Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen specific anti-leptospiral agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.020

A simple and efficient method for the synthesis of a series of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has been developed. Pyranoisoxazolones 10a-j, dihydronaphthopyran-4-one (DHNPs) class of natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six pyranoisoxazolones, 10b, 10d, 10f, 10g, 10i and 10j which displayed very good anti-leptospiral activity was taken for secondary screening against twelve strains of pathogenic and one non-pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral activity against the pathogenic serovars at MIC of 62.5–500?μg/mL. Compounds 10d, 10g and 10j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In?vivo treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires. Compound 10g was found to show cytotoxicity against THP-1?cells only at higher concentration (≥75?μg/mL). Effective binding of compound 10g with leptospiral outer membrane protein LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen specificity of compound 10g. Antibiotics acting against leptospirosis in human are very few. The results obtained from in?vitro, in?vivo and in silico study reveals that 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen specific anti-leptospiral agents.

Full text of DOI:10.1016/j.ejmech.2016.09.020

Accepted Manuscript
Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen
specific anti-leptospiral agents
Andivelu Ilangovan, Palaniappan Sakthivel, Karikalacholan Sivasankari, Charles
Solomon Akino Mercy, Kalimuthusamy Natarajaseenivasan
PII:
S0223-5234(16)30751-6
10.1016/j.ejmech.2016.09.020
EJMECH 8886
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2016
Revised Date: 6 September 2016
Accepted Date: 7 September 2016
Please cite this article as: A. Ilangovan, P. Sakthivel, K. Sivasankari, C.S.A. Mercy, K.
Natarajaseenivasan, Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of
pathogen specific anti-leptospiral agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.020.
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ACCEPTED MANUSCRIPT
Discovery of 6,7-Dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a
New Class of Pathogen Specific Anti-leptospiral Agents
a*
a
b
Andivelu Ilangovan, Palaniappan Sakthivel, Karikalacholan Sivasankari, Charles
b
b
Solomon Akino Mercy, Kalimuthusamy Natarajaseenivasan
a
School of Chemistry, Bharathidasan University, Tiruchirappalli – 620 024, Tamil Nadu, India
b
Division of Medical Microbiology, Department of Microbiology, School of Life Sciences,
Bharathidasan University, Tiruchirappalli (T.N) – 620024, India
*
E-mail: ilangovanbdu@yahoo.com
Graphical Abstract

ACCEPTED MANUSCRIPT
Discovery of 6,7-Dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a New
Class of Pathogen Specific Anti-leptospiral Agents
a*
a
b
Andivelu Ilangovan, Palaniappan Sakthivel, Karikalacholan Sivasankari, Charles
b
b
Solomon Akino Mercy, Kalimuthusamy Natarajaseenivasan
a
School of Chemistry, Bharathidasan University, Tiruchirappalli – 620 024, Tamil Nadu, India
b
Division of Medical Microbiology, Department of Microbiology, School of Life Sciences, Bharathidasan
University, Tiruchirappalli (T.N) – 620024, India
*
E-mail: ilangovanbdu@yahoo.com
Abstract
A simple and efficient method for the synthesis of a series of 6,7-dihydro-3H-pyrano[4,3-
c]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has
been developed. Pyranoisoxazolones 10a-j, dihydronaphthopyran-4-one (DHNPs) class of
natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against
pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six
pyranoisoxazolones, 10b, 10d, 10f, 10g, 10i and 10j which displayed very good anti-leptospiral
activity was taken for secondary screening against twelve strains of pathogenic and one non-
pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral
activity against the pathogenic serovars at MIC of 62.5-500 ꢀg/mL. Compounds 10d, 10g and
1
0j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a
significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In vivo
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treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against
the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires.
Compound 10g was found to show cytotoxicity against THP-1 cells only at higher concentration
(≥75 µg/mL). Effective binding of compound 10g with leptospiral outer membrane protein
LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen
specificity of compound 10g. Antibiotics acting against leptospirosis in human are very few. The
results obtained from in vitro, in vivo and in silico study reveals that 6,7-dihydro-3H-pyrano[4,3-
c]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen
specific anti-leptospiral agents.
Keywords: 5-Carboalkoxy-2,3-dihydropyranone, 6,7-Dihydro-3H-pyrano[4,3-c]isoxazol-3-one,
Anti-leptospiral, Pathogen-specific, LipL32.
1
. Introduction
Leptospirosis is a widespread and emerging infectious disease caused by the spirochete
bacteria of the genus Leptospira [1,2]. More than 5,00,000 cases of severe leptospirosis, with
fatality rates exceeding 10-20%, are reported worldwide each year [3]. The annual morbidity of
leptospirosis was estimated to be higher in developing countries of South Asia and Southeast
Asia, especially in India [4]. Leptospirosis spreads either through direct and indirect contact with
water or soil contaminated by the urine of infected rodents or animals [5]. Several animals such
as sheep, goats, dogs, swine, horses, zebrafish, pigs, raccoons, rodents, equine, buffalo and cattle
play a role as reservoir hosts [6] for leptospirosis and some of them are also affected. It is
associated with various clinical symptoms such as sudden onset of febrile illness, chills,
headache, myalgia, abdominal pain and conjunctival suffusion. Inappropriate treatment of acute
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febrile disease leads to hepatic and renal dysfunction and hemorrhagic disorders [2,7,8].
Prevention of the infection by controlling environmental factors is difficult to implement and has
considerable socio-economic implications in developing countries.
Vaccination and chemoprophylaxis are two major possible control measures for
preventing the host from leptospirosis. Vaccines for serovars like Hardjo, Pomona, Canicola,
Grippotyphosa, and Icterohaemorrhagiae have been developed, however they are associated with
disadvantages like suboptimal protection, requirement of booster doses and failure against local
serovars [1,9]. Immunization of humans with killed and attenuated whole cell vaccine (bacterins)
has generally been restricted to individuals in high-risk epidemics [10]. Currently leptospirosis in
human is being treated by using antibiotics such as erythromycin (1), amoxicillin (2), ceftriaxone
(3), doxycycline (4), penicillin G, cefotaxime, azithromycin and ampicillin (Figure 1) which are
originally developed for other bacterial infections [11-13]. Furthermore, anti-leptospiral activity
of only a few other classes of organic molecules such as quinoxaline derivatives [14], xanthones
[15], azomethines of aryl oxazole [16] and pseudo-peptides [17] were studied, however it did not
provide useful leads. Assessment of previous clinical trials and treatment revealed unsatisfactory
benefit and safety of antibiotics against leptospirosis in human [18]. Till now, there is no
approved drug, specifically designed to target leptospiral bacteria life cycle in human is
available. Anti-leptospiral drug used in animals leptospirosis are chlortetracycline (5), neomycin
sulphate (6) and oxytetracycline. Leptospiral resistance to available antibiotics is an emerging
public health threat. Therefore, the present situation emphasizes the need to develop new anti-
leptospiral drugs.
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Fig. 1. Antibiotic drugs used for treatment of leptospirosis
Compounds with pyran structural motif has attracted interest of chemists owing to their
anti-inflammatory [19], anti-cancer [20], anti-HIV [21], anti-Alzheimer’s [22], myorelaxant [23],
sex pheromones [24] and anti-microbial [25,26] activities. On the other hand, isoxazole
derivatives are important pharmacophores used extensively in the field of medicinal chemistry.
Isoxazole derivatives exhibit various biological activities such as, anti-bacterial, anti-convulsant,
anti-cholestermic, anti-cancer, anti-inflammatory, adenosine antagonist, fungicidal, herbicidal,
hypoglycemic, muscle relaxant, nematocidal, insecticidal, anti-viral and anti-microbial activities
[27-29]. Although different biological activity [20,30,31], including anti-bacterial activity [32-
3
5], of different pyrano fused hetereocycles were studied, to the best of our knowledge,
biological activity of pyranoisoxazolone derivatives has not yet been examined. This literature
background reveals that pyrans, isoxazoles and pyrano fused hetereocycles display very good
anti-bacterial activity. In continuation of our experience on utilization of 5-carboalkoxy-2,3-
dihydro-4H-pyran-4-one derivatives (5-CDHPs) [36], we envisaged that the compounds could be
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converted in to novel pyranoisoxazolone derivatives 10a-j, which may exhibit interesting anti-
bacterial viz., anti-leptospiral activity.
Furthermore, naphthalene fused pyran-4-one ring systems are known to exhibit different
biological activities including anti-bacterial activity [37-39]. We recently reported total synthesis
of phytogrowth-inhibition active dihydronaphthopyran-4-one natural products 12b and 12c and
its analogues 12a and 13a-c (Scheme 3) [36]. Interestingly, compounds 12 and 13 and the drug
doxycycline (4, Figure 1) were found to have common structural features such as the presence of
ketone and phenolic functional groups, intramolecular hydrogen bonding and linear polycyclic
system. This prompted us to investigate anti-leptospiral activity of compounds 12 and 13. Thus,
in continuation of our interest on study of biologically active organic molecules [40-41] herein,
we report synthesis of a variety of pyranoisoxazolones 10a-j and dihydronaphthopyran-4-one
(DHNP) derivatives 12 and 13 and evaluation of their anti-leptospiral activity.
2
. Results and Discussion
2
.1. Chemistry
Pyranoisoxazole derivatives are usually made through dipolar cycloaddition using nitrile
oxides or nitrones [42-44], multi-component reaction [45], and other methods [46-48]. Under the
present study, we observed that a variety of pyranoisoxazolones 10 could be made from 5-
CDHPs 9 in one pot by treating it with hydroxyl amine in ethanol. The starting materials, 5-
CDHPs 9a-j [36] and two new DHPs 9c and 9f were synthesised by adopting the procedure
reported by us [36]. Domino C-acylation of β-ketoester 7 with α,β-unsaturated acid chlorides 8 in
the presence of CaCl
2
(Scheme 1) followed by in situ 6π-oxaelectrocyclization resulted in the
formation of 5-CDHPs 9.
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Scheme 1. Synthesis of 5-carboalkoxy-2,3-dihydro-4H-pyran-4-ones: Reaction conditions: 7
(1.0 mmol), 8 (1.0 mmol), CaCl
2
(0.1 mmol), triethylamine (2.0 mmol) in DCM (5 mL) at room
temperature. Isolated yield.
Having synthesized variety of 5-carboalkoxy DHPs 9, as a next step ethyl 2,2-dimethyl-
-oxo-6-phenyl-2,3-dihydro-4H-pyran-5-carboxylate (9a) was taken as model substrate, and
4
examined its conversion to pyranoisoxazolone 10 by treatment with hydroxylamine
hydrochloride in different solvents and temperature conditions. The results obtained are
summarized in Table 1. With 1.0 equiv. of hydroxyl amine in ethanol (entry 1) the reaction at r.t.
failed. However, after refluxing the reaction for 15 h, the desired product 10a was obtained in
7
0% yield (entry 2). Pleasingly, the yield of the product 10a increased to 90% when 2.5 equiv. of
hydroxyl amine was used (entry 3). Further, when MeOH and t-BuOH was used as the solvent,
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at reflux temperature, the product 10a was obtained in 70% and 80% respectively (entry 4 and
5
1
). Based on these results the optimized condition for further study was identified as treatment of
.0 equiv. of 5-CDHP derivatives 9 with 2.5 equiv. of hydroxylamine hydrochloride in ethanol
at reflux temperature.
a
Table 1. Optimisation of the reaction conditions
o
b
Entry NH OH.HCl (eq.) Solvent Temp ( C) Time (h) Yield (%)
2
c
1
2
3
4
5
.
.
.
.
.
1.0
1.0
2.5
1.0
1.0
EtOH
EtOH
r.t.
15
15
7
NR
70
90
70
80
reflux
reflux
reflux
reflux
EtOH
MeOH
t-BuOH
10
7
a
All reactions were performed using ethyl 2,2-dimethyl-4-oxo-6-phenyl-2,3-dihydro-4H-pyran-5-
b
c
carboxylate (9a) (1.0 mmol) and hydroxylamine hydrochloride (2.5 mmol); Isolated yield; No Reaction.
Further, different 5-CDHP 9 were treated under the optimised condition, to deliver the
desired products 10 in good to excellent yield (Scheme 2). Different functional groups such as
methoxy (10b and 10c), chloro (10d and 10e) and naphthyl (10f) tolerated the reaction condition
well. Moreover, the easily polymerisable thienyl (9g) and furyl (9h) containing 5-CDHPs gave
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corresponding pyranoisoxazolones 10g and 10h in good and moderate yield respectively.
Aliphatic substrates 9i and 9j also underwent reaction to give the desired product 10i and 10j
respectively in good yield.
Scheme 2. Synthesis of substituted 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones: Reaction
conditions: DHPs 9 (1.0 mmol), NH OH.HCl (1.5 mmol) in 5 mL of EtOH at 25 °C for 5 h.
2
Isolated yield.
The DHNP analogues 12a-c and 13a-c required for the present study were synthesised
(Scheme 3) in a concise and efficient way by making use of Friedel-Crafts acylative
aromatization of 6-benzyl-5-CDHPs 11 reported by us earlier [36].
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Scheme 3. Synthesis of dihydronaphthopyran-4-one natural products and its analogues
2
2
.2. Evaluation of Biological Activity
.2.1. Preliminary Screening of 6,7-Dihydro-3H-pyrano[4,3-c]isoxazol-3-ones (10a-j) and
Dihydronaphthopyran-4-ones (12 and 13)
Having synthesised series of pyrano[4,3-c]isoxazol-3-ones, 10a-j and
a
dihydronaphthopyran-4-ones 12a-c and 13a-c, preliminary screening against pathogenic
leptospiral serovar Autumnalis strain N2 was carried out at different concentrations (500, 250,
1
25, 62.5 and 31.25 µg/mL, Table 2) by micro dilution assay. Doxycyline (4, Figure1) was used
as reference. Out of the ten pyrano[4,3-c]isoxazol-3-ones, five compounds such as 10b, 10d, 10f,
0g and 10j showed inhibition at lower concentration of 250 ꢀg/mL itself whereas, 10i showed
1
inhibition only at 500 ꢀg/mL. Autumnalis strain N2 was originally isolated from a human case of
leptospirosis earlier. Interestingly, most of these compounds were found to show activity at
concentration same as doxycyline.
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Table 2. Preliminary screening of pyrano[4,3-c]isoxazol-3-ones (10a-j) and
dihydronaphthopyran-4-ones (12a-c and 13a-c) against Leptospira interrogans serovar
Autumnalis strain N2
Minimum Inhibitory Concentration (MIC, µg/mL)
Pyranoisoxazolones
5
00
250
R
I
125
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
62.5
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
31.25
1
0a
0b
0c
0d
0e
0f
0g
0h
R
I
R
1
R
1
R
I
R
I
R
1
R
1
R
I
R
I
R
1
R
1
I
I
R
1
R
I
R
R
I
R
1
0i
R
1
0j
I
R
1
2a
2b
2c
3a
3b
3c
Doxycyline (4)
R
R
R
I
R
R
R
R
I
R
1
R
1
R
1
R
1
I
R
1
I
I
R
I
I
R
R- Resistance; I-Inhibition. All the experiments were done in triplicates.
Similarly, among naphthopyranone derivatives, compound 13a showed activity, at a
concentration of 500 µg/mL, whereas compounds 12a, 12b and 12c did not show any activity.
Interestingly, compound 13b and 13c exhibited anti-leptospiral activity at 250 µg/mL itself. This
result revealed that compounds 13b and 13c, bearing unsubstituted naphthyl ring were potential
1
0

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molecules to achieve anti-leptospiral activity compared to compounds 12a-c substituted with
OMe group. Further in order to make structure activity relationship study, six pyrano[4,3-
c]isoxazol-3-ones such as, 10b, 10d, 10f, 10g, 10i and 10j (substituted at 4 position with variety
of groups such as aryl, heteroaryl, and alkyl) were taken for secondary screening against locally
prevalent, thirteen different serovars.
2
.2.2. Secondary Screening of 6,7-Dihydro-3H-pyrano[4,3-c]isoxazol-3-ones
In the secondary screening, six potential pyrano[4,3-c]isoxazol-3-one derivatives such as
0b, 10d, 10f, 10g, 10i and 10j were screened at different concentrations of 500, 250, 125, 62.5,
1.25 µg/mL against twelve strains of pathogenic and one non-pathogenic serovars by micro
1
3
dilution assay. The results obtained with all the concentration are shown in the figure 2. The MIC
for each compound is listed out separately in table 3. The pathogenic serovars used in the study
are Australis (I, serovar Australis, strain Ballico), Autumnalis (II, serovar Autumnalis, strain
N2), Ballum (III, serovar Ballum, strain Mus 127), Bataviae (IV, serovar Bataviae, strain Swart),
Canicola (V, serovar Canicola, strain Hond Utrecht IV), Grippotyphosa (VI, serovar
Grippotyphosa, strain Moskva V), Hebdomadis (VII, serovar Hebdomadis, strain Hebdomadis),
Icterohaemorrhagiae (VIII, serovar Icterohaemorrhagiae, strain RGA), Javanica (IX, serovar
Poi, strain Poi), Pomona (X, serovar Pomona, strain Pomona), Pyrogenes (XI, serovar
Pyrogenes, strain Salinem) and Sejroe (XII, serovar Hardjo, strain Hardjoprajitno). In addition to
the pathogenic strains I-XII, a non-pathogenic strain Patoc I of serovar Semaranga (XIII) was
also included.
1
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Fig. 2. Secondary screening of anti-leptospiral activity of pyranoisoxazolones 10b, 10d, 10f, 10g, 10i and
1
0j against twelve pathogenic serovar: Australis (I); Autumnalis (II); Ballum (III); Bataviae (IV);
Canicola (V); Grippotyphosa (VI); Hebdomadis (VII); Icterohaemorrhagiae (VIII); Javanica (IX);
Pomona (X); Pyrogenes (XI); Sejroe (XII); and one non-pathogenic serovar Semaranga (XIII).
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Table 3. MICs of 6,7-dihydro-3H-pyrano[4,3-c]isxazol-3-ones for 13 serovars of Leptospira
MIC (µg/mL)
VI VII VIII IX
Compound
I
II
III
IV
V
X
XI XII XIII
1
1
0b
0d
125 250 125 250 250 250 250 250 250 250 250 250 500
a
125 250 250 250 125 250 250 250 250 250 250 250 ni
1
0f
0g
0i
0j
125 250 125 125 250 250 250 250 500 500 500 500 500
a
1
250 250 250 250 62.5 250 250 250 250 250 125 250 ni
1
500 500 500 250 250 500 250 125 500 250 250 500 500
a
1
250 250 250 125 250 250 125 250 250 250 250 250 ni
Doxycyline 200 200 100 100 200 200 200 200 200 100 200 100 100
ni: no inhibition; Tested against serovars I-XIII, mentioned in figure 2
a
Very interesting trends in inhibition of serovars by six pyrano[4,3-c]isoxazol-3-ones 10b,
1
0d, 10f, 10g, 10i and 10j was observed (Table 3). While compound 10d, 10g and 10j inhibited
only the pathogenic serovars at MIC of < 250 ꢀg/mL, the non-pathogenic serovar, semaranga
(
XIII) remained unaffected. Fascinatingly, all the six compounds inhibited the serovars Bataviae
IV), Canicola (V), Hebdomadis (VII), and Icterohaemorrhagiae (VIII) at MIC of < 250 ꢀg/mL.
(
However, compound 10f with 4-naphthyl substituent and 10i with 4-methyl substituent, showed
activity against five serovars (IX-XIII) and seven serovars (I-III, VI, IX, XII and XIII)
respectively only at highest MIC of 500 ꢀg/mL (Table 3). The compound 10g, with 4-thiphenyl
substituent showed activity against all the serovars at MIC of < 250 ꢀg/mL and particularly
against Canicola (V) at a very low MIC of 62.5 ꢀg/mL, which was much lower than the
concentration observed with the reference drug doxycycline [13,49,50]. However, in case of the
serovars Javanica (IX) and Sejroe (XII) all six compounds exhibited activity at only MIC > 250
1
3

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th
ꢀ
g/mL. Thus, structure activity relationship of compounds shows that substituent at 4 position
of pyranoisaxazolone has an important influence on the activity against leptospiral serovars, and
the compound 10g with a thienyl substituent is a potential lead for further study. In order to
establish the mechanism of action of the compound 10g, further biological studies such as real
time quantitative PCR analysis, animal survivability (in vivo), cytotoxicity (MTT), cell death
assay (PI staining), hemocompatibility, and in silico molecular docking study was carried out.
2
.2.3. Real Time Quantitative PCR Analysis of Leptospiral 16S rRNA Gene.
SYBR Green-based RT-qPCR assay, which is a highly efficient and widely used platform
amongst available real-time PCR technologies, especially for the identification of most abundant
RNA target like 16S rRNA was used under the present study to quantify the leptospiral load in
the 10g treated and untreated control [51]. The negative control was included to check the purity
of the reaction mixture (Figure 3). The Ct (Threshold clycle) value is inversely proportional to
the number of leptospires. The Ct value for 10g treated and untreated leptospires was observed as
1
8.5 and 11.3 respectively. The primers specificity was analysed by melt curve analysis. The
result shows that the leptospires growth was inhibited by pyranoisoxazolone 10g at a significant
level (P<0.05).
1
4

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Fig. 3. Real Time quantitative PCR analysis (RT-qPCR). The propagation of leptospires was significantly
less in 10g treated samples than positive control. *P<0.05.
2
.2.4. Challenge Experiments in Animal Models
Since compound 10g showed the remarkable MIC value against pathogenic leptospiral
serovars in in vitro experiments, it was selected for further in vivo studies using Cy-BALB/c
mice [52,53]. At the end of the study period, 95% of surivivabilty was observed at 20 mg/kg,
which was nearly equal to doxycycline treated groups and only 20% suvivability among 5 mg/kg
th
was observed. The untreated control leads to death from day 4 onwards. This has evidenced that
by utilising compound 10g, at a concentration of 20 mg/kg, the infected mice could be protected
completely from leptospirosis. The antibacterial activity of compound 10g might be related to its
antibiotic activity or to the presence of metabolic toxins. This suggests that the compound 10g
provides antibacterial activity against leptospires by in vivo.
1
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Fig. 4. Survival of mice treated for 3 days with compound 10g (5, 10, or 20 mg/kg one time daily).
Controls include untreated animals and those animals treated with doxycycline (5 mg/kg one time daily).
2
.2.5. Quantitative RT-PCR for leptospiral DNA from animal tissue
The qRT-PCR analysis of inspected kidney samples revealed the presence of leptospiral
renal colonization in the case of infected untreated mice (Figure 5) [51,54], and no colonization
in the mice group treated with the compound 10g at a concentration of 20 mg/kg. This finding
was also supported by the re-isolation of the leptospires in EMJH from the untreated mice and
among the treated mice group it was not successful with 20 mg/kg. RT-qPCR and reisolation in
EMJH semi-solid medium study has shown that compound 10g has the potential to inhibit
1
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leptospires by clearing renal colonization of the leptospires from the system completely. This
confirms that the compound 10g shows significant protection against virulent leptospires in Cy-
BALB/c mice model.
Fig. 5. The compound 10g treated and untreated mice kidney tissue sample were analysed by
RT-qPCR assay to determine the leptospiral load. Doxycycline was used as positive control.
2
.2.6. MTT Assay
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based
on the conversion of MTT into formazan crystals by living cells, which determines
1
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mitochondrial activity. Since for most cell populations the total mitochondrial activity is related
to the number of viable cells, this assay is broadly used to measure the in vitro cytotoxic effects
of drugs on cell lines [55]. Under the present study, in vitro cytotoxic effect of the compound 10g
against THP-1 cells were given in Figure 6. The results revealed that the compound 10g
exhibited dose dependent inhibition of the cell growth. At a lower concentration of ≤25 ꢀg/mL
no significant cytotoxic effect has been detected and it showed cytotoxic effect significantly only
≥75 ꢀg/mL. This clearly indicates that compound 10g has potential cytotoxic effect only at
higher concentration, compared to 0.1% Triton X-100 treated positive controls (Amresco, USA).
Fig. 6. Effect of compound 10g on THP-1 cells treated at 1–100
ꢀg/mL concentration for 24 h; TX-100 –
Triton X-100; UT – Untreated cells. PBS - Phosphate-buffered saline.
2
.2.7. Cell Death Assay by Propidium Iodide (PI)
Propidium lodide (PI) dye penetrates only damaged cellular membranes [56], hence used
to identify the dead cells. Intercalation complexes formed by PI with double-stranded DNA
1
8

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affects amplification of the fluorescence. Incubation of the total cell population with PI and
subsequent fluorescence detection allowed assessment of the number of non-vital cells. The THP-
1
cells treated with compound 10g, at lower concentrations of ≤25 ꢀg/mL showed no significant
cell death, but increased cell death was observed at higher concentration ≥75 µg/mL, which is
similar to the number of cells positive for PI in cells treated with tBHP (Figure 7). This reveals
that compound 10g has cytotoxicity against THP-1 cells only at higher concentration ≥75
µg/mL.
Fig. 7. PI staining of THP-1 cells at 12 h of incubations by the treatment of 10g at different
concentrations; UT – Untreated cells. tBHP – tert-butylhydroperoxide; The graph shows manual count of
apoptotic and necrotic cells in percentage. (Data are mean percent ± SD percent of triplicate each).
1
9

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2
.2.8. Hemocompatibility
To be a potential anti-leptospiral agent, it is important for an organic molecule to be non-
toxic to the normal human cells. In order to check toxicity, hemocompatibility of compound 10g
was examined at various concentration 500, 250, 125, 62.5, 31.25, 15.6, 7.8 ꢀg/mL by measuring
the damage to human RBCs (Figure 8). The mechanisms of direct hemolytic activity for different
toxic agents were found to be non-specific. According to the International Organization for
Standardization/Technical Report 7406, the admissible level of hemolysis of biological materials
is 5% [57]. As shown in Figure 8, compared to the positive control (Triton X-100), compound
1
0g exhibited very low hemoglobin release at various concentrations.
Fig. 8. In vitro hemocompatibility assay of pyranoisoxazolone 10g exhibits very less hemolytic activity.
No (0%) lysis was noticed in the negative control (NC-PBS buffer) whereas the positive control (PC- 1%
Triton X-100) shows 100% lysis.
2
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2
.2.9. In silico Molecular Docking
One of the important differences among pathogenic and non-pathogenic serovars is the
lack of outer membrane lipoprotein “LipL32”. It is reported to be absent in non-pathogenic
avirulent Leptospira [58]. It is expressed during infection and highly conserved among
pathogenic Leptospira. In silico comparison of amino acid sequences of LipL32 showed 97.8%
average sequence identity among the pathogenic species [59]. It is important to note that while
compound 10g affected all the leptospiral strains of pathogenic serovars, it did not affect non
pathogenic serovar Semaranga. Thus we were curious to know whether the lead compound 10g
could act upon leptospiral LipL32. To identify this molecular docking study was carried out for
compound 10g against LipL32 protein (PDB code: 2ZZ8) by using the Autodock4 program
(
Figure 9) [60].
2
1

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Fig. 9. (A) Docking pose of the compound 10g with amino acid sequences of LipL32 in which chain-A
shown in red color and chain-B shown in green color. (B) Docking pose of the compound 10g with
LipL32 (in zoom) (C) Line representation of selected amino acids of chain A of LipL32 interacting with
compound 10g. Yellow dotted lines indicate the hydrogen bonds. Pictures were generated by using
PyMOL.
The compound 10g upon docking was specifically surrounded by significant amino acid
residues such as Leu-214, Phe-215, Pro-216, Pro-217, Gly-218, Ile-219, Pro-220, Gly-221 and
Val-222 of the chain A and Thr-20, Ile-21, Pro-22, Gly-23, Thr-24, Asn-25, Glu-26 and Thr-27
of the chain B. Significant hydrogen bonding was observed between carbonyl O of the
2
2

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isoxazolone ring in 10g and HN of the Gly-221 of the chain A of LipL32 (O⋯HN distance = 3.3
Å). Compound 10g binds effectively to LipL32 with a binding energy of ∆G -7.2 kcal/mol. The
docking studies confirm that compound 10g has good binding affinity towards LipL32 and
explains the reason for compound 10g being specific to leptospiral strains of pathogenic
serovars.
3
. Conclusions
Ten different pyrano[4,3-c]isoxazol-3-ones and six naphthopyranones were prepared
through simple and efficient synthetic procedure. Preliminary screening of all the sixteen
compounds against Leptospira interrogans serovar Autumnalis lead to identification of six
different pyrano[4,3-c]isoxazol-3-ones 10 as prominent molecules for further study. Secondary
screening of these molecules against twelve pathogenic and one non-pathogenic serovars lead to
identification of pyrano[4,3-c]isoxazol-3-one 10g, with 4-thiphenyl substituent, as the lead
molecule. This compound achieved activity against all the leptospiral serovars at MIC of ≤250
ꢀ
g/mL and it is particularly active against Canicola at a very low MIC of 62.5 ꢀg/mL, which is
much lower than the MIC observed with the reference drug doxycyline. Interestingly,
compounds 10d, 10g and 10j targeted only pathogenic serovars and did not affect the non-
pathogenic serovar such as Semaranga. 16S rRNA gene based RT-qPCR study revealed
inhibition of leptospires growth by compound 10g at significant level. In vivo animal challenge
study carried out on Cy-BALB/c mice by treating it with the compound 10g, at a concentration
of 20 mg/kg confirmed that the infected mice has 95% of survivability against leptospirosis. The
RT-qPCR analysis of DNA extracted from kidney samples of mice treated with compound 10g
for leptospiral renal colonization revealed the absence of leptospiral specific DNA. This finding
2
3

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was also supported by the re-isolation of the leptospires from the untreated mice. MTT assay of
THP-I cells treated with compound 10g shows potential cytotoxic effect only above 75 ꢀg/mL
which was further confirmed by PI staining. Hemolytic assay convincingly proves that
compound 10g is hemocompatible. In silico molecular docking studies revealed significant
hydrogen bonding between carbonyl O of the isoxazolone ring in 10g and HN of the Gly-221, of
the chain A of LipL32. This substantiates the experimental result that compound 10g is specific
to pathogenic serovars. We have explored biological activity of pyranoisoxazolone class of
compounds for the first time, and identified it as new class anti-leptospial active molecules.
These molecules has potential for further development as pathogen specific anti-leptospiral
agents. Study on anti-leptospiral active organic molecules are otherwise rare and further study on
structural modification of this scaffold to achieve superior anti-leptospiral activity is under
progress in our group.
4
. Experimental Protocols
4
4
.1. Chemistry
.1.1. General remarks
Melting points were determined by the open capillary tube method using a Toshniwal melting
1
13
point apparatus and are uncorrected. The H and C NMR spectra were measured on a Bruker
Avance 400 (400 MHz) NMR spectrometer. Chemical shifts are reported in ppm (δ) relative to
internal standard tetramethylsilane (TMS, δ 0.00 ppm). Data are reported as follows: chemical
shift (multiplicity [singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad resonance
(br)], coupling constants [Hz], integration). All the NMR spectra were acquired at ambient
temperature. ESI-MS was recorded on Agilent 1100 LC/MSD (70 ev) spectrometer. High
2
4

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resolution mass spectra (HRMS) were recorded on a Waters Q-Tof micro mass spectrometer.
Elemental analyses were performed on a CHN analyser. Thin layer chromatography (TLC) was
performed on Merck pre-coated silica gel 60F plates and visualized by exposure to UV light.
ACME silica gel (100-200 mesh) was used for column chromatography.
4
.1.2. Preparation of 5-carboalkoxy-2,3-dihydro-4H-pyran-4-one (9)
-Carboalkoxy-2,3-dihydro-4H-pyran-4-one such as 9a, 9b, 9d, 9e, 9g, 9h, 9i and 9j
were prepared by utilizing our previous literature procedure [36].
.1.2.1. Ethyl 2,2-dimethyl-4-oxo-6-(3,5-dimethoxyphenyl)-2,3-dihydro-4H-pyran-5-
carboxylate (9c):
5
4
The reaction was carried out according to reported literature method [36] using ethyl 3-(3,5-
dimethoxyphenyl)-3-oxopropanoate (7c, 252 mg, 1.0 mmol), CaCl (11 mg, 0.1 mmol), Et N
2
3
(278 µL, 2.0 mmol), senecioyl chloride (8, 112 µL, 1.0 mmol) in DCM (4 mL) for 5 h gave 9c
o
1
(
267 mg, 80%) as a yellow solid. M.p. 98-100 C. H NMR (400 MHz, CDCl
3
): δ 6.66 (d, J =
2
6
1
.4 Hz, 2H), 6.54 (t, J = 2.2 Hz, 1H), 4.08 (q, J = 6.8 Hz, 2H), 3.77 (s, 6H), 2.63 (s, 2H), 1.54 (s,
1
3
H), 1.04 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ): δ 188.9, 170.1, 165.9, 160.6, 135.5,
3
+
11.6, 106.2, 103.5, 81.9, 61.1, 55.5, 46.9, 26.0, 13.7 ppm; HRMS (ESI): [M + Na] Calcd for
C H NaO 357.1314; found 357.1309.
18
22
6,
4
.1.2.2. Ethyl 2,2-dimethyl-4-oxo-6-(naphthalen-1-yl)-2,3-dihydro-4H-pyran-5-carboxylate
9f):
The reaction was carried out according to reported literature method [36] method A using ethyl
-(naphthalen-1-yl)-3-oxopropanoate (7f, 242 mg, 1.0 mmol), CaCl (11 mg, 0.1 mmol), Et N
(
3
2
3
(278 µL, 2.0 mmol), senecioyl chloride (8, 112 µL, 1.0 mmol) in DCM (4 mL) for 5 h gave 9f
2
5

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o
1
(
279 mg, 86%) as a yellow solid. M.p. 78-80 C; H NMR (400 MHz, CDCl
3
): δ 7.93 (d, J = 8.0
Hz, 2H), 7.88-7.86 (m, 1H), 7.56-7.45(m, 4H), 3.69 (q, J = 7.2 Hz, 2H), 2.77 (s, 2H), 1.65 (s,
1
3
6
1
H), 0.46 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl ): δ 188.5, 172.6, 164.7, 133.3, 132.0,
3
30.9, 130.3, 128.4, 127.0, 126.6, 126.4, 124.7, 124.6, 113.4, 82.7, 60.4, 47.4, 26.2, 13.1 ppm;
+
HRMS (ESI): [M + Na] Calcd for C H NaO 347.1259; found 347.1254.
2
0
20
4,
4
3
.1.3. General method A: Typical experimental procedure for the synthesis of 6,7-dihydro-
H-pyrano[4,3-c]isoxazol-3-one (10)
The mixture of 5-carboalkoxy-2,3-dihydro-4H-pyan-4-one 9 (1.0 mmol) and
2
NH OH.HCl (2.5 mmol) in 4 mL of ethanol was stirred at reflux for 5 h. The progress of the
reaction was monitored by TLC. After the completion of reaction ethanol was removed under
reduced pressure. Then the reaction mixture was quenched with water, extracted with EtOAc (3
x 8 mL) and washed with brine. The organic layer was separated, dried over anhydrous Na SO
2
4
and concentrated under reduced pressure. The crude product was purified by column
chromatography (Hexane/EtOAc = 8:2) to afford pure 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-
one 10.
4
.1.3.1. 6,6-Dimethyl-4-phenyl-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one (10a)
The reaction was carried out according to general method A using ethyl 2,2-dimethyl-4-oxo-6-
phenyl-2,3-dihydro-4H-pyran-5-carboxylate (9a, 274 mg, 1.0 mmol), NH OH.HCl (174 mg, 2.5
mmol) in ethanol (4 mL) gave the title compound 10a (206 mg, 85%) as a white solid. M.p. 158-
2
o
1
1
7
60 C; H NMR (400 MHz, CDCl ): δ 8.21 (dd, J = 8.6, 1.4 Hz, 2H), 7.64-7.60 (m, 1H), 7.53-
3
1
3
.49 (m, 2H), 2.92 (s, 2H), 1.60 (s, 6H); C NMR (100 MHz, CDCl ): δ 171.9, 169.0, 157.1,
3
2
6

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+
1
34.5, 130.1, 129.9, 128.6, 95.8, 84.2, 33.8, 26.3 ppm; HRMS (ESI): [M + H] Calcd for
C H NO 244.0974; found 244.0967.
14
14
3,
4
.1.3.2.
10b)
The reaction was carried out according to general method A using ethyl 6-(4-methoxyphenyl)-
,2-dimethyl-4-oxo-2,3-dihydro-4H-pyran-5-carboxylate (9b, 304 mg, 1.0 mmol), NH OH.HCl
4-(4-Methoxyphenyl)-6,6-dimethyl-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one
(
2
2
(174 mg, 2.5 mmol) in ethanol (4 mL) gave the title compound 10b (237 mg, 87%) as a yellow
o
1
solid. M.p. 147-149 C; H NMR (400 MHz, CDCl ): δ 8.33 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 9.2
3
1
3
Hz, 2H), 3.89 (s, 3H), 2.89 (s, 2H), 1.58 (s, 6H); C NMR (100 MHz, CDCl ): δ 171.3, 169.6,
3
+
1
64.9, 157.2, 132.7, 122.4, 114.1, 94.2, 83.5, 55.6, 33.9, 26.3 ppm; HRMS (m/z): [M + Na]
calcd. for C15 , 296.0899; found 296.0894.
.1.3.3. 4-(3,5-Dimethoxyphenyl)-6,6-dimethyl-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one
10c)
H15NNaO
4
4
(
The reaction was carried out according to general method A using ethyl 6-(3,5-
dimethoxyphenyl)-2,2-dimethyl-4-oxo-2,3-dihydro-4H-pyran-5-carboxylate (9c, 334 mg, 1.0
mmol), NH OH.HCl (174 mg, 2.5 mmol) in ethanol (4 mL) gave the title compound 10c (273
2
o
1
mg, 90%) as a yellow solid. M.p. 140-142 C; H NMR (400 MHz, CDCl ): δ 7.49 (d, J = 2.4
3
1
3
Hz, 2H), 6.72 (t, J = 2.4 Hz, 1H), 3.86 (s, 6H), 2.92 (s, 2H), 1.59 (s, 6H); C NMR (100 MHz,
CDCl ): δ 171.5, 169.0, 160.6, 157.0, 131.6, 107.7, 107.2, 96.1, 84.0, 55.7, 33.8, 26.2 ppm;
3
+
HRMS (m/z): [M + H] calcd. for C16
H18NO
5
, 304.1185; found 304.1179.
4
.1.3.4. 4-(4-Chlorophenyl)-6,6-dimethyl-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one (10d)
2
7

ACCEPTED MANUSCRIPT
The reaction was carried out according to general method A using ethyl 6-(4-chlorophenyl)-2,2-
dimethyl-4-oxo-2,3-dihydro-4H-pyran-5-carboxylate 9d (308 mg, 1.0 mmol), NH OH.HCl (174
2
mg, 2.5 mmol) in ethanol (4 mL) gave the title compound 10d (255 mg, 92%) as a yellow solid.
o
1
M.p. 156-158 C; H NMR (400 MHz, CDCl ): δ 8.23 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz,
3
1
3
2
1
2
H), 2.94 (s, 2H), 1.61 (s, 6H); C NMR (100 MHz, CDCl ): δ 170.3, 168.9, 156.9, 140.9,
3
+
31.4, 129.0, 128.3, 96.2, 84.4, 33.8, 26.3 ppm; HRMS (m/z): [M + H] calcd. for C H ClNO ,
1
4
13
3
78.0584; found 278.0575.
.1.3.5. 4-(2-Chlorophenyl)-6,6-dimethyl-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one (10e)
4
The reaction was carried out according to general method A using ethyl 6-(2-chlorophenyl)-2,2-
dimethyl-4-oxo-2,3-dihydro-4H-pyran-5-carboxylate (9e, 308 mg, 1.0 mmol), NH OH.HCl (174
mg, 2.5 mmol) in ethanol (4 mL) gave the title compound 10e (244 mg, 88%) as a white solid.
2
o
1
M.p. 140-143 C; H NMR (400 MHz, CDCl ): δ 7.54 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 4.0 Hz,
3
1
3
2
1
H), 7.39-7.35 (m, 1H), 2.96 (s, 2H), 1.64 (s, 6H); C NMR (100 MHz, CDCl ): δ 170.8, 167.8,
3
56.3, 133.5, 133.4, 131.8, 130.9, 128.5, 126.8, 98.5, 86.0, 34.1, 26.3 ppm; HRMS (m/z): [M +
+
H] calcd. for C H ClNO , 278.0584; found 278.0584.
1
4
13
3
4
.1.3.6. 6,6-Dimethyl-4-(naphthalen-1-yl)-6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one (10f)
The reaction was carried out according to general method A using ethyl 6-(naphthalen-1-yl)-
,2-dimethyl-4-oxo-2,3-dihydro-4H-pyran-5-carboxylate (9f, 324 mg, 1.0 mmol), NH OH.HCl
2
2
(174 mg, 2.5 mmol) in ethanol (4 mL) gave the title compound 10f (269 mg, 92%) as a pale
o
1
yellow solid. M.p. 177-179 C; H NMR (400 MHz, CDCl
3
): δ 8.07 (d, J = 8.4 Hz, 1H), 8.04-
8
1
.02 (m, 1H), 7.93-7.91 (m, 1H), 7.88 (dd, J = 7.2, 1.2 Hz, 1H), 7.59-7.55 (m, 3H), 3.03 (s, 2H),
1
3
.72 (s, 6H); C NMR (100 MHz, CDCl ): δ 173.7, 168.3, 156.5, 134.0, 133.7, 130.8, 130.3,
3
2
8