Design and synthesis of coumarin-glyoxal hybrids for spermicidal and antimicrobial actions: A dual approach to contraception

Article abstract of DOI:10.1039/c6ra12156j

Today there is an urgent need for safe and effective dual-purpose contraceptive agents, which can simultaneously prevent unintended pregnancies and sexually transmitted infections (STI). There are several naturally occurring antimicrobial and antibiotic drugs (novobiocin, coumermycin and chlorobiocin) reported in the literature, which are based on 4-hydroxy coumarins as the active pharmacophore. Based on these interesting reports, we designed and synthesized a library of new 4-hydroxy coumarin derivatives and evaluated them for spermicidal activity. Among the tested compounds, two compounds (2a and 2d) displayed better activity (greater than 95% sperm immobilization at 0.5 mM concentration) than the positive control nonoxynol-9 (N-9). Furthermore, all the compounds were screened for antimicrobial activity against different strains of Trichomonas vaginalis and two compounds (2c and 2h) exhibited potent activity as compared to the reference drug metronidazole. The cytotoxicity assay showed that most of these compounds were safer than the N-9 against the human cervical HeLa cell line and normal vaginal flora Lactobacillus jensenii strains. The studies have demonstrated that compound (2a) is a potential lead to develop a dually active vaginal contraceptive.

Full text of DOI:10.1039/c6ra12156j

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Design and synthesis of coumarin–glyoxal hybrids
for spermicidal and antimicrobial actions: a dual
approach to contraception†
Cite this: RSC Adv., 2016, 6, 76288
a
b
a
b
Swati Gupta, Bhavana Kushwaha, Akansha Srivastava, Jagdamba Prasad Maikhuri,
c
b
a
Satya N. Sankhwar, Gopal Gupta and Anil Kumar Dwivedi*
Today there is an urgent need for safe and effective dual-purpose contraceptive agents, which can
simultaneously prevent unintended pregnancies and sexually transmitted infections (STI). There are
several naturally occurring antimicrobial and antibiotic drugs (novobiocin, coumermycin and
chlorobiocin) reported in the literature, which are based on 4-hydroxy coumarins as the active
pharmacophore. Based on these interesting reports, we designed and synthesized a library of new 4-
hydroxy coumarin derivatives and evaluated them for spermicidal activity. Among the tested compounds,
two compounds (2a and 2d) displayed better activity (greater than 95% sperm immobilization at 0.5 mM
concentration) than the positive control nonoxynol-9 (N-9). Furthermore, all the compounds were
screened for antimicrobial activity against different strains of Trichomonas vaginalis and two compounds
Received 10th May 2016
Accepted 21st July 2016
(2c and 2h) exhibited potent activity as compared to the reference drug metronidazole. The cytotoxicity
assay showed that most of these compounds were safer than the N-9 against the human cervical HeLa
cell line and normal vaginal flora Lactobacillus jensenii strains. The studies have demonstrated that
compound (2a) is a potential lead to develop a dually active vaginal contraceptive.
DOI: 10.1039/c6ra12156j
www.rsc.org/advances
available alternatives with lesser side effects. Many research
1
Introduction

ndings have conrmed about the spermicidal properties in
5
5
The unabated increase in unintended pregnancies with high plants of Achyranthes aspera, Staphania hernandifolia, Carica
rates of STIs (Sexually Transmitted Infections) necessitates the papaya, Cestrum perqui, Hymeno cardiaaceta and Lawsonia
development of novel strategies especially in less developed enermis etc. but most of the plants used as contraceptives either
6
2
1,2
countries to help individuals in avoiding these risks. Accord- affect the fertility by decreasing spermatogenesis or alter the
7
ing to an estimate, ꢀ40% of all pregnancies worldwide are harmonic level and histoarchitecture of testis.
unintended which strongly indicates that the available methods
Sexually Transmitted Infections (STIs), the major cofactor of
3
of contraception are insufficient. Human immuno-deciency HIV, are found to increase the risk of HIV infection by 2–9
8
virus (HIV) infection and Acquired Immunodeciency times. Bacterial vaginosis, Herpes, Chlamydia, Trichomoni-
Syndromes (AIDS) have become a major cause of morbidity and asis, Gonorrhea, Hepatitis B virus, HIV and Syphilis are some of
mortality in India as well as the world. Now these have assumed
epidemic forms. Approximately 2.08 million people of the
Indian population were suffering from HIV in 2011 as per the
report of NACO (National AIDS Control Organisation).
Condoms are used to tackle this problem but their denial,
inconsistent or incorrect use and failure results in unintended
4
pregnancies. Herbal contraceptives are cost effective and easily
a
Pharmaceutics Division, CSIR-Central Drug Research Institute, Jankipuram
Extension, Sitapur Road, Lucknow, 226031, India. E-mail: anilcdri@gmail.com; Fax:
+
91-522-2623938; +91-522-2623405
b
Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow, 226031,
India
c
Urology Department, King George's Medical University, Lucknow-226003, India
†
Electronic supplementary information (ESI) available: Spectral data of
1
13
synthesized compounds ( H NMR, C NMR and HRMS and HPLC data). See
DOI: 10.1039/c6ra12156j
Fig. 1 Structure of naturally occurring antimicrobials with 4-hydroxy
coumarins scaffold.
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phytochemotherapy for treating vitiligo, psoriasis and atopic
26
dermatitis. Coumarins are also active at, benzodiazepine
receptors and on lipoxygenase and cyclooxygenase, possessing
anticancer and antimutagenic properties and have application
27
in cystic brosis treatment. It is also an important pharma-
cophore in naturally occurring biologically active compounds
like Daphnetin, Pachyrrhizine, Pimpinellin, Fraxetin, Aescule-
22,28–31
tin, Umbelliferone etc.
Anti-microbial drugs or antibiotics such as Novobiocin,
Coumermycin and Chloromycin contain 4-hydroxy coumarins
32
as their core structure. Because of this extraordinarily wide
range of biological activities, it can be stated that it has
“immense” therapeutic potential and these above interesting

ndings led us to generate a new molecule containing 4-hydroxy
coumarin as its core structure with the aim of exploring the
effect of such modication on the spermicide as well as anti-
microbial prole (Fig. 1–3).
2
Results and discussion
Fig. 2 Structure of synthesized Mannich compounds and their tartrate
salts.
2.1 Compound design and synthesis
2
.1.1 Chemistry. The tartrate salts of Mannich base bearing
0
0
0
0
0
0
0
0
coumarin derivatives (2a –2h ) (3a –3h ) (4a –4h ) (5a –5h ) have
been synthesized during this study. Designed compounds were
synthesized in three step procedure. It has been observed that
a,b unsaturation is responsible for spermicidal actions. There-
fore, we used phenyl glyoxals to increase its unsaturation for
spermicidal activity. Substituted phenyl glyoxals were prepared
by oxidation of ketones through selenium dioxide as procedure
8
the common sexually transmitted diseases. Microbicides can
be used to reduce the transmission of STIs including HIV, but
microbicides alone without contraceptive activity may not be
used with the required compliance during most of the sexual
9–11
contacts.
Membrane surfactant nonoxynol-9 (N-9) is used as
the main ingredient in most of the currently marketed vaginal
9
contraceptive products. N-9 contain multiple oligomers which
33
given in reference. These substituted phenyl glyoxals were
further reacted with 4-hydroxy coumarin and cyclic secondary
amine in the presence of methanol as solvent at reuxing
temperature for overnight. 4-Hydroxy coumarin is an important
pharmacophore responsible for its antimicrobial property.
Cyclic amines have been used to enhance its hydrophilicity,
which is an essential condition for compounds used for sper-
micidal actions as well as for its basic nature. Three different
cyclic amines i.e. piperidine, pyrrolidine and N-methyl-
have structure affinity with membrane lipids causing cell death
12
in sperm cells by disruption of its membrane integrity.
However, consistent use of N-9 or other surfactants causes
detergent-type cytotoxicity toward vaginal cells and also inacti-
vates Lactobacilli, hence increases the risk of HIV/STI trans-
mission which is the major drawback of using them despite
13
possessing potent microbicidal activity in vitro. Therefore,
there is a need to develop spermicides with microbicidal activ-
ities which are non-toxic to the membrane cells and Lactobacilli.
Coumarins, also known as benzo-a-pyrones, belong to

avonoid class of compounds. Compounds containing this
14
15
pharmacophore act as, inter alia, anti-HIV, antimitotic, anti-
fungal, anti-viral, anti-tubulin, vasodilators, anti-coagu-
lants, antioxidant, dyslipidemic, anti-malarial and anti-
hypertensive agents. Some of its derivatives are tyrosine and
16
17
18
19
20
21
22
23
24
25
protein kinase
C
inhibitors
and have been used in
Scheme 1 Reaction conditions: A ¼ substituted acetophenone (1
ꢁ
mmol), selenium(II) oxide (1.8 eq.), 1,4-dioxane, H
2
O, 80 C, reflux, 5 h,
B ¼ 4-hydroxy coumarin (1 mmol), substituted phenyl glyoxal (1.2 eq.),
cyclic amine (1 eq.), MeOH, reflux, overnight C ¼ Mannich base
product (0.5 mmol), tartaric acid (1 eq), MeOH, rt, 30 minutes.
Fig. 3 Different substitutions of phenyl glyoxal used in the reaction.
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piperazine were used during the study to check further its role metronidazole susceptible and resistant strains. 2 compounds
in spermicidal actions. The synthesized Mannich type product (2c and 2h) showed potent activity at 0.0140 mM and 0.0136 mM
were reacted with the tartaric acid in methanol for salt forma- respectively in case of metronidazole susceptible strain and
tion. Tartrate salts are formed to improve its hydrophilicity and 0.1122 mM and 0.1091 mM in case of metronidazole resistant
34
for better efficacy. Reaction of phenyl glyoxal and 4-hydroxy strains which is better than metronidazole. Most of the
coumarin gave keto–enol tautomers of Mannich reaction compounds were found safe in cytotoxicity assay (HeLa cell
product and dicoumarols (2 molecules of 4-hydroxy coumarin lines) and safety (Lactobacillus jenseii strains) studies (Table 1).
fused with phenyl glyoxal) as a side product. Saeed et al. have
reported the formation of dicoumarols which was further
conrmed by mass and NMR spectra. It was observed that
3
Conclusions
35
reaction proceeded smoothly and in good yield with electron In Conclusion, we have designed and synthesized a series of
donating groups while in the case of electron withdrawing novel 4-hydroxy coumarin and glyoxal conjugates using eco-
groups either reaction does not occur or very low yield is ob- friendly approach with good yields and evaluated them for
tained. In case of nitro as a substituent reaction, product was spermicidal and anti-microbial activity. These compounds were
not formed. When simple hydrogen was used as a substituent tested about their toxicity towards vaginal epithelial cell lines
a dimer of 4-hydroxy coumarin fused with phenyl glyoxal was and safety towards lactobacilli strains. 16 compounds showed
obtained as major product instead of our desired compound. activity comparable to N-9 in which compound 2a and 2d
When methoxy group is attached on 3, 4, 5 position then also showed signicant spermicidal activity. 18 compounds were
reaction didn't proceeded may be due to steric hindrance. To equipotential to metronidazole susceptible trichomonas cell
increase the hydrophilicity of these Mannich products, tartrate lines while 21 compounds displayed prominent activity in
salts were made using 1 equivalent of tartaric acid in methanol. comparison of metronidazole resistant trichomonas cell lines.
Reaction scheme is illustrated in gure given below. These Compound 2c and 2h were found most potent in anti-
1
13
compounds were structurally identied through H and
NMR spectroscopy and HRMS (High Resolution Mass Spec- in comparison of N-9 in cytotoxicity and safety assays. 11
trometry) to conrm their structure (Scheme 1). compounds were found active in both spermicidal and anti-
.1.2 Biology. Spermicidal activity was observed in trichomonas assays. Out of those, compound 2a, 2c and 2h
C
trichomonas assay. These compounds were found highly safe
2
comparison with the compound currently in clinical use, were observed as most potent. Furthermore, compound 2a was
nonoxynol-9 (N-9). In this experiment it was observed that in most promising in terms of spermicidal activity and thus can be
case of replacement of 4-hydroxy coumarin with 4-hydroxy-6- transformed in a lead molecule towards the development of
methyl-2H-pyran-2-one, compounds are either inactive or dually useful contraceptive microbicides.
showing very low spermicidal activity. Therefore, it can be said
that phenyl ring of 4-hydroxy coumarin is responsible for its
spermicidal activity and in the absence of this ring, spermicidal
4
Experimental procedures
activity decreased. A series of compounds was formed fused All reagents used were commercial and were used without
with piperidine, pyrrolidine and N-methyl piperazine. It was further purication. Compounds were puried on 60–120 mesh
observed that spermicidal activity was highest in case of and 100–200 mesh silica gels with methanol : chloroform as
piperidine chain, moderate in pyrrolidine and lowest in case of eluent. All reactions were monitored by silica gel TLC plated
1
13
N-methyl piperazine except few cases. In case of 4-hydroxy and with F254 uorescence. The H and C NMR spectra were
-methoxy substitution, compounds with N-methyl piperazine determined on 400 MHz and 100 MHz Bruker NMR spectrom-
was more active in comparison of pyrrolidine. Polar substitu- eters respectively using CDCl and DMSO-d as deuterated
tion like OH, OCH group on 4-position of phenyl glyoxal solvents and TMS as an internal standard. In some cases,
4
3
6
3
induces activity in the compound. When OCH3 group was increase in the no. of aromatic protons was observed in NMR
added on 3-position of these compounds, then activity spectra due to keto–enol tautomerism. Chemical shis are re-
decreased and when CH2 was inserted in methoxy group ported in parts per million. Splitting patterns are described as
2 3
(–OCH CH ) then also activity decreased. Spermicidal activity singlet (s), broad singlet (brs), doublet (d), broad doublet (brd),
was observed increasing with increase in polarity of substitu- double doublet (dd), triplet (t), quartet (q), and multiplet (m).
tion on 4-position of phenyl glyoxal. 16 compounds showed Infrared spectra were recorded on a Perkin-Elmer FT-IR RX1
ꢃ1
spermicidal activity at concentrations ranging from 0.02 to spectrophotometer (4000–450 cm range). Melting points were
1.0%. Out of these, 2 compounds (2a and 2d) showed activity taken in open capillaries on Stuart SME30 melting point appa-
better than nonoxynol-9. In case of anti-trichomonas activity ratus and are presented uncorrected. Electrospray ionization
experiment, 18 compounds showed activity at concentrations mass spectra (ESI MS) were recorded on Thermo LCQ Advantage
ranging from 0.0136 mM to 0.0638 mM in comparison of Max-IT. HRMS were recorded by Q-TOF mass spectrometer.
metronidazole susceptible trichomonas cell lines and 21 Purity analysis of compounds were characterized by HPLC
compounds showed better activity (concentrations ranging analysis (RP-18 Lichrocart ® HPLC system). The HPLC system
from 0.1089 mM to 0.2525 mM) in comparison of metronida- (Shimadzu, Kyoto, Japan) equipped with Photo Diode Array
zole resistant trichomonas cell lines. 5 compounds were detector (SPD-M20A), a pump (LC-20AD), controller (CBM-20A),
showing excellent anti-trichomonas activity in both Rheodyne (Cotati, CA, USA) model 7125 injector with a 20 ml
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loop. Data was acquired in LC solution soware (Shimadzu, Hz), 6.08 (1H, s), 4.03 (2H, s), 1.96 (4H, s), 1.75 (2H, s), 1.37–1.41
Japan). Compounds were monitored at 275 nm with a UV-Vis (4H, m), 1.27 (3H, s); d (100 MHz; CDCl ): 195.3, 167.4, 162.2,
C
3
multiple wavelength detector. Compounds with purity >95% 152.6, 131.7, 130.2, 129.0, 125.0, 123.8, 119.0, 116.5, 115.9,
were used for further experiments. 114.3, 113.7, 102.2, 63.4, 45.4, 43.0, 29.7, 22.8, 14.6; HR-MS [M +
+
H] (m/z) calculated for C24
.2.4 Compound 4-hydroxy-3-(2-(4-methoxyphenyl)-2-oxo-
-(piperidin-1-yl)ethyl)-2H-chromen-2-one (2d). Light brown
5
H25NO 408.1811 found 408.1828.
4
4.1 General procedure of phenyl glyoxal synthesis
1
SeO (6.4 mmole) was taken in a 50 ml round bottom ask with
2
solid as a mixture of keto/enol tautomers (0.291 g, 74%), mp
ꢁ
1,4-dioxane/water (10 ml/95 : 5) as solvent and heated it at 60 C
ꢁ
1
16–118 C, HPLC (purity 95.71%): t
R
¼ 14.166 min; FT-IR (KBr,
for 3 h. Substituted acetophenone (6.5 mmol) was then added
and reux the reaction mixture for 4 h. Aer completion of the
reaction, reaction mixture was ltered and lterate was
concentrated. Formation of product (1a–1h) was conrmed
ꢃ1
nmax/cm ): 3393, 3015, 1665, 1398, 1256, 1218, 1173, 765; dH
(
400 MHz; CDCl ): 8.11 (2H, d, J ¼ 8.76 Hz), 7.99 (1H, d, J ¼ 7.24
3
Hz), 7.43 (1H, t, J ¼ 7.16 Hz), 7.18 (2H, d, J ¼ 8.08 Hz), 6.86 (2H,
d, J ¼ 8.68 Hz), 6.11 (1H, s), 3.80 (3H, s), 2.13 (2H, s), 1.96 (4H, s),
33
through TLC and mass and further used without purication.
1
1
1
C 3
.40–1.80 (2H, brs); d (100 MHz; CDCl ): 192.7, 165.3, 162.5,
60.2, 152.5, 131.6, 130.1, 124.9, 123.7, 122.8, 115.8, 113.1,
4.2 General synthetic procedure for 4-hydroxy-3-(2-(4-
+
02.2, 55.0, 45.5, 30.9, 29.6, 22.7, 22.3; HR-MS [M + H] (m/z)
substitutedphenyl)-2-oxo-1-(piperidin-1-yl) ethyl)-2H-
chromen-2-one derivatives 2a–2h
calculated for C23
5
H23NO 394.1654 found 394.1646.
4.2.5 Compound 3-(2-(4-chlorophenyl)-2-oxo-1-(piperidin-
Substituted phenyl glyoxal was reuxed with 4-hydroxy 1-yl)ethyl)-4-hydroxy-2H-chromen-2-one (2e). Light brown solid
coumarin in the presence of piperidine as base and methanol as as a mixture of keto/enol tautomers (0.286 g, 72%), mp 120–122
ꢁ
solvent for 8 h. Aer completion of the reaction, it was
C, HPLC (purity 98.69%): t
R
¼ 14.587 min; FT-IR (KBr, nmax
/
ꢃ1
quenched with water and extracted with DCM. The organic layer cm ): 3388, 3018, 1603, 770, 668; d
H
(400 MHz; CDCl ): 7.9–
3
was separated and dried over sodium sulfate and concentrated 8.12 (2H, m), 7.87 (1H, d, J ¼ 8.4 Hz), 7.39 (1H, t, J ¼ 7.8 Hz),
in vacuo. Crude product was puried by column chromatog- 7.24–7.32 (1H, m), 7.0–7.2 (3H, m), 6.17 (1H, s), 3.47 (1H, s), 3.18
raphy using 100–200 mesh silica gels as adsorbent and meth- (4H, t, J ¼ 5.44 Hz), 1.81 (4H, d, J ¼ 4.88 Hz), 1.65 (2H, d, J ¼ 4.8
anol : chloroform as eluent.
Hz); d (100 MHz; CDCl ): 198.9, 175.1, 166.9, 153.9, 152.8,
C 3
4
.2.1 Compound 4-hydroxy-3-(2-(4-hydroxyphenyl)-2-oxo-1- 139.4, 138.0, 133.3, 131.2, 129.6, 129.3, 128.6, 128.2, 125.0,
(
piperidin-1-yl) ethyl)-2H-chromen-2-one (2a). Light orange 123.4, 122.9, 122.2, 116.2, 115.7, 101.3, 95.6, 79.2, 56.3, 45.0,
+
solid as a mixture of keto/enol tautomers (0.284 g, 75%), mp 22.8, 22.4; HR-MS [M + H] (m/z) calculated for C22
H20NO
4
Cl
ꢁ
1
84–186 C, HPLC (purity 95.01%): t ¼ 12.698 min; FT-IR (KBr, 398.1159 found 398.1152.
R
ꢃ1
n
max
/cm ): 3396, 3019, 2919, 1630, 1402, 1216, 1155, 1068, 770,
4.2.6 Compound 4-hydroxy-3-(2-oxo-1-(piperidin-1-yl)-2-p-
6
¼
69; d
H
(400 MHz; DMSO-d
6
): 15.8–16.0 (1H, brs), 7.77 (1H, dd, J tolylethyl)-2H-chromen-2-one (2f). Light orange solid as
ꢁ
7.76 Hz), 7.66 (2H, d, J ¼ 8.72 Hz), 7.45–7.50 (2H, dt, J ¼ 6.96 a mixture of keto/enol tautomers (0.271 g, 72%), mp 189–191 C,
ꢃ1
Hz), 7.18–7.25 (4H, m), 6.67 (2H, d, J ¼ 8.68 Hz), 6.27 (1H, s), HPLC (purity 95.65%): t
¼ 14.961 min; IR (KBr) (cm ): 3388,
R
2
1
1
.98 (4H, m), 1.50–1.70 (6H, m); d
74.2, 162.6, 154.3, 131.7, 130.8, 126.2, 125.4, 122.9, 122.2, CDCl
C
(100 MHz; DMSO-d
6
): 192.5, 1606, 1542, 1403, 1217, 1154, 1068, 771, 668; d
): 7.98–8.02 (3H, m), 7.40–7.44 (1H, m), 7.16 (4H, d, J ¼
16.4, 115.2, 90.5, 67.3, 54.3, 25.2, 23.5; HR-MS [M + H] (m/z) 6.72 Hz), 6.14 (1H, s), 2.90–3.90 (4H, br), 2.33 (3H, s), 1.97 (4H,
s), 1.72 (2H, s); d (100 MHz; CDCl ): 197.7, 170.8, 166.6, 153.1,
.2.2 Compound 4-hydroxy-3-(2-oxo-1-(piperidin-1-yl)-2-m- 142.6, 131.1, 129.1, 128.6, 128.1, 125.2, 122.9, 120.1, 115.6,
H
(400 MHz;
3
+
calculated for C H NO 380.1498 found 380.1481.
C
3
2
2
21
5
4
+
tolylethyl)-2H-chromen-2-one (2b). White solid as a mixture of 102.2, 63.3, 46.3, 31.1, 29.7, 21.3; HR-MS [M + H] (m/z) calcu-
keto/enol tautomers (0.269 g, 69%) mp 136–138 C, HPLC lated for C23
ꢁ
ꢃ1
H
23NO
4
378.1705 found 378.1690.
(purity 100%): t
R
¼ 14.271 min; FT-IR (KBr, nmax/cm ): 3393,
4.2.7 4-Hydroxy-3-(2-(4-hydroxy-3-methoxyphenyl)-2-oxo-1-
2922, 1613, 1403, 1218, 1155, 1068, 771; d
H
(400 MHz; CDCl
3
+
(piperidin-1-yl) ethyl)-2H-chromen-2-one (2g). White solid as
DMSO-d
6
): 8.73 (1H, s), 7.98 (2H, d, J ¼ 6.84 Hz), 7.69 (2H, m), a mixture of keto/enol tautomers (0.286 g, 70%), mp 213–215
ꢁ
7.37 (2H, brs), 7.17 (2H, brs), 7.05 (3H, s), 6.94 (1H, s), 6.43 (1H,
C, HPLC (purity 98.32%): t
R
¼ 12.995 min; FT-IR (KBr, nmax
/
ꢃ1
s), 2.18 (4H, m), 1.93 (4H, s), 1.72 (2H, brs), 1.27 (3H, s); d (100 cm ): 3396, 2927, 1645, 1513, 1404, 1216, 1069, 768; d
H
(400
C
MHz; CDCl + DMSO-d ): 198.4, 170.1, 166.3152.7, 137.4, 136.9, MHz; DMSO-d
6
): 16.00 (1H, s), 8.20–8.80 (1H, br), 7.78 (2H, d, J
3
6
1
1
32.6, 130.9, 128.6, 127.6, 124.9, 124.7, 123.1, 119.8, 115.6, ¼ 7.16 Hz), 7.49 (2H, t, J ¼ 7.92 Hz), 7.40 (1H, s), 7.31 (1H, d, J ¼
+
01.7, 45.0, 43.3, 22.5, 22.2, 21.2; HR-MS [M + H] (m/z) calcu- 7.84 Hz), 7.14–7.28 (4H, m), 6.69 (1H, d, J ¼ 8.24 Hz), 6.32 (1H,
lated for C23
H23NO
4
378.1705 found 378.1707.
s), 3.72 (3H, s), 3.01 (4H, s), 1.64 (4H, s), 1.56 (2H, s); d (100
C
4
.2.3 Compound 3-(2-(4-ethoxyphenyl)-2-oxo-1-(piperidin- MHz; DMSO-d
): 196.2, 168.7, 164.5, 152.9, 150.8, 147.0, 131.5,
6
1
-yl)ethyl)-4-hydroxy-2H-chromen-2-one (2c). Whitesolid as 128.5, 124.5, 123.4, 122.3, 120.0, 115.9, 115.0, 112.1, 101.8,
a mixture of keto/enol tautomers (0.289 g, 71%) mp 134–136 C, 55.8, 44.2, 43.2, 22.6, 22.0; HR-MS [M + H] (m/z) calculated for
ꢁ
+
ꢃ1
HPLC (purity 99.36%): t ¼ 12.471 min; FT-IR (KBr, nmax/cm ):
C
23
H
23NO
6
410.1603 found 410.1627.
4.2.8 Compound 3-(2-(3,4-dimethoxyphenyl)-2-oxo-1-
): 8.11 (2H, d, J ¼ 8.48 Hz), 8.00 (1H, d, J ¼ 7.00 Hz), 7.43 (piperidin-1-yl)ethyl)-4-hydroxy-2H-chromen-2-one (2h). Light
R
3
403, 3019, 1632, 1403, 1216, 1155, 1068, 770, 669; d (400 MHz;
H
CDCl
3
(
1H, t, J ¼ 7.40 Hz), 7.18 (2H, d, J ¼ 7.96 Hz), 6.84 (2H, d, J ¼ 8.32 orange solid as a mixture of keto/enol tautomers (0.313 g, 74%)
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ꢁ
+
mp 223–225 C, HPLC (purity 97.47%): t
(
R
¼ 13.230 min; FT-IR MS [M + H] (m/z) calculated for C24
H
26
N
2
O
5
423.1920 found
ꢃ1
KBr, nmax/cm ): 3408, 3019, 1654, 1403, 1268, 1216, 1154, 423.1432.
7
70; dH (400 MHz; CDCl ): 11.34 (1H, s), 8.10 (2H, dd, J ¼ 7.96
4.3.4 Compound
4-hydroxy-3-(2-(4-methoxyphenyl)-1-(4-
3
Hz), 7.63 (2H, t, J ¼ 8.52 Hz), 7.50 (1H, s), 7.37–7.45 (5H, m), 6.76 methylpiperazin-1-yl)-2-oxoethyl)-2H-chromen-2-one (3d). Light
(
(
1H, d, J ¼ 8.44 Hz), 6.13 (1H, s), 3.87 (6H, d, J ¼ 5.56 Hz), 1.58 brown solid as a mixture of keto/enol tautomers (0.314 g, 77%)
ꢁ
10H, s); d
C
(100 MHz; CDCl
3
): 195.1, 168.7, 165.1, 154.0, 152.4, mp 138–140 C, HPLC (purity 97.62%): t
R
¼ 11.689 min; IR (KBr)
ꢃ1
1
1
50.6, 148.9, 133.0, 124.9, 124.5, 122.2, 116.6, 110.9, 110.0, (cm ): 3415, 1646, 1601, 1528, 1395, 1259, 1218, 1174, 1051,
02.0, 56.0, 55.8, 42.6, 29.6, 27.3; HR-MS [M + H] (m/z) calcu- 765; d
+
H
(400 MHz; CDCl
3
): 8.14 (1H, d, J ¼ 4.8 Hz), 8.01 (1H, s),
lated for C24H25NO
6
424.1760 found 424.1746.
7.42 (1H, t, J ¼ 7.76 Hz), 7.17 (2H, d, J ¼ 7.6 Hz), 7.00 (1H, t, J ¼
8
.8 Hz), 6.85 (2H, d, J ¼ 6.68 Hz), 5.90–6.10 (1H, brs), 3.79 (3H,
s), 3.00–3.50 (2H, brs), 2.75 (2H, s), 2.30–2.50 (4H, br), 1.28 (3H,
s); d (100 MHz; CDCl ): 192.5, 175.5, 165.1, 164.9, 154.3, 131.5,
C
3
4.3 General synthetic procedure for 4-hydroxy-3-(2-(4-
1
1
30.9, 130.2, 126.9, 125.2, 123.0, 121.7, 116.4, 115.5, 113.8,
13.0, 90.9, 69.4, 55.4, 53.7, 46.1; HR-MS [M + H] (m/z) calcu-
substitutedphenyl)-2-oxo-1-(piperazin-1-yl)ethyl)-2H-
chromen-2-one derivatives 3a–3h
+
lated for C23
24 2 5
H N O 409.1763 found 409.1761.
Substituted phenyl glyoxal was reuxed with 4-hydroxy
4.3.5 Compound 3-(2-(4-chlorophenyl)-1-(4-methylpiper-
coumarin in the presence of piperazine as base and methanol as azin-1-yl)-2-oxoethyl)-4-hydroxy-2H-chromen-2-one (3e). Dark
solvent for 8 h. Aer completion of the reaction, it was orange solid as a mixture of keto/enol tautomers (0.309 g, 75%)
ꢁ
quenched with water and extracted with DCM. Organic layer mp 133–135 C, HPLC (purity 98.36%): t
¼ 14.095 min; IR (KBr)
(400
): 7.99 (1H, d, J ¼ 7.80 Hz), 7.87 (1H, d, J ¼ 8.36 Hz),
raphy using 100–200 mesh silica gel as adsorbent and meth- 7.41 (1H, t, J ¼ 7.24 Hz), 7.12–7.25 (4H, m), 6.48 (1H, s), 3.29
anol : chloroform as eluent.
(4H, q, J ¼ 7.28 Hz), 2.70–3.10 (2H, brs), 1.39 (1H, s), 1.37 (3H, s),
.3.1 Compound 4-hydroxy-3-(2-(4-hydroxyphenyl)-1-(4- 1.35 (1H, s); d (100 MHz; CDCl ): 196.9, 166.9, 152.8, 137.8,
R
ꢃ1
was separated and dried over sodium sulfate and concentrated (cm ): 3399, 1610, 1400, 1385, 1218, 1069, 771, 670; d
H
in vacuo. Crude product was puried by column chromatog- MHz; CDCl
3
4
C
3
methylpiperazin-1-yl)-2-oxoethyl)-2H-chromen-2-one (3a). pale 131.3, 130.1, 129.4, 128.9, 125.1, 123.5, 119.8, 115.7, 101.7, 68.5,
+
yellow solid as a mixture of keto/enol tautomers (0.307 g, 78%) 46.4, 43.4, 29.6; HR-MS [M + 2H] , (m/z) calculated for
ꢁ
mp 204–206 C, HPLC (purity 98.89%): t
(
R
¼ 11.339 min; IR (KBr)
C
22
H
21
N
2
O
4
Cl. 414.1346 found 414.1234.
ꢃ1
cm ): 3432, 1638, 1401, 1385, 1219, 1069, 771; d
H
(400 MHz;
4.3.6 Compound 4-hydroxy-3-(1-(4-methylpiperazin-1-yl)-2-
CDCl
3
+ DMSO-d
6
): 7.88 (1H, d, J ¼ 7.76 Hz), 7.78 (2H, d, J ¼ 8.72 oxo-2-p-tolylethyl)-2H-chromen-2-one (3f). Dark yellow solid as
ꢁ
Hz), 7.52 (1H, s), 7.34 (1H, t, J ¼ 8.0 Hz), 7.06 (2H, m), 6.65 (2H, a mixture of keto/enol tautomers (0.306 g, 78%), mp 76–78 C,
ꢃ1
d, J ¼ 8.76 Hz), 5.91 (1H, s), 2.80–3.20 (4H, brs), 2.50–2.70 (4H, HPLC (purity 95.18%): t
R
¼ 13.888 min; IR (KBr) (cm ); 3412,
brs), 2.24 (3H, s); d
C
(100 MHz; CDCl
3
+ DMSO-d
6
): 196.7, 180.1, 3019, 1617, 1467, 1404, 1385, 1215, 1069, 758, 669; d
): 7.94 (1H, d, J ¼ 7.56 Hz), 7.77 (1H, d, J ¼ 7.96 Hz), 7.33
21.0, 120.1, 93.6, 72.6, 56.5, 54.4, 50.1; HR-MS [M + H] (m/z) (1H, t, J ¼ 7.56 Hz), 7.08–7.14 (4H, m), 6.97 (1H, d, J ¼ 7.68 Hz),
395.1607 found 395.1602.
6.38 (1H, s), 3.24 (4H, q, J ¼ 7.24 Hz), 2.09 (4H, m), 1.31 (3H, t, J
(100 MHz; CDCl ): 197.7, 170.7,
H
(400 MHz;
1
1
69.6, 167.5, 158.9, 136.2, 135.4, 130.6, 130.0, 127.4, 126.5, CDCl
3
+
22 2 5
calculated for C22H N O
4
.3.2 Compound 4-hydroxy-3-(1-(4-methylpiperazin-1-yl)-2- ¼ 7.24 Hz), 1.18 (3H, s); d
C
3
oxo-2-m-tolylethyl)-2H-chromen-2-one (3b). Orangish-yellow 166.6, 152.8, 142.1, 131.0, 128.9, 128.6, 128.1, 125.2, 123.2,
solid as a mixture of keto/enol tautomers (0.298 g, 76%), mp 120.1, 115.6, 101.9, 46.5, 43.3, 30.9, 29.7, 21.5; HR-MS [M + H]
+
ꢁ
1
(
19–121 C, HPLC (purity 100%): tR ¼ 13.062 min; IR (KBr) (m/z) calculated for C23
H
24
N
2
O
4
393.1814 found 393.1805.
ꢃ1
cm ): 3412, 3019, 1617, 1467, 1404, 1385, 1215, 1069, 758, 669;
4.3.7 Compounds
4-hydroxy-3-(2-(4-hydroxy-3-methoxy-
d
H
(400 MHz; CDCl
3
): 8.00 (1H, d, J ¼ 7.68 Hz), 7.75 (1H, s), 7.68 phenyl)-1-(4-methylpiperazin-1-yl)-2-oxoethyl)-2H-chromen-2-
(
1H, d, J ¼ 6.76 Hz), 7.55 (1H, brs), 6.80–7.20 (5H, m), 6.48 (1H, one (3g). Light yellow solid as a mixture of keto/enol tauto-
ꢁ
s), 3.23 (3H, q, J ¼ 7.2 Hz), 2.18 (3H, s), 1.2–1.34 (8H, s); d
C
(100 mers (0.331 g, 78%), mp 160–162 C, HPLC (purity 99.66%): t
R
ꢃ1
MHz; CDCl
3
): 167.3, 152.6, 137.7, 136.7, 134.4, 132.9, 131.6, ¼ 11.993 min; IR (KBr) (cm ): 3410, 3019, 2927, 2400, 1646,
1
3
3
28.8, 127.7, 125.0, 124.9, 123.7, 119.2, 115.9, 102.3, 46.7, 43.2, 1401, 1385, 1215, 1070, 928, 757, 669; d
H
(400 MHz; CDCl
): 7.90 (2H, s), 7.49 (1H, d, J ¼ 3.48 Hz), 7.40 (1H, s),
3
+
+
1.4, 29.4, 22.6; HR-MS [M + H] (m/z) calculated for C H N O
93.1814 found 393.1805.
4
DMSO-d
7.32 (2H, d, J ¼ 5.6 Hz), 7.25 (2H, m), 6.58 (1H, s), 6.36 (1H, s),
.3.3 Compound 3-(2-(4-ethoxyphenyl)-1-(4-methylpiper- 3.71 (3H, s), 1.29 (3H, s), 1.18 (8H, s); d (100 MHz; CDCl
azin-1-yl)-2-oxoethyl)-4-hydroxy-2H-chromen-2-one (3c). Pale DMSO-d ): 152.7, 149.9, 146.5, 131.0, 124.9, 123.2, 122.4,
6
2
3
24 2 4
C
3
+
6
yellow solid as a mixture of keto/enol tautomers (0.317 g, 75%), 115.6, 113.9, 111.5, 102.2, 55.7, 52.0, 46.5, 43.0, 31.8, 29.5,
ꢁ
+
mp 185–187 C, HPLC (purity 98.89%): t
(
R
¼ 11.339 min; IR (KBr) 29.2, 22.5; HR-MS [M + H] (m/z) calculated for C23
H N O
24 2 6
ꢃ1
cm ): 3431, 2069, 1638, 1402, 1385, 1219, 1068, 771; d
H
(400 425.1712 found 425.1707.
MHz; CDCl
7
3
): 8.00 (1H, d, J ¼ 7.68 Hz), 7.68 (1H, d, J ¼ 6.76 Hz),
4.3.8 Compound 3-(2-(3,4-dimethoxyphenyl)-1-(4-methyl-
.41 (1H, s), 7.06–7.20 (5H, m), 6.48 (1H, s), 3.48 (2H, s), 3.24 piperazin-1-yl)-2-oxoethyl)-4-hydroxy-2H-chromen-2-one (3h).
(
1
1
3H, s), 1.29 (8H, s), 1.20 (3H, s); dC (100 MHz; CDCl ): 197.7, Pale yellow solid as a mixture of keto/enol tautomers (0.333 g,
3
ꢁ
67.3, 152.6, 137.7, 136.7, 132.9, 131.6, 128.8, 127.7, 125.0, 76%) mp 147–150 C, HPLC (purity 100%): t
R
¼ 13.062 min; IR
ꢃ1
24.9, 123.7, 115.9, 102.3, 64.5, 46.7, 43.2, 30.9, 29.6, 14.1; HR- (KBr) (cm ): 3407, 3020, 1654, 1601, 1536, 1460, 1408, 1269,
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1
215, 1025, 762, 669; d
H
(400 MHz; CDCl
3
): 8.04 (1H, s), 7.80
4.4.4 Compound 4-hydroxy-3-(2-(4-methoxyphenyl)-2-oxo-
(
(
3
1H, d, J ¼ 6.44 Hz), 7.67 (1H, s), 7.43 (1H, t, J ¼ 5.88 Hz), 7.19 1-(pyrrolidin-1-yl)ethyl)-2H-chromen-2-one (4d). Dark brown
2H, m), 6.83 (1H, d, J ¼ 6.52 Hz), 6.18 (1H, s), 3.86 (6H, s), 3.20– solid as a mixture of keto/enol tautomers (0.288 g, 76%), mp
ꢁ
.60 (4H, br), 2.70–2.90 (4H, br), 2.34 (3H, s); dC (100 MHz; 156–158 C, HPLC (purity 96.26%): t ¼ 13.299 min; IR (KBr)
R
ꢃ1
CDCl
3
): 190.4, 175.0, 164.6, 154.2, 148.8, 131.8, 126.8, 124.9, (cm ): 3396, 3018, 1653, 1603, 1541, 1395, 1217, 1175, 1033,
1
4
23.7, 123.1, 121.0, 116.5, 111.0, 110.3, 88.8, 56.0, 52.3, 50.7, 765, 668; d (400 MHz; CDCl ): 8.08 (3H, m), 7.41 (1H, t, J ¼ 7.12
H
3
+
5.2; HR-MS [M + H] (m/z) calculated for C24
H
26
N
2
O
6
439.1869 Hz), 7.15 (2H, m), 6.82 (2H, d, J ¼ 7.16 Hz), 6.16 (1H, s), 3.78 (3H,
s), 3.62 (1H, s), 3.47 (1H, s), 2.40–3.00 (2H, br), 1.80–2.20 (4H,
found 439.1863.
br); d
C
(100 MHz; CDCl
3
): 192.1, 175.5, 165.1, 164.2, 154.2, 131.5,
1
1
30.9, 130.2, 126.7, 125.2, 123.0, 121.8, 116.4, 115.5, 113.8,
+
13.1, 90.9, 69.4, 55.4, 55.0, 46.1, 24.4, 23.7; HR-MS [M + H] (m/
380.1498 found 380.1489.
.4.5 Compound 3-(2-(4-chlorophenyl)-2-oxo-1-(pyrrolidin-
-yl) ethyl)-4-hydroxy-2H-chromen-2-one (4e). Light orange
4
.4 General synthetic procedure for 4-hydroxy-3-(2-(4-
z) calculated for C22H21NO
5
substitutedphenyl)-2-oxo-1-(pyrollodin-1-yl) ethyl)-2H-
chromen-2-one derivatives 4a–4h
4
1
Substituted phenyl glyoxal was reuxed with 4-hydroxy solid as a mixture of keto/enol tautomers (0.276 g, 72%) mp
ꢁ
coumarin in the presence of pyrollidine as base and methanol 175–179 C, HPLC (purity 95.21%): t ¼ 13.286 min; IR (KBR)
R
ꢃ1
as solvent for 8 h. Aer completion of the reaction, it was (cm ): 3425, 3019, 2958, 2922, 1610, 1546, 1463, 1404, 1270,
quenched with water and extracted with DCM. Organic layer 1216, 1092, 760, 669; dH (400 MHz; CDCl ): 7.95 (2H, d, J ¼ 6.56
3
was separated and dried over sodium sulfate and concentrated Hz), 7.87 (1H, d, J ¼ 8.4 Hz), 7.39 (1H, t, J ¼ 7.8 Hz), 7.28 (1H, d, J
in vacuo. Crude product was puried by column chromatog- ¼ 8.56 Hz), 7.00–7.18 (3H, m), 6.17 (1H, s), 3.47 (1H, s), 3.18 (4H,
raphy using 100–200 mesh silica gel as adsorbent and meth- t, J ¼ 5.44 Hz), 1.70–1.90 (4H, brs), 1.50–1.70 (2H, brs); d (100
C
anol : chloroform as eluent.
MHz; CDCl ): 199.0, 175.1, 166.9, 153.9, 139.4, 131.3, 131.2,
3
4
.4.1 Compound 4-hydroxy-3-(2-(4-hydroxyphenyl)-2-oxo-1- 129.6, 129.3, 128.6, 128.2, 125.0, 123.3, 122.9, 122.2, 116.3,
+
(
pyrrolidin-1-yl)ethyl)-2H-chromen-2-one (4a). Light pink solid 115.7, 101.3, 79.2, 56.3, 22.8; HR-MS [M + H] (m/z) calculated
as a mixture of keto/enol tautomers (0.274 g, 75%) mp 224–226 for C H NO Cl 384.1002 found 384.0999.
2
1
18
4
ꢁ
ꢃ1
C, HPLC (purity 98.97%): t
400, 3021, 1643, 1600, 1514, 1391, 1216, 1072, 767, 670; d
R
¼ 12.027 min; IR (KBr) (cm ):
4.4.6 Compound 4-hydroxy-3-(2-oxo-1-(pyrrolidin-1-yl)-2-p-
(400 tolylethyl)-2H-chromen-2-one (4f). Light brown solid as
): 10.33 (1H, s), 9.30–9.80 (1H, br), 7.83 (1H, d, J a mixture of keto/enol tautomers (0.272 g, 75%), mp 200–202 C,
3
H
ꢁ
MHz; DMSO-d
6
ꢃ1
¼
7.72 Hz), 7.77 (2H, d, J ¼ 8.8 Hz), 7.44 (1H, dq, J ¼ 7.08 Hz), HPLC (purity 95.53%): t ¼ 14.282 min; IR (KBr) (cm ): 3399,
R
7
.16 (1H, dt, J ¼ 7.56 Hz), 7.10 (1H, d, J ¼ 8.16 Hz), 6.71 (2H, d, J 1606, 1385, 1217, 1156, 1069, 771, 668; d (400 MHz; CDCl ):
H
3
¼
8.8 Hz), 6.03 (1H, s), 3.00–3.30 (4H, br), 1.70–2.00 (4H, br); d
7.93 (2H, d, J ¼ 7.76 Hz), 7.75 (1H, d, J ¼ 7.32 Hz), 7.31 (1H, t, J ¼
C
(100 MHz; DMSO-d
6
): 192.5, 174.2, 162.6, 154.3, 131.7, 130.8, 6.72 Hz), 7.0–7.14 (3H, m), 6.86–7.0 (1H, brd, J ¼ 3.36 Hz), 6.39
1
26.2, 125.4, 122.9, 122.2, 116.4, 115.2, 90.4, 67.9, 23.6; HR-MS (1H, s),3.22 (4H, m), 2.09 (3H, s), 1.30 (4H, t, J ¼ 7.28 Hz); d (100
C
+
[M + H] (m/z) calculated for C21
H19NO
5
366.1341 found MHz; CDCl ): 197.8, 192.6, 170.8, 166.8, 152.8, 142.1, 134.4,
3
3
66.1335.
131.0, 128.5, 128.1, 125.1, 123.2, 123.0, 120.0, 115.6, 102.0, 46.5,
+
4.4.2 Compound 4-hydroxy-3-(2-oxo-1-(pyrrolidin-1-yl)-2-m- 43.3, 30.9, 29.7, 21.4; HR-MS [M + H] (m/z) calculated for
tolylethyl)-2H-chromen-2-one (4b). Dark red solid as a mixture C H NO 364.1549 found 364.1541.
2
2
21
4
of keto/enol tautomers, hygroscopic (0.269 g, 74%), mp 70–72
4.4.7 Compound
4-hydroxy-3-(2-(4-hydroxy-3-methoxy-
ꢁ
ꢃ1
C, HPLC (purity 97.82%): tR ¼ 13.578 min; IR (KBr) (cm ): phenyl)-2-oxo-1-(pyrrolidin-1-yl)ethyl)-2H-chromen-2-one (4g).
3
423, 1612, 1396, 1062, 762; d
H
(400 MHz; CDCl
3
): 8.00 (1H, d, J Light greyish solid as a mixture of keto/enol tautomers (0.296 g,
ꢁ
¼
7.84 Hz), 7.79 (1H, s), 7.72 (1H, d, J ¼ 7.36 Hz), 7.40 (1H, t, J ¼ 75%) mp 216–218 C, HPLC (purity 98.97%): t ¼ 12.027 min; IR
R
ꢃ1
7
.96 Hz), 7.08–7.21 (4H, m), 6.53 (1H, s), 3.31 (4H, m), 2.27 (3H, (KBr) (cm ): 3401, 3019, 1646, 1385, 1215, 1069, 758, 669; dH
(100 MHz; CDCl ): 198.3, 170.7, (400 MHz; CDCl + DMSO-d ): 7.91 (1H, s), 7.40–7.60 (3H, m),
66.6, 152.8, 132.6, 131.0, 128.7, 127.6, 125.1, 123.2, 120.0, 7.32 (1H, d, J ¼ 7.48 Hz), 7.06 (2H, d, 7.8 Hz), 6.73 (1H, d, J ¼
15.6, 101.8, 46.6, 43.4, 21.3; HR-MS [M + H] (m/z) calculated 8.32 Hz), 6.01 (1H, s), 3.78 (3H, s), 2.9 (2H, involved in dmso
peak), 2.52 (2H, s), 1.97 (4H, s); d (100 MHz; CDCl + DMSO-d ):
s), 1.37 (4H, t, J ¼ 7.28 Hz); d
C
3
3
6
1
1
+
for C H NO 364.1549 found 364.1537.
2
2
21
4
C
3
6
4
.4.3 Compound 3-(2-(4-ethoxyphenyl)-2-oxo-1-(pyrrolidin- 191.2, 165.4, 159.0, 156.2, 153.1, 136.0, 127.9, 127.4, 121.0,
+
1
-yl)ethyl)-4-hydroxy-2H-chromen-2-one (4c). Light orange solid 119.7, 116.5, 95.5, 74.5, 60.6, 28.3; HR-MS [M + H] (m/z)
as a mixture of keto/enol tautomers (0.295 g, 75%) mp 163–165 calculated for C H NO 396.1447 found 396.1436.
2
2
21
6
ꢁ
ꢃ1
C, HPLC (purity 100%): t
R
¼ 12.115 min; IR (KBr) (cm ): 3396,
4.4.8 Compound
3-(2-(3,4-dimethoxyphenyl)-2-oxo-1-
1
7
3
H 3
607, 1392, 1218, 1065, 769; d (400 MHz; CDCl ): 9.01 (1H, s), (pyrrolidin-1-yl) ethyl)-4-hydroxy-2H-chromen-2-one (4h). Pale
.70–8.00 (3H, m), 7.35 (1H, s), 6.80–7.20 (3H, m), 6.53 (2H, s), yellow solid as a mixture of keto/enol tautomers (0.301 g, 74%)
ꢁ
.71 (2H, d, J ¼ 7.48 Hz), 3.49 (3H, s), 2.05 (3H, s), 1.20 1.40 (5H, mp 150–153 C, HPLC (purity 96.30%): t ¼ 12.631 min; IR (KBr)
R
ꢃ1
m); d (100 MHz; CDCl ): 196.4, 167.4, 162.2, 152.5, 131.6, 130.2, (cm ): 3401, 3019, 1646, 1601, 1546, 1463, 1401, 1385, 1270,
C
3
1
4
28.8, 124.7, 123.7, 119.1, 115.9, 114.3, 113.6, 102.4, 53.4, 46.2, 1216, 668, 757, 769; dH (400 MHz; CDCl ): 8.04 (1H, d, J ¼ 5.84
3
+
2.9, 31.4, 29.7, 24.4, 22.6, 14.5, 14.1; HR-MS [M + H] (m/z) Hz), 7.85 (1H, d, J ¼ 8.48 Hz), 7.70 (1H, s), 7.44 (1H, t, J ¼ 6.8 Hz),
5
calculated for C23H23NO 394.1654 found 394.1640.
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ꢃ1
7
.19 (2H, m), 6.85 (1H, d, J ¼ 8.48 Hz), 6.15 (1H, s), 3.83 (6H, d, J (cm ): 3399, 3019, 1662, 1513, 1403, 1216, 1158, 1068, 771, 669;
¼
6.4 Hz), 2.30–2.80 (4H, br), 2.14 (4H, s); d (100 MHz; CDCl ):
d
H
(400 MHz; CDCl ): 8.13 (2H, d, J ¼ 8.52 Hz), 6.90 (2H, d, J ¼
C
3
3
1
1
91.6, 175.5, 165.1, 154.3, 154.0, 148.7, 131.5, 126.7, 125.1, 8.32 Hz), 5.85 (1H, s), 5.63 (1H, s), 3.85 (3H, s), 3.0–3.75 (4H, br),
23.3, 122.0, 116.5, 110.9, 110.4, 91.0, 69.7, 56.0, 56.0, 23.7; HR- 2.5–3.0 (2H, br), 2.05 (3H, s), 1.92 (3H, s), 1.68 (2H, s); d (100
C
+
MS [M + H] (m/z) calculated for C23
H
23NO
6
410.1603 found MHz; CDCl
3
): 190.1, 179.3, 166.0, 164.2, 161.1, 131.9, 131.0,
410.1594.
127.0, 113.8, 107.6, 88.2, 68.3, 55.4, 23.4, 22.3, 19.7; HR-MS [M +
+
H] (m/z) calculated for C20
5
H23NO 358.1654 found 358.1662.
4
.5.5 Compound 4-hydroxy-6-methyl-3-(2-oxo-1-(piperidin-
4
.5 General synthetic procedure for 3-(2-(4-substituted
1
-yl)-2-m-tolylethyl)-2H-pyran-2-one (5e). Dark brown solid as
a mixture of keto/enol tautomers (0.191 g, 53%) mp 150–152 C,
phenyl)-2-oxo-1-(piperidin-1-yl)ethyl)-4-hydroxy-6-methyl-2H-
pyran-2-one 5a–5f
ꢁ
ꢃ1
HPLC (purity 96.98%): t ¼ 12.824 min; IR (KBr) (cm ): 3399,
R
Substituted phenyl glyoxal was reuxed with 4-hydroxy-6- 3019, 2400, 1660, 1532, 1402, 1215, 1155, 1069, 928, 770, 669; d
H
methyl-2H-pyran-2-one in the presence of piperidine as base (400 MHz; DMSO-d
and methanol as solvent for 8 h. Aer completion of the reac- 7.42 (2H, d, J ¼ 6.96 Hz), 5.90 (1H, s), 5.56 (2H, s), 2.02 (7H, s),
tion, it was quenched with water and extracted with DCM. 1.90 (1H, s), 1.24 (3H, s); d (100 MHz; DMSO-d ): 191.4, 179.2,
6
): 15.01 (1H, s), 7.66 (2H, d, J ¼ 6.92 Hz),
C
6
Organic layer was separated and dried over sodium sulfate and 166.0, 161.4, 140.3, 132.6, 129.8, 128.8, 107.5, 88.0, 68.2, 44.7,
+
concentrated in vacuo. Crude product was puried by column 29.6, 23.1, 19.7; HR-MS [M + H] (m/z) calculated for
chromatography using 100–200 mesh silica gel as adsorbent
C
19
H20NO
4
Cl 362.1159 found 362.1152.
and methanol : chloroform as eluent.
4.5.6 Compound 4-hydroxy-6-methyl-3-(2-oxo-1-(piperidin-
4
.5.1 Compound 4-hydroxy-3-(2-(4-hydroxyphenyl)-2-oxo-1- 1-yl)-2-p-tolylethyl)-2H-pyran-2-one (5f). Dark brown solid as
piperidin-1-yl)ethyl)-6-methyl-2H-pyran-2-one (5a). White solid a mixture of keto/enol tautomers (0.174 g, 51%) mp 120–122 C,
ꢁ
(
ꢃ1
as a mixture of keto/enol tautomers (0.175 g, 50%) mp 204–206 HPLC (purity 95.29%): t
R
¼ 14.186 min; IR (KBr) (cm ): 3681,
ꢁ
ꢃ1
C, HPLC (purity 98.64%): tR ¼ 11.718 min; IR (KBr) (cm ): 3400, 3019, 2399, 1663, 1606, 1529, 1429, 1297, 1215, 1160,
3
1
(
(
(
398, 3019, 1666, 1577, 1499, 1403, 1331, 1302, 1217, 1156, 1029, 928, 759, 668, 626; d
H
(400 MHz; CDCl
3
): 7.99 (2H, d, J ¼
069, 844, 771, 669, 607; d (400 MHz; DMSO-d
H
6
): 10.00–11.00 7.48 Hz), 7.20 (2H, d, J ¼ 7.16 Hz), 5.88 (1H, s), 5.63 (1H, s), 3.24
1H, br), 7.68 (2H, d, J ¼ 8.64 Hz), 6.73 (2H, d, J ¼ 8.64 Hz), 5.68 (4H, br), 2.37 (3H, s), 2.03 (3H, s), 1.90 (4H, s), 1.66 (2H, s); d
1H, s), 5.44 (1H, s), 2.51 (3H, s), 1.93 (3H, s), 1.74 (5H, br), 1.49 (100 MHz; CDCl ): 192.2, 179.2, 166.0, 161.1, 144.8, 131.7, 129.2,
2H, br); d (100 MHz; DMSO-d
): 192.3, 178.6, 165.3, 162.6, 128.6, 107.6, 68.3, 29.6, 23.2, 22.3, 21.7, 19.7; HR-MS [M + H]
60.0, 130.6, 126.3, 115.2, 107.6, 88.2, 67.0, 22.7, 22.0, 19.7; HR- (m/z) calculated for C20 . 342.1705 found 342.1713.
C
3
+
C
6
1
H23NO
4
+
MS [M + H] (m/z) calculated for C H NO 344.1498 found
1
9
21
5
3
44.1480.
4
.6 General synthetic procedure for the formation of tartrate
4.5.2 Compound 4-hydroxy-6-methyl-3-(2-oxo-1-(piperidin-
salts of compounds 2–4(a–h) and 5a–5f
1-yl)-2-m-tolylethyl)-2H-pyran-2-one (5b). Dark brown solid as
ꢁ
Compound was dissolved in methanol and 1 equivalent tartaric
acid was added to it. This mixture was stirred for half an hour
and then methanol was evaporated in vacuo.
a mixture of keto/enol tautomers (0.177 g, 52%) mp 108–110 C,
HPLC (purity 96.73%): t
ꢃ1
R
¼ 12.605 min; IR (KBr) (cm ): 3399,
H
3019, 1678, 1403, 1215, 1157, 1068, 769, 669; d (400 MHz;
CDCl
3
): 7.99 (2H, d, J ¼ 7.4 Hz), 7.20 (2H, d, J ¼ 7.4 Hz), 5.88 (1H,
s), 5.63 (1H, s), 3.24 (4H, br), 2.37 (3H, s), 2.03 (3H, s), 1.90 (4H,
5
Biological methodology
s), 1.66 (2H, s); d (100 MHz; CDCl ): 168.7, 161.1, 138.0, 136.2,
C
3
5
.1 Spermicidal assay
1
1
34.9, 133.9, 133.3, 129.0, 128.8, 127.8, 124.9, 106.9, 104.0,
01.2, 98.4, 45.0, 41.5, 22.6, 22.3, 21.3, 21.2, 19.5; HR-MS [M + Freshly ejaculated human semen obtained by masturbation
+
H] (m/z) calculated for C20
H
23NO
4
342.1705 found 342.1704.
from healthy volunteers was collected directly into a sterile
4.5.3 Compound 3-(2-(4-ethoxyphenyl)-2-oxo-1-(piperidin- plastic tube and transported immediately to the laboratory.
1
-yl) ethyl)-4-hydroxy-6-methyl-2H-pyran-2-one (5c). Dark Prior informed consent was obtained from the donors. The
ꢁ
brown solid as a mixture of keto/enol tautomers (0.204 g, 55%) samples were allowed to liquefy at 37 C for 45 min. Semen
ꢁ
mp 162–164 C, HPLC (purity 95.05%): t ¼ 13.868 min; IR (KBr) characteristics and analysis were performed according to the
R
ꢃ1
(cm ): 3396, 3018, 1653, 1603, 1541, 1395, 1217, 1175, 1033, normal criteria as per the World Health Organization guide-
7
65, 668; d
m), 5.77 (1H, s), 5.66 (1H, s), 4.07 (2H, s), 2.51 (3H, s), 1.9–2.2 >70% motility and normal sperm morphology were used to
4H, m), 1.77 (2H, s), 1.32 (4H, s); d (100 MHz; DMSO-d ): 196.2, determine the spermicidal minimum effective concentration
65.8, 163.0, 161.8, 159.6, 129.0, 128.1, 125.9, 103.7, 99.8, 64.0, (MEC) of test compounds, in vitro. Briey, the test compounds
H 6
(400 MHz; DMSO-d ): 7.6–7.8 (2H, m), 6.8–7.0 (2H, lines. Semen samples with >65 million per ml sperm count,
(
C
6
1
6
+
3.7, 44.1, 22.7, 22.6, 19.5, 14.9; HR-MS [M + H] (m/z) calculated were dissolved in a minimum volume of DMSO and diluted with
372.1811 found 372.1795.
physiological saline (0.85% NaCl in distilled water) to make 50
.5.4 Compound 4-hydroxy-3-(2-(4-methoxyphenyl)-2-oxo- mM of test solution. 0.05 ml of liqueed human semen was
-(piperidin-1-yl)ethyl)-6-methyl-2H-pyran-2-one (5d). Dark added to 0.25 mL of the test solution and vortexed for 10 s at low
for C21H25NO
5
4
1
brown solid as a mixture of keto/enol tautomers (0.186 g, 52%) speed. A drop of the mixture was then placed on a microscope
ꢁ
mp 89–91 C, HPLC (purity 95.06%): t
R
¼ 15.444 min; IR (KBr) slide, covered with a cover glass and examined under a phase
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 76288–76297 | 76295

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36
contrast microscope. The study was approved by the Institu- test compounds were added to experimental wells, and vehicle
tional Ethics Committees of King George's Medical University was added to control wells in triplicate. Approximately 1000
and CSIR-CDRI, Lucknow, India.
CFU of L. jensenii were inoculated into each well. The plates
were incubated at 37 C in a humidied atmosphere containing
ꢁ
5.2 Trichomonas vaginalis cultures and trichomonacidal
2
5% CO for 24 h. At the end of the experiment, the cultures were
assays
mixed thoroughly, 100 ml from each well was transferred to the
corresponding well of a 96-well plate, and the number of lac-
tobacilli were estimated by measuring the turbidity (OD610).
Evaluation of antimicrobial properties of compounds was
determined by trichomonacidal assay. Trichomonads used for
assay were cultured axenically in Trypticase Yeast-Extract
Maltose (TYM) (pH 6.8) supplemented with 10% heat-
inactivated FBS, 2% vitamin mixture, 100 U of penicillin per
39
Ethical statement
ꢃ1
ꢁ
ml and 100 mg mL
anaerobic conditions (with ꢀ0.5 ml air trapped above medium). the Institutional Ethics Committees of King George's Medical
streptomycin at 37 C under partial The in vitro studies involving human samples were approved by
37
Drug susceptibility assays were conducted as detailed earlier.
University, Lucknow, and the CSIR-CDRI, Lucknow, India. The
Briey, the parasites were incubated under partial anaerobic ICMR guidelines for bio-medical research were followed.
ꢁ
condition at 37 C in culture medium containing serially diluted
test compounds or MTZ, in 48-well culture plates.0.05% DMSO
in culture medium (the highest concentration of DMSO in test-
Acknowledgements
wells) was used as vehicle in the control wells. Cell viability was
checked aer 48 h by trypan blue exclusion assay and the
minimum concentration of the test agent at which all cells were
found dead was considered as its MIC. 100% eradication was
Authors are thankful to Council of Scientic and Industrial
Research (CSIR) and Jawahar Lal Nehru University (JNU) for
providing supportive working facilities and nancial base.
Some structures were tested under the ‘PROGRAM’ project.
conrmed by transferring 100 ml of the suspension to a 15 ml
Authors are also grateful to SAIF Division, CSIR-Central Drug
ꢁ
tube with fresh medium and recording the growth at 37 C. MTZ
Research Institute to give us spectral data. CDRI communica-
(
the most widely used drug against T. vaginalis), was used as
tion No. is 9291.
38
reference standard. All experiments were repeated three times.
5
.3 Cytotoxicity of compounds toward human cervical
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RSC Adv., 2016, 6, 76288–76297 | 76297