Synthesis of the Three Mixed-ligand Metal Complexes and One Organic Salt of 3,5-Dinitrobenzoic Acid for Biopharmaceutical Optimization Through Monoethanolamine: Structures and DFT Studies of Complexes

Article abstract of DOI:10.1007/s10870-020-00864-z

Abstract: The simplest derivatives of benzoic acid show a wide spectrum of the biological action mostly being poor water soluble. In order to demonstrate on example of 3,5-dinitrobenzoic acid (DNBA) a possibility of the simultaneous enhancement of water solubility and antimicrobial activity the complexation (salt formation and metal coordination) approach has been examined. Monoethanolamine (MEA) is selected as the complexing agent. Three isomorphic mixed-ligand complexes of Cu, Ni, Co with DNBA and MEA as well one organic salt in the polymorphic form have been synthesized and their structures are characterized by the element analysis, FTIR and X-ray crystallography. Measurements of water solubility and determination of the antimicrobial activity attest that a solubility of the copper mixed-ligand complex and organic salt is increased about four and eight times with simultaneous enhancement of the bioactivity. The DFT studies by B3LYP/def2-TZVP and B3LYP/6-311G (d,p) showed that Cu, Ni, and Co complexes with DNBA and MEA have high-spin ground electronic states as a doublet, triplet and quartet, respectively. Calculated bond lengths and bond angles of all metal complexes in higher-spin ground electronic states are in good agreement (about R² = 0.97) with X-ray data. Additionally, the energies of the frontier MOs and frontier electron densities and also electrostatic potential of isostructural complexes have been analyzed by the DFT/6-311G(d,p) method. Graphic Abstract: The water solubility and bioactivity of poorly soluble 3,5-dinitrobenzoic acid are enhanced by a preparation of three isostructural metal complexes and one polymorphic organic salt with participation of monoethanolamine and DFT study of metal complexes has been carried out.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s10870-020-00864-z

Journal of Chemical Crystallography
https://doi.org/10.1007/s10870-020-00864-z
ORIGINAL PAPER
Synthesis of the Three Mixed‑ligand Metal Complexes and One
Organic Salt of 3,5‑Dinitrobenzoic Acid for Biopharmaceutical
Optimization Through Monoethanolamine: Structures and DFT
Studies of Complexes
Avazbek B. Ibragimov1 · Jamshid M. Ashurov1 · Aziz B. Ibragimov2 · Alisher G. Eshimbetov3
Received: 27 May 2020 / Accepted: 26 September 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The simplest derivatives of benzoic acid show a wide spectrum of the biological action mostly being poor water soluble. In
order to demonstrate on example of 3,5-dinitrobenzoic acid (DNBA) a possibility of the simultaneous enhancement of water
solubility and antimicrobial activity the complexation (salt formation and metal coordination) approach has been examined.
Monoethanolamine (MEA) is selected as the complexing agent. Three isomorphic mixed-ligand complexes of Cu, Ni, Co
with DNBA and MEA as well one organic salt in the polymorphic form have been synthesized and their structures are
characterized by the element analysis, FTIR and X-ray crystallography. Measurements of water solubility and determina-
tion of the antimicrobial activity attest that a solubility of the copper mixed-ligand complex and organic salt is increased
about four and eight times with simultaneous enhancement of the bioactivity. The DFT studies by B3LYP/def2-TZVP and
B3LYP/6-311G (d,p) showed that Cu, Ni, and Co complexes with DNBA and MEA have high-spin ground electronic states
as a doublet, triplet and quartet, respectively. Calculated bond lengths and bond angles of all metal complexes in higher-spin
ground electronic states are in good agreement (about R2 =0.97) with X-ray data. Additionally, the energies of the frontier
MOs and frontier electron densities and also electrostatic potential of isostructural complexes have been analyzed by the
DFT/6-311G(d,p) method.
Graphic Abstract
The water solubility and bioactivity of poorly soluble 3,5-dinitrobenzoic acid are enhanced by a preparation of three iso-
structural metal complexes and one polymorphic organic salt with participation of monoethanolamine and DFT study of
metal complexes has been carried out.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10870-020-00864-z) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Journal of Chemical Crystallography
Keywords Metal complex · Organic salt · X-ray structure · H-bond · Polymorphism · Water solubility · Bioactivity · DFT
study
of complexes were recorded using “IRTracer-100 Fourier
Transform Infrared Spectrophotometer” (Shimadzu, Japan)
with an ATR attachment. UV–Vis spectra (in the region of
200–400 nm) of the complexes 1–4 were measured on a
Shimadzu 1280 spectrometer (Japan) in an ethanol solution
using a quartz cuvette 1 cm thick.
Introduction
The simplest monoderivative of benzoic acid, 4-nitroben-
zoic acid, demonstrates a very low antimicrobial activity
against bacteria and yeasts, e.g., against flamentous fungi
M. gypseum with minimal inhibitory concentration (MIC)
of 2 mmol·l–1 [1, 2]. Biological activity of benzoic acid
binitroderivatives, for instance, 3,5-dinitrobenzoic acid
(DNBA) is even less and its MIC is equal to 3 mmol·l–1
against the same fungi [2].
Synthesis of [Cu(DNBA)2(MEA)2] (1)
To an aqueous solution (2.5 ml) of CuSO4·5H2O (0.125 g,
0.5 mmol) was slowly added an ethanol solution (4 ml) con-
taining MEA (62 μl) and DNBA (0.212 g, 1 mmol) with
constant stirring. Light-blue crystals were obtained at room
temperature by solvent evaporation after 5 days. Yield: 70%.
Elemental analysis for C18H20N6O14Cu (607.94): calcd. C
35.56; H 3.32; N 13.82%; found: C 35.50; H 3.28; N 13.77%.
FTIR (ATR), cm-1: 3394, 3242 (OH, NH2), 3120 (C–H,
Ar), 2942, 2885 (CH2), 1616 (C=O), 1587 (Ar), 1539,
1340 (NO2), 1062 (C–O), 704 (C–H out of plane). UV–Vis
(EtOH), λmax, nm: 205, 244.
The water solubility of 4-nitrobenzoic acid is 0.42 mg/ml
while the presence of two nitro groups in DNBA molecule
increases its solubility more than 3 times (1.35 mg/ml) [3],
which is still insufcient for an application of this compound
in practice. However, using of such cheap, commercially
available simple compound as an antimicrobial remedy is
of a great interest. Therefore, it is required to enhance its
biopharmaceutical properties such as water solubility and
biological activity.
In order to optimize biopharmaceutical properties of
the poorly water soluble active pharmaceutical ingredi-
ents (API), a diversity of modern approaches is used [4–6].
Among them an inorganic coordination and salt formation,
which may be considered as a particular case of the compl-
exation approach, are of a special interest. For realization of
this methodology the complexing agent has to be chosen but
it should be basic in nature and demonstrate the same type
of bioactivity. Moreover, complexing agent must coordinate
metal ions, i.e. serve as auxiliary ligand. Monoethanolamine
(MEA) is just such compound with moderate antimicrobial
activity [7, 8] and strong chelating ability [9, 10]. In the pre-
sent paper we demonstrate the simultaneous improvement
of the water solubility and biological activity of DNBA. In
order to compare data obtained in practice and theory DFT-
calculations have been performed for all obtained isostruc-
tural metal complexes.
Synthesis of [Ni(DNBA)2(MEA)2] (2)
To an aqueous solution (1.5 ml) of NiCl2·6H2O (0.119 g,
0.5 mmol) was slowly added an ethanol solution (4 ml)
containing MEA (62 μL) and DNBA (0.212 g, 1 mmol)
with constant stirring. Green color crystals of the product
were obtained by solvent evaporation at room tempera-
ture after one week. Yield: 72%. Elemental analysis for
C18H20N6O14Ni (603.07): calcd. C 35.85; H 3.34; N 13.94%;
found: C 35.80; H 3.27; N 13.87%. FTIR (ATR), cm−1
:
3352, 3286 (OH, NH2), 3089 (C–H, Ar), 2937, 2891 (CH2),
1610 (C=O), 1573 (Ar), 1539, 1342 (NO2), 1066 (C–O), 715
(C–H out of plane). UV–Vis (EtOH), λmax, nm: 205, 246.
Synthesis of [Co(DNBA)2(MEA)2] (3)
To an aqueous solution (1 ml) of Co(NO3)2 (0.091 g,
0.5 mmol) was slowly added an ethanol solution (4 ml)
containing MEA (62 μl) and DNBA (0.212 g, 1 mmol)
with constant stirring. Light-brown crystalline product was
obtained at room temperature by solvent evaporation after
4 days. Yield: 68%. Elemental analysis for C18H20N6O14Co
(603.32): calcd. C 35.83; H 3.34; N 13.93%; found: C 35.77;
H 3.28; N 13.86%. FTIR (ATR), cm−1: 3233 (OH, NH2),
3097 (C–H, Ar), 2893 (CH2), 1614 (C=O), 1575 (Ar), 1533,
1340 (NO2), 1070 (C–O), 711 (C–H out of plane). UV–Vis
(EtOH), λmax, nm: 205, 244.
Experimental
General Methods and Materials
All reagents were readily available from commercial sources
and were used as received without further purifcation. The
crystal data were collected on”Xcalibur-R CCD” (Oxford
difraction, Great Britain) difractometer. Element analysis
were carried out on “EuroVector EA3000 Series of CHNS-O
Elemental Analysers” (Eurovector, EU). The FTIR-spectra
1 3

Journal of Chemical Crystallography
Synthesis of Organic Salt (DNBA−·MEA+) (4)
program [12] integrated to OLEX2 package [13] and
refned by full-matrix least squares using the SHELXL
program [14]. Non-hydrogen atoms were refned aniso-
tropically. All H atoms were placed at idealized positions
[C–H = 0.93 to 0.97 Å, N–H = 0.89 Å and O–H = 0.82 Å]
and allowed to ride on their parent atoms with Uiso fxed
at 1.2 Ueq(C,N).
To an ethanol solution (4 ml) containing DNBA (0.212 g,
1 mmol) and MEA (62 μl) 1 mmol) was slowly added
with constant stirring. Colorless crystalline product was
obtained at 30 °C temperature by solvent evaporation after
6 days. Yield: 85%. Elemental analysis for C9H11N3O7
(273.2)–calcd: C 39.57; H 4.06; N 15.38%; found: C 39.51;
H 4.01; N 15.32%. FTIR (ATR), cm−1: 3369, 3294, 3242
(OH, NH2), 3091 (C–H, Ar), 2868 (CH2), 1618 (C=O),
1583 (Ar), 1537, 1340 (NO2), 1074 (C–O), 719 (C–H out
of plane). UV (EtOH), λmax, nm: 205, 235.
Molecular and crystal structures are plotted using MER-
CURY program package [15]. The main crystallographic
data and refnement details of structures 1–4 are given in
Table 1.
Antimicrobial Tests
X‑ray Structure Analysis
The antimicrobial activity was determined using patho-
genic test microorganisms Fusarium oxysporum because
namely these fungi cause essential damage of Uzbekistan
agricultural plants including cotton. The tests were per-
formed on solid culture media in well plates. DMSO was
used as the control. Inoculated Petri dishes were incubated
for 3–4 days in a thermostat at 30 °C and then the patho-
gen growth inhibition zone was measured in mm [16].
Refection sets were obtained at 293K on an “Xcalibur-
R CCD” automated diffractometer (CuKα–radiation,
λ = 1.54184 Å, ω-scan mode, graphite monochromator).
Experimental data were collected using the CrysAlisPro
program [11]. An absorption correction was applied by
the multi-scan method in the same program. The struc-
tures were solved by a direct method using the SHELXT
Table 1 Main crystallographic data and refnement details for structures 1–4
Data
1
2
3
4
Empirical formula
Formula weight
Crystal system
Space group
a, Å
b, Å
c, Å
C18H20N6O14Cu
607.94
monoclinic
P21/n
10.076(5)
5.520(5)
21.403(6)
90.542(5)
1190.4(13)
2
1.696
2.083
0.54×0.32×0.22
290
C18H20N6O14Ni
603.11
monoclinic
P21/n
10.377(3)
5.3850(15)
21.149(4)
92.127(2)
1181.0(5)
2
1.696
1.979
0.52×0.32×0.20
290
C18H20N6O14Co
603.33
monoclinic
P21/n
10.406(3)
5.3883(15)
21.263(4)
92.41(2)
1191.2(5)
2
1.682
6.435
0.44×0.30×0.18
290
C9H11N3O7
273.21
monoclinic
P21/n
14.660(2)
4.5532(7)
17.424(3)
100.259(14)
1144.5(3)
4
1.586
1.210
0.38×0.22×0.18
290
β, deg
V, Å3
Z
Dx, g cm−3
μ(CuKα), mm−1
Crystal size [mm]
T, K
Θ scan range, deg
Range h,k,l
Total data
4.1–66.9
−11/12, −6/6, −25/25
7450
4.2–67.0
−12/12, −6/6, −17/25
4290
4.7–66.9
−11/12, −5/6, −25/16
4402
3.6–66.9
−17/17, −3/4, −20/18
2719
Independent data
Rint
2115
0.023
2098
0.027
2096
0.053
1749
0.026
Observed data [F2 ≥2σ (F2)]
Refned parameters
Goodness-of-ft (F2)
R1, wR2 (I>2σ (I))
Δρmin/max, eÅ−3
CSD deposition number
1829
180
1.04
0.0318, 0.0916
−0.30/0.33
1,974,484
1558
179
1.02
0.0425, 0.1241
−0.27/0.53
1,974,482
1378
179
1.03
0.0744, 0.1980
−0.34/0.67
2,031,232
882
175
1.08
0.0734, 0.2723
−0.30/0.27
2,031,357
1 3

Journal of Chemical Crystallography
1–3 by Multiwfn 3.7 program package [26] on the basis of
wave function obtained by B3LYP/6-311G(d,p) method.
ESP surface and frontier electron densities of isostructural
complexes visualized by VMD 1.93 [27] and Avogadro [28]
program packages, respectively.
Measurements of Water Solubility
For this purpose, one of the simplest methods has been used
[17]. Several mg of the sample (m1) was thoroughly micro-
nized in an agate mortar for homogenization. The powder
was placed in a 50 ml measuring cylinder, then water was
added to the mark. Solution was treated with ultrasound of
diferent power for 10–15 min. The mixture was then fltered
through flter paper to collect the residue, which was washed
in ethanol. The residue was dried at 60 °C to a constant mass
and weighed again (m2). The solubility (S) was calculated
using the following formula:
Results and Discussion
The Structures of the Mixed‑ligand Complexes
[Cu(DNBA)2(MEA)2] (1), [Ni(DNBA)2(MEA)2] (2)
and [Co(DNBA)2(MEA)2] (3).
S (mg∕ml) = (m1 − m2) × 1000∕50.
(1)
Diferent metal complexes have been obtained from reaction
media containing in ethanol–water solution one of Cu(II)-,
Ni(II)- or Co (II)-salt (M-salt), DNBA and MEA. According
to results of element analysis and FTIR, two diferent ligand
molecules are coordinated to defnite metal ion in each com-
pound. Moreover, FTIR-spectra indicate to the existence of
common features in structures of metal complexes. The
X-ray crystallography data attest (Table 1) that prepared
metal complexes are isostructural with general formula of
[M(DNBA)2(MEA)2] (Fig. 1).
Each measurement was repeated three times and the mean
value was calculated.
DFT Study
DFT studies were carried out by DFT/6-311G(d,p) and DFT/
def2-TZVP methods using ORCA 4.2.0 program package
[18]. The initial geometries of the Ni(II), Cu(II) and Co(II)
complexes were obtained from X-Ray data (cif fles). Con-
formation of the Ni(II) complex was fully optimized by
both methods at the singlet, triplet and quintet electronic
spin states. In the case of Cu(II) and Co(II) complexes, the
geometries were optimized at the doublet, quartet and sextet
electronic spin states. The Hybrid method of Becke [19]
with three parameters and correlation functions of Lee, Yang
and Parr [20] was chosen as a DFT (B3LYP) method. The
B3LYP is the most popular DFT method [21–23] and it is
successfully used for coordination compounds with difer-
ent basis sets, including 6-311G(d,p) [24]. The def2-TZVP
method developed by Ahlirichs group [25] has also been
tested for description of geometrical structures of the com-
plexes. The electrostatic potential computed on surface of
The complex molecules are located on inversion cent-
ers of the crystals with space group P21/n. Metal ions are
chelated through oxygen and nitrogen atoms of two MEA
molecules which are in a neutral state. Two DNBA mol-
ecules are coordinated to the central ion by monodentate
fashion through an oxygen atom of the carboxylic group,
which are in the carboxylate form in order to compensate
positive charge of the M(II)-ion.
The coordination polyhedrons of the M-ions are distorted
octahedrons. This is especially correct for the Cu-complex,
where the O1 atom of the monodentate DNBA anion and
N3 of the bidentate MEA molecule occupy the equatorial
positions of the prolonged octahedron (i.e. bipiramid) and
O7 atom of the MEA is at the apical positions. Such type
Fig. 1 The structure of the com-
plex molecule in the isostruc-
tural mixed-ligand compounds
[M(DNBA)2(MEA)2], where
M=Cu(II), Ni(II) and Co(II) (in
example of the compound 1).
Atoms of the asymmetric part
of the unit cell are numbered.
Non-hydrogen atoms are shown
with 50%-probability level of
thermal ellipsoids. In Cu-, Ni-
and Co-complexes symmetric
halves of the complex molecule
are related by symmetry codes
2-x,-y,1-z; 2-x,-y, 2-z and 1-x,
2-y,1-z, respectively
1 3

Journal of Chemical Crystallography
coordination of the bidentate ligands occurs extremely rare,
because usually both donor atoms of the bidentate ligands
occupy the equatorial positions of a bipiramid.
direction of the b axis. This bond is longer around 0.1 Å
in other two complexes. A sufficiently strong H-bond
C(8)–H(8A)···O(4) with a length of 3.208(4)Å associates
columns into 2-D networks parallel to the (−101) plane.
Such C–H···O type bond of Ni- and Co-complexes again
shorter about 0.1 Å than in compound 1 (Table 3). One more
diference in crystal structures of the three compounds is the
existence of the very weak H-bond C(9)–H(9A)···O(4) with
length 3.512(5)Å in copper complex which is not observed
in other two isomorphs. Probably, these diferences between
crystal structures of the Cu-complex and two other isostruc-
tural compounds are appeared as a result of Jahn–Teller dis-
tortion in the molecular structure of the complex 1. Further
2-D networks are associated into crystal structure by van-
der-Waals interactions (Fig. 2).
The apical Cu–O7 distance (2.433(3) Å) is much longer
than the equatorial distances Cu–O (1.978(2) Å) and Cu–N
(2.004(3) Å). Obviously, this diference is explained by the
Jahn–Teller efect. In the Co and Ni analogs of the isostruc-
tural complexes, the coordination octahedrons of the Co and
Ni atoms are more regular and M–O and M–N bonds have
the similar distances (Table 2).
There is a sufciently strong intramolecular hydrogen
bond with a length of 2.752(3) Å and angle 151.00º in the
Cu-complex between uncoordinated oxygen atom of the car-
boxylate group and coordinated hydroxyl group of MEA.
This H-bond is stronger in Ni- and Co-complexes because
its length and angle are around 2.656(5) Å and 153º. The
crystal structures are stabilized by the H-bond with a graph-
set notation of R1 (6) [29].
In the Cambridge Structural Database (CSD) [30] two
similar copper mixed-ligand complexes on the base of
MEA and benzoic acid derivatives are registered. In the
complex with ref-code YEBPED [31] instead of DNBA
p-nitrobenzoic acid participates while in case of the com-
pound DOHWUT [32] two positions of the coordination
sphere are occupied by 4-hydroxy-3-methoxy benzoic acid.
Thus, a specifc feature of the known hexa-coordinated
mixed-ligand metal complexes on the basis of MEA and the
simplest monoderivatives of benzoic acid is (i) chelate and
monodentate coordination of ligands; (ii) relatively strong
The carboxyla1te group of the DNBA molecule is tilted
relatively to aromatic ring at 8.83(7)º. Dihedral angle
between the frst nitro-group and aromatic ring is 6.77(7)º,
while the second nitro-group is nearly coplanar with benzene
ring at angle 1.76(7)º.
The relatively a weak H-bond N(3)–H(3A)···O(7) of
the Cu-complex with a length of 2.997(4) Å and angle of
164º incorporates complex molecules into columns in the
Table 2 Bond lengths and angles of metal ions in complexes 1 -3
Bond lengths, Å
Bond
d
Bond
d
Bond
d
1
Cu(1)–O(1)
2
Ni(1)–O(1)
3
Co(1)–O(1)
1.978(2)
2.058(2)
2.069(3)
Cu(1)–O(7)
Ni(1)–O(7)
Co(1)–O(7)
2.433(3)
2.096(2)
2.141(4)
Cu(1)–N(3)
Ni(1)–N(3)
Co(1)–N(3)
2.004(3)
2.076(3)
2.124(5)
Bond angles, º
Angle
ω
Angle
ω
1
O(1)–Cu(1)–O(7)
O(1)–Cu(1)–N(3)
O(7)–Cu(1) –N(3)
2
93.62(6)
89.38(7)
79.97(8)
O(1)–Cu(1)–O(7A)
O(1)–Cu(1)–N(3A)
O(7) –Cu(1) –N(3A)
86.38(6)
90.62(7)
100.03(8)
O(1)–Ni(1)–O(7)
O(1)–Ni(1)–N(3)
O(7) –Ni(1) –N(3)
3
O(1)–Co(1)–O(7)
O(1)–Co(1)–N(3)
O(7) –Co(1) –N(3)
87.72(8)
90.04(10)
83.27(10)
O(1)–Ni(1)–O(7A)
O(1)–Ni(1)–N(3A)
O(7) –Ni(1) –N(3A)
92.28(8)
89.96(10)
96.73 (10)
88.49(15)
89.40(17)
81.27(18)
O(1)–Co(1)–O(7A)
O(1)–Co(1)–N(3A)
O(7) –Co(1) –N(3A)
91.51(15)
90.60(17)
98.73(18)
1 3

Journal of Chemical Crystallography
Table 3 Geometric parameters
of the hydrogen bonds in
structures 1–4
Bond
Distance, Å
D–H
Angle
Coordinates of atom A
D–H···A
D–H···A, º
H···A
D···A
1
O(7)–H(7)···O(2)
N(3)–H(3A)···O(7)
C(8)–H(8A)···O(4)
C(9)–H(9A)···O(4)
2
0.82
0.89)
0.97
0.97
2.010
2.130
2.530
2.56
2.752(3)
2.997(4)
3.208(4)
3.512(5)
151
164
127
166
x,1+y,z
3/2−x, −1/2+y, 3/2-z
−1/2+x, ½−y, −1/2+z
O(7)–H(7)···O(2)
N(3)–H(3B)···O(7)
C(8)–H(8B)···O(4)
3
O(7)– H(7)···O(2)
N(3) –H(3A)···O(7)
C(8)–H(8A)···O(4)
4
0.82
0.89
0.97
1.850
2.280
2.42
2.637(3)
3.106(4)
3.187(5)
160
154
136
2−x,−y,2−z
x,−1+y,z
−1/2+x, ½−y, ½+z
0.82
0.89
0.97
1.90
2.28
2.41
2.656(5)
3.105(7)
3.183(9)
153
155
136
1−x,2−y,1−z
x,1+y,z
−1/2+x,3/2−y,1/2+z
N(3)–H(3C)···O(1)
O(7)–H(7)···O(6)
N(3)–H(3A)···O(2)
N(3)–H(3B)···O(1)
O(7)–H(7)···O(7)
0.89
0.82
0.89
0.89
0.82
2.02
2.50
1.90
1.98
2.27
2.819(6)
3.159(5)
2.719(6)
2.826(6)
2.934(5)
149
139
152
158
139
1−x,1−y,1−z
x,−1+y,z
3/2−x,−1/2+y,3/2-z
Fig. 2 Packing of the complex molecules [M(DNBA)2(MEA)2] in the isostructural crystal structures on example of compound 1. H atoms not
involved in hydrogen bonding are not shown for clarity. Symmetry codes: (i) x,1+y,z; (ii) 3/2-x,-1/2+y,3/2-z; (iii) -1/2+x,1/2-y,-1/2+z
intramolecular H-bond between MEA oxygen atom and
uncoordinated oxygen atom of the carboxylate group and
(iii) occupation by complex molecules inversion centers of
crystals.
(base)—pKa (acid) ≥ 3 (rule of 3) [33]. In our case pKa
(MEA)—pKa (DNBA) is 9.50–2.82=6.68, which is essen-
tially more than 3. Therefore, according to the “rule of 3”, a
supramolecular complex between DNBA and MEA should
be in the form of organic salt (DNBA−·MEA+). Indeed, in
the obtained compound 4 the amino-group of MEA is proto-
nated due to transferring of DNBA to the carboxylate form.
There are one DNBA− and one MEA+ molecules in the
asymmetric part of the unit cell. They are associated by
The Structure of the Organic Salt (DNBA−·MEA+)
Salt formation generally requires a diference at least three
in pKa values of the acid and base components, i.e. pKa
1 3

Journal of Chemical Crystallography
two H-bonds N(3)-H(3C)···O(1) and O(7)-H(7)···O(6) into
packed to the crystal structure by van-der-Waals interac-
tions (Fig. 4).
13-membered cycles with a graph-set notation of R2 (13)
2
(Fig. 3a). A pair of H-bonds N(3)-H(3B)···O(1) and N(3)-
H(3A)···O(2) closes 12-membered centrosymmetric rings
with a graph-set notation of R4 (12) (Table 3). Centrosym-
metric units consisting of the 4two 13-membered and one
12-membered H-bonded rings are incorporated into col-
umns running in direction of axis b. Further these columns
are associated by the bond O(7)–H(7)···O(7) into 2-D net-
work parallel to the (101) plane. These 2-D networks are
The analysis of the CSD [30] attest that organic salt
between DNBA and MEA is already registered in data-
base with ref-code ICIMOX [34] but it is crystallized in
orthorhombic space group P212121 in contrast to considered
monoclinic salt. In this compound an association of anions
with cations takes place through formation of the 9-mem-
bered ring with a graph-set notation of R2 (9). Rings are
2
generated on the basis of two H-bonds between carboxylate
Fig. 3 Asymmetric part of the unit cell of organic salt (DNBA−·MEA+): monoclinic polymorph 4, obtained in the present work (a) and
orthorhombic polymorph with ref-code ICIMOX (b). Thermal ellipsoids of non-hydrogen atoms are drawn at the probability level of 50%
Fig. 4 Packing structure of organic salt (DNBA−·MEA+). Symmetry codes: (i) x,-1+y,z; (ii) 1-x,1-y,1-z; (iii) 3/2-x,-1/2+y,3/2-z
1 3

Journal of Chemical Crystallography
oxygen atoms of DNBA and hydroxyl and protonated amino-
groups of MEA (Fig. 3b). Therefore, compound 4 obtained
in the present work is a new monoclinic polymorph of
organic salt between DNBA and MEA.
which forms the 9-membered ring with a graph-set notation
of R2 (9) as in orthorhombic polymorph of the compound 4
(Fig. 23b). Therefore, in the present work a new type of the
association mode between MEA and DNBA ions is found
(Fig. 3a).
Thus, the major diference in structures of dimorphs is
in supramolecular association mode of cations with anions.
Dimeric units are characterized by diferent dimensions
in polymorphs having more volume in monoclinic form.
Moreover, anions are stronger associated with cations in
orthorhombic dimorph due to more strong intermolecular
H-bonds, for example, O–H…O bond is 2.719 Å in contrast
to 3.159 Å. In orthorhombic dimorph the highest dimension
supramolecular association of components is 3-D network.
The 7 organic salt of the simplest monoderivatives of
benzoic acid with MEA are documented in CSD [30]. In
all these compounds with ref-codes TEYTAU [35] (benzoic
acid), JAPWIJ [36] (p-aminobenzoic acid), KEZFIG [35]
(p-hydroxybenzoic acid), KEZFOM [35] (p-methoxyben-
zoic acid), LIGZUX [37] (p-nitrobenzoic acid), TEYSIB
[35] (p-iodobenzoic acid), TEYSOH [35] (p-bromobenzoic
acid) and TEYSUN [35] (p-chlorobenzoic acid) molecular
recognition of anions by cations takes place through synthon
Water Solubility and Antimicrobial Activity
Water Solubility
Water solubility measurements of API attest that in result
of metal complex formation solubility of the ligand may
enhance slightly or drastically. Poor water solubility of cur-
cumin, extensively used in oriental medicine, is improved
4 times in result of coordination with copper [38] while in
DNBA complex of cadmium nearly 40-fold increasing of
solubility is observed [39] (Table 4). In contrast to these
monoligand metal complexes, isostructural mixed-ligand
coordination complexes of DNBA with MEA, show maxi-
mum fourfold enhancement of the solubility (5.45 mg/
ml). Probably, despite of very high water solubility MEA
Table 4 Water solubility of some metal complexes, organic salt and compounds obtained in the present work
Metal complexes
Compound
Metal
Type of metal
complex
Solubility,
mg/ml
Reference
[38]
Curcumin
–
–
0.100
0.162
0.267
0.417
1.350
49.50
21.63
19.72
14.99
5.45
Fe(III)
Zn(II)
Cu(II)
–
Cd(II)
Mn(II)
Co(II)
Ni(II)
Cu(II)
Ni(II)
Co(II)
Monoligand
DNBA
DNBA
–
[39]
Monoligand
Mixed-ligand with MEA
Present
work
5.25
4.90
Organic salt
Compound
Anion
Solubility of anion or API, mg/ml
Solubility of organic salt,
mg/ml
Reference
[40]
Norfoxacin
–
0.38
–
4-hydroxy-benzoic acid
2-hydroxy-benzoic acid
3-hydroxy-benzoic acid
–
0.05
2.48
10.02
0.04
0.67
2.28
17.01
–
Ambazone
DNBA
[41]
4-aminobenzoic acid
–
MEA
6.00
1.35
849.00
0.100
–
10.25
Present
work
1 3

Journal of Chemical Crystallography
demonstrates not positive impact to solubility of the metal
complex being located in coordination sphere.
more lipophilic is API the easier is its transition through
microorganism’s membranes.
Organic salt formation demonstrates diferent infuence
on the solubility improvement. Complexation of norfoxa-
cin with hydroxybenzoic acid isomers leads to enhancement
of water solubility up to 45 times [40] whereas in case of
ambazone solubility improves only 2.5-fold [41]. However,
organic salt between DNBA and MEA shows an intermedi-
ate (eightfold) improvement of solubility (Table 4) despite of
very high one of MEA. Thus, in result of preparation of Cu-
complex with DNBA and MEA, also organic salt between
these ligands water solubility is increased 4 and 8 times, i.e.
more enhancement of solubility is reached by organic salt
formation approach.
Thus, compounds 1 and 4 show more antifungal activity
relatively to DNBA, probably due to synergism of the com-
ponents. It should be noted that an enhancement of water
solubility leads also to the improvement of the antimicrobial
activity and the more soluble is optimized API the higher
is its bioactivity.
DFT Study
From a theoretical point of view, the geometry around the
central atom and the electronic structure of transition metals
are of a great interest. In case of isomorphic metal com-
plexes theoretical calculations are of even more importance
because despite of isostructurality of the complexes their
geometric parameters and electronic properties may be dif-
ferent. Therefore, the X-ray structures of isostructural Cu(II),
Ni(II) and Co(II) complexes were optimized by the DFT/6-
311G(d,p) and DFT/def2-TZVP methods in the singlet/dou-
blet, triplet/quartet and quintet/sextet spin states. According
to (Table 5), the Cu(II), Ni(II) and Co(II) complexes have
high-spin electronic states as a doublet, triplet and quartet,
respectively, which is consistent with literature data [42].
The structures were optimized by B3LYP/def2-TZVP
and B3LYP/6-311G(d,p) methods and a mean absolute
error (MAE), largest errors (LE) and correlation coefcients
R2 were calculated in result of comparison of theoretical
bond lengths and bond angles with the experimental data
(Table S1-S6 and Table 6). The LE in bond lengths around
0.2 Å is mainly caused by the large diferences between
the experimental and theoretical C-H, N–H and O–H bond
lengths.
Antimicrobial Activity
Antimicrobial tests on fungi Fusarium oxysporum were
carried out using 1.50 mg/ml concentrations of DNBA,
MEA, [Cu(DNBA)2(MEA)2] and (DNBA−·MEA+). Inhibi-
tion zones of these compounds were 10, 13, 17 and 20 mm,
respectively (Fig. 5), i.e. organic salt demonstrates maximal
antifungal activity. In our paper [31] antimicrobial activities
against the same fungi have been determined for the copper
complex with 4-nitrobenzoic acid and MEA as well organic
salt between these ligands. Test results show that an essential
enhancement of the bioactivity takes place in both cases
but antimicrobial activity of metal complex is higher than
one of organic salt. Less antimicrobial activity of the metal
complex relatively to organic salt in the case of DNBA, may
be explained by the less lipophility of the complex molecule
1 due to the more hydrophility of the coordinated DNBA
molecules (relatively to 4-nitrobenzoic acid) because the
According to literature sources [43] the X–H bond
lengths are systematically shorter than DFT-data and it can
be explained by the low scattering factors of hydrogen atoms
in the X-ray difraction experiments. On the other hand, this
Table 5 Total energies of complexes in the low and higher spin
electronic states calculated by B3LYP/def2-TZVP and B3LYP/6-
311G(d,p)a
Complex
Total energy, kcal/mol
Low-spin state
High spin-states
1 (Cu)
2 (Ni)
3 (Co)
−2,334,053.14
−2,333,996.39
−2,333,939.63
(−2,333,817.88)
(−2,333,761.17)
(−2,333,704.44)
−2,251,113.33
−2,251,137.23
−2,251,080.27
(−2,250,875.41)
(−2,250,901.11)
(−2,250,844.14)
−2,172,344.52
−2,172,354.85
−2,172,276.44
(-2,172,118.55)
(−2,172,129.28)
(−2,172,051.31)
aB3LYP/6-311G(d,p) data are given in brackets. Spin state for Ni(II)
complex is singlet, triplet and quintet. Spin states for Cu(II), Co(II)
complexes are doublet, quartet and sextet
Fig. 5 Inhibition zones of DNBA (I, 10 mm), MEA (II, 13 mm),
[Cu(DNBA)2(MEA)2] (III, 17 mm) and (DNBA−·MEA+) (IV,
20 mm) in antimicrobial tests on fungi Fusarium oxysporum
1 3

Journal of Chemical Crystallography
Table 6 Statics for experimental and calculated by B3LYP/def2-
participation of the atom H3A in H-bonds and may be
explained as above mentioned.
TZVP bond lengths and anglesa
R2
Thus, the analysis of molecular conformations of Ni and
Co complexes by theoretical methods shows a very good
correlation between predicted and experimental data (R2 is
about 0.98). However, in case of the Cu-complex correla-
tion between calculated and experimental bond angles is
worse (R2 =0.89) but acceptable for other calculations [24]
(Table 6). Therefore, the optimized geometry by B3LYP/6-
311G(d,p) method is used for further calculations.
Complexes
MAE
LE
Bond lengths, Å
0.052 (0.049)
0.055 (0.054)
0.055 (0.055)
Bond angles,º
1.83 (1.69)
1
2
3
0.17 (0.17)
0.21 (0.21)
0.15 (0.15)
0.97 (0.98)
0.97 (0.97)
0.97 (0.98)
1
2
3
35.1 (35.1)
7.10 (6.3)
5.38 (4.6)
0.89 (0.91)
0.98 (0.98)
0.98 (0.98)
1.20(1.16)
1.25 (1.26)
To study of the electronic structure of isostructural com-
plexes, the energy of highest-occupied molecular orbital
(HOMO) and the lowest-unoccupied molecular orbital
(LUMO) as well an energy gap between orbitals have been
determined by B3LYP/6-311G(d,p) method (Table 8). Fur-
thermore, total ESP of isostructural complexes have been
analyzed using Multiwfn [26] and VMD [27] program pack-
ages. The role and application of energy of frontier MOs,
energy gap and frontier electron density are well known in
chemistry [24, 44, 45]. An ESP is useful guide to the mol-
ecule’s reactive behavior, especially in non-covalent interac-
tions [46, 47] and it indicates nucleophilic and electrophilic
sites of molecules [48].
aB3LYP/6-311G(d,p) data are given in brackets
may be due to the fact that the experimental data describe
compounds in the solid state while the calculated data cor-
respond to molecules in the gas phase.
The next LE is observed for the bond C(8)-C(9) of the
Co-complex with the values of 0.104 Å and 0.108 Å by
DFT/dev2-TZVP and DFT/6-311G(d,p) methods, respec-
tively (Table S5). Among bonds of the central ion the larg-
est diferences 0.069, 0.060 and 0.052 Å are found for bonds
M-N3 of the complexes 3, 1 and 2, respectively (Table 7).
A signifcant diference between the predicted and experi-
mental bond angles is found for the Cu-complex. Both meth-
ods DFT/def2-TZVP and DF/ 6-311G (d, p) indicate as the
largest error 35.1º for the bond angle Cu1-O7-H7. This may
be due to a reassessment of the hydrogen bond between
the oxygen atom of DNBA and OH-group of MEA in the
calculation methods because the distance between H-bond
donor (H atom) and acceptor (O-atom) is significantly
shorter than experimentally determined one. Indeed, this
distance is equal to 1.710 and 1.717 Å by DFT/def2-TZVP
and DFT/6-311G (d, p) methods, whereas its experimental
value is 2.15 Å for the Cu-complex (Table 3). As a result,
other angle C8-O7-H7 related with the H7 atom also dem-
onstrates slightly large diference of 7.56 (6.04) º.
According to Table 8, the HOMO level of Co-complex 3
is located higher relatively to other complexes and therefore
the lowest value of energy gap is observed for this complex.
This indicates its strong chemical reactivity and weak kinetic
stability relatively to complexes 1 and 2.
In Fig. 6 the αHOMO and αLUMO of 1 and also its
ESP surface are illustrated. The electron densities in the
frontier MO’s of isostructural complexes are very similar
Table 8 Calculated HOMO and LUMO energies, HOMO–LUMO
gap (EGAP) by B3LYP/6-311G(d,p) method
MO
HOMO, eV
LUMO, eV
EGAP, eV
1
2
3
αMO
βMO
αMO
βMO
αMO
βMO
−7.37
−7.32
−7.04
−7.51
−6.88
−6.88
−3.13
−3.26
−3.10
−3.10
−3.10
−3.10
4.24
4.05
3.94
4.47
3.78
3.78
In case of Ni- and Co-complexes the DFT/def2-TZVP
method shows that the LE of 7.10 and 5.38º belong to
the bond angles C9-N3-H3A and Co1-N3-H3A, respec-
tively (Table 6). These LE are also appeared due to the
Table 7 Optimized bond
Bond
Isostructural complexes
Δl, Å
lengths (Å) of the central metal
ions by B3LYP/def2-TZVP and
B3LYP/6-311G(d,p) methodsa
1
2
Δl, Å
3
Δl, Å
M – O1 2.026 (2.011) 0.048 (0.033) 2.099 (2.078) 0.041 (0.02)
2.118 (2.100) 0.045 (0.027)
M – O7 2.425 (2.375) 0.004 (0.054) 2.118 (2.102) 0.022 (0.006) 2.149 (2.134) 0.007 (0.008)
M – N3 2.064 (2.043) 0.060 (0.039) 2.127 (2.111) 0.052 (0.036) 2.193 (2.170) 0.069 (0.046)
Δl (Å)—the diference between experimental and theoretical bond lengths, M—metal ion
aB3LYP/6-311G(d,p) data are given in brackets
1 3

Journal of Chemical Crystallography
Fig. 6 Electron density on the
αHOMO (a) and αLUMO (b)
of 1 and its ESP surface (c).
Orange and blue spheres in ESP
surface correspond to maxima
and minima, respectively
(Figs. S1-S3), and the metal ion makes a large contribu-
tion to the formation of the α and βHOMOs. The α and
βLUMO are localized on DNBA (Fig. 6b). The ESP of 1,
shown in Fig. 6c, is identical for complexes 2 and 3 but
difers by the maximum and minimum ESP surface values
(Fig. S4). For example, the local surface maximum (dark
blue colored region) with high positive values ft to H atoms
of NH2 group of all 1–3 complexes. At the same time, the
1 3

Journal of Chemical Crystallography
red colored-regions are local surface minima with negative
values, which indicates electron rich groups (–NO2). The
ESP maxima (minima) value is equal to 50.29 (-26.99) kcal/
mol in the complex 1, and in the case of complexes 2 and
3—47.65 (-27.32), 47.44 (-27.32) kcal/mol. During the ESP
computing by Multiwfn program [26] according to [49], 22
surface maxima and 20 minima have been obtained for 1–3.
They are indicated as an orange (maxima) and blue (minima)
spheres in Fig. 6c. All of 22 ESP surface maxima are posi-
tive values. According to the ESP calculations, an electron
rich NO2-groups of complexes 1–3 can be participants in
non-covalent interactions of intermolecular interactions with
various molecules or in crystal packing [50].
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Afliations
Avazbek B. Ibragimov1 · Jamshid M. Ashurov1 · Aziz B. Ibragimov2 · Alisher G. Eshimbetov3
2
* Avazbek B. Ibragimov
Institute of General and Inorganic Chemistry, Uzbekistan
alex.ibragimov@inbox.ru
Academy of Sciences, Kh.Abdullaev Str., 77a, Tashkent,
Uzbekistan 100170
1
Institute of Bioorganic Chemistry, Uzbekistan
3
National University of Uzbekistan, University Str., 4,
Tashkent, Uzbekistan 100174
Academy of Sciences, Kh.Abdullaev Str., 83, Tashkent,
Uzbekistan 100125
1 3