Synthesis, structure and DNA cleavage studies of coumarin analogues of tetrahydroisoquinoline and protoberberine alkaloids

Article abstract of DOI:10.1016/j.ejmech.2010.04.041

Novel molecular matrices have been derived from coumarin-4-acetic acids and β-phenylethylamines using the Bischler-Napieralski protocol which has led to the synthesis of analogues of tetrahydropapaverine in which the dimethoxybenzene moiety has been repla

Full text of DOI:10.1016/j.ejmech.2010.04.041

European Journal of Medicinal Chemistry 45 (2010) 3575e3580
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure and DNA cleavage studies of coumarin analogues of
tetrahydroisoquinoline and protoberberine alkaloids
,
Vithal B. Jadhav a, Susanta K. Nayak b, T.N. Guru Row b, M.V. Kulkarni a
*
a Department of Chemistry, Karnatak Univeristy, Pawate Nagar, Dharwad, Karnataka 580 003, India
b Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel molecular matrices have been derived from coumarin-4-acetic acids and
b-phenylethylamines
Received 20 October 2009
Received in revised form
29 April 2010
Accepted 30 April 2010
Available online 12 May 2010
using the BischlereNapieralski protocol which has led to the synthesis of analogues of tetrahy-
dropapaverine in which the dimethoxybenzene moiety has been replaced by substituted coumarins. One
carbon homologation has led to cyclization at the C3 position of coumarin generating the protoberberine
skeleton. Structures have been confirmed by diffraction studies. The results showed that compounds 6e,
6f, 7e and 7f were found to be very effective against DNA samples of Gram positive bacterium Staphy-
lococcus aureus and fungus Aspergillus niger.
Dedicated to Professor V.D. Patil on his 75th
birthday.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Coumarin-4-acetic acids
Tetrahydroisoquinolines
Protoberberines
DNA cleavage
1. Introduction
for protoberberines and these have been the subject of several
reviews [16,17]. Coumarins with diverse structural features and
Coumarin analogues of isoquinoline alkaloids represent an
unknown class of heterocyclic templates that originate from the
basic framework of biologically active compounds, mimicking the
core groups of natural products.
Isoquinolines and protoberberines have been a part of one of the
largest groups of alkaloids and have attracted a great deal of
interest over the decades due to their potent biological activities
[1e3]. Several plants and mammalian species contain simple
1,2,3,4-tetrahydroisoquinolines (THIQs). THIQs are the constituent
of several drugs as they exhibit anti-tumor [4], cardiovascular to
versatile biological properties such as antimicrobial, anticancer,
anti-inflammatory and anti-HIV activities have been recently
reviewed [18]. DNA gyrase is mainly inhibited by quinolines and
coumarins, some of which are widely used for the treatment of
bacterial infectious diseases (e.g., ciprofloxacin) [19]. Unfortunately,
of late multi-drug resistant Gram positive bacteria have started
posing serious issues in medical science to deal with. To overcome
the limitations of the known drugs, it has become imperative to
identify a new class of compounds.
However, todate, no reports havebeen dedicated tothe synthesis
and biological evaluation of coumarin analogues of isoquinoline
alkaloids. Herein, in continuation to extend our research on poly-
cyclic compounds of coumarin [20] (Fig. 1) we designed a series of
novel coumarin analougues of 1,2,3,4-tetrahydroisoquinolines and
protoberberine alkaloids. The synthesized compounds are tested for
DNA cleavage against Gram negative Escherischia coli, Gram positive
Staphylococcus aureus and fungus Aspergillus niger.
b
-adrenergic receptor antagonistic [5], antispasmodial [6], anti-
tubercular [7], antimicrobial [8e10], antifungal [11], and non-
competitive AMPA receptor antagonism [12]. Clinically, papavarine
is employed as a vasodilator because of its relaxatory effect on
vascular smooth muscle [13]. The protoberberines, tetracyclic
isoquinoline alkaloids also display a broad spectrum of biological
activities [14,15] and feature predominantly as active components
in many folklore medicines especially in China and other Asian
countries. A wide range of synthetic methods have been reported
2. Chemistry
As shown in Scheme 1, coumarin-4-acetic acids 1, were prepared
according to literature methods [21]. A coupling component, ethyl
* Corresponding author. Tel.: þ91 836 2215286; fax: þ91 836 2747884.
E-mail address: profmvk@rediffmail.com (M.V. Kulkarni).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.041

3576
V.B. Jadhav et al. / European Journal of Medicinal Chemistry 45 (2010) 3575e3580
O
O
N
H3CO
H3CO
H3CO
NH
N
H3CO
OCH3
OCH3
O
O
O
Tetrahydropapavarine
Polycyclic coumarins
(S)-Sinactine
Fig. 1. Some biologically important coumarin and isoquinoline derivatives.
ester of coumarin-4-acetic acids 2, was prepared from 1 through
esterification. The other coupling component 2-phenylethanamine
was procured commercially. The formation of the amide bond was
accomplished by refluxing the ethyl ester 2 and 2-phenylethan-
amine in the presence of toluene to furnish 3. Alternatively, micro-
wave chemistry, a non-conventional popular technique, has been
successfully employed in the preparation of 3, in which ethyl ester 2
reacted with 2-phenylethylamines in solvent free condition in
a household microwave oven to form amides 3. The amide 3 sub-
jected to BischlereNapieralski reaction in the presence of P2O5 as
a dehydrating agent afforded 3,4-dihydroisoquinoline 4 as a bright
greenish yellow solid. Because the direct reduction of 3,4-dihy-
droisoquinoline 4 with sodium borohydride was deemed too slug-
gish, 3,4-dihydroisoquinoline 4 was converted to their corresponding
hydrochloride salts 5 to accomplish smooth reduction toyield 1,2,3,4-
tetrahydroisoquinoline 6. Further, the Mannich reaction upon
compound 6 led to one carbon homologation through nitro-
genecarbonecarbon bond formation to yield 7, which possesses the
carbon skeleton of coumarin analogues of protoberberine alkaloids.
The numbering of the skeleton 7 is shown in Fig. 2.
In summary, the synthesis of the coumarin analogues of iso-
quinoline and protoberberine alkaloids was successfully carried out
with two key steps, viz., BischlereNapieralski reaction to get 3,4-
dihydroisoquinolines 4 and at a later stage Mannich reaction to
furnish the desired final product 7. All the synthesized compounds
(Table 1) were characterized by spectral analysis like 1H, 13C, 2D-
HETCOR NMR and X-ray crystal diffraction studies wherever
necessary.
2.1. X-ray diffraction studies
Single crystals of two molecules [Scheme 1, 6a and 7a] were
obtained by slow evaporation from methanol solution. Crystals
suitable for X-ray studies were selected under a polarizing micro-
scope and then mounted on a BRUKER AXS SMART APEX CCD
diffractometer [22] with a crystal to detector distance of 6.06 cm.
The data were reduced using SAINT PLUS [22] and the structures
were solved by direct methods or SIR92 [23a] and refined using
SHELXL [23b] to R values of 0.042 and 0.049 respectively. The
packing diagrams were generated using CAMERON [24]. Hydrogen
R2
R1
O
O
O
OEt
OH
N
a
H
O
b
O
O
O
O
R
R
O
3
R
2
1
R1
R2
R1
R2
Cl
O
N
N
O
O
c
d
e
O
R
R
5
4
R1
R2
R1
R2
N
NH
H
f
O
O
O
O
R
7
R
6
R = 6-Me, 7-Me, 7,8-benzo, 5,6-methylenedioxy; R1 = R2 = H and R1 = R2 = OCH3
Scheme 1. Synthetic route for the coumarin analogues of 1,2,3,4-tetrahydroisoquinoline 6 and protoberberine alkaloids 7. Reagents and conditions: (a) ethanol, H2SO4, reflux, 24 h;
(b) 2-phenethylamines, toluene, reflux, 6 h; (c) P2O5, dry xylene, reflux, 12 h; (d) 20% HCl, heat; (e) NaBH4, methanol, RT, 1 h; (f) HCHO/AcOH, reflux, 12 h.

V.B. Jadhav et al. / European Journal of Medicinal Chemistry 45 (2010) 3575e3580
3577
10
13
9
11
12
8
N
7a
7
13b
14
6
O
O
5
1
2
4
3
Fig. 2. Numbering of the skeleton.
atoms were located by using difference Fourier map and refined
isotropically. However, in cases where location was not possible,
the H-atoms were fixed in geometrically calculated positions.
The crystallographic information of two structures is available in
CCDC [25].
Fig. 3. ORTEP diagram with the displacement ellipsoids at 30% probability level (6a).
Among the two crystals, compound 6a crystallizes in an
orthorhombic system, space group P 212121, whereas the rest of
the molecules crystallize in the centrosymmetric monoclinic
system, space group P 21/n. ORTEP [26] diagrams of both the
systems are shown in Figs. 3 and 4. All the crystal structures
are stabilized via a network of both weak and strong hydro-
pictures are shown in Figs. 5e7. The photograph (Fig. 5) shows the
molecular weight differences compared to control and is the
differentiating criterion for the DNA cleaving ability of the tested
compounds with S. aureus. Control experiments using DNA alone
does not indicate any significant cleavage of DNA even after long
exposure time. After marker M and control C, the first six lanes
correspond to tetrahydroisoquinolines and in this series the
compounds 6ae6d with methyl groups in the coumarin moiety
and corresponding to unsubstituted and 6,7-dimethoxy substi-
tuted isoquinoline moiety do not show any changes when
compared with the control. It is interesting to note that the
7,8-benzo substituted derivates 6e and 6f show difference in
molecular weight and tailing to a great extent which could be
attributed to the DNA cleavage. Even the corresponding penta-
cyclic protoberberines 7ae7d have yielded the same result. The
benzo substituted derivates 7e and 7f have clearly showed the
absence of the marker band thus providing a proof for their DNA
cleaving ability of S. aureus.
gen bonds with weak intermolecular CeH/p and p/p inter-
actions (see supplementary information; Tables S1eS2 and
Figures S1eS2).
3. Pharmacology
The DNA cleavage studies of all the reported coumarin
analogues of 1,2,3,4-tetrahydroisoquinolines (6ae6f) and the cor-
responding protoberberines (7ae7f) have been carried out against
S. aureus 6538, E. coli ATCC 35218 and fungus A. niger by agarose gel
electrophoresis method.
Gel electrophoresis technique works on the migration of DNA
under the influence of electric potential. Gel electrophoresis
It is of considerable interest to see that all the twelve
compounds 6ae6f and 7ae7f did not show any interaction with the
DNA of E. coli which shows that these compounds are inactive
against Gram negative bacterial species (Fig. 6).
Against fungal DNA of A. niger, the lanes corresponding to tet-
rahydroisoquinolines 6ae6d with methyl groups on the coumarin
Table 1
Coumarin analogues of 1,2,3,4-tetrahydroisoquinolines and protoberberine skele-
tons with two points of structural diversity.
Entry
R
R1
R2
Isolated yields (%)
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
6-CH3
6-CH3
7-CH3
7-CH3
7,8-benzo
7,8-benzo
6,7-OCH2O
6-CH3
6-CH3
7-CH3
H
OCH3
H
OCH3
H
OCH3
OCH3
H
OCH3
H
OCH3
H
OCH3
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
H
OCH3
H
OCH3
H
OCH3
OCH3
H
OCH3
H
OCH3
H
OCH3
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
83
80
82
82
76
77
75
75
50
70
77
67
71
65
74
78
70
72
74
77
77
76
70
74
72
76
7-CH3
7,8-benzo
7,8-benzo
6,7-OCH2O
6-CH3
6-CH3
7-CH3
7-CH3
7,8-benzo
7,8-benzo
2-CH3
2-CH3
3-CH3
3-CH3
3,4-benzo
3,4-benzo
Fig. 4. ORTEP diagram with the displacement ellipsoids at 30% probability level (7a).
OCH3
OCH3

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V.B. Jadhav et al. / European Journal of Medicinal Chemistry 45 (2010) 3575e3580
Fig. 5. Gel electrophoresis picture of coumarin analogues of isoquinoline alkaloids. Photograph showing the effect of representative compounds on DNA of S. aureus. Lane M: DNA
marker, Lane C: untreated DNA, Lanes 6A, 6B, 6C, 6D, 6E, 6F, 7A, 7B, 7C, 7D, 7E and 7F correspond to compounds 6a, 6b, 6c, 6d, 6e, 6f, 7a, 7b, 7c, 7d, 7e and 7f respectively.
Fig. 6. Gel electrophoresis picture of coumarin analogues of isoquinoline alkaloids. Photograph showing the effect of representative compounds on DNA of E. coli. Lane M: DNA
marker, Lane C: untreated DNA, Lanes 6A, 6B, 6C, 6D, 6E, 6F, 7A, 7B, 7C, 7D, 7E and 7F correspond to compounds 6a, 6b, 6c, 6d, 6e, 6f, 7a, 7b, 7c, 7d, 7e and 7f respectively.
ring corresponding to unsubstituted and dimethoxy substituted
isoquinolines and the corresponding protoberberines 7ae7d show
no conspicuous differences in bandwidth or molecular weight
indicating their inertness towards the fungal DNA (Fig. 7). The
corresponding benzo analogues 6e, 6f, 7e and 7f were found to
exhibit considerable molecular weight differences and tailing and
absence of marker which showed that these compounds have the
ability to cleave the fungal DNA.
In conclusion it can be seen that compounds exhibit specific
affinity for the Gram positive bacterial and fungal species. These
structure activity relationship studies also indicate that among the
tetrahydroisoquinolines 6ae6f and the polycyclic protoberberines
7ae7f, the 7,8-benzo substitution which is also naturally occurring
is associated with significant DNA cleaving activity which is prob-
ably due to the angularly fused cyclic skeleton. However, the nature
of the reactive intermediates involved in the DNA cleavage by the
compounds has not been clear. As these compounds observed to
cleave the DNA, it can be concluded that the compounds 6e, 6f, 7e
and 7f inhibit the growth of the pathogenic organisms by cleaving
the genome.
4. Conclusion
A series of novel coumarin analogues of 1,2,3,4-tetrahydoiso-
quinolines and protoberberine alkaloids 6 and 7 were synthesized
via intermediates 3, 4 and 5 were well characterized and evaluated
for DNA cleavage studies by agarose gel electrophoresis method
against Gram positive bacteria S. aureus ATCC 6538, Gram negative
bacteria E. coli ATCC 35218 and fungus A. niger. The results show
that compounds 6e, 6f, 7e and 7f show selectivity towards the
Gram positive bacteria S. aureus and A. niger. These compounds can
act as potent antibacterial agents by genomic cleavage.
5. Experimental
5.1. Analysis and instruments
The melting points were determined by open capillaries on
a Buchi-apparatus and are uncorrected. The IR spectra were
recorded on a Nicolet-Impact-410 FT-IR spectrometer, using KBr
pellets.1H NMR and 13C NMR spectra were recorded respectively,
Fig. 7. Gel electrophoresis picture of coumarin analogues of isoquinoline alkaloids. Photograph showing the effect of representative compounds on DNA of A. niger. Lane M: DNA
marker, Lane C: untreated DNA. Lanes 6A, 6B, 6C, 6D, 6E, 6F, 7A, 7B, 7C, 7D, 7E and 7F correspond to compounds 6a, 6b, 6c, 6d, 6e, 6f, 7a, 7b, 7c, 7d, 7e and 7f respectively.

V.B. Jadhav et al. / European Journal of Medicinal Chemistry 45 (2010) 3575e3580
3579
using Bruker 300 MHz and 75 MHz spectrometer. All the chemicals
purchased from SigmaeAldrich were used without further purifi-
cation. The mass spectra were recorded using Agilent Single quartz
LC MS. The elemental analysis was carried out using Heraus CHN
rapid analyzer.
5.3.3.1. 6-Methyl-4-(1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-
coumarin-2-one (6a). Mp. 140e142 ꢀC, IR (KBr) nmax/cmꢁ1: 3349,
3022, 2950,1703 cmꢁ1; 1H NMR (300 MHz, CDCl3):
d 1.7 (s (br), NH),
2.46 (s, 3H, C60-CH3), 2.89 (m, 2H, C4-CH2), 3.05 (dd, 1H, J ¼ 10.2,
4.2 Hz, C3-CH2), 3.02 (dd, 1H, J ¼ 8.1, 3.9 Hz, C
a-CH2), 3.24 (dd, 1H,
J ¼ 12.0, 6.3 Hz, C -CH2), 3.40 (dd, 1H, J ¼ 12.0, 2.4 Hz, C3-CH2), 4.41
a
5.2. X-ray studies
(dd, 1H, J ¼ 12.0, 7.8 Hz, C1-H), 6.41 (s, 1H, C30-H), 7.18e7.39 (m, 5H,
Ar-H), 7.56 (s, 1H, C50-H), 7.38 (d, 1H, C80-H); 13C NMR (75 MHz,
X-ray diffraction study was performed on a BRUKER AXS SMART
APEX CCD diffractometer.
CDCl3): d 21.5, 30.0, 39.1, 40.5, 54.9, 116.2, 117.6, 119.4, 124.6, 126.4,
126.5, 127.1, 130.1, 133.2, 134.3, 135.7, 138.1, 152.4, 153.5, 161.2; LC
MS: 306 [M þ 1]þ, 131 [100%]; Anal. cald. for C20H19NO2 (%): C,
78.66; H, 6.27; N, 4.59. Found: C, 78.84; H, 6.37; N, 4.73.
5.3. Syntheses
5.3.1. General procedure for the preparation of compound (3)
Ethyl coumarin-4-acetate (0.002 mol, 1 equiv) was taken in
10 mL of dry toulene. Added 2-phenylethanamine (0.006 mol,
3 equiv) and refluxed for 6 h. Solvent removed under reduced
pressure. The residue obtained was taken in a small quantity of
alcohol and on cooling a colorless solid separated out. Filtered and
the solid was washed several times with cold ethanol. Further the
pure product was obtained by recrystallization in hot ethanol.
5.3.4. General procedure for the synthesis of (7)
6-Methyl-4-(1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-coum-
arin-2-one (1 g, 0.003 mol) was refluxed with 20 mL of formalin and
1 mL of acetic acid for 12 h. Reaction mixturewas cooled and basified
with liquor ammonia carefully. Violet colored solid separated was
column purified using hexane/ethyl acetate (8/2) to afford the
colorless crystalline solid 2-methyl-8,9,13b,14-tetrahydro-7H-5-
oxa-7a-aza-dibenzo[a,j] anthracene-6-one (0.8 g, 77%).
5.3.1.1. 2-(6-Methyl-2-oxo-2H-coumarin-4-yl)-N-phenethyl-acet-
amide (3a). Mp. 178e180 ꢀC, IR (KBr) nmax/cmꢁ1: 3284 (NH), 3082,
2918, 1732 (nC¼O lactone), 1640 (nC¼O amide), 1574, 1498, 908,
5.3.4.1. 2-Methyl-8,9,13b,14-tetrahydro-7H-5-oxa-7a-aza-dibenzo[a,
j]anthracene-6-one (7a). Mp. 173e176 ꢀC; IR (KBr) nmax/cmꢁ1
:
3056, 2927, 1710; 1H NMR (300 MHz, CDCl3):
d 2.44 (s, 3H, C2-CH3),
826 cmꢁ1; 1H NMR (300 MHz, CDCl3):
d
2.42 (s, 3H, C6-CH3), 2.75 (t,
2.73e2.90 (m, 3H, C9-H, C14-H, C8-H), 3.18e3.26 (m, 2H, C9-H, C8-
H), 3.42e3.51 (m, 2H, C14-H, C7-H), 3.76e3.79 (m, 1H, C13b-H),
4.06e4.11 (m, 1H, C7-H), 7.19e7.38 (m, 7H, Ar-H); 13C NMR
2H, J ¼ 6.5 Hz, Ar-CH2), 3.53 (t, 2H, J ¼ 6.5 Hz, N-CH2), 3.64 (s, 2H,
coumarin-C4-CH2), 5.81 (s (br), 1H, NH, D2O exchangeable), 6.28 (s,
1H, C3-H), 7.43 (s, 1H, C5-H), 6.98e7.39 (m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3): d 21.4, 29.7, 33.4, 51.0, 53.8, 58.4, 117.0, 119.5,121.8,
(75 MHz, CDCl3):
d
21.4, 35.7, 40.9, 41.2, 117.1, 117.4, 118.8, 125.0,
123.4, 125.7, 126.6, 126.9, 129.5, 132.2, 134.3, 135.0, 137.1, 146.0,
150.9, 160.6; LC MS: 317 [Mþ], 132 [100%]; Anal. cald. for C21H19NO2
(%): C, 79.47; H, 6.03; N, 4.41. Found: C, 79.62; H, 6.18; N, 4.53.
127.0, 128.9, 128.9, 129.0, 129.0, 133.7, 134.8, 138.7, 149.6, 152.2,
160.9, 167.6; LC MS: 322 [M þ 1]þ; Anal. cald. for C20H19NO3: C,
74.75; H, 5.96; N, 4.36. Found: C, 74.84; H, 5.78; N, 4.48.
5.4. Bioassay conditions
5.3.2. General procedure for the synthesis of (4)
2-(2-Oxo-2H-coumarin-4-yl)-N-phenethyl-acetamide (2 g) was
taken in 50 mL of dry xylene and 10 g of P2O5 as dehydrating agent.
This mixture was refluxed for 10 h on an oil bath at 150 ꢀC. Reaction
mixture was cooled and carefully poured over 1000 mL ice-cold
water. Separated solid was filtered and strongly basified with
ammonia. In some cases product was soluble in water then aqueous
layer was washed with benzene (20 mL ꢂ 2) and strongly basified
with ammonia to afford the bright yellow colored solid which was
filtered, dried and purified over column chromatography.
5.4.1. DNA cleavage experiment
5.4.1.1. Preparation of culture media. DNA cleavage experiments
were done according to the literature [27]. Nutrient broth [peptone,
10; NaCl, 10 and Yeast extract, 5 (g/L)] was used for culturing of E.
coli (Gram negative) and S. aureus (Gram positive) and potato
dextrose broth [potato, 250; dextrose, 20; in g/L] was used for the
culture of A. niger. The 50 mL media was prepared, autoclaved for
15 min at 121 ꢀC, 15 lb pressure. The autoclaved media were inoc-
ulated with the seed culture. E. coli and S. aureus were incubated for
24 h and A. niger for 48 h at 37 ꢀC.
5.3.2.1. 4-(3,4-Dihydro-isoquinoline-1-ylmethyl)-6-methyl-
coumarin-2-one (4a). Mp. 148e150 ꢀC, IR (KBr) nmax/cmꢁ1
:
5.4.1.2. Isolation of DNA. The fresh bacterial culture (1.5 mL) is
centrifuged to obtain the pellet which is then dissolved in 0.5 mL of
lysine buffer (100 mM Tris pH 8.0, 50 mM EDTA, 10% SDS). To this
0.5 mL of saturated phenol was added and incubated at 55 ꢀC for
10 min. Then centrifuged at 10,000 rpm for 10 min and to the
supernatant, equal volume of chloroform:isoamyl alcohol (24:1)
and 1/20th volume of 3 M sodium acetate (pH 4.8) were added.
Then centrifuged at 10,000 rpm for 10 min and to the supernatant,
3 volumes of chilled absolute alcohol was added. The precipitated
DNA was separated by centrifugation and the pellet was dried and
dissolved in TAE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and stored
in cold condition.
1711 cmꢁ1(C¼O for lactone carbonyl), 1661 cmꢁ1 (C¼N); 1H NMR
(300 MHz, CDCl3):
d
2.43 (s, 3H, C60-CH3), 2.77 (t, 2H, J ¼ 7.2 Hz, C3-
CH2,), 3.78 (t, 2H, J ¼ 7.2 Hz, C4-CH2), 4.21 (s, 2H,
a-CH2), 6.22 (s, 1H,
C30-H), 7.26e7.86 (m, 7H, Ar-H); LC MS: 304 [M þ 1]þ; Anal. cald.
for C20H17NO7 (%): C, 79.19; H, 5.65; N, 4.62. Found: C, 79.87; H,
5.78; N, 4.73.
5.3.3. General procedure for the synthesis of (6)
Compound 4 (5 g) was treated with 20% HCl (100 mL) and on
heating clear solution was resulted which was filtered and allowed
to cool. Colorless to brown crystals separated out in the form of
their corresponding hydrochlorides 5. These were used further
without purification.
5.4.1.3. Agarose gel electrophoresis. Cleavage products were ana-
To the hydrochloride of 5a (2 g, 0.0058 mol) in methanol (30 mL)
was added sodium borohydride (1 g) gradually during 10 min
interval. The reaction mixture was stirred at room temperature for
1 h (TLC monitored). The colorless solid separated was filtered,
washed with cold water, dried and recrystallized from ethanol.
lysed by agarose gel electrophoresis method [27]. Test samples
(1 mg/mL) were prepared in DMF. The samples (50
to isolated DNA of S. aureus, E. coli and A. niger. The samples were
incubated for 2 h at 37 ꢀC and then 20
L of the DNA sample (mixed
with bromophenol blue dye at 1:1 ratio) was loaded carefully into
mg) were added
m

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Acknowledgements
We are thankful to Astra-Zeneca Research Foundation, Banga-
lore for the financial support for this work. One of the authors VBJ
thanks Karantak University Dharwad for University Research
Studentship. We also thank USIC, Karnatak University Dharwad for
analytical data and DST-IRHPA for the CCD X-ray facility at IISc,
Bangalore.
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Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2010.04.041.
References and notes
ratory, University of Oxford, England, 1993.
[25] Crystal data 6a: C20H19N1O2, M ¼ 305.4, orthorhombic, space group P 212121,
a ¼ 8.357(1) Å, b ¼ 12.208(2) Å, c ¼ 16.021(3) Å, V ¼ 1634.6(1) Å3, Z ¼ 4,
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rcalcd ¼ 1.24 g cmꢁ3, F(0 0 0) ¼ 647.9,
m
¼ 0.080 mmꢁ1, R ¼ 0.042, wR ¼ 0.101,
GOF ¼ 1.087 for 1375 reflections with I > 2
s(I), CCDC 671669. 7a: C21H19N1O2,
M ¼ 317.4, monoclinic, space group P 21/c, a ¼ 10.428(1) Å, b ¼ 20.159(2) Å,
c ¼ 7.704(1) Å,
(0 0 0) ¼ 671.9,
b
m
¼ 94.731(2)ꢀ, V ¼ 1614.1(1) Å3, Z ¼ 4, rcalcd ¼ 1.31 g cmꢁ3, F
¼ 0.084 mmꢁ1, R ¼ 0.049, wR ¼ 0.117, GOF ¼ 1.072 for 2260
reflections with I > 2s(I), CCDC 671672. The CIF files have been submitted to
the Cambridge Crystallographic Data Centre. These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,
UK; fax: þ44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk).
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