-methyloximes 8a, 8b, and 8c proved to be quite active with
IC50 ≤5 nM. O-Ethanolamine 9 also showed promising activity
with an IC50 value of 20 nM.
The introduction of hydrophilic groups in place of the isoprenyl
also resulted in improvements in mouse liver microsomal stability,
which was a potential liability for 5k and 5r (Table 5). The ketone
derivative 6c was very resistant to oxidative degradation in
microsomes. The oximes demonstrated a range of stabilities, with
7c having a t1/2 of 190 min. While the O-methyloxime 8a was
less stable, the aminoethyleneoxime derivative 9 showed excellent
mouse liver microsomal stability with t1/2 > 2 h. All of the
compounds examined demonstrated excellent mouse plasma
stability.
Evaluation of Antiparasitic Activity. Toxoplasma gondii is
an intracellular parasite closely related to Cryptosporidium that,
unlike C. parvum, can be continuously cultured in hTERT
immortalized human foreskin fibroblasts. We engineered a
T. gondii strain (Toxo/CpIMPDH) that relies on CpIMPDH,
and is sensitive to CpIMPDH inhibitors.21 In contrast, the wild-
type T. gondii strain RH (Toxo/WT) contains a typical eukaryotic
IMPDH and is resistant to CpIMPDH inhibitors. Therefore,
Finally, based on the encouraging results obtained with 6a
the ketone functional group was transformed into hydrophilic
oximes and O-methyloximes (Table 4). Gratifyingly, the
oximes (7a, 7b, 7c, 7d, and 7e) demonstrated excellent
CpIMPDH inhibitory potencies, with IC50 ≤1 nM. Likewise,
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b
Table 3. SAR of the Urea Moiety
a
b
One determination. n.d., not determined.
a
Table 4. SAR of Oxime and Methyloxime Analogues
Table 5. Mouse Liver Microsome and Plasma Stability
mouse microsomal stability
mouse plasma stability
compound
(t1/2, min)
(t1/2, min)
5k
5r
6c
7a
7b
7c
7e
8a
9
25
5.9
>700
88
>120
>120
>120
n.d.
IC50 (nM)
ID
X
R
(-) BSA
(+) BSA
11
n.d.
7a
7b
7c
7d
7e
8a
8b
8c
9
N−OH
N−OH
N−OH
N−OH
N−OH
N-OMe
N-OMe
N-OMe
3-CF3,4-Cl-Ph
3-NO2,4-Cl-Ph
3-CONH2, 4-Cl-Ph
2-Naphthyl
1.0 0.1
4
2
190
45
>120
>120
n.d.
0.66 0.08
2.0 0.4
5
1
6
2
33
1.0 0.2
0.9 0.2
2.3 0.6
1.2 0.3
121
n.d
7-Quinolyl
a
3-CF3, 4-Cl-Ph
3-CONH2, 4-Cl-Ph
2-Naphthyl
5
5
1
2
18
5
3
1
4
n.d., not determined.
1.6 0.1
20 3.4
13
Compounds 7a and 7e are also very promising candidates,
with EC50 values of 10 nM and 58 nM, respectively, and selec-
tivity ≥230.
N−O(CH2)2NH2 3-NO2,4-Cl-Ph
24 17
Toxo/WT serves as a proxy for host cell toxicity, as well as
provides important target validation.
CONCLUSION
■
Approximately thirty compounds with good inhibitory activity
against CpIMPDH were tested in the Toxoplasma model. Eleven
compounds had EC50 values ≤200 nM with selectivity ≥100-fold
over Toxo/WT. Gratifyingly, 7b, our best inhibitor in the
enzyme assay, also displayed the best antiparasitic activity with
EC50 of 6 nM and 670-fold selectivity over Toxo/WT (Table 6).
In this study, the SAR of the CpIMPDH inhibitor 1 was
investigated. Initially, the anilide portion of the molecule was
explored. Several derivatives containing 3,4-disubstitution or
3,4-ring fusion were found to significantly increase inhibitory
potency. The aniline nitrogen atom of the urea was necessary for
potent activity, while the other nitrogen could be replaced with
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a
septet at 39.5 ppm for samples in DMSO-d6. Coupling constants
(J values) are reported in hertz. Column chromatography was carried
out on SILICYCLE SiliaFlash silica gel F60 (40−63 μm, mesh 230−
400). High-resolution mass spectra were obtained using a Q-Tof Ultima
mass spectrometer (University of Illinois Urbana−Champaign, Urbana,
IL 61801). All melting points were taken in glass capillary tubes and are
uncorrected. All test compounds reported in this manuscript had a
purity ≥95% as determined by high-performance liquid chromatog-
raphy (HPLC) analyses using an instrument equipped with a quaternary
pump and a Zorbax SB-C8 column (30 × 4.6 mm, 3.5 μm). UV
absorption was monitored at λ = 254 nm. The injection volume was
5 μL. HPLC gradient went from 5% acetonitrile and 95% water to 95%
acetonitrile and 5% water (both solvents contain 0.1% trifluoroacetic
acid) over 1.9 min with a total run time of 2.5 min and a flow rate of
3.0 mL/min.
Table 6. Antiparasitic Activity of Selected Urea Compounds
EC50 (μM)
b
compound
Toxo/WT
Toxo/CpIMPDH
selectivity
5a
5b
5c
5d
5g
5h
5j
6
>25
>25
4
0.77 0.06
0.18 0.02
0.2 0.01
0.38 0.01
8
>140
>120
1.0
3
0.402 0.002
c
c
20
4
2
2
7.2 0.4
0.22 0.04
20
c
c
2.3 0.2
0.018 0.002
127
65
5k
5l
15
19
9
3
6
1
1
0.23 0.04
0.18 0.03
0.09 0.03
0.2 0.2
0.20 0.03
0.02 0.01
<0.2
100
100
50
5m
5n
5o
5q
5r
Synthesis of 3-Acetyl-α,α-dimethylbenzyl Isocyanate (3). A
solution of 3-isopropenyl-α,α-dimethyl benzyl isocyanate (2.04 g,
10.14 mmol) in dichloromethane (40 mL) was cooled to −78 °C and
then treated with dry ozone in oxygen until a blue color persisted.
Excess ozone was flushed off with oxygen. Dimethyl sulfide (0.74 mL,
10.14 mmol) was added to the reaction mixture, which was then stirred
overnight at room temperature. Excess Me2S was removed by evaporated
on a water bath placed inside a fume hood. Water (30 mL) was added to
the reaction mixture, which was then extracted with dichloromethane.
The combined organic layers were washed with brine (30 mL) and dried
over anhydrous MgSO4. The mixture was filtered and the filtrate
concentrated. The residue was purified by silica gel column chromatog-
raphy using ethyl acetate/hexane (1:10) as an eluent to furnish 5b
(1.19 g, 58%). 1H NMR (CDCl3, 400 MHz) δ 1.76 (s, 6H), 2.63 (s, 3H),
7.47 (t, J = 8 Hz, 1H), 7.67 (dd, J1 = 7.6 Hz, J2 = 2 Hz, 1H), 7.86 (dt, J1 =
7.6 Hz, J2 = 1.2 Hz, 1H), 8.04 (t, J = 2 Hz, 1H); 13C NMR (CDCl3, 100
MHz) δ 26.9, 33.2, 60.9, 124.2, 127.6, 129.0, 129.4, 137.4, 146.6, 198.1.
General Procedure for the Preparation of Urea Derivatives 5:
Exemplified for the preparation of 1-(4-Chlorophenyl)-3-(2-(3-(prop-
1-en-2-yl)phenyl)propan-2-yl)urea (5b). To a solution of 3-isopro-
penyl α,α-dimethylbenzyl isocyanate 2 (473 mg, 2.35 mmol) in
dichloromethane (6 mL) at 0 °C was added 4-chloroaniline (300 mg,
2.35 mmol) in dichloromethane (3 mL). The reaction was stirred until
complete consumption of starting materials. The precipitated product
was collected by filtration and washed with dichloromethane to give 5b
8
>25
>120
≥80
>15
2.3
0.9
16
7.6 0.8
3.0 0.1
c
5s
2
3
1
1
1.1 0.4
3.4 0.3
0.2 0.1
5t
6b
6c
7a
7b
7c
7d
7e
8a
8b
8c
9
3.2 0.7
>25
c
0.38 0.05
>66
250
670
>12
63
2.7 0.2
0.01 0.01
0.006 0.005
1.9 0.6
4
>25
5
2
4
6
0.08 0.01
0.058 0.003
0.016 0.008
0.2 0.1
14
230
86
1.4 0.4
>25
>120
180
20
2.3 0.8
10.1 1.9
0.013 0.009
0.51 0.007
0.9 0.4
10
17
23
36a
37
19
8
6
4
5
3
6
20
2.3 0.6
4
7
11
3
0.6
30
6
0.21 0.09
0.6 0.2
9
15
Assays as described in Methods.21 Unless otherwise stated, all values
are the average 3 independent determinations. Selectivity is the ratio
of EC50 Toxo/CpIMPDH to EC50 Toxo/WT. Two determinations.
a
1
b
(852 mg, 80%). mp 234−236 °C. Yield 80%; H NMR (DMSO-d6,
c
400 MHz) δ 1.58 (s, 6H), 2.08 (s, 3H), 5.06 (s, 1H), 5.36 (s, 1H), 6.64
(s, 1H), 7.20 (d, J = 6.4 Hz, 2H), 7.23−7.32 (m, 5H), 7.47 (s, 1H), 8.55
(s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.24, 30.30, 55.0, 113.06,
119.50, 122.35, 123.69, 124.86, 124.95, 128.65, 129.09, 140.11, 140.73,
143.60, 148.95, 154.53; ESI-HRMS for C19H22N2OCl (M+H)+ calcd.
329.1421, found 329.1418.
oxygen. The gem-dimethyl group was also found to be best for
CpIMPDH inhibition. The 3-isopropenyl substituent could be
replaced with a number of hydrophobic or hydrophilic groups,
but those with sp2-hybridization (e.g., phenyl, acetyl, oximes, or
O-methyloximes) were best. Eleven compounds displayed
potent and selective in vitro antiparastic activity, including two
that also demonstrated good in vitro metabolic stability. These
compounds appear to possess the necessary properties for
evaluation in an animal model of cryptosporidiosis in order to
determine the optimal pharmacological profile necessary for in
vivo efficacy.
1-Phenyl-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)urea (5a).
1
mp 188−190 °C; Yield 81%; H NMR (DMSO-d6, 400 MHz) δ 1.58
(s, 6H), 2.08 (s, 3H), 5.06 (s, 1H), 5.36 (s, 1H), 6.59 (s, 1H), 6.83 (t, J =
7.2 Hz, 1H), 7.15 (t, J = 8 Hz, 2H), 7.24−7.31 (m, 5H), 7.47 (s, 1H),
8.39 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.25, 30.39, 54.97,
113.05, 118.00, 121.55, 122.36, 123.65, 124.87, 128.64, 129.28, 140.70,
141.12, 143.62, 149.11, 154.71; ESI-HRMS for C19H23N2O (M+H)+
calcd. 295.1810, found 389.1815.
1-(4-Bromophenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-
urea (5c). Yield 78%; 1H NMR (DMSO-d6, 400 MHz) δ 1.61 (s, 6H),
2.10 (s, 3H), 5.08 (s, 1H), 5.38 (s, 1H), 6.66 (s, 1H), 7.28−7.35 (m,
5H), 7.49 (s, 1H), 8.57 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ
22.23, 30.29, 55.07, 112.76, 113.06, 119.96, 122.35, 123.70, 124.85,
128.65, 131.97, 140.53, 140.74, 143.62, 148.94, 154.50; ESI-HRMS for
C19H22N2OBr (M+H)+ calcd. 373.0915, found 373.0915.
1-(3-Chlorophenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-
urea (5d). Yield 83%; 1H NMR (DMSO-d6, 400 MHz) δ 1.59 (s, 6H),
2.09 (s, 3H), 5.07 (s, 1H), 5.37 (s, 1H), 6.70 (s, 1H), 6.89 (d, J = 7.6 Hz,
1H), 7.05 (d, J = 7.6 Hz 1H), 7.19 (t, J = 8 Hz, 1H), 7.28−7.30 (m, 3H),
7.48 (s, 1H), 7.60 (s, 1H), 8.63 (s, 1H); 13C NMR (DMSO-d6, 100
MHz) δ 22.23, 30.26, 55.08, 113.08, 116.41, 117.32, 121.15, 122.32,
123.71, 124.83, 128.67, 130.88, 133.78, 140.74, 142.63, 143.60, 148.88,
EXPERIMENTAL SECTION
■
Chemistry Materials and Methods. Unless otherwise noted, all
reagents and solvents were purchased from commercial sources and
used without further purification. All reactions were performed under
nitrogen atmosphere unless otherwise noted. The NMR spectra were
1
obtained using a 400 MHz spectrometer. All H NMR spectra are
reported in δ units ppm and are reference to tetramethylsilane (TMS) if
conducted in CDCl3 or to the central line of the quintet at 2.49 ppm for
samples in DMSO-d6. All chemical shift values are also reported with
multiplicity, coupling constants, and proton count. All 13C NMR spectra
are reported in δ units ppm and are reference to the central line of the
triplet at 77.23 ppm if conducted in CDCl3 or to the central line of the
7765
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Journal of Medicinal Chemistry
Article
154.44; ESI-HRMS for C19H22N2OCl (M+H)+ calcd. 329.1421, found
329.1430.
2-Chloro-N,N-dimethyl-5-(3-(2-(3-(prop-1-en-2-yl)phenyl)-
1
propan-2-yl)ureido)benzamide (5m). Yield 72%; H NMR (CDCl3,
400 MHz) δ 1.66 (s, 6H), 2.13 (s, 3H), 2.86 (s, 3H), 3.07 (s, 3H), 5.05
(s, 1H), 5.33 (s, 1H), 6.02 (s, 1H,), 6.88 (d, J = 2.4 Hz, 1H), 7.11 (d, J =
8.8 Hz, 2H), 7.25−7.35 (m, 4H), 7.50 (s, 1H), 8.01 (s, 1H); 13C NMR
(CDCl3, 100 MHz) δ 22.17, 29.89, 30.16, 34.94, 38.49, 55.13, 112.51,
116.93, 120.92, 121.29, 122.18, 123.81, 124.24, 128.33, 129.97, 135.15,
139.83, 141.27, 143.81, 147.93, 154.48, 169.80; ESI-HRMS for
C22H27N3O2Cl (M+H)+ calcd. 400.1792, found 400.1787.
1-(2-Chlorophenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-
urea (5e). Yield 65%; 1H NMR (DMSO-d6, 400 MHz) δ 1.62 (s, 6H),
2.1 (s, 3H), 5.08 (s, 1H), 5.38 (s, 1H), 6.91 (t, J = 7.2 Hz, 1H), 7.16 (t, J =
8.8 Hz, 1H), 7.30−7.32 (m, 3H), 7.37 (dd, 1H, J = 7.2 Hz, J2 = 3.2 Hz),
7.49−7.52 (m, 2H), 8.02 (dd, J1 = 7.8 Hz, J2 = 2 Hz), 8.09 (d, J = 2 Hz,
1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.22, 30.29, 55.17, 113.06,
121.08, 121.50, 122.32, 122.86, 123.69, 124.86, 127.99, 128.68, 129.67,
137.38, 140.71, 143.59, 148.92, 154.22; ESI-HRMS for C19H22N2OCl
(M+H)+ calcd. 329.1421, found 329.1430.
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(2-(3-(prop-1-en-2-yl)-
1
phenyl)propan-2-yl)urea (5n). Yield 75%; H NMR (DMSO-d6, 400
MHz) δ 1.59 (s, 6H), 2.10 (s, 3H), 4.15 (d, J = 7.6 Hz, 4H), 5.08 (s, 1H),
5.37 (s, 1H), 6.50 (s, 1H), 6.67 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 7.29−
7.31 (m, 3H), 7.49 (s, 1H), 8.22 (s, 1H); 13C NMR (DMSO-d6, 100
MHz) δ 22.22, 30.42, 54.93, 64.44, 64.82, 107.22, 111.36, 113.0, 117.31,
122.35, 123.60, 124.85, 128.60, 134.89, 138.31, 140.68, 143.62, 143.65,
149.17, 154.81; ESI-HRMS for C21H25N2O3 (M+H)+ calcd. 353.1865,
found 353.186.
1-(4-Methoxyphenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-
yl)urea (5f). Yield 85%; 1H NMR (DMSO-d6, 400 MHz) δ 1.59 (s, 6H),
2.10 (s, 3H), 3.66 (s, 3H), 5.08 (s, 1H), 5.37 (s, 1H), 6.50 (s, 1H), 6.77
(d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.29−7.31 (m, 3H), 7.49 (s,
1H), 8.22 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.23, 30.46,
54.92, 55.74, 113.01, 114.47, 119.64, 122.36, 123.60, 124.88, 128.61,
134.28, 140.67, 143.63, 149.25, 154.40, 154.95; ESI-HRMS for
C20H25N2O2 (M+H)+ calcd. 325.1916, found 325.1919.
1-(4-(tert-Butyl)phenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-
yl)urea (5g). Yield 87%; 1H NMR (DMSO-d6, 400 MHz) δ 1.20 (s, 9H),
1.58 (s, 6H), 2.09 (s, 3H), 5.06 (s, 1H), 5.36 (s, 1H), 6.54 (s, 1H), 7.15−
7.19 (m, 4H), 7.27−7.30 (m, 3H), 7.48 (s, 1H), 8.31 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 22.25, 30.45, 31.93, 34.42, 54.95, 113.02,
117.88, 122.37, 123.62, 124.87, 125.84, 128.62, 138.52, 140.70, 143.63,
143.79, 149.19, 154.82; ESI-HRMS for C23H31N2O (M+H)+ calcd.
351.2436, found 3512433.
1-(Naphthalen-2-yl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-
1
urea (5o). mp 201−203 °C; Yield 75%; H NMR (DMSO-d6, 400
MHz) δ 1.64 (s, 6H), 2.11 (s, 3H), 5.08 (s, 1H), 5.39 (s, 1H), 6.72 (s,
1H), 7.27−7.39 (m, 6H), 7.53 (d, J = 1.2 Hz, 1H), 7.68 (d, J = 8 Hz, 1H),
7.75 (d, J = 8.4 Hz, 2H), 7.98 (s, 1H), 8.64 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz) δ 22.23, 30.35, 55.04, 112.83, 113.06, 119.92,
122.37,123.69, 124.12, 124.90, 126.85, 127.38, 128.01, 128.67, 128.89,
129.29, 134.49, 138.73, 140.75, 143.63, 149.09, 154.78; ESI-HRMS for
C23H25N2O (M+H)+ calcd. 345.1967, found 345.1964.
1-(3,4-Dichlorophenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-
1-(Naphthalen-1-yl)-3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-
1
1
yl)urea (5h). mp 198−200 °C Yield 80%; H NMR (DMSO-d6, 400
urea (5p). mp 206−208 °C; Yield 75%; H NMR (DMSO-d6, 400
MHz) δ 1.61 (s, 6H), 2.10 (s, 3H), 5.09 (s, 1H), 5.38 (s, 1H), 6.79 (s,
1H), 7.13 (t, J = 4.8 Hz, 1H), 7.31−7.34 (m, 3H), 7.42 (d, J = 7.6 Hz,
1H), 7.49 (s, 1H), 7.79 (s, 1H), 8.79 (s, 1H); 13C NMR (DMSO-d6, 100
MHz) δ 22.23, 30.21, 55.14, 113.03, 118.13, 118.95, 122.31, 123.75,
124.82, 128.69, 131.07, 131.58, 140.76, 141.31, 141.34, 143.58, 148.79,
154.33; ESI-HRMS for C19H21N2OCl2 (M+H)+ calcd. 363.1031, found
363.1029.
MHz) δ 1.66 (s, 6H), 2.10 (s, 3H), 5.08 (s, 1H), 5.39 (s, 1H), 7.13 (s,
1H), 7.29−7.38 (m, 4H), 7.50−7.55 (m, 4H), 7.90 (d, J = 7.6 Hz, 1H),
7.87 (d, J = 8 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.54 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 22.24, 30.42, 55.16, 113.05, 116.51, 121.79,
122.40, 123.68, 124.92, 125.80, 126.01, 126.36, 126.52, 128.68, 129.06,
134.33, 135.77, 140.74, 143.61, 149.15, 155.02; ESI-HRMS for
C23H25N2O (M+H)+ calcd. 345.1967, found 345.1976.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-(3-(prop-1-en-2-yl)-
1-(2-(3-(Prop-1-en-2-yl)phenyl)propan-2-yl)-3-(quinolin-6-yl)urea
(5q). Yield 81%; 1H NMR (DMSO-d6, 400 MHz) δ 1.65 (s, 6H), 2.11 (s,
3H), 5.08 (s, 1H), 5.39 (s, 1H), 7.10 (s, 1H), 7.29−7.37 (m, 2H), 7.54
(s, 1H), 7.67−7.74 (m, 2H), 8.04 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 2 Hz,
1H), 8.33 (s, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.87 (d, J = 4 Hz, 1H), 9.39
(s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.25, 30.31, 55.14, 112.04,
113.07, 122.37, 122.45, 123.68, 124.87, 125.70, 125.75, 128.65, 130.06,
138.77, 138.79, 140.72, 140.75, 143.58, 145.04, 148.96, 154.68; ESI-
HRMS for C22H24N3O (M+H)+ calcd. 346.1919, found 346.1925.
1-(2-(3-(Prop-1-en-2-yl)phenyl)propan-2-yl)-3-(quinolin-7-yl)urea
(5r). Yield 72%; 1H NMR (DMSO-d6, 400 MHz) δ 1.64 (s, 6H), 2.11 (s,
3H), 5.08 (s, 1H), 5.39 (s, 1H), 6.79 (s, 1H), 7.28−7.37 (m, 4H), 7.47−
7.53 (m, 2H), 7.80 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 2 Hz, 1H), 8.18 (d, J =
1
phenyl)propan-2-yl)urea (5i). Yield 82%; H NMR (DMSO-d6, 400
MHz) δ 1.61 (s, 6H), 2.10 (s, 3H), 5.09 (s, 1H), 5.38 (s, 1H), 6.81 (s,
1H), 7.30−7.33 (m, 3H), 7.42−7.44 (m, 1H), 7.49−7.53 (m, 2H), 8.01
(s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 22.22, 30.22, 55.20, 113.11,
116.35, 116.40, 121.90, 122.12, 122.30, 122.78, 123.77, 124.81, 128.70,
132.50, 140.66, 140.78, 143.58, 148.73, 154.36; ESI-HRMS for
C20H21N2OClF3 (M+H)+ calcd. 397.1295, found 397.1296.
1-(4-Chloro-3-methoxyphenyl)-3-(2-(3-(prop-1-en-2-yl)phenyl)-
propan-2-yl)urea (5j). Yield 89%; 1H NMR (DMSO-d6, 400 MHz) δ
1.59 (s, 6H), 2.09 (s, 3H), 3.73 (s, 3H), 5.07 (s, 1H), 5.37 (s, 1H), 6.66
(s, 1H), 6.72 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.8 Hz,1H), 7.28−7.32 (m,
4H), 7.48 (s, 1H), 8.59 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ
22.23, 30.34, 55.03, 56.28, 102.50, 110.59, 112.95, 113.10, 122.33,
123.72, 124.85, 128.70, 130.16, 140.75, 141.47, 143.59, 148.97, 154.53,
155.10; ESI-HRMS for C20H24N2O2Cl (M+H)+ calcd. 359.1529, found
359.1532.
2-Chloro-5-(3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)ureido)-
benzamide (5k). Yield 72%; 1H NMR (DMSO-d6, 400 MHz) δ 1.57 (s,
6H), 2.06 (s, 3H), 5.04 (s, 1H), 5.34 (s, 1H), 6.65 (s, 1H), 7.22−7.27
(m, 5H), 7.44−7.48 (m, 3H), 7.75 (s, 1H), 8.61 (s,1H); 13C NMR
(DMSO-d6, 100 MHz) δ 22.23, 30.27, 55.08, 113.08, 117.61, 119.67,
121.36, 122.31, 123.71, 124.82, 128.66, 130.33, 137.89, 139.95, 140.73,
143.59, 148.89, 154.45, 168.85; ESI-HRMS for C20H23N3O2Cl (M+H)+
calcd. 372.1479, found 372.1485.
2-Chloro-N-methyl-5-(3-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-
yl)ureido)benzamide (5l). Yield 72%; 1H NMR (CDCl3, 400 MHz) δ
1.64 (s, 6H), 2.17 (s, 3H), 2.89 (d, J = 4.8 Hz, 3H), 5.05 (s, 1H), 5.32 (s,
1H), 5.97 (s, 1H,), 6.61 (d, J = 4.4 Hz, 1H), 7.10−7.13 (m, 2H), 7.27−
7.31 (m, 2H), 7.49−7.51 (m, 3H), 7.81 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) δ 22.25, 26.56, 30.26, 55.08, 113.09, 117.76, 119.75, 121.57,
122.31, 123.71, 124.82, 128.66, 130.32, 137.80, 140.01, 140.74, 143.59,
148.90, 167.41, 189.22; ESI-HRMS for C21H25N3O2Cl (M+H)+ calcd.
386.1635, found 386.1639.
8.4 Hz, 1H), 8.75 (dd, J1 = 4.4 Hz, J2 = 1.6 Hz, 1H), 8.85 (s, 1H); 13
C
NMR (DMSO-d6, 100 MHz) δ 22.23, 30.30, 55.16, 113.09, 113.66,
119.69, 120.23, 122.37, 123.73, 124.88, 128.69, 128.98, 136.03, 140.77,
142.06, 143.61, 148.92, 149.48, 151.27, 154.55; ESI-HRMS for
C22H24N3O (M+H)+ calcd. 346.1919, found 346.1915.
1-(2-(3-(Prop-1-en-2-yl)phenyl)propan-2-yl)-3-(quinolin-3-yl)urea
(5s). Yield 69%; 1H NMR (DMSO-d6, 400 MHz) δ 1.65 (s, 6H), 2.11
(s, 3H), 5.09 (s, 1H), 5.39 (s, 1H), 6.88 (s, 1H), 7.31−7.38 (m, 3H),
7.49−7.55 (m, 3H), 7.79 (d, J = 7.2 Hz, 1H), 7.88 (d, J = 7.4 Hz, 1H),
8.41 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.91 (s, 1H); 13C NMR(DMSO-d6,
100 MHz) δ 22.24, 30.25, 55.17, 113.11, 119.44, 122.35, 123.75, 124.89,
127.27, 127.49, 127.81, 128.71, 128.90, 129.10, 134.88, 140.77, 143.60,
143.82, 144.47, 148.88, 154.72; ESI-HRMS for C22H24N3O (M+H)+
calcd. 346.1919, found 346.1924.
1-(2-(3-(prop-1-en-2-yl)phenyl)propan-2-yl)-3-(quinolin-2-yl)urea
(5t). mp 126−128 °C; Yield 75%; 1H NMR (CDCl3, 400 MHz) δ 1.90
(s, 6H), 2.13 (s, 3H), 5.05 (s, 1H), 5.37 (s, 1H), 6.54 (d, J = 8.8 Hz, 1H),
7.25−7.38 (m, 3H), 7.48 (d, J = 7.6 Hz, 1H), 7.57−7.67 (m, 4H), 7.83
(d, J = 8.8 Hz, 1H), 9.69 (s, 1H), 10.92 (s, 1H); 13C NMR (CDCl3, 100
MHz) δ 22.20, 30.11, 55.62, 113.19, 114.22, 122.47, 123.95, 124.71,
124.84, 124.89, 126.57, 128.42, 128.82, 130.71, 139.03, 140.94, 143.57,
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145.55, 148.65, 153.54, 154.10; ESI-HRMS for C22H24N3O (M+H)+
calcd. 346.1919, found 346.1917.
11.18 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 12.4, 30.13, 55.12,
113.84, 116.26, 122.42, 122.86, 124.27, 125.84, 128.79, 132.28, 137.36,
141.11, 148.14, 148.67, 153.74, 154.12; ESI-HRMS for C18H20N4O4Cl
(M+H)+ calcd. 391.1173, found 391.1172.
1-(4-Chlorophenyl)-1-methyl-3-(2-(3-(prop-1-en-2-yl)phenyl)-
propan-2-yl)urea (5u). Yield 56%; 1H NMR (DMSO-d6, 400 MHz) δ
1.50 (s, 6H), 2.06 (s, 3H), 3.11 (s, 3H), 5.04 (s, 1H), 5.32 (s, 1H), 6.11
(s, 1H), 7.21−7.25 (m, 5H), 7.34−7.40 (m, 3H); 13C NMR (DMSO-d6,
100 MHz) δ 22.25, 30.28, 37.49, 55.72, 112.90, 122.26, 123.40, 124.81,
127.92, 128.49, 129.50, 129.58, 140.54, 143.64, 144.18, 149.37, 156.06;
ESI-HRMS for C20H24N2OCl (M+H)+ calcd. 343.1577, found
343.1570.
General Procedure for the Preparation of Urea Derivatives 6:
Exemplified for the Preparation of 5-(3-(2-(3-acetylphenyl)propan-
2-yl)ureido)-2-chlorobenzamide (6c). To a solution of 3-acetyl-α,α-
dimethyl isocyanate 3 (118 mg, 0.584 mmol) in THF at room tem-
perature was added 5-amino-2-chlorobenzamide (100 mg, 0.584 mmol).
The reaction was heated to 70 °C for 6 h. Volatiles were removed under
reduced pressure, and the residue was purified by column chromatog-
raphy using methanol/chloroform as eluent to obtain urea derivative 6c
(156 mg, 72%). 1H NMR (DMSO-d6, 400 MHz) δ 1.61 (s, 6H), 2.58
(s, 3H), 6.80 (s, 1H), 7.26 (s, 2H), 7.45−7.52 (m, 3H), 7.66 (d, J =
7.2 Hz, 1H), 7.79−7.83 (m, 2H), 7.94 (s, 1H), 8.68 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 27.44, 30.19, 54.89, 117.63, 119.72, 121.43,
124.57, 127.12, 129.11, 130.42, 137.24, 137.89, 139.87, 149.54, 154.40,
168.87, 198.76; ESI-HRMS for C25H16N3O (M+H)+ calcd. 374.1293,
found 374.1283.
(E)-1-(2-(3-(1-(Hydroxyimino)ethyl)phenyl)propan-2-yl)-3-(naph-
1
thalen-2-yl)urea (7d). Yield 85%; H NMR (DMSO-d6, 400 MHz) δ
1.69 (s, 6H), 2.21 (s, 3H), 6.81 (s, 1H), 7.35−7.50 (m, 6H), 7.72−7.80
(m, 4H), 8.05 (s, 1H), 8.70 (s, 1H), 11.22 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) δ 12.40, 30.32, 54.90, 112.75, 119.86, 122.49, 124.11, 124.16,
125.93, 126.85, 127.37, 128.00, 128.74, 128.90, 129.26, 134.48, 137.32,
138.70, 149.12, 153.77, 154.66; ESI-HRMS for C22H24N3O2 (M+H)+
calcd. 362.1869, found 362.1872.
(E)-1-(2-(3-(1-(Hydroxyimino)ethyl)phenyl)propan-2-yl)-3-(quino-
lin-7-yl)urea (7e). mp 164−166 °C; Yield 85%; 1H NMR (pyridine-d5,
400 MHz) δ 1.68 (s, 6H), 2.28 (s, 3H), 6.99−7.02 (m, 2H), 7.25 (t, J =
8 Hz, 1H), 7.53−7.62 (m, 3H), 7.88 (t, J = 9.6 Hz, 2H), 8.16
(s, 1H), 8.63 (s, 1H), 8.77 (d, J = 1.6 Hz, 1H), 9.54 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 12.41, 30.29, 55.03, 113.61, 119.70, 120.20,
122.50, 123.78, 124.22, 125.93, 128.77, 129.01, 136.04, 137.35, 142.06,
148.97, 149.4, 151.29, 153.77, 154.47; ESI-HRMS for C21H23N4O2
(M+H)+ calcd. 363.1821, found 363.1825.
General Procedure for the Preparation of Alkoxy Oxime
Derivatives 8: Exemplified for the Preparation of (E)-2-chloro-5-(3-
(2-(3-(1-(methoxyimino)ethyl)phenyl)propan-2-yl)ureido)-
benzamide (8b). Methoxyamine hydrochloride (30 mg, 0.362 mmol)
was added to a solution of 6b (100 mg, 0.302 mmol) in 3 mL of pyridine.
The reaction solution was heated to 90 °C for 2 h, and then, the pyridine
was removed by evaporation under reduced pressure. The residue was
dissolved in methanol and purified by column chromatography eluting
1-(2-(3-Acetylphenyl)propan-2-yl)-3-(4-chlorophenyl)urea (6a).
1
Yield 85%; H NMR (DMSO-d6, 400 MHz) δ 1.57 (s, 6H), 2.52 (s,
3H), 6.71 (s, 1H), 7.17 (d, J = 9.2 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 7.42
(t, J = 7.6 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.91
(s, 1H), 8.54 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 29.8, 32.6,
57.2, 121.9, 127.0, 127.4, 129.4, 131.5, 131.55, 132.8, 139.7, 142.4, 152.0,
156.9, 201.1; ESI-HRMS for C18H19ClN2O2 (M+H)+: calcd. 331.1213,
found 331.1214.
1-(2-(3-Acetylphenyl)propan-2-yl)-3-(4-chloro-3-(trifluoromethyl)-
phenyl)urea (6b). Yield 69%; 1H NMR (DMSO-d6, 400 MHz) δ 1.62
(s, 6H), 2.51 (s, 3H), 6.91 (s, 1H), 7.42−7.53 (m, 3H), 7.68 (d, J =
7.2 Hz, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.95 (s, 1H), 8.01 (s, 1H),
8.95 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 27.45, 30.14, 54.99,
116.36, 116.41, 116.47, 121.96, 122.83, 124.56, 127.17, 129.15, 130.42,
132.50, 137.28, 140.58, 149.37, 154.31, 198.71; ESI-HRMS for
C19H19N2O2ClF3 (M+H)+ calcd. 399.1087, found 399.1089.
1
with methanol/chloroform to obtained 8b (99 mg, 82%). H NMR
(CDCl3, 400 MHz) δ 1.55 (s, 6H), 2.12 (s, 3H), 3.91 (s, 3H), 6.18 (s,
1H), 6.59 (s, 1H), 6.86 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.15 (s, 1H),
7.25 (d, J = 6.8 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.6 Hz,
2H), 7.67 (1H, s), 7.97 (s, 1H); 13C NMR (CDCl3, 100 MHz) δ 13.29,
30.23, 54.97, 62.17, 117.68, 119.75, 121.42, 122.77, 124.49, 126.46,
128.83, 130.33, 136.30, 137.89, 139.93, 149.10, 154.43, 155.037, 168.88;
ESI-HRMS for C20H24N4O3Cl (M+H)+ calcd. 403.1537, found
403.1532.
(E)-1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-(3-(1-
(methoxyimino)ethyl)phenyl)propan-2-yl)urea (8a). 1H NMR
(DMSO-d6, 400 MHz) δ 1.62 (s, 6H), 2.1 (s, 3H), 3.90 (s. 3H), 6.84
(s, 1H), 7.36 (dd, J1 = 7.6 Hz, J2 = 1.6 Hz, 1H), 7.43−7.48 (m, 3H), 7.52
General Procedure for the Preparation of Oxime Derivatives 7:
Exemplified for the Preparation of (E)-2-Chloro-5-(3-(2-(3-(1-
(hydroxyimino)ethyl)phenyl)propan-2-yl)ureido)benzamide (7c).
Hydroxylamine hydrochloride (25 mg, 0.362 mmol) was added to a
solution of 6 (100 mg, 0.302 mmol) in 3 mL of pyridine. The reaction
solution was heated to 90 °C for 2 h. The reaction was allowed to cool to
room temperature, and then, the pyridine was removed by evaporation
under reduced pressure. The resulting residue was dissolved in methanol
and purified by column chromatography eluting with methanol/
(dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.67 (s, 1H), 8.01 (s, 1H), 8.93 (s, 1H); 13
C
NMR (DMSO-d6, 100 MHz) δ 12.80, 29.86, 55.30, 62.04, 117.77,
117.82, 117.88, 122.29, 122.87, 124.88, 124.93, 125.63, 128.82, 131.77,
136.96, 137.96, 147.31, 154.63, 154.91; ESI-HRMS for
C20H22N3O2F3Cl (M+H)+ calcd. 428.1353, found 428.1353.
(E)-1-(2-(3-(1-(Methoxyimino)ethyl)phenyl)propan-2-yl)-3-(naph-
1
thalen-2-yl)urea (8c). Yield 58%; H NMR (DMSO-d6, 400 MHz) δ
1.65 (s, 6H) 2.19 (s, 3H), 3.90 (s, 3H), 6.78 (s, 1H), 7.30−7.42 (4H, m),
7.48 (d, J = 7.2 Hz, 2H), 7.68−7.72 (m, 2H), 7.76 (d, J = 8.4 Hz, 2H),
8.00 (s, 1H), 8.67 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 13.30,
30.30, 54.91, 62.16, 112.8, 119.9, 122.8, 124.1, 124.4, 126.5, 126.8, 127.3,
128.0, 128.83, 128.89, 129.28, 134.47, 136.29, 138.68, 149.28, 154.71,
155.06; ESI-HRMS for C23H26N3O2 (M+H)+ calcd. 376.2025, found
376.2026.
Preparation of (E)-1-(2-(3-(1-((2-Aminoethoxy)imino)ethyl)-
phenyl)propan-2-yl)-3-(4-chloro-3-nitrophenyl)urea (9). (E)-1-(4-
Chloro-3-nitrophenyl)-3-(2-(3-(1-(hydroxyimino)ethyl)phenyl)-
propan-2-yl)urea (7b, 100 mg, 0.25 mmol) in 3 mL dry DMF was added
dropwise at 0 °C to sodium hydride dispersion in mineral oil (15.3 mg,
0.50 mmol). The resulting mixture was stirred at 0 °C for 30 min.
2-Chloroethylamine hydrochloride (29.6 mg, 0.25 mmol) in 2 mL DMF
was added and the reaction mixture was stirred at room temperature for
2 h, and then the volatiles were removed under reduced pressure. The
residue was dissolved in a minimal amount of methanol and purified by
column chromatography using methanol/chloroform to obtained 9
(49 mg, 46%). 1H NMR (DMSO-d6, 400 MHz) δ 1.61 (s, 6H), 2.19 (s,
3H), 2.79 (t, J = 5.6 Hz, 2H), 4.05 (t, J = 5.6 Hz, 2H), 6.95 (s, 1H), 7.34
(t, J = 7.6 Hz, 1H), 7.42−7.47 (m, 3H), 7.56 (d, J = 8.8 Hz, 1H), 7.65 (s,
1H), 8.20 (d, J = 2 Hz, 1H), 9 (s, 1H); 13C NMR (DMSO-d6, 100 MHz)
1
chloroform to obtained 7c (88 mg, 85%). H NMR (DMSO-d6, 400
MHz) δ 1.54 (s, 6H), 2.11 (s, 3H), 6.67 (s, 1H), 7.20−7.49 (m, 7H),
7.67 (s, 1H), 7.76 (s, 1H), 8.62 (s, 1H), 11.13 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 12.42, 30.27, 54.98, 117.59, 119.67, 121.38,
122.44, 124.21, 125.87, 128.76, 130.35, 137.33, 137.90, 139.96, 148.95,
153.79, 154.39, 168.90; ESI-HRMS for C19H22N4O3Cl (M+H)+ calcd.
389.1380, found 389.1384.
(E)-1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-(3-(1-
(hydroxyimino)ethyl)phenyl)propan-2-yl)urea (7a). mp 194−196 °C;
Yield 71%; 1H NMR (DMSO-d6, 400 MHz) δ 1.61 (s, 6H), 2.15 (s, 3H),
6.82 (s, 1H), 7.34 (d, J = 8 Hz, 1H), 7.39−7.46 (m, 3H), 7.52 (d, J =
9.2 Hz, 1H), 7.70 (s, 1H), 8.01 (s, 1H), 8.91(s, 1H), 11.17 (bs, 1H); 13C
NMR (DMSO-d6, 100 MHz) δ 12.39, 30.19, 55.07, 116.31, 116.37,
116.42, 121.91, 122.44, 122.76, 124.25, 125.84, 128.78, 132.50, 137.36,
140.64, 149.77, 153.74, 154.29; ESI-HRMS for C19H20N3O2ClF3
(M+H)+ calcd. 414.1196, found 414.1191.
(E)-1-(4-Chloro-3-nitrophenyl)-3-(2-(3-(1-(hydroxyimino)ethyl)-
phenyl)propan-2-yl)urea (7b). mp 183−185 °C; Yield 80%; 1H NMR
(DMSO-d6, 400 MHz) δ 1.62 (s, 6H), 2.16 (s, 3H), 6.91 (s, 1H), 7.35 (t,
J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.41−7.47 (m, 3H), 7.58 (d,
J = 8.8 Hz, 1H), 7.71 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 9.04 (s, 1H),
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δ 13.3, 30.1, 41.6, 55.4, 76.6, 113.8, 116.2, 122.7, 122.9, 124.5, 126.3,
128.8, 132.2, 136.5, 141.1, 148.1, 148.8, 154.2, 154.9; ESI-HRMS for
C20H24Cl N5O4 (M+H)+ calcd. 434.1595, found 434.1595.
heated at 80 °C for 2 h. The reaction mixture was concentrated under
reduced pressure. The resulting acid chloride was used without further
purification.
A solution of the acid chloride (84 mg, 0.40 mmol) in 3 mL dry
acetone was added to a solution of sodium azide (325 mg, 5 mmol) in
2 mL of water at 0 °C over 10 min. The reaction mixture was stirred for
2 h at 25 °C, and then poured into ice and extracted with ether (2 ×
10 mL). The organic extracts were combined, washed with brine, dried
over anhydrous MgSO4, filtered, and concentrated to give acyl azide 15,
which was used without further purification.
1-(3-(1-Isocyanatoethyl)phenyl)ethanone (16). Acyl azide 15
(80 mg, 0.42 mmol) was refluxed in benzene (5 mL) of 1.5 h, and
then the solvent was removed under vacuum to give isocyanate 16 in
quantitative yield, which was used without further purification.
1-(1-(3-Acetylphenyl)ethyl)-3-(4-chlorophenyl)urea (17). Com-
pound 17 (71%) was prepared following the general procedure for 6.
1H NMR (DMSO-d6, 400 MHz) δ 1.35 (d, J = 6.8 Hz, 3H), 2.55 (s, 3H),
4.95 (pent, J = 6.8 Hz, 1H), 5.68 (d, J = 7.2 Hz, 1H), 7.12−7.17 (m, 4H),
7.32−7.36 (m, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H),
7.84 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 23.20, 26.97, 49.77,
120.94, 124.96, 127,84, 128.14, 129.14, 129.16, 131.40, 137.47, 137.58,
145.23, 155.23, 199.09; ESI-HRMS for C17H18N2O2Cl (M+H)+ calcd.
317.1057, found 317.1060.
3-(2-Methyl-1,3-dioxolan-2-yl)benzonitrile (19). To the mixture of
ethylene glycol (0.22 mL, 4 mmol), 3-acetylbenzonitrile 18 (300 mg,
2.04 mmol), and benzene (10 mL) in a Dean−Stark apparatus was
added a catalytic amount of p-TSA (0.1 equiv). The reaction mixture
was heated at 110 °C for 4 h. The benzene was removed under reduced
pressure, and the residue was purified by column chromatography using
ethylacetate−hexane as an eluent to give 19 (293 mg, 76%). 1H NMR
(CDCl3, 400 MHz) δ 2.62 (s, 4H), 7.59 (t, J = 7.6 Hz, 1H), 7.82 (d, J =
7.6 Hz, 1H)), 8.15 (d, J = 8 Hz, 1H), 8.21 (s, 1H).
1-(4-Chlorophenyl)-3-(2-(3-(1-hydroxyethyl)phenyl)propan-2-yl)-
urea (10). A solution of 1-(2-(3-acetylphenyl)propan-2-yl)-3-(4-
chlorophenyl)urea (25 mg, 0.07 mmol) in THF was cooled in an ice
bath to 0 °C. Lithium aluminum hydride solution (2 M in THF, 0.8 equiv)
was added dropwise over 5 min, and then the reaction was continued
for approximately 1 h at 0 °C until the starting material disappeared.
The reaction was carefully quenched with a solution of sodium sulfate.
The reaction mixture was filtered through a sintered funnel and the
supernatant washed with dichloromethane. Combined organic fractions
were concentrated under reduced pressure. The residue was purified by
column chromatography using chloroform−methanol as eluent to give 10
(19 mg, 74%). 1H NMR (DMSO-d6, 400 MHz) δ 1.25 (d, J = 6 Hz, 3H),
1.54 (s, 6H), 4.64 (pent, J = 4.4 Hz, 1H), 5.09 (d, J = 4 Hz, 1H), 6.56 (s,
1H), 7.11−7.19 (m, 5H), 7.28 (s, 1H), 7.31 (d, J = 9.2 Hz, 1H), 8.51 (s,
1H); 13C NMR (DMSO-d6, 100 MHz) δ 26.78, 30.33, 30.44, 55.06, 68.94,
119.47, 122.42, 123.70, 124.88, 128.28, 129.10, 140.18, 147.68, 148.65,
154.48; ESI-HRMS for C18H22N2O2Cl (M+H)+ calcd. 333.1370, found
333.1378.
1-(2-(3-Isopropylphenyl)propan-2-yl)-3-(naphthalen-2-yl)urea
(11). A solution of 1-(naphthalen-2-yl)-3-(2-(3-(prop-1-en-2-yl)-
phenyl)propan-2-yl)urea (5o, 25 mg, 0.072 mmol) in methanol (3 mL)
containing a catalytic amount of 10% Pd/C was placed under an atmos-
phere of hydrogen. After 1 h, the reaction mixture was filtered through
a short silica gel column and concentrated to give 11 (24 mg, 98%).
1H NMR (DMSO-d6, 400 MHz) δ 1.17 (d, J = 4.4 Hz, 6H), 1.60 (s, 6H),
2.84 (hept, J = 6.8 Hz, 1H), 6.66 (s, 1H), 7.03−7.36 (m, 7H), 7.66 (d, J =
8 Hz, 1H), 7.75 (d, J = 8 Hz, 2H); 13C NMR (DMSO-d6, 100 MHz) δ
24.69, 30.42, 34.26, 55.09, 122.75, 118.22, 119.91, 123.01, 123.58, 124.08,
124.31, 126.84, 127.36, 128.01, 128.59, 128.87, 129.25, 134.49, 138.80,
149.00, 154.78; ESI-HRMS for C23H27N2O (M+H)+ calcd. 347.2123,
found 347.2126.
3-(2-Methyl-1,3-dioxolan-2-yl)phenylmethanamine (20). A solu-
tion of cyano ketal 19 (290 mg, 1.53 mmol) in dry THF (10 mL) was
cooled to 0 °C under a nitrogen atmosphere. Then, a solution of 2 M
lithium aluminum hydride (3 mmol) in THF was added over a 10 min
period. The reaction mixture was stirred for 1.5 h, and then ethyl acetate
was added followed by slow addition of water to decompose the excess
LAH. The reaction mixture was concentrated under reduced pressure.
The residue was dissolved in chloroform, washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated. The residue was purified
by column chromatography using chloroform and methanol as an eluent
2-(3-Acetylphenyl)propanenitrile (13). Thionyl chloride (10 mL)
was added at 0 °C to 3-(1-cyanoethyl)benzoic acid (12, 500 mg,
2.85 mmol). The reaction mixture was heated at 80 °C for 2 h. The
excess thionyl chloride was removed to give 3-(1-cyanoethyl)benzoyl
chloride, which was used without further purification.
Next, to a solution of Meldrum’s acid (408 mg, 2.84 mmol) in
dichloromethane (15 mL) at 0 °C was added pyridine (0.457 mL,
5.68 mmol). The resulting mixture was stirred for 15 min and then
3-(1-cyanoethyl)benzoyl chloride (482.5 mg, 2.5 mmol) was added.
The reaction mixture was stirred at 0 °C for 30 min and then for 1 h at
room temperature. The reaction mixture was diluted with dichloro-
methane and washed with 1 N HCl. The organic layer was dried over
anhydrous magnesium sulfate, filtered, and concentrated under reduced
pressure. The crude product was dissolved in AcOH−H2O (1:2) and
heated at reflux for 4 h. The reaction mixture was diluted with water and
extracted with ethyl acetate (3 × 30 mL). The combined organic extracts
were washed with NaHCO3 solution and brine, then dried over
anhydrous magnesium sulfate, filtered, and concentrated. The residue
was purified by column chromatography using hexane−ethyl acetate
(9:1) as eluent to give 13 (138 mg, 28%). 1H NMR (CDCl3, 400 MHz)
δ 1.68 (d, J = 7.2 Hz 3H), 2.63 (s, 3H), 3.99 (q, J = 7.2 Hz, 1H), 6.60 (d,
J = 7.6 Hz, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.91 (t,
J = 2 Hz, 1H), 7.94 (s, 1H).
2-(3-Acetylphenyl)propanoic Acid (14). To a solution of 13 (100 mg,
0.57 mmol) in 2 mL of 1,4-dioxane was added conc. HCl (1.5 mL), and
then the resulting mixture was refluxed for 5 h. After the mixture was
allowed to cool to room temperature, the volatiles were removed under
reduced pressure. The residue was diluted with water (10 mL) and
extracted with dichloromethane (3 × 10 mL). The organic extracts were
combined, washed with brine (2 × 10 mL), dried over anhydrous
magnesium sulfate, filtered, and concentrated to give 14 as a white solid
(86 mg, 78% yield). 1H NMR (CDCl3, 400 MHz) δ 1.57 (d, J = 7.2 Hz,
3H), 2.38 (s, 3H), 3.83 (q, J = 7.2 Hz, 1H), 7.42−7.50 (m, 2H), 7.61
(d, J = 7.6 Hz, 1H), 7.75 (s, 1H).
1
to give 20 (176 mg, yield 60%). H NMR (CDCl3, 400 MHz) δ 1.66
(s, 3H), 3.79 (t, J = 6 Hz, 2H), 3.88 (s, 2H), 4.04 (t, J = 6 Hz, 2H), 7.24−
7.42 (m, 4H).
4-Nitrophenyl 3-(2-Methyl-1,3-dioxolan-2-yl)benzylcarbamate
(21). To a solution of benzylamine 20 (176 mg, 0.911 mmol) and
N,N-diisopropylethylamine (313 μL, 1.8 mmol) in 4 mL of 1:1 CH2Cl2/
THF was added a solution of 4-nitrophenylchloroformate (366 mg,
1.82 mmol) in 2 mL of 1:1 CH2Cl2/THF. After stirring the reaction
mixture at room temperature for 24 h, it was diluted with dichloro-
methane and washed sequentially with saturated NaHCO3, water, and
brine. The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified by column chromatography
eluting with ethylacetate/hexane to give 21 (267 mg, 82%).
1-(4-Chlorophenyl)-3-(3-(2-methyl-1,3-dioxolan-2-yl)benzyl)urea
(22). 4-Nitrophenyl-N-benzylcarbamate 21 (267 mg, 0.767 mmol) was
added to a solution of 4-chloroaniline (97 mg, 0.767 mmol) and
triethylamine in dichloromethane (5 mL). The mixture was stirred at
room temperature until starting materials were consumed. The reaction
mixture was then diluted with dichloromethane (50 mL) and washed
with aq. NaOH, water, and brine. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated. The residue was purified
by column chromatography eluting with methanol/chloroform to give
22 (165 mg, 71%). 1H NMR (CDCl3, 400 MHz) δ 1.58 (s, 3H), 3.71 (t,
J = 6.8 Hz, 2H), 3.99 (t, J = 6.8 Hz, 2H), 4.36 (d, J = 5.6 Hz, 2H), 6.78 (s,
1H), 7.16−7.18 (m, 4H), 7.23−7.26 (m, 1H), 7.35 (d, J = 5.6 Hz, 2H).
1-(3-Acetylbenzyl)-3-(4-chlorophenyl)urea (23). A 2 N HCl
solution was added to a solution of 22 (165 mg, 0.479 mmol) in THF
(2 mL). The mixture was refluxed for several hours until the starting
2-(3-Acetylphenyl)propanoyl Azide (15). Thionyl chloride (2 mL)
was added to 14 (86 mg, 0.40 mmol) at 0 °C. The mixture was then
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materials were consumed. The reaction mixture was allowed to cool to
room temperature, quenched with solid NaHCO3, and then the volatiles
were removed under reduced pressure. The residue was diluted with
ethyl acetate and then washed with water. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated. The residue was
chromatography eluting with ethyl acetate/hexane to obtained 26
(26 mg, ∼30%). 1H NMR (CDCl3, 400 MHz) δ 1.55 (s, 6H), 2.55 (s,
3H), 7.37 (t, J = 8 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.76 (d, J = 7.6 Hz,
1H), 8.04 (s, 1H). 13C NMR (CDCl3, 400 MHz) δ 26.9, 31.9, 72.5,
124.4, 126.9, 128.6, 129.6, 137.1, 150.0, 198.8.
1
purified by chromatography to give 23 (144 mg, 100%). H NMR
2-(3-Acetylphenyl)propan-2-yl(4-chlorophenyl)carbamate (28).
To a solution of 3-acetyl-α,α-dimethylbenzylalcohol (27, 21 mg,
0.117 mmol) in 2 mL dry benzene was added 4-chlorophenyl isocyante,
followed by 17 μL triethyamine. The reaction mixture was heated
to 70 °C for 3 h. The reaction mixture was allowed to cool to room
temperature and then diluted with ethyl acetate (20 mL), washed with
1 N HCl, brine, and water. The organic layer was dried over anhydrous
magnesium sulfate, filtered, and evaporated. The residue was purified by
column chromatography eluting with ethyl acetate/hexane yielding 28
(23 mg, 62%). 1H NMR (CDCl3, 400 MHz) δ 1.84 (s, 6H), 2.60 (s, 3H),
6.71 (s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.45 (t, J =
8.0 Hz), 7.62 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 8.02 (s, 1H);
13C NMR (CDCl3, 100 MHz) δ 26.96, 29.08, 81.73, 119.8, 127.74,
128.90, 129.15, 129.26, 129.92, 136.67, 137.39, 146.71, 151.89, 198.29;
ESI-HRMS for C18H18ClNO3 (M+Na)+ calcd. 354.0873, found
354.0872.
3-Phenyl α,α-dimethylbenzylamine (30). To a room temperature
stirring solution of 3-cyanobiphenyl (200 mg, 1.11 mmol) in 5 mL of
diethyl ether was added 1 M methylmagnesium bromide in butyl ether
(3.33 mmol). The reaction mixture was stirred for 30 min and then
326 μL (1.11 mmol) of (i-PrO)4Ti was added. The solution became
dark brown and was stirred overnight before being treated with 10%
solution NaOH (4 mL). The suspension was filtered to remove
precipitated inorganic material, which washed with dichloromethane.
The organic layer was separated and the aqueous layer was extracted with
dichloromethane. The combined organic solutions were washed with
water and brine, and then dried over anhydrous MgSO4, filtered, and
evaporated to dryness. The residue was purified by column chromatog-
raphy eluting with methanol/chloroform to give 30 (133 mg, 57%).
1H NMR (CDCl3, 400 MHz) δ 1.57 (s, 6H), 2.61 (bs, 2H), 7.34−7.50
(m, 6H), 7.60 (d, J = 7.2 Hz, 2H), 7.73 (s, 1H); 13C NMR (CDCl3, 400
MHz) δ 32.7, 53.0, 123.97, 123.98, 125.4, 127.4, 127.5, 128.9, 141.4,
141.7, 150.4.
(DMSO-d6, 400 MHz) δ 4.44 (d, J = 5.6 Hz, 2H), 5.37 (t, J = 3.7 Hz,
1H), 6.83 (s, 1H), 7.20−7.26 (5H, m), 7.40 (t, J = 7.6 Hz, 1H), 7.50 (d,
J = 7.2 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.85 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) δ 27.45, 43.13, 119.85, 125.21, 127.11, 127.59,
129.13, 129.37, 132.61, 137.49, 140.07, 141.68, 155.73, 198.58; ESI-
HRMS for C16H16N2O2Cl (M+H)+ calcd. 303.0900, found 303.0907.
3-Acetyl-α,α-dimethylbenzylamine (24). 3-Acetyl-α,α-dimethyl-
benzyl isocyanate 3 (1000 mg, 4.92 mmol) in 8 N HCl (30 mL) were
refluxed for 30 min. The reaction mixture was cooled to 0 °C and then
washed with diethyl ether. The aqueous portion was neutralized with
a 10% NaOH solution and extracted with diethyl ether (3 × 20 mL).
The combined organic layers were washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated. The residue was purified
by chromatography eluting with methanol/chloroform to give 24 as
a yellow oil (478 mg, 55%). 1H NMR (CDCl3, 400 MHz) δ 1.57 (s, 6H),
2.61 (s, 3H), 3.72 (s, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz,
1H), 7.82 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H).
4-Chlorophenyl (2-(3-acetylphenyl)propan-2-yl)carbamate (25).
4-Chlorophenyl chloroformate (317 μL, 2.24 mmol) in dichloro-
methane (2 mL) was added to a mixture of 3-acetyl-α,α-dimethylbenzyl-
amine 24 and diisopropylethylamine (390 μL, 2.24 mmol). The reaction
mixture was stirred for 2 h. It was then diluted with dichloromethane,
washed with 1 N HCl, and then brine. The organic layer was dried over
anhydrous magnesium sulfate, filtered, and concentrated. The residue
was purified by column chromatography using hexane/ethyl acetate as
eluent to give 25 (594 mg, 81%). 1H NMR (CDCl3, 400 MHz) δ 1.73 (s,
6H), 2.60 (s, 3H), 5.65 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.25 (d, J =
6 Hz, 2H), 7.44 (t, J = 8 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.82 (d, J =
7.6 Hz, 1H), 8.07 (1H, s); 13C NMR (DMSO-d6, 100 MHz) δ 27.45,
29.79, 55.50, 124.23, 124.55, 127.31, 129.28, 129.55, 129.79, 130.39,
137.30, 148.70, 150.32, 152.97, 198.64; ESI-HRMS for C18H19NO3Cl
(M+H)+ calcd. 332.1053, found 332.1059.
N-(2-(3-Acetylphenyl)propan-2-yl)-2-(4-chlorophenyl)acetamide
(26). 4-Chlorophenylacetylchloride (106 mg, 0.56 mmol) in dichloro-
methane was added to a solution of 3-acetyl-α,α-dimethylbenzylamine
24 (100 mg, 0.56 mmol) and triethylamine (120 μL, 0.86 mmol) in
dichloromethane over a period of 5 to 10 min at 0 °C. The reaction
mixture was stirred at room temperature for 2 h. The mixture was diluted
with dichloromethane (20 mL) and washed with 1 N HCl, water, and
brine. The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified by column chromatography
using hexane/ethyl acetate as eluent to give 26 (146 mg, 80%). 1H NMR
(CDCl3, 400 MHz) δ 1.61 (s, 6H), 2.53 (s, 3H), 3.46 (s, 2H), 5.68 (s,
1H), 7.19 (d, J = 7.2 Hz, 2H), 7.30−7.38 (m, 3H), 7.46 (d, J = 8 Hz, 1H),
7.75 (d, J = 7.2 Hz, 1H), 7.87 (s, 1H); 13C NMR (DMSO-d6, 100 MHz)
δ 27.26, 29.89, 42.54, 55.34, 124.71, 126.76, 128.74, 128.98, 130.23,
131.45, 131.63, 136.22, 137.19, 148.96, 169.54, 198.59; ESI-HRMS for
C19H21NO2Cl (M+H)+ calcd. 330.1261, found 330.1270.
3-Phenyl α-Cyclopropylbenzylamine (31). Ethyl magnesium bro-
mide (3 M) in diethyl ether (1.841 mmol, 2.2 equiv) was added at
−78 °C to a solution of 3-cyanobiphenyl (150 mg, 0.838 mmol) and
Ti(Oi-Pr)4 (0.269 mL, 0.920 mmol) in 5 mL of Et2O. The yellow
solution was stirred for 10 min, and then the solution was allowed to
warm to room temperature and continued to be stirred for 1 h before
BF3·OEt2 (0.206 mL, 1.76 mmol) was added. Stirring was continued for
an additional hour. The reaction mixture was quenched with 3 mL of 1 N
HCl and then washed with ether (15 mL). The aqueous layer was
basified with 10% NaOH (10 mL) and extracted with diethyl ether.
The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel eluting with methanol/chloroform
to yield 31 (98 mg, 52%). 1H NMR (CDCl3, 400 MHz) δ 1.1 (t, J = 4 Hz,
2H), 1.04 (t, J = 4 Hz, 2H), 7.25−7.79 (m, 1H), 7.35−7.46 (m, 5H),
7.52 (s, 1H), 7.58 (d, J = 7.2 Hz,1H).
3-Acetyl-α,α-dimethylbenzylalcohol (27). A solution of 3-acetyl-
α,α-dimethylbenzylamine (500 mg, 2.82 mmol) in chloroform was
cooled to 0 °C. Chlorosulfonic acid (109 mg, 0.94 mmol) was added
dropwise over a period of 15 min. During this time, a white precipitate
formed. Stirring was continued for another 10 min. The reaction mixture
was filtered to yield the corresponding substituted N-benzylsulfonic acid
(253 mg, 70% with respect to chlorosulfonic acid), which was used
without further purification.
The N-benzylsulfonic acid (169 mg, 0.658 mmol) was suspended in
2 mL of water and then KNO2 (168 mg, 1.97 mmol, 3 equiv) was added.
An almost clear solution was immediately formed. To this solution was
added 2 mL sulfate buffer (pH = 2.8) and the resulting mixture was
stirred for 2 h. The reaction mixture was diluted with 4 mL water and
extracted with ethyl acetate (3 × 10 mL). The combined organic layers
were washed with water. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated. The residue was purified by column
4-Nitrophenyl (2-([1,1′-biphenyl]-3-yl)propan-2-yl)carbamate
1
(33). Prepared following the same procedure as 21. Yield 71%. H
NMR (CDCl3, 400 MHz) δ 1.62 (s, 6H), 6.81 (d, J = 8.8 Hz, 2H), 7.26−
7.66 (m, 10H), 8.11 (d, J = 8.8 Hz, 2H).
4-Nitrophenyl (1-([1,1′-biphenyl]-3-yl)cyclopropyl)carbamate
1
(34). Prepared following the same procedure as 21. Yield 68%. H
NMR (CDCl3, 400 MHz) δ 1.39−1.40 (m, 2H), 1.42−1.45 (m, 2H),
6.89 (d, J = 9.2 Hz, 2H), 7.31−7.49 (m, 6H), 7.54−7.58 (m, 3H), 8.16
(d, J = 9.2 Hz, 2H).
4-Nitrophenyl (2-phenylpropan-2-yl)carbamate (35). Prepared
1
following the same procedure as 21. Yield 62%. H NMR (CDCl3,
400 MHz) δ 1.73 (s, 6H), 5.6 (bs, 1H), 6.79 (d, J = 8.8 Hz, 2H), 7.23−
7.25 (m, 2H), 7.34 (t, J = 7.6 Hz, 1H), 8.09 (d, 2H, J = 9.2 Hz), 8.16 (d,
J = 8.8 Hz, 1H).
5-(3-(2-([1,1′-Biphenyl]-3-yl) propan-2-yl)ureido)-2-chlorobenza-
mide (36a). Prepared following the same procedure as 22. Yield 45%.
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dx.doi.org/10.1021/jm3007917 | J. Med. Chem. 2012, 55, 7759−7771

Journal of Medicinal Chemistry
Article
1H NMR (CDCl3, 400 MHz) δ 1.74 (s, 6H), 5.63 (s, 1H), 5.96 (s, 1H),
AUTHOR INFORMATION
Corresponding Author
*E-mail: gdcuny@central.uh.edu. Tel no.: 1-713-743-1274.
Notes
■
6.62 (s, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 2.4, 1H), 7.31−7.35
(m, 1H), 7.39−7.48 (m, 6H), 7.55 (d, J = 7.2 Hz, 2H), 7.68 (s, 1H), 7.77
(d, J = 8.8 Hz, 1H); 13C NMR (CD3OD, 100 MHz) δ 29.1, 54.9, 118.3,
120.6, 122.8, 123.4, 123.7, 124.8, 126.8, 127.0, 128.5, 130.1, 132.5, 136.0,
141.3, 141.6, 155.1, 171.0; ESI-HRMS for C23H23N3O2Cl (M+H)+
calcd. 408.1479, found 408.1485.
The authors declare no competing financial interest.
1-(2-([1,1′-Biphenyl]-4-yl)propan-2-yl)-3-(4-chlorophenyl)urea
ACKNOWLEDGMENTS
■
(36b). Synthesized following the synthetic procedure in Scheme 6
1
This work was supported by funding from the National Institute
of Allergy and Infectious Diseases (U01AI075466) to LH. GDC
thanks the New England Regional Center of Excellence for
Biodefense and Emerging Infectious Diseases (NERCE/BEID)
and the Harvard NeuroDiscovery Center for financial support.
BS is a Georgia Research Alliance Distinguished Investigator.
starting with 4-phenylbenzonitrile. Yield 68%. H NMR (CDCl3, 400
MHz) δ 1.73 (s, 6H), 5.53 (s, 1H), 6.76 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H),
7.23 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 7.2 Hz, 1H), 7.35−7.38 (m, 2H),
7.45−7.53 (m, 6H), 8.06 (d, J = 8.8 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H);
13C NMR (DMSO-d6, 100 MHz) δ 30.28, 54.79, 116.49, 119.52, 126.07,
126.89, 126.99, 127.18, 129.11, 129.58, 138.48, 140.02, 140.66, 148.20,
154.48; ESI-HRMS for C22H22N2OCl (M+H)+ calcd. 365.1421, found
365.1428.
5-(3-(1-([1,1′-Biphenyl]-3-yl)cyclopropyl)ureido)-2-chlorobenza-
mide (37). Prepared following the same procedure as 22. Yield 62%.
1H NMR (DMSO-d6, 400 MHz) δ 1.23 (t, 2H, J = 6.8 Hz), 1.30 (t, 2H,
J = 8 Hz), 7.22 (d, J = 7.2 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 7.35−7.47
(m, 5H), 7.53 (t, J = 2.4 Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.80 (s, 1H),
8.70 (s, 1H), 8.70 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) δ 19.2, 34.9,
118.1, 121.6, 123.7, 124.6, 124.8, 127.0, 127.4, 128.0, 129.3, 129.5, 130.3,
137.8, 139.8, 140.7, 141.1, 145.5, 155.5, 168.8; ESI-HRMS for
C23H20N3O2Cl (M+H)+ calcd. 406.1322, found 406.1323.
1-(2-Oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-3-(2-phenylpro-
pan-2-yl)urea (38). 1.52 (s, 6H), 6.38 (d, J = 6 Hz, 1H), 6.56 (t, J =
7.2 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 7.10−7.15 (m, 2H), 7.21−7.25 (m,
2H), 7.31−7.33 (m, 2H), 8.16 (s, 1H), 10.26 (s, 1H), 10.35 (s, 1H));
13C NMR (DMSO-d6, 100 MHz) δ 30.4, 54.8, 94.6, 99.9, 108.9, 110.7,
ABBREVIATIONS USED
■
Cp, Cryptosporidium parvum; BSA, bovine serum albumin;
DIPEA, diisopropylethylamine; hTERT, human telomerase
reverse transcriptase; IMP, inosine 5′-monophosphate; IMPDH,
inosine 5′-monophosphate dehydrogenase; N.D., not determined;
p-TSA, p-toluenesulfonic acid; TEA, triethylamine; Toxo,
Toxoplasma; XMP, xanthosine 5′-monophosphate
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed information for HPLC methods and compound purity
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
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