Benzofused N-substituted cyclic enediynes: Activation and DNA-cleavage potential

Article abstract of DOI:10.1016/j.bmc.2008.02.044

The effect of electron withdrawal on the reactivity of N-substituted cyclic enediynes has been studied. These were synthesized via an intramolecular Mitsunobu reaction. The electron withdrawing effect of the nitro groups or the positive charge on the free

Full text of DOI:10.1016/j.bmc.2008.02.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4532–4537
Benzofused N-substituted cyclic enediynes:
Activation and DNA-cleavage potential
Amit Basak* and Moumita Kar
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
Received 28 December 2007; revised 12 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—The effect of electron withdrawal on the reactivity of N-substituted cyclic enediynes has been studied. These were synthe-
sized via an intramolecular Mitsunobu reaction. The electron withdrawing effect of the nitro groups or the positive charge on the
free ammonium salts was found to lower the cyclization temperature for Bergman cyclization. The ammonium salts cleave ds-DNA
at nanomolar concentrations.
Ó 2008 Published by Elsevier Ltd.
1. Introduction
electron withdrawing groups in the sulfonamide aryl
ring (Type 1), (ii) by attaching electron withdrawing
groups in the aromatic ring,7 which is a part of the ened-
iyne system (Type 2) and (iii) by keeping the N atom in
the protonated amine form (Type 3). All these ap-
proaches are expected to lower the activation barrier
to BC. The electron withdrawing effect (mainly I) from
the substituents in the aryl ring or from the positively
charged N atom reduces the repulsion between the in-
plane alkyne p-orbitals (Koga–Morokuma hypothesis)8
thus helping the process of cycloaromatization leading
to a planar aromatic ring. Moreover, the electron with-
drawal lowers the singlet–triplet gap9 and thus favors
the triplet state. The triplet, being a better H-abstractor,
then produces cleavage of DNA. The first strategy was
attempted earlier10 and the results indicated that incor-
poration of electron withdrawing groups in the aryl ring
of the sulfonamide had some influence upon the BC
kinetics. However, the effect is not sufficient enough to
have the BC occurring under ambient conditions. Thus,
we became interested to study the activity of molecules
belonging to Type 2 and 3 categories. In this communi-
cation, we report our results.
Various structural changes are believed to lower the
activation barrier for the Bergman cycloaromatization1
process either by bringing the terminal acetylenic carbon
atoms (c, d distance)2 closer or by easing the overall con-
formational restrictions.3 It is difficult to judge the effect
of one factor independent of the other on the overall
kinetics of the reaction and quite often, both c, d dis-
tance and strain factors act synergistically to drive the
process of diradical generation at physiologically rele-
vant temperature. In reality, both the factors need to
be considered while designing new enediynes as possible
therapeutic agents. Cyclic aliphatic enediynes with a ring
size of 10 have low half-life4 and as such are not easy to
handle. Thus, elaboration of these molecules into a drug
candidate will pose problems because of limited shelf-
life. Aromatic enediynes, on the other hand, are stable
at room temperature. For example,5 the aliphatic
N-substituted enediyne 1 cyclizes spontaneously at room
temperature of 30 °C with a half-life of 36 h. The corre-
sponding aromatic counterparts 2a–2c are stable under
ambient conditions and cyclize only when heated at
65–70 °C6 (Scheme 1). Our long standing objective was
to activate aromatic fused cyclic enediynes towards BC
to bring down the cyclization temperature. In principle,
this can be achieved in three ways: (i) by incorporating
The synthesis of Type 2 molecules starting from the ni-
tro substituted dibromobenzene11 is shown in Scheme 2.
The salient features of the synthesis are mentioned be-
low: (a) nitration of dibromo benzene11 using a mixture
of concd HNO3 and H2SO4 (1:3 by volume) at 0 °C for
15 min yielded the 4-nitro-1,2-dibromo benzene 9a as
the major product, isolated by chromatography. This
on further nitration and fractional crystallization from
hexane–CH2Cl2 furnished the dinitro derivative 9b; (b)
Keywords: Enediynes; Mitsunobu reaction; Bergman cyclization; DNA
cleavage; pH.
*
Corresponding author. Tel.: +91 3222283300; fax: +91
3222282252; e-mail: absk@chem.iitkgp.ernet.in
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.02.044

A. Basak, M. Kar / Bioorg. Med. Chem. 16 (2008) 4532–4537
4533
X
X
H
H
N
N
Y
O
SO2Ar
N
S
Salt
O
Type 2
Type 1
Type 3
X
X = electron withdrawing
group (NO2)
R2
O
O
R2
S
O
H
H
O
S
O
R1
heat
S
H
H
N
t 1/2 = 36 h
30 o
N
O
S
R1
N
O
N
O
C
1
1a
2a-2c
3a-3c
Scheme 1. Reactivity of N-substituted enediynes. Reagents and conditions: (a) R1 = Me, R2 = H, t1/2 = 72 h, 65 °C; (b) R1 = R2 = NO2, t1/2 = 28 h,
70 °C (c) R1 = NO2, R2 = H, t1/2 = 49 h, 70 °C.
OTHP
OTHP
OMs
R2
R1
NaN3
R2
R1
N3
(Not obtained)
12a
11a
OH
NHSO2Ar
R2
R1
OTHP
R2
R1
Br
Br
R2
OTHP
i
iii
R2
R1
ii
60 %
55%
Br
R1
50%
NHSO2Ar
9a-9b
14a-14b
14a-14b
10a-10b
13a-13b
4a-4b
OMs
NHSO2Ar
vi
R2
R1
R2
R1
iv
SO2Ar
v
N
100%
15a-15b
yield 10% for 4a
yield 0% for 4b
Ar =
NHSO2Ar
16
For a, R1 = NO2, R2 = H
For b, R1 = R2 = NO2
NO2
Scheme 2. Synthesis of enediynes via intramolecular N-alkylation from mesylate. Reagents and conditions: (i) Propargyl OTHP ether, Pd (0)/CuI/
Et3N/5 h (for 10a), 1 h (for 10b)/rt; (ii) 16, Pd (0)/CuI/Et3N/THF (3:1)/reflux, 1 h (for 13a)/rt, 5 h (for 13b); (iii) PPTS/Dry EtOH/40 °C/12 h; (iv)
MsCl/CH2Cl2/0 °C/20 min; (v) K2CO3/Dry DMF/2 h; (vi) PPh3/DEAD/THF/30 min/rt.
Compound 9a was subjected to sequential Sonogashira
coupling12 with THP-protected propargyl alcohol and
3-butyn-1-ol. Treatment with mesyl chloride in the pres-
ence of Et3N gave mesylate 11a; (c) attempted displace-
ment of the mesylate with NaN3 in DMF met with
failure. The azide probably underwent cycloaddition13
with the acetylenic arm in conjugation to the nitro
group; so the N-4-nitrophenyl sulfonamido homoprop-
argyl amine 16 was used as the second coupling14 part-
ner; (d) the basic condition usually employed to effect
cyclization via intramolecular N-alkylation led to for-
mation of side products. This is probably due to the
base-induced isomerization of propargylic hydrogen
(which is more acidic because of the nitro group) that al-
lowed its decomposition via Myers–Saito pathway.15 In
case of the mononitro derivative, only a 10% yield of the
cyclized product was obtained while for the dinitro sys-
tem, we could not isolate any cyclized product, the reac-
tion producing insoluble polymeric material (Scheme 2).
Thus, the final cyclization needed avoidance of strongly
basic conditions. Previously, Reitz et al.16 have reported
the synthesis of benzo fused N-substituted cyclic enediy-
nes via an intramolecular Mitsunobu reaction. Based on
that report,16 as well as by others,17 sulfonamide was
treated with PPh3 and diethylazido dicarboxylate
(DEAD) (2.5 equiv) in THF, for 30 min. Gratifyingly,
only intramolecular cyclization took place and the cyclic
sulfonamide was isolated in up to 60% yield after col-
umn chromatography over Si-gel. The reaction also
worked with other systems and the yields range from
60% to 70% (Table 1).
The chemical reactivity of sulfonamides 4a–4b was then
checked to find out the effect of the electron withdraw-
ing nitro group on the onset temperature and kinetics
Table 1. Intramolecular Mitsunobu reaction
OH
R
2
PPh
R
2
3
NSO Ar
2
DEAD
R
NHSO Ar
R
1
1
2
2c, 4a-4b
14a-14c
Compound R1
R2
Ar
4-Tolyl
4-Nitrophenyl 4a
Product Yield
14c
14a
14b
H
NO2
H
H
2c
60
70
67
NO2 NO2 4-Nitrophenyl 4b

4534
A. Basak, M. Kar / Bioorg. Med. Chem. 16 (2008) 4532–4537
of BC. First, the DSC (differential scanning calorimetry)
theromogram18 was recorded for 4a which showed a
strong exothermic peak starting at around 166 °C which
can be regarded as the onset temperature for BC. For
the corresponding nonnitrobenzene fused enediyne 2c,
the onset temperature as recorded in DSC was found
to be 240 °C thus showing significant lowering of onset
temperature for BC in case of the nitroenediyne. Kinetic
studies in solution were carried out by heating a DMSO-
tography (Scheme 3). Use of basic conditions produced
poor yield of 5a. Deprotection of the dinitroenediyne
system 4b was not successful, we could not isolate any
free amine and that precluded any reactivity study using
5b. DSC measurement on the tosylate 7c indicated sig-
nificant lowering of the cyclization temperature (Table
2). Solution phase kinetics for 7c in DMSO-d6 showed
the half-life of 130 h at 50 °C, close to the biological
temperature. Indeed, at 30 °C it showed a measurable
rate of BC (half-life of 770 h). The tosylate salt of amine
5a could not be isolated as the electron withdrawal by
both the positively charged nitrogen and the nitro group
made it reactive under ambient conditions. Even the free
amine slowly decomposed in CDCl3 at room tempera-
ture leading to unidentifiable products. However, gener-
ation of signals in the aromatic region is suggestive of
occurrence of BC. It may be noted that sulfonamides
4a–4b did not produce any cyclization product at
50 °C even when kept for 7 days. Inspired by these re-
sults, we then checked the DNA-cleavage activity of
amines 5a and 5c at pH 8.0 in which the amine should
be mostly present in the conjugate acid form. Both these
compounds now showed cleavage of plasmid DNA
(pBR 322) at micromolar concentrations (Fig. 1). Most
interestingly, amine 5c even showed the generation of
Form III with pBR 322 which was not observed with
p-BlueScript SK+ plasmid.20 If the ammonium salt is
really responsible for the cleavage, then the cleavage effi-
ciency should depend upon the extent of conjugate acid
formation which in turn should depend upon the pH.
Thus, the DNA-cleavage study was carried out with
the tosylate salt 7c at three different pHs (7.5, 8.0, and
8.5). The gel pattern (Fig. 2) clearly showed increase
of cleavage efficiency as the pH is lowered thus support-
ing the involvement of the ammonium salt as the key
1
d6 solution of enediyne at 70 °C and checking the H
NMR at different time points. The singlet at d 4.44
was slowly replaced by a broad singlet at d 4.58 typical
of a methylene adjacent to the isoquinoline derivative,
thus pointing to the formation of cycloaromatized prod-
uct 6a which was also proved by the appearance of
peaks at m/z 415, 414, 413 (major) in the ESI mass spec-
trum corresponding to the abstraction of two deuterium,
one deuterium one hydrogen, and two hydrogen atoms,
respectively. The cyclization followed first order kinetics
like the benzene fused analogue. The half-life for 4a at
70 °C was found to be 12 h as compared to 48 h for 2c
at the same temperature. The half-life of 4a was also re-
corded at 60 °C, which came out to be 138 h. For the di-
nitro enediyne 4b, the onset temperature was further
lowered to 128 °C and the half-life at 70 °C was found
to be 9.5 h.
Thus, by incorporating electron withdrawing nitro
group(s) we could significantly enhance the reactivity to-
wards Bergman cyclization as revealed in DSC as well as
in the rate of solution phase cyclization. However, the
molecules still remain stable at room temperature and
hence, did not show any DNA cleavage under biological
conditions. It may be noted that the reactivity trend ob-
tained from DSC measurements was in agreement with
that obtained from solution phase kinetic studies.
Table 2. Results of DSC studies
Having failed to bring down the temperature for the BC
to the level of biological conditions for nitroaromatic
systems, we turned our attention to the cyclic enediynes
in the protonated form (Type 3). Thus, both the cyclic
enediynes 4a and 2c were deprotected19 using thiophenol
and K2CO3 in DMF and the free amines 5a (yield 20%)
and 5c (yield 70%) were isolated pure by Si-gel chroma-
Compound
Onset temperature
(T1) for BC (°C)
Difference in onset
temperature (DT)
4a
4b
2c
7c
166
128
240
110
74 (T12c À T14a)
112 (T12c À T14b)
—
130 (T12c À T17c)
H
R2
R1
SO2Ar
R2
R1
SO2Ar
N
N
ii
H
4a-4b, 2c
6a-6b, 3c
H
4a, 2c
H
H
iv
N
iii
N
-
5c
i
H
H
-
OTs
R2
R1
OTs
NH
H
7c
8c
Ar = 4-nitrophenyl
5a, 5c
For a, R1 = NO2, R2 = H
For b, R1 = R2 = NO2
For c, R1 = R2 = H
Scheme 3. BC of various enediynes. Reagents and conditions: (i) PhSH, K2CO3, DMF, rt, 30 min (20% for 5a, 70% for 5c); (ii) DMSO-d6, 70 °C; (iii)
4-toluene sulphonic acid, ether, MeOH, 90%; (iv) DMSO-d6, 50 °C.

A. Basak, M. Kar / Bioorg. Med. Chem. 16 (2008) 4532–4537
4535
phine (0.15 mmol) in 10 mL of dry tetrahydrofuran,
and the solution was stirred under a dry nitrogen atmo-
sphere for 30 min at room temperature. The solvent was
concentrated and worked up with EtOAc and water.
The title compounds were isolated by column chroma-
tography (hexane/Ethyl acetate 4:1) followed by crystal-
lization from CHCl3–hexane.
(5a, 20 μM, 24 h)
(5a, 20 μM, 48 h)
A
C
B
D
1
2
1
2
(5c, 20 μM, 12 h)
(5c, 20μM, 12 h)
2
1
1
2
2.1.1. 1-[4-Nitrophenylsulphonyl]-[5,6]-[4-nitrobenz]-1-aza-
cyclodec-3,7-diyne (4a). Yield 70%; white solid, mp
125 °C; mmax (KBr, cmÀ1) 3430, 1526, 1348, 1159,
1095, 852, 744, 607; dH (DMSO-d6) 8.38 (d,
J = 8.8 Hz, 2H), 8.17 (d, J = 8.4 Hz, 4H), 7.67 (d,
J = 8.4 Hz, 1H), 4.44 (s, 2H), 3.55 (t, J = 4.6 Hz, 2H),
2.85 (t, J = 4.8 Hz, 2H); dC (DMSO-d6) 150.0, 146.7,
143.1, 133.7, 131.7, 129.8, 128.8, 128.8, 124.7, 123.1,
122.6, 101.6, 99.9, 85.4, 81.2, 54.9, 50.7, 41.9; Mass
(ES+) 412.06 (MH+); HRMS calculated for
C19H13N3O6S + H+: 412.06042; found: 412.06040.
(5c, 20 μM, 48 h)
(5c, 20 μM, 24 h)
E
F
1
2
2
1
Figure 1. DNA cleavage with compounds 5a and 5c at 37 °C; for A, B,
C, E pBR 322 and for D, F pBluscript+ were, respectively, used: lane
1, DNA in TAE buffer (pH 8.0, 0.4 lm bp) (7 ll) + CH3CN (5 ll); for
A: lane 2, DNA in TAE buffer (pH 8.0, 0.4 lm bp) (7 ll) + 5a (20 lM)
in CH3CN (5 ll); for B: lane 2, DNA in TAE buffer (pH 8.0, 0.4 lm
bp) (7 ll) + 5a (20 lM) in CH3CN (5 ll); for C: lane 2, DNA in TAE
buffer (pH 8.0, 0.4 lm bp) (7 ll) + 5c (20 lM) in CH3CN (5 ll); for D:
lane 2, DNA in TAE buffer (pH 8.0, 0.4 lm bp) (7 ll) + 5c (20 lM) in
CH3CN (5 ll); for E: lane 2, DNA in TAE buffer (pH 8.0, 0.4 lm bp)
(7 ll) + 5c (20 lM) in CH3CN (5 ll); for F: lane 2, DNA in TAE buffer
(pH 8.0, 0.4 lm bp) (7 ll) + 5c (20 lM) in CH3CN (5 ll).
2.1.2. 1-[4-Nitrophenylsulphonyl]-[5,6]-[3,4-dinitrobenz]-
1-aza cyclodec-3,7-diyne (4b). Yield 67%; white solid,
mp 120 °C, mmax (KBr, cmÀ1) 3676, 3295, 2370, 1605,
1529, 1421, 1350, 1305, 1159, 1085, 854, 799, 734, 683,
619, 464; dH (acetone-d6) 8.44 (d, J = 8.8 Hz, 2H), 8.25
(d, J = 8.8 Hz, 2H), 8.16 (s, 1H), 8.11 (s, 1H), 4.56 (s,
2H), 3.74 (t, J = 5.2 Hz, 2H), 3.00 (t, J = 5.2 Hz, 2H);
dC (acetone-d6) 143.9, 142.5, 134.7, 133.3, 132.3, 130.4,
128.8, 124.5, 124.5, 124.3, 106.1, 100.8, 83.9, 80.6,
50.8, 42.1, 22.1; Mass (ES+) 456 (M+), 380
(M+ÀNOÀNO2), 326 (M+ÀNO); HRMS calcd for
C19H12N4O8S + H+: 457.04549; found: 457.04530.
1
2
3
4
G (7c, 20 μM, 24 h)
2.2. General procedure for deprotection of sulfonamides:
synthesis of free amines 5a and 5c
Figure 2. pBR 322 DNA cleavage with 7c at 37 °C after 24 h; lane 1,
DNA in TAE buffer (pH 8.0, 0.4 lm bp) (7 ll) + CH3CN (5 ll); for
lane 2, DNA in TAE buffer (pH 7.5, 0.4 lm bp) (7 ll) + 7c (0.4 lM) in
CH3CN (5 ll); lane 3, DNA in TAE buffer (pH 8.0, 0.4 lm bp)
(7 ll) + 7c (0.4 lM) in CH3CN (5 ll); lane 4, DNA in TAE buffer (pH
8.5, 0.4 lm bp) (7 ll) + 7c (0.4 lM) in CH3CN (5 ll).
To a solution of compound (0.1 mmol) (2c, 4a) in DMF
(15 mL), thiophenol (1.2 equiv) and K2CO3 (3 equiv)
were added and the mixture was stirred for 40 min at
room temperature. After partitioning between water
and EtOAc, the organic layer was evaporated using li-
quid N2 in the vacuum pump and the free amine (5a/
5c) was isolated by column chromatography (Si-gel,
20% CH3OH in CH2Cl2).
cleaving agent.21 The stabilizing ionic interaction be-
tween the negatively charged DNA and the positively
charged ammonium salt may be aiding to the efficiency
of cleavage.
In conclusion, we have demonstrated that by simply
using N-substituted enediyne as protonated amine, one
can bring down the activation barrier for BC to such
an extent that the molecules can inflict damage to ds-
DNA under ambient conditions. This observation is
important as we can now think of better selectivity of
these molecules in showing cytotoxic activity against
cancer cells, which has much lower pH22 as compared
to the normal cell.
2.2.1. [5,6]-Benz-1-azacyclodec-3,7-diyne (5c). Yield
70%; brown oil; dH 7.24–7.20 (m, 2H), 7.15–7.12 (m,
2H), 3.78 (NH), 3.58 (s, 2H), 3.18 (t, J = 5.4 Hz, 2H),
2.48 (t, J = 5.4 Hz, 2H); Mass (ES) m/z 182 (MH+).
2.2.2. [5,6]-(4-Nitrobenz)-1-azacyclodec-3,7-diyne (5a).
Yield 20%; brown oil; dH 8.09 (s, 1H), 8.01 (d,
J = 8.4 Hz, 2H), 7.26 (d,J = 8.4 Hz, 1H), 3.45 (s, 2H),
3.11 (bs, 2H), 2.46 (t, J = 5.2 Hz, 2H); Mass (ES) m/z
227 (MH+).
2. Experimental
2.3. Synthesis of [5,6]-benz-azacyclodec-3,7-diyne 4-tolu-
ene sulfonate (7c)
2.1. General procedure for intramolecular Mitsunobu
reaction
The diethyl ether solution of the amine compound (5a, 5c)
was added drop wise to the solution of p-toluene sulfonic
acid in diethyl ether with few drops of methanol mixture.
The reaction mixture was stirred for few minutes until the
Diethyl azodicarboxylate (0.15 mmol) was added to a
solution of 14a–14c (0.06 mmol) and triphenylphos-

4536
A. Basak, M. Kar / Bioorg. Med. Chem. 16 (2008) 4532–4537
103, 4091; (d) Mayer, J.; Sondheimer, F. J. Am. Chem.
Soc. 1966, 88, 602.
2. (a) Nicolaou, K. C.; Dai, W. M. Angew. Chem. Int. Ed.
1991, 30, 1387; (b) Schreiner, P. R. J. Am. Chem. Soc.
1998, 120, 4184.
3. (a) Magnus, P.; Parry, D.; Iliadis, T.; Eisenbeis, S. A.;
Fairhurst, R. A. Chem. Commun. 1994, 1543; (b) Magnus,
P.; Fortt, S.; Pitterna, T.; Snyder, J. P. J. Am. Chem. Soc.
1990, 112, 4986.
4. Nicolaou, K. C.; Zuccarello, G.; Oogawa, Y.; Schweiger,
E. J.; Kumazawa, T. J. Am. Chem. Soc. 1988, 10, 4866.
5. Basak, A.; Khamrai, U. K.; Shain, J. C. Tetrahedron Lett.
1997, 38, 6067.
white precipitate appeared. The solid material was iso-
lated by washing with diethyl ether several times; Yield:
100%, white solid, mp 235 °C (decomposed); mmax (neat):
3464, 2979, 2924, 2786, 2629, 2524, 1461, 1213, 1154, 1030
and 1006; dH (DMSO-d6) 9.39 (br s, 2H), 7.50–7.39 (m,
6H), 7.09 (d, J = 8 Hz, 2H), 4.20 (s, 2H), 3.46 (t,
J = 5.1 Hz, 2H), 2.82 (t, J = 5.1 Hz, 2H), 2.27 (s, 3H);
dC(DMSO-d6) 145.5, 137.8, 129.2, 128.5, 128.1, 128.0,
127.8, 126.9,125.5, 98.2, 90.3, 88.1, 83.8, 48.7, 39.5 (ob-
scured by DMSO), 20.8, 18.7; Mass (ES+) m/z 182.11
(MH+), 153.08 (M+ÀCH2@NH); HRMS calcd for
C13H12N: 182.0971; found: 182.0977.
6. Basak, A.; Shain, J. C.; Khamrai, U. K.; Rudra, K.;
Basak, A. J. Chem. Soc. Perkin Trans. 1 2000, 1955.
7. The effect is most pronounced at the ortho position (ortho
effect). For reference see (a) Pickard, F. C.; Shepherd, R.
L.; Gillis, A. E.; Dunn, M. E.; Feldgus, S.; Kirschner, K.
N.; Shields, G. C.; Manoharan, M.; Alabugin, I. V.
J. Phys. Chem. A 2006, 110, 2517; (b) Zeidan, T. A.;
Kovalenko, S. V.; Manoharan, M.; Alabugin, I. V. J. Org.
Chem. 2006, 71, 962.
2.4. Spectral data of cycloaromatized products
For 6a: dH (DMSO-d6) 8.5–8.2 (9H, m), 4.58 (2H, m),
3.56 (2H, m), 3.10 (2H, m); Mass 413 (M+).
For 6b: dH (DMSO-d6) 8.6–8.2 (8H, m), 4.60 (2H, m),
3.58 (2H, m), 3.14 (2H, m); Mass 458 (M+).
8. Koga, N.; Morokuma, K. J. Am. Chem. Soc. 1991, 113,
1907.
9. (a) Chen, P.; Hoffner, J.; Schottelius, M. J.; Feichtinger,
D. J. Am. Chem. Soc. 1998, 120, 376; (b) Logan, C. F.;
Chen, P. J. Am. Chem. Soc. 1996, 118, 2113.
For 8c: dH (DMSO-d6) 8.95 (1H, br s), 7.35 (8H, m),
7.05 (2H, d, J = 8.0 Hz), 4.40 (2H, m), 3.40 (2H, ob-
scured), 3.12 (2H, m), 2.14 (3H, s); Mass 184 (MH+).
10. Basak, A.; Bdour, H. M.; Shain, J. C. J. Ind. Chem. Soc.
1999, 76, 679.
2.5. Differential calorimetric studies
11. (a) Singh, R.; Just, G. J. Org. Chem. 1989, 54, 4453; (b)
Zeidan, T. A.; Kovalenko, S. V.; Manoharan, M.;
Alabugin, I. V. J. Org. Chem. 2006, 71, 962.
DSC measurements were recorded in Perkin-Elmer Jade
instrument (Jade DSC). The rate of heating was fixed at
10 °C/min. Heating was carried out in an atmosphere of
nitrogen (99.49% purity) in aluminum crucibles. Cali-
bration was done with indium.
12. (a) Sonogashira, K.; Tohoda, Y.; Hagihara, N. Tetrahe-
dron Lett. 1975, 16, 4467; (b) Takahashi, S.; Kuroyama,
Y.; Sonogashira, K.; Hagihara, N. Synthesis 1980, 627.
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M. R. Org. Lett. 2003, 5, 2773; (b) von Riesen, C.;
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2.6. DNA-cleavage experiment
Sample solution (5 ll) in acetonitrile was mixed with the
DNA solution (7 ll) in TAE (Tris–Acetate–EDTA) buf-
fer (pH 8.0) and was incubated at 37 °C for 12–48 h.
The solution was then mixed with aqueous sucrose
(40%, 20 ll)) and bromophenol blue (0.25%, 5 ll). From
the above mixture, 15 ll was loaded on 0.7% agarose gel
and was subjected to electrophoresis in a horizontal slab
gel apparatus in TAE buffer for 1 h under 75 V. After elec-
trophoresis, the bands were visualized under UV-transil-
luminator and photographed using GELDOC. The
cleavage efficiency was measured by densitometry using
image processing software (Kodak 1D version V.3.6.3).
Acknowledgments
M.K. is grateful to CSIR for the fellowship and A.B.
thanks, DST for a research grant. Dr. Subrata Mandal
is thanked for carrying out some initial studies. DST is
also thanked for creation of 400 MHz facility under
IRPHA programme.
20. Although 5a is more reactive than 5c in the protonated
form, the lower basicity of the former may be responsible in
generating less number of cleavage events. This might
explain the DNA-cleavage result observed with the amines.
21. Considering the pKa of the cyclic amine 5c to be ꢀ8.6
(assumed to be close to N-ethyl N-propargyl amine), it has
References and notes
1. (a) Jones, R. G.; Bergman, R. G. J. Am. Chem. Soc. 1972,
9, 660; (b) Bergman, R. G. Acc. Chem. Res. 1973, 6, 25; (c)
Lockhart, T. P.; Bergman, R. G. J. Am. Chem. Soc. 1981,

A. Basak, M. Kar / Bioorg. Med. Chem. 16 (2008) 4532–4537
4537
been estimated that the percentage of protonated form of
the amine 5c at pH 7.5, 8.0 and 8.5 to be approximately
92, 80, and 55, respectively. Thus, there is a definite
correlation between extent of cleavage and percentage of
protonated form. The pKa of N-ethyl N-propargyl amine
has been obtained from SciFinder search which used ACD
software for calculating the pKa.
22. (a) Adams, G. E.; Stratford, I. J. Int. J. Radiat. Oncol.
Biol. Phys. 1994, 29, 231; (b) Priyadarsini, K.; Dennis, L.
M. F.; Naylor, M. A.; Stratford, M. R. L.; Wardman, P.
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(d) Lyons, J. C.; Song, C. W. Radiat. Res. 1995, 141, 216.