Conformations and interactions in pasiniazid: A spectroscopic and computational characterization

Article abstract of DOI:10.1016/j.molstruc.2016.11.095

In this work, the conformations of isonicotinic acid hydrazide 4-aminosalicylate (pasiniazid) has been comprehensively investigated by analyzing the potential binding site on isoniazid and para-aminosalicylic acid. The powder X-ray diffraction and vibrational spectroscopy analysis with quantum chemical calculation are utilized to characterize the appropriate conformation of pasiniazid. Two hydrogen-bond systems are found in pasiniazid and the relevant vibrational modes have been assigned with the help of potential energy distribution analysis. Natural bond orbital analysis is implemented for evaluating interactions in pasiniazid. The result indicates that the intermolecular interaction is responsible for the stabilization of this complex. Furthermore, molecular electrostatic potential provides an intuitive vision for intermolecular interaction between isoniazid and para-aminosalicylic acid. The chemical stability is estimated by the frontier molecular orbitals analysis.

Full text of DOI:10.1016/j.molstruc.2016.11.095

Accepted Manuscript
Conformations and interactions in pasiniazid: A spectroscopic and computational
characterization
Zhen Li, Yude Wang, Sheng Chen, Linwei Li, Tiemin Sun
PII:
S0022-2860(16)31286-8
10.1016/j.molstruc.2016.11.095
MOLSTR 23196
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 7 July 2016
Revised Date: 30 November 2016
Accepted Date: 30 November 2016
Please cite this article as: Z. Li, Y. Wang, S. Chen, L. Li, T. Sun, Conformations and interactions in
pasiniazid: A spectroscopic and computational characterization, Journal of Molecular Structure (2017),
doi: 10.1016/j.molstruc.2016.11.095.
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ACCEPTED MANUSCRIPT
Conformations and Interactions in Pasiniazid: A Spectroscopic and
Computational Characterization
Zhen Li, Yude Wang, Sheng Chen, Linwei Li, Tiemin Sun
*
Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang Pharmaceutical
University, Ministry of Education. Shenyang 110016, PR China
Corresponding author. Tel. /fax: +86-24-23986398.
E-mail address: suntiemin@126.com (Tiemin. Sun).
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Abstract
In this work, the conformations of isonicotinic acid hydrazide 4-aminosalicylate (pasiniazid)
has been comprehensively investigated by analyzing the potential binding site on isoniazid and
para-aminosalicylic acid. The powder X-ray diffraction and vibrational spectroscopy analysis
with quantum chemical calculation are utilized to characterize the appropriate conformation of
pasiniazid. Two hydrogen-bond systems are found in pasiniazid and the relevant vibrational
modes have been assigned with the help of potential energy distribution analysis. Natural bond
orbital analysis is implemented for evaluating interactions in pasiniazid. The result indicates that
the intermolecular interaction is responsible for the stabilization of this complex. Furthermore,
molecular electrostatic potential provides an intuitive vision for intermolecular interaction
between isoniazid and para-aminosalicylic acid. The chemical stability is estimated by the
frontier molecular orbitals analysis.
Keywords: Pasiniazid; Electrostatic potential surface analysis; X-ray powder diffraction;
Vibrational assignments; Natural bond orbital analysis;
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1. Introduction
Pasiniazid, isonicotinic acid hydrazide 4-aminosalicylate, is an efficient drug for
tuberculosis (TB) treatment, especially for drug-resistant TB [1-3]. Generally, pasiniazid is
produced by cooling down the mixture solution of two classical anti-TB drugs, isoniazid (INH)
and para-aminosalicylic acid (PAS), in the molar ratio of 1:1. However, there are differences
between pasiniazid and mixture of INH and PAS in physicochemical aspect, such as solubility. In
addition, compared with taking INH and PAS separately, pasiniazid reveals prominent curative
efficacy and pharmacokinetic advantages in TB treatment, which also indicates that pasiniazid
cannot be considered simply as a mixture of INH and PAS but a pharmaceutical complex in
pharmacological aspect [4, 5]. The pharmacological mechanism and clinical application of
pasiniazid have been investigated extensively [4-6]. According to literature, the major metabolic
pathway of INH is attributed to the acetylation through the livers prevailingly by the
polymorphic enzyme acetyltransferase-2 (NAT-2) [7-9]. Some suggest PAS is able to retard the
acetylation pathway of INH, especially in rapid acetylators, resulting in increasing plasma levels
of the anti-tuberculosis agent and extending effective time [11, 12]. Since numerous complex
drugs are found showing pharmacological advantages due to the interaction among the
monomeric drug molecules, we speculate the interaction between INH and PAS is the key to
explain the interesting pharmacokinetic property of pasiniazid. To the best of our knowledge, the
geometry and interaction of pasiniazid have not been fully described in detail. Therefore, it’s
necessary to implement a comprehensive conformation investigation on pasiniazid, which can
provide a visual method to understand the interactions in pasiniazid. In this circumstance,
vibrational spectroscopy combined with quantum chemical calculations are often carried out as a
useful way of studying the structural properties and the interactions.
The aim of this work is not only to seek an appropriate conformation of pasiniazid but also
to characterize the interaction in pasiniazid. Hence, density functional theory (DFT) method is
employed to seek the conformations for pasiniazid and obtain thermodynamic information.
Spectroscopic (PXRD, IR and Raman) analysis combining computational and experimental data
is presented to characterize structural properties. The vibrational modes related to the
hydrogen-bond system are pointed out for discussion. Natural bond orbital analysis is carried out
to verify the potential intra- and inter-molecular interactions between the units that are capable to
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form the hydrogen bond relationships. The chemical stability of pasiniazid is evaluated by
molecular electrostatic potential surface and frontier molecular orbitals analysis.
2. Materials and Methods
2.1. Synthesis
INH was blended with PAS (in the molar ratio of 1:1) dissolving in ethanol at reflux
temperature for 0.5 h. Then, crystalline powder of pasiniazid was generated after cooling the
reaction to room temperature. The powder has been dried for spectrums measurement.
1
DSC (5 °C min-1): 142.16 °C (mp). H NMR (300 MHz, [D6]DMSO): δ (ppm) 8.72 (dd,
J=4.5; 1.6 Hz, 2H), 7.74 (dd, J=4.5; 1.6 Hz, 2H), 7.44 (d, J=8.6 Hz, 1H), 6.11 (dd, J=8.6; 2.0 Hz,
1H), 5.99 (d, J=2.0 Hz, 1H). 13C NMR (75 MHz, [D6]DMSO): δ (ppm) 172.3, 164.2, 163.7,
+
+
156.0, 150.4, 140.5, 131.7, 121.2, 106.4, 100.4, 98.8. GC-MS (EI, m/z): 137 (M1 ), 153 (M2 ).
2.2. Instruments
13
1H and C NMR spectroscopic data were recorded on a BRUKER ARX-300 spectrometer
with tetramethylsilane (TMS) as an internal standard and [D6]DMSO as the solvent. Mass
analysis was performed on a gas chromatography-mass spectrometry (GC-EI/MS,
GCMS-QP5050A, Shi madzu Corporation, Kyoto). Differential scanning calorimetry (DSC)
scans were performed using a DSC-60 (Shimadzu, Kyoto, Japan) calorimeter. The sample was
heated in sealed aluminum pans under nitrogen flow (30 ml min-1) at a scanning rate of 5 °C
min-1 from 30 to 220°C. The powder X-ray diffraction (PXRD) pattern was measured using a
Bruker powder diffractometer D8 Advance with Cu Kα radiation (λ=1.54056 Å) at the scan rate
0.01° s-1 for the 2θ angular range of 10-80°. According to the main peaks of diffraction pattern,
the range of PXRD pattern presented in this article will be in 10-50°. The infrared spectrum of
the sample was recorded between 4000 and 400 cm-1 on a BRUKER IFS-55V IR spectrometer
which was calibrated by polystyrene. The sample was prepared as a KBr disc. The Raman
spectrum in the 3500-50 cm-1 range was measured on a Thermo DXR dispersive Raman
microscope with a 532 nm lasers. The resolution was 1.0 cm-1 both in IR and Raman studies.
2.3. Theoretical Methods
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Spartan08 [10] was employed to acquire the conformers distribution of INH and PAS by
means of MMFF94 method [11]. All further structural optimization and vibrational calculation
were performed using Gaussian 09 software package [12]. The density functional theory method
was carried out. And the Becke three-parameter hybrid exchange functional and the
Lee-Yang-Parr correlation functional (B3LYP) were utilized in the calculation with the
6-311++G (2d, p) basis set [13-15]. In order to offset the systematic errors caused by basis set
incompleteness, neglect of electron correlation and vibrational anharmonicity [16], the least
square method was carried out to obtain scaling factor (0.992) with comparison of the major
vibrational modes at experimental and computational IR spectrum.
The lattice parameters of pasiniazid was calculated by EXPO2013 program [17] following
the Le Bail algorithm and using a split type pseudo-Voigt peak profile function [18, 19]. And the
Crystallographic Calculations program provided by FullProf_Suite software package [20] was
carried out to simulate the pattern based on the experimental crystallographic data and the
conformation we achieved in this article.
The computational Raman activities (Si) have been converted to related Raman intensities (Ii)
using the following relationship according to the basic theory of Raman scattering:[21, 22]
f (ν 0
−
ν i )4 Si
− hc
（1）
Ii =
ν
ν i[1− exp(
i )]
kT
Where νo is the exciting frequency (in cm-1 unit);
νi the vibrational wave number of the ith normal mode;
Si is the Raman scatting activity of the normal mode νi;
h, k, c and T are Planck and Boltzmann constants, speed of light and temperature in
Kelvin, respectively;
f is the suitably chosen common scaling factor for all the peak intensities.
The potential energy distribution (PED) was estimated by VEDA4 (Vibrational Energy
Distribution Analysis) program [23]. The vibrational modes were assigned by combining the
result of Gaussview 5.08 [24] and PED analysis.
The natural bond orbital (NBO) analysis was carried out by means of NBO 3.1 program [25]
in the Gaussian 09 package at the same DFT level. And the energy of hyper conjugative
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interactions E (2) value, which represents the extent of electron delocalization between donor
NBO (i) and acceptor NBO (j), is estimated by the second order Fock matrix:[26]
F(i, j)2
E(2) = ∆Eij = qi
(2)
ε j −εi
Where qi is the donor orbital occupancy;
εi, εj is the diagonal elements (orbital energies);
F (i,j) is the off diagonal NBO Fock matrix element.
3. Results and discussion
3.1. Conformational analysis
For a comprehensive conformations investigation, a full scan of potential energy surfaces is
required, which would lead to a gigantic computation cost. In order to lower the cost without
missing possible conformers, the probable conformers for pasiniazid are preferred to be
investigated by speculating the potential interactions between INH and PAS. As a matter of fact,
INH and PAS can be detected separately in a low-concentration solution of pasiniazid, which
reveals that the non-covalent interactions represent the major interactions in pasiniazid. And as a
strong non-covalent interaction, hydrogen bond (HB) makes a significant contribution for
stabilizing conformation [27]. Because of these, the electrostatic potential analysis is carried out
to present the charges distribution of pasiniazid intuitively for revealing the potential interactive
site.
Two stable conformers are obtained both for monomeric INH and PAS. After that, Mulliken
charges and electrostatic potential surfaces for 4 conformers are obtained to give a preliminary
prediction of the potential interactive sites shown in Fig. 1. The color range of the electrostatic
potential map range from -5.507 e-2 (the deepest red) to 5.507 e-2 (the deepest blue). Mulliken
charges analysis reveals the negative and positive charges concentrate on the chain side
comparing with aromatic rings. As a consequence, the potential binding site is located on the
hydrazine group from INH and carboxyl group from PAS.
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Figure 1. Electrostatic potential surfaces for INH (y1 and y2) and PAS (s1 and s2). Mulliken
charges of potential binding regions are given in red and blue fonts.
Figure 2. Eight conformers of pasiniazid and Gibbs free energy difference comparing with
conformer 1.
As plotted in Fig. 1, the red region denotes electron concentrating area and the blue region
shows a shortage of electrons. The red region can be considered as an acceptor and the blue
region is the donor. In addition, more formations of interactions between acceptor and donor
make conformer more stable. In this circumstances, two hydrogen bonds can be most formed
between INH and PAS. For INH, both of y1 and y2, the carboxyl and hydrazine group can be
regarded as acceptors due to the possession of lone pair electrons. And the hydrogen from
hydrazine group is qualified to be a donor. On the other side, both conformers of PAS, s1 and s2,
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have one acceptor and donor around carboxylic group, which is capable to interact with INH.
With permutation and combination, eight conformers of pasiniazid are generated and optimized
at the same level as mentioned before in Fig. 2. And the thermodynamics analysis show the
Gibbs free energy of pasiniazid is much lower than summation of PAS and INH, which suggests
the combination between PAS and INH is a spontaneous process in thermodynamics and the
generated conformation is stable. Compared the Gibbs energy of eight conformers, conformer 1
is the most stable conformer with 95.8% Boltzmann contribution. Therefore, conformer 1 is
capable of representing the reasonable vision and interactive relationship for pasiniazid, which is
chosen for further study.
3.2. Powder X-ray diffraction investigation
Despiting it is difficult to acquire structure directly from the data of powder X-ray
diffraction, PXRD still contains structural information of the solid powder. Therefore, PXRD
analysis is carried out by means of comparing the experimental and calculated pattern to give a
preliminary evaluation on the reliability of the conformation carried out by conformational
analysis. Based on the experimental PXRD data, the crystallographic data of pasiniazid is
calculated by Expo2013 program [17] showed in Table 1. The result shows a triclinic cell with
a=16.66 Å, b=7.786 Å, c=6.299 Å, α=98.42°, β=99.89°, γ=82.31° and V=791.2 Å3. The most
probable space group is determined as P-1 by means of the FINDSPACE module in the
integrated package EXPO2013 [17]. Then, the theoretical powder X-ray diffraction pattern is
simulated by combining the calculated conformation and crystallographic data, shown in Fig. 3.
Result reveals that the major peaks of simulation pattern show great agreement with the
experimental data. As a consequence, we believe the appropriate conformer for pasiniazid is
found by computational simulation.
Figure 3. Experimental and calculated X-ray powder diffraction patterns of pasiniazid.
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Table 1 Calculated crystallographic data for pasiniazid
Formula
Mol.wt.
Crystal group
Space group
2θ range(°)
Step(2θ)(°)
λ(Å)
a(Å)
b(Å)
c(Å)
V(Å3)
C13H14N4O4
290.27
triclinic
P-1
10-50
0.01
1.54056
16.66
7.786
6.299
791.2
1
0.6094
298K
0.595
0.194
0.868
Z
ρcalc. (g cm-3)
T
Rwp
Rp
RF
Rwp =[ ∑w(Io – Ic)2/∑(wIo )]1/2; Rp = ∑│Io – Ic│/∑Ic ; RF = <│Fo│-│Fc│>/<│Fo│>.
2
3.3. Hydrogen bond description and Mulliken charges analysis
The plot of optimal conformation is distinctively shown in Fig. 4. Some geometrical
parameters related to potential interaction sites with Mulliken atomic charges are listed in Table
2. Obviously, there are three interactive connections in pasiniazid. Intramolecularly, the phenolic
hydroxyl interacts with carbonyl O atom as well as in monomeric PAS. The hydrazino group
from INH is involved in the intermolecular interaction with carbonyl group from PAS. The
distance of H16…N15, H14…O27 and H32…O27 is 1.764 Å, 1.934 Å and 1.716 Å respectively.
And the angle of O28-H16…N15, N8-H14…O27 and O25-H32…O27 is 177.7°, 151.6° and 147.6°.
Based on the distance, the strength of the hydrogen bond can be approximately defined as strong
(below 2.5 Å), normal (2.5-2.7 Å) and weak (above 2.7 Å). Therefore, all these are qualified to
establish strong hydrogen bonds. In Table 2, the difference of Mulliken charges between
monomeric INH and PAS and pasiniazid reveals the significant tendency of charges transfer
between INH and PAS. As shown in the Fig. 4, N15 is restricted by interacting with O28-H16,
which might decrease chemical activity on N15 and block the acetylation pathway of INH. The
particular evaluation of this interaction will be discussed in NBO analysis section.
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Figure 4. The optimal conformation of pasiniazid.
Table 2 Mulliken charges and geometrical parameters of hydrogen bond
Parameters Mulliken charges (e-2)
Parameters
O28-H16…N15
N8-H14…O27
O25-H32…O27
Distance(
Å
)
Angle(°)
177.7
N15
H16
O27
H14
H32
-0.844
0.605
-0.695
0.350
0.420
(-0.467)
(0.307)
(-0.607)
(0.254)
(0.378)
1.764
1.934
151.6
1.716
147.6
The numbers in brackets signify the Mulliken charges from INH (y1) and PAS (s1) respectively.
3.4. Vibrational analysis
Vibrational spectroscopy has been proven as an efficient tool for characterizing functional
groups. And it is also extensively utilized in studying interaction in complex compounds [28-30].
Pasiniazid contains 35 atoms which declare 99 normal modes of vibrations under C1 point group
symmetry, according to non-linear molecular vibrational mode formula (3N-6). All these
vibrations are both IR and Raman active. The theoretical vibration spectrum is scaled by the
factor (0.992) acquired from the least squares calculation. The correlation coefficient of the
linear regression between theoretical and experimental data is 0.9996, which demonstrates the
computational conformer can be appropriately used to explain the interaction between INH and
PAS. For an intuitive view, the comparison figure of experimental and calculated IR spectrum is
plotted in Fig. 5. And the comparison of Raman spectrum is presented in Fig. 6. The theoretical
and experimental wavenumbers along with intensity and approximate assignments are listed in
Table 3. Since the vibrational wavenumbers in fingerprint region are too close to being
distinguished, the influence on the single peak in this region is driven by multiple structural
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features, which is difficult to unravel. Therefore, the discussion of fingerprint region is excluded
in this section.
Figure 5. Experimental and theoretical IR spectrum of pasiniazid.
Figure 6. Experimental and theoretical Raman spectrum of pasiniazid.
Apparently, there are two hydrogen-bond systems appear in pasiniazid as plotted in Fig. 4.
The phenolic hydroxyl (O25-H32) interacts with the carbonyl group (C24=O27) resulting in a
six-membered HB system. Usually, the stretching vibration of hydrogen-bonded phenolic
hydroxyl appears around 3200 cm-1 in IR spectrum. However, in our present work, the carbonyl
group is involved in another hydrogen bond with the N8-H14, leading to an increase of the OH
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stretching wavenumber by more than 100 cm-1 [31]. As a consequence, the corresponding
stretching vibration is observed at 3365 cm-1 in IR spectrum. The computed wavenumber for this
mode of vibration at 3369 cm-1 shows a great agreement with experimental observation. And
according to the PED analysis, the band observed at 1416 cm-1 in IR spectrum is most
contributed by the bending vibration of this phenolic hydroxyl, corresponding to 1416 cm-1 in
calculated spectrum. Generally, the stretching vibration of carboxylic carbonyl group is expected
around 1700 cm-1 with a very intense and narrow peak, which can be easily distinguished from
different vibration modes [28]. The stretching vibration of carbonyl C24=O27 is observed at
1657 cm-1 in IR and 1639 cm-1 in Raman as strong band. The theoretical wavenumber at 1669
cm-1 confirms this vibration mode with good match. The wavenumber of this stretching mode is
lower from normal value. This down shift of carbonyl stretching vibration is mainly attributed to
the fact that this carbonyl group not only forms the intramolecular interaction with O25-H32, but
it also involves in an intermolecular interaction with N8-H14.
Table 3 Selected experimental and calculated vibrational wavenumber (in cm-1) and vibrational
assignments of pasiniazid.
Mode
Experimental
Calculated
No.
IR
Raman
Unscaled
Scaled
IIR
IRaman
Assignments, PED (%) ≥10%
1
3682
3579
3492
3437
3424
3396
3205
3205
3192
3180
3170
3156
3153
3046
1718
1692
1682
1663
1649
3653
3550
3464
3410
3397
3369
3179
3179
3166
3155
3145
3131
3128
3022
1704
1678
1669
1650
1636
25.4
55.2
15.8
343.0
158.3
905.8
3.0
71.9
336.3
77.7
176.4
454.6
7.9
νasH33-N26-H34(100)
2
νsH33-N26-H34(100)
3
νasH17-N15-H35(100)
4
3440 m
3365 s
3433 w
3349 w
νsH17-N15-H35(49) + νN8-H14(46)
νsH17-N15-H35(50) + νN8-H14(42)
νO25-H32(87) + νN8-H14(11)
νC18-H29(95)
5
6
7
85.5
93.4
113.7
95.0
104.4
133.5
108.6
354.6
49.6
4.9
8
2.7
νC2-H11(97)
9
4.9
νC4-H12(95)
10
11
12
13
14
15
16
17
18
19
3.9
νC21-H31(99)
8.1
νC19-H30(95)
5.9
νC1-H10(56) + νC5-H13(40)
νC5-H13(56) + νC1-H10(42)
νO28-H16(93)
3096 w
1680 s
1657 s
1626 s
27
3082 s
1639 s
2532.3
147.1
68.5
787.7
110.2
78.6
νC7=O9(93)
δH17-N15-H35(51) + νC7=O9(17)
νC21-C22(21) + νC24=O27(13) + δC22-O25-H32(12)
δH33-N26-H34(62)
264.5
5.1
27.9
δH33-N26-H34(23) + νC24=O27(20) + νC18-C19(10)
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20
21
1632
1609
1619
4.6
48.1
νC4-C5(28) + νC2-C3(11)
νC19-C20(16) + δC22-O25-H32(15) + νC20-C21(11) +
νC18-C23(10)
1581 s
1596
118.9
5.7
νC2-C3(27)
+
νC1-N6(25)
+
δC4-C5-N6(12)
+
22
1596
1583
30.8
3.8
δC1-C2-C3(11)
23
24
25
26
27
1543
1537
1520
1509
1468
1531
1525
1508
1497
1456
30.2
8.5
δC7-N8-H14(16) + δC20-C19-H30(11)
δC7-N8-H14(17)
1527 m
1502 m
429.6
198.4
140.6
2.1
23.8
32.9
30.9
26.2
δC7-N8-H14(20) + δN6-C1-H10(15) + δN6-C5-H13(15)
δC24-O28-H16(21)
νC24=O27(21) + νC21-C22(10)
δN6-C5-H13(28) + δN6-C1-H10(23) + νC1-C2(12) +
νC4-C5(11)
28
1443
1431
8.6
7.4
29
30
1416 w
1356 m
1427
1373
1416
1362
105.5
312.7
14.8
δC22-O25-H32(35) + νC20-C21(16)
νC23-C24(12)
1359 m
189.1
Abbreviations: s - strong; m - medium; w - weak.
v - stretching vibration; νas - asymmetric stretching vibration; νs - symmetric stretching vibration;
δ - in-plane bending vibration;
The atoms are labeled in Fig. 4. PED is evaluated by VEDA4 program.
The scale factor is 0.992 calculated by the least square method.
IIR, IR intensity / K mmol-1; IRaman, Raman intensity / a.u..
On the other hand, the interaction between the hydrazine group and the carboxyl group
establish an intermolecular seven-membered HB system. The stretching vibration of N8-H14 is
observed at 3440 cm-1 in IR and 3433 cm-1 in Raman, consisting with the theoretical
wavenumber at 3410 cm-1. And the corresponding bending vibration is located at 1527 cm-1 in IR
in a great agreement with the calculated wavenumber at 1525 cm-1. The band observed at 3082
cm-1 in Raman spectrum is assigned to the stretching vibration of hydroxyl O28-H16
corresponding to the theoretical wavenumber at 3022 cm-1 in DFT calculation. The stretching
vibration of monomeric carboxylic hydroxyl usually appears around 3500 cm-1 as a narrow peak.
The formation of strong hydrogen bond can lower the O-H strength frequency. And the in-plane
bending vibration of O28-H16 is observed at 1502 cm-1 in IR spectrum, confirmed by the
theoretical wavenumber at 1497 cm-1.
To study the influence of hydrogen-bonded interaction in the vibrational aspect, vibrational
modes of monomeric INH and PAS are calculated respectively at the same DFT level. To
compare with the theoretical wavenumbers of monomeric INH and PAS, the correlate vibrational
wavenumbers of N8-H14, O28-H16 and O25-H32 confirmed by experimental spectrums are
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presented in Table 4. The carbonyl C24=O27 in PAS is involved in another interaction with
N8-H14 after the combination of INH and PAS. As a consequence, the hydrogen bond between
O25-H32 and C24=O27 is weakened resulting in the down shift of bend vibration of O25-H32
[31]. The unchanged wavenumber of corresponding stretching vibration is probably because of
mixing with the vibration of N8-H14, which is also revealed by the theoretical PED analysis.
The down-shifted stretching vibration and up-shifted bend vibration of N8-H14 also confirm
presence of the intermolecular interaction between C24=O27 and N8-H14. In the
seven-membered HB system, another hydrogen bond is established between O28-H16 and N15.
The significant down shift of stretching vibration of O28-H16 is observed by comparing the
theoretical wavenumber between pasiniazid and PAS. And due to the formation of this hydrogen
bond, the double bond character of carbonyl group is decreased with lowering wavenumber of
stretching vibration of C24=O27 subsequently.
Table 4 Theoretical vibrational difference between pasiniazid and monomeric INH (y1) and PAS
(s1).
No.
ν
δ
N8-H14
O28-H16
O25-H32
C24=O27
3437
3046
3396
1682
(3610)
(3765)
(3396)
(1707)
1537
1509
1427
(1496)
(1402)
(1441)
The numbers in brackets signify the wavenumber of functional groups from monomeric INH (y1)
and PAS (s1). ν, stretching vibration; δ, in-plane-bending vibration;
3.5. Natural bond orbital analysis
Natural bond orbital analysis has been generally accepted in the investigation of hyper
conjugative interactions in molecular system. Furthermore, it’s also an efficient tool for studying
electron density transfer from the filled lone pair electrons [32-36]. In energetic terms, hyper
conjugation can be described as an overlap between an occupied Lewis-type natural bond orbital
(donor) and a non-Lewis-type orbital (acceptor). This electronic delocalization demonstrates a
charge transfer from an electron donor, with decreasing of its occupancy, to an electron acceptor,
resulting in the stabilization of the system. The energy of hyper conjugative interactions E (2)
value obtained from the result of NBO calculation represents the lowering of orbital energy
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causing by the interaction between doubly occupied orbitals and unoccupied ones. The larger the
value, the more stable the donor-acceptor system is.
Table 5 The energy of hyperconjugative interactions for pasiniazid using NBO basis by
B3LYP/6-311++G (2d, p) method.
ED
E (i)
ED
E(2)a
(KJ mol-1)
124.36
24.87
Donor(i)
Accpetor(i)
E (j) (a.u.)
E(i)-E(j)b (a.u.) F(i,j)c (a.u.)
(e)
(a.u.)
(e)
LP1 N15 1.90413 -0.40198 σ*(O28-H16) 0.07910
LP1 O27 1.95868 -0.73138 σ*(N8-H14) 0.04433
LP2 O27 1.85736 -0.30400 σ*(N8-H14) 0.04433
LP1 O27 1.95868 -0.73138 σ*(O25-H32) 0.04966
LP2 O27 1.85736 -0.30400 σ*(O25-H32) 0.04966
0.36369
0.39662
0.39662
0.40295
0.40295
0.77
1.13
0.70
1.13
0.71
0.14
0.07
0.05
0.06
0.10
17.85
18.73
65.67
ED (e) is the electron density of donor and acceptor of NBO orbitals.
a E(2) means the energy of hyperconjugative interactions; cf. Eq (2).
b Energy difference between donor (i) and acceptor (j) NBO orbitals.
c F(i,j) is the Fock matrix element between i and j NBO orbitals
In present work, we focus on the atoms involving in intra- and inter-molecular interactions
which have been revealed by spectroscopy analysis. The E (2) value of corresponding interaction
is obtained by the NBO analysis enumerated in Table 5. The intermolecular hydrogen bond
O28-H16…N15 is formed by the orbital overlap between the LP1 N15 and σ*(O28-H16),
resulting in the intermolecular charge transfer and subsequently stabilizing the HB system. The
stabilization energy E (2) associated with hyper conjugative interaction between LP1 N15 and
σ*(O28-H16) is obtained as 124.36 KJ mol-1, which is qualified for establishing a strong
hydrogen bonding between O28-H16 and N15. This interaction is responsible for a significant
decrease of the electron density on N15, which can be confirmed by the decrease of
corresponding Mulliken charges compared with monomeric INH. As a consequence, the
nucleophilicity of N15 is decreased leading to a retard of N-acetylation on INH. Another
important contribution to the stabilization of pasiniazid is provided by the interaction due to the
orbital overlap between the lone pair of O27 and anti-bonding orbitals of N8-H14. The E (2)
value for [LP (1) O27-σ*(N8-H14)] and [LP (2) O27-σ*(N8-H14)] is 24.87 and 17.85 KJ mol-1
respectively. Due to O27 participating in the formation of another interaction, O25-H32…O27,
there is a competitive relationship on the charges from O27. The stabilization energy of [LP (1)
O27-σ*(O25-H32)] and [LP (2) O27-σ*(O25-H32)] is 18.73 and 65.67 KJ mol-1 respectively.
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The difference in E (2) value demonstrates that the intramolecular hydrogen bond
O25-H32…O27 is stronger than N8-H14…O27. The possible explanation for this situation is that
a conjugated system is formed between carboxyl group and phenolic hydroxyl, in which the
charge transfer between O27 and O25-H32 is facilitated through the benzene ring. And because
of the conjugation relationship between carboxyl group and the benzene ring in PAS, the electron
delocalization is easily conducted between carboxyl group and the benzene ring, leading to the
decrease of electron density on O27. Consequently, the strength of hydrogen bond N8-H14…O27
is weaken by this conjugative interaction. As a matter of fact, the E (2) of N8-H14…O27 is much
lower than O28-H16…N15, which also demonstrates that this hyper conjugative interaction could
not be formed as strong as O28-H16…N15.
3.6. Molecular electrostatic potential (MEP) analysis
Figure 7. Molecular electrostatic potential surfaces of pasiniazid.
Molecular electrostatic potential describes the electrostatic effect around a molecule
produced by total contribution of electron and proton. Moreover, MEP is usually carried out for
evaluation of correlated dipole moments, partial charges, electro-negativity and chemical
reactivity of the molecules [37-39]. The MEP of pasiniazid is described in Fig. 7 based on
theoretical conformation carried out in this article. The color range is set to be as same as before
(-5.507 e-2 to 5.507 e-2) in order to compare with the MEP of INH and PAS intuitively. As plotted
in Fig 7, the electrons overlapping area indicates hyper conjugative relationship between PAS
and INH. The fading of overlap region between hydrazine group and carboxyl group indicates
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that the electron density transfer through the interactive region and the chemical activity of this
area is restricted. Therefore, the nucleophilicity of N15 is decreased due to the interaction with
O28-H16, which provides a visual evidence for understanding the retard of metabolic activities
on INH.
3.7. Frontier molecular orbitals (FMOs) investigation
Frontier molecular orbitals investigation frequently shows important in reactive prediction
[40, 41]. The highest occupied molecular orbital (HOMO), which is regarded as an electrons
donor, is related to potential electron delocalization directly. The energy of HOMO reveals
ability of charges transfer. On the other hand, the lowest unoccupied molecular orbital (LUMO)
describes an area where electrons can be accepted, and the energy of LUMO is regarded as
electron affinity [42]. The energy of band gap expresses the energy difference between this two
important FMOs which signifies the stability of molecular structure.
Figure 8. Frontier molecular orbitals for HOMO and LUMO of pasiniazid.
The counter of two FMOs we mentioned above is described in Fig. 8. The HOMO is
localized on PAS and the LUMO is localized on INH, which indicates that the tendency of
charges transfer is from PAS to INH. Therefore, the intermolecular relationship between two
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molecules has been verified. The energy of two FMOs is enumerated in Fig. 8. The band gap
equals to 4.27 eV, which shows a great agreement of Gibbs energy investigation of stabilization
of molecular conformation. Furthermore, the ionization energy and electron affinity can be
expressed as: I= -EHOMO= 6.25 eV, A= -ELUMO=1.98 eV. The hardness, which can be denoted as:
η= (I-A)/2, indicates the resistance toward the deformation of the electron cloud of chemical
systems under small perturbation encountered during chemical process [43-45]. The hardness of
pasiniazid is 2.14 eV. Such chemical hardness is attributed to the chemical activities for
pasiniazid are limited due to the restriction of potential reactive sites, carboxyl and hydrazine
group, caused by the formation of intermolecular interaction between PAS and INH.
4. Conclusion
The appropriate conformer for pasiniazid have been found by theoretical investigation based
on the quantum chemical approach by DFT B3LYP method with 6-311++G (2d, p) basis set. The
spectroscopy characterization analysis, such as PXRD, IR, Raman, has been performed. There is
a good agreement between experimental and computational results. Moreover, two
hydrogen-bond systems with three hydrogen bonds were characterized by spectroscopy and
NBO analysis. Result reveals that the intermolecular charge transfer is mainly conducted by the
orbitals overlap between hydrazine group and carboxyl group resulting in stabilization of the
system. MEP surface provides a visual guide for understanding that the N-acetylation on INH is
restricted due to interaction with the carboxyl group from PAS. The frontier orbitals investigation
for HOMO and LUMO demonstrates the stabilization of the conformer.
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Acknowledgments
This work has been financially supported by the National Fund for Talent Training
in Basic Science (No.J1103606), Program for Innovative Research Team of the Ministry of
Education and Program for Liaoning Innovative Research Team in University. The theoretical
calculations were performed by the ScGrid and Deepcomp7000 the Supercomputing Center,
Computer Network Information Center of Chinese Academy of Sciences.
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1. The appropriate conformer of pasiniazid was found.
2. Theoretical PXRD and vibration spectrums consisted with experimental data.
3. The interactions in pasiniazid were studied by spectroscopy and NBO analysis.
4. MEP analysis was performed.
5. DFT method was carried out for theoretical investigation.