Cytotoxic activity and QSAR of N,N′-diarylalkanediamides

Article abstract of DOI:10.1016/S0223-5234(01)01260-0

The one- and two-step syntheses of N,N′-(dinitrophenyl)alkanediamides and N,N′-(diaminophenyl)alkanediamides from 4-nitroaniline were carried out. These compounds were subjected to cytotoxic evaluation against different tumoural cell lines. A QSAR analysi

Full text of DOI:10.1016/S0223-5234(01)01260-0

Eur. J. Med. Chem. 36 (2001) 731–736
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01260-0/SCO
Short communication
Cytotoxic activity and QSAR of N,N%-diarylalkanediamides
Luis Chaco´n-Garc´ıa, Roberto Mart´ınez*
Instituto de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Circuito Interior, Ciudad Universitaria, Coyoaca´n,
04510 Mexico DF, Mexico
Received 28 February 2001; revised 10 July 2001; accepted 11 July 2001
Abstract – The one- and two-step syntheses of N,N%-(dinitrophenyl)alkanediamides and N,N%-(diaminophenyl)alkanediamides from
4-nitroaniline were carried out. These compounds were subjected to cytotoxic evaluation against different tumoural cell lines. A
QSAR analysis for the aminated series, introducing a QSAR descriptor (DCL) designed herein for groove binders, reveals that the
´
activity in the K562 cell line is related to the charge, polarisability, volume and length of the molecules. © 2001 Editions scientifiques
et me´dicales Elsevier SAS
cytotoxic activity / QSAR / N,N%-diarylalkanediamides
1. Introduction
these portions against tumoural cell lines, using
amino 1a–g and nitro 2a–g groups instead of the
chromophore groups.
Among compounds with cytotoxic activity, the
most important groups are those that interact re-
versibly with DNA without forming covalent bonds,
such as DNA intercalators or groove binders. Some
of the former are drugs used for cancer therapy, e.g.
2. Chemistry
Daunorubicin, employed in leukaemia treatment.
The N,N%-diarylalkanediamides were obtained from
Amides have attracted attention in medicinal chem-
p-nitroaniline. In this method, 4-nitroaniline (2
istry not only because of their antitumoural activity
equiv.) was condensed with the corresponding dia-
[1] but also in the treatment of other illnesses. In
cylchlorides (1 equiv.) in acetone while being cooled
particular, some N,N%-diaryldiamides have been in-
in an ice bath, leading to the formation of the respec-
vestigated as antibacterial agents [2] or HIV inhibitors
tive N,N%-(4,4%-dinitrophenyl)alkanediamides. The
[3] but not, to our knowledge, as cytotoxic agents. In
products obtained were precipitated, filtered and
a recent study we described the synthesis of bis-benza-
washed with acetone. Chromatographic purification
cridinones in which the linker portion was a diaryldi-
was not necessary. Yields varied from 50 to 65%, with
amide chain (figure 1) [4].
the exception of the reaction with succinyl chloride,
Taking into account the important role of the
for which the yield was very low (18%). In neither
linker in the DNA bis-intercalators in recognising the
case were the reactions carried out in the presence of
double helix, and then increasing the affinity for this
an additional base.
[5, 6], we decided to explore the cytotoxic activity of
The N,N%-(4,4%-diaminophenyl)alkanediamides were
obtained by reduction of the nitrated compounds
with hydrazine and palladium/carbon in ethanol at
reflux temperature and recrystallised from methanol
with yields over 80% in all the cases (figure 2).
* Correspondence and reprints
E-mail address: robmar@servidor.unam.mx (R. Mart´ınez).

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L. Chaco´n-Garc´ıa, R. Mart´ınez / European Journal of Medicinal Chemistry 36 (2001) 731–736
3. Pharmacological results and discussion
due to these increased interactions that almost all com-
pounds in this group present cytotoxicity. One obvious
hypothesis for these systems would be that the inhibition
of the growth is proportional to the length of the chain.
However, it is easily seen from the results (K562) that in
the first three compounds (2a–c) there is a decrease in
the percentage of inhibition followed by an increase in
compounds 2e–f (see figure 3).
3.1. Cytotoxic activity
Compounds 1a–f and 2a–f were evaluated for cyto-
toxic activity in cultures of PC-3, U251 and K562 cells.
The results are given in table I.
For the purpose of this study, and because of the
structure of the diaryldiamides described herein, we
consider the cytotoxic activity of these compounds to be
due to an interaction with DNA as groove ligands [7].
In the group of nitrated compounds studied, the most
active was 1f in the PC3 and U251 cell lines followed by
1c. The IC50 of 1f was 34.78 ( 5.9) mM in PC-3 and 3.84
( 0.65) mM in U251 and of 1c 32 ( 0.7) mM in U252.1
Although this result is moderate, 1f is considered to be
active. The potency of this is high if compared with
Distamicyn A, a known groove binder, whose IC50 in
U251 is over 100 mM.
3.2. QSAR
The above results indicate that the activity cannot be
explained only in terms of liposolubility, chain length or
molecular weight. To address this point, a QSAR analy-
sis of these compounds was carried out.2 Since the
number of compounds was small, the study was made
for the purpose of interpolation, not for extrapolation.
Due to the high correlation between almost all 16 of the
descriptors used by the program, it was not possible to
carry out the QSAR analysis with the remaining descrip-
tors, and it was necessary to incorporate a new descrip-
tor (DCL) related to the topological recognition of DNA.
This descriptor was obtained from the length of the
molecule geometrically optimised using PM3, assuming
that a specific length is required for an optimal recogni-
tion of the double helix, as shown in figure 4. The
distance within the molecule between the extreme
groups (NH2–NH2), divided by 4.202 or 3.4/cos 36,
should in principle be the number of gaps (or base pairs
plus one) that the cross-linker will recognise. Therefore,
if the closest whole number to this value is subtracted
from its value, the resulting difference will be the separa-
tion of the optimal (zero) or the worst (0.5) relative
recognition. This can be appreciated in figure 4 and Eq.
(1).
Compound 1a exhibited no activity in any line, while
compounds 1b, 1d, and 1e showed almost negligible
activity in all entries.
With respect to the group of the aminated com-
pounds, 2a (n=2), 2b (n=3), 2c (n=4), 2e (n=6) and
2f (n=7) showed medium activity in K562 (2d was not
probed in this line) and very little cytotoxicity in all the
others. This set of compounds, in contrast with the
preceding, could have additional intermolecular interac-
tions with DNA due to hydrogen bonds between the
amine group at the end of the molecule and base pairs,
either directly or through water molecules. It is probably
DCL=ꢀ(NH2−NH2 distance/4.202)−Nꢀ
(1)
where N is the closest whole number.
Considering this descriptor it was possible to formu-
late a mathematical expression. Eq. (2) was found to be
the best expression, containing the topological (DCL),
polarisability/volume (Sp.Pol) and charges (ABSQ)
descriptors.3
Figure 1. Structure of bis-benzacridines with the linker portion
2 QSAR analysis was made by multiple linear regressions. All
descriptors considered (with exception of DCL, see below) were
taken from SCIQSAR, version 3.0, SciVision, Inc., 1998. Statis-
tical criteria for avoidance chance were carefully taken into
account.
shown.
1 IC50 of this compound was evaluated by duplicate in three
different experiments using concentrations of 3.1, 10, 31 and
100 mM.
3 Linear estimation and Student’s t-distribution (h=0.05) be-
tween these descriptors discarded H0.

L. Chaco´n-Garc´ıa, R. Mart´ınez / European Journal of Medicinal Chemistry 36 (2001) 731–736
733
Figure 2. Synthesis of the N,N%-diarylalkanediamides 1a–f and 2a–f.
Table I. Percentage of inhibition of the growth (at 31 mM concentration). a
Comp.
n
M.p.
PC-3 (prostate)
U251 (CNS)
K562 (leukaemia)
b
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2
3
4
5
6
7
2
3
4
5
6
7
286–288
278–280
280–285
206–208
220–222
155–158
232–234
228–230
238–240
203–204
211–212
195–197
0
0
0
5
0
50
\50
23
20
50.7
b
b
b
2
\50
16.8
14.8
4.7
0
21.3
0
82
43.8
67.4
59.3
15.4
23.9
23.9
0
20.5
18.2
51.6
b
54.2
54.9
a A concentration of 31 mM was chosen because it represents the medium point between log −4 and log −5, which is the range
where inhibition of the growth was found.
b Activity was not measured.
log(% growth)= −20.55×10−2DCL+70.92×10−2ABSQ
+29.617Sp.Pol−2.107, n=5, r2=0.99,
F=31 782(1,3),h=0.05, s=0.045
(2)
where DCL (see Eq. (1)); ABSQ=ꢁi ꢀQiꢀ, where Qi is the
charge on the ith atom; Sp.Pol=ꢁAꢀhAꢀ/Vol, where hA
is the average atomic hybrid polarisability for atom A
and Vol is the molecular volume of the molecule.
The good correlation between Eq. (2) and the experi-
mental data can be seen in table II.
Figure 3. Behaviour of the inhibition of the growth in K562
cell vs. the number of methylenes (n) in the alkyl chain of the
amino series.

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L. Chaco´n-Garc´ıa, R. Mart´ınez / European Journal of Medicinal Chemistry 36 (2001) 731–736
Figure 4. Schematic representation of DCL
.
Table II. Comparison of the calculated data from Eq. (2) with experimental.
Comp.
log (%inhib)
Exp.
Dist. NꢀN a
DCL
ABSQ
Sp.Pol
Calc.
D (×10−4
)
2a
2b
2c
2e
2f
1.8286
1.7730
1.7126
1.7339
1.7395
1.8287
1.7728
1.7127
1.7340
1.7396
1
2
1
1
1
16.85
17.56
19.32
21.8
0.00939
0.17833
0.40287
0.18722
0.30619
4.496668
4.560416
4.663736
4.875879
5.268802
0.122193
0.121323
0.120606
0.11932
22.3
0.119391
a Distance between amines groups, taken from the geometry optimised using PM3. ALCHEMY 2000, version 2.05, Tripos, Inc., 1998.
However, the descriptor DCL can be used in this
and any other structurally different cross-linker and
could be very useful in the design of DNA bis-interca-
lating compounds in the spacer region.
4. Conclusions
The molecules described in this study are easily
obtained and the significant activity and high selectiv-
ity displayed by some of them is noteworthy.
The results from Eq. (1) are in agreement with the
molecular mechanism of action proposed for these
compounds. Firstly, the relative length between the
cross-linker and the number of DNA base pairs that
it will recognise through H-bonds, directly or by
water molecules, is given by DCL. It is noteworthy
that the length in DNA groove ligands is still continu-
ing to be a topic in the design of this kind of
molecules [8–10]. Secondly, as is expected for cross-
linker agents, charges (ABSQ) and polarisability
(Sp.Pol) are directly related to repulsive and/or at-
tractive interactions with the DNA grooves. Finally,
volume contributes to the correct docking of the
molecule.
5. Experimental protocols
5.1. Chemistry
All melting points are uncorrected. The IR spectra
were recorded in a Nicolet FT-55X spectrophotometer.
The 1H-NMR spectra were determined in a Varian
FT-200 instrument. All NMR spectra were obtained
with the pulse sequence as part of the spectrometer’s
software and were determined in DMSO-d6. Chemical
shifts are expressed in l (ppm) relative to TMS as
internal standard and coupling constants J in Hz.
Unfortunately, the presence of more than seven
methylenes in the alkyl chain could present additional
factors that affect the activity, such as additional
degrees of freedom, many more than one low energy
conformation, the marked effect of the partition co-
efficient, etc. In these cases another mathematical
expression should be sought.
5.1.1. General procedure for the preparation of the
N,N%-(4,4%-dinitrophenyl)alkanediamides
Diacyl chloride (0.72 mmol) was added to a solution
of 4-nitroaniline (1.44 mmol) in 15 mL of acetone at
5 °C. After stirring for 2 h, the mixture was filtered and
washed with acetone to afford 1a–c.

L. Chaco´n-Garc´ıa, R. Mart´ınez / European Journal of Medicinal Chemistry 36 (2001) 731–736
735
5.1.1.1. N,N%-Bis(4-nitrophenyl)ethanediamide (1a)
M.p. 286–288 °C, 18% yield, 1H-NMR: l 2.75 (s,
2H), 2.32 (s, 2H), 7.8 (d, J=9 Hz, 4H), 8.19 (d, J=9
Hz, 4H), 10.65 (s, 2H).
5.1.2.3. N,N%-Bis(4-aminophenyl)butanediamide (2c)
M.p. 238–240 °C, 93% yield, 1H-NMR: l 1.56 (t,
J=7 Hz, 4H), 2.21 (t, J=7 Hz, 4H), 4.81 (s, 4H), 6.46
(d, J=9 Hz, 4H), 7.19 (d, J=9 Hz, 4H), 9.43 (s, 2H).
5.1.1.2. N,N%-Bis(4-nitrophenyl)propanediamide (1b)
M.p. 278–280 °C, 59% yield, 1H-NMR: l 1.92 (q,
J=7 Hz, 2H), 2.45 (t, J=7 Hz, 4H), 7.8 (d, J=9 Hz,
4H), 8.18 (d, J=9 Hz, 4H), 10.52 (s, 2H).
5.1.2.4. N,N%-Bis(4-aminophenyl)pentanediamide (2d)
M.p. 203–204 °C, 83% yield, H-NMR: l 1.26–1.31
(m, 2H), 1.55 (q, J=7 Hz, 4H), 2.19 (t, J=7 Hz, 4H),
4.79 (s, 4H), 6.46 (d, J=9 Hz, 4H), 7.17 (d, J=9 Hz,
4H), 9.40 (s, 2H).
1
5.1.1.3. N,N%-Bis(4-nitrophenyl)butanediamide (1c)
M.p. 280–285 °C, 63% yield, 1H-NMR: l 1.64 (q,
J=6 Hz, 4H), 2.42 (s, J=6 Hz, 4H), 7.82 (d, J=9 Hz,
4H), 8.19 (d, J=9 Hz, 4H), 10.53 (s, 2H).
5.1.2.5. N,N%-Bis(4-aminophenyl)hexanediamide (2e)
1
M.p. 211–212 °C, 87% yield, H-NMR: l 1.25–1.35
(m, 4H), 1.56 (q, J=7 Hz, 4H), 2.2 (t, J=7 Hz, 4H),
4.74 (s, 4H), 6.47 (d, J=9 Hz, 4H), 7.19 (d, J=9 Hz,
4H), 9.39 (s, 2H).
5.1.1.4. N,N%-Bis(4-nitrophenyl)pentanediamide (1d)
1
M.p. 206–208 °C, 57% yield, H-NMR: l 1.34 (m,
2H), 1.62 (q, J=7 Hz, 4H), 2.38 (t, J=7 Hz, 4H), 7.79
(d, J=8 Hz, 4H), 8.16 (d, J=8 Hz, 4H), 10.46 (s, 2H).
5.1.2.6. N,N%-Bis(4-aminophenyl)heptanediamide (2f)
M.p. 195–197°C, 88% yield, 1H-NMR: l 1.25–1.3
(m, 6H), 1.54 (q, J=7 Hz, 4H), 2.19 (t, J=7 Hz, 4H),
4.79 (s, 4H), 6.46 (d, J=9 Hz, 4H), 7.18 (d, J=9 Hz,
4H), 9.39 (s, 2H).
5.1.1.5. N,N%-Bis(4-nitrophenyl)hexanediamide (1e)
1
M.p. 220–222 °C, 49% yield, H-NMR: l 1.26–1.38
(m, 4H), 1.60 (q, J=7 Hz, 4H), 2.37 (t, J=7 Hz, 4H),
7.8 (d, J=9 Hz, 4H), 8.17 (d, J=9 Hz, 4H), 10.46 (s,
2H).
5.2. Cytotoxic activity
5.1.1.6. N,N%-Bis(4-nitrophenyl)heptanediamide (1f)
M.p. 155–158 °C, 52% yield, H-NMR: l 1.24–1.36
(q, J=7 Hz, 6H), 1.58 (q, J=7 Hz, 4H), 2.36 (t, J=7
Hz, 4H), 7.81 (d, J=9 Hz, 4H), 8.18 (d, J=9 Hz, 4H),
10.46 (s, 2H).
The tumoural cell lines were supplied by the National
Cancer Institute. The cytotoxicity assays were carried
out at 5000–7500 cells mL−1 as reported by Skehan et
al. [11] and Monks et al. [12] using the sulforhodamine
B (SRB) protein assay to estimate cell growth. The
percentage growth was evaluated spectrophotometrically
in a Bio kinetics reader spectrophotometer.
1
5.1.2. General procedure for the preparation of the
N,N%-(4,4%-diaminophenyl)alkanodiamides
Ethanol (10 mL), Pd/C 5% (0.046 g), hydrazine (0.818
mL, 25.9 mmol), water (0.93 mL) and 1a–c (2.59 mmol)
were mixed in a round bottom flask. The mixture was
refluxed for 2 h. The resulting solid was dissolved in
methanol with heat and filtered at vacuum. Methanol
was eliminated upon precipitation of a solid that was
filtered and crystallised from methanol to afford 2a–c.
Acknowledgements
We thank CONACYT (32633-E) and DGAPA-
UNAM (IN206598) for financial support. L.C.G.
thanks CONACYT for a doctoral fellowship support.
We also thank M.T. Ram´ırez Apan for obtaining the
biological data. Contribution No. 1737 from Instituto
de Qu´ımica, UNAM.
5.1.2.1. N,N%-Bis(4-aminophenyl)ethanediamide (2a)
M.p. 232–234 °C, 92% yield, 1H-NMR: l 2.52 (s,
4H), 4.78 (s, 4H), 6.47 (d, J=9 Hz, 4H), 7.20 (d, J=9
Hz, 4H), 9.51 (s, 2H).
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J=7 Hz, 2H), 2.25 (t, J=7 Hz, 4H), 4.79 (s, 4H), 6.47
(d, J=10 Hz, 4H), 7.20 (d, J=10 Hz, 4H), 9.44 (s, 2H).
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