Synthesis, characterization, molecular structures, cytotoxic and antibacterial activities of N,N′-diaryl-o-phenylenediamines

Article abstract of DOI:10.1016/j.molstruc.2011.08.040

The molecular structures of N,N′-2,6-dimethylphenyl-o- phenylenediamine (1), N,N′-2,4,6-trimethylphenyl-o-phenylenediamine (2), N,N′-2,6-diisopropylphenyl-o-phenylenediamine 3a and 3b (dimorph of 3a) have been elucidated by X-ray diffraction as well as characterized by FT-IR, HRMS, ¹H, ¹³C NMR and 2D experiments. The cytotoxic and antibacterial activities of the N,N′-diaryl-o-phenylenediamines (1-3) were tested against human tumor cell lines A431 and MOLT4 and Salmonella typhimurium and Staphylococcus aureus respectively. Compounds 1 and 2 showed higher biological activities than compound 3 in cell line A431 and against S. typhimurium. Tumor cell growing was inhibited with 1 and 2 1.0 μg/mL and 0.5 μg/mL, respectively. The minimal inhibitory concentration against S. typhimurium and S. aureus for both compounds was 0.35 μg/mL. However, all compounds presented very similar activities in cell line MOLT4 (1-3: 0.5 μg/mL) and S. aureus (1-3: 0.35 μg/mL). A decrease of steric hindrance on phenylenediamine derivatives might influence on biological activity.

Full text of DOI:10.1016/j.molstruc.2011.08.040

Journal of Molecular Structure 1031 (2013) 168–174
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, characterization, molecular structures, cytotoxic and antibacterial
activities of N,N0-diaryl-o-phenylenediamines
a
a
a
Víctor M. Jiménez-Pérez a, , Marisol Ibarra-Rodríguez , Blanca M. Muñoz-Flores , Alberto Gómez ,
Rosa Santillan b, Eugenio Hernández Fernadez a, Sylvain Bernès a, Noemi Waksman c,
Rosalba Ramírez Duron c
⇑
a Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Pedro de Alba s/n, C.P. 66451, Nuevo León, Mexico
b Departamento de Química, Centro de Investigación y de Estudios Avanzados del IPN, A.P. 14-740, C.P. 07000, D.F., Mexico
c Departamento de Química Analítica, Facultad de Medicina, PO Box 2316, Sucursal Tecnológico, 64841 Monterrey, Nuevo León, Mexico
a r t i c l e i n f o
a b s t r a c t
The molecular structures of N,N0-2,6-dimethylphenyl-o-phenylenediamine (1), N,N0-2,4,6-trimethyl-
phenyl-o-phenylenediamine (2), N,N0-2,6-diisopropylphenyl-o-phenylenediamine 3a and 3b (dimorph
of 3a) have been elucidated by X-ray diffraction as well as characterized by FT-IR, HRMS, 1H, 13C NMR
and 2D experiments. The cytotoxic and antibacterial activities of the N,N0-diaryl-o-phenylenediamines
(1–3) were tested against human tumor cell lines A431 and MOLT4 and Salmonella typhimurium and
Staphylococcus aureus respectively. Compounds 1 and 2 showed higher biological activities than com-
pound 3 in cell line A431 and against S. typhimurium. Tumor cell growing was inhibited with 1 and 2
Article history:
Received 4 June 2011
Received in revised form 22 August 2011
Accepted 23 August 2011
Available online 17 September 2011
Keywords:
N,N0-diaryl-o-phenylenediamine
X-ray
NMR
1.0
and S. aureus for both compounds was 0.35
ities in cell line MOLT4 (1–3: 0.5 g/mL) and S. aureus (1–3: 0.35
on phenylenediamine derivatives might influence on biological activity.
Ó 2012 Published by Elsevier B.V.
l
g/mL and 0.5
l
g/mL, respectively. The minimal inhibitory concentration against S. typhimurium
g/mL. However, all compounds presented very similar activ-
g/mL). A decrease of steric hindrance
l
l
l
Biological activity
1. Introduction
been reported. However, there is not a clear structure–activity
relationship [12]. On the other hand, Hartwig and Buchwald have
been developed an efficient synthetic methods to carry out a cou-
pling reaction between 1,2-dibromoarenes and anilines [13,14]. In
order to carry out a structural modification on phenylene diamines,
Harlan et al. reported a simple catalyzed coupling reaction to iso-
lated N,N-diaryl-o-phenylenediamine derivatives [15]. Base on
that, in this paper, we describe the molecular structure and their
biological activity of a short series of N,N0-aryl-o-phenylenediam-
ines (Scheme 2) such as N,N0-2,6-dimethylphenyl-o-phenylenedi-
amine (1), N,N0-2,4,6-trimethylphenyl-o-phenylenediamine (2),
and N,N0-2,6-diisopropylphenyl-o-phenylenediamine (3).
o-Phenylenediamines (o-PDA) derivatives have been shown as
an important class of synthons in several heterocyclic [1], organic
[2], organometallic [3], and coordination complexes [4]. Some
phenylenediamines derivatives have been showed important bio-
logical effects such as antifungal [5], anticancer [6], and antimuta-
genic [7]. Also, the PDA derivatives can act as antiozonant [8], hair
coloring [9], anticorrosion material [10], and nitric oxide sensor
[11] (Scheme 1). The search and design of new drugs is a continu-
ous need nowadays mainly because of the development of multiple
resistance mechanisms in both bacteria and cancer cells. Besides
cytotoxic activity, any chemical compound designated to be used
as a new pharmacologic active substance or to improve treatment
strategies, must offer possibilities in its molecular structure to be
adapted in regard of enhancement of solubility, chemical stability
or any other attribute focused on improving its mechanism of
action.
2. Experimental
2.1. Reagents and measurements
All manipulations were performed under nitrogen atmosphere
by using standard Schlenk techniques [16] or inside a glovebox.
Toluene was dried with Na/benzophenone prior to use under N2.
Chemicals were commercial available and used as received. The
compounds 1 and 3 were readily synthesized by coupling reaction
of the corresponding aryl amines with 1,2-dibromobenzene [15].
Recently Hahn et al. reported the synthesis of 2 by different
Recently, the cytotoxicity activity against Salmonella typhimuri-
um TA 102 bacteria of a short series of phenylendiamines deriva-
tives with a structural modification on the aromatic ring has
⇑
Corresponding author. Tel.: +52 8183294000; fax: +52 8183760570.
E-mail address: vjimenez@fcq.uanl.mx (V.M. Jiménez-Pérez).
0022-2860/$ - see front matter Ó 2012 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.molstruc.2011.08.040

V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
169
2.3. In vitro cytotoxicic assay
Heterocycle
chemistry
Synthon of
organic compunds
Human epidermoid carcinoma ATCC cell line A431 and human
lymphoblastic leukemia MOLT-4 were employed to test the cyto-
toxic effects of compounds 1–3. A431 cells were maintained in
R
NH
Dulbecco’s Modified Eagle Media (DMEM) with 4 mM
and supplemented with 10% Foetal Bovine Serum (Gibco), 100 IU/
mL penicillin and 100 g/mL streptomycin. Culture of both cell
L-glutamine
Organometallic
compounds
Coordination
compounds
l
NH
R
lines was carried out at 37 °C in an Incubator (Shell Lab, TC-
2323) with a 95% air and 5% CO2 atmosphere. Leukemia cells were
treated equally but cultured in RPMI culture media. Cytotoxicity of
compounds 1–3 was tested against both cell lines at 0.5, 1.0, 5.0
and 10 ppm. Tumor cells were seeded in 24-well tissue culture
plates with 5 ꢁ 104 cells/well and 24 h after platting they were
supplemented by triplicate with each compound. All dilutions
were prepared with fresh culture media and plates were incubated
for additional 24 h. Cells in plates were washed with PBS pH = 7.4
to remove death cells. Surviving cells were measured by the MTS
method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium] and related to the mock
cell population by measuring absorbance at 590 nm to establish
cell viability with previous microscopic analysis for morphological
changes exploration in cells.
R = alkyl, aryl, acyl, etc.
Biological
compounds
Cosmetic
chemistry
Scheme 1. N,N0-disubstituted-o-phenylenediamines derivatives on different fields.
R´
Pd(OAc)2 , tBu3P
R
R
R´
R
NaOtBu
toluene, 110 oC
NH
NH
Br
Br
+
2
R
R
2.4. Antibacterial activity
R
NH2
The antibacterial activity of the reported compounds was
proved against S. typhimurium ATCC 14028 and Staphylococcus aur-
eus ATCC 25923. Bacterial strains were tested in a Minimal Inhibi-
tory Concentration assay (MIC) to determine the lowest
concentration obtained by serial dilutions showing to inhibit visi-
ble growth of a microorganism after incubation. Cultures were pre-
pared in TSB media in 96 well plates and immediately after
preparation they were incubated at 37 °C during 24 h. Compounds
1–3 were tested against both bacterial strains from 50 ppm to
0.5 ppm. Once incubation time elapsed plates were read at
540 nm. The minimal concentration for growth inhibition was cal-
culated by reference to each strain positive control.
R´
1: R = CH3, R´ = H
2: R = R´ = CH3
3: R = iPr, R´ = H
Scheme 2. Synthesis of N,N0-aryl-o-phenylenediamines 1–3.
synthetic route [17]. Mass spectra in the EI mode were recorded at
20 eV on a Hewlett-Packrad HP 5989 spectrometer. High resolu-
tion mass spectra were obtained by LC/MSD TOF on an Agilent
Technologies instrument with APCI as ionization source. The
C6D6 and CDCl3 were used without further purification. Melting
points were obtained on a Mel-Temp II apparatus. Assignment of
1H and 13C data was performed using 2D 1H/13C HMBC and
1H/13C HSQC experiments (Supplementary data). Spectra were re-
corded by using Bruker 300: 1H (300.13 MHz), 13C (75.47 MHz);
Bruker 400: 1H (399.78 MHz), 13C (100.52 MHz) and JEOL 400: 1H
(400.13 MHz), 13C (100.61 MHz). IR spectra were obtained with
FT-IR 1600 Perkin Elmer.
2.5. Chemical synthesis of 1–3
2.5.1. Synthesis of compound (1) N,N0-2,6-dimethylphenyl-o-
phenylenediamine
A Schlenk flask of 500 mL was charged with Pd(OAc)2 (0.22 g,
0.98 mmol) and solid tBu3P (0.61 g, 3.27 mmol) in toluene
(50 mL), then it was stirred until the Pd(OAc)2 was dissolved.
Dibromobenzene (5.9 g, 25 mmol) was added followed by 2,6-di-
methyl aniline (9.1 g, 75 mmol) and NaOtBu (7.2 g, 75 mmol).
The reaction mixture was heated to 110 °C for 14 h. After several
hours a precipitate was formed. The flask was opened and the reac-
tion mixture was quickly quenched with an aqueous 25 mL NH4Cl.
The toluene layer was separated and washed 2 ꢁ 80 mL water. The
toluene layer was dried over MgSO4 and it was flashed through sil-
ica gel (hexane/ethyl acetate (8:2)) to yield a dark green solution
then concentrated until crystals began to form. The crystals were
isolated by filtration and dried under vacuum; the crystals were
washed with cold methanol. Yield: 7.9 g 87%. m. p. 144–146 °C.
1H NMR (300.13 MHz, CDCl3): d = 7.18 (d, 3J = 7 Hz, 4H, H-2 and
H-4), 7.09 (t, 3J = 7 Hz, 2H, H-3), 6.73 (dd, 3J = 6, 4J = 3.3 Hz, 2H,
H-10), 6.36 (dd, 3J = 6, 4J = 3.3 Hz, 2H, H-9), 5.25 (s, 2H, H-7), 2.28
(s, 12H, H-11). 13C NMR (75.45 MHz, CDCl3): d = 139.7 (C-6),
135.1 (C-8), 133.4 (C-1 and C-5), 128.8 (C-2 and C-4), 124.6 (C-
3), 120.4 (C-10), 114.7 (C-9), 18.3 (C-11). HSQC correlation [dH/
dC]: 2.28/18.3 [H-11/C-11], 6.36/114.7 [H-9/C-9], 6.73/120.4 [H-
10/C-10], 7.09/128.8 [H-3/C-3], 7.18/133.4 [H-2, H-4/C-2, C-4].
HMBC correlation [dH/dC]: 5.25/135.1 [H-7/C-8], 6.36/120.4/135.1
2.2. X-ray crystallography
Diffraction data were collected at 298 K using a Siemens P4 dif-
fractometer equipped with the Mo K
a radiation (k = 0.71073 Å),
using standard procedure [18], and the structures refined with
SHELXL [19]. Amine H atoms were found in difference maps and
refined freely, with fixed isotropic displacement parameters.
C-bonded H atoms were placed in idealized position for ordered
crystals 1 and 3b, and refined as riding to their carrier atoms. For
2, aromatic H atoms were calculated, while methyl H atoms were
refined freely with H sites splitted over two disordered equally
occupied positions. Methyl geometry was however regularized
with suitable soft restraints for C–H and Hꢀ ꢀ ꢀH distances (see
archived CIF). For 3a, two of four isopropyl groups are disordered.
Site occupation factors for disordered C atoms were refined and
C–C bond lengths involved in disordered parts were restrained
to 1.540(15) Å. All C-bonded H atoms were placed in idealized
positions and refined as riding atoms.

170
V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
[H-9/C-10/C-8], 6.73/114.7/135.1 [H-10/C-9/C-8], 7.18/133.4/139.7
[H-3/C1 and C-5/C-6], 7.18/128.8/139.7 [H-3/C2 and C-4/C-6]. MS
(TOF): m/z [M+H+] calcd. for C22H25N2: 317.2012 a.m.u.; found:
317.2017 a.m.u. (error = 1.4960 ppm). MS (DIP 20 eV): m/z (%):
phatic groups respectively (Table 1). In the 1H NMR spectrum of
compound 3 there are three interesting features. Firstly, the H-9
protons are shifted to lower frequency than the o-phenylenedi-
amine might be explained on the basis of the proximity to p-elec-
316 (100) [M]+, 194 (20). IR (KBr)
m
(cmꢂ1): 3356 (wk and shp),
tron cloud of bulky aromatic ring. Secondly, septet of isopropyl
methines (d = 3.25 ppm) are shifted to high frequencies due to
strong hydrogen bound with the lone pairs on the nitrogen atoms
as is confirmed by X-ray diffraction, with a distance C–Hꢀ ꢀ ꢀN
2.401(3) Å (vide infra). Finally, compounds 3 shown two doublets
with the same intensity at d = 1.25 (d, 3J = 7 Hz, 12H) and 1.19 (d,
3J = 7 Hz, 12H) of methyl groups probably due to rapid exchange
of trans and cis-conformations in solution.
3337 (wk and shp), 2941 (wk), 2911 (wk), 1587 (shp), 1471 (shp
and str), 1397 (shp), 1217 (shp and str), 769 (shp and str), 747
(shp and str).
2.5.2. Synthesis of compound (2) N,N0-2,4,6-trimethylphenyl-o-
phenylenediamine
The procedure was similar to the synthesis phenylendiamine 1.
Pd(OAc)2 (0.264 g, 1.18 mmol); tBu3P (0.71 g, 3.52 mmol); 1,2-di-
bromo-benzene (6.9 g, 29.4 mmol); 2,4,6-trimethylaniline (7.9 g,
58.6 mmol); NaOtBu (8.46 g, 87.9 mmol). The reaction mixture
was heated to 110 °C for 14 h. After several hours a precipitate
was formed. The flask was opened and the reaction mixture was
quickly quenched with an aqueous 25 mL NH4Cl. The toluene layer
was separated and washed 2 ꢁ 80 mL water. The toluene layer was
dried over MgSO4 and it was flashed through silica gel (hexane/
ethyl acetate (8:2)) to yield a dark green solution then concen-
trated until crystals began to form. The crystals were isolated by
filtration and dried under vacuum; the crystals were washed with
cold methanol. Yield: 7.1 g, 71%. m. p. 219 °C. 1H NMR
(399.78 MHz, C6D6): d = 6.89 (s, 4H, H-2 and H-4), 6.70 (dd,
3J = 5.6, 4J = 3.6 Hz, 2H, H-10), 6.46 (dd, 3J = 6, 4J = 3.3 Hz, 2H, H-
9), 4.78 (s, 2H, H-7), 2.25 (s, 6H, p-CH3), 2.15 (s, 12H, o-CH3). 13C
NMR (100.52 MHz, C6D6): d = 137.26 (C-6), 135.43 (C-8), 134.04
(C-3), 133.76 (C-1 and C5), 129.58 (C-2 and C-4), 120.42 (C-10),
114.12 (C-9), 20.81 (p-CH3), 17.96 (o-CH3). MS (TOF): m/z [M+H+]
calcd for C24H29N2: 345.500 a.m.u.; found: 345.2323 a.m.u. IR
Assignment of 13C data of compounds 1–3 was based on HSQC
experiments (Table 2). In order to carry out an unambiguously
assignment of quaternary carbon atom C-8 in compound 1, the
2D Heteronuclear Multiple Bond Correlation (HMBC) experiment
was performed. The HMBC spectrum exhibit two correlations of
H-9 with C-8 and C-10. Also, the experiment indicated correlation
between N–H protons (d = 5.35 ppm) and
a resonance at
135.1 ppm of C-8 (see Supplementary data).
Comparison of the IR stretching vibration of the N–H in 1–3 (1:
3356, 2: 3331 and 3: 3352 cmꢂ1) and the 2,6-diphenylaniline [20],
clearly indicate the presence of an intramolecular hydrogen bridge
towards the nitrogen atoms. The high resolution mass analyses
show the molecular ions of compounds 1 [M]+ 317.2012 (100), 2
[M]+ 345.2323 (100), and 3 [M]+ 429.3006 (100) as peak base. In
both cases, the first loss observed corresponds to the bulky groups
bonded to the nitrogen atom, indicating that the phenylenedi-
amine is very stable.
(KBr)
m
(cmꢂ1): 3331 (wk and shp), 2914 (w), 1597 (w), 1496
Table 1
1H NMR data of 1–3.
(str), 1482 (str), 1398 (w), 1256 (shp), 1227 (shp), 739 (shp and
str).
10
2.5.3. Synthesis of compound (3) N,N0-2,6-diisopropylphenyl-o-
phenylenediamine
9
8
The procedure was similar to the synthesis phenylendiamine 1.
Pd(OAc)2 (0.25 g, 1.12 mmol); tBu3P (0.68 g, 3.37 mmol); 1,2-di-
bromo-benzene (6.6 g, 28 mmol); 2,6-diisopropylaniline (9.90 g,
56 mmol); NaOtBu (8.06 g, 84 mmol). The toluene was removed
under vacuum to yield a dark solid. Methanol was added and the
mixture sonicated for 30 min, to yield an off-white solid that was
isolated by filtration, the solid was washed with cold methanol
(2 ꢁ 10 mL) and dried under vacuum to produce an off white solid.
Yield: 7.2 g, 60%. m. p. 114–116 °C. 1H NMR (400.13 MHz, CDCl3):
d = 7.28–7.24 (m, 6H, H-2-H-4), 6.67–6.65 (dd, 3J = 6, 4J = 3.6 Hz,
2H, H-10), 6.32–6.29 (dd, 3J = 6, 4J = 3.6 Hz, 2H, H-9), 5.24 (s, 2H,
H-7), 3.25 (sept, 3J = 7 Hz, 4H, H-11), 1.25 (d, 3J = 7 Hz, 12H, H-
12), 1.19 (d, 3J = 7 Hz, 12H, H-13). 13C NMR (100.61 MHz, CDCl3):
d = 145.5 (C-8), 137.1 (C-6), 136.9 (C-1 and C5), 126.3 (C-3), 124
(C-2 and C-4), 119.5 (C-10), 114.7 (C-9), 28 (C-11), 24.0 (C-12),
23.4 (C-13). MS (TOF): m/z [M+H+] calcd. for C30H41N2: 429.3264
a.m.u.; found: 429.3261 a.m.u. (error = ꢂ0.7595 ppm). MS (DIP
20 eV): m/z (%): 428 (100) [M]+, 342 (10) [M-C6H14]+, 250 (25),
R
R
5
4
6
NH
HN
3
7
R´
´R
1
R
11
R
2
1 (CDCl3)
2 (C6D6)
3 (CDCl3)
H-2 and H-4
H-3
7.18
7.09
5.25
6.36
6.74
2.28
6.89
7.27
H-7
H-9
H-10
H-11
4.78
6.46
6.70
2.15
2.25
5.24
6.32–6.29
6.67–6.65
(CH(CH3)2) 3.25
(CH(CH3)2) 1.25, 1.19
H-12
Table 2
13C NMR data of 1–3.
208 (15), 177 (7). IR (KBr)
m
(cmꢂ1): 3352 (wk), 2963 (str), 2926
(wk), 2868 (wk), 1602 (wk), 1500 (wk), 1441(shp), 1263 (br and
1 (CDCl3)
2 (C6D6)
3 (CDCl3)
str), 741 (shp and str).
C-1 and C-5
C-2 and C-4
C-3
C6
C-8
C-9
C-10
C-11
C-12
133.4
128.8
124.6
139.7
135.1
114.7
120.4
18.3
133.7
129.5
134.0
137.2
135.4
114.1
120.4
17.96
20.81
136.9
124.0
126.3
137.1
145.5
114.77
119.5
24.0
3. Results and discussion
3.1. Spectroscopic analyses
The 1H NMR (at 25 °C) spectra of 1–3 exhibit resonances in the
range from d = 7.28 to 6.29 and 3.25 to 1.19 for aromatic and ali-
23.4

V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
171
Fig. 1. Molecular structures of bulky N,N0-phenylenediamines derivatives 1–3.
Table 3
Crystal data and structure refinement for compounds 1–3a,b.
1
2
3a
3b
CCDC
803473
22H24N2
316.43
803474
C24H28N2
344.48
803475
C30H40N2
428.64
803476
C30H40N2
428.64
300(1)
0.71073
0.50 ꢁ 0.30 ꢁ 0.20
Monoclinic
P21/c
Empirical formula
Formula weight
Temperature, K
Wavelength
C
300(1)
299(1)
299(1)
0.71073
0.6 ꢁ 0.4 ꢁ 0.4
Monoclinic
P21/c
0.71073
0.6 ꢁ 0.2 ꢁ 0.2
Triclinic
P-1
0.71073
0.4 ꢁ 0.4 ꢁ 0.2
Monoclinic
P21/c
Cryst size, mm3
Crystal system
Space group
Cell parameters
a, Å
b, Å
c, Å
a
12.783(3)
8.888(2)
15.811(9)
90
96.23(4)
90
8.434(7)
8.850(5)
13.972(7)
85.96(2)
88.21(8)
73.00(6)
994.7(11)
12.489(2)
12.737(4)
17.079(5)
90
99.34(2)
90
13.512(4)
22.371(7)
8.693(3)
90
96.62(3)
90
b
c
0
1785.8(12)
2680.8(12)
2610.1(14)
V, ÅA3
Z, Z0
4,1
1.177
0.069
2, 1
1.150
0.067
4, 1
1.062
0.061
4, 1
1.091
0.063
q
l
calc, g cmꢂ3
, mmꢂ1
Data collection
2h range for data
collection
5.18 – 50.00°
4.82 – 50.10°
4 – 50.4°
3.54 – 50.08°
Index ranges
ꢂ15 6 h 6 5, ꢂ10 6 k 6 1,
ꢂ10 6 h 6 9, ꢂ10 6 k 6 10,
ꢂ14 6 h 6 9, ꢂ15 6 k 6 15,
ꢂ16 6 h 6 16, ꢂ26 6 k 6 1,
ꢂ18 6 l 6 18
ꢂ16 6 l 6 16
ꢂ20 6 l 6 20
ꢂ10 6 l 6 4
No. of reflns collected
No. of indep reflns
5260
3125
11,199
3516
12,743
4719
7762
4604
Refinement
[Rint
Goodness of fit
R1, wR2 (I > 2
]
6.45%
2.429
7.78%, 21.61%
11.32%, 23.95%
0.208, ꢂ0.352
7.54%
1.658
6.02%, 15.12%
7.50%, 16.63%
0.242, ꢂ0.131
5.89%
1.750
6.61%, 16.43%
12.33%, 19.93%
0.177, ꢂ0.192
4.37
1.591
5.97%, 14.69%
10.96%, 17.49%
0.179, ꢂ0.215
r(I))
R1, wR2 (all data)
Largets diff peak/hole,
0
e ÅAꢂ3
3.2. X-ray analysis
The molecular conformation in the series is characterized by an
arrangement of phenylamine groups nearly orthogonal to the cen-
tral benzene ring, regardless of the nature of substituents in exter-
nal phenyl rings. In 1, the dihedral angles between the central
benzene ring C8ꢀ ꢀ ꢀC13 and phenyl groups C1ꢀ ꢀ ꢀC6 and C15ꢀ ꢀ ꢀC20
(see Fig 2 for numbering scheme) are 82.49(10)° and 86.44(11)°.
Similar angles are found in 2, 3a and 3b, in the range 78.74(7)–
86.24(7)°. As a consequence, peripheral phenyl rings are expected
to lie in nearly parallel planes. Compounds 1 and 2 match this fea-
ture, as reflected by dihedral angles formed by phenyl planes,
4.1(2)° and 2.06(13)°, respectively. However, some deviation is
o-Phenylenediamines 1, 2 and 3 are built up on a common 1,2-
diaminobenzene core, with nitrogen atoms substituted by 2,6-
dimethylphenyl (1), mesityl (2) or 2,6-diisopropylphenyl (3) groups
(Fig. 1). Although they potentially display a twofold symmetry axis
(C2v maximum point group) and crystallize in Laue group 2/m or ꢂ1,
in all cases the asymmetric unit contains a single molecule lying in
general position. In the case of 3, a dimorphism is observed at room
temperature, in a single space group. Each form, 3a and 3b, has been
characterized by X-ray diffraction (Tables 3–5).

172
V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
Table 4
Selected bond distances (°) and angles (°) for 1–3a and b.
1
2
3a
3b
N(14)–H(14)
C(15)–N(14)
N(14)–C(13)
C(8)–N(7)
0.93(4)
0.88(2)
0.77(3)
0.84(3)
1.419(4)
1.411(4)
1.393(4)
0.88(4)
1.429(2)
1.403(2)
1.412(2)
0.92(2)
1.438(3)
1.409(3)
1.405(3)
0.87(3)
1.431(3)
1.409(3)
1.402(3)
0.83(3)
N(7)–H(7)
N(7)–C(6)
1.424(3)
1.431(2)
1.427(3)
1.433(3)
C(15)–N(14)–H(14)
H(14)–N(14)–C(13)
C(8)–N(14)–C(13)
C(13)–C(8)–N(7)
112(2)
119(2)
108(2)
117.7(3)
117.8(14)
108.9(14)
108.9(13)
118.13(14)
116(3)
112(2)
112.9(9)
116.66(19)
110(2)
120(2)
110(2)
117.56(19)
C(15)–N(14)–C(13)–C(12)
N(14)–C(13)–C(8)–N(7)
23.6(4)
ꢂ0.4(4)
ꢂ3.0(3)
15.0(4)
0.2(3)
9.0(3)
ꢂ0.2(3)
0.5(2)
Table 5
Hydrogen bonds.
1
2
3a
3b
N7ꢀ ꢀ ꢀH14
N14ꢀ ꢀ ꢀH7
2.282
2.765
2.290
2.424
2.372
2.560
2.319
2.522
observed for the most bulky system, with angles as large as
13.45(13)° for form 3a and 27.89(10)° for form 3b. It seems that
these molecules take advantage of both a degree of tetrahedral dis-
tortion for amine N atoms and restricted rotation of phenyl rings,
to minimize steric hindering. Such arrangement was previously
observed in related 1,2,4,5-tetrakis(amino)benzene derivatives
[21], as well as in organometallics bearing N-heterocyclic carbene
derivatives [22,23] or diimine ligands [24]. The non-parallel
arrangement typical of 2,6-diisopropylphenylamine derivatives is,
for example, patent in X-ray structures reported for boryllithium
salts [25].
It is rather obvious that steric restrictions induced by phenyl
substituents determinate the relative orientation for aromatic
rings in these systems. In contrast, non-restricted diamine deriva-
tives may adopt a non-orthogonal conformation, in order to favor
Fig. 2. ORTEP view of 1 with displacement ellipsoids for non-H atoms at the 25%
probability level. The inset (right) is a spacefill representation of the same molecule,
showing the trans configuration of amine NH groups.
intermolecular stabilizing interactions, like
p
ꢀ ꢀ ꢀ
p
or C–Hꢀ ꢀ ꢀ
p con-
tacts involving phenyl groups. An example of such a behavior has
been described for 4,5-dianilinophthalamide [26], which is stabi-
lized in the solid state with an asymmetric propeller-shaped
conformation.
An important geometric feature related to the reactivity of com-
pounds 1–3 is the relative orientation of lone pairs on tetrahedral
N atoms. In the present study, the positions of amine H atoms H7
and H14 were determined from X-ray data, and refined freely. De-
spite of the limited resolution of diffraction patterns, N–H groups
converged to a sensible geometry, similar along the series of stud-
ied bisamines. N–H bond lengths vary from 0.77(3) to 0.93(4) Å,
and N atoms are clearly in a sp3 hybridization state, with the larg-
est bond angles being systematically C–N–C, ca. 120°, due to the
steric repulsion between aromatic system substituting N atoms.
Lone pairs are placed trans with respect to the central benzene
ring, as reflected by the positions of H atoms, which point toward
Fig. 3. Best fit between the four characterized bisamines. The overlay has been
carried out using 1 as base, and by fitting aromatic C atoms and N atoms (20 atoms
for each molecule) in 2, 3a and 3b. C-bonded H atoms have been omitted in the
figure for clarity. Color code: red: 1, blue: 2, gray, 3a, magenta: 3b. (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
the
a and b faces of the benzene plane (Fig. 2, inset). This arrange-
ment, invariably found in secondary 1,2-diaminobenzene deriva-
tives [21,22,26], avoids Hꢀ ꢀ ꢀH short contacts. In 1–3, amine Hꢀ ꢀ ꢀH
separations are however close to the sum of van der Waals radii
or even shorter: the shortest Hꢀ ꢀ ꢀH separation is 1.96 Å for 2 and
the longest 2.34 Å for 1. The barrier to N inversion in such diamines
should be high, and N inversion is thus very unlikely to occur in
solution at room temperature.
The four structures here characterized display common geo-
metric features, and have similar conformations in the solid state.
An overlay [27] carried out on non-H atoms and excluding substit-
uents in peripheral phenyl rings shows that all the molecules in-
deed fit a mean conformation (Fig. 3): using geometry of 1 as a
reference, root mean square deviations for fitted molecules 2, 3a
and 3b are 0.31, 0.17 and 0.35 Å, respectively.

V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
173
Table 6
Cytotoxicity assay on A431 and MOLT4 cells.
Compound
Activitya on
A431 cells
Morphological Changes in cells
Activitya on
MOLT4 cells
Morphological Changes in cells
(
lg/mL)
(lg/mL)
1
2
3
1.0
0.5
5.0
Nuclei swelling and cytoplasm disturbance
Diffuse nuclei swelling and cytoplasm disturbance
Minor cytoplasm disturbances
0.5
0.5
0.5
Only cell debris were observed
Only cell debris were observed
Only cell debris were observed
a
Minimal concentration showing toxic effects on both cell lines.
4. Conclusions
Table 7
Minimal inhibitory concentration for bacteria.
In summary, we report the characterization and molecular
structures of three N,N0-diaryl-o-phenylenediamine derivatives. In
all cases the arrangement of phenylamine groups nearly orthogo-
nal to the central benzene ring. In the case of the more sterically
demanding phenylenediamine (3) showed dimorphism. A decrease
of steric hindrance on o-phenylendiamines might influence on bio-
logical activity.
Compound
Bacterial strain ATCC (lg/mL)
Salmonella typhimurium 14028
Staphylococus aureus 25923
1
2
3
0.35
0.35
0.75
0.35
0.35
0.35
Finally, this geometry dictated mainly by steric factors, is unfa-
vorable for efficient packing in the crystals. Instead, molecules are
well separated, and no significant intermolecular interactions are
available for crystal stabilization. The packing index [28] computed
for 1, 2 and 3b (non-disordered crystals) is consistent with poorly
packed molecules: 0.669 (1), 0.699 (2) and 0.646 (3b). This charac-
teristic also explains why these compounds afford poorly diffract-
ing samples, and why methyl groups have disordered H atoms
positions, due to a degree of free rotation about their C–C bonds.
In the present case, this disorder has been modeled for all methyl
groups in 2, although it is also probably present in 1 and 3. The
dimorphism observed in 3 may also be rationalized on the basis
of non-flexible, poorly packed molecules. Dynamic motions for iso-
propyl substituents allow to stabilize [29] disordered (3a) and non-
disordered (3b) concomitant crystals, which display different crys-
tal structures.
Acknowledgements
This work was supported by CONACYT (Grant: 82605) and PAI-
CYT-UANL (IT-160-09). M. I.-R. thanks for scholarship from
CONACYT.
Appendix A. Supplementary material
High resolution mass spectrometry and spectroscopic data for
compounds 1–3. X-ray crystallographic data in the CIF format of
the reported structures has been deposited with the Cambridge
Crystallographic Data Centre. CCDC reference numbers
803473(1), 803474(2), 803475(3a) and 803476(3b). Copies of the
data can be obtained free of charge on application to CCDC, 12 Un-
ion Road, Cambridge CB21EZ, UK (fax: +44 1223 336 033; e-mail:
deposit@ccdc.cam.ac.uk). Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.molstruc.2011.08.040.
3.3. Biological activity
The in vitro cytotoxic assay showed that compounds 1–3 have a
toxic effect against A431 cancer cells and MOLT4 leukemic cells.
The resume of cytotoxic activities of all compounds is given in Ta-
References
[1] C.N. Raut, S.M. Bagul, R.A. Janrao, S.D. Vaidya, V.B.S. Kumar, P.P. Mahulikar, J.
Heterocycl. Chem. 47 (2010) 582.
[2] X.H. Zeng, M. Liu, M.W. Ding, H.W. He, Synth. Commun. 40 (2010) 1453.
[3] A.V. Zabula, F.E. Hahn, Eur. J. Inorg. Chem. (2008) 5165.
[4] Y. -B Huang, G.-R. Tang, G.-Y. Jin, G.-X. Jin, Organometallics 27 (2008) 259.
[5] G.J. Niemann, J. Dekker, Neth. J. Plant Pathol. 72 (1966) 213.
[6] I. Bouabdallah, L.A. MBarek, A. Zyad, A. Ramdani, I. Zidane, A. Melhaoui, Nat.
Prod. Res. 20 (2006) 1024.
[7] E.E. Obaseiki-Ebor, K. Odukoya, H. Telikepalli, L.A. Mitscher, D.M. Shankel,
Mutat. Res. 302 (1993) 109.
[8] R.H. Krüger, C. Boissiére, K. Klein-Hartwig, H.J. Kretzschmar, Food Addit.
Contam. 22 (2005) 968.
[9] [a] I.J. Kang, M.H. Lee, Contact Dermatitis 55 (2006) 26;
[b] F. Hueber-Becker, G.J. Nohynek, W.J. Meuling, F. Benech-Kieffer, H. Toutain,
Food Chem. Toxicol. 42 (2004) 1227.
ble 6. On A431 cells, compound 1 started to be active at 1.0 lg/mL.
Some morphological changes and nuclei swelling and cytoplasm
disturbance were observed. Similar results were found with com-
pound 2 where a more diffuse nuclei swelling and cytoplasm dis-
turbances were evident and clear. It appears to be more active
´
starting to be cytotoxic at 0.5
of compound 3 is lower compared to the former two compounds
(5.0 g/mL) regardless of the slight effects on cell morphology.
lg/mL. In contrast, cytotoxic activity
l
Similar results were obtained for the three compounds when
tested on MOLT4 cells, but in this cell line each compound showed
activity at 0.5 lg/mL.
The synthesized compounds where screened by a turbidimetric
method and all of them showed antibacterial activity. The corre-
sponding MIC values for compounds 1 and 2 against S. typhimurium
and S. aureus were established at 0.35 lg/mL. Compound 3 also
presented activity against both bacterial strains but with different
[10] H.P. Sachin, M.H. Moinuddin Khan, N.S. Bhujangaiah, Int. J. Electrochem. Sci. 4
(2009) 134.
[11] M.N. Friedemann, S.W. Robinson, G.A. Gerhardt, Anal. Chem. 68 (1996) 2621.
[12] H. Yu, M. Foreman, J. Choi, S. Wang, Food Chem. Toxicol. 46 (2008) 3780.
[13] J.F. Hartwig, Angew. Chem. Int. Ed. 37 (1998) 2046.
[14] J.P. Wolfe, S. Wagaw, J.-F. Marcoux, S.L. Buchwald, Acc. Chem. Res. 31 (1998)
805.
[15] T. Wenderski, K.M. Light, D. Ogrin, S.G. Bott, C.J. Harlan, Tetrahedron Lett. 45
(2004) 6851.
[16] D.F. Shriver, M.A. Drezdon, The Manipulation of Air-Sensitive Compounds, 2nd
ed., Wiley, New York, 1986.
[17] J.V. Dickschat, S. Urban, T. Pape, F. Glorius, F.E. Hahn, Dalton Trans. 39 (2010)
11519.
[18] XSCANS (Version 2.31). Siemens Analytical X-ray Instruments Inc., Madison,
Wisconsin, USA, 1996.
MIC values. For S. typhimurium the growth inhibition was reached
at 0.75 lg/mL meanwhile S. aureus was inhibited at 0.35 lg/mL
(Table 7). Based on these cytotoxic and antibacterial results, it
seems that the steric effect of N-aryl groups on o-phenylendiam-
ines is a significant factor in cell line A431 and against S. typhimu-
rium. However, all compounds presented very similar activities in
cell line MOLT4 (1–3: 0.5
lg/mL) and S. aureus (1–3: 0.5
l
g/mL).
[19] G.M. Sheldrick, Acta Cryst. A 64 (2008) 112.

174
V.M. Jiménez-Pérez et al. / Journal of Molecular Structure 1031 (2013) 168–174
[20] M. Tanabiki, K. Tsuchiya, Y. Kumanomido, K. Matsubara, Y. Motoyama, H.
Nagashima, Organometallics 23 (2004) 3976.
[21] D.M. Khramov, A.J. Boydston, C.W. Bielawski, Org. Lett. 8 (2006) 1831.
[22] H. Sakurai, K. Sugitani, T. Moriuchi, T. Hirao, J. Organomet. Chem. 690 (2005)
1750.
[26] U. Trinks, E. Buchdunger, P. Furet, W. Kump, H. Mett, T. Meyer, M. Müller, U.
Regenass, G. Rihs, N. Lydon, P. Traxler, J. Med. Chem. 37 (1994) 1015.
[27] C.F. Macrae, I.J. Bruno, A.J. Chisholm, P.R. Edgington, P. McCabe, E. Pidcock, L.
Rodríguez-Monge, R. Taylor, J. van de Streek, P.A. Wood, J. Appl. Cryst. 41
(2008) 466.
[23] S. Urban, M. Tursky, R. Fröhlich, F. Glorius, Dalton Trans. (2009) 6934.
[24] Y.B. Huang, G.R. Tang, G.Y. Jin, G.X. Jin, Organometallics 27 (2008) 259.
[25] Y. Segawa, Y. Suzuki, M. Yamashita, K. Nozaki, J. Am. Chem. Soc. 130 (2008)
16069.
[28] A.L. Spek, J. Appl. Cryst. 36 (2003) 7.
[29] S.L. Price, Acc. Chem. Res. 42 (2009) 117.