Experimental and theoretical structure characterization of two isoniazid derivatives: 2,4-Difluoro-N′-isonicotinoylbenzohydrazide and 2,4-dichloro-N′-isonicotinoylbenzohydrazide hydrochloride

Article abstract of DOI:10.1016/j.molstruc.2005.11.023

An X-ray and a theoretical study of the structure of the isoniazid derivatives, 2,4-difluoro-N′-isonicotinoylbenzohydrazide, 3, and 2,4-dichloro-N′-isonicotinoylbenzohydrazide hydrochloride, [4H⁺, Cl^-, 2(H₂O)], are reported. Quantum chemical calculations as well as conformational analysis are presented with the isolated cations [3H⁺] and [4H⁺], where the former was protonated in silico to allow direct comparison of results. Supermolecule calculations were also carried out with the asymmetric unit of [4H⁺, Cl^-, 2(H₂O)], which is comprised of two independent cations (4H⁺), two chloride ions and four water molecules. Our results indicate that the crystal structures, although clearly representing accessible conformations, are highly distorted in comparison to the predicted lower energy conformers in the gas-phase. These distortions are most probably imposed by polar and electrostatic interactions within the crystal packing. In general, the calculated potential energy surfaces (PES) for both isoniazid derivatives are fairly flat, a feature confirmed by the low energetic difference calculated for several conformers corresponding to local minima in PES. Noteworthy, the fluorinated compound [3H⁺] shows an important additional energy barrier for rotation around the bond connecting the halogenated ring to the proximate carbonyl due to a strong internal hydrogen bond involving the fluorine atom.

Full text of DOI:10.1016/j.molstruc.2005.11.023

Journal of Molecular Structure 788 (2006) 63–71
www.elsevier.com/locate/molstruc
Experimental and theoretical structure characterization of two isoniazid
derivatives: 2,4-Difluoro-N0-isonicotinoylbenzohydrazide and
2,4-dichloro-N0-isonicotinoylbenzohydrazide hydrochloride
c
d
c
Floriano P. Silva Jr a,b, , Javier Ellena , Marcelle de Lima Ferreira , Yvonne P. Mascarenhas ,
*
Marcus V.N. de Souza d, Thatyana R.A. Vasconcelos d, James L. Wardell b,
Solange M.S.V. Wardell d
a
´ ´ ´ ´
Laboratorio de Bioquımica de Proteınas e Peptıdeos, Instituto Oswaldo Cruz, FIOCRUZ 21045-900, RJ, Brazil
b
´
Instituto de Quımica, Universidade Federal do Rio de Janeiro, 21945-970 Rio de Janeiro, RJ, Brazil
c
´
Instituto de Fısica de Sa˜o Carlos, Universidade de Sa˜o Paulo, 13560-970 Sa˜o Carlos, SP, Brazil
d
´
Instituto de Tecnologia em Farmacos, Far-Manguinhos, FIOCRUZ, 21041-250 Rio de Janeiro, RJ, Brazil
Received 5 August 2005; received in revised form 14 November 2005; accepted 16 November 2005
Available online 4 January 2006
Abstract
An X-ray and a theoretical study of the structure of the isoniazid derivatives, 2,4-difluoro-N0-isonicotinoylbenzohydrazide, 3, and 2,4-dichloro-
N0-isonicotinoylbenzohydrazide hydrochloride, [4HC, ClK, 2(H2O)], are reported. Quantum chemical calculations as well as conformational
analysis are presented with the isolated cations [3HC] and [4HC], where the former was protonated in silico to allow direct comparison of results.
Supermolecule calculations were also carried out with the asymmetric unit of [4HC, ClK, 2(H2O)], which is comprised of two independent cations
(4HC), two chloride ions and four water molecules. Our results indicate that the crystal structures, although clearly representing accessible
conformations, are highly distorted in comparison to the predicted lower energy conformers in the gas-phase. These distortions are most probably
imposed by polar and electrostatic interactions within the crystal packing. In general, the calculated potential energy surfaces (PES) for both
isoniazid derivatives are fairly flat, a feature confirmed by the low energetic difference calculated for several conformers corresponding to local
minima in PES. Noteworthy, the fluorinated compound [3HC] shows an important additional energy barrier for rotation around the bond
connecting the halogenated ring to the proximate carbonyl due to a strong internal hydrogen bond involving the fluorine atom.
q 2005 Elsevier B.V. All rights reserved.
Keywords: Isoniazid derivatives; X-ray structure; Supermolecule calculation; Density functional theory; RHF; Potential energy surfaces; Electronic energy
1. Introduction
drug-resistant (MDR) organisms, which would lead to an
ineffective treatment. Several factors have prompted the
scientific community to develop new drugs to fight tubercu-
losis. The most significant of these are (i) the rapid spread of
MDR–TB strains resistant to all major anti-tuberculosis drugs
in the market and (ii) the association of TB with human
immunodeficiency virus (HIV) infection in AIDS. In this
paper, we report the X-ray crystallographic and molecular
modeling study of two new isoniazid derivatives, obtained
from the reactions of isoniazid with 2,4-X2C6H3COCl (1: XZ
F; 2: XZCl), see Scheme 1. The compound, 2,4-difluoro-N0-
isonicotinoylbenzohydrazide, (3), and the salt, 2,4-dichloro-N0-
isonicotinoylbenzohydrazide hydrochloride [4HC, Cl], pre-
sented minimal inhibitory concentration (MIC) of 10 mg/mL
when tested against Mycobacterium tuberculosis strain H37Rv
(ATCC 27294). A paper with the biological activities of these
Tuberculosis (TB) is currently a worldwide problem. This
contagious disease kills four people every minute somewhere
in the world and amounts to more than two million deaths per
year [1]. At present, the accepted treatment of TB involves a
combination of the first-line drug, isoniazid, 4-(H2-
NNHCO)pyridine, along with pyrazinamide, ethambutol and
rifampicin given in combination over 6–9 months [2,3].
Combinations are used to limit the emergence of multiple
*
Corresponding author. Address: Fundao Instituto Oswaldo Cruz, Bioqui-
mica e Biologia Molecular, Av. Brasil 4365, Pav. 26, sl. 311, 21045-900 Rio de
Janeiro, Brazil. Tel.: C55 21 3865 8157; fax: C55 21 25903 495.
E-mail address: floriano@ioc.fiocruz.br (F.P. Silva).
0022-2860/$ - see front matter q 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2005.11.023

64
F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
Scheme 1.
compounds and their analogues has been submitted to a
specialist journal [4] and hence these are not reported here.
7.45 (1H; dt; JZ2.5, 6.0 Hz; H3); 7.26 (1H; ddd; JZ2.0, 8.5
and 15.0 Hz; H5) ppm. 13C NMR (125.0 MHz, DMSO-d6) d:
163.9; 162.5; 163.7 (dd; JZ11.1, 248.6 Hz); 159.8 (dd; JZ
11.0, 159.8 Hz); 150.4; 139.2; 122.7; 121.2; 118.6 (d; JZ
14.5 Hz); 112.0 (d; JZ21.0 Hz); 104.8 (t; JZ26 Hz) ppm. IR
(cmK1; KBr): 3191 (NH); 1610 (CO); 1605 (CO).
2. Experimental, theoretical and computational methods
2.1. Synthesis
Crystals for X-ray diffraction study were obtained by slow
crystallization from heptane.
2.1.1. General
Melting points were determined on a Buchi apparatus and
are uncorrected. Infrared spectra were recorded on a Thermo
Nicolet Nexus 670 spectrometer in KBr pellets and frequencies
are expressed in cmK1. Mass spectra (CG/MS) were recorded
on a Agilent Tecnologies 61530A/5792A mass spectrometer.
NMR spectra were recorded on a Bruker Avance 500
spectrometer operating at 500.00 MHz (1H) and 125.0 MHz
(13C), in deuterated dimethyl sulfoxide. Chemical shifts are
reported in ppm (d) relative to tetramethylsilane. Proton and
carbon NMR spectra were obtained at room temperature.
2.1.3. Preparations of 2,4-dichloro-N0-isonicotinoylbenzohy-
drazide hydrochloride [4HC, ClK]
This was prepared from 2 and isoniazid by a similar
procedure as used for 3, except that triethylamine was not used.
Yield: 78%; m.p.: 221–222 8C. 1H NMR (500.00 MHz,
DMSO-d6) d: 11.42 (1H; s; NH); 10.84 (1H; s; NH); 9.03 (2H;
d; JZ6.0 Hz; H2/ and H6/); 8.24 (2H; d; JZ6.0 Hz; H3/ and
H5/); 7.79 (1H; s; H3); 7.61 (1H; d; JZ11.0 Hz; H5 or H6); 7.60
(1H; d; JZ10.0 Hz; H5 or H) ppm. 13C NMR (125.0 MHz,
DMSO-d6) d: 164.5; 162.5; 145.9; 143.6; 135.4; 133.0; 131.7;
130.6; 129.5; 127.4; 123.6 ppm. IR (cmK1; KBr): 3137 (NH);
1703 (CO); 1668 (CO).
2.1.2. Preparations of 2,4-difluoro-N0-
isonicotinoylbenzohydrazide
Suitable crystals for the X-ray study were grown from
methanol solution and were shown to be the dihydrate [4HC,
ClK$2H2O].
The carboxylic acid chloride derivative (1) was prepared by
treating 2,4-F2C6H4CO2H (1 g) with thionyl chloride
(3 equiv.), N,N-dimethylformamide (0.1 equiv.) in dichloro-
methane (20 mL) at room temperature, under stirring and
nitrogen atmosphere. After 6 h, the excess of thionyl chloride
was removed and the crude product was used as such in the
next stage.
2.2. X-ray crystallographic study of 3 and [4HC, ClK$2(H2O)]
The measurements were made at 150 K for 3 and at room
temperature for [4HC, ClK$2(H2O)], on an Enraf-Nonius
KAPPA CCD diffractometer (95 mm CCD camera on
k-goniostat) with graphite monochromated Mo Ka (lZ
Reaction between (1), isoniazid and triethylamine (equi-
molar) in tetrahydrofuran (20 mL) at 70 8C, under nitrogen
atmosphere and stirring for 6h led to the crude product 3
(Scheme 1). Purification by column chromatography on silica
gel (0.063–0.2 mm mesh size), eluting with a hexane:ethyl
acetate gradient, afforded pure 3.
Yield: 76%; m.p.: 188–189 8C. GC/MS: m/z [M]C : 277; 1H
NMR (500.00 MHz, DMSO-d6) d: 10.96 (1H; s; NH); 10.54
(1H; s; NH); 8.80 (2H; d; JZ6.0 Hz; H2/ and H6/); 7.83 (2H; d;
JZ6.0 Hz; H3/ and H5/); 7.76 (1H; dd; JZ8.5, 15.0 Hz; H6);
˚
0.71703 A) radiation. Data collection (f scans and u scans
with k offsets) was made using the COLLECT Program [5];
final unit cell parameters based on all reflections, integration
and scaling of the reflections intensities were performed with
the HKL DENZO SCALEPACK system of programs [6]. The
structures were solved using direct methods and refined by full-
matrix least squares procedure on F2 with SHELXS-97 [7].

F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
65
The asymmetric unit of 3 consists of a single molecule,
exhibiting disorder of the ortho-fluoro group in the disub-
stituted phenyl ring. The disorder corresponds to equal
occupation of the two possible ortho positions, which enable
designations to be used of syn and anti carbonyl oxygen(O1)–
ortho-fluorine orientations, see Fig. 1. Atom arrangements and
the crystallographic numbering scheme for the syn arrange-
ment are shown in Fig. 2.
The asymmetric unit of [4HC, ClK, 2(H2O)] is comprised of
two independent cations, two chloride ions and four water
molecules. The major difference between the two cations is the
orientation of the o-Cl group of the disubstituted phenyl ring
relative to the nearest carbonyl oxygen. Atom arrangements
and the crystallographic numbering scheme are shown in
Fig. 3. Again syn and anti designations [as indicated by Cl22–
O21, and Cl12–O11, respectively] can be used here (see
Fig. 1).
Fig. 2. Atom arrangements and atom numbering scheme for one of the
disordered forms [syn-conformer] of 3.
assigned as Nam (amide nitrogen) types and the pyridinium ring
nitrogen as Npl (trigonal planar nitrogen).
2.3.2. Quantum chemical calculations
All quantum chemical calculations were performed within
Gaussian 03, Revision B.01 [10], running on the computational
The program WINGX [8] was used to analyze and prepare
the data for publication. Crystal data collection procedures and
refinement results are presented in Table 1. The molecular
representations were prepared using ORTEP [9].
´
´
´
ˆ
facilities in the Laboratorio de Fısico-quımica Organica e
Modelagem Molecular (Dr Ricardo Bicca De Alencastro,
DQO, IQ, Universidade Federal do Rio de Janeiro). Con-
formers corresponding to symmetry-unique local minima in
[3HC] and [4HC] PES were subjected to preliminary full
optimization with AM1 semi-empirical method and sub-
sequently optimized with Restricted Hartree–Fock theory
level and 6-31G* basis set (RHF/6-31G*). Geometries
corresponding to RHF/6-31G* local minima were further
optimized using density functional theory (DFT) with Becke’s
three-parameter functional and the correlation functional of
Lee–Yang–Parr (B3LYP) using the standard 6-31G* basis set
(DFT-B3LYP/6-31G*). The resulting geometries also had their
electronic energies calculated with a larger basis set (DFT-
B3LYP/6-311CCG**//DFT-B3LYP/6-31G*) for validation
of results. Frequency calculations were accomplished for each
conformer representing a local minimum in the PES in order to
correct for zero-point energy (ZPE) deviation. A single-point
supermolecule calculation was attempted based on the
2.3. Computational methods
2.3.1. Conformational analysis
To allow direct theoretical comparison of the different
conformations of the cation [4HC] and the molecule 3, the later
was protonated at the pyridine nitrogen in silico, to give [3HC]
prior to computational analysis. The anti conformations of
molecule [3] and cation [4HC], determined in the X-ray
crystallographic study, were used as starting geometries for the
conformational analysis: that in [4HC, ClK$2(H2O)] was
denoted as [4HC]-a and that in 3 as 3-c.
The ‘search’ module of Sybyl v6.8 modelling package
(Tripos, Inc., Illinois, MA) was used to perform a systematic
analysis, using a 308 step, in torsion angles 1, 2 and 3 (q1, q2 and
q3, respectively) as identified in Fig. 1. Molecular mechanics
(MM) energies for each of the 1728 conformers for [3HC] and
[4HC] were calculated in order to evaluate minima in the
potential energy surface (PES). Accordingly, the conformers
described in Table 5 along with Fig. 5 are designated by
numbers reflecting the sequential numeration from 1 through
1728 received during the conformational analysis. MM
energies were obtained using the Tripos force field parameters
and partial atomic charges accommodated from the electro-
static potential, which was calculated for the X-ray derived
geometry with the AM1 Hamiltonian. Also for the purpose of
MM calculations, the central hydrazide nitrogen atoms were
Fig. 1. The syn and anti forms of 3, 4, [3HC] and [4HC].
Fig. 3. Atom arrangements and atom numbering scheme for [4HC,ClK$2(H2O)].

66
F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
Table 1
Crystal data and structure refinement for 3 and [4HC, ClK$2(H2O)]
Data
3
[4HC, ClK$2(H2O)]
Empirical formula
Formula weight
Temperature
C13H7F2N3O2
275.22
C13H18Cl3N3O6
382.62
150(2) K
˚
0.71073 A
293(2) K
˚
0.71073 A
Wavelength
Crystal system
Space group
Monoclinic
Monoclinic
P21
P21/n
˚
˚
Unit cell dimensions
aZ4.709(3) A
aZ7.2069(3) A
˚
˚
bZ24.398(16) A
cZ5.281(4)
bZ48.5142(18) A
˚
cZ9.4688(3) A
aZgZ908
aZgZ908
bZ92.54(5)8
606.1(7) A
bZ106.658(1)8
3171.7(2) A
3
˚
3
˚
Volume
Z
2
8
Absorption coefficient
F(000)
0.126 mmK1
0.601 mmK1
280
0.24!0.12!0.04 mm3
3.86–20.008
1568
0.34!0.20!0.12 mm3
3.07–25.008
Crystal size
Theta range for data collection
Index ranges
K4!h!4
K8!h!8
K23!k!23
K54!k!57
K5!l!4
919
K10!l!11
14,610
Reflections collected
Independent reflections
Completeness
919 [R(int)Z0.0000]
94.0% (to qZ20.008)
0.9950 and 0.9705
Full-matrix least-squares on F2
919/1/185
5525 [R(int)Z0.0466]
99.0% (to qZ25.008)
0.9314 and 0.8219
Full-matrix least-squares on F2
5525/0/415
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [IO2sigma(I)]
R indices (all data)
1.078
1.044
R1Z0.0728, wR2Z0.1932
R1Z0.0902, wR2Z0.2101
R1Z0.0380, wR2Z0.0904
R1Z0.0981, wR2Z0.0904
K3
K3
˚
0.215 and K0.196 e A
˚
0.443 and K0.287 e A
Largest diff. peak and hole
asymmetric unit of [4HC, ClK$(H2O)] using DFT-B3LYP/6-
31CG* either in vacuo or using the IPCM solvation model.
The supermolecule was built from eight fragments: two
cations, two chloride ions and four water molecules. In
vacuo, supermolecule calculations were accompanied by
counterpoise correction.
bonding network and the geometric parameters associated with
the H-bonds are listed in Table 3.
3.2. Comparison between crystallographic and theoretically
optimized structures
Structural optimizations of [3HC] and [4HC] were initially
carried out. The ab initio calculations were performed using the
‘standard’ double-split basis set, 6-31G*, within two levels of
theory: the usual Hartree–Fock treatment (RHF) and DFT. In
Table 4 are listed the main torsional angles q1, q2 and q3, and
the electronic energies calculated for the different conformers
of [4HC] either observed experimentally (‘crystal’, single
point calculations) or obtained after full geometric optimiz-
ations (optimized). The data show that none of the theoretical
methods led to an equilibrium structure strictly identical to that
obtained by crystallography. This is clearly perceived from the
superposition of the [4HC] experimental conformers and their
geometry-optimized counterparts in Fig. 4. Such discrepancy
suggests that the crystallographic conformers of [4HC] are
stabilized by inter-species interactions involving the cations,
chloride anions and water molecules. A detailed comparison of
experimental and optimized geometries for the ‘anti’ and ‘syn’
cations in [4HC, ClK$2(H2O)] is presented in Table 2. The
rmsd of bond lengths between theory and experiment within
the ‘anti’ conformer geometry, as predicted by RHF and DFT-
3. Results and discussion
3.1. X-ray structure determinations
As stated in Section 2, the ortho-fluorine atom in 3 is
disordered over two sites, which leads to syn and anti
designations: the former is shown as an ORTEP [9]
representation in Fig. 2. As also indicated in Section 2, the
two independent cations in the asymmetric unit of [4HC,
ClK$2(H2O)] also exhibit different carbonyl-O o-F orien-
tations (see Fig. 3). Bond distances and angles are as expected
in both 3 and the cations of [4HC, ClK$2(H2O)]. Comparison
of the bond lengths and angles determined by X-ray
crystallography for [4HC] can be made with calculated values,
in Table 2. Bond angles and lengths for 3 are not included here,
but have been deposited, as well as anisotropic thermal
parameters, in the CCDC, registry number 280895. Related
data for [4HC, ClK$2(H2O)] have also been deposited under
number 280896. The water molecules and the chloride ions in
[4HC, ClK$2(H2O)] participate in an extensive hydrogen
˚
B3LYP methods, are very similar (0.015 and 0.014 A,

F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
67
Table 2
Comparison of calculated structural parameters and experimental (X-ray) for anti- and syn-[4HC
]
˚
(a) Bond lengths (A)
Calculated
X-ray values
Bond
Anti
Syn
Anti
Syn
RHF/6-31G*
B3LYP/6-31G*
RHF/6-31G*
B3LYP/6-31G*
ClX1–CX01
ClX2–CX03
OX1–CX07
OX2–CX08
NX1–NX2
1.730
1.745
1.197
1.185
1.367
1.368
1.365
1.334
1.339
1.382
1.383
1.383
1.393
1.393
1.499
1.379
1.517
1.392
1.393
1.373
1.369
1.741
1.764
1.235
1.229
1.373
1.375
1.349
1.353
1.355
1.394
1.396
1.393
1.410
1.409
1.492
1.386
1.505
1.406
1.406
1.382
1.379
1.731
1.734
1.191
1.186
1.374
1.387
1.364
1.334
1.339
1.384
1.382
1.382
1.393
1.391
1.496
1.382
1.516
1.392
1.393
1.373
1.369
1.744
1.747
1.216
1.215
1.391
1.416
1.378
1.351
1.355
1.395
1.395
1.394
1.409
1.406
1.490
1.390
1.515
1.403
1.404
1.384
1.380
1.736(2)
1.739(2)
1.229(3)
1.225(3)
1.384(3)
1.339(3)
1.342(3)
1.341(3)
1.337(3)
1.386(4)
1.382(4)
1.381(3)
1.396(3)
1.392(3)
1.506(3)
1.378(3)
1.506(3)
1.397(3)
1.388(3)
1.372(3)
1.377(3)
1.741(3)
1.733(2)
1.216(3)
1.225(3)
1.379(3)
1.357(3)
1.346(3)
1.334(3)
1.336(3)
1.380(3)
1.380(3)
1.384(3)
1.391(3)
1.391(3)
1.503(3)
1.387(3)
1.511(3)
1.396(3)
1.381(3)
1.372(3)
1.373(3)
NX1–CX07
NX2–CX08
NX3–CX11
NX3–CX12
CX01–CX02
CX01–CX06
CX02–CX03
CX03–CX04
CX04–CX05
CX04–CX07
CX05–CX06
CX08–CX09
CX09–CX10
CX09–CX13
CX11–CX10
CX13–CX12
(b) Bond angles (8)
Calculated
X-ray values
Anglea
Anti
Syn
Anti
Syn
RHF/6-31G*
B3LYP/6-31G*
RHF/6-31G*
B3LYP/6-31G*
ClX1–CX01–CX02
ClX1–CX01–CX06
CX12–CX03–CX02
CX12–CX03–CX04
OX1–CX07–NX1
OX1–CX07–CX04
OX2–CX08–NX2
OX2–CX08–CX09
NX2–NX1–CX07
NX1–NX2–CX08
NX1–CX07–CX04
NX2–CX08–CX09
CX12–NX3–CX11
NX3–CX11–CX10
NX3–CX12–CX13
CX02–CX01–CX06
CX01–CX02–CX03
CX01–CX06–CX05
CX02–CX03–CX04
CX03–CX04–CX05
CX03–CX04–CX07
CX05–CX04–CX07
CX04–CX05–CX06
CX08–CX09–CX10
CX08–CX09–CX13
CX13–CX09–CX10
CX09–CX10–CX11
CX09–CX13–CX12
119.2
119.9
116.1
122.8
120.0
122.0
125.8
119.1
118.2
117.7
118.0
115.0
123.1
119.7
119.7
121.0
119.4
118.8
121.1
117.9
127.4
114.7
121.8
123.2
116.9
119.9
118.8
118.9
119.2
120.0
115.0
123.5
117.4
122.3
123.3
120.7
115.2
119.6
120.4
116.0
122.8
119.6
119.4
120.8
119.5
118.8
121.5
116.9
129.2
113.8
122.5
125.5
115.6
118.8
119.6
119.8
119.2
118.7
117.3
121.1
124.0
123.3
124.2
120.8
119.6
119.0
112.7
115.0
122.7
120.1
119.5
122.1
118.4
118.4
121.6
118.2
122.9
118.8
121.4
123.0
117.7
119.3
118.8
119.6
119.1
119.7
117.4
121.9
120.4
125.4
125.2
119.2
116.4
117.6
114.1
115.6
123.1
119.7
119.7
121.1
119.5
118.8
120.6
118.5
122.4
119.0
121.5
123.3
116.7
119.9
118.7
118.8
118.5(2)
119.9(2)
118.46(19)
119.91(17)
124.0(2)
121.9(2)
124.0(2)
120.7(2)
120.99(19)
119.14(19)
114.0(2)
115.3(2)
122.8(2)
120.0(2)
119.4(2)
121.6(2)
118.3(2)
119.2(2)
121.6(2)
118.3(2)
122.6(2)
119.0(2)
121.0(2)
123.0(2)
117.6(2)
119.4(2)
118.9(2)
119.5(2)
119.25(19)
118.67(19)
117.24(19)
121.17(19)
124.0(2)
123.3(2)
124.2(2)
120.8(2)
119.62(19)
119.03(19)
112.7(2)
115.1(2)
122.7(2)
120.0(2)
119.4(2)
122.1(2)
118.4(2)
118.4(2)
121.6(2)
118.2(2)
122.9(2)
118.9(2)
121.4(2)
123.0(2)
117.7(2)
119.3(2)
118.8(2)
119.6(2)
a
XZ1 for anti-[4HC] and 2 for syn-[4HC].

68
F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
Table 3
Hydrogen bonding parameters for [4HC, ClK$2(H2O)]
˚
˚
˚
D–H/A
D–H (A) D/A (A)
H/A (A)
D–H/A (8)
N11–H11–Cl2
0.860
0.860
0.860
0.855
0.759
0.949
0.941
0.860
0.860
0.860
0.881
0.912
0.881
0.948
0.831
3.119
2.741
2.791
2.992
3.237
3.278
2.861
3.017
3.174
2.797
3.196
2.908
3.196
3.257
2.996
2.133
1.963
2.050
2.156
2.494
2.340
2.020
2.408
2.483
2.098
2.317
2.065
2.317
2.310
2.177
160
150
144
166
167
170
148
128
138
138
175
153
175
178
16,981
N12–H12/O2w
N22–H22/O3w
O1w–H11w/O12
O3w–H31w/Cl2
O3w–H32w/Cl1
O4w–H41w/O21
N13–H13/O1wi
N13–H13/Cl2i
Fig. 4. Comparison between experimental and optimized [4HC] “anti” (A) and
“syn” (B) conformations. Experimental structures are colored in black while
structures optimized with either RHF/6-31G* or DFT-B3LYP/6-31G* are
colored in light gray or dark gray, respectively. Some of the atoms are labeled
to allow for correct orientation.
N23–H23/O4wii
O2w–H22w/Cl1vi
O1w–H12w/O11x
O2w–H22w/Cl1vi
O2w–H21w/Cl2vi
O4w–H42w/O22x
3.3. Potential energy surfaces (PES)
A systematic conformational analysis of [3HC] and [4HC]
was accomplished over each of the torsion angles identified in
Fig. 1. Variations in amide torsional angles were not
considered due to the fact that cis-amide geometries were not
observed for either 3 or [4HC] in the X-ray diffraction study.
The interdependence of each dihedral angle analyzed was
studied from pair-wise contour plots and PES derived for each
of the three possible combinations of torsion angles.
Comparison of the pattern for each torsion angle in all the
generated contour plots indicated a fair degree of independence
for q1, q2 and q3. As expected, the PESs for each pair-wise
combination of torsional angles were very similar for [3HC]
and [4HC]. In general, the q1 torsion presented a symmetric
potential energy profile with an energy barrier around q1Z08
and no marked minimum while assuming most stable values at
G20–408, G100–1208, G140–1808. A more interesting profile
is observed for [3HC] where a clear barrier also exists for q1Z
1808 and another minor barrier at ca. q1ZG908. The common
barrier around q1Z08 can be ascribed to the steric clashes
between H–N1 and H–C6 when the planes for the phenyl ring
and neighboring amide bond are aligned. On the other hand, the
energy profile for the two derivatives indicates differences at
q1Z1808. In [3HC], q1 torsion angle near to 1808 places the H–
N1 of the adjacent amide linkage in perfect position to make a
strong hydrogen bonding interaction with F2. This stabilizing
interaction is not so effective in [4HC], due to the greater size
and lower electronegativity of the chlorine atom. Regarding q2,
for each of [3HC] and [4HC], a deep minimum in the energetic
profile is observed at 08. In this conformation, the molecule’s
carbonyl moieties point in the same direction. With respect to
q3, a profile similar, but even more featureless, than that for q1
is obtained for both analogs.
Symmetry operations: (i) xC1, Cy, CzC1; (ii) x,Cy, CzK1; (vi) xC1, Cy,
Cz; (x) xK1, Cy, Cz.
respectively). However, a comparison of the rmsd of the bond
2
angles clearly indicates the RHF method (rmsd 2.0 A ) to be
˚
2
˚
better than the DFT-B3LYP (rmsd 2.9 A ) approach in
predicting the compound planar geometries. Similar results
were obtained for the ‘syn’ conformers.
Considering again the data in Table 4, one can clearly note
that after optimization, energy could be reduced by as much as
0.2 hartree (w125 kcal/mol). This large improvement in
stabilization was completely unexpected, since crystallo-
graphic structures usually correspond to local minima in the
PES. The origin of this discrepancy could reside in the
intermolecular interactions between symmetry-related [4HC],
in the crystal, obviously not captured by calculations made
with the isolated conformers in vacuo. As will be discussed in
the following sections, this issue was assessed using different
methodologies, such as conformational analysis for both
[3HC] and [4HC] and supermolecule calculations on [4HC].
Table 4
Electronic energies for the experimental and theoretically optimized
conformers of [4HC
]
Conformer
q1 (8)
q2 (8)
q3 (8)
Eela (hartrees)
Anti-RHF
Crystal
K66.5
K37.5
85.7
159.9
K1729.70098
K1729.92628
Optimized
Anti-B3LYP
Crystal
70.4
147.5
3.4. Characterization of minimum energy conformers
K66.5
0.4
85.7
159.9
168.9
K1736.00397
K1736.25814
Optimized
Syn-RHF
Crystal
172.3
From the analysis of the PESs for each of [3HC] and [4HC],
we selected seven conformers at q2Z08 and varying q1 and q3
to be subjected to geometry optimizations with RHF/6-31G*.
Results are summarized in Table 5. From the calculations for
the conformers of [3HC], only three unique equilibrium
geometries were found: [3HC_0080], [3HC_0217] and
[3HC_0794]. Noteworthy, none of the equilibrium geometries
found for [3HC] were representative of either crystallographic
K131.6
K135.2
K82.6
K66.3
K153.9
K146.1
K1729.70110
K1729.92428
Optimized
Syn-B3LYP
Crystal
K131.6
K145.2
K82.6
K71.2
K153.9
K155.3
K1736.00432
K1736.24923
Optimized
a
Energies calculated using the standard 6-31G* basis-set with the methods
(RHF or DFT-B3LYP) indicated in the table. Eel values under ‘crystal’ rows
were obtained from single-point calculations with X-ray geometries.

F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
69
Table 5
Relative stabilities of minimum energy conformers for [3HC] and [4HC
]
a
EZPE (hartree)
b
Erel (kcal/mol)
Conformer
q1 (8)
q2 (8)
q3 (8)
E (hartree)
[3HC
RHF
]
0000c
K3.8
6.0
147.8
K71.3
69.2
K56.1
K34.6
57.4
K1009.84209
K1009.84129
K1009.83493
K1009.83320
0.23926
0.23924
0.23918
0.23893
0.0
0.5
4.4
5.4
0217
0080
67.3
0222
K141.9
K93.6
K33.3
DFT-B3LYP
0000c
0217
K5.5
8.4
K71.1
75.5
K57.0
K27.2
14.2
K1015.53918
K1015.53785
K1015.53396
K1015.53122
0.22172
0.22156
0.22095
0.22167
0.0 (0.0)
0.7 (0.4)
2.8 (2.4)
5.0 (4.5)
0222
K154.2
152.1
K156.8
67.3
0080
58.7
[4HC
RHF
1082
0227
1230
0798
]
K37.5
40.4
70.3
68.0
147.5
K34.6
146.7
K1729.92627
K1729.92638
K1729.92466
K1729.92428
0.23568
0.23599
0.23556
0.23587
0.0
0.1
0.9
1.4
K117.3
K135.2
69.0
K66.3
K146.1
DFT-B3LYP
0227
21.6
21.6
75.5
75.4
K27.0
K27.1
154.3
K1736.25280
K1736.25279
K1736.24890
K1736.24923
0.21840
–
0.0 (0.0)
0.0 (–)
1082
1230
K129.0
K145.1
72.1
0.21812
0.21842
2.3 (2.0)
2.3 (2.2)
0798
K71.3
K155.1
a
Computed by frequency calculations using the standard basis-set 6-31G* and the methods (RHF or DFT-B3LYP) indicated in the table.
b
Energy relative to the most stable conformer; figures given in parenthesis correspond to the results of DFT-B3LYP/6-311CCG**//DFT-B3LYP/6-31G*
calculations.
c
Conformer 0000 represents the RHF/6-31G* optimized geometry of [3HC]-c.
geometry found by X-ray diffraction data. Moreover, in none of
the three equilibrium geometries found for [3HC] were the
amide linkages trans as found in the X-ray structure but these
are gauche where a higher dipole moment would be expected.
These experimental geometries are characterized by a mostly
planar conformation with the amide linkages nearly co-planar
with both phenyl rings and the central carbonyls trans to each
other. These differences are somewhat expected since X-ray
geometries refer to the anti and syn conformations of the free
base, 3. Accordingly, for [4HC], both conformations given by
X-ray diffraction analysis could be found among the four
minimum energy conformers obtained by the gas-phase
calculations ([4HC_0227], [4HC_0798], [4HC_1082] and
[4HC_1230]). The [4HC_1082] conformer presented a similar
geometry to the ‘anti’ conformer present in the asymmetric
unit cell, while conformer [4HC_0798] was equivalent to the
‘syn’ conformer (Table 5).
[3HC_0222] that shows significant deviations in q2 and q3
upon optimization at the DFT-B3LYP theory level. The
relative energies reported in Table 5 also reflect this behavior.
The conformer [3HC_0000] is predicted to be more stable than
the conformers 0217 and 0080 by almost the same amounts
with RHF and DFT-B3LYP energy models (0.5–0.7 and 4.4–
5.0 kcal/mol, respectively). This result is maintained despite
the employment of a much larger basis-set, discarding possible
basis-set effects. On the other hand, the results for [3HC_0222]
differ by more than 1.5 kcal/mol when comparing calculations
performed at the two theory levels. This discrepancy actually
inverts the stability order regarding [3HC] conformers 0080
and 0222 when going from RHF to DFT-B3LYP calculations.
Fig. 5 shows the structures for the symmetry-unique
[3HC_0222] conformers optimized at the DFT-B3LYP
(B3LYP) and RHF levels of theory. It can be noted that the
conformer obtained after optimization with DFT-B3LYP
presents a quite planar structure contrasting with the ‘packed’
geometry, which is furnished by the RHF method.
Regarding [4HC], results in Table 5 demonstrate an even
more pronounced disagreement in geometries obtained by
calculations performed with RHF and DFT-B3LYP energy
models. The extreme case is that of the [4HC_1082] conformer
whose deviation in q1 dihedral angle amounts to almost 608
upon optimization with the DFT-B3LYP method. Indeed, the
[4HC_1082] conformer obtained after DFT-B3LYP geometry
optimization is equivalent to the geometry of the 0227
conformer obtained at the same theory level. Interestingly,
these conformers are also nearly energetically equivalent at the
RHF level, although presenting different geometries as
Each of the symmetry-unique equilibrium geometries
encountered for [3HC] and [4HC] using the RHF/6-31G*
calculation was subjected to further optimization within the
DFT-B3LYP theory level while maintaining the same basis-
set. This model would take some account of electron
correlation effects, which are completely neglected by the
RHF method, at a reasonable computational cost. The
electronic and relative energies (corrected for ZPE) for the
RHF and DFT-B3LYP optimized final geometries are listed in
Table 5. In the case of [3HC], most of the conformers retained
their geometries after optimization within DFT-B3LYP
method, including conformer [3HC_0000] (the RHF/6-31G*
optimized geometry of [3HC]-c). The exception is

70
F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
Fig. 5. Comparison of selected minimum energy conformers encountered for [3HC] and [4HC] at RHF/6-31G* (RHF) and DFT-B3LYP/6-31G* (B3LYP) levels.
depicted by the values of the dihedral angles reported in
Table 5 and by the structures in Fig. 5. The results of the
calculations with RHF and DFT-B3LYP also disagree over the
relative stabilities of the conformers by about 1 kcal/mol,
although roughly preserving the relative stability order.
energy is relative to the calculation performed in vacuo and
that a much smaller interaction energy should be expected in a
solvated system, mainly due to shielding of the electrostatic
interactions. We have actually attempted to make the same
supermolecule calculation employing iterated and non-iterated
polarization continuum methods (IPCM and PCM, respect-
ively) to estimate, quantum chemically, the solvation
enthalpies involved but calculations failed to converge with
[4HC, ClK$2(H2O)].
3.5. Supermolecule calculation
Supermolecule calculations are commonly employed to
estimate the magnitude of non-covalent interactions in
molecular complexes. Accordingly, we have performed this
type of calculation with the complex found in the asymmetric
unit of crystalline [4HC,ClK$2(H2O)] using DFT-B3LYP/6-
31CG*. The polarization function was introduced to estimate
more properly the interaction energies in the presence of
chloride anions while the basis-set superposition error (BSSE)
was treated by applying counterpoise correction. The inter-
molecular interaction energy in [4$HC,ClK$2(H2O)] was
estimated to be K175.2 kcal/mol (corrected for BSSE) and
the BSSE energy was calculated as 7.0 kcal/mol. The
magnitude of this stabilization energy is comparable to the
difference between Eel in crystallographic and theoretically
optimized [4HC] conformers (around 0.2 hartree) and gives
support to our previous discussion about the origin of such a
discrepancy. It should be clear that this huge stabilization
4. Concluding remarks
X-ray crystallography and quantum chemical calculations
using a standard medium-sized basis-set within two model
chemistries were employed to characterize the structures of
two halogenated isoniazid derivatives active against the
tuberculosis bacilli. In general, DFT-B3LYP model furnished
[4HC] conformations in better agreement with experiment than
the simpler RHF method, possibly due to some account of
polarization effects by the former. The results of these
calculations also evidenced a high degree of distortion in the
geometries of crystallized compounds compared to theoretical
gas-phase structures. Supermolecule calculations within the
asymmetric unit of crystalline [4$HC, ClK$2(H2O)], using
DFT-B3LYP/6-31CG*, suggest that such distorted structures

F.P. Silva et al. / Journal of Molecular Structure 788 (2006) 63–71
71
result from polar and electrostatic interactions within the
solvent and other symmetry-related species in the crystal. The
characterization of the PES considering three significant
dihedral angles showed that the studied isoniazid derivatives
present highly flexible structures. In general, an energetic
barrier could not be directed for restricted rotation of any of the
varied torsion angles at room temperature, even though the
fluorinated compound [3HC] showed an important energy
barrier for rotation around q1 due to a strong internal hydrogen
bond between F4 and H–N6. Differences in electronic energies
for the most stable gas-phase conformers were found to amount
to a maximum of 2–5 kcal/mol.
fruitful discussions on quantum chemical calculations and Dr
Ricardo Bicca for making Gaussian03 available. FPS Jr was a
doctoral fellowship recipient from the CNPq. JLW thanks
CNPq for financial support.
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Sa˜o Paulo and Rio de Janeiro State Foundations for the
Support of Research (FAPESP and FAPERJ) and the National
Council for Scientific Research and Development (CNPq) for
[11] D.A. Rozwarski, G.A. Grant, D.H.R. Barton, W.R. Jacobs Jr.,
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´
financial support. FPS Jr thanks to Dr Pierre Mothe Esteves for