Design, synthesis and reactivity of C2-symmetric azobenzene-based amino acid-bis(propargyl sulfones)

Article abstract of DOI:10.1016/j.bmcl.2010.08.085

C₂-Symmetric azobenzene-amino acid linked bis(propargyl sulfones) 1 and 2 containing stable E azo moiety have been synthesized. Upon irradiation with long wavelength UV these compounds isomerized to the Z-form, whose thermal reisomerization to

Full text of DOI:10.1016/j.bmcl.2010.08.085

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6831–6835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and reactivity of C2-symmetric azobenzene-based amino
acid-bis(propargyl sulfones)
Debarati Mitra a, Deb Ranjan Banerjee a, Amit Kumar Das b, Amit Basak a,
⇑
a Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India
b Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 May 2010
Revised 23 July 2010
Accepted 18 August 2010
Available online 23 September 2010
C2-Symmetric azobenzene-amino acid linked bis(propargyl sulfones) 1 and 2 containing stable E azo moi-
ety have been synthesized. Upon irradiation with long wavelength UV these compounds isomerized to
the Z-form, whose thermal reisomerization to the E-isomer slowed down considerably Under basic pH,
the compounds showed DNA cleavage in
lmolar concentrations with the Z-isomers showing better
cleaving efficiency. The difference in cleaving efficiency between the Z and the E-isomer is more than
the corresponding pair of sulfones without amino acid linker.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Azobenzene
Amino acid
Propargyl sulfones
DNA cleavage
Irradiation
Bispropargyl sulfones constitute an important class of
DNA-cleaving agent.1 Various modifications have been made on
their structures to bring selectivity in their DNA-cleaving action.2
The mechanism involves isomerisation to the allenic sulfones
followed by conjugate addition of a DNA base. This generates a
positive charge on the DNA base, resulting in a Maxam–Gilbert type
cleavage.3 An alternative mechanism involving the formation of
diradical via Garatt–Braverman rearrangement,4 has also been pro-
posed. Subsequently, Kerwin reported the synthesis of crown-ether
based cyclic bispropargyl sulfone.5 In alkaline pH, the compound
cleaved supercoiled DNA via a Maxam–Gilbert type of alkylation
pathway. Dai et al.6 has reported the synthesis of propargyl sulfones
with DNA-intercalating moieties. These compounds showed
enhanced DNA-cleaving property with respect to non-intercalating
counterpart. Previously7 we have reported the DNA cleavage
exhibited by E- and Z-isomers of azobenzene-based bispropargyl
sulfones. Although, the cleavage efficiency of the Z-isomer was
higher than the E-isomer, the difference was not significant mainly
due to the rapid thermal Z to E-isomerization. Keeping this in mind,
we were inspired in synthesizing E-azobenzene based sulfones 1
and 2 with amino acid linker. The amino acids are incorporated in
order to stabilize8 the Z-isomers 3 and 4 via weak stabilizing inter-
without the amino acid linker. The results are described in this
communication.
Docking studies10 using AutoDock 4.2 were first carried out. The
energy minimized docked conformations of E- and Z-sulfones 1
and 3 are shown in Figure 2. It was observed that the Z-isomer 3
closely fits itself in the major groove of DNA (Fig. 3). It docked in
such a way that the terminal propargyl sulfone units are directed
towards DNA base pairs. It exhibited one hydrogen bond involving
oxygen atom of amide group of one side chain to deoxy adenine
(DA16) of B-chain with a distance of 2.8 Å. It also showed van
der Waal’s interaction (hydrophobic) between the benzene ring
(sulfone unit) of hydrogen bonded side chain with DT17 of B-chain.
The distance between the closest center of aforesaid two units is
3.93 Å. The distance between the midcenter of propargyl unit of
one side chain with the nearby deoxy adenine (DA16) residue is
3.93 Å which favors the proposed alkylation reaction due to close
proximity of the reaction center. But E azo sulfone molecule is
loosely bound to the major groove of DNA. It docked in such a
way that both terminal propargyl sulfone moeities are directed
outwards from DNA base pairs and overall docked energy is com-
paratively much higher than the Z-isomer. It has no hydrogen
bonding interactions. The Zazo sulfone molecule also predicted to
have lower free energy of binding (ꢀ12.15 kcal/mol) than the E
azo sulfone molecule (ꢀ7.53 kcal/mol). In other words, Z-azo sul-
fone molecule is predicted to have the higher binding affinity to-
wards the binding site of DNA as compared to the E-isomer.
The synthesis of target sulfones required assembling of three
units: azobenzene, amino acids and butyn-1, 4-diol. This involved
actions like H-bonding,
shown in Figure 1. Their chemical, photochemical and DNA cleav-
age activities were also studied and compared with the sulfones
p
-stacking etc.9 The target sulfones are
⇑
Corresponding author. Tel.: +91 322 228 3300; fax: +91 322 228 2252.
E-mail address: absk@chem.iitkgp.ernet.in (A. Basak).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.085

6832
D. Mitra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6831–6835
O
H
N
SO2Ph
SO2Ph
O
O
O
O
H
N
O
N
N
O
SO2Ph
O
N
N
PhO2S
O
O
O
O
N
O
H
O
O
N
H
O
1
3
O
H
N
SO2Ph
O
O
O
O
O
H
Ph
N
N
N
Ph
O
O
O
O
SO2Ph
N
N
Ph
O
PhO2S
O
O
O
SO2Ph
Ph
N
H
O
N
H
O
2
4
Figure 1. Target sulfones.
Irradiation of dichloromethane solution of the compound 1 for
3 h under high pressure Hg lamp at 354 nm produced a mixture
of E- and Z-isomers (1 and 3) in 1:2 ratio (Scheme 2). Compound
2 produced a photo stationary state of E- and Z-forms in 1:1.5 ratio
under similar condition. The thermal reisomerisation carried out at
30 °C for both the compounds followed first order kinetics. The
kinetics was followed by taking 1H NMR at various time points.
The half lives for reisomerisation were found to be 72.8 h for com-
pound 3 and 69.2 h for compound 4. These half lives are much lar-
ger than that of bissulfone 13 without amino acid linker.7 Thus
incorporation of amino acids in the bissulfone framework has con-
siderably slowed down the thermal reisomerisation at room
temperature.
Figure 2. Energy minimized docked conformation.
The chemical reactivity of the sulfones 1 and 2 under basic con-
dition was evaluated by studying the kinetics of isomerisation
from propargyl to allene (Scheme 3). For this, a CDCl3 solution of
the compound 1 was taken and triethylamine (3 equiv) was added
and the progress of reaction was followed by monitoring the 1H
NMR profile. New peaks started to appear at d 6.34 and d 5.93
which were assigned to the olefinic protons of bisallene 14.11 At
equilibrium, which was reached after 30 min, the ratio of propargyl
to allene was 1:1. This ratio did not alter even after keeping the
solution for 12 h at 25 °C. Similar results were observed in case
of the corresponding Z-isomer 3. The relative reactivities of the
compounds 2 and 4 under basic conditions were also checked. In
this case, however, the ratio of propargyl to allene became 1.5:1
at equilibrium. Here again, the extent of allene formation in both
Z and E forms for both the pairs was more as compared to what
was observed for the bissulfones 12 and 13.7
DNA-cleaving efficiency of the compounds was then studied.
Since we could not isolate the Z-isomers in pure form, we had to
check the cleavage with pure E and a mixture of E- and Z-isomers
(with excess of Z-form). The experiments were carried out at 37 °C
using pBR322 supercoiled plasmid DNA at pH 8.5. The greater half
lives of the Z-isomers (3 and 4) allowed us to extend the time of
incubation up to 48 h. The gel electrophoresis patterns are shown
in Figure 4. Densitometric analysis12 indicated the extent of cleavage
for the compounds (Table 1). From the gel pattern, we can conclude
that the mixture containing predominantly the Z-form has better
cleaving efficiency as compared to the pure E-isomer. This is true
for both the valine and phenyl alanine based sulfones. Moreover,
the difference in the extent of DNA cleavage for E- and Z-isomers
for these amino acid inked azobenzene systems is much more signif-
icant as compared to the compounds without amino acid linker 12
and 13 as reported earlier. Incidentally, the same pBR322 DNA was
used to study the cleavage potency of 12 and 13.
Figure 3. Interaction of DNA–ligand complexes, Z-isomer (a) and E-isomer (b); here
DNA is represented as surface.
initial linking between 2,20–dihydroxy azobenzene and the bromo-
acylated amino acid benzyl ester (5a–b). The resulting alkylated
products (6a–b) upon hydrolysis produced corresponding diacids
(7a–b). These were converted to the dipotassium salts (8a–b)
which were then coupled with (4-bromo-but-2-ynyl sulfanyl)-
benzene (11) derived from 4-phenyl sulfanyl-but-2-yn-1-ol (10),
to produce 1 and 2. The synthetic strategy is described in the
Scheme 1. All the compounds were characterized by spectral
analysis.

D. Mitra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6831–6835
6833
COOK
NH
COOH
COOCH2Ph
R
O
R
R
NH
NH
O
O
O
O
N N
O
R
O
N N
Br
b
a
c
N
COOCH2Ph
N N
H
5a-b
O
O
O
O
R
O
R
O
R
HN
HN
HN
KOOC
HOOC
PhH2COOC
7a-b
8a-b
6a-b
SPh
SPh
O
SPh
HO
O
f
R
O
NH
O
Br
e
10
11
d
NN
1, 2
(f) CBr4, PPh3, CH2Cl2, 0 oC addition, 6 h, 95%
O
O
HN
R
O
O
For a, R = -iPr, for b, R = -CH2Ph
PhS
9a-b
Scheme 1. Synthesis of target sulfones. Reagents and conditions: (a) 2,20-dihydroxyazobenzene, anhyd Cs2CO3, dry CH3CN, 40–50 °C, 12 h, 50%; (b) LiOH, THF/H2O, 16–18 h,
60%; (c) KHCO3, MeOH/H2O, 2 h, 80%; (d) (4-bromo-but-2-ynyl sulfanyl)-benzene, dry DMF, 24 h, 65% (R = –iPr), 60% (R = –CH2Ph); (e) m-CPBA, CH2Cl2, 0 °C addition, 1.5 h,
75%.
SO2Ph
SO2Ph
O
Ph
O
O
N N
NN
O
NH
O
O
O
O
O
NH
O
O
O
O
O
O
HN
NH
light
heat
HN
NH
Ph
Ph
O
O
O
O
O
O
O
N N
light
heat
O
O
NN
O
O
O
O
PhO2S
SO2Ph
3
PhO2S
HN
HN
O
SO2Ph
O
O
4
Ph
O
2
PhO2S
PhO2S
1
SO2Ph
N=N
O
light
heat
O
O
N=N
O
PhO2S
13
Scheme 2. Light induced E–Z isomerization study.
SO2Ph
PhO2S
12
The results are according to our prediction as the design was
based on the expected better binding of the Z-isomer with ds
DNA along with greater half life. DNA-binding studies13 did indi-
cate one order of magnitude higher binding of the Z-isomer
(Table 2).
In conclusion, we have successfully synthesized acyclic azo
amino acid linked bis(propargyl sulfones). The Z-isomers showed
higher cleaving efficiency than the corresponding E-isomers as
was expected from its higher half life, greater extent of allene
formation and better DNA binding. Currently, we are engaged

6834
D. Mitra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6831–6835
N N
O
O
SO2Ph
SO2Ph
O
O
C
HN
NH
O
O
O
light
O
O
O
O
O
NH
heat
NH
O
O
PhO2S
SO2Ph
O
O
3
N N
N N
Et3N
Et3N
O
O
O
O
HN
N N
HN
O
O
O
O
O
O
O
O
HN
NH
14
O
O
O
O
C
C
PhO2S
PhO2S
1
C
SO2Ph
PhO2S
15
Scheme 3. Base induced isomerization study.
in the synthesis of water soluble analogs based on similar
framework.
Spectral data of selected compounds: IR data taken in KBr pellet
and expressed in cmꢀ1 1H NMR and 13C NMR were recorded at
,
400 MHz and 100 MHz, respectively, in CDCl3:
For 9a:
m
max (neat, cmꢀ1) 2078, 1633, 1242, 686; dH 7.90 (2H, d,
Figure 4. DNA cleavage studies with pBR322: For (A), DNA cleavage experiment of
compounds and mixture containing predominantly Z-isomer after 48 h
incubation at 37 °C; lane 1: control DNA in TAE buffer (pH 8.5, 7 L) + CH3CN (5
lane 2: DNA in TAE buffer (pH 8.5, 7 L) + E-sulfone 1 (0.02 mM) in CH3CN (5
lane 3: DNA in TAE buffer (pH 8.5, 7
CH3CN (5 L); For (B), DNA cleavage experiment of compounds 2 and a mixture
containing predominantly Z-isomer 4 after 48 h incubation at 37 °C; lane 1: control
DNA in TAE buffer (pH 8.5, 7 L) + CH3CN (5 L); lane 2: DNA in TAE buffer (pH 8.5,
L) + E-sulfone 2 (0.02 mM) in CH3CN (5 L); lane 3: DNA in TAE buffer (pH 8.5,
L) + Z- and E-sulfones 4 and 2 (1.5:1, 0.02 mM) in CH3CN (5 L).
J = 7.6 Hz), 7.59 (2H, d, J = 9.2 Hz), 7.46–7.38 (6H, m), 7.31–7.26
(4H, m), 7.21 (2H, t, J = 7.2 Hz), 7.11–7.05 (4H, m), 4.78–4.59 (10H,
m), 3.61 (4H, s, –CH2SPh), 2.21–2.13 (2H, m), 0.90 (6H, d,
J = 6.8 Hz), 0.81 (6H, d, J = 6.8 Hz); dC 170.6, 168.0, 155.0, 142.5,
134.8, 132.8, 129.9, 129.0, 126.9, 122.5, 117.6, 114.6, 83.2, 77.4,
68.3, 56.4, 53.0, 31.3, 22.7, 18.9, 17.6; mass (ES+): m/z 849 (MH+);
HRMS Calcd for C46H48N4O8S2 + H+ 849.2992 found 849.2997.
1
a
3
l
l
l
L);
L);
l
l
L) + Z and E-sulfones 3 and 1 (2:1, 0.02 mM) in
l
l
l
l
7
7
l
l
l
For 9b: m
max (CHCl3, cmꢀ1) 2353, 1217, 1644, 1183, 754; dH 7.66–
7.60 (4H, m), 7.51 (2H, d, J = 3.6 Hz), 7.42–7.41 (6H, m), 7.32–7.20
(6H, m), 7.10–7.01 (12H, m), 5.01–4.47 (10H, m), 3.66 (4H, s), 3.13
(2H, dd, J = 8.4, 5.6 Hz, –CH2Ph), 2.99 (2H, q, J = 6.8 Hz, –CH2Ph); dC
170.2, 167.9, 155.1, 142.5, 135.3, 132.7, 130.1, 129.2, 129.0, 128.5,
128.4, 127.1, 127.0, 122.1, 115.1, 114.1, 83.3, 77.3, 68.9, 53.2, 52.6,
37.8, 22.7; mass (ES+): m/z 945 (MH+); HRMS Calcd for
C54H48N4O8S2 + H+ 945.2992 found 945.2998.
Table 1
Extent of DNA-cleavage by densitometry
Compound no
% Cleavage after 48 h of incubation
1
ꢁ20
ꢁ65
ꢁ10
ꢁ45
ꢁ25
ꢁ35
For 1: mmax (neat, cmꢀ1) 2077, 1642, 1139, 749; dH 7.94 (4H, d,
J = 8.0 Hz), 7.81 (2H, d, J = 8.0 Hz), 7.67–7.62 (2H, m), 7.56 (6H, t,
J = 7.6 Hz), 7.47 (2H, t, J = 8.0 Hz), 7.10–7.07 (4H, m), 4.79–4.59
(10H, m), 3.97 (4H, s), 2.19–2.11 (2H, m), 0.90 (6H, d, J = 6.8 Hz),
0.81 (6H, d, J = 6.8 Hz); dC 170.5, 168.0, 154.9, 142.5, 137.5, 134.3,
132.9, 129.2, 128.8, 122.5, 117.6, 114.7, 81.3, 74.9, 68.4, 56.4,
52.4, 48.5, 31.2, 18.9, 17.6; mass (ES+): m/z 935.42 (MNa+); HRMS
Calcd for C46H48N4O12S2 + H+ 913.2788 found 913.2794.
3 + 1 (2:1)
2
4 + 2 (1.5:1)
12
13 + 12 (3:1)
For 2: mmax (neat, cmꢀ1) 2353, 1642, 1166, 771; dH (400 MHz),
7.96 (4H, d, J = 6.0 Hz), 7.77–7.53 (10H, m), 7.43 (2H, t,
J = 7.6 Hz), 7.24–6.92 (14H, m), 4.97–4.37 (10H, m), 3.97 (4H, s),
3.09 (2H, dd, J = 12.0, 6.0 Hz), 2.96 (2H, q, J = 6.8 Hz); dC 170.1,
167.9, 155.1, 142.6, 137.5, 135.2, 134.3, 132.8, 129.3, 129.2,
129.0, 128.8, 127.1, 122.8, 117.7, 115.3, 81.1, 75.1, 68.9, 52.6,
48.5, 37.8, 37.3; mass (ES+): m/z 1031.59 (MNa+); HRMS Calcd for
Table 2
DNA–ligand binding constant (the estimated error is within +5%)
Binding
constant
Binding constant
of compound
Binding
constant
Binding constant of
compound
of compound 3 + 1 (2:1) (ꢂ105) of compound 4 + 2 (1.5:1)
1 (ꢂ104) Mꢀ1 Mꢀ1
2 (ꢂ104) Mꢀ1 (ꢂ105) Mꢀ1
9
11
16
15
C
54H48N4O12S2 + H+ 1009.2788 found 1009.2795.

D. Mitra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6831–6835
6835
15458; (c) Hunter, C. A. Chem. Soc. Rev. 1994, 101; (d) Hoeben, F. J. M.;
Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. Chem. Rev. 2005, 105, 1491;
Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives; VCH:
Weinheim, Germany, 1995. pp 139–147; (f) Sokolov, A. N.; Friscic, T.;
MacGillivray, L. R. J. Am. Chem. Soc. 2006, 128, 2806; (g) Bhosale, S.; Sisson, A.
L.; Sakai, N.; Matile, S. Org. Biomol. Chem. 2006, 4, 3031.
Acknowledgments
The authors A.B. and D.M./D.R. are grateful to CSIR, Government
of India for research funding and fellowship, respectively. DST is
thanked for the funds for 400 MHz NMR facility under the IRPHA
program.
10. The computer simulated automated docking study was performed using
AutoDock4.2. The B-DNA form of DNA decamer d(GGCCAATTGG) (PDB ID:
432D) was used for docking study with E azo and Z azo sulfone molecule.
Water molecules were removed from DNA PDB file and polar hydrogen atoms
were added with Gesteiger charges. The coordinates of ligands in its Z and E
configuration were obtained from Dundee PRODRG server.2 The docking was
carried out with Lamarckian Genetic Algorithm (GA). Some key references:
Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235; Schuettelkopf, A.
W.; Van Aalten, D. M. F. Acta Crystallogr. 2004, D60, 1355; Morris, G. M.;
Goodsell, D. S.; Huey, R.; Olson, A. J. J. Comput. Aided Mol. Des. 1996, 10, 293;
Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
References and notes
1. Nicolaou, K. C.; Wendeborn, S.; Maligres, P.; Isshiki, K.; Zein, N.; Ellestad, G.
Angew. Chem., Int. Ed. Engl. 1991, 30, 418.
2. Basak, A.; Khamrai, U. K. Tetrahedron Lett. 1995, 36, 7913.
3. (a) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499; (b) Maxam, A. M.
Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 560.
4. (a) Garratt, P. J.; Neoh, S. B. J. Org. Chem. 1979, 44, 2667; (b) Cheng, Y. S. P.;
Garratt, P. J.; Neoh, S. B.; Rumjanek, V. H. Isr. J. Chem. 1985, 26, 101; (c)
Braverman, S.; Duar, Y.; Seggev, D. Tetrahedron Lett. 1979, 3181.
5. Kerwin, S. M. Tetrahedron Lett. 1994, 35, 1023.
11. The allene will surely be a mixture of diastereomers. However, these could not
be distinguished by 1H NMR.
6. (a) Dai, W.-M.; Fong, K. C. Tetrahedron Lett. 1995, 36, 5613; (b) Dai, W.-M.; Fong,
K. C.; Danjo, H.; Nishimoto, S.-i.; Solow, M.; Mak, W. l.; Yeung, M. L. Bioorg. Med.
Chem. Lett. 1996, 6, 1093; (c) Dai, W.-M.; Li, Q.; Fong, K. C.; Chow, C. W.; Zhou,
L.; Hamaguchi, W.; Nishimoto, S.-i. Bioorg. Med. Chem. Lett. 1998, 8, 169; (d) Dai,
W.-M.; Chow, C. W.; Zhou, L.; Ishii, A.; Lau, C. W.; Li, Q.; Hamaguchi, W.;
Nishimoto, S.-i. Bioorg. Med. Chem. Lett. 1999, 9, 2789; (e) Haruna, K.-i.; Tanabe,
K.; Ishii, A.; Dai, W.-M.; Hatta, H.; Nishimoto, S.-i. Bioorg. Med. Chem. 2003, 11,
5311.
7. Mitra, D.; Kar, M.; Pal, R.; Basak, A. Bioorg. Med. Chem. Lett. 2007, 17, 4514.
8. Amino acid linkers have previously been used to stabilize Z-azo enediynes:
Basak, A.; Mitra, D.; Kar, M.; Biradha, K. Chem. Commun. 2008, 3067.
9. Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, 1984. pp
261–267; (b) McGaughey, G. B.; Gagne, M.; Rappe, A. K. J. Biol. Chem. 1998, 272,
12. The cleavage efficiency was determined by checking the relative UV-absorbance
of the bands at 260 nm. The cleavage efficiency was also measured by
densitometry using image processing software (Kodak 1D version V.3.6.3) and
similar results were obtained. The identity of the bands has been ascertained
from the control DNA which has form I as a major band. It is to be noted that the
control DNA specimen is usually contaminated with some nicked form (form II).
The reason for having alkaline pH 8.5 was to aid the propargyl to allene
isomerization.
13. The DNA-binding studies were carried out by absorption titrations which
involved addition of a solution of Calf Thymus DNA to a fixed concentration of
the probe. A small red shift of the absorption maxima for both the E-sulfones
(375 nm) and Z-sulfones (a shoulder near 438 nm) was observed.