A Garratt-Braverman cyclization route towards the synthesis of phenanthridine derivatives and their DNA-binding studies

Article abstract of DOI:10.1016/j.tet.2019.02.020

Garratt-Braverman cyclization has been employed to synthesize a series of dihydroisofuran fused phenanthridine derivatives. The established protocol proposes a simpler synthetic alternative to have access to these therapeutically relevant cytotoxic scaffo

Full text of DOI:10.1016/j.tet.2019.02.020

Accepted Manuscript
A Garratt-Braverman cyclization route towards the synthesis of phenanthridine
derivatives and their DNA-binding studies
Arundhoti Mandal, Prabuddha Bhattacharya, Amit K. Das, Amit Basak
PII:
S0040-4020(19)30170-X
https://doi.org/10.1016/j.tet.2019.02.020
TET 30143
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 November 2018
Revised Date: 6 February 2019
Accepted Date: 8 February 2019
Please cite this article as: Mandal A, Bhattacharya P, Das AK, Basak A, A Garratt-Braverman cyclization
route towards the synthesis of phenanthridine derivatives and their DNA-binding studies, Tetrahedron
(2019), doi: https://doi.org/10.1016/j.tet.2019.02.020.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A Garratt-Braverman Cyclization Route towards the
Leave this area blank for abstract info.
Synthesis of Phenanthridine Derivatives and their
a+
b+
a+
DNA-Binding Studies Arundhoti Mandal* , Prabuddha Bhattacharya* , Amit Kumar Dasc and Amit Basak*
O
Endo
OH
O
R
R
N
N
N
H, Me, CH2OH, CHO,
OH
,
R
,
N
ORTEP view of crystal
structure of Endo Product
Hypochromic shift in UV-Vis titration curve
O
N
OMe
O
R
R
N
N
Exo
H, Me, CH2OH, CHO,
,
R
,
N
ORTEP view of crystal
structure of Exo Product
Fluorescence quenching in
Ethidium Bromide displacement assay
OMe

ACCEPTED MANUSCRIPT
A Garratt-Braverman Cyclization Route towards the
Synthesis of Phenanthridine Derivatives and their
DNA-Binding Studies
Arundhoti Mandal*a+, Prabuddha Bhattacharya*b+, Amit K. Dasc and Amit Basak*a+
aDepartment of Chemistry, c Department of Biotechnology, Indian Institute of Technology Kharagpur
721302 India
bDepartment of Chemistry, Adamas University, Kolkata 700126 India
+ equal contribution
* Corresponding author
+arundhoti@chem.iitkgp.ernet.in; prabuddha.bhattacharya@adamasuniversity.ac.in;
absk@chem.iitkgp.ernet.in
Abstract
Garratt-Braverman cyclization has been employed to synthesize a series of dihydroisofuran fused
phenanthridine derivatives. The established protocol proposes a simpler synthetic alternative to have
access to these therapeutically relevant cytotoxic scaffolds. Single crystal X-ray data unambiguously
confirmed the structures of the synthesized phenanthridine derivatives. UV-Vis absorption titration with
calf-thymus DNA followed by fluorescence-based competitive ethidium bromide displacement assay
established the synthesized target compounds as potent DNA-intercalating agents with intrinsic binding
constant of the range 103-105. Results obtained from the molecular docking further justified the
spectroscopically obtained results.
Keywords: Phenanthridine, Garratt-Braverman cyclization, UV-Vis absorption, Fluorescence, Ethidium
bromide, Docking
Introduction
Phenanthridines represent a privileged class of angularly fused heterocyclic compounds having high
significance in medicinal chemistry and materials science. Many naturally occurring bioactive alkaloids1a-
f
such as trispheridine, asiaticumine A, nornitidine, decarine, norchelerythrine, noravicine,
norsanguinarine (1-7) contain phenanthridine skeleton (Figure 1). Phenanthridine derivatives exhibit a
broad spectrum of biological activities such as antibacterial, antitumoral, antileukemic, SPECT scan
1

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tracer (ligands for the brain imaging of 5-HT4 receptors), Bcl-XL inhibitors, and other DNA-binding
cytotoxic activities.2a-f Phenanthridinium moiety is extensively used as gold-standard DNA- and RNA-
fluorescent markers (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide known as ethidium
bromide), and probes for cell viability (propidium iodide).3a-g Owing to their extensive π-conjugated
electron system, fused phenanthridine derivatives have also found application in the development of new
organic light-emitting materials and new semiconductors.4a-e
Figure 1. Alkaloids with phenanthridine scaffold
Because of the above reasons, extensive research has been done to develop simple, cost-effective and
selective methodology for the synthesis of phenanthridines. Recently several methods5a-s for synthesizing
phenanthridine moiety are reported for improving reaction condition and yield. These works have been
summarized by Keller.5a Some recent works to synthesize phenanthridine derivatives involve palladium
catalyzed cascade transition-metal-catalyzed cyclization of imines, annulations using arynes, applying
UV radiation to iminyl radicals, photochemical processes, and cyclizations using microwave.5a-s In spite
of all these developments, the overall yield of these newly developed methods are less than satisfactory.
Bicyclization reactions involving radicals or ions have always been an important synthetic tool to
construct diverse structural scaffolds.6 Recent example includes bicyclization involving diynes leading to
the stereoselective formation of naphtho[1,2-c]thiophene 2,2-dioxides as reported7 by Wang et al.
Incidentally, Garratt-Braverman (GB) cyclization8a-r has emerged to be synthetically simple, base
mediated bicyclization reaction involving the formation of a new 6-membered ring fused to a 5-
membered heterocycle. Herein, we report a simple high yielding synthesis of dihydrofuran fused
2

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phenanthridine using GB) cyclization as the key cycloaromatization step. The reaction goes via either a
mono-allene or a bis-allene intermediate depending upon the nature of substrate. Which of the two rings
attains aromaticity (leading to the formation of products 9 or 10) depends on the pKa difference of the
propargylic hydrogens (indicated in structure 8 by the arrows in Figure 2) in the two arms.9 In our earlier
work, we have reported10 the formation of furan fused dihydrocarbazole derivatives (the new 6-membered
ring is non-aromatic) via the GB cyclization of indolyl propargyl ethers involving a bis-allene
intermediate. Substituting the electron donating indole moiety with electron withdrawing quinoline or
isoquinoline should widen the gap in acidity of propargyl hydrogens in the two arms and hence fully
aromatic phenanthridines with a fused dihydroisofuran moiety are expected to be formed via GB
cyclization involving mono-allene. Our assertion was proved to be correct and in the process, several
phenanthridine derivatives have been synthesized. The fused isofuran moeity may act as an additional
handle for functionalization and may help in further modification leading to other bioactive motifs (e.g.
oxidation to butyrolactone or butyrolactol).8o,11a-d The synthesized compounds also showed good DNA-
binding affinities via predominantly intercalative mode. The synthesis and DNA binding affinity
including in silico docking studies are described in this paper.
O
R1
R1
O
O
R
R1
8
9
10
R
R
R1 = 3-indolyl
R1 = quinolinyl/isoquinolinyl
(Obatined for R = H)
(Obtained for R = H/alkyl/aryl)
Figure 2. Fate of GB cyclization of mono-heteroaryl propargyl ethers
Results and discussion
Retrosynthetic analysis revealed that phenanthridine derivatives (endo 11 and exo-isomers 15) can be
obtained using GB cyclization of bis-propargylic ethers (12 and 16) which in turn can be synthesized
using base mediated O-alkylation of the corresponding propargyl alcohols (13 and 17). Sonogashira
coupling12 of 3-bromo quinoline (14)/4-bromo isoquinoline (18) could afford the desired propargyl
alcohols (Scheme 1).
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O
R
O
OH
Br
i
ii
iii
R
13
12
N
N
11
N
14
Br
N
endo-isomer
O
O
OH
i
ii
iii
R
16
17
N
R
N
N
18
15
N
i) GB cyclization ii) O-alkylation iii) Sonogashira coupling
exo-isomer
Scheme 1. Retrosynthetic analysis of dihydroisofuran fused phenanthridine derivatives
A careful analysis revealed that for bis-propargyl ethers of 3-substituted quinoline derivative (12), the GB
cyclization could take place either in angular fashion via path ‘a’ (involving C3-C4 double bond) to
afford the phenanthridine derivative (11), or in linear fashion via path ‘b’ (involving C2-C3 double bond)
to afford acridine derivative (25) (Scheme 2). It was expected that the phenanthridine derivative (formed
via path ‘a’ involving C3-C4 double bond) would be obtained selectively in these cases. This regio-
selectivity expected during the cycloaromatization could be explained from the canonical structures of
quinoline moiety (12, 26 and 27). These canonical structures indicate that double bond character of the
C3-C4 bond is higher than C2-C3 bond. So the cyclization was expected to occur in angular fashion
(involving the C3-C4 bond with greater double bond character) resulting in the formation of
phenanthridine moiety. Another way to explain this regioselectivity is to invoke the loss of resonance
energy in forming the intermediates which will be much more in case of involvement of the C2-C3
double bond. Cyclization involving C3-C4 bond was also obtained for the 4-subtituted isoquinoline
derivative (16) which could be also rationalized on the basis of similar argument (greater double bond
character of C3-C4 as evident from the canonical structures 16, 28 and 29). However it is worth
mentioning that unlike 12, for 16 there are no other possible sites for the GB cyclization to occur other
than through C3, hence no issue of regio-selectivity arises for the isoquinoline derivative 16.
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Scheme 2. Regioslectivity observed during Garratt-Braverman cyclization
Accordingly, we synthesized a series of quinoline (36, 37, 38, 39a-b, 40) and isoquinoline (43, 44, 45,
46a-b, 47) based bis-propargyl ether derivatives (as GB precursors) following the synthetic protocol
shown in Scheme 3 and Scheme 4. The first step involved Sonogashira coupling between 3-bromo
quinoline/4-bromo isoquinoline (14 and 18 respectively) and propargyl alcohol to afford the
corresponding alcohols 13 and 17 (Scheme 3). The various propargyl bromide derivatives 32a, 32b, 35
were synthesized separately according to Scheme 3. Subsequent O-propargylation of the alcohols (13 and
17) with the suitable propargyl bromides in presence of NaH in dry THF at room temperature (Scheme 4)
afforded the mono-quinoline/mono-isoquinoline based bis-propargyl ether derivatives (36, 38, 39a-b, 40,
43, 45, 46a-b, 47). For the synthesis of the bis-quinoline/bis-isoquinoline based bis-propargyl ether
derivatives (37 and 44 respectively), Sonogashira coupling was carried out between mono-
quinoline/mono-isoquinoline bis-propargyl ether derivatives (36 and 43 respectively) and 3-
bromoquinoline/4-bromo isoquinoline (14 and 18 respectively). Finally these synthesized bis-propargyl
ether derivatives (36, 37, 38, 39a-b, 40, 43, 44, 45, 46a-b, 47) were subjected to GB cyclization by
5

t
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treatment with KO Bu in toluene, under refluxing conditions for 2-3 h which furnished the
dihydroisofuran fused phenanthridine derivatives exclusively in high yield (41, 48, 50, 51, 52, 53a-b, 55,
56, 57, 58a-b). The exclusive formation of phenanthridine derivatives justified our idea regarding the
regioselectivity observed in GB cyclization. To increase the structural diversity, aldehyde substituted
phenanthridines 54 and 59 were also synthesized (via the alcohols 42 and 49 as intermediates) as shown
in Scheme 4.
Scheme 3. Synthesis of the required propargyl alcohols (13, 17) and bromides (32a, 32b, 35)
NOESY spectra of compound 52 confirmed the angular cyclization of the propargyl ether to furnish
phenanthridine moiety (Figure 3). The through-space interaction between methyl proton and aromatic
proton Hf confirmed their structural proximity as found in compound 52. This type of interaction should
be absent for the analogous acridine derivative (25, with R=Me).
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iii
O
N
90%
O
50
OH
O
O
i
iii
37
36
38
N
N
N
78%
72%
87%
N
N
CH3
R
13
O
CH3
iii
iv
O
N
N
51
52
N
92%
72%
v
53a, 53b
R=H, 53a
O
iii
39a, 70%
39b, 74%
39 a, 39b
53a, 92%
53b, 87%
R=OMe, 53b
N
N
R
R=H, 39a
R=OMe, 39b
O
O
N
O
O
HO
OTHP
iii
vi
viii
90%
vii
OHC
80%
OTHP
88 %
95%
O
40
N
N
N
Br
41
H3C
42
54
1-bromo-2-butyne
iii
60
89%
OH
O
O
55
N
O
O
i
iii
92%
73%
N
70%
N
N
43
44
N
N
O
17
iv
iii
N
O
N
56
57
CH3
CH3
75%
45
90%
N
v
58a, 58b
iii
O
O
R=H,58a
R=OMe, 58b
46a, 73%
46b, 70%
58a, 91%
58b, 88%
N
46a, 46b
N
R=H, 46a
R=OMe, 46b
R
O
O
OH
R
O
CHO
vi
viii
iii
93%
vii
O
OTHP
N
98%
92%
N
78%
N
48
N
47
59
49
OTHP
Reagents and conditions: i) NaH, Dry THF, 00 C- r.t, 6h, propargyl bromide; ii) 14 (for the synthesis of 37)/18 (for the synthesis
of 44), Pd(PPh3)2Cl2,nBuNH2, 900 C, 3h; iii) KOtBu, toluene, reflux, 2h; iv) NaH, Dry THF, 00 C- r.t, 6h, 1-bromo-2-buyne (60 shown
in the inset); v) NaH, Dry THF, 00 C- r.t, 6h, 32a/32b; vi) NaH, Dry THF, 00 C- r.t, 6h, 35; vii) PPTS, EtOH, 550 C; viii) Dess-Martin
Periodinane, Dry DCM, r.t
Scheme 4. Synthesis of dihydroisofuran fused phenanthridine derivatives using Garratt-Braverman cyclization
The structures were further confirmed from X-ray crystallography. The single crystal X-ray diffraction
was obtained for compounds 52, 53a, 58a, 58b. ORTEP view of the crystal structures of these
compounds are shown in Figure 4. Crystal structures also established the planar geometry of the
synthesized phenanthridine skeletons.
7

’’
H / Hc’/ Hd/ Hd
Me proton
ACCEPTEcD MANUSCRIPT
He
Hh
Hg
Hf
Hi
Hj
Interaction CH3
and Hf
Interaction between
Me protons and Hf
'
Hc
Hc
O
Hd
Hd
'
H3C
Hf
Hi
Hj
Hh
N
He
Hg
5.053
Interaction between Me protons and H
Interaction between CH and H
f
3
f
Figure 3. NOESY spectra of compound 52 to establish the regioslectivity observed during Garratt-Braverman cyclization
Compound 53a
Compound 52
Compound 58a
Compound 58b
Figure 4. ORTEP view of single-crystal structures of compound 52, 53a, 58a, 58b
DNA Binding Studies: Since the crystallographic studies suggested a planar structure for the synthesized
phenanthridine derivatives, we expected the compounds to show potent cytotoxic acitivities via DNA-
intercalation.13a-b To validate our idea we went forward to study their interaction with DNA through
UV/Vis absorption titration14a-b,13a,8o and fluorescence based competitive ethidium bromide displacement
8

assay15a-d,13a,8o. All studies were carried out using Tris-HCl buffer (pH 7.2). In case of UV–Vis absorption
ACCEPTED MANUSCRIPT
titration study, the concentration of the compounds (42, 49, 50, 51, 52, 53a, 53b, 54, 55, 56, 57, 58a and
58b) (dissolved in acetonitrile-buffer) were kept fixed, while known concentrations of CT DNA solution
were added into both the cuvettes in increasing amounts until saturation in hypochromism was observed
(Figure 5 shows the hypochromic shifts observed for compounds 53a, 53b and 42). The concentration of
DNA used during titration varied in the range 2.7 µM - 100 µM. Absorbance values were recorded after
each successive addition of DNA solution and equilibration. The observed hypochromic shift was
14a-b,13a, 8o
suggestive of DNA-intercalation.
Compound 59 could not be studied for its DNA-binding
ability because of solubility problems. The data were fitted to modified Benesi-Hildebrand equation16
(Equation 1) obtain the intrinsic binding constant (Figure 6 shows the linear-fits and the corresponding
binding constants obtained for compounds 53a, 53b and 42).
1
1
1
1
1
1
1
=
(Equation 1)
+
[compound]
(ɛb- ɛf) [compound]0
(A0-A)
[DNA]
(ɛb- ɛf)
Kb
0
In this equation, A and A0 are the absorbances of the compound in the presence and absence of DNA,
respectively, ɛb and ɛf are the molar extinction coefficient of compound-DNA complex and free
compound, [compound]0 is the concentration of the compound used and [DNA] is the concentration of
CT-DNA added. Ratio of intercept to slope from the plot of 1/(A0-A) vs. 1/[DNA] gave us the intrinsic
binding constant (Kb).
Compound 53a
Compound 53b
[DNA]: 2.7-99.1 µM
Compound 42
Compound 53a
Compound 53b
[DNA]: 2.7-21.6 µM
2.7-21.6 µM DNA
[DNA]: 2.7-24.3 µM
2.7-24.3 µM DNA
Compound 42
2.7-99.1 µM DNA
Figure 5. UV-Vis absorption titration spectra of the compounds (denoted on their respective spectrum) in the presence of CT
DNA. In all cases, the uppermost spectrum represents the compound alone in the absence of DNA. The lower spectra
(hypochromic shift) were obtained by the gradual increase in the concentration of DNA for a fixed concentration of compound.
Decrease in absorbance is indicated by the downward arrow. Concentration range of DNA used during the titration has been
9

shown in each titration spectra for the corresponding compounds. Concentration of compounds in all the cases were kept fixed
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at 1x10-5M. (Refer to the SI for the corresponding spectrum obtained for compounds 49, 50, 51, 52, 55, 56, 57, 58a, 58b)
Compound 53a
Compound 42
Compound 53b
Kb =3.7 x 105 M-1
Kb =2.9 x 105 M-1
Kb =3.6 x 103 M-1
Figure 6. Double reciprocal plots are shown for the binding of different compounds with CT DNA. The linear fits were
obtained by plotting 1/ (A0-A) vs. 1/[DNA]. Intrinsic binding constants (Kb) were obtained from the ratio of intercept to slope.
The experiments were performed three times and mean value of the binding constant was reported. (Refer to the SI for the
corresponding plots obtained for compounds 49, 50, 51, 52, 55, 56, 57, 58a, 58b)
Compounds 51, 53a, 53b with pendant aryl group (endo-isomers) showed best binding interaction with
DNA with 105 order of the intrinsic binding (Table 1). It was observed that substitution by –CH2OH
(hydroxymethyl) group (compounds 42 and 49) decreased the DNA-binding as the order of the binding
constant dropped to 103. Remaining compounds 50, 52, 55, 56, 57, 58a and 58b showed moderate
binding interactions with DNA, with 104 order of binding constant. The study further revealed that the
aryl substitution in the endo position of phenanthridine (51, 53a, 53b) showed higher binding interaction
(105 order of binding constant) as compared to the analogue having exo-substituted aryl group with 104
order of binding constant (56, 58a, 58b). Compound 54 showed no binding interaction with DNA as it
showed no change in the absorption spectra with progressive addition of DNA.
Table 1. Intrinsic binding constants (Kb) obtained from the Benesi-Hildebrand plots.
Compound
Intrinsic binding constant (Kb)
3.6 x 103 M-1
42
49
4.6 x 103 M-1
50
1.8 x 104 M-1
1.1 x 105 M-1
51
4.8 x 104 M-1
52
2.9 x 105 M-1
53a
53b
3.7 x 105 M-1
10

2.3 x 104 M-A1 CCEPTED MANUSCRIPT
55
56
4.4 x 104 M-1
5.1 x 104 M-1
8.9 x 104 M-1
4.2 x 104 M-1
57
58a
58b
Since hypochromic shift was observed in UV-Vis absorption titration which is indicative of DNA-
intercalation14a-b,13a,8o, so to further ascertain the mode of compound–DNA interaction, competitive
ethidium bromide (EB) displacement assay was carried out by using fluorescence emission
spectroscopy.15a-d,13a,8o The concentration of the DNA and EB (in buffer) were kept fixed, while known
concentrations of the compound solutions were added into the cuvette in increasing amounts quenching of
fluorescence intensity was observed for compounds 42, 49, 50, 51, 52, 53a-b, 55, 56, 57, 58a-b (Figure 7
shows the fluorescence quenching for compounds 53a, 53b and 42).
Compound 42
Compound 53a
Compound 53b
Figure 7. Emission spectra of the CT DNA–EB system in tris–HCl buffer based on the titration with compounds (denoted on
their respective spectra). Quenching of the fluorescence is indicated by the arrow. [EB] = 20 µM, [DNA] = 26 µM, in buffer
(150 mM NaCl and 1 mM Tris-HCl at pH 7.2), emission wavelength = 588 nm. (Refer to the SI for the corresponding
spectrum obtained for compounds 49, 50, 51, 52, 55, 56, 57, 58a, 58b)
Figure 8. Relative quenching in fluorescence intensity observed upon gradual increase in the concentration of the compound
11

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A plot of F/Fo versus the concentration of compounds was made to depict the relative fluorescence
quenching of EB, induced by these compounds (Figure 8). Thereafter, the Stern-Volmer constant (KSV)
was determined (Table 2) for these compounds to evaluate their quenching efficiency using Equation
2.17a-b
F0/F = 1 + KSV.[Q]
( Equation 2)
[Q] is the quencher concentration. The Stern–Volmer plots for the compounds 53a, 53b and 42 have been
shown in Figure 9. The Stern–Volmer plot of DNA–EB reveals that the quenching of EB bound to DNA
by each compound is in good agreement (R = 0.99) with the linear Stern–Volmer equation (Equation 2).
Compound 28b
Compound 17
Compound 28a
Ksv = 7.5 x 103 M-1
Ksv = 3.5 x 103 M-1
Ksv = 7.6 x 103 M-1
Figure 9. Stern–Volmer quenching plot of EB bound to CT-DNA by titrating with compounds (denoted on their respective
spectra). The corresponding Stern-Volmer constants (KSV) were obtained from the slopes. The experiments were performed
three times and mean value of the binding constant was reported. (Refer to the SI for the corresponding plots obtained for
compounds 49, 50, 51, 52, 55, 56, 57, 58a, 58b)
Table 2. Stern Volmer constants (KSV) obtained from the Stern Volmer plots.
Compound
Stern Volmer constant (KSV)
3.5 x 103 M-1
42
49
3.4 x 103 M-1
50
5.2 x 103 M-1
6.9 x 103 M-1
51
4.2 x 103 M-1
52
7.5 x 103 M-1
53a
53b
55
7.6 x 103 M-1
5.3 x 103 M-1
5.6 x 103 M-1
56
4.0 x 103 M-1
57
12

6.2 x 103 M-A1 CCEPTED MANUSCRIPT
6.0 x 103 M-1
58a
58b
It can be concluded that the reduction in fluorescence intensity is occurring due to partial substitution of
EB bound to DNA by the compounds via DNA-intercalation.15,13a,8o A comparative study of Table 1 and
2 reveals that the trend observed in the competitive EB-displacement assay (both in terms of Stern-
Volmer constant and the relative fluorescence quenching) is in accordance with the corresponding trend
of DNA-binding obtained from UV-Vis absorption titration.
Although the order of the magnitude varies (103 to 105 in case of UV, and 103 in case of fluorescence),
such variations have been reported in the past in literature. There are reported cases18a-b where for a given
compound, KSV is greater in order than corresponding Kb, while there are also reports19a-b on the reverse
situation, that is, Kb is greater in order than the corresponding KSV. The rationale behind such variation is
probably arising from the fact that the binding constant obtained from UV-Vis Absorption titration takes
account of every possible binding interaction that the compound has with DNA (intercalation,
electrostatic, co-operative interactions etc), whereas, EB displacement assay only considers the
intercalative mode of binding. This might be the reason for the observed difference between the
magnitudes of two binding constants (Kb and KSV).
To further understand the interaction of these compounds with DNA, molecular docking studies were
carried out using Hex 8.0.0 docking server20. The pdb file used for ds DNA was downloaded from RCSB
protein data bank (PDB Id: 1XRW). As evident from the docked images (Figure 10), the planar, π-
electron rich phenanthridine skeletons intercalated through stacking in-between the nucleobase pairs.
Thus the docking results were in accordance with the spectroscopically obtained results. It is known21 that
π−π stacking between the π-skeleton of DNA nucleobases and the π-skeleton of the molecular probe, is
one of the major stabilizing forces responsible for intercalation of a probe with ds DNA. It is reported22a-b
that for DNA-intercalation the interplanar distance between the aryl rings of the probe and that of the
13

22b
ACCEPTED MANUSCRIPT
adjacent nucleobase should be ~ 3.4 Å (which is also a favourable factor for π−π stacking ).
Accordingly, we tried to analyse the DNA-binding interactions of compound 53b (which showed the
highest intrinsic binding constant of the order 105) and 42 (which showed the lowest intrinsic binding
constant of the order 103) based on the docking images. These images revealed that the compounds
showed selectivity towards CG complementary base pairs. The average interplanar distance (between the
fused aryl rings of synthesized phenanthridine derivatives and the nearest heterocyclic rings of the
nucleobases) in case of compound 53b is 3.55 Å (Figure 10A), whereas for 42, interplanar distance is
3.85 Å (Figure 10C). Lesser the interplanar distance, stronger is the intercalation.22a In case of 53b,
although the pendant aryl ring is not oriented in exactly parallel position with the π-skeleton of the
nucleobase aryl rings (Figure 10B), however some kind of weak interaction might still be possible as the
pendant heterocyclic rings is at an average interplanar distance of ~ 4.95 Å. Probably all these factors
together contribute towards the higher binding constant obtained for compound 53b. Overall, we can
conclude that probably some kind of synergestic co-operative effect23a-b of two conjoint aryl systems is
responsible for the greater DNA-intercalating ability of the compounds 51, 53a, and 53b. However
further studies are needed to be carried out to explain the comparative binding affinities in greater details.
A
B
C
14

Figure 10. Docking images of compounds 53b and 42 with ds DNA. A shows the interplanar distances between the aryl rings
ACCEPTED MANUSCRIPT
of the fused phenanthridine moiety of compound 53b and the adjacent nucleobases; B shows the interplanar distances between
the aryl rings of the pendant p-methoxy phenyl moeity of compound 53b and the adjacent nucleobases; C shows the interplanar
distances between aryl rings of the fused phenanthridine moiety of compound 42 and adjacent nucleobases
Conclusion
We have successfully developed a simple, straightforward and cost-effective methodology for
synthesizing dihydroisofuran fused phenanthridine derivatives using Garratt-Braverman cyclization in
moderate to good yields. The current work established a newer synthetic alternative to functionalize the
phenanthridine scaffold with greater potential bioactivity. All the synthesized compounds showed DNA-
intercalation (except compounds 54 and 59) with three compounds (51, 53a, 53b) having binding
constant of the order of 105. The regioselectivity obtained during the cyclization step provides a deeper
insight in the manifestation of ‘double bond fixation’ upon cycloaromatization transformations. This
relatively simpler synthetic protocol would lead to easier access to these cytotoxic scaffolds which in turn
would facilitate their application as molecular probes to understand DNA-small molecule interactions.
Further work on synthesizing a more diverse library of DNA-binding heterocyclic cores is going on in our
laboratory.
Experimental
Synthesis and spectral data of compounds
All 1H and 13C NMR spectra for all the compounds were recorded at 400/500/600 and 100/125/150 MHz
respectively. The spectra were recorded in deuterochloroform (CDCl3) as solvent at room temperature
unless mentioned otherwise. The following abbreviations are used to describe the peak patterns where
appropriate: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, ABq = AB quartet, dd =
doublet of doublet, app. = apparent, and b = broad signal. All coupling constants (J) are given in Hz. High
resolution mass spectra were recorded in ESI+ mode (ion trap) while LCMS were recorded under low
resolution. All the dry solvents used for reactions were purified according to the standard protocols. n-
butyl amine was distilled from calcium hydride. Solvents used for column chromatography were distilled
15

ACCEPTED MANUSCRIPT
prior to use. In most of the column chromatographic purifications, ethyl acetate (EtOAc) and hexane of
boiling range 60−80 °C were used as eluents. Columns were prepared with silica gel (Si-gel, 60−120 and
230−400 flash, SRL).
General procedure for Sonogashira Coupling
Synthesis of compounds (37, 44, 31a, 31b, 13, 17). To a solution of bromo aryl derivative (1 mmol) in
dry degassed n-butylamine (7 mL), PdCl2 (PPh3)2 (3 mol %), corresponding alkyne compound (1.2 equiv)
in were added under an inert atmosphere, and the mixture was refluxed for 4 h. The mixture was then
poured into ethyl acetate, and the organic layer was washed with brine. The organic layer was then dried
over anhydrous Na2SO4 and evaporated, and the purified product was obtained via flash chromatography
by using hexane−ethyl acetate as eluent.
3-phenylprop-2-yn-1-ol (31a). Following the general procedure, pure product 31a was isolated by flash
chromatography on silica gel (hexane:EtOAc 2:1). State: liquid; yield: 103 mg, 78%; Experimental data
properly matched with previously reported one.24
3-(4-methoxyphenyl)prop-2-yn-1-ol (31b). Following the general procedure, pure product 31b was
isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: liquid; yield: 133 mg, 82%;
Experimental data were properly matched with previously reported one.25
3-(quinolin-3-yl)prop-2-yn-1-ol (13). Following the general procedure, pure product 13 was isolated by
flash chromatography on silica gel (hexane:EtOAc 2:1). State: liquid; yield: 142 mg, 78%; Experimental
1
data were properly matched with previously reported one. H NMR (400 MHz, CDCl3) δ 9.027 (s, 1H),
8.25 (1H, s), 8.12 (d, J= 8.4 Hz, 1H), 7.79 ( d, J= 8.4 Hz, 1H), 7.748 (t, J= 7.2 Hz, 1H), 7.58 (t, J= 8 Hz,
1H), 4.58 (s, 2H); (100 MHz, CDCl3) δ 151.9, 146.2, 139.1, 130.5, 128.7, 127.7, 127.6, 127.3, 117.0,
92.1, 82.1, 51.0. Calcd for C12H10NO (M+H) + 184.0762, found 184.0764.
16

ACCEPTED MANUSCRIPT
3-(isoquinolin-4-yl)prop-2-yn-1-ol (17). Following the general procedure, pure product 17 was isolated
by flash chromatography on silica gel (hexane:EtOAc 2:1). State: liquid; yield: 152 mg, 83%; 1H NMR
(400 MHz, DMSO) δ 9.29 (s, 1H), 8.60 (s, 1H), 8.18 (d, J = 3.3 Hz, 1H), 8.16 (d, J = 3.1 Hz, 1H), 7.89 (t,
J = 7.6 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 5.51 (t, J = 6.0 Hz, 1H), 4.45 (d, J = 5.9 Hz, 2H); 13C NMR (100
MHz, DMSO) δ 152.24, 145.75, 134.74, 131.72, 128.33, 128.26, 127.37, 124.25, 114.83, 97.55, 78.55,
49.67; Calcd for C12H10NO (M+H) + 184.0762, found 184.0766.
3-(3-(3-(quinolin-3-yl)prop-2-ynyloxy)prop-1-ynyl)quinoline (37). Following the general procedure,
pure product 37 was isolated by flash chromatography on silica gel (hexane:EtOAc 1:1). State: liquid;
yield: 250 mg, 72%; 1H NMR (400 MHz, CDCl3) δ 8.92 (d, J = 1.5 Hz, 2H), 8.23 (s, 2H), 8.07 (d, J = 8.4
Hz, 2H), 7.88 – 7.60 (m, 4H), 7.53 (t, J = 7.5 Hz, 2H), 4.64 (s, 4H); 13C NMR (100 MHz, CDCl3) δ 152.1,
147.1, 138.9, 130.4, 129.5, 127.7, 127.5, 127.2, 116.6, 87.7, 84.4, 57.8; HRMS: Calcd for C24H17N2O
(M+H) + 349.1341, found 349.1341.
4-(3-(3-(isoquinolin-4-yl)prop-2-ynyloxy)prop-1-ynyl)isoquinoline (44). Following the general
procedure, pure product 44 was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1).
State: yellow liquid; yield: 243 mg, 70%; 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 2H), 8.72 (s, 2H), 8.26
(d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.1 Hz, 2H), 7.76 (t, J = 7.5 Hz, 2H), 7.65 (t, J = 7.5 Hz, 2H), 4.80 (s,
13
4H); C NMR (125 MHz, CDCl3) δ 152.5, 147.0, 135.8, 131.4, 128.1, 128.0, 127.8, 125.0, 115.2, 91.8,
82.4, 57.9; Calcd for C24H17N2O (M+H)+ 349.1341, found 349.1348.
General procedure for OTHP Protection of alcohol
Synthesis of compounds (34). To a solution of but-2-yne-1,4-diol (1mmol) in 200 ml dry DCM, 3,4-
dihydro-pyran (0.5 equiv) was added and mixture was stirred at room temperature for 4h. The mixture
was then poured into DCM, and the organic layer was washed with brine. The organic layer was then
17

ACCEPTED MANUSCRIPT
dried over anhydrous Na2SO4 and evaporated, and the purified product was obtained via flash
chromatography by using hexane−ethyl acetate as eluent.
4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-yn-1-ol (34). Following the general procedure, pure product 34
was isolated by flash chromatography on silica gel (hexane:EtOAc 5:1). State: liquid; yield: 110 mg, 65
%; Experimental data were properly matched with previously reported one.26
General procedure for Bromination of alcohol
Synthesis of compounds (32a, 32b, 35). To a solution of alcohol derivative (0.75 mmol) in dry DCM,
CBr4 (1.2 equiv), PPh3 (1.5 equiv) were added under an inert atmosphere at 00 C, and the mixture was
stirred at room temperature for 2h. The mixture was then poured into DCM, and the organic layer was
washed with brine. The organic layer was then dried over anhydrous Na2SO4 and evaporated, and the
purified product was obtained via flash chromatography by using hexane−ethyl acetate as eluent.
(3-bromoprop-1-yn-1-yl)benzene (32a). Following the general procedure, pure product 32a was isolated
by flash chromatography on silica gel (hexane:EtOAc 4:1). State: liquid; yield:, 113 mg, 82%;
Experimental data were properly matched with previously reported one.27
1-(3-bromoprop-1-yn-1-yl)-4-methoxybenzene (32b). Following the general procedure, pure product 32b
was isolated by flash chromatography on silica gel (hexane:EtOAc 4:1). State: liquid; yield: 141 mg,
84%; Experimental were data properly matched with previously reported one.28
2-((4-bromobut-2-yn-1-yl)oxy)tetrahydro-2H-pyran (35). Following the general procedure, pure product
34 was isolated by flash chromatography on silica gel (hexane:EtOAc 5:1). State: liquid; yield: 142 mg,
82 %; Experimental data were properly matched with previously reported one.26
18

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General Procedure for O-Propargylation
Synthesis of compounds (36-40 and 43-47): To an ice-cold solution of alcohol 13/17 (0.5 mmol) in dry
THF (6 mL) was added NaH (2 equiv, 60% suspension in mineral oil), and the solution was stirred for 30
min at room temperature under N2 atmosphere. After the alkoxide was generated, corresponding
propargyl bromide (1.2 equiv) was added dropwise at 00 C, and the mixture was stirred for 1 h at room
temperature. It was then partitioned between ethyl acetate and water. The organic layer was washed with
brine, and the organic layer was dried with anhydrous Na2SO4. The solvent was removed, and the crude
residue was purified by flash chromatography (Si-gel, hexane−ethyl acetate mixture as eluent).
3-(3-(prop-2-ynyloxy)prop-1-ynyl)quinoline (36). Following the general procedure, pure product 36 was
isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: liquid; yield: 86 mg, 78%; 1H
NMR (400 MHz, CDCl3) δ 8.89 (d, J = 1.2 Hz, 1H), 8.22 (d, J = 1.5 Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H),
7.82 – 7.63 (m, 2H), 7.55 (d, J = 7.8 Hz,1H), 4.54 (s, 2H), 4.35 (d, J = 2.3 Hz, 2H), 2.50 (t, J = 2.3 Hz,
13
1H); C NMR (100 MHz, CDCl3) δ 152.0, 146.9, 139.0, 130.4, 129.4, 127.7, 127.5, 127.2, 116.6, 87.7,
84.1, 78.9, 75.4, 57.3, 56.9; HRMS: C15H12NO (M+H)+ 222.0919, found 222.0919.
3-(3-(but-2-ynyloxy)prop-1-ynyl)quinoline (38). Following the general procedure, pure product 38 was
isolated by flash chromatography on silica gel (hexane:EtOAc 5:1). State: liquid; yield: 85 mg, 72%; 1H
NMR (400 MHz, CDCl3) δ 8.91 (d, J = 1.9 Hz, 1H), 8.24 (s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.78 (d, J =
8.1 Hz, 1H), 7.73 (t, J = 8.3 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 4.53 (s, 2H), 4.31 (s, 2H), 1.89 (s, 3H); 13C
NMR (125 MHz, CDCl3) δ 152.0, 146.9, 138.6, 130.1, 129.3, 127.5, 127.2, 127.0, 116.6, 83.7, 83.4, 74.3,
57.4, 57.0, 3.6; HRMS: Calcd for C16H14NO (M+H) + 236.1075, found 236.1077.
3-(3-(3-phenylprop-2-ynyloxy)prop-1-ynyl)quinoline (39a). Following the general procedure, pure
product 39a was isolated by flash chromatography on silica gel (hexane:EtOAc 4:1). State: liquid; yield:
19

1
ACCEPTED MANUSCRIPT
104 mg, 70%; H NMR (400 MHz, CDCl3) δ 8.93 (d, J = 2.0 Hz, 1H), 8.25 (d, J = 1.5 Hz, 1H), 8.09 (d, J
= 8.5 Hz, 1H), 7.85 – 7.69 (m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.39 – 7.31 (m, 3H),
4.62 (s, 2H), 4.59 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 152.1, 147.0, 138.9, 131.9, 130.3, 129.4, 128.7,
128.4, 127.7, 127.4, 127.2, 122.5, 116.7, 88.0, 87.2, 84.2, 84.1, 57.8, 57.5; HRMS: Calcd for C21H16NO
(M+H)+ 298.1232, found 298.1244.
3-(3-(3-(4-methoxyphenyl)prop-2-ynyloxy)prop-1-ynyl)quinoline(39b).
Following
the
general
procedure, pure product 39b was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1).
1
State: liquid; yield: 121 mg, 74%; H NMR (400 MHz, CDCl3) δ 8.92 (d, J = 2.0 Hz, 1H), 8.23 (s, 1H),
8.08 (d, J = 8.4 Hz, 1H), 7.83 – 7.65 (m, 2H), 7.55 (t, J = 7.5 Hz, 1H), 7.49 – 7.32 (m, 2H), 6.83 (d, J =
8.8 Hz, 2H), 4.60 (s, 2H), 4.56 (s, 2H), 3.79 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.0, 152.2, 147.1,
138.9, 133.5, 130.4, 129.5, 127.7, 127.4, 127.2, 116.8, 114.6, 114.1, 88.1, 87.2, 84.0, 82.9, 58.0, 57.4,
55.4; HRMS: Calcd for C22H18NO2 (M+H)+ 328.1338, found 328.1335.
3-(3-(4-(tetrahydro-2H-pyran-2-yloxy)but-2-ynyloxy)prop-1-ynyl)quinoline (40). Following the general
procedure, pure product 40 was isolated by flash chromatography on silica gel (hexane:EtOAc 4:1).
1
State: liquid; yield: 134 mg, 80%; H NMR (400 MHz, CDCl3) δ 8.91 (d, J = 1.7 Hz, 1H), 8.25 (s, 1H),
8.09 (d, J = 8.4 Hz, 1H), 7.82 – 7.69 (m, 2H), 7.57 (t, J = 7.5 Hz, 1H), 4.83 (t, J = 3.3 Hz, 1H), 4.54 (s,
2H), 4.45 – 4.25 (m, 4H), 3.90 – 3.79 (m, 1H), 3.59 – 3.49 (m, 1H), 1.98 – 1.50 (m, 6H); 13C NMR (100
MHz, CDCl3) δ 152.2, 147.1, 139.0, 130.4, 129.5, 127.8, 127.5, 127.8, 116.7, 97.8, 87.9, 84.1, 83.5, 81.2,
62.2, 57.4, 57.3, 54.4, 30.4, 25.5, 19.2. HRMS: Calcd for C21H22NO3 (M+H)+ 336.1600, found 336.1627.
4-(3-(prop-2-ynyloxy)prop-1-ynyl)isoquinoline (43). Following the general procedure, pure product 43
was isolated by flash chromatography on silica gel (hexane:EtOAc 4:1). State: liquid; yield: 81 mg,
73%; 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.70 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 8.2
20

ACCEPTED MANUSCRIPT
Hz, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 4.66 (s, 2H), 4.41 (d, J = 2.3 Hz, 2H), 2.52 (t, J
13
= 2.3 Hz, 1H); C NMR (100 MHz, CDCl3) δ 152.5, 147.0, 147.0, 135.8, 131.3, 128.0, 127.9, 125.1,
115.2, 91.7, 82.2, 79.0,75.4, 57.5, 56.9; HRMS: Calcd for C15H12NO (M+H)+ 222.0919, found 222.0917.
4-(3-(but-2-ynyloxy)prop-1-ynyl)isoquinoline (45). Following the general procedure, pure product 45
was isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: yellow liquid; yield: 88
mg, 75%; 1H NMR (400 MHz, CDCl3) δ 9.15 (d, J = 1.8 Hz, 1H), 8.65 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H),
7.93 (dd, J = 7.8, 2.8 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 4.59 (s, 2H), 4.33 (s, 2H),
1.86 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 152.3, 146.8, 135.6, 131.2, 127.9, 127.7, 125.0, 115.2, 92.1,
83.5, 81.7, 74.3, 57.5, 57.3, 3.7; HRMS: Calcd for C16H14NO (M+H)+ 236.1075, found 236.1073.
4-(3-(3-phenylprop-2-ynyloxy)prop-1-ynyl)isoquinoline (46a). Following the general procedure, pure
product 46a was isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: yellow liquid;
1
yield: 108 mg, 73%; H NMR (400 MHz, CDCl3) δ 9.28 (s, 1H), 8.78 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H),
7.99 (d, J = 8.1 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.49-7.47 (dd, J = 7.3, 2.1 Hz,
2H), 7.41 – 7.20 (m, 3H), 4.72 (s, 2H), 4.63 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 152.3, 146.8, 135.8,
132.0, 131.8, 131.4, 128.8, 128.5, 128.4, 128.1, 128.1, 125.2, 122.5, 92.1, 87.2, 84.3, 82.1, 57.8, 57.6;
HRMS: Calcd for C21H16NO (M+H)+ 298.1232, found 298.1233.
4-(3-(3-(4-methoxyphenyl)prop-2-ynyloxy)prop-1-ynyl)isoquinoline (46b). Following the general
procedure, pure product 46b was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1).
State: yellow liquid; yield: 114 mg, 70%; 1H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H), 8.70 (s, 1H), 8.24
(d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.41 (d, J =
8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 4.70 (s, 2H), 4.61 (s, 2H), 3.79 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 160.0, 152.4, 147.0, 135.8, 133.4, 131.3, 128.0, 128.0, 127.8, 125.1, 115.3, 114.6, 114.1, 92.1,
87.2, 82.9, 82.0, 57.9, 57.5, 55.4; HRMS: Calcd for C22H18NO2 (M+H)+ 328.1338, found 328.1335.
21

ACCEPTED MANUSCRIPT
4-(3-(4-(tetrahydro-2H-pyran-2-yloxy)but-2-ynyloxy)prop-1-ynyl)isoquinoline (47).
Following the general procedure, pure product 47 was isolated by flash chromatography on silica gel
(hexane:EtOAc 3:1). State: liquid; yield: 131 mg, 78%; 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.69
(s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.79 (t, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz,
1H), 4.83 (t, J = 3.3 Hz, 1H), 4.64 (s, 2H), 4.49 – 4.23 (m, 4H), 3.87-3.81 (m, 1H), 3.68 – 3.34 (m, 1H),
1.90 – 1.48 (m, 6H); 13C NMR (126 MHz, CDCl3) δ 152.4, 146.8, 135.7, 131.3, 128.0, 128.0, 127.8,
125.0, 115.2, 97.0, 91.8, 83.4, 82.0, 81.1, 62.1, 57.5, 57.2, 54.3, 30.3, 25.4, 19.1; HRMS: Calcd for
C21H22NO3 (M+H)+ 336.1600, found 336.1589.
General Procedure for the Garratt–Braverman Cyclization: Synthesis of compounds (50-53, 41, 55-
58, 48). To a solution of bispropargyl ether (0.2 mmol) in toluene (3 mL), KOBut (2.5 equiv) was added,
and the mixture was refluxed at 110ꢀ˚C for 6 h. The reaction mixture was then partitioned between ethyl
acetate and water. The organic layer was washed with brine, and the combined organic layer was dried
with anhydrous Na2SO4. The solvent was removed, and the crude residue was purified by flash
chromatography (Si-gel, hexane−ethyl acetate mixture as eluent).
8,10-dihydrofuro[3,4-j]phenanthridine (50). Following the general procedure, pure product 50 was
isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: white solid; mp 169-173°C;
1
yield: 40 mg, 90%; H NMR (600 MHz, CDCl3) δ 9.24 (s, 1H), 8.52 (d, J = 8.1 Hz, 1H), 8.43 (s, 1H),
8.19 (d, J = 8.1 Hz, 1H), 7.86 (s, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 5.33 (s, 2H), 5.30
13
(s, 2H); C NMR (151 MHz, CDCl3) δ 153.45, 144.71, 143.60, 139.66, 132.66, 130.27, 128.82, 127.24,
126.33, 124.34, 122.23, 120.80, 114.00, 73.46, 73.11; HRMS: C15H12NO (M+H)+ 222.0919, found
222.0925.
8,10-dihydro-11-(quinolin-3-yl)furo[3,4-j]phenanthridine (51). Following the general procedure, pure
product 51 was isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: white solid;
22

1
ACCEPTED MANUSCRIPT
mp 128-130 °C; yield: 61 mg, 87%; H NMR (600 MHz, CDCl3) δ 9.29 (d, J = 5.2 Hz, 1H), 8.85 (s, 1H),
8.26 (d, J = 8.5 Hz, 1H), 8.22 – 8.09 (m, 2H), 7.95 (d, J = 7.0 Hz, 1H), 7.92 – 7.79 (m, 2H), 7.66 (t, J =
7.5 Hz, 1H), 7.55 (t, J = 8.3 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.7 Hz, 1H), 5.38 (s, 2H), 5.01
(d, J = 13.7 Hz, 1H), 4.88 (d, J = 13.6 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 153.9, 150.4, 147.7,
146.0, 145.1, 139.0, 134.9, 134.1, 130.6, 130.3, 129.8, 129.0, 128.4, 128.2, 128.1, 128.0, 127.7, 126.5,
126.3, 124.4, 121.7, 73.9, 73.8; HRMS: Calcd for C24H17N2O (M+H) + 349.1341, found 349.1343.
11-methyl-8,10-dihydrofuro[3,4-j]phenanthridine (52). Following the general procedure, pure product
52 was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: liquid; yield: 43 mg,
92%; 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.82 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.79
– 7.73 (m, 2H), 7.67 (t, J = 7.2 Hz, 1H), 5.38 (s, 4H), 2.95 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 154.1,
145.5, 143.6, 143.6, 138.0, 138.0, 131.0, 130.3, 130.3, 127.7, 126.2, 125.4, 118.9, 73.8, 73.7, 21.6;
HRMS: Calcd for C16H14NO (M+H) + 236.1075, found 236.1086.
8,10-dihydro-11-phenylfuro[3,4-j]phenanthridine (53a). Following the general procedure, pure product
53a was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid; mp 130-
134°C; yield: 55 mg, 92%; 1H NMR (500 MHz, CDCl3) δ 9.23 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.84 (s,
1H), 7.59-7.52 (m, 5H), 7.30 (d, J = 6.3 Hz, 2H), 7.13 (t, J = 7.6 Hz, 1H), 5.35 (s, 2H), 4.92 (s, 2H); 13C
NMR (125 MHz, CDCl3) δ 153.9, 145.8, 144.1, 140.8, 138.6, 133.0, 130.2, 129.8, 128.3, 128.2, 128.1,
127.9, 126.6, 126.0, 124.7, 120.8, 73.8; HRMS: Calcd for C21H16NO (M+ H)+ 298.1232, found 298.1231.
8,10-dihydro-11-(4-methoxyphenyl)furo[3,4-j]phenanthridine (53b). Following the general procedure,
pure product 53b was isolated by flash chromatography on silica gel (hexane:EtOAc 1:1). State: white
solid; mp 154-156°C; yield: 57 mg, 87%; δ 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.14 (d, J = 7.9
Hz, 1H), 7.89 (s, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.29 – 7.15 (m, 3H), 7.08 (d, J =
23

13
ACCEPTED MANUSCRIPT
8.6 Hz, 2H), 5.38 (s, 2H), 4.96 (s, 2H), 3.94 (s, 3H); C NMR (125 MHz, CDCl3) δ 159.4, 158.4, 153.9,
148.0, 146.0, 145.0, 138.4, 132.8, 132.7, 130.4, 130.1, 129.3, 127.9, 127.8, 126.5, 126.0, 124.8, 120.5,
115.2, 73.9, 73.8, 55.4;. HRMS: Calcd for C22H18NO2 (M+H) + 328.1338, found 328.1334.
8,10-dihydro-11-((tetrahydro-2H-pyran-2-yloxy)methyl)furo[3,4-j]phenanthridine (41). Following the
general procedure, pure product 41 was isolated by flash chromatography on silica gel (hexane:EtOAc
1
2:1). State: liquid; yield: 59 mg, 88%; H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.87 (d, J = 8.4 Hz,
1H), 8.22 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.77 (t, J = 7.5 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 5.49 (s, 2H),
5.35-5.32 (m, 3H), 5.00 (d, J = 11.8 Hz, 1H), 4.91 (t, J = 3.1 Hz, 1H), 4.04-3.99 (m, 1H), 3.67-3.64 (m,
1H), 1.93 – 1.54 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 153.9, 145.8, 145.7, 139.1, 132.5, 130.1, 128.5,
128.1, 127.4, 127.3, 126.9, 124.5, 121.5, 98.8, 73.7, 73.4, 66.6, 62.8, 30.8, 25.5, 19.5; HRMS: Calcd for
C21H22NO3 (M+H)+ 336.1600, found 336.1595.
8,10-dihydrofuro[3,4-b]phenanthridine (55). Following the general procedure, pure product 55 was
isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid; mp 115-118 °C;
1
yield: 39 mg, 89%; H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 8.55 (d, J = 8.3 Hz, 1H), 8.39 (s, 1H),
8.06 – 7.99 (m, 2H), 7.85 (t, J = 8.2 Hz, 1H), 7.70 (t, J = 7.4 Hz, 1H), 5.32 (s, 4H); 13C NMR (101 MHz,
CDCl3) δ 153.44, 144.45, 140.95, 139.21, 132.71, 131.14, 128.92, 127.58, 126.41, 123.73, 122.06,
121.87, 114.09, 73.35, 73.34; HRMS: Calcd for C15H12NO (M+H)+ 222.0919, found 222.0916.
8,10-dihydro-7-(isoquinolin-4-yl)furo[3,4-b]phenanthridine (56). Following the general procedure, pure
product 56 was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid;
mp 178-180 °C; yield: 64 mg, 92%; 1H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H), 9.11 (s, 1H), 8.69 (d, J =
8.3 Hz, 1H), 8.61-8.56 (m, 2H), 8.09 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.91 (t, J = 7.7 Hz,
1H), 7.73 (t, J = 7.4 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H),
24

13
ACCEPTED MANUSCRIPT
5.46 (s, 2H), 5.10 (d, J = 13.3 Hz, 1H), 4.81 (d, J = 13.3 Hz, 1H); C NMR (125 MHz, CDCl3) δ 153.4,
152.7, 143.3, 143.3, 141.4, 138.8, 134.9, 132.7, 131.3, 130.7, 129.9, 129.0, 128.2, 127.9, 127.4, 126.2,
125.2, 124.3, 122.1, 114.6, 74.0, 73.3; Calcd for C24H17N2O (M+H)+ 349.1341, found 349.1340.
8,10-dihydro-7-methylfuro[3,4-b]phenanthridine (57). Following the general procedure, pure product 57
was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid; mp 162-165
1
°C; yield: 42 mg, 90%; H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.56 (d, J = 8.3 Hz, 1H), 8.27 (s,
1H), 8.05 (d, J = 7.9 Hz, 1H), 7.83 (t, J = 8.1 Hz, 1H), 7.69 (t, J = 7.4 Hz, 1H), 5.35 (s, 2H), 5.30 (s, 2H),
2.74 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 151.7, 142.6, 139.3, 137.9, 132.8, 130.6, 130.1, 128.6,
127.2, 125.9, 123.5, 121.8, 111.2, 74.0, 73.2, 14.7; HRMS: Calcd for C16H14NO (M+H)+ 236.1075, found
236.1086.
8,10-dihydro-7-phenylfuro[3,4-b]phenanthridine (58a). Following the general procedure, pure product
58a was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid; mp 145-
149 °C; yield: 54 mg, 91%; 1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 8.64 (d, J = 8.3 Hz, 1H), 8.48 (s,
1H), 8.04 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 7.62 – 7.34 (m, 5H), 5.41
(s, 2H), 5.17 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 153.0, 142.1, 139.9, 138.7,138.0, 135.1, 132.8,
131.0, 130.1, 128.8, 128.3, 127.7, 127.6, 126.14, 124.3, 122.1, 113.4, 74.0, 73.6; HRMS: Calcd for
C21H16NO (M+H)+ 298.1232, found 298.1234.
8,10-dihydro-7-(4-methoxyphenyl)furo[3,4-b]phenanthridine (58b). Following the general procedure,
pure product 58b was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white
1
solid; mp 160-165 °C; yield: 58 mg, 88%; H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.63 (d, J = 8.3
Hz, 1H), 8.44 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.87 (t, J = 7.7 Hz, 1H), 7.71 (t, J = 7.4 Hz, 1H), 7.43 (d, J
13
= 8.7 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 5.40 (s, 2H), 5.19 (s, 2H), 3.89 (s, 3H); C NMR (125 MHz,
25

ACCEPTED MANUSCRIPT
CDCl3) δ 159.0, 152.7, 142.0, 139.7, 138.47, 134.5, 132.6, 131.2, 130.8, 130.0, 129.0, 127.4, 125.9,
124.1, 121.9, 113.7, 112.9, 73.8, 73.5, 55.3; HRMS: Calcd for C22H18NO2 (M+H)+ 328.1338, found
328.1335.
8,10-dihydro-7-((tetrahydro-2H-pyran-2-yloxy)methyl)furo[3,4-b]phenanthridine (48). Following the
general procedure, pure product 23 was isolated by flash chromatography on silica gel (hexane:EtOAc
1
3:1). State: liquid; yield: 62 mg, 93%; H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.58 (d, J = 8.3 Hz,
1H), 8.38 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 7.4 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 5.79 – 5.26
(m, 6H), 4.88 (t, J = 3.3 Hz, 1H), 4.11 – 3.87 (m, 1H), 3.62-3.59 (m, J = 10.8, 4.7 Hz, 1H), 2.02 – 1.48
13
(m, 6H); C NMR (100 MHz, CDCl3) δ 152.2, 141.7, 139.6, 139.2, 132.7, 130.9, 130.5, 128.7, 127.4,
126.0, 123.4, 121.9, 113.0, 98.8, 73.8, 73.0, 64.8, 62.2, 30.7, 25.6, 19.5; HRMS: Calcd for C21H22NO3
(M+H)+ 336.1600, found 336.1595.
General Procedure for OTHP deprotection: Synthesis of compounds (42 and 49). To a solution of
OTHP (0.15 mmol) protected alcohol in ethanol, PPTS (0.5 equiv) was added, and the mixture was stirred
at 50ꢀ˚C for 12 h. After that, ethanol was evaporated and crude residue was purified by column
chromatography (Si-gel, hexane−ethyl acetate mixture as eluent).
(8,10-dihydrofuro[3,4-j]phenanthridin-11-yl)methanol (42). Following the general procedure, pure
product 42 was isolated by flash chromatography on silica gel (hexane:EtOAc 1:1). State: white solid;
mp 199-203 °C; yield: 36 mg, 95%; 1H NMR (400 MHz, DMSO) δ 9.31 (s, 1H), 9.07 (d, J = 8.4 Hz, 1H),
8.14-8.12 (m, 2H), 7.80 (t, J = 7.4 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 5.84 (t, J = 5.2 Hz, 1H), 5.42 (s, 2H),
5.26 (s, 2H), 4.95 (d, J = 5.2 Hz, 2H); 13C NMR (125 MHz, DMSO) δ 154.0, 145.2, 144.9, 139.1, 131.1,
130.4, 129.6, 128.4, 127.7, 127.6, 126.7, 124.0, 121.2, 72.6, 72.3, 60.2; HRMS: Calcd for C16H14NO2
(M+H)+ 252.1025, found 252.1032.
26

ACCEPTED MANUSCRIPT
(8,10-dihydrofuro[3,4-b]phenanthridin-7-yl)methanol (49). Following the general procedure, pure
product 49 was isolated by flash chromatography on silica gel (hexane:EtOAc 3:1). State: white solid;
mp 154-157 °C; yield: 37 mg, 98%; 1H NMR (500 MHz, CDCl3) δ 9.17 (s, 1H), 8.53 (d, J = 8.3 Hz, 1H),
8.29 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 5.31 (d, J = 4.9
13
Hz, 4H), 5.12 (s, 2H); C NMR (125 MHz, CDCl3) δ 151.9, 143.0, 139.0, 138.2, 132.9, 131.4, 131.3,
129.1, 127.7, 126.0, 124.2, 122.0, 113.2, 73.6, 72.5, 62.0; HRMS: Calcd for C16H14NO2 (M+H)+
252.1025, found 252.1035.
General Procedure for oxidation of alcohol to aldehyde: Synthesis of compounds (54 and 59). To a
solution of alcohol in Dry DCM (0.10 mmol), Dess-Martin-Periodinane reagent (0.1 mmol) was added at
0ꢀ˚C and stirred at room temperature for 1 h. The reaction mixture was then partitioned between DCM and
water. The organic layer was washed with saturated sodium thiosulpahate solution and sodium
bicarbonate solution. The organic layer was washed with brine solution, and the combined organic layer
was dried with anhydrous sodium sulfate. The solvent was removed, and the crude residue was purified
by column chromatography (Si-gel, hexane−ethyl acetate mixture as eluent).
8,10-dihydrofuro[3,4-j]phenanthridine-11-carbaldehyde (54). Following the general procedure, pure
product 54 was isolated by flash chromatography on silica gel (hexane:EtOAc 2:1). State: white solid;
mp 135-140 °C; yield: 22 mg, 90%; 1H NMR (600 MHz, CDCl3) δ 10.65 (s, 1H), 9.23 (s, 1H), 8.24 (d, J
= 8.1 Hz, 1H), 8.16 – 7.91 (m, 2H), 7.81 (m, 1H), 7.69 (t, J = 8.2 Hz, 1H), 5.46 (s, 2H), 5.28 (s, 2H); 13C
NMR (150 MHz, CDCl3) δ 191.4, 153.1, 146.5, 145.9, 140.4, 132.6, 130.4, 129.8, 128.2, 127.5, 127.4,
127.2, 125.1, 122.6, 74.8, 72.4; HRMS: Calcd for C16H12NO2 (M+H)+ 250.0868, found 250.0864.
8,10-dihydrofuro[3,4-b]phenanthridine-7-carbaldehyde (59).
27

ACCEPTED MANUSCRIPT
Following the general procedure, pure product 59 was isolated by flash chromatography on silica gel
(hexane:EtOAc 3:1). State: white solid; mp 244-246 °C; yield: 23 mg, 92%; 1H NMR (400 MHz, CDCl3)
δ 11.60 (s, 1H), 9.39 (s, 1H), 8.92 – 8.45 (m, 2H), 8.14 (d, J = 7.5 Hz, 1H), 7.93 (t, J = 6.9 Hz, 1H), 7.79
(t, J = 6.9 Hz, 1H), 5.62 (s, 2H), 5.33 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 194.3, 154.1, 144.6, 143.3,
140.0, 132.3, 131.7, 129.2, 128.3, 126.3, 124.1, 121.9, 119.8, 75.0, 72.3; HRMS: Calcd for C16H12NO2
(M+H)+ 250.0868, found 250.0875.
DNA binding assay
a) UV based assay: UV-visible absorption titration was carried out using Jasco V spectrophotometer.
Solution of Calf thymus DNA (CT DNA) was prepared in 1mM Tris-HCl buffer (pH= 7.2). The ratio of
UV absorbances at 260 nm and 280 nm was found to be 1.89. Concentration of DNA solution was
determined from its extinction coefficient (6600 M-1 at 260 nm). UV absorption titration experiment was
carried out in Tris-HCl buffer medium at a fixed concentration of compound solution (1 x 10-5 M) upon
addition of incremental amount of known concentration (2.7 to 100 ꢁM) of Calf Thymus (CT) DNA
solution into both the cuvettes. Progressive addition of CT-DNA solution was continued until saturation
in hypochromism was observed. Absorbance values were recorded after each successive addition of CT-
DNA solution.
b) Ethidium Bromide displacement assay: EB displacement fluorescence assay was carried out to
verify DNA intercalation. The CT DNA–EB complex was prepared by adding 20 µM EB and 26 µM CT
DNA in buffer (150 mM NaCl and 1 mM Tris-HCl Buffer at pH 7.4). Fluorescence emission spectra
(excitation wavelength = 546 nm) were recorded at 300C. The assay was performed by gradual addition
of certain amount of a solution of the compound into the solution of the DNA–EB complex. The
influence of the addition of each compound to the DNA–EB complex solution has been obtained by
recording the variation of fluorescence emission spectra. A plot of F/Fo versus the concentration of
28