Synthesis of carboxyimidamide-substituted benzo[c][1,2,5]oxadiazoles and their analogs, and evaluation of biological activity against Leishmania donovani

Article abstract of DOI:10.1039/c5md00119f

A facile synthesis route to carboxyimidamide-substituted benzoxadiazoles and related derivatives was developed. A total of 25 derivatives were synthesized. They were evaluated for antileishmanial activity by inhibition of Leishmania donovani axenic amastigote growth using a fluorescent viability microplate assay. The most promising derivative (14) demonstrated an antileishmanial EC₅₀ of 4.0 μM, and it also showed activity in infected macrophages (EC₅₀ 5.92 μM) without signs of cytotoxicity.

Full text of DOI:10.1039/c5md00119f

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CONCISE ARTICLE
Synthesis of carboxyimidamide-substituted
benzo[c][1,2,5]oxadiazoles and their analogs, and
evaluation of biological activity against Leishmania
donovani
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
a
a
a
Leena Keurulainen,
Mikko Heiskari,
Satu Nenonen,
Abedelmajeeda
b
b
a
Nasereddin, Dmitry Kopelyanskiy, Teppo O. Leino, Jari Yli-Kauhaluoma,
DOI: 10.1039/x0xx00000x
b
a
Charles L. Jaffe, and Paula Kiuru
www.rsc.org/
A facile synthesis route to carboxyimidamide-substituted benzoxadiazoles and related
derivatives was developed. A total of 25 derivatives were synthesized. They were evaluated
for antileishmanial activity by inhibition of Leishmania donovani axenic amastigote growth
using a fluorescent viability microplate assay. The most promising derivative (14
)
demonstrated an antileishmanial EC50 of 4.0 µM, and it also showed activity in infected
macrophages (EC50 5.92 µM) without signs of cytotoxicity.
1
. Introduction
In addition, poor efficacy in the case of visceral
leishmaniasis/HIV coinfection has raised the need for new
Leishmaniasis is a spectrum of human diseases caused by at antileishmanial treatments.3
least 20 different species of protozoan parasites. These parasites In
our previous study,
are responsible for three major diseases: cutaneous, visceral, benzo[c][1,2,5]oxadiazole derivatives showed activity against
2-arylbenzimidazole
and
1
and mucocutaneous. Over 300 million people are at risk.
Visceral leishmaniasis (VL), caused primarily by L. donovani pneumoniae. In that study, a similarity-based model of C.
anthroponotic) or L. infantum/L. chagasi (zoonotic), is fatal if pneumoniae dimethyladenosine transferase was used to
the intracellular Gram-negative bacterium Chlamydia
4
(
left untreated. There are an estimated 300 000 cases of VL per virtually screen compounds from commercially available
year, and 20 000 to 40 000 deaths. Over 90% of new cases databases against C. pneumoniae. The 2-arylbenzimidazoles
5
occur in six countries: Bangladesh, Brazil, Ethiopia, India, found were then tested and proven active against L. donovani.
South Sudan, and Sudan. Although two new drugs, miltefosine This prompted us to consider benzo[c][1,2,5]oxadiazole-
and paromomycin, are currently in use and combination therapy derived compounds as a new family of antileishmanial agents.
2
is becoming more common, an urgent need for new In addition, benzoxadiazoles and their N-oxides have been
6
antileishmanial chemotypes persists. The major issues to be previously studied as antibiotic and antiparasitic agents. In this
confronted in Leishmania treatment are the toxicity of primary study, a facile synthesis route to carboxyimidamide-substituted
medicines and the development of parasite resistance.
benzoxadiazoles
was
developed,
a
set
of
25
benzo[c][1,2,5]oxadiazole derivatives and other structurally
related compounds was synthesized and evaluated as
Faculty of Pharmacy, Division of Pharmaceutical Chemistry and antileishmanial agents.
a
Technology, University of Helsinki, Viikinkaari 5 E, P. O. Box 56, FI-
b
0
0014 Helsinki, Finland. Department of Microbiology and Molecular 2. Results and discussion
Genetics, IMRIC, Hebrew University-Hadassah Medical School, P. O.
Box 12272, Jerusalem 9112102, Israel.
2
.1 Chemistry
Electronic Supplementary Information (ESI) available: Synthesis Initially, benzo[c][1,2,5]oxadiazole-5-carbonitrile
procedures for compounds 13 15 18 21 39 See synthesized
from 5-chloro-
DOI: 10.1039/b000000x/
bromobenzo[c][1,2,5]oxadiazole in
reaction using NaCN, CuCN, or K [Fe(CN) ], but these
4
or
was to be
5-
6
-
,
-
,
-
.
a
microwave-assisted
7
4
6
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Scheme 1. Synthetic procedures and chemical structures of the products
7%; b) P(OEt) , EtOH, 70 °C, 17 h, 67%; c) SOCl , reflux, 4 h, 97%; d) NH
rt, 2 h, 94%; g) substituted phenyl isocyanate, THF or CHCl , rt, 3-21 or 96-120 h, 27-70%; h) MeOH, 0 °C, 0.5 h then H
isocyanate, 57%; j) PhCOCl/PhOCOCl/PhCH COCl, Et N, THF, 70 °C, 2-4 h, 14-63%; k) amine/benzylamine, Et N, THF, rt, 2 h, 29-53%.
2
-
24. Reagents and conditions: a) KOH, EtOH, H
2
O, 70 °C, 2 h, then NaOCl, 0 °C rt, 21 h,

8
3
2
3
aq., 1,4-dioxane, 0 °C, 1.5 h, 90%; e) TFAA, Et N, THF, rt, 3 h, 79%; f) H
3
2
NOH∙HCl, Et N, EtOH,
3
3
2
NOH∙HCl, MeOH, rt, 1.5 h, 57%; i) THF, phenyl
2
3
3
24) was carried out in the presence of the substituted phenyl
isocyanates in THF or CHCl . The products 21-23 were
3
synthesized in a similar manner by using a solution of Et N in
3
THF. Compound 20 was synthesized from the acyl chloride
2
via the hydroxamic acid 19. The final products 33 39 were
-
synthesized from the commercially available nitriles by using
the methods described above (Scheme 2). Finally, the related
benzimidazole derivative 25 was formed from 3,4-
1
1
diaminobenzonitrile in refluxing formic acid.
Scheme 2. Synthetic procedures and chemical structures of the products 25
Reagents and conditions: a) H NOH∙HCl, Et N or Na CO , EtOH or H O, rt/100 °C,
.5-24 h, 38-98%; b), phenyl isocyanate, THF, CHCl or 1,4-dioxane, rt, 2-5 h or 72
-39.
2
3
2
3
2
2
.2 Evaluation of antileishmanial activity
3
3
h, 6-85%.
The activity of the new compounds was determined by using a
fluorescent viability microplate assay with L. donovani axenic
1
2
syntheses failed to produce the target nitrile. Instead the amastigotes and alamarBlue. All compounds were initially
synthesis of benzo[c][1,2,5]oxadiazole derivatives was started screened at 50 µM, and the most active derivatives sequentially
from the commercially available 4-amino-3-nitrobenzoic acid tested at lower concentrations (15 and 5 µM).
(
Scheme 1). It was converted to benzo[c][1,2,5]oxadiazole-5-
carboxylic acid ( ) by using a two-step procedure via the N- with different substitution patterns in the “Eastern” phenyl ring
oxide intermediate that was produced in the presence of sodium of the structure, were synthesized and assayed. The derivative
hypochlorite in an alkaline EtOH-H O solution and with no additional substituents (compound
) showed 70% and
Next, 24% inhibition of L. donovani proliferation at 50 µM and 15
via the acyl µM, respectively, whereas meta-Cl substitution (compound 14
in the presence of aqueous ammonia in 1,4-dioxane. increased growth inhibition to 95% and 87% at 50 µM and 15
The reaction of the obtained primary amide with µM, respectively (Table 1). Overall, meta substitution including
trifluoroacetic anhydride and Et N in THF gave the Cl-, OMe-, Me-, and CF -substituted derivatives seems to be
First, a set of benzo[c][1,2,5]oxadiazole derivatives 6-18,
1
2
6
8
subsequently reduced to the carboxylic acid
compound was converted to the primary amide
chloride
1.
1
3
)
2
3
9
3
3
corresponding nitrile
by using hydroxylamine hydrochloride in the presence of
Et N in EtOH. The final step to obtain the derivatives (
4, which was converted to the amidoxime beneficial for antileishmanial activity.
1
0
5
Replacing HN of the heteroatom containing chain with
6
-
18, oxygen or carbon atom (compounds 22 and 23) significantly
3
2
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reduced antileishmanial activity to 26.5 and 22.4%, 15 µM, respectively). Good inhibition of compound 34 can be
respectively, at 50 µM. Similarly, removal of the aniline NH hypothesized to result from increased lipophilicity of the
(
compound 21) reduced activity, but this compound still compound. Moreover, the phenyl derivative 33 is a very
showed more activity (51% growth inhibition) than carboxy 22 interesting compound, because the shorter synthetic route is
or benzylic 23 derivatives. Moreover, the cyclohexane likely to increase the prospects for further preparation of new
derivative 24 displays leishmanial inhibition in the absence of antileishmanial derivatives of this chemotype.
an aniline moiety. Replacement of the amino group in the
Table 2. EC50 of the selected derivatives on Leishmania donovani axenic
imidamide 20 with a carbonyl group increased inhibition to
amastigotes and intracellular amastigotes in L. donovani infected
macrophages; cytotoxicity on THP-1 macrophages and NIH/3T3
fibroblasts; and respective selectivity indices [SI].
9
6% and 68% at concentrations of 50 µM and 15 µM,
respectively.
Axenic
amastigotes
EC50 ± SEM
Intracellular
amastigotes
EC50 ± SEM
(n=3) (µM)
THP-1
EC50
(µM)
Table 1. Inhibition of Leishmania donovani axenic amastigote growth.
Fibroblasts
EC50 (µM)
(n=2) [SI]
Compound
a
%
Inhibition
(
n=2) (µM)
(n=2)[SI]
Compound
50 µM
70.1
15 µM
24.2
―
5 µM
―
7
9.9±5.4
19.0]
>300
[>71.4]
6
7
8
9
14
20
4.2±0.2
5.92±1.7
[
42.0
―
3
3.7±5.0
4.2]
>300
[>37.0]
67.2
44.6
―
―
8.1±0.9
―
[
64.1
―
Ampho-
tericin B
0.09-0.7
17
2.1-7.6
18
b
0.11-0.26
2.2
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
0
1
2
3
4
3
4
5
6
7
8
9
38.0
―
―
12, 14-16
15, 16
71.4
46.0
31.8
―
―
―
Further evaluation of the most promising derivatives 14 and
revealed low EC50 values on axenic amastigotes, 4.2 and 8.1
µM respectively; moderate cytotoxicity on the human THP-1
macrophage cell line, 79.9 µM and 33.7 respectively; and no
cytotoxicity against murine fibroblasts at the highest
concentration tested, 300 µM (Table 2). Values for
b
29.6
94.8
44.0
42.1
51.4
51.7
96.2
51.1
26.5
22.4
―
―
2
0
87.2
―
43.4
―
―
―
―
―
―
―
amphotericin B, a reference compound used to treat
68.2
―
54.6
―
leishmaniasis, are given for comparison. When tested at 5 µM
on infected macrophages, a concentration non-toxic for the
macrophage cell line, both compounds, 14 and 20, were still
active, 33.1 and 19.9% amastigote growth inhibition
respectively, albeit lower than that observed with axenic
amastigotes. The EC50 for 14, the most active and least
cytotoxic derivative, on intracellular amastigotes in infected
macrophages was 5.92 µM similar to that observed using
axenic amastigotes.
―
―
―
―
b
43.9
78.4
98.8
72.0
97.9
95.2
91.9
―
―
5.4
24.9
―
―
―
―
35.2
48.0
21.6
―
―
3
. Conclusion
―
The developed synthesis route to carboxyimidamide-substituted
benzo[c][1,2,5]oxadiazoles and related derivatives facilitates
access to a compound library and determination of the
93.0
33.9
―
c
Amphotericin B
―
―
99.7
a
b
c
±
SEM: at 50 µM 0.1-2.3%, 15 µM 0.2-1.9%, and 5 µM 2.4-5.9%.
precipitates at 50 µM.
amphotericin B is positive control and was tested at 1 µM.
structure-activity
relationships
for
this
antileishmanial heterocyclic chemotype. Carboxyimidamide-
substituted benzoxadiazole derivative 14 is the most promising
compound of this study, demonstrating good antileishmanial
inhibition activity in infected macrophages and, remarkably, no
signs of cytotoxicity. Although mechanism of action of the
compounds is not known yet, it is worth continuing the
development of these compounds to explore further prospects
of these compounds.
Finally, a second set of related heterocyclic compounds was
synthesized with replacements of the benzo[c][1,2,5]oxadiazole
moiety. These heterocycles (benzothiophene 36, benzofuran 37
,
indole 38, and 1,3-benzodioxole 39) showed an increase in
antileishmanial activity at 50 µM compared to the benzoxazole
derivative 6. This suggests that the benzo[c][1,2,5]oxadiazole
ring is not crucial to antileishmanial activity. In addition, the
phenyl and naphthalene derivatives 33 and 34 significantly
inhibited growth of L. donovani amastigotes (78% and 5% for
33, and 99% and 25% for 34, at concentrations of 50 µM and
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4
. Experimental section
benzo[c][1,2,5]oxadiazole-5-carboxylic acid 1-oxide (1.90 g,
0.5 mmol) in absolute EtOH (25 mL) was heated to 70 °C, and
triethyl phosphite (2.80 mL, 16.0 mmol, 1.5 equiv) was added
1
4
.1 Chemistry
Unless otherwise stated, reactions were carried out in oven- dropwise to the mixture. The resulting mixture was heated to
dried glassware under an argon atmosphere. All reagents were reflux and the reflux was maintained for 17 h. The solvent was
commercially available and were acquired from Fluka (Buchs, evaporated, and the residual black oil was treated with H
Switzerland), Aldrich (Schnelldorf, Germany), Riedel-de Haën extracted twice with EtOAc. The organic phase was extracted
Seelze, Germany), and Alfa Aesar (Karlsruhe, Germany). THF with a 2 M aqueous solution of NaOH, and the aqueous phase
was distilled over sodium/benzophenone ketyl. The progress of was washed three times with EtOAc. The aqueous phase was
chemical reactions was monitored by
thin-layer cooled to 0 °C and acidifed (pH <1) with a 6 M aqueous
O and
2
(
chromatography on silica gel 60-F254 plates acquired from E. solution of HCl resulting in the formation of a light brown
Merck (Darmstadt, Germany). The eluent consisted of EtOAc precipitate, which was filtered and dried in vacuo to yield the
1
and n-hexane, or EtOAc and MeOH, and detection was compound
conducted at 254 or 366 nm. The products were purified by MHz, DMSO-d
flash chromatography on silica gel with a Biotage SP1 8.13 (dd, J = 9.4, 1.1 Hz, 1H), 7.97 (dd, J = 9.4, 1.1 Hz, 1H).
1
(1.10 g, 67%) as a brown solid. H NMR (300
) δ 13.7 (br s, 1H), 8.63 (t, J = 1.1 Hz, 1H),
6
1
3
purification system (Uppsala, Sweden) using 25+M cartridges
or SNAP 10 g, 25 g, or 50 g cartridges, detection at 254 nm. 131.4, 119.4, 116.6. H spectral data is consistent with those
C NMR (75 Hz, DMSO-d ) δ 165.6, 149.3, 148.9, 134.7,
6
1
1
4
Melting points were measured using an IA9100 digital melting reported earlier.
point apparatus (Electrothermal Engineering, Essex, UK) and Benzo[ ][1,2,5]oxadiazole-5-carbonyl chloride (2). A mixture
are uncorrected. IR spectra were recorded on a Bruker Vertex of benzo[c][1,2,5]oxadiazole-5-carboxylic acid (1.82 g, 11.1
0 FT-IR spectrometer (Ettlingen, Germany) with ATR mmol) and thionyl chloride (18 mL) was refluxed for 4 h. The
technique. The synthesized compounds were analyzed by NMR resulting mixture was concentrated and dried in vacuo to yield
c
1
7
1
on a Varian Mercury 300 MHz spectrometer (Palo Alto, CA,
USA). H and C NMR were recorded as solutions in DMSO-
2
d
(2.0 g, 97%) as a brown solid. H NMR (300 MHz, DMSO-
1
13
) δ 8.62 (t, J = 1.1 Hz, 1H), 8.12 (dd, J = 9.4, 1.1 Hz, 1H),
d6 (Aldrich). Chemical shifts () are given in parts per million 7.96 (dd, J = 9.4, 1.1 Hz, 1H). C NMR (75 Hz, DMSO-d
6
1
3
) δ
6
(
ppm) relative to the NMR solvent signals (DMSO-d 2.50 and 165.4, 149.3, 148.9, 134.6, 131.3, 119.4, 116.6.
6
1
13
3
9.51 ppm for H and C NMR, respectively). LC-MS analyses Benzo[
c
][1,2,5]oxadiazole-5-carboxamide (3).
A
25%
were performed with a HP1100 instrument (Agilent, Palo Alto, aqueous solution of ammonia (4.8 mL) was cooled in an ice
USA) with UV detector ( 210 nm) and an Esquire LC bath. To this solution, compound (0.700 g, 3.80 mmol) in 1,4-
2
spectrometer (Bruker Daltonik, Bremen, Germany) with ESI dioxane (8 mL) was added dropwise. The resulting mixture was
ion source. Signal separation was carried out by the use of a stirred at the same temperature for 1.5 h and then evaporated in
Waters XBridge C18 column (2.1 mm  50 mm, 2.1 m) with a vacuo. The residue was dissolved in EtOAc and washed with
Waters XBridge C18 guard column (Milford, MA, USA) (2.1
mm × 10 mm, 2.5 µm). The eluent consisted of water (+ 0.1% combined organic phases were dried over anhydrous Na
HCO H) and acetonitrile (+ 0.1% HCO H) (gradient run 80:20 evaporated, and dried in vacuo to yield (0.56 g, 90%) as a
5:95). Purity of all tested compounds was >95%. High light orange solid. H NMR (300 MHz, DMSO-d ) δ 8.55 (t, J
resolution mass spectra (HRMS) were measured on a Synapt = 1.2 Hz, 1H), 8.32 (br s, 1H), 8.12 (dd, J = 9.4, 1.2 Hz, 1H),
H
2
O. The aqueous phase was extracted twice with EtOAc. The
SO
,
4
2
3
2
2
1

6
1
3
G2 HDMS Q-TOF-instrument (Milford, MA, USA) with 7.97 (dd, J = 9.4, 1.2 Hz, 1H), 7.79 (br s, 1H). C NMR (75
positive mode ESI.
MHz, DMSO-d ) δ 166.0, 149.1, 148.9, 137.8, 131.4, 116.2,
Benzo[
][1,2,5]oxadiazole-5-carboxylic acid (1). Potassium 115.8.
hydroxide (2.02 g, 36.0 mmol, 2.4 equiv) was dissolved in Benzo[
equimixture of EtOH and H O (40 mL). 4-Amino-3- (1.50 g, 9.20 mmol) was dissolved in anhydrous THF (30 mL),
6
c
c
][1,2,5]oxadiazole-5-carbonitrile (4). Compound 3
2
nitrobenzoic acid (2.73 g, 15.0 mmol) was added to this and distilled Et N (2.80 mL, 20.2 mmol, 2.2 equiv) was added
3
solution, and the resulting mixture was heated at 70 °C for 2 h to this solution. The solution was cooled in an ice bath, and
and cooled to 0 °C. A 13% aqueous solution of sodium trifluoroacetic anhydride (1.50 mL, 11.0 mmol, 1.2 equiv) in
hypochlorite (80 mL) was added dropwise to this solution THF (8 mL) was added dropwise. The resulting mixture was
during 30 min. The resulting mixture was slowly allowed to stirred at rt for 3 h. H
O was added and the volatiles were
2
warm to rt and stirred for 21 h. H O (35 mL) was added at 0 °C, removed in vacuo. The residue was dissolved in CH
Cl and
2
2
2
and the mixture was acidified (pH <1) with a 6 M aqueous washed twice successively with a 0.1 M aqueous solution of
solution of HCl resulting in the formation of a yellow HCl and a 0.1 M aqueous solution of NaOH. The organic phase
precipitate. Sodium chloride (1.9 g) was added, and the mixture was dried over anhydrous Na
was further stirred for 1.5 h, filtered, and dried in vacuo to yield vacuo. The crude product was purified by flash column
benzo[c][1,2,5]oxadiazole-5-carboxylic acid 1-oxide (2.4 g, chromatography (EtOAc/n-hexane 1:2) to give (1.10 g, 79%)
) δ 8.98 (t, J =
br s, 1H), 7.84 (br s, 1H), 7.73 (br s, 1H). A mixture of 1.2 Hz, 1H), 8.28 (dd, J = 9.3, 1.2 Hz, 1H), 7.82 (dd, J = 9.3,
SO , evaporated, and dried in
2
4
4
1
1
8
(
7%) as a yellow solid. H NMR (300 MHz, DMSO-d )  8.06 as a pink solid. H NMR (300 MHz, DMSO-d
6
6
4
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1
3
1
1
N
.2 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 148.4, 147.9, mL) was stirred at ice-water bath temperature for 30 min. The
6
31.8, 125.7, 118.3, 117.2, 115.4.
'-Hydroxybenzo[ ][1,2,5]oxadiazole-5-carboximidamide
5). A mixture of compound
H NOHHCl (750 mg, 10.8 mmol, 1.5 equiv) and distilled Et N (0.12 g, 57%) as a light brownish solid. H NMR (300 MHz,
resulting mixture was let warm to rt, stirred for 3 h, evaporated,
c
and dried in vacuo. The crude product was purified by flash
(
4 (1.05 g, 7.20 mmol), SiO column chromatography (CH Cl /MeOH 19:1) to yield 20
2 2 2
1
2
3
(
1.50 mL, 10.8 mmol) in EtOH (45 mL) was stirred at rt for 2 h. DMSO-d ) δ 12.79 (br s, 1H), 10.38 (br s, 1H), 8.55 (t, J = 1.2
6
The solvent was evaporated and the residue was dissolved in Hz, 1H), 8.22 (dd, J = 9.5, 1.2 Hz, 1H), 7.92 (dd, J = 9.5, 1.2
EtOAc and brine. The organic phase was washed with brine, Hz, 1H), 7.52-7.49 (m, 2H), 7.37-7.32 (m, 2H), 7.11-7.05 (m,
1
3
dried over anhydrous Na SO , evaporated, and dried in vacuo 1H). C NMR (75 MHz, DMSO-d ) δ 163.0, 152.0, 149.1,
2
4
6
1
to yield
5 (1.2 g, 94%) as a yellowish solid. H NMR (300 148.6, 138.0, 134.9, 130.6, 129.0, 123.3, 118.5, 117.1, 116.7.
MHz, DMSO-d ) δ 10.38 (s, 1H), 8.29 (t, J = 1.1 Hz, 1H), 8.03 LC-MS: [M+H] 299.2 m/z (t = 5.2 min). FT-IR (ATR, cm ):
+
-1
6
r
(
dd, J = 9.6, 1.1 Hz, 1H), 7.96 (dd, J = 9.6, 1.1 Hz, 1H), 6.13 (s, 3321, 3224, 1759, 1663, 1535, 1203, 888. HRMS (ESI): m/z
H). C NMR (75 MHz, DMSO-d ) δ 149.2, 149.0, 148.7, calcd for C H N O [M+H] 299.0780, found 299.0779.
6 14 11 4 4
1
3
+
2
1
36.4, 130.9, 115.2, 110.7.
'-[[(3-
Chlorophenyl)carbamoyl]oxy]benzo[
carboximidamide (14). A mixture of
4
.2 Biology
Antileishmanial evaluation using axenic amastigotes L.
(89.0 mg, 0.500 mmol), donovani (MHOM/SD/1962/1S-Cl2d) was used in all
N
c
5
][1,2,5]oxadiazole-5-
and isocyanate (0.550 mmol, 1.1 equiv) in dry THF (5 mL) was bioassays. Screening of the compounds for antileishmanial
stirred at rt for 4 h. The solvent was evaporated. The crude activity using axenic amastigotes was carried out using the
product was purified by flash SiO column chromatography alamarBlue (AbD Serotec, Oxford, UK) viability assay, as
2
(
EtOAc/n-hexane 1:2) to give 14 (0.10 g, 60%) as a white previously described, similar to that reported for leishmanial
1
12
powder. H NMR (300 MHz, DMSO-d ) δ 9.68 (s, 1H), 8.57 (s, promastigotes. Axenic amastigotes were grown at 37 C in a
6
1
1
1
1
1
H), 8.14-8.13 (m, 2H), 7.73-7.71 (m, 1H), 7.52 (dd, J = 8.4, 5% CO incubator in complete RPMI 1640 containing 20%
2
1
3
.2 Hz), 7.36 (t, J = 8.4 Hz, 1H), 7.18 (s, 2H), 7,12 (dd, J = 8.4, fetal calf serum, pH 5.5. Compounds to be assayed were
1
3
.2 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 153.6, 152.1, diluted in the complete amastigote medium containing 1%
6
48.9, 148.8, 140.0, 134.9, 133.1, 131.4, 130.4, 122.8, 118.8, DMSO to twice the final concentration used in the assays, and
+
17.8, 116.0, 114.6. LC-MS: [M+H] 332.0 m/z (t = 6.9 min). were aliquoted in triplicate (125 µL/well) into 96-well flat-
r
-
1
FT-IR (ATR, cm ): 3440, 3349, 1739, 1647, 1274. HRMS bottom plates (Nunc, Roskilde, Denmark). For determination of
+
(
ESI): m/z calcd for C H ClN O [M+H] 332.0550, found the EC , final concentrations in triplicate spanning 300 to 0.14
14 10 5 3 50
5
3
N
32.0551.
-Hydroxybenzo[
Compound (183 mg, 1.00 mmol) was stirred in MeOH (5 CO incubator. The alamarBlue viability indicator was added
μM were used. Amastigotes (5.0×10 cells/mL; 125 µL/well)
c][1,2,5]oxadiazole-5-carboxamide (19). were added to each well and incubated for 24 h at 37 C in a 5%
2
2
mL) in an ice bath for 30 min. The solvents were evaporated in (25 µL/well) and the plates incubated for an additional 24 h at
vacuo to give methyl benzo[c][1,2,5]oxadiazole-5-carboxylate. which time the fluorescence (ex = 544 nm; em = 590 nm) was
A solution of hydroxylamine in MeOH was prepared by measured in a microplate reader (Fluoroskan Ascent FL,
dissolving H NOHHCl (542 mg, 7.80 mmol, 7.8 equiv) in Finland). Complete medium, both with and without DMSO,
2
MeOH (5 mL) in a water-ice bath. KOH (645 mg, 11.5 mmol, was used as negative controls (0% inhibition of amastigote
11.5 equiv) in MeOH (10 mL) was added, resulting in growth). Amphotericin B (Sigma-Aldrich, St. Louis MO), a
formation of a precipitate. The resulting mixture was stirred in drug used to treat VL, was included as a positive control on
an ice-water bath for 30 min, and the precipitate was filtered off each plate and gave >90% inhibition of parasite growth at 1.5
to yield a solution of N-hydroxycarboxamide in MeOH. The µM.
methyl ester (vide supra) was dissolved in this solution, stirred Cytotoxicity EC50 was determined on NIH/3T3 fibroblasts or
for 1.5 h, neutralized with acetic acid, and evaporated. The THP-1 monocyte cells using the alamarBlue viability indicator
resulting residue was dissolved in EtOAc and washed with as follows. Cells in DMEM medium plus 10% fetal calf serum
brine. The aqueous phase was further extracted with EtOAc. and antibiotics were aliquoted in triplicate (fibroblast or THP-1
5
4
The combined organic phases were dried over with anhydrous at 8×10 or 6.4×10 cells/well respectively; 125 µL/well) into
Na SO , evaporated, and dried in vacuo. The crude product was 96-well plates (Nunc, Roskilde, Denmark). Compounds to be
2
4
purified by flash SiO column chromatography (CH Cl /MeOH assayed were diluted in the same medium containing 1%
2
2
2
1
9
:1) to yield 19 (0.10 g, 57%) as a light solid. H NMR (300 DMSO at twice the final concentration used in the assays (300
MHz, DMSO-d ) δ 11.56 (br s, 1H), 9.40 (br s, 1H), 8.37 (s, to 0.14 μM for fibroblasts or 150 to 0.10 μM for THP-1), added
6
1
1
1
H), 8.14 (dd, J = 9.4, 1.0 Hz, 1H), 7.88 (dd, J = 9.4, 1.0 Hz, to the cells (125 µL/well), and the plates incubated for 48 h at
1
3
H). C NMR (75 MHz, DMSO-d ) δ 161.7, 149.0, 148.7, 37 C in a 5% CO2 incubator. The alamarBlue viability
6
39.6, 131.0, 116.5, 114.8.
indicator was added (25 µL/well) and the plates incubated for
N
-[(Phenylcarbamoyl)oxy]benzo[
c
][1,2,5]oxadiazole-5-
an additional 3-5 h at which time the fluorescence (ex = 544
carboxamide (20). A mixture of 19 (128 mg, 0.70 mmol) and nm; em = 590 nm) was measured. Complete medium both with
phenyl isocyanate (87 µL, 0.77 mmol, 1.1 equiv) in dry THF (5
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and without DMSO was used as negative controls (0%
inhibition).
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Cerecetto, M. González, Eur. J. Med. Chem., 2008, 43, 2229–2237;
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Activity on intracellular amastigotes Inhibition of amastigote
growth in infected THP-1 cells was measured as previously
5
described. In brief, THP-1 cells in complete RPMI-1640 containing
1
0% fetal calf serum and antibiotics were treated for 24 h with 25
7
R. K. Arvela and N. E. Leadbeater, J. Org. Chem., 2003, 68, 9122–
9125; D. Wang, L. Kuang, Z. Li and K. Ding, Synlett, 2008, 69–72;
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Tetrahedron Lett., 2009, 50, 4595–4597.
ng/mL phorbol 12-myristate-13-acetate (PMA, Sigma-Aldrich, St.
Louis, MO). PMA and non-adherent cells were removed with excess
warm
medium,
and
stationary-phase
Ld:pSSU-int/LUC
promastigotes expressing luciferase (5:1 parasite/macrophage ratio)
used to infect the macrophages. Twenty-four hours later the adherent
cells were washed 4-5 times with warm RPMI-1640 alone, and
8
9
US Pat. 0122861, 2012.
E. A. A. Wallén, J. A. M. Christiaans, M. M. Forsberg, J. I.
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,
infected macrophages detached by treating with Trypsin EDTA.
4
Infected macrophages (5×10 cells/100 µL/well) were dispensed in 10 H. Käsnänen, M. J. Myllymäki, A. Minkkilä, A. O. Kataja, S. M.
triplicate into white 96-well flat bottom plates (NUNC, Denmark)
and the compounds diluted in complete RPMI-1640 containing 1%
Saario, T. Nevalainen, A. M. P. Koskinen and A. Poso,
ChemMedChem, 2010, , 213–231.
5
DMSO final concentration and added in triplicate (50 μL/well). 11 WO Pat., 069949, 2010.
Cultures were incubated for a further 48 h (37 °C, 5% CO ), lysed by 12 O. Shimony and C. L. Jaffe, J. Microbiol. Methods, 2008, 75, 196–
2
the addition of Bright-Glo Luciferase Assay substrate (100 µL/well,
Promega, MT, U.S.A.), and chemiluminescence measured using a 13. A. Debrabant, M. B. Joshi, P. F. P. Pimenta and D. M. Dwyer, Int. J.
microplate reader (Fluoroskan Ascent, Thermo Scientific).
Parasitol., 2004, 34, 205–217.
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200.
included as a positive control. Complete medium both with and
without DMSO were used as negative controls. Experiments were
Cos and L. Maes, Antimicrob. Agents Chemother., 2009, 53, 3855-
3859.
repeated three times. Calculation of the EC 's and statistical analysis 15. M. de Rycker, I. Hallyburton, J. Thomas, L. Campbell, S. Wyllie, D.
5
0
were carried out using GraphPad Prism Version 6.0b (GraphPad
Software, Inc. San Diego, CA).
Joshi, S. Cameron, I. H. Gilbert, P. G. Wyatt, J. A. Frearson, A. H.
Fairlamb and D. W. Gray, Antimicrob. Agents Chemother., 2013, 57,
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913-2922.
Acknowledgements
We thank Ms. Anna Keltikangas née Takala for the synthesis
assistance. This study was supported by the Academy of
16. S. Oh, B. Kwon, S. Kong, G. Yang, N. Lee, D. Han, J. Goo, J. L.
Siqueira-Neto, L. H. Freitas-Junior, M. Liuzzi, J. Lee and R. Song,
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Finland (projects 264020 and 265481 to JYK). CLJ holds the 17. K. Seifert, P. Escobar and S.L. Croft, J. Antimicrob. Chemother.,
Michael and Penny Feiwel Professorial Chair of Dermatology.
2010, 65, 508-511.
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8. Y. S. Rizk, A. Fischer, M. de Castro Cunha, P. O. Rodrigues, M. C. S.
Marques, M. de Fatima C. Matos, M. C. T. Kadri, C. A. Carollo and
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Table of contents entry (graphical abstract and 20-30 words of text)
Facile synthesis route to carboxyimidamide-substituted benzo[c][1,2,5]oxadiazole and related derivatives was
developed, and 25 derivatives were synthesized. Antileishmanial evaluation revealed promising inhibition
activity.