Title of Invention

VOILOGEN LINKED ACRIDINE BASED MOLECULE

Abstract The invention relates to Viologen linked acridine based molecule of the general formula 1(1a,1b,1cand 1d) la wherein Y=-(CH2)n" wherein n = 1-11 R=-MV2+ -(CH2)m"CH3 2Xe or -pyr2* -(CH2)m"CH3 2X9 wherein m=1-13 Ib wherein Y=-(CH2)n- wherein n = 1-11 R= -MV2+ -(CH2)m-Acr 2Xe or -pyr2+ -(CH2)m"Acr 2Xe wherein m=1-11 Ic wherein Y= ortho or para tolyl R=_MV2+ -(CH2)m-CH3 2Xe or -pyr2* -(CH2)m-CH3 2Xe wherein m=1-13 Id wherein Y=-(CH2)n-wherein n=1-10 R= -Acr+-R1 Xe/2Xe wherein N in the acridine main ring is also quarternised by alkyl group R1=-(CH2)m-CH3 and -(CH2)m-C6H4-(CH2)m-(para) Wherein rn =0-13 Formula 1 The molecules are useful for the stabilization of DNA including duplex, triplex and quadruplex structures through intercalation and/or bisintercalation and groove binding interactions.
Full Text VIOLOGEN LINKED ACRIDINE BASED MOLECULE
Field of the invention
The present invention relates to viologen linked acridine based molecule of the
general formula 1 (la, Ib, Ic and Id) below
(Figure Remove) la wherein Y = -(CH2)n- wherein n = 1-11
R = -MVCH-CH2X® or
-Pyr2+-(CH2)m-CH3 2Xe wherein m = 1-13
\b wherein Y = -(CH2)n- wherein n = 1-1 1
R = -MV2+-.CCH2)nr-Acr 2X® or
-Pyr2+-(CH2)m-Acr 2X® wherein m = 1-11
. wherein Y = ortho or para tolyl
R = -MVCHArCHZX® or
2Xe wherein m = 1-13
wherein Y = -(CH2)n- wherein n = 1-10
R= -Aer^R1 XS/ [email protected]
wherein N in the acridine main ring is also quarternised by alkyl group
R1 = -(CH2)m-CH3 and -(CH2)m-C6H4-(CH2)m- (para),
wherein m = 0-13
MV2+ = -- X = Cl, Br, I, CF3SO3
Acr = N Acr+ =
(Figure Remove)and/or pharmaceutically acceptable derivatives thereof, useful as phototherapeutical
and catalytic photoactivated DNA cleaving agents. The novel molecules of the
invention are useful for the stabilization of DNA including duplex, triplex and
quadruplex structures through intercalation and/or bisintercalation and groove binding
interactions. The novel molecules of the invention are also for the catalytic
photoactivated cleavage of DNA purely through cosensitization with selectivity at
guanine (G) sites in duplex and AG two base bulge containing sequences.
The present invention also relates to a series of bifunctional molecules of the
general formula 1 (la, Ib, and Ic) and derivatives thereof, are also useful as
photocatalysts for the oxidation of water to generate hydrogen in industrial
applications.
Background of the invention
Design of functional molecules that bind selectively to nucleic acids (DNA or
RNA) and are capable of cleaving duplex or single stranded nucleic acids is an active
area of research that has important biochemical and biomedical applications. Some of
these effective agents have been extremely useful in the treatment of various diseases
and also as probes for understanding DNA structures and DNA-protein interactions.
While natural restriction enzymes have been very useful in many of these applications,
their large size and/or limited range of sequence recognition capabilities prevent their
general use. Hence, synthetic functional molecules that cause site-selective or sequence
specific modifications of DNA and offer a clean and efficient way of cutting DNA at
sites that are not recognized by conventional restriction enzymes are highly required. /
In this context, a large number of synthetic ligands have been developed, which
have ability to recognize and bind to specific sequences or structural domains in DNA
and exhibit nucleolytic activity under physiological conditions (chemical nucleases) or
upon photoactivation (photonucleases). Some of these include, 1,10-phenanthrolinecopper,
ferrous-EDTA, bleomycin, enediyene antibiotics and anthraquinones. For
examples, references may be made to US patent No. 5,985,557; No. 6,090,543; No.
5,739,022; No. 5,556,949; No. 5,552,278; No. 5,504,075; No. 4,942,227; Nielsen, P. E.
J. Mol. Recog.. 1990, 3, 1; Papavassiliou, A. G. Eiochem. J. 1995, 305, 345; Sigman,
D. S.; Graham, D. R; D'Aurora, V.; Stern, A. M. J. Biol. Chem. 1979, 254, 12269;
Pope, L. E.; Sigman, D. S. Proc. Natl. Acad. Sci. USA. 1984, 81, 3; Tullius, T. D.;
Dombroski, B. A. Proc. Natl. Acad. Sci. USA. 1986, 83, 5469; Hertzberg, R. P.;
Dervan, P. B. J. Am. Chem. Soc. 1982, 104, 313; Hecht, S. M.; Bleomycin: Chemical,
Biochemical and Biological aspects, Ed.; Springer Verlag: New York, 1979; Sausville,
E. A.; Peisach, J.; Horwitz, S. B. Biochemistry, 1978, 17, 2740. However, synthetic
ligands that are versatile and mimic the conventional restriction enzymes are yet to be
developed.
Of the several classes of DNA cleaving systems reported, the photoactivated
cleaving agents have been found to posses significant practical advantages over the
reagents that cleave DNA under thermal conditions. An interesting aspect of the
photoactivated DNA cleaving agent is that it allows the reaction to be controlled
spatially and temporally by combining all of the components of the reaction mixture
before the irradiation. Excitation of the reaction mixture with an appropriate light
source initiates the reaction, which continues until the light is shut off. The ability to
control light, in both spatial and temporal sense would be advantageous for applications
ranging from the time resolved probing of various biochemical processes such as
transcription and translation to genomic analysis and therapeutic agents. For selected
examples, reference may be made to US patent No. 5,994,410; No. 5,734,032; No.
5,650,399; No. 5,607,924; No. 6,087,493; No. 6,057,096; 5,767,288: No. 5,439,794;
Armitage, B. Chem. Rev. 1998, 98, 1171 and references sited therein; Kochevar, I. E.;
Dunn, D. A. Bioorg. Photochem. 1990,1, 273 and references sited therein; Paillous, N;
Vicendo, P. J. Photochem. Photobiol, B 1993, 20, 203; Nielsen, P. E.; Jeppesen, C.;
Buchardt, O. FEBS left. 1988, 235, 122; Chow, C. S.; Barton, J. K. Methods Enzymol.
1992, 212, 219; Chang, C. -H.; Meares, C. F. Biochemistry 1982, 21, 6332; Riordan, C.
G.; Wei, P. J. Am. Chem. Soc. 1994, 116, 2189; Thorp, H. H. Angew. Chem., Int. Ed
Eng. 1991, 30, 1517; Armitage, B.; Yu, C.; Devadoss, C.; Schuster, G. B. J. Am.
Chem. Soc. 1994, 116, 9847; Adam, W.; Cadet, J.; Dall'Acqua, F.; Epe, B.; Ramaiah,
D.; Saha-Moller, C. R. Angew. Chem. Int. Ed Engl. 1995, 34, 107; Uesawa, Y.;
Kuwahara, J.; Sugiura, Y. Biochem. Biophys. Res. Commun. 1989, 164, 903; Ito, K.;
Inoue, S.; Yamamoto, K.; Kawanishi, S. J. Biol. Chem. 1993, 268, 13221; Saito, I.;
Takayama, M.; Matsuura, T.; Matsugo, S.; Kawanishi, S. J. Am. Chem. Soc. 1990,112,
883; Sako, M.; Nagai, K.; Maki, Y. J. Chem. Soc. Chem. Commun. 1993, 750.
These photoactivated cleaving agents found to cleave DNA (i) by generation of
diffusible (singlet oxygen) and non-diffusible (hydroxyl radicals) reactive
intermediates, (ii) hydrogen atom abstraction and (iii) electron transfer. Most of the
systems reported so far, initiate photocleavage by more than one mechanism. Though
the damage induced by all these mechanisms lead to the initial modification of either
sugar or nucleobase, which then results in phosphodiester cleavage, serious efforts are
in progress to develop reagents which cleave DNA purely by one mechanism and also
to target these cleaving agents to specific sequences or domains in DNA. References
may be made to Cadet, J.; Teoule, R. Photochem. Photobiol. 1978, 28, 661; Croke, D.
T.; Perrouault, L.; Sari, M. A.; Battioni, J. P.; Mansuy, D.; Magda, D.; Wright, M. M;
Miller, R. A.; Sessler, J. L.; Sansom, P. I. J. Am. Chem. Soc. 1995, 117, 3629;
Theodorakis, E.; Wilcoxen, K. M. Chem. Commun. 1996, 1927; Suenaga, H.;
Nakashima, K.; Hamachi, I.; Shinkai, S. Tetrahedron Lett. 1997, 38, 2479; Cullis, P.
M.; Malone, M. E.; Merson-Davies, L. A. J. Am. Chem. Soc. 1996,118, 2775; Sies, H.;
Schulz, W. A.; Steenken, S. J. Photochem. Photobiol. B 1996, 32, 97; Saito, L;
Takayama, M.; Sugiyama, H.; Nakamura, T. In DNA and RNA Cleavers and
Chemotherapy of Cancer and Viral Diseases; Meunier, B., Ed.; Kluwer: Netherlands,
1996, pp 163-176.
Recently, there has been growing interest in designing molecules, which cleave
DNA effectively through photoinduced electron transfer mechanism involving purely
by the oxidation of nucleobases. A unique feature of this mechanism is that one can
have reasonable control over the cleavage. It has been observed that DNA cleavage by
this mechanism occurs at guanine (G), since guanine is the most easily oxidizable base
of the nucleic acids because of its low ionization potential. A large number of organic
as well as inorganic systems have been reported which cause DNA cleavage by
photoinduced electron transfer mechanism. However, most of these reagents were
found to be less efficient with the cleavage efficiency in the order of 10~8. References
may be made to Sevilla, M. D.; D'Arcy, J. B.; Morehouse, K. M.; Englehardt, M. L.
Photochem. Photobiol. 1979, 29, 37; Blau, W.; Croke, D. T.; Kelly, J. M.; McConnel,
D. J.; OhUigin, C.; Van der Putten, W. J. M. J. Chem. Soc. Chem. Commun. 1987, 751;
Sage, E.; Le Doan, T.; Boyer, V.; Helland, D. E.; Kittler, .; Helene, C; Moustacchi, E.
./. Mol. Biol. 1989, 209, 297; Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1991, 773,
8153; Ly, D.; Kan, Y.; Armitage, B.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118,
8747: Hall, D. B.; Holmlin, R. E.;Barton, J. K. Nature 1996, 382, 731; Gasper, S. M.;
Schuster, G. B. J. Am. Chem. Soc. 1997, 779, 12762. Therefore, efficient
photoactivated DNA cleaving agents based on electron transfer mechanism are highly
desired for biological applications.
In the case of the photoactivated DNA cleaving agents by electron transfer
mechanism, the efficiency of the reaction depends on the reduction potential of the
cleaving agent, excited state energy and the oxidation potential of the ground state base,
in addition to other conditions. For an efficient reaction to occur, the rate of the forward
electron transfer from the donor to an acceptor must be greater than the back electron
transfer process. Therefore, the inefficiency associated with the photoactivated DNA
cleaving agents can be attributed to the existence of an efficient back electron transfer
between the resultant oxidized base and the reduced sensitizer. To overcome the
drawback of the back electron transfer process associated with such systems, a few
examples based on cosensitization mechanism have been developed (Dunn, D. A.; Lin,
V. H.; Kochevar, I. E. Biochemistry 1992, 31, 11620; Atherton, S. I; Beaumont, P. C.
J. Phys. Chem. 1987, 91, 3993; Fromherz, P.; Rieger, B. J. Am. Chem. Soc. 1986,108,
5361). These systems consists of a sensitizer, which is also an intercalator, transfers an
electron upon photoactivation to a cosensitizer (electron acceptor), bound on the
surface of DNA. The photosensitization involving the cosensitizer that bound far away
from the sensitizer is expected to inhibit the back electron transfer and thereby increase
the DNA cleavage. However, in reality, only marginal improvement in DNA cleavage
efficiency (in the order of 10~7) was observed in these systems owing to the
complications with respect to the concentration, distance and DNA binding affinities of
the sensitizer and cosensitizers.
Therefore, development of small compounds which are, soluble in aqueous
medium, overcome the inefficiency due to the back electron transfer, undergo strong
binding interactions with DNA, selective and effective in inducing DNA cleavage
purely through electron transfer mechanism are highly desired for biological
applications including clean and efficient way of cutting DNA at sites that are not
recognized by the conventional restriction enzymes.
It is an objective of the present investigation to provide functional molecules
which bind strongly and selectively with DNA and act as selective and effective
photoactivated DNA cleaving agents which function purely through cosensitization
mechanism.
Objects of the invention
The main objective of the present invention is to provide novel bifunctional
molecules based on viologen linked acridines or derivatives thereof, which can be used
as phototherapeutical and catalytic photoactivated DNA cleaving agents.
Another objective of the present invention is to provide bifunctional molecules
based on viologen linked acridines or derivatives thereof, which can act as probes for
various DNA structures (single strand, duplex, triplex and quadruplex) of biological
significance and with high selectivity.
Yet another objective of the present invention is to provide bifunctional
molecules based on viologen linked acridines or derivatives thereof, which can act as
catalytic photoactivated DNA cleaving agents of duplex and base bulges containing
DNA, purely through cosensitization mechanism.
Still another objective of the present invention is to provide bifunctional
molecules based on viologen linked acridines or derivatives thereof, which can act as
photocatalysts for the oxidation of water in industrial applications.
Summary of the invention
Accordingly the present invention relates to viologen linked acridine based
molecule of the general formula 1 (la, Ib, Ic and Id) below
id t wherein Y = -(CH2)tt- wherein n = 1-11
R =-MV2+-(CH2)m-CH32Xe or
-Pyr2+-(CH2)m-eH3 2Xe wherein m = 1-13
^ wherein Y =-(CH2)n- wherein n = 1-11
R =-MV2+-(CH2)m-Acr2Xe or
-PyT2+-(CH2ym-A.ci 2X® wherein m = 1-11
wherein Y = ortho or para tolyl
R =-MV2+-(CH2)m.CH3.2Xe or
-Pyr2+-(CH2)m-CH3 2X® wherein m = 1-13
wherein Y =-(CH2)n - wherein n = 1-10
R = —Acr+-R'Xe/2X
wherein N in the erktiae-min-rifrg fs a4so quartermsed by alkyl group
R1 = -(CH2)m-CH3 and -(CH2)m-C6H4-(CH2)m- (para),
wherein m =0-13
MV2+ = - 3 - £ - X=Cl,Br, I,CF3S03
Acr =
(Figure Remove)The present invention also relates to a process for the preparation of the novel
bifunctional molecule based on viologen linked acridines, bisacridines and acridinium
salts of the general formula 1 (la, Ib, Ic and Id) above, said process comprising
forming a solution of o-(acridin-9-yl)-a-bromoalkanes and/or l-alkyl-4,41-
bipyridinium bromides in dry acetonitrile in the ratio of 1:1, stirring the solution at a
temperature in the range of 20-50 °C for a time period in the range between 8-24 h to
obtain a precipitate, filtering, and washing the precipitate with dry acetonitrile and
dichloromethane to remove any unreacted starting materials, purifying the solid so
obtained to give obtain compound of formula 1 (la, Ib, Ic and Id).
In one embodiment of the invention, the compounds of formula 1 are preferably
recrystallized from a mixture (1:4) of methanol and acetonitrile.
Brief description of the accompanying drawings
FIG. 1 shows the fluorescence enhancement of the compound of formula la
(wherein n = 1, R,= -MV2+-(CH2)3-CH3 and X = Br) in presence of various
concentrations of poly(dA).poly(dT).
FIG. 2 shows the effect on thermal denaturation temperature of DNA duplex by
various concentrations of compound of formula la wherein n = 1, R = -
MV2+-(CH2)3-CH3 and X = Br and formula Ib wherein n = 1, R = -MV2+-
CH2-Acr and X = Br.
FIG. 3 shows the hypochromocity observed in DNA duplex absorption in presence
of various concentrations of compound of formula la wherein n = 1, R = -
MV2+-(CH2)3-CH* and X = Br and formula Ib wherein n = 1, R = -MV2+-
CH2-Acr and X = Br.
FIG. 4 shows the DNA damage profiles showing single strand breaks and various
endonuclease-sensitive modifications induced in PM2 DNA by compound of
formula la (250 nM, 18 kJ/m2) wherein n = 1, R = -MV2+-(CH2)3-CH3 and X
- Br and at (1 nH 9 kJ/m2) wherein n = 11, R = -MV2+-(CH2)3-CH3 and X =
Br.
FIG. 5 shows the time dependence of DNA modifications, single strand breaks (SSB)
(O) and formamidopyrimidine-DNA glycosylase (FPG protein) sensitive
modifications (A) induced in PM2 DNA by compound of formula la wherein
n = 1, R = -MV2+-(CH2)3-CH3 and X = Br (0.25 uM, 0 °C) upon UV
irradiation with 360 nm light (4.6 kJ/m2).
FIG. 6 shows the time dependence of DNA modifications, single strand breaks (SSB)
(O) and formamidoglycosylase (FPG protein) sensitive modifications (A)
induced in PM2 DNA by compound of formula la wherein n = 11, R = -
MV2+-(CH2)3-CH3 and X = Br (0.30 jiM, 0 °C) upon UV irradiation with 360
nm light (4.6 kJ/m2).
FIG. 7 shows the autoradigram of a 10% denaturing polyacrylamide gel showing
photocleavage of duplex DNA (10 uM) induced by compound of formula la
wherein n = 1, R = -MV2+-(CH2)3-CH3 and X = Br and formula Ib wherein n
= 1, R = -MV2+-CH2-Acr and X = Br with piperidine treatment (90°C, 30
min). Lane 1: DNA (3) (control). Lane 2: DNA (3) and 50 fjM of compound
of formula la wherein n = 1, R = -MV2+-(CH2)3-CH3 and X = Br. Lane 3:
DNA (3) and 50 uM of compound of formula Ib wherein n = 1, R = -MV2+-
CHj-Acr and X = Br. Lane 4: DNA (5) and 50 uM of compound of formula
la wherein n - 1, R = -MV2+-(CH2)3-CH3 and X = Br. Lane 5: DNA (5) and
50 uM of compound of formula Ib wherein n = 1, R = -MV2+-CH2-Acr and
X = Br. Lane 6: DNA (6) and 50 uM of compound of formula la wherein n
= 1, R = -MV2+-(CH2)3-CH3 and X = Br. Lane 7: DNA (6) and 50 uM of
compound of formula Ib wherein n = 1, R = -MV2+-CH2-Acr and X = Br.
The Maxam-Gilbert sequencing lanes are marked with A/G and T.
FIG. 8 shows the formation of reduced methyl viologen upon irradiation of
compound of formula la wherein n = 11, R = -MV2+-(CH2)3-CH3 and X = Br
in presence of DNA (sacrificial electron donor), demonstrating the catalytic
properties of viologen linked acridines.
Detailed description of the invention
In the present invention, the novel bifunctional molecules incorporate a DNA
intercalator and cosensitizer linked through rigid to flexible spacer groups. The
intercalator such as acridine moiety is capable of absorption in the visible region and
acting as an electron-donor (sensitizer) in the excited state. The cosensitizer, methyl
viologen moiety, on the other hand, is a very good electron acceptor and is capable of
undergoing groove-binding interactions with DNA. The rigid to flexible spacer groups
play a major role in controlling the rate of electron transfer in these systems through the
alteration of distance and relative orientation between the intercalator and cosensitizer.
In the present invention, a series of novel viologen linked acridine, bisacridine
and bisacridinium salts of the general formula 1 (la, Ib, Ic and Id) have been
synthesised, having flexible to rigid spacer groups and their photophysical properties
examined under different conditions. Further the DNA binding and DNA stabilization
properties of these molecules have been examined employing calf thymus DNA and
synthetic oligonucleotides. The photoactivated DNA cleaving properties were
investigated and the sequence specific cleavage induced by these compounds by
employing plasmid DNA, the synthetic duplex and base bulge containing DNA
sequences were also analysed. Further the mechanism of their biological and catalytic
activities have been evaluated.
As a result, it is observed that compounds of the general formula 1 (la, Ib, Ic
and Id) and their derivatives thereof posses good solubility at physiological pH
conditions and exhibit good absorption properties. Further protonation of the acridine
ring leads to the formation of corresponding acridinium salts which show quite
different and interesting photophysical and redox properties. The absorption and
fluorescence studies confirm the existence of through bond and through space
interactions between viologen and acridine moieties. Further, efficient quenching of
fluorescence yields was observed and which indicate the mechanism of quenching is
through electron transfer mechanism with rates in the order of 1010.
Interestingly, these bifunctional molecules exhibit strong binding with DNA
through intercalation and or bisintercalation and groove binding interactions and with
unusually high affinity for the poly(dA).poly(dT) sequence. Upon excitation, these
molecules cleaved DNA very effectively and with high selectivity at guanine (G) sites
and predominance of 5'-G of GG step. Moreover, these molecules are found to be very
attractive for sequences containing base bulges and cleave upon photoactivation
specifically at the G sites of the AG base bulge. The DNA cleavage induced by these
compounds is purely through the electron transfer mechanism, where the excited
acridine moiety transfers an electron to methyl viologen (MV2+) and lead to the radical
cation of acridine and radical cation of methyl viologen (MV+ ). The radical cation of
acridine once formed, can oxidize DNA base at the site of binding and ultimately
resulted in efficient cleavage of DNA at the G sites, as expected. Moreover, these
molecules are found to be recyclable and act as catalysts in presence of sacrificial
electron donors. Therefore, the present systems are not only bifunctional with
interesting photophysical properties but also recyclable and act as effective
10
photoactivated DNA cleaving and phototherapeutical agents and photocatalysts for the
oxidation of water in industrial applications.
Table 1 shows DNA association constants of bifunctional molecules based on
viologen linked acridines compound of formula la wherein n = 1, R = -
MV2+-(CH2)3-CH3 and X = Br and wherein n = 11, R - -MV2+-(CH2)3-CH3
and X = Br and bisacridine system (compound of formula Ib wherein n = 1,
R = -MV2+-CH2-Acr and X = Br) in buffer containing 1 mM EDTA and 2 or
IQOmMNaCl.
Table 2 shows the structures of oligonucleotides used for DNA melting studies.
Table 3 shows the structures of oligonucleotides used for DNA cleaving studies.
The present invention accordingly provides a process for the preparation of
bifunctional molecules represented by compound of formula 1 (la, Ib, Ic and Id) and
derivatives thereof.
Another embodiment of the invention provides bifunctional molecules of the
general formula 1 (la, Ib, Ic and Id) and pharmaceutically acceptable derivatives
thereof for the stabilization of DNA structures including duplex, triplex and quadruplex
DNA
Yet another embodiment of the present invention relates to bifunctional
molecules of the general formula 1 (la, Ib, Ic and Id) and pharmaceutically acceptable
derivatives thereof for the photocatalytic cleavage of DNA at G sites of duplex and AG
two base bulges containing DNA purely through cosensitization mechanism.
In another embodiment of the invention, bifunctional molecules of the general
formula 1 (la, Ib, Ic and Id) and pharmaceutically acceptable derivatives thereof are
used as DNA targeted diagnostic or phototherapeutic agents. These sensitizers are to be
used for diagnosis or treatment of human beings or animals.
In another embodiment of the present invention the bifunctional molecules of
the invention are used for the stabilization of DNA including duplex, triplex and
quadruplex structures through intercalation, bisintercalation and groove binding.
Yet another embodiment of the present invention is the use of the bifunctional
molecules of general formula 1 (la, Ib, Ic and Id) for the photocatalytic cleavage of
DNA with selectivity at G sites of duplex and AG two base bulges and hence can be
used as probes for these structures.
Still another embodiment of the present invention is that the bifunctional
molecules and derivatives thereof of the general formula 1 (la, Ib, and Ic) are used as
photocatalysts for the oxidation of water in industrial applications.
The following examples are given by way of illustration and therefore should
not be construed to limit the scope of present investigation.
Examples 1-4 represent typical synthesis of compounds of general formula 1
(la, Ib, Ic and Id) and Examples 5-11 represent photophysical and in vitro DNA
binding and cleaving properties of bifunctional viologen linked acridine based
molecules.
EXAMPLE 1
General procedure for the preparation of formulae represented by compound of
formula la (wherein Y = -(CH^, wherein n = 1 - 11; R = -MV2+-(CH2)m-CH32XB or -
Pyr2+-(CIfe)m-CH32XB). A solution of o-(acridin-9-yl)-a-bromoalkanes (1-10 mmol)
and l-alkyl-4,4'-bipyridinium bromides (1-10 mmol, which in turn were obtained in 93-
98% yields by the reaction of 4,4'-bipyridine with the corresponding a-bromoalkanes
in the molar ratio of 3:1.) in the ratio of 1:1 in dry acetonitrile (30-50 mL) was stirred at
20°C for 10 h. The precipitated solid thus obtained was filtered, washed with dry
acetonitrile and dichloromethane to remove any unreacted starting materials. The solid
was further purified by soxhlet extraction with dichloromethane to give the compound
of formula la in 70-95% yields. These compounds were recrystallized from a mixture
(1:4) of methanol and acetonitrile.
The physiochemical properties of l-[(acridin-9-yl)methyl]-l'-butyl-4,4'-
bipyridinium dibromide (compound of formula la wherein n = 1, R = -MV2+-(CH2)3-
CH3 anc} X = Br): melting point: 260-261 °C; Molecular Weight: MS (FAB): m/z 484
(M+BO; 'H NMR (300 MHz, DMSO-ck): 8 - 0.91 (X 3H), 1.26-1.33 (m, 2H), 1.89-
1.94 (m, 2H), 4.66 (t, 2H), 7.11 (s, 2H), 7.77 (t, 2H), 7.97 (t, 2H), 8.31 (d, 2H), 8.53 (d,
2H), 8.59 (d, 2H), 8.66 (d, 2H), 9.19 (d, 2H), 9.31 (d, 2H); 13C NMR (75 MHz, DMSOd6,)
8= 149.78-121.41, 61.05, 55.44, 33.01, 19.10, 13.66; Nature: Pale yellow powder.
The physiochemical properties of l-[3-(acridin-9-yl)propyl]-r-butyl-4,4'-
bipyridinium dibromide (compound of formula la wherein n = 3, R = -MV2+-(CH2)3-
CH3 and X = Br): melting point: 253-254 °C; Molecular Weight: MS (FAB): m/z 433
(M+); !H NMR (300 MHz, DMSO-ck) 8 = 0.95 (t, 3H), 1.28-1.40 (m, 2H), 1.92-2.02
(m, 2H), 2.46-2.51 (m, 4H), 3.95 (t, 2H), 4.74 (t, 2H), 7.79 (t, 2H), 8.05 (t, 2H), 8.28 (d,
2H), 8.83-8.86 (m, 6H), 9.45 (d, 2H), 9.57 (d, 2H); 13C NMR (75 MHz, DMSO-d6) 8 =
149.45-124.96, 61.44, 60.73, 33.21, 33.15, 24.79, 19.27, 13.80; Nature: Pale yellow
powder.
The physiochemical properties of l-[ll-(acridin-9-yl)undecyl]-r-butyl-4,4'-
bipyridinium dibromide (compound of formula la wherein n = 11, R = -MV2+-(CH2)3-
CH3 and X = Br): melting point: 248 -249 °C; Molecular Weight: MS (FAB): m/z 545
(M*); 1H NMR (300 MHz, DMSO-de): 8 - 0.95 (t,3HX 1.23-1.71 (m, 18H), 1.95-1.98
(m, 4H), 3.65 (t, 2H), 4.73 (t, 4H), 7.65 (t, 2H), 7.85 (t, 2H), 8.14 (d, 2H), 8.38 (d, 2H),
8.82 (d, 4H), 9.43 (d, 4H); 13C NMR (75 MHz, DMSO-d6): 148.97-124.71, 61.25,
61.04, 33.07, 31.64, 31.15, 29.68, 29.26, 29.13, 28.76, 27.13, 25.79, 19.15, 13.71;
Nature: Pale yellow powder.
EXAMPLE 2
General procedure for the preparation of formulae represented by compound of
formula Ib (wherein Y = -(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-Acr2Xa or -
Pyr2+-(CH2)m-Acr2XB). A solution of o-(acridin-9-yl)-a-bromoalkanes (2-10 mmol)
and 4,4'-bipyridine (1-5 mmol) in the ratio of 2:1 in dry acetonitrile (50-150 mL) was
stirred at 35°C for 20h. Precipitated solid was filtered and washed with
dichloromethane and acetonitrile. Soxhlet extraction of the solid with dichloromethane
gave compound of formula Ib in 65-90% yields.
The physiochemical properties of bis-l,r-[(acridin-9-yl)methyl]-4,4'-bipyridinium
dibromide (Ib wherein n - 1, R = -MV2+-CH2-Acr and X = Br): melting point: >400
°C; Molecular Weight: MS (FAB): m/z 619 (M^Bf); *H NMR (300 MHz, DMSO-d6):
8 = 7.09 (s, 4H), 7.77-7.95 (m, 8H), 8.21-8.55 (m, 16H), 9.05-9.16 (m, 8H); 13C NMR
(75 MHz, DMSO- d6) 8 = 150.85-124.51, 58.52; Nature: Pale yellow powder.
The physiochemical properties of bis-l,r-[3-(acridin-9-yl)propyl]-4,4'-
bipyridinium dibromide (Ib wherein n = 3, R = -MV2+-(CHj)3-Acr and X = Br):
melting point: 223-224 °C; Molecular Weight: MS (FAB): m/z 596 (M+); 1H NMR
(300 MHz, D2O): 8 = 2.50-2.60 (m, 4H), 3.95 (t, 4H), 4.9 (t, 4H), 7.79-7.83 (m, 6H),
8.01-8.07 (m, 8H), 8.26 (d, 2H), 8.46-8.48 (m, 4H), 8.69 (d, 2H), 8.93 (d, 2H); 13C
NMR (75 MHz, DMSO-d6): 8 = 150.82-121.86, 60.26, 32.36, 24.30; Nature: Pale
yellow powder.
The physiochemical properties of bis-!,!'-[ 11-(acridin-9-yl)undecyl]4,4'-
bipyridinium dibromide (Ib wherein n = 11, R = -MV2+-(CH2)n-Acr and X = Br):
melting point: 152-153 °C; Molecular Weight: MS (FAB): m/z 820 (M+); 1H NMR
(300 MHz, DMSO-d6): 8 = 1.22-1.94 (m, 36H), 3.63 (t, 4H), 4.64 (t, 4H), 7.65 (t, 4H),
7.85 (t, 4H), 8.04 (d, 4H), 8.15 (d, 4H), 8.37 (d, 2H), 8.63 (d, 2H), 8.87 (d, 2H), 9.24 (d,
2H); 13C NMR (75 MHz, DMSO-de): 5 = 151.36-122.30, 60.83, 31.63, 31.07, 29.69,
29.26, 29.11, 28.75, 27.13, 25.79; Nature: Pale yellow powder.
EXAMPLE 3
Synthesis of compound of formula Ic (wherein Y = ortho or para tolyl; R = -
MV2+-(CH2)m-CH32Xa or -Pyr2+-(CH2)m-CH32XH). A solution of 9-(2-
bromomethylphenyl)acridine (1-5 mmol) and l-butyl-4,4'-bipyridinium bromide (1-5
mmol) in the ratio of 1:1 in dry acetonitrile (30-120 mL) was stirred at 50°C for 15h.
The precipitated solid thus obtained was filtered, washed with dry acetonitrile and
dichloromethane to remove any unreacted starting materials. The solid was further
purified by soxhlet extraction with dichloromethane to give compound of formula Ic
(wherein n = 1, R = -MV2+-(CH2)3-CH3, X = Br and acridine is at the ortho position) in
35-55% yield and the product was further purified by recrystallization from ethyl
acetate. Similarly, the reaction of 9-(4-bromomethyl phenyl)acridine (1-5 mmol) and 1-
butyl-4,4'-bipyridinium bromide (1-5 mmol) in the ratio of 1:1 in dry acetonitrile (30-
120 mL) gave compound of formula Ic (wherein n = 1, R = -MV2+-(CH2)3-CH3, X = Br
and acridine is at the para position) in 55-70% yield and the product was further
purified by recrystallization from ethyl acetate.
The physiochemical properties of l-[(2-(acridin-9-yl)-l-methyl)phenyl]-l'-
butyl-4,4'-bipyridinium dibromide (Ic wherein n = 1, R - -MV2+-(CH2)3-CH3, X = Br
and acridine is at the ortho position): melting point: 224-225 °C; Molecular Weight:
MS (FAB): m/z : 561 (M+Br'); 1H NMR (300 MHz, DMSO-d6): 8 = 0.95 (t, 3H), 1.31-
1.39 (m, 2H), 1.95-2.00 (m, 2H), 4.73 (t, 2H), 5.56 (s, 2H), 7.21 (d, 2H), 7.42 (t, 2H),
7.5 (d, 2H), 7.75-7.89 (m, 4H), 8.20-8.23 (m, 4H), 8.41 (d, 2H), 8.55 (d, 2H), 9.4 (d,
2H); 13C NMR (75 MHz, DMSQ-ds): 8 = 148.49-124.24, 62.01, 60.64, 32.67, 18.78,
13.35; Nature: Yellow powder.
The physiochemical properties of l-[(4-(acridin-9-yl)-l-methyl)phenyl]-l'-butyl-
4,4'-bipyridinium dibromide (Ic wherein n = 1, R = -MV2+-(CH2)3-CH3, X = Br and
acridine is at the para position): melting point: 268-269 °C; Molecular Weight: MS
(FAB): m/z 561 (M+Br'); JH NMR (300 MHzr DMSO-d6): 8 = 0.94 (t, 3H), 1.31-1.38
(m, 2H), 1.90-1.95 (m, 2H), 4.72 (t, 2H), 6.18 (s, 2H), 7.57- 7.65 (m, 6H), 7.88-7.92
(m, 4H), 8.24 (d, 2H), 8.86 (d, 2H), 8.88 (d, 2H), 9.43 (d, 2H), 9.69 (d, 2H); 13C NMR
(75 MHz, DMSO-d6): 8 - 149.23-124.22, 62.86, 60.63, 32.70, 18.76, 13.32; Nature:
Yellow powder.
EXAMPLE 4
General procedure for the preparation of formulae represented by compound of
formula Id (wherein Y = -(CH2)n, wherein n = 1 - 10; R = -Acr+-R1XB/2Xs wherein N
in the acridine ring main ring is also quarternised by alkyl group. R1 = -(CH2)m-CH3 and
-(CH2)m - C6H4-(CH2)m-(/w#) wherein m = 0 - 13: A solution of a,«-bis(9-
acridinyl)alkane (1-5 mmol) and alkyl halide (3-15 mmol) in the ratio of 1:3 in dry
acetonitrile (30-150 mL) was refluxed for 8-24 h. The precipitated solid was filtered
and washed with dry acetonitrile and dichloromethane in small portions to remove
unreacted starting materials. The solid was further purified by recrystallization from a
mixture (1:4) of ethyl acetate and acetonitrile to give compound of formula Id in 65-
95% yields.
The physiochemical properties of compound of formula Id (wherein n = 5, R = -
Acr+-CH3 and X = I): melting point: 222-223 °C; Molecular Weight: MS (FAB): m/z
561 (M+Bf) !H NMR (300 MHz, DMSO-d6): 8 - 1.88 (m, 6H), 3.99 (t, 4H), 4.82 (s,
6H), 8.01 (t, 4H), 8.35 (t, 4H), 8.78 (d, 4H), 8.87 (d, 4H); 13C NMR (75 MHz, DMSOd6):
8 - 164.38-20.01, 32.60, 32.22, 30.19, 29.37; Nature: Yellow powder.
The physiochemical properties of compound of formula Id (wherein n = 10, R = -
Acr+-CH3 and X = I): melting point: 230-232 °C; *H NMR (300 MHz, DMSO-de): 8 =
1.26-1.74 (m, 16H), 3.97 (t, 4H), 4.82 (s, 6H), 8.03 (t, 4H), 8.43 (t, 4H), 8.77 (d, 4H),
8.9 (d, 4H); 13C NMR (75 MHz, DMSO-d6): S = 164.65-120.03, 33.75, 33.05, 29.94,
29.44,29.32, 24.97; Nature: Yellow powder.
EXAMPLE 5
DNA binding efficiency. DNA binding affinities of acridine-viologen bifunctional
molecules represented by compound of formula la and Id were analyzed using calf
thymus DNA, in NaCl buffer at different salt concentrations (2 mM and 100 mM). Test
solutions containing different concentrations of calf thymus DNA in NaCl buffer were
incubated at room temperature for one hour to complete the complexation and were
analyzed using absorption and fluorescence techniques. Strong hypochromicity in
absorption and effective quenching of fluorescence emission yields of viologen linked
acridines were observed in presence of DNA. The binding constants of viologen linked
acridines with DNA were determined according to the reported procedures. References
may be made to Peacocke, A. R.; Skerrett, J. N. H. Trans. Faraday Soc. 1956, 52, 261;
McGhee, J. D.; von Hippel, P. H. J. Mol. Biol. 1974, 86, 469; Scatchard, G. Ann. N.Y.
Acad Sci 1949, 51, 660; Adam, W.; Cadet, J.; Dall'Acqua, F.; Epe, B.; Ramaiah, D.;
Saha-Moller, C. R. Angew. Chem. Int. Ed Engl. 1995, 34, 107). These molecules
exhibited appreciable binding affinity (in the order of 106) for calf thymus DNA and
were found to be one order less at higher salt concentration (Table 1). Further
fluorescence lifetimes of these compounds were also examined in presence and absence
ofDNA.
Compound Ionic strength K (M"1)
la wherein n=l; R = -MV2+-(CH2)a- 2mMNaCl 9.24 x 105
CH3;X-Br lOOmMNaCl 1.01 x 105
la wherein n=l 1; R = -MV2+-(CH2)3- 2 mM NaCl 5.24 x 106
CH3; X = Br 100 mM NaCl 1.64 x 105
Ib wherein n=l;R = -MV2+-CH2-Acr; 2mMNaCl 1.30 x 106
X = Br 100 mM NaCl 3.90 x 105
These results indicate that the planar acridine ring can intercalate between the
base pairs of calf thymus DNA in a position perpendicular to the helix axis. At the
same time, the viologen moiety interacts electrostatically with the phosphate backbone
of the DNA. The strong dependence of the binding constants on ionic strength of the
buffer medium is a strong indication that these systems interact with DNA through
intercalation as well as by groove binding, indicating their bifunctional in character.
EXAMPLE 6
Demonstration of special affinity for poly(dA).poly(dT) sequence. In order to
have a better understanding on the binding site of acridine chromophore in viologen
linked acridines in DNA, the absorption and fluorescence properties of these molecules
16
ere examined in presence of various polynucleotides. The gradual addition of
poly(dA).poly(dT) to the buffered solutions of viologen linked acridines led to a
gradual decrease in absorption intensity with a strong enhancement in their
fluorescence emission yields (FIG. 1). However, no significant changes were observed
when a solution of poly(dG).poly(dC) was added. These results indicate the fact that
the viologen linked acridines examined herein posses special affinity for A.T sequence
and hence can have potential applications as probes for the detection of such sequences
in DNA.
EXAMPLE 7
Enhancement in thermal stability of DNA. Intercalation of small ligands into
DNA duplex is known to increase the DNA melting temperature (Tm), i.e. the
temperature at which the double helix denatures into single stranded DNA (Gasparro,
F. P. Psoralen DNA photobiology, CRC Press Inc.; Boca Raton, Florida, 1988; Patel,
D. J.; Cannel, L. Proc. Natl. Acad. Sci. USA 1976, 73, 674). To examine the effect of
the present systems on the thermal denaturation of DNA, experiments were carried out
employing calf thymus DNA and synthetic oligonucleotides listed in Table 2. In 10
mM phosphate
TABLE 2
DNA(l)
DNA(2)
DNA sequence
5'-CGT GGA CAT TGC ACG GTA C-3'
5'-GTA CCG TGC AAT GTC CAC G-3'
buffer, both compound of formula la and Ib (wherein Y = -(CH2)n, wherein n - 1 - 11;
R = -MV2+-(CH2)m-CH32XB or -Pyr2+-(CH2)m-CH32XB and wherein Y = -(CH2)n,
whereirj n - 1 - 11; R = -MV2+-(CH2)m-Acr2XB or -Pyr2+-(CH2)m-Acr2XB
respectively) were found to stabilize the DNA and the extent of stabilization increases
with the increasing in concentration of viologen linked acridines as shown in FIG. 2.
At all concentrations of the ligand only one transition temperature was observed, in
each case, thereby indicating that only one type of binding with DNA is responsible for
such behavior. The extent of stabilization was found to be nearly 20 °C in the case of at
40 uM of compound of formula la (wherein Y = -(CH2)n, wherein n = l - l l ; R = -
MV2+-(CH2)m-CH32XH or -Pyr2+-(CH2)m-CH32XBX whereas 20 ^iM of compound of
formula Ib (wherein Y = -(CH2k wherein n = 1 - 11;R = -MV2+-(CH2)ra-Acr2X& or -
Pyr2+-(CH2)m-Acr2Xa)showed nearly 18 °C stability.
In addition to the significant thermal stability, unusually large hypochromicity
(around 80%) was observed upon binding of these molecules with DNA as shown in
FIG. 3. These results indicate the existence of strong 7t-stacking interactions between
the ligand molecules and DNA bases. This result in, only partial separation of DNA
strands on melting, leading to a decrease in absorbance change. The large stabilization
and significant hypochromicities offered by the structures of formula la and Ib prove
further their strong interaction with DNA and potential use in biology for the detection
of various DNA structures, stabilization of triplex and G-quadruplex structures.
EXAMPLE 8
Demonstration as photoactivated DNA cleaving agents. Cleavage of plasmid
DNA was followed by monitoring the conversion of supercoiled (Form I) to open
circular relaxed (Form II) and linear (Form III). Plasmid DNA cleavage is a very
sensitive technique and when combined with several repair endonucleases, it can serve
as a kind of fingerprint of the species directly responsible for the damage. Induction of
one single strand break (SSB) by a compound converts Form I to Form II and the
quantification of which indicates its efficiency of DNA cleavage. A DNA relaxation
assay was used to quantify SSB and endonuclease-sensitive modifications (Epe, B.;
Helger, J.; Wild, D. Carcinogenesis 1989,10, 2019 and Epe, B.; Mftzel, P.; Adam, W.
Chem. Biol. Interactions 1988, 67, 149). This assay makes use of the fact that Form I
when converted by either a single strand break (SSB) or the incision by a repair
endonuclease leads to Form II, which migrates separately in agarose gel
electrophoresis.
Phosphate-buffered (pH 7.0), air-saturated solutions of PM2 DNA (10 ug/mL) at 0
°C were irradiated with 360 nm near-UV irradiation in the presence of various concentrations
of acridine-viologen bifunctional molecules represented by compound of formula la and
Ib (wherein Y = -(CH2)n, wherein n - 1 - 11; R = -MV2+-(CH2)m-CH32XB or -Pyi*+-
(CH2)m-CH32XH and wherein Y = -(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-
Acr2XH or -4>yr2+-(CH2)m-Acr2XB respectively). Subsequently, the DNA was analyzed
for the following types of modifications: (i) DNA single and double strand breaks; (ii)
sites of base loss (AP sites) recognized by exonuclease III; (iii) base modifications plus
AP sites sensitive to the T4 endonuclease V; (iv) base modifications plus AP sites
sensitive to the endonuclease III and (v) base modifications plus AP sites sensitive to
formamidopyrimidine-DNA glycosylase (FPG protein). DNA damage profile induced
by the acridine-viologen bifunctional molecules are presented in FIG. 4.
It is evident from the damage profiles that both the compounds induced very
few AP sites and few modifications sensitive to endonuclease III, but a large number of
base modifications sensitive to FPG protein were observed. Further, no significant DNA
damage was observed either by irradiation of PM2 DNA alone or in the dark in presence of
viologen linked acridines at the highest concentrations, thereby indicating that the damage
observed is purely initiated by the photoactivation of these compounds. Hence these
compounds can be used as efficient photoactivated DNA cleaving agents.
EXAMPLE 9
Efficiency of DNA cleavage. Since the major damage induced by the acridineviologens
is recognized by the FPG protein (FIG. 4), we have investigated the effect of
irradiation time and concentration of these systems on the formation of FPG sensitive
modifications and SSB. FIG. 5 and FIG. 6 show the irradiation time dependent formation
of single-strand breaks (SSB) and FPG sensitive modifications induced by the acridineviologen
bifunctional derivatives compound of formula la (wherein n = 1, R = -MV2+-
(CH2)3-CH3 and X = Br and wherein n = 11, R = -MV2+-(CH2)3-CH3 and X = Br),
respectively. As can be seen from these figures the damage sensitive to FPG protein
increases, in each case, with increase in the time of irradiation, indicating the catalytic
property of these compounds. No significant increase in the generation of SSB was
observed, even after the irradiation for 30 min. Similarly, increase in DNA damage was
observed with the increase in concentration as shown in the inset of FIG. 6.
These results clearly demonstrate that the acridine-viologen bifunctional
derivatives induce large number of base modifications sensitive to the repair
endonuclease FPG protein, with little damage recognized by repair endonuclease III.
FPG protein is known to recognize modifications such as 8-oxoguanosine and
formamido-pyrimidines, ring-opened products of purines (Boiteux, S.; Gajewski, E.;
Laval, J.; Dizdarough, M. Biochemistry, 1992, 31, 106). Both 8-oxoguanosine and
formamidopyrimidines can be generated in DNA by hydroxyl radicals (Epe, B.; Haring,
M.; Ramaiah, D.; Stopper, H.; Abou-Elzahab, M.; Adam, W.; Saha-Moller, C. R.
Carcinogenesis 1993, 14, 2271; Epe, B.; Pflaum, M.; Haring, M.; Hegler, J.; Rudiger,
H. Toxicol. Lett. 1993, 67, 57), singlet oxygen and by electron transfer mechanism (von
Sonntag, C. The Chemical Basis of Radiation Biology, Taylor and Francis, London,
1987).
The involvement of both hydroxyl radicals and singlet oxygen can be ruled out
since the damage profiles shown in FIG. 4 are different from those induced by the
ionizing radiation and disodium salt of l,4-etheno-2,3-benzoxlioxin-l,4-dipropanoic
acid (Aruoma, O. I.; Halliwell, B.; Dizdaroglu, M. J. Biol. Chem. 1989, 264, 13024;
Muller, E.; Boiteux, S.; Cunningham, R. P.; Epe, B. Nucl. Acids Res. 1990,18, 5969). In
addition, DNA cleavage studies were examined in presence of various additives.
Results of these studies indicate that acridine-viologen bifunctional molecules can be
used as reagents for induction of DNA damage purely through photoinduced electron
transfer particularly for the modification (oxidation) of guanine base in DNA.
EXAMPLE 10
Demonstration of cleavage at guanine (G), preferential cleavage of5'-G ofGG
sequence andGofaAG bulge. In order to examine the selectivity in cleavage and also
to find out whether if there is any preferential reaction of the viologen linked acridines
towards base bulges, we have analyzed the cleavage reactions using a few end labeled
synthetic oligonucleotides (Table 3) by polyacrylamide gel electrophoresis (PAGE).
DNA sequence
(Table Remove)Oligonucleotides DNA(3), DNA(5) (CC-bulge) and DNA(6) (AG-bulge) were
radiolabeled at 5'-OH using [y-32P]ATP and bacterial T4 polynucleotide kinase
according to the standard procedure (Sambrook, J.; Fritsch, E. F.; Maniatis, T.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New
York, 1989) and hybridized with DNA(4), incubated with various concentrations of
viologen linked acridines and irradiated with a light source in microcentrifuge tubes in
a Rayonet Photoreactor (RPR) containing eight 350 nm lamps according the reported
procedure (Ramaiah, D.: Kan, Y.; Koch, T.; Qrum, H.; Schuster, G. B. Proc. Natl.
Acad. Sci. USA 1998, 95, 12902; Ramaiah, D.; Koch, T.; Qrum, H.; Schuster, G. B.
20
Nucl. Acids Res. 1998, 26, 3940). After irradiation, the samples were precipitated with
buffer, washed two times with cold 70% aqueous ethanol, treated with hot piperidine
for 30 min at 90 °C, lyophilized under vacuum and analyzed by PAGE. Maxam-Gilbert
A/G, T and C- specific reactions were performed by routine protocols (Maxam, A. M.;
Gilbert, W. Methods in Enzymol. 1980, 65, 499; Ramaiah, D.: Kan, Y.; Koch, T.;
Qrum, H.; Schuster, G. B. Proc. Natl. Acad. Sci. USA 1998,95,12902).
The selectivity of cleavage pattern of 5'-labeled DNA (3), DNA (5) and DNA
(6), caused by the bifunctional acridine-viologen derivatives of compound of formula
la and Ib (wherein Y = -(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-CH32X® or -
Pyr2+-(CH2)m-CH32Xe> and wherein Y - -(CHak wherein n = 1 - 11; R = -MV2+-
(CH2)m-Acr2X®or -Pyr2+-(CH2)m-Acr2Xai'respectively) is shown in FIG. 7. Both these
compounds were found to cleave DNA(3), at the GG sites with a significant preference
for the 5'-G over the 3'-G (lanes 2 and 3 of FIG. 7). Small amount of cleavage Ws also
observed at the G site. Similar observations were made with DNA(5) (CC-bulge) by
both of compound of formulae la and Ib (lanes 4 and 5 of FIG. 7). Practically no
cleavage at the CC bulge site was observed. Irradiation of DNA(6) (AG-bulge) in
presence of compound of formula la and Ib (wherein Y = -(CH2)n, wherein n = 1 - 11;
R = -MV2"-(CH2)m-CH32XK> or -Pyr2+-(CH2)m-CH32X® and wherein Y - -(CH2)n,
wherein n = 1 - 11; R = -MV2+-(CH2)m-Acr2X® or -Pyr2+-(CH2)m-Acr2XE>
respectively), caused remarkably selective cleavage at the G of the AG bulge (lanes 6
and 7 of FIG. 7). Compound of formula la (wherein Y = -(CH2)n, wherein n = 1 - 11;
R = -MV2+-(CH2)m-CH32Xs'or -Pyr2+-(CH2)m-CHj2Xs) was found to be more efficient
in cleaving the duplex DNA structures and also duplexes containing the base bulges.
These results indicate that these molecules can be used for the photoactivated selective
cleavage of 5'-G of the GG sites and G of the AG two base bulges in DNA and also for
their recognition.
EXAMPLE 11
Demonstration of catalytic activity. Since the systems under investigation
posses high DNA association constants and found to cleave efficiently at G sites in
DNA duplex and base bulges upon irradiation, we further demonstrated their catalytic
properties so that they can have potential applications in biology and industry. Direct
laser excitation of the viologen linked acridine of formula la, Ib and Ic (wherein Y = -
(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-CH32X® or -Pyr2+-(CH2)m-CH32X^
wherein Y - -(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-Acr2X® or -Pyr2+-
21
(CH2)m-Acr2X® and wherein Y = ortho or para tolyl; R - -MV2+-(CH2)m-CH32Xs>or -
Pyr2+-(CH2)m-CH32Xs> respectively) in water or buffer showed no absorptions
assignable to transient intermediates. However, when the laser excitation of viologen
* ,
linked acridine of formula la (wherein Y = -(CH2)n, wherein n = l - l l ; R = -MV -
(CH2)m-CH32X®or -Pyr2+-(CH2)m-CH32Xs>was carried out in water or buffer in the
presence of external electron donors such as N,N-dimethylaniline (10 mM) or
guanosine (1.8 mM) a transient species with absorption maxima at 395 and 610 nm,
was observed. The spectral features of this transient were similar to those of methyl
viologen radical cation (MV'+), reported in the literature (Watanabe, T.; Honda, K. J.
Phys. Chem. 1982, 86, 3661; Kelly, L. A.; Rodgers, M. A. J. J. Phys. Chem. 1994, 98,
6377). Similarly, laser excitation of viologen linked acridine of formula la (wherein Y
- -(CH2)n, wherein n = 1 - 11; R = -MV2+-(CH2)m-CH32X^or -Pyr2+-(CH2)m-CH32X^
in the presence of DNA in water or buffer, gave a transient species assignable to the
reduced methyl viologen (FIG. 8).
These results clearly show that the excited acridine chromophore transfers an
electron to the viologen moiety leading to the formation of radical cation of acridine
and reduced methyl viologen. The radical cation of acridine once formed, oxidizes the
electron donor present in the medium (the formation of methyl viologen radical cation
(MV'+) is facilitated by external sacrificial electron donors) and reverts back to
acridine for re-absorption of photons. The reduced methyl viologen can in turn transfer
an electron to the molecular oxygen thereby reverting back to methyl viologen and
generating superoxide radical anion. Therefore, these bifunctional molecules and
derivatives thereof can function as catalytic photoactivated DNA cleaving agents in
presence of sacrificial electron donors and as catalysts for the oxidation of water under
appropriate conditions to generate hydrogen in industrial applications.
Bifunctional molecules of the present invention posses excellent properties of a
photosensitizer for phototherapeutical as well as catalytic photoactivated DNA cleaving
and industrial applications. The main advantages of the present systems include:
1. Compounds represented by formula 1 (la, Ib, Ic and Id) are pure single
substances.
2. Their synthetic methodology is very economical.
3. They are very stable, highly soluble in aqueous medium and exist in the neutral
form under physiological conditions.
22
4. These compounds posses good absorption properties and are very stable in the
dark and under irradiation conditions.
5. They constitute an effective donor-acceptor system and their redox properties
can be tuned as a function of pH and spacer group.
6. They form a novel class of compounds with high affinity to DNA and interact
with DNA through intercalation, bisintercalation and groove binding.
7. These systems stabilize various structures of DNA including duplex DNA, base
bulges, triplex and quadruplex DNA structures.
8. These systems can be easily covalently linked to oligonucleotides for the
stabilization of triplex DNA and also for the selective photocleavage of DNA.
9. These systems posses special affinity for A.T sequence and hence can have
potential applications as probes for the detection of such sequences in DNA
10. They form a novel class of compounds, which cleave DNA in a catalytic way
under irradiation conditions and act as catalytic photoactivated DNA cleaving
agents.
11. They cleave DNA purely through the photoinduced electron transfer mechanism
and selectively at guanine sites and with excellent selectivity at 5'-G of GG
sequence in DNA.
12. They cleave duplex DNA containing AG base bulges selectively at G sites and
hence act as probes for the detection of AG bulge containing DNA sequence.
13. These systems form a novel class of photosensitizers, which upon irradiation in
presence of external donors results in an effective charge separation and hence
these systems and derivatives thereof can act as photocatalysts in industrial
applications including in the photoinduced hydrogen generation from water.





We claim :
1. Viologen linked acridine based molecule of the general formula 1(1a,1b, 1c and (Formula Removed)


la
Ib
Ic
Id

wherein Y=-(CH2)n~ wherein n = 1-11 R=-MV2+ -(CH2)m CH3 2Xe or
-pyr2+ -(CH2)m"CH3 2X9 wherein m=1-13 wherein Y=-(CH2)n- wherein n = 1-11 R= _MV2+ -(CH2)m-Acr 2Xe or
-pyr2+ -(CH2)m'Acr 2Xe wherein m=1-11 wherein Y= ortho or para tolyl
R=-MV2+ -(CH2)m-CH3 2Xe or
-pyr2+ -(CH2)m-CH3 2Xe wherein m=1-13 wherein Y=-(CH2)n-wherein n=1-10 R= -Acr+-R1 X9/2Xe

wherein N in the acridine main ring is also quarternised by alkyl group R1=-(CH2)m-CH3 and -(CH2)m-C6H4-(CH2)m-(para) Wherein m=0-13

(Formula Removed) Formula 1
2. Viologen linked acridine molecule of the general formula 1 (1a, 1b, 1c and 1d) substantially as herein before described and with reference to the foregoing examples and accompanying drawings

Documents:

0234-del-2001-abstract.pdf

0234-del-2001-claims.pdf

0234-del-2001-complete specification (granted).pdf

0234-del-2001-correspondence-others.pdf

0234-del-2001-description (complete).pdf

0234-del-2001-drawings.pdf

0234-del-2001-form-1.pdf

0234-del-2001-form-18.pdf

0234-del-2001-form-2.pdf

234-DEL-2001-Abstract-(19-03-2009).pdf

234-DEL-2001-Claims-(19-03-2009).pdf

234-DEL-2001-Correspondence-Others-(19-03-2009).pdf

234-DEL-2001-Description (Complete)-(19-03-2009).pdf

234-DEL-2001-Form-3-(19-03-2009).pdf

abstract.jpg


Patent Number 235787
Indian Patent Application Number 0234/DEL/2001
PG Journal Number 36/2009
Publication Date 04-Sep-2009
Grant Date 27-Aug-2009
Date of Filing 28-Feb-2001
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 DANABOYINA RAMAIAH EMPLOYED AT REGIONAL RESEARCH LABORATORY (COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH) THIRUVANANTHAPURAM 695019, KERALA, INDIA
2 NADUKKUDY VARGHESE ELDHO EMPLOYED AT REGIONAL RESEARCH LABORATORY (COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH) THIRUVANANTHAPURAM 695019, KERALA, INDIA
3 JOSHY JOSEPH EMPLOYED AT REGIONAL RESEARCH LABORATORY (COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH) THIRUVANANTHAPURAM 695019, KERALA, INDIA
PCT International Classification Number A61K 31/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA