Synthesis, biological evaluation, and structure-activity relationships of N -benzoyl-2-hydroxybenzamides as agents active against P. falciparum (K1 strain), trypanosomes, and leishmania

Article abstract of DOI:10.1021/jm2015183

In our efforts to identify novel chemical scaffolds for the development of new antiprotozoal drugs, a compound library was screened against Toxoplasma gondii tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2- hydroxybenzamide 1a against T. gon

Full text of DOI:10.1021/jm2015183

Article
pubs.acs.org/jmc
Synthesis, Biological Evaluation, and Structure−Activity
Relationships of N-Benzoyl-2-hydroxybenzamides as Agents Active
against P. falciparum (K1 strain), Trypanosomes, and Leishmania
Jozef Stec,†,# Qingqing Huang,†,#,○ Marco Pieroni,†,◆ Marcel Kaiser,‡,§ Alina Fomovska,∥ Ernest Mui,∥
William H. Witola,∥,¶ Samuel Bettis,⊥ Rima McLeod,*,∥ Reto Brun,*,‡,§ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South
Wood Street, Chicago, Illinois 60612, United States
‡Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
§University of Basel, Petersplatz 1, CH-4003, Basel, Switzerland
∥Department of Ophthalmology and Visual Sciences, Pediatrics (Infectious Diseases), Committees on Genetics, Immunology, and
Molecular Medicine, Institute of Genomics and Systems Biology, and The College, The University of Chicago, Chicago, Illinois
60637, United States
⊥Cellular Screening Center Institute for Genomics and Systems Biology, The University of Chicago Center for Integrative Sciences,
929 East 57th Street, Chicago, Illinois 60637, United States
ABSTRACT: In our efforts to identify novel chemical
scaffolds for the development of new antiprotozoal drugs, a
compound library was screened against Toxoplasma gondii
tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2-
hydroxybenzamide 1a against T. gondii as described elsewhere.
Synthesis of a compound set was guided by T. gondii SAR with
1r found to be superior for T. gondii, also active against Thai
and Sierra Leone strains of Plasmodium falciparum, and with
superior ADMET properties as described elsewhere. Herein,
synthesis methods and details of the chemical analysis of the
compounds in this series are described. Further, this series of N-benzoyl-2-hydroxybenzamides was repurposed for testing against
four other protozoan parasites: Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and P. falciparum (K1
isolate). Structure−activity analyses led to the identification of compounds in this set with excellent antileishmanial activity
(compound 1d). Overall, compound 1r was the best and had activity 21-fold superior to that of the standard antimalarial drug
chloroquine against the K1 P. falciparum isolate.
infects about 225 million people and causes nearly 800000
deaths, mostly in children, annually.5 Overall, in the absence of
vaccines and with few chemotherapeutics available, some with
reduced efficacy and adverse effects, diseases caused by
protozoan parasites still contribute significantly to morbidity
and mortality. Thus, discovery and development of effective,
safe and affordable antiprotozoal agents remains an urgent
need.
In our efforts to identify novel chemical scaffolds as potent
antiprotozoal drugs, a small compound library (ASDI) was
screened initially against Toxoplasma gondii tachyzoites as
described elsewhere.1 Moderate activity was found for N-(4-
ethylbenzoyl)-2-hydroxybenzamide 1a (Figure 1) against the T.
gondii parasites.1 Interestingly, some salicylimides, among other
compounds, have been reported to possess antimalarial activity
ascribed to their targeting the hemozoin detoxification protein
(HDP), a protein known to be vital for P. falciparum.6 It is also
INTRODUCTION
■
Protozoan parasites cause diseases such as malaria, amoebiasis,
giardiasis, toxoplasmosis, leishmaniasis, and trypanosomiases
and are among the most serious public health problems in
developing countries. Some of these parasites also cause major
health problems in the U.S., Europe, and Japan.1 According to
the World Health Organization (WHO), parasitic protozoan
diseases plague billions of people worldwide and kill millions
annually.2 Nevertheless, few medicines are available to treat
those serious and sometimes lethal infections. Human African
trypanosomiasis (HAT, sleeping sickness), caused by Trypano-
soma brucei rhodesiense (T. b. rhodesiense) and T. b. gambiense,
threatens about 50 million people living in sub-Saharan Africa
and causes an estimated 25000 deaths per year.3 There are an
estimated 10 million people suffering from American
trypanosomiasis (Chagas disease), which is caused by
Trypanosoma cruzi (T. cruzi).4 Visceral leishmaniasis is caused
by Leishmania donovani (L. donovani) and currently threatens
350 million persons around the world.4 Plasmodium falciparum
(P. falciparum) is one of the causative agents of malaria and
Received: November 10, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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Further modifications of the linker resulted in synthesis of N-
[(4-ethylphenyl)carbamoyl]-2-hydroxybenzamide (16) and 1-
(4-ethylphenyl)-3-(2-hydroxyphenyl)urea (19) (Scheme 4).
The intermediate 15 was readily obtained from salicylamide
following a known procedure.15 Standard hydrogenolysis of 15
gave the compound 16 in 69% yield (Scheme 4). The
compound 19 bearing a urea linker was prepared by the
reaction of isocyanate 17 with 4-ethylaniline and subsequent
deprotection (Scheme 4).16
Several compounds 21a−c bearing linkers that are chemically
more stable than an imide function were analyzed. The
compounds were prepared from the corresponding imide/
amine precursors (20a−c) as shown in Scheme 5 by following
the standard procedure described above. The oxadiazole 23 was
prepared from 1a by cyclodehydration in the presence of
hydroxylamine hydrochloride and sodium acetate (Scheme
5).17 Reaction of 2-(benzyloxy)aniline (24) with ethyl 3-chloro-
3-oxopropanoate gave the intermediate 25, which was readily
hydrolyzed to the carboxylic acid 26.13 This intermediate was
converted to the final bis-amide 28 through amide bond
formation13 with 4-ethylaniline and subsequent deprotection
(Scheme 5).18
Figure 1. Hit compound 1a.
known that certain salicylamide derivatives, such as salicylylhy-
droxamic acid, nitazoxanide, and other thiazolides, display
antiprotozoal activity.7−11 Herein, we report the details of the
synthesis, modification, and biological evaluation of this set of
N-benzoyl-2-hydroxybenzamides and related compounds, as
potent, and in some cases selective, agents against the K1
isolate of P. falciparum and L. donovani. We furthermore studied
their effects on T. b. rhodiense and T. cruzi.
RESULTS AND DISCUSSION
CHEMISTRY
■
■
In search of compounds with improved activity and ADMET
properties,1 we modified the hit 1a at three sites: the phenol
ring (A), 4-ethylphenyl ring (B), and the imide linker (Figure 1).1
ADMET studies, described in detail elsewhere,1 revealed
metabolic instability to be the main concern for 1a.1 Briefly,
the compound was found to be quickly metabolized in human
liver microsomes (HLM), and only 0.5% of the compound
remained after incubation for 60 min in the presence of
NADPH.1 However, in the absence of NADPH, 53% of the
compound was left unchanged after the incubation.1 The
potassium ion channel coded by hERG was not inhibited by 1a
at compound concentration as high as 300 μM.1 Among the
recombinant cytochrome P450 (CYP450) isozymes, 3A4, 2D6,
and 2C9, only the latter was inhibited to the extent of 71% at a
compound concentration of 10 μM.1 The aqueous solubility in
phosphate-based buffer of compound 1a was 2.76 μM at pH
4.0 and 50.8 μM at pH 7.4, respectively (for comparison, the
control compound diclofenac exhibits the following solubility:
9.10 μM at pH 4.0 and 303 μM at pH 7.4).1 The tested
compound presented a similar permeability as the control drugs
testosterone and methotrexate.1 However, the protein binding
in human plasma of 1a was high (99.9%) in comparison with
the control drug, fluconazole (23.7%), and both its intrinsic
clearance and its metabolism rate in HLM (282 μL/min/mg
proteins) were faster than that of the control verapamil (38.8
μL/min/mg proteins).1 Nonetheless, these results encouraged
us to modify the hit 1a to generate a lead compound, with the
improvement of both antiprotozoal potency and metabolic
stability as the primary goal.1
The desired N-benzoyl-2-hydroxybenzamides (1a−ab) were
generated by the reaction of salicylamide (2) with various acid
chlorides (3a−ab) in refluxing pyridine (Scheme 1). The crude
a
Scheme 1. Preparation of N-Benzoyl-2-hydroxybenzamides
a
Reagents and conditions: (a) pyridine, reflux, 4 h.
products (mostly solids) were subsequently purified by
crystallization or preparative HPLC to give the pure
compounds in yields ranging from modest to very good
(Table 1).
The hydroxybenzamides 1k and 1ab were further elaborated
as shown in Scheme 2. Catalytic hydrogenation over palladium-
on-carbon of compounds 1k and 1ab provided corresponding
aniline 4a and phenol 4b derivatives in good yields (Scheme 2).
Reaction of compound 4b with methyl 3-(chlorocarbonyl)-
propanoate provided compound 5 in modest yield (Scheme 2).
The hydroxamic acid 8 was prepared in a three-step procedure
starting with protection of the hydroxyl group in 4a as its silyl
ether.12 Subsequent reaction with 8-[(benzyloxy)amino]-8-
oxooctanoyl chloride13 gave the intermediate 7, which was
deprotected to provide compound 8 in low yield (Scheme 2).
To better understand the importance of the imide linker for
biological activity, modification of this moiety was investigated.
Syntheses are shown in Scheme 3. The N-methyl imide 10 was
prepared in high yield from 4-ethylbenzoyl chloride (3a) and
N-methylsalicylamide (9)14 by following the standard proce-
dure described above. In the same manner, the acylated
salicylhydrazide 12 was prepared from 4-ethylbenzoyl chlo-
ride (3a) and salicylhydrazide (11) (Scheme 3). Reaction of
the original hit compound 1a with Lawesson’s reagent in
refluxing chlorobenzene provided the thioimide derivative 13
(Scheme 3).
All of the compounds 1a−ab, 4a, 4b, 5, 8, 10, 12, 13, 16, 19,
21a−c, 23, and 28 were evaluated for in vitro activity against
four pathogenic protozoa: T. b. rhodesiense (trypomastigote
stage, STIB 900 strain), T. cruzi (intracellular amastigote stage,
Tulahuen C4 strain), L. donovani (axenic amastigote stage,
MHOM-ET-67/L82), and P. falciparum (erythrocytic stage, K1
strain, which is a chloroquine and pyrimethamine resistant
strain). The toxicity of the designed compounds was assessed
against a mammalian primary cell line derived from rat skeletal
myoblasts (L6). The following drugs were used as the reference
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Table 1. Variation of the Ring B in N-Benzoyl-2-hydroxybenzamides (1)
a
Isolated yield of product exhibiting >95% HPLC purity.
compounds: melarsoprol (T. b. rhodesiense), benznidazole (T.
cruzi), miltefosine (L. donovani), chloroquine (P. falciparum),
and podophyllotoxin (L6). The IC50 values presented below
indicate the concentration of the tested compound in μg/mL
that is necessary to inhibit 50% of the parasite growth. The
displayed numbers are the average values collected from two
replicate experiments. The selectivity index is the ratio between
the IC50 for rat skeletal myoblasts (L6) and the IC50 for the
respective protozoan parasite. The screening data are presented
in three tables. Table 2 shows compounds bearing various para-
substituents on the aromatic B ring (1a−s, 4a, 5, and 8). Table
3 presents compounds with other modification of the B ring
(1t−aa, 4b), and Table 4 provides results for the structures
with modifications in the linker and the A ring (10−28).
We were pleased to find that many of the synthesized
analogues were more potent against the tested parasites than
the hit 1a, showing that various substituents in the para-
position of the B ring improved the desired antiparasitic activity
(Table 2). Compounds 1n, 1o, and 1r, bearing ethoxy, n-
butoxy, and N,N-diethylamino substituents, respectively,
showed reasonable activity against T. cruzi, good activity
against L. donovani, and very good activity against P. falciparum
(K1 strain). Compound 1r exhibited the best IC50 value against
P. falciparum (K1 strain) among all the tested compounds. The
simple replacement of ethyl in 1a by tert-butyl in 1d led to good
and very good activity against L. donovani and P. falciparum (K1
strain), respectively. The potency of compound 1d against L.
donovani was comparable to that of miltefosine. Compounds
bearing other alkyl substituents (ethyl 1a, isopropyl 1c) or
aromatic groups (phenyl 1e, benzyl 1p) displayed similar
potency against T. cruzi, L. donovani, and P. falciparum (K1
strain). Compounds with methyl (1b), fluoro (1f), methoxy
(1m), and amino (4a) groups also had similar activity as
compounds 1a,c,e,p, however, only against L. donovani and P.
falciparum (K1 strain). Compound 1q possessing a para-N,N-
dimethylamino substitutent turned out to be not only potent
but very selective against P. falciparum (K1 strain). It was
unexpected for us to see such dramatic change in the selectivity
for compounds 1q,r and 4a by introducing such minor
structural modifications. The morpholinyl-substituted com-
pound 1s showed reasonable activity and selectivity against
malaria parasites. To our surprise, compound 8 bearing the
octanedioic acid amide hydroxyamide side chain was modestly
active only against P. falciparum (K1 strain). The benzoic acid
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all tested parasites. Against T. cruzi, modest inhibitory activity
was displayed by several compounds already mentioned above
(1a,c−e,n,p,r). Unfortunately, none of the compounds
presented in Table 2 were active against T. b. rhodesiense.
Summarizing the SAR data shown in Table 2, we observe a
clear relationship between structural modifications at the para
position of the B ring and antileishmanial and anti-P. falciparum
(K1 strain) activity. Some of the most active compounds
against P. falciparum (K1 strain) are those with bulky alkyl
substituents such as tert-butyl (1d). Reduction of the
substituent size to ethyl (1a), methyl (1b), or even isopropyl
(1c) resulted in decreased potency. This finding would suggest
the presence of a large hydrophobic space in the binding pocket
able to accommodate branched substituents in the area
contiguous to the para position of the B ring. In comparison
with the preferred tert-butyl group, a phenyl substituent
(compound 1e) causes reduction in activity, therefore
suggesting the preference for an alkyl group in the binding
site. Antiplasmodial potency is enhanced by introduction of
bulky and strongly electron-donating N or O substituents.
Compound 1r with N,N-diethylamino substitution turns out to
be the most active and selective in this series against malaria
parasites. Its N,N-dimethylamino analogue 1q and the
morpholinyl-substituted compound 1s both exhibit good
potency and moderate selectivity against the same parasite.
The high potency of these compounds not only reiterates the
significance of hydrophobic interactions involving lipophilic
partial structures of the 4-substituents but also points to
potential dipolar interactions or H-bonding between the ligand
and the binding pocket. The aniline 4a, which lacks the ability
to engage in hydrophobic interactions via its 4-substituent, is far
less active against P. falciparum (K1 strain) than compounds
bearing a dialkylated nitrogen atom (1q,r,s). Potency enhance-
ment by large, aliphatic, heteroatom-containing groups is also
apparent among alkoxy-substituted compounds. High potency
is observed for compounds 1n and 1o with ethoxy and n-
butoxy substituents, respectively. However, the relatively small
trifluoromethoxy (compound 1h) and methoxy (compound
1m) groups along with the large, aromatic benzyloxy
substituent (compound 1p) are less potent in comparison
with 1n,o. Enhanced potency is also observed for analogues
bearing small electron-withdrawing groups such as fluoro
(compound 1f) and trifluoromethyl (compound 1g). Other
electron-withdrawing groups such as cyano (compound 1i),
methanesulfonyl (compound 1j), and nitro (compound 1k)
lead to loss of potency; as an exception, the methoxycarbonyl-
substituted compound 1l maintains fair potency against P.
falciparum (K1 strain). The modest activity of compound 8
bearing the octanedioic acid amide hydroxyamide moiety in
comparison with the inactive compound 5 that contains a
succinamic acid methyl ester side chain would suggest an
alternative mode of action for the former compound against P.
falciparum (K1 strain). On the basis of these results, we can
characterize the binding site for N-benzoyl-2-hydroxybenza-
mides para-substituted in their B ring as a fairly large
hydrophobic site with preference for branched alkyl sub-
stituents as well as strongly electron-donating groups, such as
dialkylamino and alkoxy, that are able to accept H-bonds. All in
all, the excellent potency of compound 1r along with its
negligible cytotoxicity against the L6 cell line (selectivity index,
SI = 1640) make this compound a promising lead for the
further development of antimalarial drugs. This compound has
been evaluated for its ADMET properties elsewhere,1 which
Scheme 2. Elaboration of Selected N-Benzoyl-2-
hydroxybenzamides
a
a
Reagents and conditions: (a) Pd/C, H2, MeOH, room temp, 18 h;
(b) methyl 3-(chlorocarbonyl)propanoate, pyridine, CH2Cl2, room
temp, 1 h; (c) TIPSCl, Et3N, DMAP, DMF, room temp, 3.5 h; (d) 8-
[(benzyloxy)amino]-8-oxooctanoyl chloride, pyridine, CH2Cl2, room
temp, 2 h.
Scheme 3. Modifications of the Linker in N-Benzoyl-2-
a
hydroxybenzamides
a
Reagents and conditions: (a) pyridine, reflux, 4 h; (b) Lawesson’s
reagent, chlorobenzene, 135 °C, 3 h.
methyl ester analogue 1l gave similar results. In contrast to
these results, compound 5 containing a succinamic acid methyl
ester side chain was inactive against all species. On the other
hand, the analogue 1g containing a trifluoromethyl group is the
only compound in this series that is potent and at the same
time reasonably selective against L. donovani. Compound 1h
bearing a trifluoromethoxy group also appeared to be selective
against L. donovani although significantly less potent (around
10-fold) than compound 1g. The methanesulfonyl group in
compound 1j caused complete loss of inhibitory activity against
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a
Scheme 4. Synthesis of the Carbamoylsalicylamide 16 and the Urea 19
a
Reagents and conditions: (a) BnBr, K2CO3, acetone, reflux, 16 h; (b) (1) (COCl)2, CH2Cl2, room temp to 50 °C, 3 h, (2) 4-ethylaniline, MeCN,
room temp, 18 h; (c) 10% Pd/C, H2, MeOH, room temp, 24 h; (d) 4-ethylaniline, CH2Cl2, room temp, 19 h; (e) BBr3, CH2Cl2, −78 to 0 °C.
a
Scheme 5. Preparation of Compounds with Modified Linkers
a
Reagents and conditions: (a) pyridine, reflux, 4 h; (b) 200 °C, 1 h; (c) NH2OH·HCl, NaOAc, absolute EtOH, 16 h; (d) pyridine, CH2Cl2, ethyl 3-
chloro-3-oxopropanoate, room temp, 18 h; (e) THF, LiOH, room temp, overnight; (f) DIPEA, BOP-Cl, 4-ethylaniline, room temp, 22 h; (g)
Pd(OH)2/C (25 wt %), EtOH−cyclohexene (2:1), reflux, 2.5 h.
turned out to be more favorable than those of the hit 1a.1
Additionally, compounds 1q and 1s have reasonable selectivity
index values (110 and 272, respectively) and can thus also be
regarded as attractive leads.
result may assist in designing further structural modifications
aimed at providing potent and selective compounds against L.
donovani.
In analogy to the SAR for P. falciparum (K1 strain) and L.
donovani, the most active compounds against T. cruzi were
analogues with branched and electron-donating substituents
(1a,c,d,n, and r). However, all of them were less potent against
this protozoan than against P. falciparum (K1 strain) and L.
donovani. On the other hand, compounds bearing electron-
withdrawing groups resulted in the complete loss of
antiparasitic activity (1f,g,i−l). Surprisingly, the aromatic
substituents in 1e and 1p retained moderate activity against
T. cruzi, indicating possible π−π interaction in the binding
pocket. None of the preceding compounds turned out to be
active against T. b. rhodesiense.
In the final series of compounds, we probed the influence of
other modifications of the B ring. Encouragingly, we observed
an improvement in the antiparasitic activity for some of the
synthesized compounds (Table 3). The 2-naphthyl analogue 1t,
the 6-methoxy-2-naphthyl analogue 1u, and the 2,3,4-
trimethoxyphenyl analogue 1y were the most active against P.
Similar trends in the SAR were noticed for L. donovani,
suggesting that this protozoan possesses a homologous
molecular target for this series of compounds. The most active
compounds were the tert-butyl analogue 1d, the trifluoromethyl
analogue 1g, the ethoxy and n-butoxy compounds 1n and 1o, as
well as the N,N-dimethylamino compound 1r. Decreased steric
bulk of the substituents resulted in a decrease (compounds
1a,b,c,h,m) or complete loss (compound 1q) of potency.
Reduced activity was also observed for compounds 1e,p
containing aromatic substituents and for the morpholinyl
derivative 1s. This would suggest a strict cutoff for the size of
the para substituent at the L. donovani binding site. Complete
loss of activity was observed for the compounds bearing large
and strongly electron-withdrawing groups (1i,j,k,l), or bearing
long polar chains (5 and 8). The L. donovani binding site
tolerates small electron-withdrawing groups (compounds 1f,g)
as well as or better than that of P. falciparum (K1 strain). This
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Table 2. Effect of Variation in the para-Substituent of the B Ring on Biological Activity
T. b. rhodesiense
a
T. cruzi
L. donovani axe.
IC50 SI
9.3
14
P. falciparum (K1)
L6
b
compd
1a
IC50
81
SI
IC50
SI
15
IC50
SI
IC50
0.86
<0.46
0.29
0.038
>1.2
0.60
0.19
1.3
4.8
38
7.4
5.6
12
33
70
46
19
2.3
1b
1c
1d
1e
1f
>100
1.2
3.2
1.4
65
61
2.0
6.0
7.7
51
9.5
1.3
14
17
1.50
0.029
3.9
12
0.38
0.14
7.4
80
87
>13
>14
23
>26
25
>100
64
0.75
0.26
1.2
1.6
1.5
38
13
1g
1h
1i
70
50
0.57
5.3
23
3.8
3.4
65
72
16
22
3.8
3.9
2.7
2.4
84
>100
>100
>100
>50
50
<0.55
1.0
100
0.55
12
4.7
14
38
22
55
1j
51
56
20
31
>2.0
>100
11
1.0
4.9
>100
53
1k
1l
<0.53
<0.83
0.78
0.28
0.23
1.0
0.94
2.1
1.3
4.1
1.2
>50
<0.83
23
2.9
14
42
1m
1n
1o
1p
1q
1r
1.7
1.6
24
17
39
60
4.1
11
4.4
37
2.8
0.45
0.94
1.5
37
1.0
17
55
14
0.26
4.1
50
13
62
15
42
16
64
81
0.66
<0.16
1.0
1.4
2.9
1.0
0.98
1.0
18
0.91
16
7.8
22
>100
0.19
3.0
0.49
0.005
0.18
3.9
110
1640
>272
10
54
>50
>50
46
9.1
8.2
1s
>50
40
>3.1
5.0
>50
39
4a
5
0.84
1.0
>50
65
>50
49
>2.3
1.0
21
>2.4
>29
>50
>100
8
>1.5
>2.1
3.5
c
d
e
f
g
standard
0.005
0.48
0.11
0.004
a
Values indicate the inhibitory concentration of a compound or standard in μg/mL that is necessary to achieve 50% growth inhibition (IC50). Data
b
shown in the table represent mean values from two independent determinations. Selectivity index = (IC50 for L6)/(IC50 for the respective
protozoan parasite). Standard compound: melarsoprol. Standard compound: benznidazole. Standard compound: miltefosine. Standard
compound: chloroquine. Standard compound: podophyllotoxin.
c
d
e
f
g
Table 3. Effect of Other Modifications of the B Ring in N-Benzoyl-2-hydroxybenzamides on Biological Activity
T. b. rhodesiense
a
T. cruzi
L. donovani axe.
P. falciparum (K1)
L6
b
compd
1a
IC50
SI
IC50
SI
15
2.4
11
IC50
SI
IC50
SI
12
IC50
81
26
0.86
0.78
4.8
8.5
5.1
7.4
9.3
5.6
70
1t
1.0
20
0.22
0.98
4.4
94
57
21
56
1u
42
1.3
1.4
40
11
1v
20
0.087
<0.62
<0.47
0.26
17
0.10
1.1
0.16
0.40
1.8
1w
1x
>100
>50
79
58
27
40
16
36
14
11
>50
3.2
5.5
14
4.4
5.6
62
24
21
55
16
39
0.86
0.51
3.4
<0.47
6.5
4.2
1y
0.78
26
1z
>100
48
<0.55
0.33
6.0
9.2
13
4.4
3.4
1aa
4b
0.44
2.8
2.0
7.8
4.7
62
0.63
1.9
21
1.1
36
c
d
e
f
g
standard
0.005
0.48
0.19
0.11
0.004
a
Values indicate the inhibitory concentration of a compound or standard in μg/mL that is necessary to achieve 50% growth inhibition (IC50). Data
b
shown in the table represent mean values from two independent determinations. Selectivity index = (IC50 for L6)/(IC50 for the respective
protozoan parasite). Standard compound: melarsoprol. Standard compound: benznidazole. Standard compound: miltefosine. Standard
compound: chloroquine. Standard compound: podophyllotoxin.
c
d
e
f
g
falciparum (K1 strain). Compound 1t showed activity at low
concentration (only 2-fold less potent than the standard)
against malaria parasites. Also compounds 1u and 1y displayed
potency within the same range as 1t against the same
protozoan. We also observed modest potency of these
compounds (1t,u,y) against L. donovani. Compound 1aa with
2-methyl-3-furyl as the B ring, and the symmetric analogue 4a
showed dual activity against malaria and leishmania parasites.
However, both were less active than 1t,u,y. The piperonyl-
substituted compound 1x turned out to be weakly active only
against P. falciparum (K1 strain) and thus is somewhat selective
against this protozoan. Compounds with 2,4-dichlorophenyl
(1w) or cyclohexyl (1z) as their B rings were inactive against P.
falciparum (K1 strain).
Many of the analogues presented in Table 3 showed
moderate (corresponding to micromolar) activity against L.
donovani, and the 3-chloromethyl analogue 1v even had activity
slightly better than the antileishmanial drug miltefosine.
However, the selectivity index of compound 1v was only 11,
which makes this compound less viable for further develop-
ment. Analogues 1z with a cyclohexyl B ring and 1aa with a 2-
methyl-3-furyl B ring turned out to be moderately active only
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Table 4. Effect of Variations in the Linker and A Ring on Biological Activity
T. b. rhodesiense
a
T. cruzi
L. donovani axe.
IC50
P. falciparum (K1)
L6
b
compd
1a
IC50
SI
IC50
4.8
SI
15
SI
IC50
5.6
SI
12
IC50
81
>100
17
0.86
<0.094
0.28
0.28
2.0
7.4
9.1
6.2
5.3
9.3
70
9.4
4.8
17
70
9.6
26
23
8.2
10
29
2.2
10
74
7.0
8.8
28
8.9
39
20
0.48
0.33
2.2
1.0
28
27
4.2
26
2.1
6.9
23
8.5
27
12
0.11
0.33
0.18
4.1
12
0.77
3.3
13
61
1.7
16
36
0.95
1.4
100
0.70
2.6
2.8
19
18
0.54
0.31
<0.46
1.0
3.7
1.9
4.6
21a
21b
21c
23
83
3.0
13
3.7
>50
<0.81
0.93
>2.6
0.76
>50
3.0
>100
13
0.19
<0.46
2.8
1.0
8.2
0.96
61
35
>1.6
0.44
>1.0
1.2
>3.7
1.3
>100
16
0.004
28
c
d
e
f
g
standard
0.005
a
Values indicate the inhibitory concentration of a compound or standard in μg/mL that is necessary to achieve 50% growth inhibition (IC50). Data
b
shown in the table represent mean values from two independent determinations. Selectivity index = (IC50 for L6)/(IC50 for the respective
protozoan parasite). Standard compound: melarsoprol. Standard compound: benznidazole. Standard compound: miltefosine. Standard
compound: chloroquine. Standard compound: podophyllotoxin.
c
d
e
f
g
against L. donovani. Compounds 1w and 1x were completely
inactive against leishmania parasites. The fact that compounds
with B rings other than benzene still retained moderate activity
against L. donovani when compared to other protozoa provides
valuable guidance for the design of further potential
antileishmanial agents. Besides the hit compound 1a, the
naphthyl derivatives 1t and 1u were the only compounds
moderately active against T. cruzi. In accord with the previous
observations, none of these compounds were active against T. b.
rhodesiense parasites.
Concluding the SAR for 2-hydroxybenzamides presented in
Table 3, we can state that the identity of the B ring as benzene
seems to be important for the anti-P. falciparum (K1 strain)
activity as presented by 1t,u,v,y. However, cyclohexane
(compound 1z) and furan (compound 1aa) substitutes retain
moderate antileishmanial activity. Free phenol functionality
(compound 4b) is tolerated, but alkoxy substituents (com-
pounds 1x,y) provide a better contribution to the compounds’
antimalarial activity. Although significantly cytotoxic, the
(chloromethyl)phenyl analogue 1v provided excellent potency
against L. donovani, whereas 2,4-dichlorophenyl (compound
1w) gave a completely inactive analogue. These conclusions are
in good agreement with the initial SAR described for the para
position of N-benzoyl-2-hydroxybenzamides (Table 2) and
confirm the importance of the aromatic nature of the ligand’s B
ring, preferably bearing electron-donating groups attached
through a heteroatom.
At the same time, we also prepared analogues with modified
linker and A ring moieties. A series of 10 compounds was
prepared and screened for antileishamania, -trypanosome, and
-plasmodium (K1 strain) activity (Table 4). We found that
most of the modifications caused a decrease in anti-P.
falciparum (K1 strain) activity. Only the sulfur analogue 13
and urea-linked compound 19 were slightly more potent
against malaria parasites than the hit compound 1a.1 On the
other hand, the benzoxazinanedione 21a and indazolylbenza-
mide 21c were slightly less active against the same parasites
than the hit 1a. Although anti-P. falciparum (K1 strain) activity
was retained by compounds 13, 19, and 21a,c, their small
selectivity indices suggest safety problems. The above
observations support the notion that the H-bond donor−
acceptor pattern in the imide linker is crucial for antiparasitic
activity. This was additionally supported by methylation of the
nitrogen atom in the imide linker, which gave the inactive
compound 10.
Pleasingly, the present set of structural modifications
provided some compounds with improved potency against L.
donovani. Good antileishmanial activity was shown by
compounds which had already proven activity against P.
falciparum (K1 strain) (13, 19, and 21a,c). In addition to
these compounds, the N-methylated compound 10 and the
acylated salicylhydrazide 12 showed modest potency against L.
donovani. As in the case of compounds active against malaria
parasites, all the compounds active against leishmania parasites
suffered from lack of selectivity (SI < 5). Four compounds, the
acylated salicylhydrazide 12, the urea-linked compound 19, the
benzoxazinanedione 21a, and the indazolylbenzamide 21c,
were only moderately active against T. cruzi. The only
compound, which inhibited at moderate concentration the
growth of T. b. rhodesiense was 21c. Despite its moderate
activity, this compound was 10-fold more potent than the
parent compound 1a. Contrary to this, compounds which had
chemically stable linkers, i.e., the acylated urea 16, indolylben-
zamide 21b, diphenyloxadiazole 23, and bis-amide 28, turned
out to be inactive across all four parasites. Summarizing the
SAR for this series of compounds, we can state that the imide
linker with an unsubstituted N atom is crucial for antimalarial
activity. However, we were pleased to see that compounds
bearing other linker/A ring combinations, such as the urea 19,
the benzoxazinanedione 21a, and the indazolylbenzamide 21c,
retained moderate antileishmanial activity.
CONCLUSIONS
■
This research provides solid ground for the discovery of new
and effective agents to treat protozoan infections that cause
malaria and leishmaniasis. The hit compound 1a was
discovered by initial HTS screening against T. gondi,1 and the
preliminary ADMET results, while encouraging, revealed this
agent to be metabolically unstable in HLM.1 The metabolic
instability is ascribed to the imide linker, and therefore
extensive modifications of this moiety as well as the B-ring
were pursued.1 Several promising lead compounds active
against the K1 strain of P. falciparum and L. donovani were
identified. Among the tested parasites P. falciparum (K1 strain)
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(Micromass). TLC was performed on Merck 60 F254 silica gel plates.
Column chromatography was performed using Merck silica gel (40−
60 mesh). Preparative HPLC was carried out on an ACE 5 AQ column
(150 mm × 20 mm), with detection at 254 and 280 nm on a Shimadzu
SPD-10A VP detector; flow rate = 17 mL/min; gradient from 8% to
100% methanol in water (both solvents containing 0.05 vol% of
CF3COOH). Purities of final compounds were established by
analytical HPLC, which was carried out on an Agilent 1100 HPLC
system with a Synergi 4 μ Hydro-RP 80A column, with detection at
254 nm on a variable wavelength detector G1314A; flow rate = 1.4
mL/min; gradient from 10% to 100% methanol in water (both
solvents containing 0.05 vol% of CF3COOH) within 20 min. All the
final compounds were of purity ≥95% as determined by the method
described above.
General Procedure for the Synthesis of Imide Compounds.
To a solution of amide (5 mmol) in pyridine (6 mL) was added an
acyl chloride (5 mmol). After refluxing for 4 h, the resulting mixture
was diluted with Et2O (30 mL)/H2O (20 mL) or H2O (20 mL)/1.0
M HCl (5 mL). The resulting precipitate was collected by filtration,
washed with water, and dried, or the resulting mixture was diluted with
EtOAc, washed with H2O (20 mL) and brine (20 mL), dried over
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatography on silica gel or recrystallization from
MeOH. Noncommercial acyl chlorides were obtained from the
corresponding carboxylic acid by the following procedure: to a
solution of acid (5 mmol) in anhydrous CH2Cl2 (6 mL) was added
(COCl)2 (6 mmol) and 1 drop of DMF at 0 °C. The resulting mixture
was allowed to warm to room temperature and stirred for 2 h. After
evaporation of the solvent, the residue was used without purification in
the next step. Alternatively, the acid was dissolved in SOCl2 and heated
at 80 °C for 1.5−3 h before cooling to room temperature and
evaporating the volatiles in vacuo. The residue was used in the next
step without further purification.
was the most sensitive to these N-benzoyl-2-hydroxybenza-
mides. Compound 1r is 21-fold more potent against the K1
isolate of malaria than the standard drug, chloroquine.
Moreover, it exhibits an SI value of 1640, suggesting both
high selectivity and low toxicity for this compound. The SAR
for T. gondii reported elsewhere is similar to that described for
P. falciparum K1 isolate.1 Efficacy of compound 1a was found to
be much lower for other malaria isolates, and 1r was also found
less effective in different types of assays1 for other P. falciparum
or other plasmodial isolates.
Additionally, the electron-donating diethylamino group in
compound 1r stabilizes the imide linker, thus improving the
chemical and metabolic stability of this analogue. With its
excellent antiplasmodial potency, this highly selective agent
may be used as a possible lead for the further development of
antimalarial agents. Although analogue 1g is 4 times less potent
than 1d, it shows better selectivity against L. donovani parasites
than malaria, therefore both compounds are attractive leads for
further research including assessment of activity in infected
macrophages. Compound 1v, with a 3-chloromethyl substituent
on the B ring, might also be regarded as an interesting
compound for the development of antileishmanial agents.
Moreover, the fact that the L. donovani binding site better
tolerates small electron-withdrawing groups provides a good
starting point for the design of more potent and selective agents
to combat this protozoan. Compounds 1c and 12 are the most
potent members of this series against T. cruzi. Because of its
hydrolytic stability, the latter compound can be considered an
attractive scaffold for further work. T. b. rhodesiense was
generally resistant to the compounds used in this study, and
compound 21c was the only one showing weak activity against
this parasite. Studies to determine IC90s and whether the
compounds are cidal will be important in the future. While the
data generated in this research may be useful in the design and
synthesis of novel agents with improved potencies and safety
margins to treat infections caused by L. donovani and P.
falciparum (K1 strain), we call attention to the fact that it will
be essential to study the potential teratogenicity of these
imides, as they do bear some structural relationship to
teratogen thalidomide.
Hydrogenation reactions were carried out with 10% Pd/C (10−15
wt %) and a H2 balloon in MeOH as the solvent for 18 h at room
temperature.
N-(4-Ethylbenzoyl)-2-hydroxybenzamide (1a). The general
procedure was used with salicylamide (2) and 4-ethylbenzoyl chloride
(3a) as reactants. Purification of the crude material by preparative
1
HPLC gave the title compound as a white solid (51%). H NMR
(DMSO-d6) δ 11.70 (s, 1H), 11.60 (s, 1H), 7.72−7.88 (m, 3H), 7.28−
7.42 (m, 3H), 6.88−6.98 (m, 2H), 2.62 (q, J = 8.0 Hz, 2H), 1.13 (t, J =
8.0 Hz, 3H). 13C NMR (DMSO-d6) δ 164.8, 164.2, 156.4, 149.4,
134.3, 131.3, 131.1, 128.4, 127.9, 120.0, 119.1, 117.1, 28.8, 15.3. HPLC
purity 99.4%. MS (ESI) m/z 270 (M + H)+. HRMS (ESI) calcd for
C16H16NO3 270.1125 (M + H)+, found 270.1131.
EXPERIMENTAL SECTION
2-Hydroxy-N-(4-methylbenzoyl)benzamide (1b). The general
procedure was used with salicylamide (2) and 4-methylbenzoyl
chloride (3b) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (85%). 1H
NMR (DMSO-d6) δ 11.88 (s, 1H), 11.77 (s, 1H), 7.88 (dd, J = 8.0, 2.0
Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.37
(d, J = 8.0 Hz, 2H), 6.96−7.08 (m, 2H), 2.36 (s, 3H). 13C NMR
(DMSO-d6) δ 164.7, 164.1, 156.4, 143.3, 134.3, 131.1, 131.0, 129.5,
127.8, 120.0, 119.1, 117.1, 99.6, 21.1. HPLC purity 97.6%. HRMS
(ESI) calcd for C15H14NO3 256.0968 (M + H)+, found 256.0978.
2-Hydroxy-N-(4-isopropylbenzoyl)benzamide (1c). The gen-
eral procedure was used with salicylamide (2) and 4-isopropylbenzoyl
chloride (3c) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (22% over
■
Antiprotozoal Assays. The in vitro activities against T. b.
rhodesiense, T. cruzi, L. donovani, and P. falciparum (K1 strain) were
determined as described earlier.19 The following strains and parasite
forms were used: T. b. rhodesiense, STIB900, trypomastigote form; T.
cruzi, Tulahuen C2C4, amastigote form in L6 rat myoblasts; L.
donovani, MHOM/ET/67/L82, axenic amastigote form; P. falciparum,
K1 (chloroquine and pyrimethamine resistant) erythrocytic stage.
Chemistry. All chemicals and solvents were purchased from Sigma-
Aldrich and/or Fisher Scientific and were used without further
purification. Anhydrous THF and CH2Cl2 were obtained by
distillation over sodium wire or CaH2, respectively. All nonaqueous
reactions were carried out under argon atmosphere with exclusion of
1
moisture from reagents, and all reaction vessels were oven-dried. H
1
NMR and 13C NMR spectra were recorded on a Bruker spectrometer
at 400 and 100 MHz, respectively, and were referenced to the
residual peaks of CHCl3 at 7.26 ppm or DMSO-d6 at 2.50 ppm (1H
NMR) and CDCl3 at 77.23 ppm or DMSO-d6 at 39.51 ppm (13C
NMR). Chemical shifts were reported in parts per million downfield of
TMS and the following abbreviations used to denote coupling
patterns: s = singlet; d = doublet, t = triplet, q = quartet, br = broad, fs
= fine splitting. Mass spectra were measured in the ESI mode at an
ionization potential of 70 eV with an LC-MS MSD (Hewlett-Packard).
HRMS experiments were performed on a Q-TOF-2TM instrument
two steps). H NMR (CD3OD/CDCl3) δ 8.09 (d, J = 8.0 Hz, 1H),
7.90 (d, J = 8.4 Hz, 2H), 7.46 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.4 Hz,
2H), 7.00 (m, 2H), 3.00 (q, J = 6.8 Hz, 1H), 1.28 (d, J = 6.8 Hz, 6H).
13C NMR (CD3OD/CDCl3) δ 167.1, 166.2, 158.0, 156.1, 136.0, 132.8,
132.3, 128.9, 128.1, 121.6, 119.3, 117.9, 35.4, 24.0. HPLC purity
97.6%. MS (ESI) m/z 284 (M + H)+.
N-(4-tert-Butylbenzoyl)-2-hydroxybenzamide (1d). The gen-
eral procedure was used with salicylamide (2) and 4-tert-butylbenzoyl
chloride (3d) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (95%). 1H
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1
NMR (DMSO-d6) δ 11.94 (s, 1H), 11.91 (s, 1H), 7.84−7.92 (m, 3H),
7.57 (d, J = 8.0 Hz, 2H), 7.43 (dt, J = 8.0, 2.0 Hz, 1H), 6.94−7.05 (m,
2H), 1.31 (s, 9H). 13C NMR (DMSO-d6) δ 164.8, 164.2, 156.4, 156.0,
134.3, 131.1, 127.7, 125.8, 120.0, 119.1, 117.1, 34.9, 30.9.; HPLC
purity 98.0%. HRMS calcd for C18H19NO3 297.1365 (M)+, found
297.1369.
HPLC gave the title compound as a white solid (42%). H NMR
(DMSO-d6) δ 11.8 (s, 1H), 11.7 (br s, 1H), 8.36 (d, J = 8.0 Hz, 2H),
8.11 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz,
1H), 6.99 (m, 2H). 13C NMR (DMSO-d6) δ 164.5, 164.1, 156.6,
149.6, 139.4, 134.3, 130.9, 129.1, 123.7, 119.7, 118.5, 117.0. HPLC
purity 99.8%. MS (ESI) m/z 287 (M + H)+.
N-(2-Hydroxybenzoyl)-[1,1′-biphenyl]-4-carboxamide (1e).
The general procedure was used with salicylamide (2) and biphenyl-
4-carbonyl chloride (3e) as reactants. Purification of the crude material
by preparative HPLC gave the title compound as a white solid (54%
Methyl 4-[(2-hydroxybenzoyl)carbamoyl]benzoate (1l). The
general procedure was used with salicylamide (2) and 4-
(chlorocarbonyl)benzoic acid methyl ester (3l, prepared from the
corresponding carboxylic acid) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a white solid
(200 mg, 33%). 1H NMR (DMSO-d6) δ 11.75 (br s, 2H), 8.11 (d, J =
8.3 Hz, 2H), 8.03 (d, J = 8.3 Hz, 2H), 7.86 (dd, J = 7.7, 1.1 Hz, 1H),
7.46, (td, J = 7.5, 1.4 Hz, 1H), 7.04−6.97 (m, 2H), 3.90 (s, 3H). 13C
NMR (DMSO-d6) δ 165.5, 164.6, 164.5, 137.8, 134.3, 133.0, 131.0,
129.5, 128.1, 119.9, 119.0, 117.0, 52.5. HPLC purity 99.5%. MS (ESI)
m/z 300 (M + H)+.
2-Hydroxy-N-(4-methoxybenzoyl)benzamide (1m). The gen-
eral procedure was used with salicylamide (2) and 4-methoxybenzoyl
chloride (3m) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (37%). 1H
NMR (DMSO-d6) δ 11.81 (s, 1H), 11.72 (s, 1H), 7.80−7.91 (m, 3H),
7.46 (dt, J = 8.4, 2.0 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H) 6.90−7.05 (m,
2H), 3.85 (s, 3H). 13C NMR (DMSO-d6) δ 164.3, 163.0, 156.4, 134.2,
131.1, 129.2, 125.9, 119.9, 119.2, 117.1, 114.2, 99.6, 55.7. HPLC purity
95.0%. HRMS (ESI) calcd for C15H14NO4 272.0917 (M + H)+, found
272.0927.
N-(4-Ethoxybenzoyl)-2-hydroxybenzamide (1n). The general
procedure was used with salicylamide (2) and 4-ethoxybenzoyl
chloride (3n) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (77%). 1H
NMR (DMSO-d6) δ 11.87 (s, 1H), 11.56 (s, 1H), 7.90−8.05 (m, 3H),
7.46 (dt, J = 8.4, 2.0 Hz, 1H), 6.93−7.05 (m, 4H), 4.13 (d, J = 4.0 Hz,
2H), 1.30 (t, J = 4.0 Hz, 3H). 13C NMR (DMSO-d6) δ 164.2, 162.3,
156.4, 134.2, 131.1, 129.9, 125.6, 120.0, 119.2, 117.0, 114.6, 63.7, 14.6.
HPLC purity 99.6%. HRMS (ESI) calcd for C16H14NO4 284.0928 (M
− H)+, found 284.0936.
N-(4-Butoxybenzoyl)-2-hydroxybenzamide (1o). The general
procedure was used with salicylamide (2) and 4-butoxybenzoyl
chloride (3o) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (62%). 1H
NMR (CDCl3) δ 8.12 (d, J = 8.4 Hz, 2H), 7.90 (m, 1H), 7.50 (m,
1H), 7.32 (m, 1H), 7.20 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H),
6.48 (br, 1H), 6.33 (br, 1H), 4.05 (d, J = 6.4 Hz, 2H), 1.81 (m, 2H),
1.52 (m, 2H), 0.99 (d, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 167.6,
164.8, 164.2, 148.6, 132.6, 132.4, 130.7, 127.7, 126.4, 123.5, 120.8,
114.8, 68.3, 31.2, 19.3, 14.0. HPLC purity 99.8%. MS (ESI) m/z 314
(M + H)+.
1
over two steps). H NMR (DMSO-d6) δ 11.8 (s, 1H), 11.7 (s, 1H),
8.02 (d, J = 4.0 Hz, 2H), 7.89 (t, J = 8.0 Hz, 3H), 7.77 (d, J = 8.0 Hz,
2H), 7.49 (m, 4H), 7.01 (q, J = 8.0 Hz, 2H). 13C NMR (DMSO-d6) δ
164.6, 164.3, 156.4, 144.4, 138.8, 134.2, 132.5, 131.0, 129.1, 128.4,
127.1, 127.0, 120.0, 119.1, 117.0, 99.5. HPLC purity 98.9%. MS (ESI)
m/z 318 (M + H)+.
N-(4-Fluorobenzoyl)-2-hydroxybenzamide (1f). The general
procedure was used with salicylamide (2) and 4-fluorobenzoyl chloride
(3f) as reactants. Purification of the crude material by preparative
1
HPLC gave the title compound as a white solid (17%). H NMR
(DMSO-d6) δ 11.82 (s, 2H), 7.90−8.05 (m, 2H), 7.84 (dd, J = 7.6, 2.0
Hz, 1H), 7.36−7.48 (m, 3H), 6.94−7.02 (m, 2H). 13C NMR (DMSO-
d6) δ 166.0, 164.3 (d, J = 172 Hz), 163.6, 156.4, 134.3, 131.0, 130.7 (d,
J = 38 Hz), 130.3 (d, J = 12 Hz), 119.9, 119.2, 117.1, 116.1, 115.9.
HPLC purity 96.0%. HRMS (ESI) calcd for C14H11FNO3 260.0717
(M + H)+, found 260.0720.
2-Hydroxy-N-(4-(trifluoromethyl)benzoyl)benzamide (1g).
The general procedure was used with salicylamide (2) and 4-
(trifluoromethyl)benzoyl chloride (3g) as reactants. Purification of the
crude material by preparative HPLC gave the title compound as a
1
white solid (29%). H NMR (DMSO-d6) δ 11.64 (s, 1H), 11.46 (s,
1H), 8.09 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.0 Hz, 2H), 7.84 (dd, J =
8.0, 2.0 Hz, 1H), 7.46 (dt, J = 8.0, 2.0 Hz, 1H), 6.90−7.04 (m, 2H),
3.85 (s, 3H). 13C NMR (DMSO-d6) δ 164.8, 164.6, 156.6, 137.8,
134.4, 132.4 (q, J = 32 Hz), 131.0, 129.2 (2C), 125.8 (q, J = 4 Hz),
123.5 (q, J = 270 Hz), 119.9, 119.0, 117.1. HPLC purity 98.0%. HRMS
(ESI) calcd for C15H11F3NO3 310.0685 (M + H)+, found 310.0689.
2-Hydroxy-N-(4-(trifluoromethoxy)benzoyl)benzamide (1h).
The general procedure was used with salicylamide (2) and 4-
(trifluoromethoxy)benzoyl chloride (3h) as reactants. Purification of
the crude material by preparative HPLC gave the title compound as a
white solid (38%). 1H NMR (DMSO-d6) δ 11.7 (br s, 2H), 8.04 (d, J
= 8.0 Hz, 2H), 7.85 (dd, J = 8.0, 4.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H),
7.46 (t, J = 8.0 Hz, 1H), 7.01 (m, 2H). 13C NMR (DMSO-d6) δ 164.7,
164.3, 156.5, 151.2 (q, J = 1.8 Hz), 134.3, 133.0, 131.0, 130.3 (2C),
121.1 (2C), 119.9 (q, J = 258 Hz), 119.8, 119.2, 117.1. HPLC purity
99.0%. MS (ESI) m/z 326 (M + H)+.
N-(4-Cyanobenzoyl)-2-hydroxybenzamide (1i). The general
procedure was used with salicylamide (2) and 4-cyanobenzoyl chloride
(3i) as reactants. Purification of the crude material by preparative
N-[4-(Benzyloxy)benzoyl]-2-hydroxybenzamide (1p). The
general procedure was used with salicylamide (2) and 4-(benzyloxy)-
benzoyl chloride (3p) as reactants. Purification of the crude material
by preparative HPLC gave the title compound as a white solid (39%).
1H NMR (DMSO-d6) δ 11.7 (br s, 1H), 11.6 (br s, 1H), 7.87 (m, 3H),
7.41 (m, 6H), 7.17 (d, J = 8.0 Hz, 2H), 7.00 (m, 2H), 5.22 (s, 2H).
13C NMR (DMSO-d6) δ 164.2, 164.1, 162.0, 156.3, 136.5, 134.2,
131.0, 129.8, 128.5, 128.1, 127.8, 126.0, 119.9, 119.2, 117.0, 115.0,
69.6. HPLC purity 98.3%. MS (ESI) m/z 348 (M + H)+.
1
HPLC gave the title compound as a white solid (91%). H NMR
(DMSO) δ 11.73 (s, 2H), 8.04 (app s, 4H), 7.84 (dd, J = 7.6, 2.0 Hz,
1H), 7.46 (dt, J = 7.6, 2.0 Hz, 1H), 6.95−7.05 (m, 2H), 2.36 (s, 3H).
13C NMR (DMSO) δ 164.7, 164.4, 156.6, 137.9, 134.4, 132.6, 131.0,
128.5, 119.9, 119.0, 118.4, 117.5, 114.9. HPLC purity 96.4%. HRMS
(ESI) calcd for C15H9N2O3 265.0619 (M − H)+, found 265.0629.
2-Hydroxy-N-[4-(methylsulfonyl)benzoyl]benzamide (1j).
The general procedure was used with salicylamide (2) and 4-
(methylsulfonyl)benzoyl chloride (3j, prepared from the correspond-
ing carboxylic acid) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a pale-pink solid (90
mg, 14%). 1H NMR (DMSO-d6) δ 11.73 (br s, 1H), 11.61 (br s, 1H),
8.14−8.09 (m, 4H), 7.84 (dd, J = 7.9, 1.7 Hz, 1H), 7.47 (ddd, J = 8.5,
7.3, 1.8 Hz, 1H), 7.03−6.98 (m, 2H), 3.31 (s, 3H). 13C NMR
(DMSO-d6) δ 164.8, 164.6, 156.5, 144.0, 138.4, 134.3, 130.9, 128.8,
127.4, 119.8, 119.0, 117.0, 43.2. HPLC purity 99.7%. MS (ESI) m/z
320 (M + H)+.
N-[4-(Dimethylamino)benzoyl]-2-hydroxybenzamide (1q).
The general procedure was used with salicylamide (2) and 4-
(dimethylamino)benzoyl chloride (3q) as reactants. Purification of the
crude material by preparative HPLC gave the title compound as yellow
1
solid (31%). H NMR (DMSO-d6) δ 11.8 (s, 1H), 11.5 (s, 1H), 7.90
(d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H),
7.02 (m, 2H), 6.77 (d, J = 8.0 Hz, 2H), 3.03 (s, 6H). 13C NMR
(DMSO-d6) δ 164.6, 164.4, 156.7, 153.6, 134.5, 131.5, 130.0, 120.3,
120.0, 119.6, 117.5, 111.5, 40.8. HPLC purity 98.3%. MS (ESI) m/z
285 (M + H)+.
2-Hydroxy-N-(4-nitrobenzoyl)benzamide (1k). The general
procedure was used with salicylamide (2) and 4-nitrobenzoyl chloride
(3k) as reactants. Purification of the crude material by preparative
N-[4-(Diethylamino)benzoyl]-2-hydroxybenzamide (1r). The
general procedure was used with salicylamide (2) and 4-
(diethylamino)benzoyl chloride (3r, prepared from the corresponding
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1
carboxylic acid) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as yellow solid (8% over
two steps). 1H NMR (DMSO-d6) δ 11.8 (br s, 1H), 11.5 (s, 1H), 7.90
(d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H),
7.00 (m, 2H), 6.73 (d, J = 8.0 Hz, 2H), 3.41 (q, J = 8.0 Hz, 4H), 1.12
(t, J = 8.0 Hz, 6H). 13C NMR (DMSO-d6) δ 164.6, 164.4, 156.8,
151.2, 134.5, 131.5, 130.2, 120.3, 119.6, 119.0, 117.5, 110.9, 44.3, 12.7.
HPLC purity 98.5%. MS (ESI) m/z 313 (M + H)+.
2-Hydroxy-N-(4-morpholinobenzoyl)benzamide (1s). The
general procedure was used with salicylamide (2) and 4-
morpholinobenzoyl chloride (3s, prepared from the corresponding
carboxylic acid) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (25% over
two steps). 1H NMR (DMSO-d6) δ 11.8 (br s, 1H), 11.5 (s, 1H), 7.89
(d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.46 (t, J = 8.0 Hz, 1H),
7.00 (m, 4H), 3.75 (t, J = 4.0 Hz, 4H), 3.30 (t, J = 4.0 Hz, 4H). 13C
NMR (DMSO-d6) δ 164.8, 164.5, 156.7, 154.4, 134.5, 131.5, 129.8,
122.8, 120.4, 119.6, 117.5, 113.8, 66.3, 47.3. HPLC purity 98%. MS
(ESI) m/z 327 (M + H)+.
N-(2-Hydroxybenzoyl)-2-naphthamide (1t). The general pro-
cedure was used with salicylamide (2) and naphthalene-2-carbonyl
chloride (3t, prepared from the corresponding carboxylic acid) as
reactants. Purification of the crude material by preparative HPLC gave
the title compound as a white solid (170 mg, 29% over two steps). 1H
NMR (DMSO-d6) δ 11.89 (br s, 2H), 8.58 (s, 1H), 8.10 (m, 2H), 8.04
(d, J = 7.6 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H),
7.71−7.64 (m, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H),
7.01 (d, J = 7.5 Hz, 1H). 13C NMR (DMSO-d6) δ 165.1, 164.4, 156.6,
134.8, 134.2, 132.1, 131.1, 131.0, 129.2, 128.7, 128.6, 128.5, 127.7,
127.2, 123.8, 119.8, 119.1, 117.1. HPLC purity 100%. MS (ESI) m/z
292 (M + H)+.
(234 mg, 41% over two steps). H NMR (DMSO-d6) δ 11.75 (br s,
1H), 11.57 (s, 1H), 7.87 (dd, J = 7.8, 1.4 Hz, 1H), 7.52 (dd, J = 8.1,
1.5 Hz, 1H), 7.47−7.41 (m, 2H), 7.08 (d, J = 8.2 Hz, 1H), 7.03−6.97
(m, 2H), 6.16 (s, 2H). 13C NMR (DMSO-d6) δ 164.2, 163.8, 156.3,
151.2, 147.9, 134.2, 131.0, 127.7, 123.3, 119.9, 119.1, 117.0, 108.3,
107.6, 102.2. HPLC purity 98.7%. MS (ESI) m/z 286 (M + H)+.
N-(2-Hydroxybenzoyl)-3,4,5-trimethoxybenzamide (1y). The
general procedure was used with salicylamide (2) and 3,4,5-
trimethoxybenzoyl chloride (3y) as reactants. Purification of the
crude material by preparative HPLC gave the title compound as a
1
white solid (33%). H NMR (DMSO-d6) δ 11.63 (s, 1H), 11.60 (s,
1H), 7.86 (dd, J = 7.6, 2.0 Hz, 1H), 7.23 (s, 1H), 6.96−7.10 (m, 2H).
13C NMR (DMSO-d6) δ 164.2, 164.1, 156.3, 141.4, 134.2, 131.1,
128.8, 120.0, 119.5, 117.1, 105.3, 60.2, 56.2. HPLC purity 96.9%.
HRMS (ESI) calcd for C17H18NO6 332.1129 (M + H)+, found
332.1135.
N-(Cyclohexanecarbonyl)-2-hydroxybenzamide (1z). The
general procedure was used with salicylamide (2) and cyclo-
hexanecarbonyl chloride (3z) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a white solid
(354 mg, 48%). 1H NMR (DMSO-d6) δ 11.44 (s, 1H), 10.80 (s, 1H),
7.81 (dd, J = 7.9, 1.7 Hz, 1H), 7.44 (ddd, J = 8.3, 7.2, 1.7 Hz, 1H), 6.98
(dd, J = 8.2, 0.9 Hz, 1H), 6.94 (d, J = 7.1, 1.1 Hz, 1H), 2.87 (tt, J =
11.0, 3.3 Hz, 1H), 1.89−1.86 (m, 2H), 1.75−1.72 (m, 2H), 1.66−1.63
(m, 1H), 1.39−1.13 (m, 5H). 13C NMR (DMSO-d6) δ 176.2, 164.8,
156.7, 134.2, 130.7, 119.7, 118.6, 117.0, 44.6, 28.5, 25.4, 25.1. HPLC
purity 100%. MS (ESI) m/z 248 (M + H)+.
N-(2-Hydroxybenzoyl)-2-methylfuran-3-carboxamide (1aa).
The general procedure was used with salicylamide (2) and 2-
methylfuran-3-carbonyl chloride (3aa) as reactants. Purification of the
crude material by preparative HPLC gave the title compound as a
1
N-(2-Hydroxybenzoyl)-6-methoxy-2-naphthamide (1u). The
general procedure was used with salicylamide (2) and 6-
methoxynaphthalene-2-carbonyl chloride (3u, prepared from the
corresponding carboxylic acid) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a pale-
white solid (83%). H NMR (DMSO-d6) δ 11.65 (s, 1H), 11.24 (s,
1H), 7.84 (d, J = 7.6 Hz, 1H), 7.68, (s, 1H), 7.45 (t, J = 7.6 Hz, 1H),
6.82−7.05 (m, 2H), 6.77 (s, 1H), 2.50 (s, 3H). 13C NMR (DMSO-d6)
δ 164.1, 161.0, 158.8, 156.2, 141.6, 134.2, 131.0, 119.9, 119.2, 117.0,
115.9, 108.8, 13.5. HPLC purity 98.7%. HRMS (ESI) calcd for
C13H12NO4 246.0761 (M + H)+, found 246.0765.
1
yellow solid (180 mg, 28% over two steps). H NMR (DMSO-d6) δ
11.79 (s, 1H), 11.76 (s, 1H), 8.51 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H),
7.97 (d, J = 8.8 Hz, 1H), 7.92 (dd, J = 3.4, 1.8 Hz, 1H), 7.90 (m, 1H),
7.50−7.45 (m, 2H), 7.29 (dd, J = 8.9, 2.5 Hz, 1H), 7.05 (d, J = 8.1 Hz,
1H), 7.01 (t, J = 7.7 Hz, 1H), 3.93 (s, 3H). 13C NMR (DMSO-d6) δ
165.0, 164.4, 159.3, 156.4, 136.7, 134.2, 131.1, 130.9, 128.6, 127.4,
127.3, 124.4, 119.9, 119.8, 119.2, 117.1, 106.0, 55.5. HPLC purity
97.2%. MS (ESI) m/z 322 (M + H)+.
2-(Benzyloxy)-N-[(2-hydroxyphenyl)carbonyl]benzamide
(1ab). The general procedure was used with salicylamide (2) and 2-
(benzyloxy)benzoyl chloride (3ab) as reactants. The reaction was
carried out on 4 mmol scale and provided crude material as a brown
oil (423 mg, 30%), which was pure enough to be used in the next step
without further purification. Purification of a portion of the crude
material by preparative HPLC gave the title compound as a white solid
1
N-[3-(Chloromethyl)benzoyl]-2-hydroxybenzamide (1v). The
general procedure was used with salicylamide (2) and 3-
(chloromethyl)benzoyl chloride (3v) as reactants. Purification of the
crude material by preparative HPLC gave the title compound as a
(60 mg). H NMR (DMSO-d6) δ 11.68 (br s, 1H), 11.39 (br s, 1H),
7.79 (dd, J = 7.9, 1.6 Hz, 1H), 7.73 (dd, J = 7.7, 1.7 Hz, 1H), 7.52
(ddd, J = 8.5, 7.5, 1.8 Hz, 1H), 7.44 (ddd, J = 8.3, 7.3, 1.8 Hz, 1H),
7.38−7.35 (m, 2H), 7.25−7.21 (m, 4H), 7.09 (td, J = 7.6, 0.5 Hz, 1H),
6.96 (dd, J = 8.3, 0.5 Hz, 1H), 6.93 (td, J = 8.0, 0.8 Hz, 1H), 5.29 (s,
2H). 13C NMR (DMSO-d6) δ 164.8, 164.4, 156.8, 155.8, 136.3, 134.4,
133.4, 130.9, 130.8, 128.4 (2C), 127.9, 127.3 (2C), 123.8, 121.1, 119.8,
118.4, 117.0, 113.6, 69.9. HPLC purity 99.2%. MS (ESI) m/z (M +
H)+.
N-(4-Aminobenzoyl)-2-hydroxybenzamide (4a). Compound
4a was prepared by standard hydrogenolysis of compound 1i as a
pale-yellow solid (2.468 g, 96%). 1H NMR (DMSO-d6) δ 11.77 (br s,
1H), 11.44 (s, 1H), 7.90 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H),
7.45 (t, J = 7.3 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.99 (t, J = 7.4 Hz,
1H), 6.62 (d, J = 8.2 Hz, 2H), 6.05 (br, 2H). 13C NMR (DMSO-d6) δ
164.1, 163.9, 156.3, 153.5, 134.0, 131.0, 129.7, 119.9, 119.5, 119.1,
117.0, 112.8. HPLC purity 97.7%. MS (ESI) m/z 257 (M + H)+.
2-Hydroxy-N-(2-hydroxybenzoyl)benzamide (4b). Com-
pound 4b was prepared by standard hydrogenolysis of compound
1ab as a white solid (76%). 1H NMR (DMSO-d6) δ 11.54 (br s, 3H),
7.82 (dd, J = 7.8, 1.5 Hz, 2H), 7.45 (td, J = 8.5, 1.6 Hz, 2H), 7.02−6.96
(m, 4H). 13C NMR (DMSO-d6) δ 164.6, 156.9, 134.3, 130.7, 119.7,
119.0, 117.1. HPLC purity 98.5%. MS (ESI) m/z 258 (M + H)+.
Methyl 4-[[4-[(2-Hydroxybenzoyl)carbamoyl]phenyl]-
amino]-4-oxobutanoate (5). To a suspension of 4a (244 mg,
0.95 mmol) and pyridine (0.085 mL, 1.05 mmol) in CH2Cl2 (2 mL)
1
white solid (24%). H NMR (DMSO-d6) δ 11.85 (s, 2H), 7.99 (s,
1H), 7.84−7.90 (m, 2H), 7.73 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz,
1H), 7.46 (dt, J = 7.6, 1.6 Hz, 1H), 6.90−7.02 (m, 2H), 4.87 (s, 2H).
13C NMR (DMSO-d6) δ 164.7, 164.4, 156.5, 138.7, 134.3, 134.2,
133.2, 131.1, 129.3, 128.3, 127.5, 120.0, 119.1, 117.1, 45.5. HPLC
purity 96.2%. HRMS (ESI) calcd for C15H13ClNO3 290.0578 (M +
H)+, found 290.0584.
2,4-Dichloro-N-(2-hydroxybenzoyl)benzamide (1w). The
general procedure was used with salicylamide (2) and 2,4-
dichlorobenzoyl chloride (3w) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a white solid
1
(41%). H NMR (DMSO-d6) δ 11.60 (s, 2H), 7.68−7.82 (m, 2H),
7.60 (d, J = 7.6 Hz, 1H), 7.54 (dd, J = 7.6, 2.0 Hz, 1H), 7.47 (dt, J =
7.6, 2.0 Hz, 1H), 6.90−7.02 (m, 2H). 13C NMR (DMSO-d6) δ 166.2,
165.0, 157.3, 135.2, 134.9, 130.7, 130.6, 129.9, 129.2, 127.6, 119.8,
117.8, 117.2. HPLC purity 98.8%. HRMS (ESI) calcd for
C14H8Cl2NO3 307.9887 (M − H)+, found 307.9891.
N-(2-Hydroxybenzoyl)benzo[d][1,3]dioxole-5-carboxamide
(1x). The general procedure was used with salicylamide (2) and
benzo[d][1,3]dioxole-5-carbonyl chloride (3x, prepared from the
corresponding carboxylic acid) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a white solid
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was added a solution of methyl 3-(chlorocarbonyl)propanoate (150
mg, 1.0 mmol) in CH2Cl2 (2 mL) at 0 °C. The mixture was stirred in
the cold bath for 30 min and then at room temperature for 1 h before
quenching with HCl (0.1M) to give a pale-green precipitate, which
was filtered off, washed with water, and dried. The crude material was
recrystallized from MeOH and purified further by HPLC to give the
title compound as a white solid (90 mg, 26%). 1H NMR (DMSO-d6) δ
11.72 (s, 1H), 11.61 (s, 1H), 10.38 (s, 1H), 7.88 (d, J = 8.5 Hz, 3H),
7.75 (d, J = 8.7 Hz, 2H), 7.46 (td, J = 7.5, 1.8 Hz, 1H), 7.03 (d, J = 8.2
Hz, 1H), 7.00 (t, J = 8.2 Hz, 1H), 3.60 (s, 3H), 2.69−2.60 (m, 4H).
13C NMR (DMSO-d6) δ 172.8, 170.5, 164.2, 156.4, 143.3, 134.2,
7.5, 1.6 Hz, 1H), 7.60−7.53 (m, 3H), 7.46 (d, J = 8.3 Hz, 2H), 7.42−
7.31 (m, 4H), 7.22−7.11 (m, 3H), 5.29 (s, 2H), 2.57 (q, J = 7.5 Hz,
2H), (t, J = 7.6 Hz, 3H); 13C NMR (DMSO-d6) δ 167.5, 156.2, 150.3,
139.2, 136.1, 135.2, 133.8, 130.3, 128.5, 128.2, 128.1, 127.6, 122.0,
121.0, 119.8, 113.7, 70.3, 27.5, 15.6; MS (ESI) m/z 375 (M + H)+.
Standard hydrogenolysis of the intermediate 15 afforded the final
compound 16 as a clean product (254 mg of a white powdery solid,
1
69%). H NMR (DMSO-d6) δ 11.83 (br s, 1H), 10.69 (s, 1H), 10.57
(s, 1H), 7.96 (dd, J = 7.9, 1.4 Hz, 1H), 7.50 (dd, J = 8.0, 1.4 Hz, 1H),
7.48 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.2 Hz,
1H), 7.01 (t, J = 7.6 Hz, 1H), 2.57 (q, J = 7.5 Hz, 2H), 1.16 (t, J = 7.6
Hz, 3H). 13C NMR (DMSO-d6) δ 166.7, 156.8, 150.4, 139.3, 135.2,
135.0, 130.8, 128.2, 120.0, 119.9, 117.2, 117.0, 27.6, 15.7. HPLC purity
99.8%. MS (ESI) m/z 285 (M + H)+.
131.0, 128.9, 127.6, 119.9, 119.1, 118.4, 117.0, 51.4, 31.0, 28.3. HPLC
purity 97.7%. MS (ESI) m/z 371 (M + H)+.
N-Hydroxy-N′-[4-[[(2-hydroxyphenyl)carbonyl]carbamoyl]-
phenyl]octanediamide (8). Compound 8 was prepared by standard
hydrogenolysis of 7 as a pale-yellow solid (15 mg, 7% over two steps).
Compound 7 was obtained from 6 by following the same methodology
as employed for the preparation of 5 from 4a, with the exception of
using 8-[(benzyloxy)amino]-8-oxooctanoyl chloride as the acylating
agent and extending the reaction time to 2 h. 1H NMR (DMSO-d6) δ
11.73 (br s, 1H), 11.62 (br s, 1H), 10.33 (br s, 1H), 10.25 (br s, 1H),
8.65 (br s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.77
(d, J = 8.8 Hz, 2H), 7.46 (td, J = 7.5, 1.6 Hz, 1H), 7.04−6.98 (m, 2H),
2.35(t, J = 7.5 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.59 (t, J = 7.5 Hz,
2H), 1.49 (t, J = 7.0 Hz, 2H), 1.29 (br, 4H). 13C NMR (DMSO-d6) δ
171.9, 169.1, 164.2, 156.3, 143.5, 134.2, 131.0, 128.8, 127.6, 119.9,
119.1, 118.5, 117.0, 36.4, 32.2, 28.4, 25.0, 24.8. HPLC purity 96.0%.
MS (ESI) m/z 428 (M + H)+.
1-(4-Ethylphenyl)-3-(2-hydroxyphenyl)urea (19). To a sol-
ution of 17 (0.27 mL, 2.0 mmol) in CH2Cl2 (4 mL) was added
dropwise 4-ethylaniline (0.25 mL, 2.0 mmol). The resulting mixture
was stirred for 19 h at room temperature, and the resulting precipitate
was then filtered off and washed with CH2Cl2 (25 mL) to give the
1
desired crude 18 as a pale-yellow solid (507 mg, 94%). H NMR
(DMSO-d6) δ 9.20 (s, 1H), 8.17 (s, 1H), 8.12 (dd, J = 7.8, 1.7 Hz,
1H), 7.35 (d, J = 8.4 Hz, 2H), 7.1 (d, J = 8.4 Hz, 2H), 7.01 (dd, J =
7.9, 1.4 Hz, 1H), 6.93 (td, J = 7.7, 1.8 Hz, 1H), 6.88 (td, J = 7.7, 1.5
Hz, 1H), 3.87 (s, 3H), 2.54 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz,
3H). 13C NMR (DMSO-d6) δ 152.4, 147.6, 137.5, 137.1, 128.8, 128.0,
121.6, 120.5, 118.2, 118.1, 110.7, 55.8, 27.5, 15.8. HPLC purity 99.7%.
MS (ESI) m/z 271 (M + H)+. To a solution of 18 (270 mg, 1.0 mmol)
in CH2Cl2 (25 mL) was added dropwise BBr3 (4.0 mL of a 1.0 M
solution in CH2Cl2, 4.0 mmol) at −78 °C. The resulting mixture was
allowed to warm to 0 °C within 2 h. After stirring for further 2 h at 0
°C, the reaction mixture was quenched with MeOH (25 mL) and
washed with satd NaHCO3 (2 × 50 mL) and H2O (2 × 50 mL). The
organic phase was dried over Na2SO4, filtered, and concentrated to
give the crude product which was dissolved in MeOH to yield 19 as
N-(4-Ethylbenzoyl)-2-hydroxy-N-methylbenzamide (10). The
general procedure was used with 2-hydroxy-N-methylbenzamide (9)14
and 4-ethylbenzoyl chloride (3a) as reactants. Purification of the crude
material by preparative HPLC gave the title compound as a white solid
1
(84% over two steps). H NMR (CDCl3) δ 8.08 (d, J = 8.0 Hz, 2H),
7.75 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz,
2H), 7.27 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.58 (br s, 1H),
2.79 (d, J = 4.8 Hz, 3H), 2.73 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz,
3H). 13C NMR (CDCl3) δ 166.4, 165.1, 151.1, 148.2, 131.6, 130.4,
129.9, 128.9, 128.4, 126.4, 126.2, 123.2, 29.1, 26.7, 15.2. HPLC purity
98.7%. MS (ESI) m/z 284 (M + H)+.
N′-(4-Ethylbenzoyl)-2-hydroxybenzhydrazide (12). The gen-
eral procedure was used with salicylhydrazide (11) and 4-ethylbenzoyl
chloride (3a) as reactants. Purification of the crude material by
preparative HPLC gave the title compound as a white solid (36%). 1H
NMR (DMSO-d6) δ 12.0 (br s, 1H), 10.7 (br s, 1H), 10.6 (s, 1H),
7.93 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.46 (t, J = 8.0 Hz,
1H), 7.36 (d, J = 8.0 Hz, 2H), 6.97 (m, 2H), 2.68 (q, J = 8.0 Hz, 2H),
1.21 (t, J = 8.0 Hz, 3H). 13C NMR (DMSO-d6) δ 168.2, 165.9, 159.8,
148.6, 134.6, 130.2, 128.7, 128.3, 128.1, 119.5, 117.8, 115.0, 28.5, 15.7.
HPLC purity 97.5%. MS (ESI) m/z 285 (M + H)+.
1
pale-yellow crystals (175 mg, 68%). H NMR (DMSO-d6) δ 9.89 (s,
1H), 9.19 (s, 1H), 8.11 (s, 1H), 8.03 (dd, J = 7.6, 1.3 Hz, 1H), 7.35 (d,
J = 8.3 Hz, 2H), 7.10 (d, J = 8.3 Hz, 2H), 6.83 (dd, J = 7.6, 1.4 Hz,
1H), 6.78 (td, J = 7.2, 1.5 Hz, 1H), 6.73 (td, J = 7.3, 1.4 Hz, 1H), 2.54
(q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.5 Hz, 3H). 13C NMR (DMSO-d6) δ
152.6, 145.6 137.6, 137.0, 128.0, 127.9, 121.6, 119.1, 118.5, 118.0,
114.4, 27.5, 15.8. HPLC purity 100%. MS (ESI) m/z 257 (M + H)+.
3-(4-Ethylbenzoyl)-2H-benz[e][1,3]oxazine-2,4(3H)-dione
(21a). The general procedure was used with 2H-benz[e][1,3]oxazine-
2,4(3H)-dione (20a) and 4-ethylbenzoyl chloride (3a) as reactants.
Purification of the crude material by preparative HPLC gave the title
compound as a white solid (29%). 1H NMR (DMSO-d6) δ 8.17 (d, J =
8.4 Hz, 2H), 8.00 (d, J = 6.8 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.52
(m, 2H), 7.44 (d, J = 8.0 Hz, 2H), 2.72 (q, J = 7.6 Hz, 2H), 1.21 (t, J =
7.6 Hz, 3H). 13C NMR (DMSO-d6) δ 167.5, 160.4, 153.3, 153.0,
145.8, 137.0, 131.3, 128.9, 128.4, 127.1, 125.7, 116.8, 114.3, 28.4, 15.0.
HPLC purity 97.4%. MS (ESI) m/z 296 (M + H)+.
N-(1-Acetyl-1H-indol-3-yl)-4-ethylbenzamide (21b). To a
suspension of 1-(1H-indol-3-yl)ethanone (796 mg, 5 mmol) in 95%
ethanol (6 mL) was added NH2OH·HCl (417 mg, 6 mmol) and
NaOAc (492 mg, 6 mmol). After refluxing for 2 h, the mixture was
cooled, diluted with EtOAc (50 mL), washed with H2O (20 mL) and
brine (20 mL), dried over Na2SO4, and concentrated in vacuo. The
crude product was purified by column chromatography on silica gel
using hexane−EtOAc (2:1) as the eluent to give (E)-1-(1H-indol-3-
yl)ethanone oxime as a white solid (797 mg, 92%).20 The oxime was
dissolved in ethanol (6 mL) followed by addition of conc H2SO4
(several drops). The mixture was refluxed for 4 h, concentrated in
vacuo, and then diluted with H2O (40 mL) and washed with CHCl3 (3
× 20 mL). The aqueous layer was basified with 2N NaOH to pH 9
and extracted with EtOAc (3 × 40 mL). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by column
chromatography on silica gel using CHCl3/MeOH (10:1) as the
eluent to give 1-(3-amino-1H-indol-1-yl)ethanone (20b)20 as a dark-
green solid (310 mg, 51%). Then the final compound 21b was
4-Ethyl-N-(2-hydroxythiobenzoyl)benzamide (13). To a
suspension of compound 1a (231 mg, 0.86 mmol) in chlorobenzene
(3 mL) was added Lawesson’s reagent (522 mg, 1.29 mmol) under
argon. The resulting mixture was refluxed for 3 h to produce a clear
dark-red solution. After evaporation of the bulk of solvent, the crude
product was directly purified by column chromatography on silica gel
using hexane−EtOAc (2:1) as the eluent to give 13 as a yellow solid
1
(16.5 mg, 7%). H NMR (CDCl3) δ 8.10 (d, J = 8.0 Hz, 2H), 7.86
(dd, J = 8.0, 1.6 Hz, 1H), 7.64 (br, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.41
(m, 4H), 7.16 (d, J = 8.0 Hz, 1H), 2.75 (q, J = 8.0 Hz, 2H), 1.28 (t, J =
8.0 Hz, 3H). 13C NMR (CDCl3) δ 200.6, 166.0, 151.4, 145.6, 134.6,
131.6, 130.7, 130.6, 128.4, 126.4, 126.1, 123.1, 29.1, 15.2. HPLC purity
97.8%. MS (ESI) m/z 286 (M + H)+.
N-[(4-Ethylphenyl)carbamoyl]-2-hydroxybenzamide (16).
The intermediate 14 was prepared on 20 mmol scale to give the
crude product as a white solid. The crude material was recrystallized
from acetone to provide the product as a white crystals (3.0 g, 66%),
which was subsequently converted into compound 15.15 The latter
was obtained as a white solid (540 mg, 72%) containing an
unidentified impurity (17% by HPLC). NMR data for the product
15: 1H NMR (DMSO-d6) δ 10.59 (s, 1H), 10.52 (s, 1H), 7.77 (dd, J =
3098
dx.doi.org/10.1021/jm2015183 | J. Med. Chem. 2012, 55, 3088−3100

Journal of Medicinal Chemistry
Article
obtained as a dark-green solid (380 mg, 25% over three steps)
following the general procedure. 1H NMR (DMSO-d6) δ 11.7 (s, 1H),
8.33 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 4.0 Hz, 1H), 8.02 (d, J = 8.0 Hz,
2H), 7.45 (m, 3H), 7.20 (m, 2H), 2.72 (q, J = 8.0 Hz, 2H), 2.52 (s,
3H), 1.23 (t, J = 8.0 Hz, 3H). 13C NMR (DMSO-d6) δ 163.6, 161.1,
150.3, 137.5, 130.9, 129.7, 128.8, 127.1, 124.5, 123.4, 123.1, 121.2,
112.3, 111.1, 28.7, 15.7, 14.5. HPLC purity 98.8%. MS (ESI) m/z 307
(M + H)+.
4-Ethyl-N-(1H-indazol-3-yl)benzamide (21c). To a solution of
2-fluorobenzonitrile (0.54 mL, 5 mmol) in n-BuOH (6 mL) was added
NH2NH2·H2O (0.78 mL, 25 mmol). After refluxing for 4 h, the
mixture was cooled, diluted with EtOAc (50 mL), washed with H2O
(20 mL) and brine (20 mL), dried over Na2SO4, and concentrated in
vacuo. The resulting crude 1H-indazol-3-amine (20c)21 was converted
without purification following the general procedure to compound
21c, which was obtained as a white solid (465 mg, 35% over two
steps). 1H NMR (CDCl3) δ 9.76 (s, 1H), 8.73 (s, 1H), 8.12 (d, J = 8.0
Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.39 (m, 2H), 7.33 (d, J = 8.0 Hz,
2H), 7.19 (m, 1H), 2.73 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H).
13C NMR (CDCl3) δ 165.5, 149.1, 141.8, 141.6, 131.2, 128.3, 127.7,
123.4, 120.8, 116.2, 109.6, 28.9, 15.3. HPLC purity 98.1%. MS (ESI)
m/z 266 (M + H)+.
2-[3-(4-Ethylphenyl)-[1,2,4]oxadiazol-5-yl]phenol (23). Neat
compound 1a (22 mg, 0.08 mmol) was heated to its melting point
(∼200 °C) for 1 h. The resulting product 22 was cooled to room
temperature and suspended in absolute ethanol (2 mL). Solid NaOAc
(9 mg, 0.12 mmol) and NH2OH·HCl (8.4 mg, 0.12 mmol) were
added. The mixture was stirred for 16 h, diluted with EtOAc (50 mL),
washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4,
and concentrated in vacuo.17 The crude product was purified directly
by column chromatography on silica gel using hexane−EtOAc (10:1)
as the eluent to give 7 as a white solid (15 mg, 69% over two steps).
1H NMR (CDCl3) δ 10.5 (s, 1H), 8.06 (d, J = 8.0 Hz, 2H), 8.03 (dd, J
139.2, 136.9, 136.4, 128.4 (2C), 128.0 (2C), 127.8, 127.7, 127.4 (2C),
124.2, 120.9, 120.8, 119.4 (2C), 112.7, 69.7, 44.6, 27.7, 15.8. HPLC
purity >95%. MS (ESI) m/z 411 (M + Na)+.
The final compound 28 was obtained as a yellow solid by following
a literature procedure.18 Purification by preparative HPLC provided
the desired compound as a white solid (110 mg, 55% on a 0.67
1
mmolar scale). H NMR (DMSO-d6) δ 9.68 (br s, 1H), 9.43 (br s,
1H), 9.20 (br s, 1H), 9.48 (dd, J = 8.0, 1.0 Hz, 1H), 7.03 (d, J = 8.3
Hz, 2H), 6.69 (d, J = 8.3 Hz, 2H), 6.46 (td, J = 8.0, 1.3 Hz, 1H), 6.41
(dd, J = 8.0, 1.3 Hz, 1H), 6.30 (td, J = 8.0, 1.3 Hz, 1H), 3.10 (s, 2H),
2.09 (q, J = 7.6 Hz, 2H), 0.69 (t, J = 7.5 Hz, 3H). 13C NMR (DMSO-
d6) δ 166.0, 165.3, 147.2, 139.1, 136.4, 128.0 (2C), 126.4, 124.3, 121.1,
119.4 (2C), 119.0, 115.2, 45.0, 27.6, 15.7. HPLC purity 97.7%. MS
(ESI) m/z 299 (M + H)+.
AUTHOR INFORMATION
■
Corresponding Author
*For A.P.K.: phone, +1-312-996-7577; fax, +1-312-996-7107;
E-mail, kozikowa@uic.edu. For R.B.: phone, +41-61-284-8231;
fax, +41-61-284-8101; E-mail, reto.brun@unibas.ch. For R.M.:
phone, 773-834-4152; fax, 773-834-3577; E-mail, rmcleod@
midway.uchicago.edu.
Present Addresses
○Institute of Drug Discovery and Development, /i/AIR,
Shanghai Engineering Research Center of Molecular Ther-
apeutics and New Drug Development, East China Normal
University, Shanghai 200062, PR China.
◆Centro Interdipartimentale Biopharmanet-Tec, Universita
̀
di
Parma, Via GP Usberti 27/A-Campus Universitario, Parma, Italy.
¶Department of Agricultural and Environmental Science,
Tuskegee University, 312 Milbank Hall Building, Tuskegee,
Alabama 36088, United States.
= 8.0, 4.0 Hz, 1H), 7.56 (dd, J = 8.0, 4.0 Hz, 1H), 7.38 (d, J = 8.0 Hz,
2H), 7.17 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 2.76 (q, J = 8.0
Hz, 2H), 1.31 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3) δ 174.1, 167.1,
158.1, 148.4, 135.1, 128.5, 127.8, 127.6, 123.1, 120.1, 117.7, 108.2,
28.9, 15.3. HPLC purity 99.8%. MS (ESI) m/z 267 (M + H)+.
N-(4-Ethylphenyl)-N′-(2-hydroxyphenyl)propanediamide
(28). Compound 25 was obtained from 24 by following the same
methodology as employed for the preparation of 5 from 4a, with the
exception of using ethyl 3-chloro-3-oxopropanoate as the acylating
agent and extending the reaction time to 18 h. The crude material was
subsequently purified by column chromatography on silica gel with
hexane−EtOAc (gradient from 0 to 30% EtOAc) as the eluent to
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the financial support provided by NIAID
grant U01 AI082180, The Cornwell−Mann Family Founda-
tion, The Pritzker Family Foundation, and The Rooney Alden
and Mussillami Families. Special acknowledgment is addressed
1
provide pure compound 25 as a pale-yellow oil (497 mg, 75%). H
NMR (CDCl3) δ 9.53 (br s, 1H), 8.38 (dd, J = 7.9, 1.5 Hz, 1H), 7.50
(d, J = 7.0 Hz, 2H), 7.42−7.32 (m, 3H), 7.04 (td, J = 7.7, 1.5 Hz, 1H),
6.99−6.95 (m, 2H), 5.16 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.47 (s,
2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3) δ 169.1, 162.8,
147.5, 136.4, 128.5 (2C), 128.1, 127.7, 127.4 (2C), 124.1, 121.3, 120.3,
111.7, 70.7, 61.6, 42.5, 14.0. HPLC purity 94%. MS (ESI) m/z 314 (M
+ H)+.
to Dr. W. Tuckmantel and Drs. K. El Bissati, D. Jacobus, J.
̈
McCall, C. Eid, M.-F. Cesbron-Delauw, J. Rogers, and N.
Grihalde for useful discussion and comments.
Hydrolysis13 of the ester 25 gave the desired acid 26 as orange
crystals (260 mg, 91% on a 1.0 mmolar scale). The crude product was
pure enough to be used in the next step without further purification.
1H NMR (DMSO-d6) δ 9.63 (br s, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H),
7.51 (d, J = 7.0 Hz, 2H), 7.40−7.29 (m, 3H), 7.09 (dd, J = 8.2, 1.2 Hz,
1H), 7.03 (td, J = 7.8, 1.5 Hz, 1H), 6.90 (td, J = 7.9, 1.3 Hz, 1H), 5.22
(s, 2H), 3.50 (s, 2H). 13C NMR (DMSO-d6) δ 170.1, 164.6, 148.2,
137.0, 128.4 (2C), 127.8, 127.6, 127.3 (2C), 124.4, 121.7, 120.6, 112.9,
69.6, 43.1. HPLC purity 97.8%. MS (ESI) m/z 284 (M + H)+.
Compound 27 was obtained as a yellow oil (300 mg, 85%) by
following the literature procedure.13 The crude product was of
adequate purity to be used in the next step without further purification.
1H NMR (DMSO-d6) δ 10.21 (br s, 1H), 9.99 (br s, 1H), 8.12 (d, J =
7.5 Hz, 1H), 7.58 (d, J = 6.9 Hz, 2H), 7.52 (d, J = 8.3 Hz, 2H), 7.38−
7.30 (m, 3H), 7.18−7.13 (m, 3H), 7.04 (t, J = 7.4 Hz, 1H), 6.92 (t, J =
7.6 Hz, 1H), 5.22 (s, 2H), 3.59 (s, 2H), 2.57 (q, J = 7.5 Hz, 2H), 1.16
(t, J = 7.5 Hz, 3H). 13C NMR (DMSO-d6) δ 166.4, 165.0, 147.8,
ABBREVIATIONS USED
■
ADMET, absorption, distribution, metabolism, excretion,
toxicity; CYP450, cytochrome P450; DMSO-d6, deuterated
dimethyl sulfoxide; hERG, human ether-a-go-go-related gene;
̀
HLM, human liver microsomes; HPLC, high-performance
liquid chromatography; HRMS, high-resolution mass spec-
trometry; HTS, high-throughput screening; L. donovani,
Leishmania donovani; L6, toxicity of the tested compounds
assessed against a mammalian primary cell line derived from rat
skeletal myoblasts; NADPH, nicotinamide adenine dinucleotide
phosphate; SAR, structure−activity relationships; SI, selectivity
index; T. b. rhodesiense, Trypanosoma brucei rhodesiense; T. cruzi,
Trypanosoma cruzi; T. gondii, Toxoplasma gondii; TLC, thin
layer chromatography; TMS, tetramethylsilane; P. falciparum,
Plasmodium falciparum
3099
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Article
(18) Hanessian, S.; Liak, T. J.; Vanasse, B. Facile cleavage of benzyl
ethers by catalytic transfer hydrogenation. Synthesis 1981, 396−397.
(19) Orhan, I.; Sener, B.; Kaiser, M.; Brun, R.; Tasdemir, D.
Inhibitory activity of marine sponge-derived natural products against
parasitic protozoa. Mar. Drugs 2010, 8, 47−58.
REFERENCES
■
(1) Fomovska, A.; Huang, Q.; Bissati, K. E.; Mui, E. J.; Witola, W. H.;
Cheng, G.; Zhou, Y.; Sommerville, C.; Roberts, C. W.; Bettis, S.;
Prigge, S. T.; Afanador, G. A.; Hickman, M. R.; Lee, P. J.; Leed, S. E.;
Auschwitz, J. M.; Pieroni, M.; Stec, J.; Muench, S. P.; Rice, D. W.;
Kozikowski, A. P.; McLeod, R. Novel N-benzoyl-2-hydroxybenzamide
disrupts unique parasite secretory pathway. Antimicrob. Agents
Chemother. 2012, DOI: 10.1128/AAC.06450-11.
(2) Working to Overcome the Global Impact of Neglected Tropical
Diseases: First WHO Report on Neglected Tropical Diseases; World
Health Organization: Geneva, 2010.
(3) Brun, R.; Blum, J.; Chappuis, F.; Burri, C. Human African
trypanosomiasis. Lancet 2010, 375, 148−159.
(4) Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gurtler, R. E.;
McKerrow, J.; Reed, S.; Tarleton, R. Kinetoplastids: related protozoan
pathogens, different diseases. J. Clin. Invest. 2008, 118, 1301−1310.
(5) Malaria Fact sheet No. 94; World Health Organization: Geneva,
2011.
(20) Qu, J.; Kumar, N.; Alamgir, M.; Black, D. S. A versatile synthetic
route to 11H-indolo[3,2-c]isoquinolines. Tetrahedron Lett. 2009, 50,
5628−5630.
(21) Vasudevan, A.; Souers, A. J.; Freeman, J. C.; Verzal, M. K.; Gao,
J.; Mulhern, M. M.; Wodka, D.; Lynch, J. K.; Engstrom, K. M.; Wagaw,
S. H.; Brodjian, S.; Dayton, B.; Falls, D. H.; Bush, E.; Brune, M.;
Shapiro, R. D.; Marsh, K. C.; Hernandez, L. E.; Collins, C. A.; Kym, P.
R. Aminopiperidine indazoles as orally efficacious melanin concen-
trating hormone receptor-1 antagonists. Bioorg. Med. Chem. Lett. 2005,
15, 5293−5297.
̈
(6) Rathore, D.; Jani, D.; Nagarkatti, R. Novel therapeutic target for
protozoal diseases. U.S. Patent US20070148185 (A1), 2007.
(7) Adagu, I. S.; Nolder, D.; Warhurst, D. C.; Rossignol, J. F. In vitro
activity of nitazoxanide and related compounds against isolates of
Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis.
J. Antimicrob. Chemother. 2002, 49, 103−111.
(8) Cortes, H. C.; Mueller, N.; Esposito, M.; Leitao, A.;
Naguleswaran, A.; Hemphill, A. In vitro efficacy of nitro- and
bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoi-
tia besnoiti infection in Vero cells. Parasitology 2007, 134, 975−985.
(9) Esposito, M.; Moores, S.; Naguleswaran, A.; Muller, J.; Hemphill,
A. Induction of tachyzoite egress from cells infected with the
protozoan Neospora caninum by nitro- and bromo-thiazolides, a class
of broad-spectrum anti-parasitic drugs. Int. J. Parasitol. 2007, 37,
1143−1152.
(10) Fonseca-Salamanca, F.; Martinez-Grueiro, M. M.; Martinez-
Fernandez, A. R. Nematocidal activity of nitazoxanide in laboratory
models. Parasitol. Res. 2003, 91, 321−324.
(11) Roberts, C. W.; Roberts, F.; Henriquez, F. L.; Akiyoshi, D.;
Samuel, B. U.; Richards, T. A.; Milhous, W.; Kyle, D.; McIntosh, L.;
Hill, G. C.; Chaudhuri, M.; Tzipori, S.; McLeod, R. Evidence for
mitochondrial-derived alternative oxidase in the apicomplexan parasite
Cryptosporidium parvum: a potential antimicrobial agent target. Int. J.
Parasitol. 2004, 34, 297−308.
(12) Cai, Z. R.; Jabri, S. Y.; Jin, H.; Kim, C. U.; Lansdown, R. A.;
Metobo, S. E.; Mish, M. R.; Pastor, R. M. Integrase inhibitor
compounds. Patent WO 2006/125048 A2, 2006.
(13) Wittich, S.; Scherf, H.; Xie, C.; Brosch, G.; Loidl, P.; Gerhauser,
̈
C.; Jung, M. Structure−activity relationships on phenylalanine-
containing inhibitors of histone deacetylase: in vitro enzyme
inhibition, induction of differentiation, and inhibition of proliferation
in Friend leukemic cells. J. Med. Chem. 2002, 45, 3296−3309.
(14) Hossain, N. Novel tricyclic spiroderivatives as modulators of
chemokine receptor activity. Patent WO 2005/037814 (A1), 2005.
(15) Qiao, C.; Gupte, A.; Boshoff, H. I.; Wilson, D. J.; Bennett, E. M.;
Somu, R. V.; Barry, C. E.; Aldrich, C. C. 5′-O-[(N-Acyl)sulfamoyl]-
adenosines as antitubercular agents that inhibit MbtA: an adenylation
enzyme required for siderophore biosynthesis of the mycobactins. J.
Med. Chem. 2007, 50, 6080−6094.
(16) Adler, T.; Bonjoch, J.; Clayden, J.; Font-Bardia, M.; Pickworth,
M.; Solans, X.; Sole, D.; Vallverdu, L. Slow interconversion of
enantiomeric conformers or atropisomers of anilide and urea
derivatives of 2-substituted anilines. Org. Biomol. Chem. 2005, 3,
3173−3183.
(17) Kline, T.; Fromhold, M.; McKennon, T. E.; Cai, S.; Treiberg, J.;
Ihle, N.; Sherman, D.; Schwan, W.; Hickey, M. J.; Warrener, P.; Witte,
P. R.; Brody, L. L.; Goltry, L.; Barker, L. M.; Anderson, S. U.; Tanaka,
S. K.; Shawar, R. M.; Nguyen, L. Y.; Langhorne, M.; Bigelow, A.;
Embuscado, L.; Naeemi, E. Antimicrobial effects of novel siderophores
linked to beta-lactam antibiotics. Bioorg. Med. Chem. 2000, 8, 73−93.
3100
dx.doi.org/10.1021/jm2015183 | J. Med. Chem. 2012, 55, 3088−3100