Synthesis and immunopotentiating activity of novel isoxazoline functionalized coumarins

Article abstract of DOI:10.1016/j.ejmech.2016.07.026

A novel series (13) of isoxazoline functionalized coumarins was synthesized through 1,3-dipolar cyclization of nitrile oxides with Allylated coumarins. Synthesis of effective and target selective immunostimulators through conjugation of diversely substitu

Full text of DOI:10.1016/j.ejmech.2016.07.026

Accepted Manuscript
Synthesis and immunopotentiating activity of novel isoxazoline functionalized
coumarins
Tabasum Ismail, Syed Shafi, Swarn Singh, Tabasum Sidiq, Anamika Khajuria, Abdul
Rouf, Mahipal Yadav, Varma Saikam, Parvinder Pal Singh, Mohammad Sarwar Alam,
Nasarul Islam, Kalicharan Sharma, Halmuthur Mahabalarao Sampath Kumar
PII:
S0223-5234(16)30574-8
10.1016/j.ejmech.2016.07.026
EJMECH 8744
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 January 2016
Revised Date: 29 June 2016
Accepted Date: 12 July 2016
Please cite this article as: T. Ismail, S. Shafi, S. Singh, T. Sidiq, A. Khajuria, A. Rouf, M. Yadav,
V. Saikam, P.P. Singh, M.S. Alam, N. Islam, K. Sharma, H.M. Sampath Kumar, Synthesis and
immunopotentiating activity of novel isoxazoline functionalized coumarins, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.026.
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Synthesis and Immunopotentiating activity of novel Isoxazoline functionalized
Coumarins
Tabasum Ismail*,1, Syed Shafi2, Swarn singh1, Tabasum Sidiq1, Anamika Khajuria1, Abdul Rouf1,
Mahipal Yadav1, Varma Saikam1, Parvinder Pal Singh1, Mohammad Sarwar Alam2, Nasarul Islam3,
Kalicharan Sharma4 and Halmuthur Mahabalarao Sampath Kumar*,5
1Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Jammu, India
2Department of Chemistry, Jamia Hamdard University, New Delhi, India
3Department of Chemistry, Guru Nanak Dev University, Amritsar, India
4Department of Pharmaceutical chemistry, Jamia Hamdard University, New Delhi, India
5Vaccine Immunology Laboratory, NPC Division, Indian Institute of Chemical Technology,
Hyderabad, India
1
*Corresponding author : Email: tabsismail@gmail.com3Email: hmskumar@gmail.com; Tel: +91-
9912901010
Abstract
A novel series (13) of isoxazoline functionalized coumarins was synthesized through 1,3-
dipolar cyclization of nitrile oxides with Allylatedcoumarins. Synthesis of effective and target
selective immunostimulators through conjugation of diversely substituted isoxazolines and 7-
hydroxycoumarins is the focus of the present article. The proposed synthetic scheme was
observed to be highly regiospecific yielding attempted conjugates in good yield (>90%).
Kinetic resolution of the racemates was carried out by employing lipase B from Candida
antarctica (CALB). The synthesized compounds were screened in vitro and in vivo for their
biological activities viz. toxicity and impact on splenocyte proliferation (T- and B-cell
proliferation), antibody production (HA titer), delayed-type hypersensitivity reaction (DTH),
T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4) and NO
(macrophage) production. Our results establish that isoxazoline functionalized coumarins
exhibit excellent immune potentiating activity especially compounds 2, 4 and 8 whose
activity is more than that of Levimasole as standard. The structure activity relations are
explained in light of the structural/functional aspects of tested compounds. To the best of our
knowledge the presented work is first of its kind and is presaged to prove very useful for the
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design and synthesis of bis-heterocycle based novel, therapeutically selective and effective
immunopotentiators.
Keywords: 7-hydroxycoumarin; 2-isoxazoline; Bis-heterocycles; kinetic resolution;
Immunopotentiation.
Introduction
Encouraging results from immunomodulation based immunopharmacological approaches as a
cure for many biological malfunctions has stimulated an intense research activity towards
design of appropriate, less toxic and effective immunopotentiators. As an outcome of this
intense research activity, a huge library of immunopotentiators, of natural and synthetic origin
is available today [1-6]. Among the currently known immunopotentiators like, muramyl
dipeptide derivatives [7], levamisole [8], niridazole [9], and those from the groups of
interferons and interleukins, many have been found to express undesirable side effects and/or
high toxicity during the clinical trials [10-13].The presaged potential of immunomodulation
based therapies and the safety and therapeutic effectiveness concerns about currently known
immunopotentiators, makes studies related to design of novel, safe to use and effective
immunopotentiators indispensable. The development of natural product based low molecular
weight immunopotentiators is considered as one of the promising approaches in this direction
[6].
Aromatic heterocycles have served as valuable synthetic templates for the preparation of
new compounds with specific biological or material properties. Biological activities of bis-
heterocycles, especially those with nitrogen and oxygen as heteroatoms are well documented
in literature [14-16]. In addition to their use in synthetic chemistry, these compounds have
been found to express cytotoxic, immunostimulatory, antimicrobial, tuberculostatic, plant
growth regulative properties and various other biological activities [17]. Since recent years,
attention is increasingly being paid towards the synthesis of aromatic bis-heterocyclic
compounds. The diverse functional groupings and the synergistic activity of the fused
pharmacophores make bis-heterocyclic compounds divalent dual target interacting with
improved pharmacological functions. Individually both coumarins and isoxazolines in view
of their immunostimulatory effects have been employed for diverse applications and
pharmacological activities [14-16, 18-25]. Coumarins have been demonstrated to exhibit a
broad spectrum of pharmacological activities like antidepressant, antimicrobial, antioxidant,
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antitumor, antihistaminic, antiviral, CNS stimulant and anticoagulant [26-34]. Similarly
isoxazolines; the O- and N-containing heterocycles have been extensively explored for
synthetic and biological applications [35-37]. Isoxazolines have been demonstrated to exhibit
interesting and diverse biological properties like antiinflamatory, antagonistic, anticancer, anti
HIV, antidepressant and are currently used as pharmacophores of many clinically approved
therapeutic agents [25, 38]. The compounds with two or more heterocycles play a vital role in
natural and synthetic bioactive compounds [39-40]. Also it has been established that
biological efficacy of coumarins can be desirably altered through incorporation of another
heterocyclic moiety and the resulting compound often exhibiting promising and
unprecedented properties. These above cited facts and literature documented biological
expressions of coumarins and isoxazolines imply that conjugation of isoxazolines with
coumarins can serve as a promising synthetic strategy for the design of effective and target
selective immunostimulators; the same is demonstrated through the present work. Herein we
report a simple scheme for the synthesis of novel immunopotentiators through conjugation of
bioactive pharmacophoric isoxazolines with 7-hydroxycoumarin (7-OHC). A novel series of
13 isoxazoline functionalized 7-OHC (Table 1) was synthesized and tested in vitro and in
vivo to probe the immune potentiating potential and relevant structure activity relationship
(SAR) of these conjugates. The structures of new compounds were confirmed by chemical
1
and spectroscopic methods like IR, H NMR, 13C NMR, and mass spectrometry. The
synthesized conjugates were found to express excellent immunopotentiating activity. To the
best of our knowledge the presented work is first of its kind and is presaged to prove very
useful for the design and synthesis of bis-heterocycle based novel, therapeutically selective
and effective immune potentiators.
Results and discussion
Chemistry
For the synthesis of aimed conjugates, the strategy of 1,3-dipolar cyclization between an
Allylatedcoumarin and various substituted nitrile oxides (Scheme 1) was followed.
Dipolarophile was obtained by alkylating 7-OHC with allylbromide using K2CO3.
Nitrileoxides used as 1,3-dipoles, were synthesized according to the literature procedures
[41]. The novel isoxazoline conjugates of 7-OHC synthesized in the ensuing work are
presented in Table 1. Formation of 3,5-disubstituted isoxazolines possessing O-allyl
derivative of 7-OHC at 5th position, was established through the characteristic chemical shift
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values with multiplets at 4.00-4.26 ppm for the C5 protons and 3.00-3.65 ppm for C4 protons.
Similarly, characteristic chemical shift values for CH2 group of O-allyl derivative were
established with double-doublets/multiplets in the range of 4.06-4.30 ppm and 5.05-5.29 ppm
for the two non equivalent protons. The details of synthetic and characterization procedures
are given in the experimental section.
Scheme 1: Synthesis of bis-heterocycles encompassing 7-hydroxycoumarin and isoxazolines.
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Table 1: Synthesis of various isoxazoline derivatives of umbelliferone
aIsolated yields after chromatographic purification
The isoxazoline derivatives as presented in table 1, possess one chiral centre in the
isoxazoline ring thereby raising the possibility that derivatives synthesized as per Scheme 1,
would be a mixture of two possible enantiomers. In order to investigate the impact of
stereochemistry of bis-heterocyclic conjugates on their immune potentiating potential, kinetic
resolution of the racemate by employing lipase B from Candida Antarctica (CALB) was
carried out. The details of the kinetic resolution procedures are presented in the experimental
section.
Evaluation of biological activity
The isoxazoline-coumarin conjugates synthesized for the present work (Table 1) were tested
for their biological expressions viz. lymphocyte proliferation, antibody titre, delayed type
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hypersensitivity response, spleenocyte proliferation, cytokine release, macrophage production
and toxicity following procedures whose details given elsewhere [6] are also included in the
supporting information. To begin with, all the synthesised compounds were tested for their
impact on lymphocyte proliferation under in vitro conditions.The significant activity observed
for compounds 2, 4 and 8 in in vitro investigations motivated us for their further in vivo
investigations. In the in vivo investigations, compounds 2, 4 and 8 were tested for their impact
on primary and secondary antibody and DTH response, spleen cell subtyping and
proliferation, cytokine (IL-2, IFN-γ, and IL-4) and NO production. The efficacy of tested
compounds was compared to the results observed for Levamisole (Lev), a commonly used
standard for such investigations [8]. Lev was administered orally at a dose of 2.5 mg/kg body
weight as a positive control. In view of the encouraging immunopotentiating activity of
compounds 2, 4 and 8 noticed during in vivo investigations these were also tested for their
toxicity.
Effect of test compounds on in vitro lymphocyte proliferation by MTT assay
The results recorded during the in vitro screening of isoxazoline functionalized coumarins
synthesised for the present study, for their impact on lymphocyte proliferation are presented
as Figure 1. As is evident from these results, the screened compounds exhibit a dose
dependent response. Compounds 2, 3, 4, 5, 7 and 8 were observed to significantly enhance
the immunostimulation, especially compounds 2, 4 and 8 whose immunostimulating potential
even at lower doses (0.1 µg/mL) seems significantly higher than that observed for levamisole
(Lev) and 7-OHC. Immunostimulatory effect, as evident from Figure 1 was found to decrease
with increase in dosage from 0.1-10 µg/mL in compounds 9-13. These compounds did not
enhance the lymphocyte proliferation significantly.
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Figure 1: Effect of test compounds on lymphocyte proliferation in vitro. The proliferation was
calculated based on MTT assay. Absorbance was recorded at 570 nm. Values are expressed as mean
*
**
±
S.E. of three observations. P < 0.05; P < 0.01; ***P < 0.001 as compared to control determined
by one-way Anova (Bonferroni correction multiple comparison test)
.
Effect on antibody titre
The compounds 2, 4 and 8 were tested for their impact on B- cell activation. For this we
monitored the impact of these compounds at dose level of 0.001 mg/kg, 0.01 mg/kg and 0.1
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mg/kg on primary and secondary antibody response through estimation of total antibody titre.
Assuming that the response recorded on day 7 corresponds to primary antibody titre and that
recorded on day 14 corresponds to secondary antibody titre; the results presented as Figure 2
suggest that in comparison to standard, compounds 2, 4 and 8 enhance the primary and
secondary antibody synthesis by a significantly higher extent. In view of the above cited
assumption it may be inferred that the extent of increase in antibody at 14 day (usually
attributed to IgG titre) seems to be more than that of antibody response on day 7 (usually
attributed to IgM titre). Comparatively, the immunostimualation activity of compound 2 at a
dose of 0.1 mg/kg is highest.
Figure 2: Effect of compounds 2, 4 and 8on antibody titres in mice. Data are mean ± S.E. of six
*
**
animals. P < 0.05; P < 0.01; ***P < 0.001 when compared with control group determined by one-
way ANOVA (Bonferroni correction multiple comparison test).
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Delayed type hypersensitivity (DTH) response
Compounds 2, 4 and 8 were administered at varying doses to mice and the impact on SRBC-
induced DTH reaction was monitored. The results from these studies presented as Figure 3
clearly indicate a significantly higher DTH response than that observed with Lev even at
doses as low as 0.001 mg/kg. The DTH response was observed to decrease with time (24 hr >
48 hr > 72 hr) with compound 2 being the most effective.
Figure 3: Effect of compounds 2, 4 and 8 on DTH response. Data are expressed as mean ± S.E. of five
*
observations of left hind foot pad thickness measured at 24, 48 and 72 hr. P < 0.05; **P < 0.01;
***P < 0.001 as compared to control determined by one-way Anova (Bonferroni correction multiple
comparison test).
Effect on spleenocyte proliferation ex vivo (T and B cell proliferation)
The results from ex vivo splenocyte proliferation investigations wherein compounds 2, 4 and
8 were administered at various doses to mice are depicted as Figure 4. Cell proliferation
showed a regular increase with increase in the dose for all these compounds. As is evident
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from Figure 4, compound 2 resulted in a significant increase in T and B cell proliferation at
all doses in comparison to Lev and the other two compounds. Compound 4 showed
proliferation equivalent to Lev at a dose of 0.01 mg/kg. Compound 8 also increased cell
proliferation to a significant extent.
Figure 4: Effect of compounds 2, 4 and 8 on T and B cell proliferation.Splenocyte proliferation
*
expressed as the absorption at 570 nm ex vivo. Data are mean ± SE of six animals. P < 0.05;
**P < 0.01; ***P < 0.001 compared with control group determined by one-way ANOVA (Bonferroni
correction multiple comparison test).
Effect on spleen T-cell subtyping
The impact of compounds 2, 4 and 8 on T-cell subtyping (CD4+/CD8+) in spleen single cell
suspension was monitored by flow cytometry. For these investigations fluoresceine-
isothiocyanate (FITC) conjugated anti-CD4 and phycoerthyrin (PE) conjugated CD8
monoclonal antibodies were employed. The total amounts of CD4 and CD8 subtypes were
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calculated by multiplying their estimated ratios with the total spleen cell contents and the
results are enlisted in Table 2. In these studies compound 2 seems to be very effective
yielding 40.2% CD4+ and 21.6% CD8+ T cells at a dose of 0.1 mg/kg which is much better
than that observed for the control values of 20.7% of CD4+ and 13.3% of CD8+ T cells under
similar conditions. Even with an oral dose of 2.5 mg/kg Lev stimulated CD4+ and CD8+ T
cells only to the extent of 30.8% and 18.3% respectively.
Spleen CD4+
Spleen CD8+
Treatment
Dose
CD4/CD8
Ratio
CD4+ T-Cell
(%)
CD8+ T-Cell
(%)
Content
Content
(×107)
(mg/kg)
(×107)
Control
20.7 ± 0.90
13.3 ± 0.34
1.56 ± 0.09
2.70 ± 0.12
1.47 ± 0.04
(vehicle)
Levamisole
2.5
30.8 ± 1.30a 18.3 ± 0.53a 1.68 ± 0.06a 1.48 ± 0.10a 1.38 ± 0.10a
30.3 ± 0.58
34.2 ± 0.62
40.2 ± 0.70
20.3 ± 0.63
24.2 ± 0.59
31.1 ± 0.59
20.3 ± 0.58
27.2 ± 0.59
16.4 ± 0.37
19.8 ± 0.39
21.6 ± 0.46
15.4 ± 0.35
15.3 ± 0.38
18.4 ± 0.39
15.4 ± 0.34
16.2 ± 0.38b
1.32 ± 0.06
1.72 ± 0.06
1.86 ± 0.07
1.32 ± 0.07
1.58 ± 0.07
1.69 ± 0.07
1.32 ± 0.06
1.67 ± 0.06
1.77 ± 0.07
1.23 ± 0.06
1.61 ± 0.06
1.85 ± 0.07
1.02 ± 0.07
1.31 ± 0.06
1.51 ± 0.06
1.16 ± 0.05
1.26 ± 0.03
1.35 ± 0.03
1.48 ± 0.04
0.88 ± 0.04
1.18 ± 0.03
1.37 ± 0.03
1.19 ± 0.03
0.001
Compound
0.01
0.1
2
0.001
Compound
0.01
0.1
4
0.001
Compound
1.54 ± 0.06b 1.29 ± 0.03b
1.67. ± 0.07 1.41 ± 0.04b
0.01
0.1
8
35.3 ± 0.64b 19.9 ± 0.42b
Table 2: Effect of different doses of compounds 2, 4 and 8on spleen T cell subtypes
Number of observation =6
(a) P < 0. 01; (b) P < 0.05
Effect on cytokine release (IL-2, IFN-γ& IL-4)
The impact of compounds 2, 4 and 8 on the release of cytokines IL-2, IFN-γ, and IL-4 was
monitored to point out their specific effects on cytokine profiles. The results presented as
Figure 5, indicate a dose dependent impact on the release of IL-2, IFN-γ, and IL-4 wherein
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IL-4 release was always the least one. Compound 2 seems to be the most effective followed
by compound 8. At a dose of 0.01 mg/kg the observed effect of compound 4 is comparable to
that of Lev while at dose of 0.1 mg/kg it proves to be more effective than the later.
Figure 5: The bars in the depicted graphs are a mean± SE of results from three different experiments
carried under similar conditions. Mouse spleen cells (2 × 106 cells/mL) were stimulated for 48 h with
and without (control) 2.5 µg/well Con-A in the presence of each of these compounds after which the
cell supernatant was collected and tested for IL-2, IFN- γ and IL-4, by employing commercially
available standard kits (Quantikine, R&D Systems).Impact of isoxazoline functionalized coumarins 2,
4 and 8 on IL-2, IFN-γ and IL-4 cytokine production at changing doses.
Effect on NO production (macrophage production)
To test the impact of compounds 2, 4 and 8 on macrophage production, NO production in
response to different doses (0.001, 0.01 and 0.1 mg/kg) of these compounds and the standard
under similar conditions was monitored. The statistically analysed results from these
experiments are depicted as Figure 6. As evident from the presented results, the NO
production (an index of macrophage production) in response to administration of 2, 4 and 8 is
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significantly more than that of standard, and the production increases with the increase in
dosage. Comparatively compound 2 seems to be the most active followed by 4 and 8.
Figure 6: Effect of compounds 2, 4 and 8 on NO production.Results are expressed in µM. Data are
*
mean ± SE of six animals. P < 0.05; **P < 0.01; ***P < 0.001 compared with control group
determined by one-way ANOVA (Bonferroni correction multiple comparison test).
Toxicity studies
The synthesised compounds were tested for possible cellular toxic effects on spleen cells by
MTT assay [42]. Our observations vis-a-vis toxicity studies suggest that even at a dose of as
high 100 µg/mL the test compounds do not exhibit any toxic effect after 72 h incubation.
Docking Results
In this study we carried out the molecular docking studies, in which Levamisole, compounds
2, 4 and 8 were docked into ATP binding site of Acetylcholine binding protein at 2.38
resolution (PDB code: 4MU1). The docking studies were performed with Glide (Grid-
Based Ligand Docking with Energetics) program [43, 44] incorporated in the Schrödinger
molecular modeling package (Schrödinger, LLC, New York, NY, 2015). The binding poses
found by maestro 9.0 for Levamisole, compounds 2, 4 and 8 are given in Figure 7 (also see
figure S1). All the four compounds were successfully docked into the active site of 4MU1.
On analyzing the docking poses the positioned compounds shows interactions with try 89 and
ser186. In addition, we have observed a relatively consistent interaction of carbonyl group
with trp 143. In order to validate the interaction results we superimposed our ligand with
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standard (levamisole) ligand and observed that our ligands and the levamisole shares similar
binding orientation against the 4MU1 (Figure 7 also see figure S2). Results from the docking
studies indicate that binding affinity of the most active compounds follows the order 2> 8 > 4,
and with almost similar binding affinity of R and S isomers for their respective enantiomers.
(a)
(b)
(c)
Figure 7: Binding interactions of (a) levamisole, (b) comp-2, with against acetylcholine
binding protein and (c) superimposition of compd-2 (green) with levamisole
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Discussion
Our main interests for the work presented in current MS were; 1) Designing of a simple
synthetic strategy for conjugation of isoxazolines and coumarins for the synthesis of novel
isoxazoline derivatized coumarins as novel immune potentiators. 2) To explore the SAR of
the resulting conjuagtes vis-à-vis their immunopotentiating potential. A major challenge in
the synthetic approaches involving the conjugation of isoxazoline and coumarin is to ensure
the stability of the chemically very sensitive functional groups present on each of these
counterparts [45-47]. To ensure the same we followed a simple low cost but efficient and
chemically less demanding strategy for the functionalization of hydroxyl coumarins with
isoxazolines. The synthetic scheme involved 1,3-dipolar cyclization between an
Allylatedcoumarin and in situ generated variedly substituted nitrile oxides. For all the
compounds synthesized in present work we observed the formation of only one regioisomer
(see Table 1), this suggests that nitrile oxide cycloaddition to allyliccoumarin is highly
regiospecific. For exploration of SAR in isoxazoline conjugates of 7-OHC as
immunopotentiators, correlation of their activity with the substitution pattern in the basic
skeleton seems quite informative. To decipher such information, for present work we
attempted varied type of substitutions on the aryl ring attached to isoxazoline from simple H,
electron donating (OCH3 and CH3) and electron withdrawing groups (F, Cl, Br and NO2) to
bulky aryl (naphthyl) groups for the synthesis of coumarin-isoxazoline conjugates.
In the biological experiments compounds (1-13) showed no or little toxicity in comparison to
the cell control and compounds 2, 4 and 8 showed excellent dose dependent Con-A
proliferative activity (Figure. 1). The activity observed for compounds 2, 4 and 8 under in
vitro conditions motivated us to test these for in vivo. In in vivo studies levamisole was used
as standard, different doses of compounds 2, 4, 8 and the standard were administered to the
animals and the blood collected at day 7 and 14 was tested for HA titre. Significant HA titre
observed at day 14 (Figure 2) after administration of compounds 2, 4, 8 suggests that these
compounds appreciably enhance the production of antibody against SRBC’s. Similarly these
compounds were also observed to enhance the secretion of cell-secreted cytokines, such as
IL-2, IL-4, and IFN-γ and splenocyte proliferation significantly more than the administration
of standard. The observations vis a vis immunophenotyping expressions like CD4 and CD8
(Table 2), DTH responses and NO production (Figure 3 and 6) also support observations
regarding the cytokine expression. The well-known synergism between the cytokines in iNOS
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expression [42], suggests that the induction of cytokines observed after treatment with
compounds 2, 4, 8 can be attributed to the release of NO. In the in vivo experiments,
compound 2 exhibited highest activity at all doses in comparison to control, levamisole and
compounds 4 and 8. At lower doses compound 4 showed activities almost similar to that of
standard; however at higher doses the activity was found to increase with increase in dosage
per kg body weight.
Our observations vis a vis the production of primary and secondary antibodies (Figure 2, IgG
> IgM) suggests that administration of compounds 2, 4, 8 leads to generation of a good
number of memory cells. The observations about the impact of compounds 2, 4 and 8 on
spleen T-cell sub typing and the relative CD4+ and CD8+ counts (Table 2) are in conformity
with their antibody response. The observed enhancement in CD4+ values for these
compounds clearly implies that the immunogenic response follows MHC-class II pathway.
It is important to mention that in addition to the electronic effects the substituents used for the
current investigations are also expected to change the lipophilicity and hence the membrane
permeation properties of the coumarins. To know the impact of the substituents on the
lipophilicity of coumarins, we estimated the Log P values for the compounds 1-13. The
estimated Log P values (Table S2 Supporting Information) were found to vary in the order
13>12>11>10>5>6>7>9>2>8>3>4>1. Relative Log P values of the investigated coumarins in
light of their observed immune potentiating activities clearly indicate that the substituent
effects of the attempted substitutions in the present study are dominated by the electronic
effects of the substituents.
It is pertinent to mention that the isoxazoline-coumarin conjugates presented in table 1,
possess one chiral centre in the isoxazoline ring thereby raising the possibility that derivatives
synthesized as per Scheme 1, would be a mixture of two possible enantiomers. In order to
investigate the impact of stereochemistry of bis-heterocyclic conjugates on their immune
potentiating potential, kinetic resolution of the racemate of the most active of all these
compounds viz. (2) was carried out following the steps depicted in Scheme 2. The lipase
showed excellent differential enantioselectivity in catalysing the hydrolysis of racemic ester.
It was observed that the enzyme in presence of S-isomer selectively hydrolysed the R-
counterpart without affecting S-isomer, producing the corresponding enantiopure alcohol with
high enantiomeric excesses (>99% ee) in accordance with the Kazlauskas rule. However, in
the absence of R-counterpart, the ester with S-configuration at chiral carbon in the ring is
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slowly hydrolysed to the corresponding enantiopure alcohol with high enantiomeric excesses
(>99% ee). The enantiomeric excesses of acetate (S)-2c, alcohols [(S)-2d, (R)-2d] and bromo
compounds [(S)-2e, (R)-2e] were determined by chiral column chromatography. The absolute
configurations of compounds (S)-2c,(S)-2d, (R)-2d, (S)-2e and (R)-2e were determined
comparing the specific rotation signs measured for the products with that reported in the
literature [48-51]. The spectroscopic data (1H-NMR, MS and IR) and melting point of
compounds 2c, 2d and 2e matches well with the data reported in the literature [49, 51-52].
The known absolute configuration of enantiomers (S)-2d, (R)-2d, (S)-2e and (R)-2e provided
the way to determine the absolute configurations of the bis-heterocycles [(R)-2 and (S)-2].
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Scheme 2: Stereoselective synthesis of R and S enantiomers of bis-hetrocycle (2)
Both the compounds [(R)-2 and (S)-2] as well as their racemate were explored for their in
vitro lymphocyte proliferation potential. It was observed that both the tested enantiomers as
well as their racemate show excellent dose-related increase of titre (Figure 8) even at lower
doses (0.1 µg/mL) in comparison to levamisole. It was noticed that the activity of the
enantiomers in their pure form as well as their racemate form is almost similar at all doses.
We attribute this stereo-independent biological activity of the tested bis-heterocycles to their
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flexible molecular structure. On prima facie the results from the in vitro lymphocyte
proliferation studies, the toxicity studies of the racemate and the docking studies on the R and
S isomers of compound 2 indicate that these isomers can be expected to exhibit similar
stereo-independent activity under in vivo conditions, however a thorough investigation on the
toxicity of these isomers needs to be carried out to support the said claim.
Figure 8: Effect of enatiomers for compound 2 in [(R)-2, (S)-2] and their racemate on lymphocyte
proliferation. The depicted bars present mean
± S.E. of three observations. The proliferation was
calculated based on MTT assay. Absorbance was recorded at 570 nm. Values are expressed as.
*P < 0.05; **P < 0.01; ***P < 0.001 as compared to control determined by one-way Anova (Bonferroni
correction multiple comparison test).
Analysis of the results from biological experiments in light of the structure activity relations
(SAR) points to some interesting conclusions about the immune potentiating activity of
coumarin based immunopotentiators. The immune-stimulating potential of isoxazoline
functionalized coumarins synthesized for the present study is evidently more than that
observed for the control. The results of preliminary assays and structures of synthesized
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conjugates indicate that isoxazolines possessing electron donating groups on the aryl ring (2-
8) are stronger immunopotentiators (4-OCH3> 2,4-OCH3> 2-OCH3 > 3-OCH3 > 4-CH3 > 2-
CH3> 3-CH3) in comparison to those possessing electron withdrawing groups (9-12) (4-Cl >
4-Br > 4-F ≈ 4-NO2). The results further indicate that the immunostimulatory activity is
enhanced by increasing dosage of electron rich isoxazoline derivatives. Introducing a bulky
aryl (naphthyl) group (13) decreases the immnostimulating potential with increase in dose.
This may be attributed to high molecular mass and overcrowding of aryl rings, which
probably inhibit the effective interaction of the pharmacophore with its corresponding binding
receptors. The attachment of 7-OHC at 5th position via an O-allyl intermediate formation on
the isoxazoline ring together with appropriately functionalized aromatic ring at the 3rd
position of isoxazoline ring seems to impart immune-enhancing activity to the molecule
(compounds 2, 4 and 8).
Coumarins are reported to interact with ubiquitous intracellular receptor proteins through
binding of their aromatic hydrocarbons [53]. From the data observed for present study it can
be inferred that electron donating groups at ortho or para positions with respect to
isoxazoline ring increase the overall electron density and hence lead to their stronger
interaction with the receptor binders. The possible single bond rotations between an aryl ring
and isoxazoline ring in the conjugates ensure that presence of later does not impede the
binding of coumarin unit to its relevant receptors. The presence of electron donating groups
seems to facilitate the kinetics and thermodynamics for the binding of these conjugates with
the relevant intracellular receptor proteins.This is well in agreement with the results we
obtained in the current study. Compounds with OCH3 and CH3 groups on the phenyl rings
ortho/para to isoxazoline ring (compounds 2, 4, 5 and 7) displayed significantly higher
activity than OCH3 and CH3 groups meta to isoxazoline ring (compounds 3 and 6). Conjugate
occupied with OCH3 group at both ortho and para positions with respect to isoxazoline ring
(compound 8) also resulted in better activity. The difference observed in activity of 2 and 4
may be attributed to the fact that compound 2 being para substituted is more symmetrical and
stable, that leads to its better interaction with the receptor binders in comparison to compound
4. In contrast, compound 8 being slightly bulkier and less symmetrical than 2 may show
lesser interaction with the receptors. This is further supported by the results from the docking
studies where binding affinity and Log P value of 2 was observed to be higher than that of
compound 4 and 8.
20

ACCEPTED MANUSCRIPT
The results from biological activity experiments suggest that compounds 2, 4 and 8 are a
good addition to the library of till date known immunostimulators that need to be tested for
their prophylactic and therapeutic potential. Results from our present work suggest that
isoxazoline functionalized coumarins can serve as promising lead compounds for the design
of clinically safe and effective immunepotentiators. In view of the short half-life of the
coumarins, it seems that multiple oral or intravenous administrations of these compounds may
be required for threshold saturation of the relevant receptors for the optimal activation of
lymphocytes and macrophages.
Conclusion
A simple synthetic scheme was attempted for the synthesis of novel isoxazoline
functionalized coumarins. The kinetic resolution of the racemate was carried out by
employing lipase B from Candida antarctica (CALB). The synthesized compounds when
tested for their immune potentiating activity exhibited appreciable activity, especially
compounds 2, 4 and 8.The observed immune potentiating activity for compounds 2, 4 and 8
in our opinion makes them a promising addition to the library of till date known
immunostimulators that need to be tested for their prophylactic and therapeutic potential. The
results from our presented work suggest that isoxazoline functionalized coumarins can serve
as useful immnopotentiators whose thearupatic efficacy can be tuned through appropriate
substitutions in the fused heterocyclic moieties.
Experimental
Chemistry
All solvents were dried and freshly distilled prior to use. IR spectra were recorded on a
1
Bruker Vector 22 instrument using KBr pellets and in CHCl3. H and 13C NMR were
recorded on a Bruker DPX 500 instrument in CDCl3 using TMS as internal standard for
protons. Mass spectra were recorded on ESI-esquire 3000 Bruker Daltonics instrument and
MALDI TOF-TOF. Elemental analysis was carried out using Elemental Vario EL III
elemental analyser. Chemicalshift values are mentioned in δ (ppm) and coupling constants (J)
are given in Hz. Mass-spectrometric (MS) data is reported in m/z. Elemental analysis data is
reported in % standard. The progress of all reactions was monitored by TLC on 2 cm × 5 cm
pre-coated silica gel 60 F254 plates of thickness 0.25 mm (Merck). The chromatograms were
21

ACCEPTED MANUSCRIPT
visualized under UV 254-366 nm and iodine. Melting points were determined on Buchi B-
542 apparatus by an open capillary method and are uncorrected. Optical rotations were
measured on Perkin-Elmer 241 polarimeter at 25 °C using sodium D light. Enantiomeric
excess (ee%) was determined by chiral HPLC on OJH and ADH chiral columns. Chemicals
were purchased from M/s Aldrich Chemicals, Mumbai. Reagents and solvents used were
mostly of LR grade.
7-(Allyloxy)-2H-chromen-2-one (1c):
In a typical procedure, 1b (0.50 g, 3.08 mmol) was dissolved in dry acetone (20 mL). K2CO3
(4.26 g, 30.85 mmol) was added and the reaction mixture stirred for 5 min at ambient
temperature. 1a (0.44 g, 3.70 mmol) was charged to the above reaction mixture and the
reaction mixture allowed to reflux for 3 h. After completion of the reaction (monitored by
TLC), the reaction mixture was allowed to attain room temperature, filtered and the filtrate
concentrated under vacuum to afford 1c in pure form (100%).
1H NMR (500 MHz, CDCl3): δ 4.61 (d, 2H, J = 5.27 Hz), 5.31-5.37 (m, 1H), 5.40-5.49 (m,
1H), 5.96-6.12 (m, 1H), 6.26 (d, 1H, J = 9.48 Hz), 6.86 (d, 1H, J = 9.58 Hz), 6.89 (s, 1H),
7.38 (d, 1H, J = 8.36 Hz), 7.64 (d, 1H, J = 9.49 Hz); Mass (ESI-MS): 224.8 (M+ + Na); C, H
analysis for C12H10O3: Calculated C, 71.28; H, 4.98. Found C, 71.24; H, 4.93.
Phenylhydroxymoyl chloride (1d):
Phenylhydroxymoyl chloride was synthesized as per the literature procedure [54]. In a typical
procedure, NH2OH.HCl (0.50 g, 7.22 mmol) was dissolved in water and neutralized with
NaOH. To the neutralized solution of hydroxylamine hydrochloride, benzaldehyde (1.00 g,
9.43 mmol) was added and the reaction mixture stirred for 1 h at ambient temperature. After
completion of the reaction (monitored by TLC), excess of water was added to the reaction
mixture and the organic compound extracted with EtOAc (2 × 50 mL). The combined organic
layers were dried over anhydrous Na2SO4 and concentrated under vacuum to afford pure
oxime in 99% yield. Benzaldoxime (1.00 g, 6.42 mmol) was dissolved in DMF (20 mL). N-
chlorosuccinimide (0.95 g, 7.18 mmol) was added to the above solution and the reaction
mixture stirred for 8-10 h. After completion of the reaction (monitored by TLC), excess of
water was added to the reaction mixture and organic compound extracted with Et2O (3 × 50
mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated
under vacuum to afford pure 1d (74%).
22

ACCEPTED MANUSCRIPT
1H NMR (200 MHz, CDCl3): δ 7.42 (m, 3H), 7.85 (m, 2H); Mass (ESI-MS): 155.58 (M+); C,
H, N analysis for C7H6ClNO: Calculated C, 54.04; H, 3.89; N, 9.00. Found C, 53.98; H, 4.00;
N, 9.11.
7-{(3-Phenyl-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (1):
In a typical procedure, 1d (0.10 g, 0.04 mmol) was dissolved in THF (4 mL) and to it was
added Et3N (0.02 g, 0.02 mmol). A solution of 1c (0.05 g, 0.02 mmol) in THF (4 mL) was
added to the above solution. The reaction mixture was stirred at ambient temperature for 4 h.
After completion of the reaction (monitored by TLC), the reaction mixture was diluted with
80 mL of water and extracted with EtOAc (2 × 30 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under vacuum to afford crude product which
was subjected to column chromatography [silica gel 230-400 mesh as stationary phase,
hexane: EtOAc; (7:3) as mobile phase] to yield the pure 7-{(3-Phenyl-4,5-dihydroisoxazol-
5-yl)methoxy}-2H-chromen-2-one (1) as a white solid (90%).
o
1
White solid; mp: 132-133 C; H NMR (500 MHz, CDCl3): δ 3.39-3.42 (dd, 1H, J1 = 15.54
Hz, J2 = 6.93 Hz), 3.54-3.59 (dd, 1H, J1 = 15.55 Hz, J2 = 10.82 Hz), 4.16-4.22 (m, 2H), 5.04-
5.09 (m, 1H), 6.15 (d, 1H, J = 8.78 Hz), 6.27 (d, 1H, J = 8.46 Hz), 6.83 (s, 1H), 6.86 (m,
3H), 7.38 (d, 1H, J = 8.56 Hz), 7.63 (d, 1H, J = 9.47 Hz), 7.71 (d, 2H, J = 9.61 Hz); 13C NMR
(500 MHz, CDCl3): δ 37.50, 69.03, 78.35, 101.75, 112.80, 113.05, 113.56, 126.79, 128.81,
128.89, 129.11, 129.57, 130.39, 143.29, 155.74, 156.42, 161.06, 161.50, 176.86; IR (KBr,
cm-1): 693, 761, 853, 913, 1124, 1230, 1280, 1355, 1402, 1448, 1507, 1558, 1614, 1723,
2358, 2853, 2924, 3386. ESI-MS: 321.8 (M+); C, H, N analysis for C19H15NO4: Calculated C,
71.02; H, 4.71; N, 4.36. Found C, 71.04; H, 4.74; N, 4.30.
7-{(3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (2):
o
1
White solid; mp:140-141 C; H NMR (500 MHz, CDCl3): δ 3.25-3.29 (dd, 1H, J1 = 16.45
Hz, J2 = 6.82 Hz), 3.42-3.47 (dd, 1H, J1 = 16.65 Hz, J2 = 10.75 Hz), 3.88 (s, 3H), 4.06-4.09
(m, 1H), 4.12-4.15 (dd, 1H, J1 = 9.75 Hz, J2 = 4.98 Hz), 5.05-5.08 (dd, 1H, J1 = 10.93 Hz, J2 =
5.44 Hz), 6.20 (d, 1H, J = 9.47 Hz),6.75 (s, 1H), 6.79 (d, 1H, J = 8.56 Hz), 6.90 (d, 2H, J =
8.64 Hz), 7.31 (d, 1H, J = 8.52 Hz), 7.52 (d, 1H, J = 8.46 Hz), 7.56 (d, 2H, J = 9.52 Hz); 13C
NMR (500 MHz, CDCl3): δ 39.69, 55.72, 69.10, 79.15, 101.77, 112.83, 113.07, 113.57,
114.98, 117.69, 124.20, 128.90, 129.22, 131.41, 143.29, 155.78, 156.56, 158.29, 161.04,
161.56; IR (KBr, cm-1): 669, 810, 837, 897, 1022, 1063, 1095, 1126, 1232, 1275, 1356, 1402,
23

ACCEPTED MANUSCRIPT
1507, 1558, 1615, 1650, 1714, 2337, 2361, 2852, 3389; MALDI-mass: 352 (M+ + H); C, H,
N analysis for C20H17NO5: Calculated C, 68.37; H, 4.88; N, 3.99. Found C, 68.32; H, 4.85; N,
3.94.
7-{(3-(3-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (3):
White solid; mp: 96-97 oC; 1H NMR (500 MHz, CDCl3): δ 3.48-3.51 (dd, 1H, J1 = 16.50 Hz,
J2 = 6.93 Hz), 3.65-3.69 (dd, 1H, J1 = 14.64 Hz, J2 = 10.54 Hz), 3.82 (s, 3H), 4.10-4.14 (m,
2H), 5.13-5.16 (dd, 1H, J1 = 11.68 Hz, J2 = 6.72 Hz), 6.27 (d, 1H, J = 9.47 Hz), 6.85-6.95
(m, 3H), 7.20 (d, 1H, J = 8.56 Hz), 7.30-7.41 (m, 3H), 7.64 (d, 1H, J = 9.50 Hz);13C NMR
(500 MHz, CDCl3): δ 37.81, 55.39, 69.18, 78.11, 101.85, 112.79, 113.35, 114.25, 114.83,
115.44, 124.53, 128.35, 128.89, 129.40, 143.26, 155.10, 155.97, 160.34, 161.31, 161.61; IR
(KBr, cm-1): 759, 835, 908, 1027, 1124, 1231, 1291, 1377, 1402, 1462, 1507, 1557, 1613,
1728, 2360, 2850, 2921, 2955, 3416; Mass (ESI-MS): 374 (M+ + Na);C, H, N analysis for
C20H17NO5: Calculated C, 68.37; H, 4.88; N, 3.99. Found 68.39; H, 4.84; N, 3.91.
7-{(3-(2-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (4):
White solid; mp: 142-143 oC;1H NMR (500 MHz, CDCl3): δ 3.41-3.44 (dd, 1H, J1 = 16.56
Hz, J2 = 7.05 Hz), 3.62-3.67 (dd, 1H, J1 = 16.00 Hz, J2 = 10.80 Hz), 3.86 (s, 3H), 4.12-4.18
(m, 2H), 5.08 (m, 1H), 6.27 (d, 1H, J = 9.45 Hz), 6.83 (s, 1H), 6.86-6.89 (m, 2H), 7.26-7.39
(m, 3H), 7.64 (d, 1H, J = 9.50 Hz), 7.74 (d, 1H, J = 9.40 Hz); 13C NMR (500 MHz, CDCl3):δ
39.80,55.95, 69.26, 78.67, 101.75, 111.82, 112.76, 112.89, 113.53, 117.88, 121.11, 128.93,
129.09, 131.12, 143.39, 155.10, 155.97, 160.34, 161.31, 161.61; IR (KBr, cm-1): 755, 808,
835, 891, 1023, 1096, 1124, 1231, 1277, 1340, 1401, 1460, 1487, 1556, 1614, 1727, 2284,
2853, 2924, 3405; Mass (ESI-MS): 374 (M+ + Na); Anal Calcd. for C20H17NO5: Calculated C:
68.37, H: 4.88, N: 3.99; found C: 68.33, H: 4.82, N: 3.93.
7-{(3-p-Tolyl-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (5):
o
1
White solid; mp: 158-159 C; H NMR (500 MHz, CDCl3): δ 2.39 (s, 3H), 3.38-3.39 (m,
2H), 3.49-3.54 (dd, 1H, J1 = 15.15 Hz, J2 = 10.86 Hz), 4.14-4.17 (dd, 1H, J1 = 12.39 Hz, J2 =
4.94 Hz), 5.13-5.14 (m, 1H), 6.27 (d, 1H, J = 9.50 Hz), 6.83 (s, 1H), 6.87 (d, 1H, J = 8.60
Hz), 7.23 (d, 2H, J = 7.62 Hz), 7.38 (d, 1H, J = 8.59 Hz), 7.59 (d, 2H, J = 7.08 Hz), 7.69 (d,
1H, J = 8.78 Hz); 13C NMR (500 MHz, CDCl3): δ 20.44, 36.63, 68.11, 77.17, 100.78,
111.77, 112.03, 112.52, 125.28, 125.77, 125.79, 127.85, 128.22, 128.48, 139.65, 142.35,
154.74, 155.34, 160.02, 160.54; IR (KBr, cm-1): 634, 818, 837, 887, 920, 986, 1027, 1123,
24

ACCEPTED MANUSCRIPT
1184, 1231, 1280, 1532, 1394, 1448, 1506, 1616, 1710, 2049, 2854, 2922, 3444; Mass (ESI-
MS): 357.9 (M+ + Na);C, H, N analysis for C20H17NO4: Calculated C, 71.63; H, 5.11; N,
4.18. Found C, 71.66; H, 5.15; N, 4.14.
7-{(3-m-Tolyl-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (6):
White solid; mp: 133-134 oC; 1H NMR (500 MHz, CDCl3): δ 2.34 (s, 3H), 3.48-3.51 (dd, 1H,
J1 = 11.56 Hz, J2 = 6.63 Hz), 3.65-3.69 (dd, 1H, J1 = 14.49 Hz, J2 = 10.07 Hz), 4.20 (m, 2H),
5.13-5.18 (dd, 1H, J1 = 12.01 Hz, J2 = 5.27 Hz),6.27 (d, 1H, J = 9.41 Hz), 6.84 (s, 1H), 6.90-
13
6.95 (m, 3H), 7.19-7.41 (m, 3H), 7.64 (d, 1H, J = 9.36 Hz); C NMR (500 MHz, CDCl3):
δ23.96, 37.36, 69.10, 78.74, 101.85, 112.85, 113.03, 113.65, 127.80, 128.06, 128.33, 129.05,
129.15, 134.65, 141.33, 143.30, 160.77, 161.38; IR (KBr, cm-1): 694, 757, 787, 835, 912,
1025, 1123, 1158, 1231, 1280, 1349, 1401, 1507, 1615, 1711, 2109, 2850, 2918, 3441; Mass
(ESI-MS): 358 (M+ + Na);C, H, N analysis for C20H17NO4: Calculated C, 71.63; H, 5.11; N,
4.18. Found C, 71.64; H, 5.19; N, 4.20.
7-{(3-o-Tolyl-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (7):
White solid; mp: 92-93 oC; 1H NMR (500 MHz, CDCl3): δ 2.51 (s, 3H), 3.00-3.04 (dd, 1H,
J1 = 15.32 Hz, J2 = 9.71 Hz), 3.32-3.36 (dd, 1H, J1 = 16.42 Hz, J2 = 7.41 Hz), 4.18-4.20 (m,
2H), 5.16-5.19 (dd, 1H, J1 = 12.15 Hz, J2 = 5.60 Hz), 6.26 (d, 1H, J = 9.22 Hz), 6.83 (s, 1H),
7.25-7.44 (m, 4H), 7.60-7.69 (m, 3H); 13C NMR (500 MHz, CDCl3): δ 19.57, 37.50, 69.01,
78.41, 101.79, 112.78, 113.10, 113.61, 122.56, 123.03, 126.46, 128.69, 128.89, 143.24,
155.77, 161.00, 161.49; IR (KBr, cm-1): 615, 718, 759, 835, 892, 989, 1031, 1123, 1230,
1279, 1347, 1377, 1400, 1456, 1493, 1507, 1556, 1613, 1726, 2110, 2853, 2869, 2924,
2956, 3384; Mass (ESI-MS): 357.9 (M+ +Na); C, H, N analysis for C20H17NO4: Calculated
C, 71.63; H, 5.11; N, 4.18. Found C, 71.61; H, 5.17; N, 4.13.
7-{(3-(2,4-Dimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one
(8):
White solid; mp: 141-142 oC; 1H NMR (500 MHz, CDCl3): δ 3.55-3.59 (dd, 1H, J1 = 13.34
Hz, J2 = 6.76 Hz), 3.72-3.76 (dd, 1H, J1 = 14.65 Hz, J2 = 10.74 Hz), 3.90 (s, 3H), 3.91 (s,
3H), 4.17 (m, 1H), 4.18-4.21 (dd, 1H, J1 = 12.46 Hz, J2 = 5.20 Hz), 5.14-5.16 (m, 1H), 6.27
(d, 1H, J = 9.46 Hz),6.84 (s, 1H), 6.85- 6.90 (m, 3H), 7.27 (d, 1H, J = 8.22 Hz), 7.39 (d, 1H,
J = 8.58 Hz), 7.64 (d, 1H, J = 9.48 Hz); 13C NMR (500 MHz, CDCl3): δ 39.84, 56.27, 69.16,
79.15, 101.74, 112.19, 112.89, 113.16, 113.54, 113.90, 124.20, 128.93, 129.30, 143.38,
25

ACCEPTED MANUSCRIPT
155.78, 156.39, 156.56, 158.29,161.04, 161.56; IR (KBr, cm-1): 664, 792, 841, 868, 934,
990, 1052, 1129, 1161, 1172, 1210, 1232, 1255, 1281, 1430, 1457, 1518, 1548, 1618, 1735,
2049, 2927, 3452; Mass (ESI-MS): 381 (M+); C, H, N analysis for C21H19NO6: Calculated
C, 66.14; H, 5.02; N, 3.67. Found C, 66.19; H, 5.06; N, 3.62.
7-{(3-(4-Fluorophenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (9):
White solid; mp: 114-115 oC; 1H NMR (500 MHz, CDCl3): δ 3.34-3.39 (dd, 1H, J1 = 17.21
Hz, J2 = 7.04 Hz), 3.51- 3.56 (m, 1H), 4.15-4.18 (dd, 1H, J1 = 9.86 Hz, J2 = 4.84 Hz), 4.21-
4.23 (dd, 1H, J1 = 9.68 Hz, J2 = 4.75 Hz), 5.14-5.17 (m, 1H), 6.26 (d, 1H, J = 9.44 Hz), 6.82
(s, 1H), 6.85 (d, 1H, J = 8.54 Hz), 7.12-7.15 (m, 1H), 7.37-7.46 (m, 4H), 7.63 (d, 1H, J =
9.44 Hz); 13C NMR (500 MHz, CDCl3): δ 38.71, 70.38, 80.18, 103.16, 114.26, 114.55,
115.00, 115.05, 118.71, 124.02, 130.38, 131.88, 131.94, 144.76, 157.07, 157.18, 162.51,
162.87, 165.22; IR (KBr, cm-1): 615, 718, 759, 835, 892, 989, 1031, 1123, 1230, 1279,
1347, 1377, 1400, 1456, 1493, 1507, 1556, 1613, 1726, 2110, 2853, 2869, 2924, 2956,
3384; Mass (ESI-MS): 361.8 (M+ + Na); C, H, N analysis for C19H14FNO4: Calculated C,
67.25; H, 4.16; N, 4.13. Found C, 67.22; H, 4.19; N, 4.18.
7-{(3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (10):
White solid; mp: 111-112oC; 1H NMR (500 MHz, CDCl3): δ 3.27-3.32 (dd, 1H, J1 = 17.93
Hz, J2 = 7.08 Hz), 3.44-3.50 (dd, 1H, J1 = 19.04 Hz, J2 = 10.87 Hz), 3.53 (m, 1H), 4.08-4.11
(dd, 1H, J1 = 10.12 Hz, J2 = 4.93 Hz), 5.13-5.15 (m, 1H), 6.12 (d, 1H, J = 8.95 Hz), 6.75 (s,
13
1H), 6.81 (d, 1H, J = 9.48 Hz), 7.30-7.34 (m, 3H),7.56-7.58 (m, 3H); C NMR (500 MHz,
CDCl3): δ 37.02, 68.74, 78.40, 101.85, 111.56, 112.85, 113.65, 127.37, 128.43, 128.63,
128.81, 128.90, 129.22, 131.41, 143.00, 155.10, 155.97, 160.34, 161.31; IR (KBr, cm-1): 666,
760, 919, 1049, 1093, 1159, 1230, 1351, 1402, 1558, 1615, 1650, 1714, 2360, 2853, 2924,
3416, 3672; Mass (ESI-MS): 355.3 (M+); C, H, N analysis for C19H14ClNO4: Calculated C,
64.14; H, 3.97; N, 3.94. Found C, 64.12; H, 3.93; N, 3.91.
7-{(3-(4-Bromophenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (11):
Brown solid; mp: 134-135 oC; 1H NMR (500 MHz, CDCl3):δ 3.28-3.32 (dd, 1H, J1 = 13.95
Hz, J2 = 5.93 Hz), 3.50-3.56 (dd, 1H, J1 = 18.45 Hz, J2 = 11.04 Hz), 3.64 (m, 1H), 4.26 (m,
1H), 5.26 (m, 1H), 6.29 (d, 1H, J = 9.17 Hz), 6.90 (s, 1H), 6.95 (d, 1H, J = 6.79 Hz), 7.17
(m, 2H), 7.46 (d, 2H, J = 8.43 Hz), 7.74 (d, 2H, J = 9.15 Hz); 13C NMR (500 MHz, CDCl3):
δ 36.23, 67.89, 77.69, 100.67, 111.78, 112.51, 112.56, 116.22, 116.39, 121.55, 128.89,
26

ACCEPTED MANUSCRIPT
129.39, 129.45, 142.27, 158.43, 159.64, 163.70, 164.24; IR (KBr, cm-1): 683, 835, 896,
1032, 1125, 1158, 1231, 1281, 1351, 1403, 1512, 1556, 1614, 1726, 2361, 2852, 2923,
3411; Mass (ESI-MS): 401 (M+ + H); C, H, N analysis for C19H14BrNO4: Calculated C,
57.02; H, 3.53; N, 3.50. Found C, 57.10; H, 3.58; N, 3.52.
7-{(3-(4-Nitrophenyl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (12):
Yellow solid; mp: 130-131 oC; 1H NMR (500 MHz, CDCl3): δ 3.40-3.45 (dd, 1H, J1 = 17.01
Hz, J2 = 7.25 Hz), 3.55-3.61 (dd, 1H, J1 = 19.21 Hz, J2 = 10.71 Hz), 4.23 (m, 2H), 5.20-5.29
(m, 1H), 6.28 (d, 1H, J = 9.48 Hz), 6.83 (s, 1H), 7.39 (d, 1H, J = 8.20 Hz), 7.60-7.67 (m,
13
2H), 8.13 (d, 2H, J = 7.85 Hz), 8.30 (d, 2H, J = 7.40 Hz); C NMR (500 MHz, CDCl3): δ
37.68, 73.35, 76.92, 101.44, 102.20, 109.68, 109.75, 115.05, 122.73, 126.83, 127.17,
132.58,136.99, 137.01, 151.39, 151.44, 157.30; IR (KBr, cm-1): 679, 738, 854, 983, 1097,
1191, 1236, 1298, 1345, 1404, 1490, 1523, 1533, 1557, 1623, 1730, 2058, 2927, 3450; Mass
(ESI-MS): 389 (M+ + Na); C, H, N analysis for C19H14N2O6: Calculated C, 62.30; H, 3.85;
N, 7.65. Found C, 62.38; H, 3.82; N, 7.69.
7-{(3-(Naphthalen-1-yl)-4,5-dihydroisoxazol-5-yl)methoxy}-2H-chromen-2-one (13):
White solid; mp: 119-120 oC; 1H NMR (500 MHz, CDCl3): δ 3.56-3.60 (dd, 1H, J1 = 13.86
Hz, J2 = 6.13 Hz), 3.75-3.80 (dd, 1H, J1 = 15.32 Hz, J2 = 10.92 Hz), 4.11-4.24 (m, 2H), 5.16
(m, 1H), 6.26 (d, 1H, J = 9.15 Hz), 6.84 (s, 1H), 7.36 (d, 1H, J = 8.04 Hz), 7.50 (d, 1H, J =
13
7.46 Hz), 7.56-7.63 (m, 4H), 7.89-7.93 (m, 3H), 8.99 (d, 1H, J = 8.01 Hz); C NMR (500
MHz, CDCl3): δ 37.45, 69.30, 77.31, 104.44, 115.47, 115.72, 116.19, 126.49, 126.80,
127.45, 128.90, 129.15, 129.60, 130.30, 130.42, 133.31, 133.72, 136.68, 145.95, 158.43,
159.64, 163.70, 164.24; IR (KBr, cm-1): 615, 751, 775, 800, 834, 1026, 1122, 1230, 1279,
1348, 1401, 1508, 1612, 1724, 2341, 2361, 2852, 2923, 2955, 3423; Mass (ESI-MS): 394
(M+ + Na); C, H, N analysis for C23H17NO4: Calculated C, 74.38; H, 4.61; N, 3.77. Found C,
74.32; H, 4.67; N, 3.74.
{3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl}methanol (2b):
In a typical procedure, 2a (0.20 g, 1.07 mmol) was dissolved in THF (5 mL) and to it was
added Et3N (0.054 g, 0.534 mmol) at 0 ºC. A solution of allyl alcohol (0.062 g, 1.07 mmol)
in THF (4 mL) was added to the above solution. The reaction mixture was stirred for 8 h
between 0-5 ºC. After completion of the reaction (monitored by TLC), the reaction mixture
was diluted with 60 mL of water and extracted with EtOAc (2 × 30 mL). The combined
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ACCEPTED MANUSCRIPT
organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to afford
crude product which was subjected to column chromatography [silica gel 230-400 mesh as
stationary phase, hexane: EtOAc; (7:3) as mobile phase] to yield the pure racemic {3-(4-
methoxyphenyl)-4,5-dihydroisoxazol-5-yl}methanol (90%).
o
1
White solid; mp: 166-167 C; H NMR (200 MHz, CDCl3): δ 3.18-3.31 (m, 2H), 3.57-3.63
(dd, 1H, J1 = 6.00 Hz, J2 = 4.00 Hz), 3.74-3.80 (m, 1H), 3.87 (s, 3H), 4.76-4.82 (dd, 1H, J1 =
10.00 Hz, J2 = 8.00 Hz), 6.85 (d, 2H, J = 8.00 Hz), 7.47 (d, 2H, J = 8.00 Hz); IR (KBr, cm-1):
626, 707, 814, 906, 955, 1018, 1063, 1178, 1258, 1274, 1300, 1343, 1415, 1459, 1508, 1560,
1604, 2849, 2925, 3391; Mass (ESI-MS): 208 (M+ + H); C, H, N analysis for C11H13NO3:
Calculated C, 63.76; H, 6.32; N, 6.76. Found C, 63.62; H, 6.41; N, 6.69.
{3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl}methyl acetate (2c):
2b (0.19 g, 0.917 mmol) was dissolved in DCM (5 mL). Acetic anhydride (0.093 g, 0.917
mmol) was charged to the above solution followed by the addition of DMAP (catalytic
amount). The reaction mixture was then allowed to stir at ambient temperature for 3 h. After
completion of the reaction (monitored by TLC), the reaction mixture was quenched with
NaHCO3 solution and extracted with DCM (2 × 50 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under vacuum. The semisolid left behind
was recrystallized from hexane-ethyl acetate to yield racemic compound 2c in pure form
(100%).
White solid; mp: 82-83 oC; 1H NMR (200 MHz, CDCl3): δ 2.02 (s, 3H), 2.98-3.09 (dd, 1H, J1
= 14 Hz, J2 = 8.00 Hz), 3.39-3.38 (dd, 1H, J1 = 8.00 Hz, J2 = 6.00 Hz), 3.74-3.78 (m, 1H),
3.88 (s, 3H), 4.16-4.19 (m, 1H), 4.83-4.94 (dd, 1H, J1 = 14 Hz, J2 = 10.00 Hz), 6.87 (d, 2H, J
= 8.00 Hz), 7.49 (d, 2H, J = 10 Hz); IR (KBr, cm-1): 626, 706, 816, 904, 1045, 1064, 1103,
1175, 1249, 1274, 1301, 1344, 1362, 1441, 1462, 1510, 1605, 1742, 2855, 2930, 3458; Mass
(ESI-MS): 288 (M+ + K); C, H, N analysis for C13H15NO4: Calculated C, 62.64; H, 6.07; N,
5.62. Found C, 62.59; H, 6.17; N, 5.33.
Typical lipase-catalyzed kinetic resolution of (±)-2c:
The racemic acetate (.100 g, .401 mmol), aqueous phosphate buffer (4 mL, 0.1 M, pH. 7.0),
toluene (300 µL) and immobilized lipase B from Candida antarctica (80 mg) were shaken
(150 rpm) continuously at 25 ± 1 °C for 7 min. After a certain degree of conversion ( 50%)
as indicated by high performance liquid chromatography (HPLC), the reaction was terminated
by adding ethyl acetate and centrifuging the mixture at 10,000–15,000 g to remove the
28