Accepted Manuscript

Structural, spectral, theoretical and anticancer studies on new co-crystal of the

drug 5- Fluorouracil

S. Suresh Kumar, S. Athimoolam, B. Sridhar

PII: S0022-2860(18)30898-6

DOI: 10.1016/j.molstruc.2018.07.079

Reference: MOLSTR 25484

To appear in: Journal of Molecular Structure

Received Date: 12 March 2018

Accepted Date: 24 July 2018

Please cite this article as: S. Suresh Kumar, S. Athimoolam, B. Sridhar, Structural, spectral,
theoretical and anticancer studies on new co-crystal of the drug 5- Fluorouracil, Journal of
Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.07.079

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 ACCEPTED MANUSCRIPT

Structural, spectral, theoretical and anticancer studies on new co-crystal of the drug
5- Fluorouracil
S. Suresh Kumara, S. Athimoolama* and B. Sridharb
a Department of Physics, University College of Engineering Nagercoil, Anna University,
Nagercoil 629 004, India.
b Laboratory of X-ray Crystallography, Indian Institute of Chemical Technology, 500 007,

Hyderabad, India
*E-mail: athi81s@yahoo.co.in
Abstract

New co-crystal containing 5-Fluorouracil (an anticancer drug) as an active pharmaceutical

ingredient (API) was prepared by the liquid-assisted grinding method. The prepared co-crystal
namely, 5-fluorouracil hydroquinone (5FUHYQ) was studied through single X-ray diffraction. It
reveals that the crystal packing is supported by N–H···O and O–H···O classical hydrogen bonds
forming a sheet-like molecular architecture. The drug molecules are connected themselves
through three N–H∙∙∙O intermolecular hydrogen bonds leading to two dimeric ring R22(8) motifs.
These two ring motifs are placed adjacently forming two chain C21(6) motifs which are running
along the a-axis of the unit cell, oppositely. The drug molecules are sandwiched at y = 1/2 and
the coformers are placed at y = 0 and 1 making the supramolecular assembly. These ring and
chain motifs to form a hydrogen-bonded sheeted. The supramolecular sheets are parallel to
(0 4 2) and (0 4 2) planes of the unit cell. This molecular aggregation is replicated in the
diffraction pattern as a strong intensity X-ray peaks. The 5FUHYQ has lower calculated density
than the parent drug which favour the improved membrane permeability of the drug. Geometrical
optimizations of the compound were done by Hartree-Fock (HF) and Density Functional Theory
(DFT) using the B3LYP function with the 6-311++G(d,p) basis sets. The optimized molecular
geometries and computed vibrational spectra are compared with their experimental counterparts.
The Frontier Molecular Orbital (FMO) analysis shows that the present compound has lower band
gap value which reiterates the possible pharmaceutical activity of the compound. The anticancer
activity of the parent drug is retained against human cervical cancer cell line (HeLa) in this new
co-crystalline form.

Keywords: XRD, Anticancer activity, FT-IR, FT-Raman, DFT and FMO

Introduction
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Recently, the fluorine based compounds received the attention of many pharmaceutical scientist.
Because, the presence of fluorine as a functional element in the pharma drug highly alter its
physicochemical and biological properties. Fluorine has significant influence on the chemical
reactivity due to its physical properties viz., size, lipophilicity, electronegativity, omniphobicity
and electrostatic interactions [1–4]. Generally, one-third of the drugs available in the market
contain fluorine in their structure [5]. It is proposed that fluorine derivatives play important role
in the pharmaceutical industry.
5-Fluorouracil (5FU) is one of the fluorine-based derivatives and a well-known
anticancer drug. It was first prepared in 1957 [6] and used in the treatment of solid tumors, such
as colorectal, breast, gastrointestinal and ovarian cancers [7-9]. Also, it is used as an ointment,
particularly in skin cancer [10-13]. It is one of the antimetabolite drugs and also the pyrimidine
analogue [14]. From the crystal engineering perspective, the 5FU has hydrogen bonding donors
(two N−H) and hydrogen bonding acceptors (two C=O) in the molecular structure [15]. It act as
strong hydrogen bonding synthon with neighbouring molecules in crystalline assembly. In this
point of view, number of co-crystals were attempted with 5FU, with the coformers of cytosine
[16], 1-methylcytosine [17], theophylline [18], urea, thiourea, 2,2′-bipyridine & 4,4′-bipyridine
[19], acridine, phenazine & 4,4-bispyridylethene [20], adipic, succinic, terephthalic, benzoic &
malic acid [21], 4-hydroxybenzoic acids [22] and 3-hydroxybenzoic acid, 4-aminobenzoic acid
& cinnamic acid [23] etc. The present work was initiated with the series of hydroxyl aromatic
compounds like catechol, resorcinol and hydroquinone as a coformers. In which, the co-crystal
of hydroquinone was successfully crystallized with the 5FU using the liquid-assisted grinding
method and reported here as a first time in literature (scheme 1). Further, the several
characterizations, such as quantum chemical, Mulliken charge analysis, vibrational, Frontier
Molecular Orbital (FMO) analysis and cytotoxicity studies were also done to describe the
properties of new co-crystal.
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Scheme 1 Structures of 5-Fluorouracil (5FU) and Hydroquinone (HYQ)

Experimental

Material preparation

The co-crystal of 5-fluorouracil hydroquinone (5FUHYQ) was synthesized by the liquid-assisted

grinding method. The experiment was performed by adding an equimolar of 5FU (0.130 g) and
the hydroquinone (0.110 g) with few drops of distilled water to make the paste using mortar
pestle. Finally, this paste was dissolved in the methanolic solution to allow the slow evaporation
at room temperature. After two week period, the needle-shaped crystal was harvested.

X-ray crystallography

The necessary crystallographic calculations, i.e., the unit cell parameters of 5FUHYQ and full
data collections were done from single-crystal X-ray diffraction studies by Bruker SMART
APEX CCD area detector diffractometer (graphite- monochromated, MoK = 0.71073 Å) [24].
Crystallographic data, details of the data collection and refinement statistics are given in Table 1.
The structure was solved by direct methods using SHELXL 2014 [25]. All the H atoms were
positioned geometrically and refined using riding model approximation, with C−H = 0.86-0.93 Å
and Uiso(H) = 1.2 Ueq (parent atom). The R-factor (0.035 %) of the compound confirms the
convergence of the crystal structure.

Computational details

All the theoretical calculations were carried out using Gaussian 09 program [26]. The geometry
optimization was carried out free of any constraint on the symmetry [27-29]. Initially, the
molecular geometry of the present compound was taken from the single crystal X-ray studies and
it was optimized by Hartree−Fock (HF) and Density Functional Theory (DFT) levels using the 6-
311++G(d,p) basis set. Further, the optimization methods of the calculated molecular geometries
of present compound was combined with the calculation of vibrational frequencies like IR and
Raman. Finally, the results analyzed with the help of GAUSSVIEW program [30]. Also, the
Frontier Molecular Orbital (FMO) calculation was carried out with same basis sets.

Vibrational spectra
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The FT-IR spectrum of the present compound was recorded in the KBr phase in the frequency
region of 4000–400 cm-1 using a Perkin Elmer Spectrometer at a resolution of 1cm-1. The FT-
Raman spectrum was recorded in the frequency range of 4000-50 cm-1 using a BRUKER RFS 27
FT-Raman Microscope module with the resolution of 2 cm-1. The Nd:YAG Laser source was
operated at 1064 nm for the excitation. The plots comparing the experimental and theoretical
counterparts were drawn in the range of 4000-400 cm-1 for IR and Raman spectra.

Anticancer activity Studies

Cell culture

The cytotoxicity of the grown crystal (5FUHYQ) was investigated against human cervical cancer
cell line (HeLa) and compared with the parent drug 5FU. The above cell line was acquired from
the National Centre for Cell Science (NCCS), Pune. It was grown in Eagles Minimum Essential
Medium, which is containing 10% Fetal Bovine Serum (FBS). These cells were sustained at
37 °C, 5% CO2, 95% air and 100% relative humidity.

Cytotoxicity assessment using MTT assay

The monolayer cells of the HeLa were separated by trypsin-ethylenediamine tetraacetic acid
(EDTA) to create a single cell suspension. The possible cells were counted through
hemocytometer and it was diluted with 5% FBS to give the final density of 1×105 cells/ml. The
10,000 cells/well were seeded in 96-well plates using culture medium and incubated to allow for
cell attachment at relative humidity. After 24 hr the cells were treated with serial concentrations
of the test samples. The grown sample of 5FUHYQ was dissolved in phosphate buffered saline.
The aliquot of the sample solution was diluted twice to obtain the desired final maximum test
concentration with serum-free medium. Additionally, four serial dilutions were made to provide
a total of five sample concentrations. Aliquots of 100 µl of these different sample dilutions were
added to the appropriate wells already containing 100 µl of the medium, resulting in the required
final sample concentrations. Following sample addition, the plates were incubated for an
additional 48 hr at 37 °C with 5% of CO2, 95% of air and 100% relative humidity. The medium
without samples was served as control and triplicate was maintained for all concentrations. After
the incubation, MTT assay was performed. MTT is a yellow water-soluble tetrazolium salt. A
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mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring,

converting the MTT to an insoluble purple formazan. Hence, the amount of formazan produced
is directly proportional to the number of viable cells. After 48 h of incubation, 15µl of MTT
(5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for
4h. The medium with MTT was then flicked off and the formed formazan crystals were
solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using a micro plate
reader. The % cell inhibition was determined using the following formula [31].

absorbance of the sample

% Cell Inhibition = 100- ( absorbance of the control ) × 100

The graph was plotted between % Cell inhibition and Log concentration and the IC50 was
calculated using Graph Pad Prism software.

Result and discussion

molecular geometry

The compound crystallized in triclinic centrosymmetric unit cell containing two 5-Fluorouracil,
a hydroquinone and two lattice water. Interestingly the centroid of the hydroquinone is situated
at the inversion centre owing to its symmetrical molecular geometry. Hence, the asymmetric
part contains a 5-Fluorouracil, half of hydroquinone and a lattice water (Fig. 1). The
pharmacokinetically important C–F bond length in the drug molecule is observed to be
1.341 (2) Å in single crystal XRD studies and the corresponding bond length is well matched
with the theoretically calculated value. This bond has higher bond energy than the C–Br and
C–Cl bonds and has higher chemical stability. The fluorinated compound oppose the oxidation
processes in metabolic decomposition [32].

The optimized C–N bond distance varies from 1.364 to 1.387 Å and 1.380 to
1.412 Å in HF and DFT/B3LYP methods, respectively. The solid state values are observed to be
1.351 (2), 1.365 (2), 1.375 (2) and 1.367 (2) Å (Table 2). The deviations are due to the fact that
the theoretical calculations were carried out with molecules in a gaseous phase, whereas the
experimental results correspond to the molecule is in the solid crystalline state. The calculated
density of the 5FUHYQ has lower value than parent compound. The dense packing of 5FU is
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disturbed due to the formation of co-crystallization and looser its molecular packing specificity.
Hence, it may improve the permeability of the drug in biological systems.

Hydrogen bonding features

Fluorines are involved in the hydrogen-bonding interactions with lower energy than an X–H∙∙∙O
interactions [33] which is very significant in bio-activity of a drug molecule. This hydrogen
bonding interactions are the robust influence in binding affinity, bioavailability and
pharmacokinetic properties of a drug molecule.

The crystal structure of 5FUHYQ is stabilized by three-dimensional hydrogen bonding

network formed through N–H∙∙∙O and O–H∙∙∙O interaction (Table 3). 5FU drug molecules are
connected themselves through three N–H∙∙∙O interaction leading to two ring R22(8) motifs. These
two ring motifs are placed adjacently forming two chain C21(6) motifs. These chains are running
along the a-axis of the unit cell, oppositely (Fig 2). The drug molecules are sandwiched at y = 1/2
and the coformers are placed at y = 0 and 1. The coformers are connected to the drug molecule
through lattice water by O–H∙∙∙O hydrogen bonds. The lattice water molecule is acting as a donor
and an acceptor for two O–H∙∙∙O hydrogen bonds and hence connecting the centrosymmtrically
related drug ring motifs. This leads to a chain C55(21) motif extending along the bc-diagonal of
the unit cell (Fig. 3). These ring and chain motifs are assembled like hydrogen-bonded sheets.
These sheets are parallel to the (0 4 2) and (0 4 2) planes of the unit cell. This molecular
aggregations is replicated in the diffraction pattern as a strong intensity X-ray peaks. These
molecular sheets are interlinked through weak water-water hydrogen bond (O1W-H1W∙∙∙O1W).
The crystal packing features a C–H∙∙∙F interaction between the drug and coformer.

Mulliken atomic charge analysis

In theoretical calculation, the charge distribution on the drug molecule contains a crucial role in
the polarizability, dipole moment and electronic structure [34]. The Mulliken charges are
predicted at the HF and DFT/ B3LYP methods with the 6-311++G(d,p) basis sets and the values
are tabulated in Table 4. The corresponding graph is shown in Fig 4. The entire hydrogen atoms
are positive in nature. Additionally, it is observed that the hydrogen, H12 (0.605 e and 0.506 e
for HF and B3LYP methods respectively) atom has larger positive nature than the other
hydrogen atoms. Because this hydrogen atom situated between a donor N atom of the drug and
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an acceptor O atom in the coformer. Moreover, this N–H∙∙∙O interaction form a dimeric ring
motif in solid crystalline state. The electronegative fluorine atom has the charge value of -0.235 e
and -0.175 e in HF and DFT/ B3LYP methods, respectively and act as the acceptor for the
C–H∙∙∙F intermolecular interaction to form a chain motif.

Vibrational analysis

Vibrational spectroscopy was used here to identify the strength of the hydrogen bonds from the
alteration in vibrational modes of the functional groups, which is responsible for the creation of
the co-crystal. The experimental FT-IR and FT-Raman spectra of the 5FUHYQ are shown in the
Figs.5 and 6, respectively. The predicted vibrational frequencies by DFT/B3LYP and HF have
been compared with experimental results. The vibrational modes with their corresponding
assignments are shown in Table 5.

The observed vibrational bands in the experimental spectra are hypsochromic or

bathochromic shift with their theoretically predicted wavenumbers. The larger the shifting shows
the stronger the hydrogen bonds in its crystalline environment. The stretching modes are
bathochromic shifted and the bending modes are hypsochromic shifted for the vibrational bands
of -NH and -OH functional groups. Apart from the shifting of bands due to the hydrogen bonds,
strong intensity peaks are observed for intermolecular interaction around the wavenumber of
2500 cm-1 in theoretical spectra and in experimental, the phenomena is observed as broad band
due to multiple number of such bonds.

5FUHYQ has 26 atoms and 72 normal modes of vibration and it has various functional
groups such as –N–H, –C–H, –C–C, –C=O and –OH. Generally, the –OH stretching vibration is
occurred at 3600 cm-1 for phenols [35]. The two O–H stretching vibrations are calculated at
4082/4193 cm-1 and 3659/3843 cm-1 in HF and DFT/ B3LYP levels, respectively. The
corresponding stretching vibration is overlapped with the other stretching frequency in IR spectra
and this stretching is not present in Raman spectra. The N–H stretching vibration is normally
observed in the range of 3000–3230 cm-1 in many co-crystals [36]. In the present case, the strong
intensity peak observed at 3392 cm-1 in IR spectra is attributed to the N–H stretching vibration.
This stretching vibration is predicted at 3752/3882 cm-1 and 3637/3517 cm-1 in HF and DFT/
B3LYP levels, respectively. The bathochromic shifting of more than 200 cm-1 compared to
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theoretical prediction clearly shows the presence of strong N–H∙∙∙O interactions in the solid state.
Generally, the strong C=O stretching band occurs in the range 1800-1700 cm-1 [37]. 5FUHYQ
co-crystal, the C=O stretching vibration appears at 1894 cm-1 in IR spectra and this band is
absent in Raman spectrum. The same stretching vibration is computed at 1939/1987 cm-1 and
1767/1793 cm-1 in HF and DFT/ B3LYP methods, respectively. The characteristic absorption
peaks of N–H and C=O bending vibrations in 5FU is observed in hypsochromic shift due to the
intermolecular hydrogen bonds.

In the heterocyclic aromatic ring, the C–H stretching vibration occurs in the region of
3100-3000 cm-1 [38]. Generally, the bands are not affected in this region by the nature of the
substituent. The weak bands at 3012/3113 cm-1 in IR spectra and the corresponding Raman
spectra at 3060/3100 cm-1 are observed due to the C-H stretching vibration. This same vibration
is computed at 3310/3319 and 3151/3171 cm-1 in HF and DFT/B3LYP levels, respectively. The
medium band appearing at 1261/1658 cm-1 in the IR and 1243/1686 cm-1 in Raman spectrum is
assigned to C–H in-plane bending vibration. The corresponding bending vibration predicted at
1817 cm-1 in HF level and 1664 cm-1 in DFT/ B3LYP level. Another C–H in-plane bending
vibration appears at 1522 cm-1 in IR spectrum. This vibration is well correlated with the
theoretically calculated value. The weak band at 1335 cm-1 in IR spectra and 1336 cm-1 in
Raman spectra is due to the C–C stretching vibration. This stretching vibration is well matched
with the theoretically predicted data. The lower wavenumber region (1000 - 200 cm-1) was
assigned to the N-H and C–H out-of-plane bending vibrations. In this region, the experimental
and theoretical results are matching to each other.

HOMO and LUMO band gap

The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbitals
(LUMO) are termed as frontier molecular orbitals (FMOs). FMOs participate crucial role in the
intramolecular charge transfer between molecules as well as in electronic spectra of the molecule
[39]. The energy gap between HOMO and LUMO determines the chemical hardness,
electronegativity and chemical potential of a molecule, as present in Table 6. The chemical
hardness is a good indicator of the chemical stability. The chemical hardness value of the drug is
small in both DFT/ B3LYP and HF levels calculations showing the soft nature of the molecule.
The frontier orbital of HOMO and LUMO plots of the 5FUHYQ is shown in the Fig. 7.
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By DFT/ HF levels, the calculated energy values of HOMO and LUMO in DFT/ HF
levels are -0.297/0.211 a.u and 0.030 /-0.079 a.u and the energy gap value between the frontier
molecular orbitals are 0.267/0.132 a.u, respectively. These calculations show that 5FUHYQ has
the lower value of frontier orbital energy gap. The small energy gap is associated with a high
chemical reactivity and low kinetic stability. Further, the lower value of HOMO and LUMO
energy gap explains that a large number of intramolecular charge transfer takes place within the
molecule. This large charge transfer influences the biological activity of the molecule.

Cytotoxicity studies

The Anticancer activity for the grown co-crystal of 5FUHYQ was examined on human cervical
cancer cell line (HeLa) by means of MTT assays. The present compound examined with the
human cervical cancer cell line (HeLa) with different concentrations in the range of 0.1–100 μM
and the corresponding plots are shown in the Fig. 8. The result shows the good inhibitory effect
on the growth of human cervical cancer cell line. The IC50 value of the present compound
(5FUHYQ) is 72.05 μM which is comparable with the parent drug 5FU. The result proves that
the 5FUHYQ retains its activity against cancer cell line.

Conclusion

New co-crystal of 5-fluorouracil hydroquinone (5FUHYQ) was prepared by the liquid-assisted

grinding method. The molecular structure of the present compound is confirmed by single crystal
X-ray diffraction technique. The crystal packing is stabilized through the three-dimensional
network of intricate N–H∙∙∙O and O–H∙∙∙O classical hydrogen bonds forming sheet-like
architecture. This sheets are parallel to the (0 4 2) and (0 4 2) planes of the unit cell. The drug
molecules are situated midpoint of the sheet and the coformers are placed at y = 0 and 1. The
present compound has lower calculated density than the parent drug which favour the improved
membrane permeability of the drug. The experimental and theoretical molecular geometries were
compared. The shifting of vibrational bands due to the intermolecular hydrogen bonds were also
analyzed. The energy gap between the HOMO and LUMO give the valuable information about
the chemical stability and chemical reactivity of the drug. The anticancer activity of the present
compound was analyzed against the human cervical cancer cell line (HeLa). This study reveals
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that 5FUHYQ retains its activity against human cervical cancer cell line (HeLa) with possible
improved physiochemical and pharmacokinetic properties.

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Fig. 1. (a) The molecular structure of the 5FUHYQ with atom numbering scheme and 50% probability displacement ellipsoids and (b) Optimized
Structure with atomic numbering for HF/6-311++G(d,p) (c) Optimized Structure with atomic numbering for DFT/6-311++G(d,p). The double
dotted line indicates hydrogen bond interaction
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Fig. 2. Molecular aggregations formed in 5FUHYQ showing ring and chain motifs. Hydrogen bonds are
shown dashed lines.
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Fig. 3. Packing diagram for 5FUHYQ viewed along the a- axis showing the sheet-like molecular
architecture. H-bonds are shown as dashed lines.
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Fig. 4. The atomic charges of the optimized molecular structures for 5FUHYQ by (a) HF/ 6-311++G(d,p)
and (b) DFT/ 6-311++G(d,p) levels
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Fig 5. Comparative representations of FT-IR spectra for 5FUHYQ

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Fig. 6. Comparative representations of Raman spectra for 5FUHYQ

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Fig. 7. Calculated HOMO-LUMO energy values for 5FUHYQ

Fig. 8. Cytotoxic activity of 5FUHYQ and 5FU towards human cervical cancer cell line (HeLa)
are plotted in the above graph
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HIGHLIGHTS
 Co-crystal of 5-fluorouracil hydroquinone (5FUHYQl) was prepared by the liquid-
assisted grinding method.
 Classical hydrogen bonds forming a sheet-like molecular architecture
 Geometrical optimizations of the compound were done by Hartree-Fock (HF) and
Density Functional Theory (DFT)
 Anticancer activity of the parent drug is retained against human cervical cancer cell line
 The Frontier Molecular Orbital (FMO) analysis shows that the present compound has
lower band gap value
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Table 1
Crystallographic data and Structure refinement Parameters of 5FUHYQ
Empirical formula (C4H3FN2O2).(C6H6O2).H2O
Formula weight 258.21
Temperature 294(2) K
Wavelength 0.71073 Å
Crystal system, space group Triclinic, P1
Unit cell dimensions a = 7.043 (1) Å α = 82.4 (3)°
b = 7.595 (2) Å β = 88.8 (3)°
c = 8.377 (2) Å γ = 77.2 (3)°
Volume 433.1 (2) Å3
Z, Calculated density 2, 1.558 Mg/m3
Absorption coefficient 0.141 mm-1
F(000) 268
Crystal size 0.21 x 0.19 x 0.20 mm3
Theta range for data collection 2.453 to 24.998 °
Limiting indices -8≤h≤8, -9≤k≤9, -9≤l≤9
Reflections collected / unique 4175 / 1518 [R(int) = 0.0123]
Completeness to theta = 25.242 97.40%
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 1518 / 0 / 143
Goodness-of-fit on F2 1.021
Final R indices [I>2sigma(I)] R1 = 0.0352, wR2 = 0.1044
R indices (all data) R1 = 0.0383, wR2 = 0.1078
Largest diff. peak and hole 0.277 and -0.155 e. Å-3
CCDC No 1560138
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Table 2
Important optimized molecular geometrical parameters for 5FUHYQ

HF/6- DFT/6-
Atoms connected Experimental
311++(d,p) 311++(d,p)
Bond length (Å)
C1–O10 1.236 (2) 1.197 1.222
C1–N6 1.351 (2) 1.364 1.382
C1–N9 1.365 (2) 1.367 1.380
C2–O11 1.224 (2) 1.183 1.208
C2–N9 1.375 (2) 1.387 1.412
C2–C3 1.439 (2) 1.468 1.464
C3–C4 1.331 (2) 1.319 1.342
C3–F8 1.341 (2) 1.315 1.338
C4–N6 1.367 (2) 1.379 1.381
C13–O18 1.376 (2) 1.357 1.366
C13–C14 1.378 (2) 1.382 1.396
C13–C23 1.383 (2) 1.385 1.4
C23–C14#1 1.381( 2) 1.384 1.392
Bond Angle (°)
O10–C1–N6 122.7 (1) 122.3 122.1
O10–C1–N9 121.7 (1) 123.2 124.1
N6–C1–N9 115.6 (1) 114.5 113.8
O11–C2–N9 121.0 (1) 122.2 121.9
O11–C2–C3 126.1 (1) 125.1 126
N9–C2–C3 112.9 (1) 112.8 112.1
C4–C3–F8 121.4 (1) 121.9 121.1
C4–C3–C2 122.2 (1) 121.1 121.8
F8–C3–C2 116.4 (1) 117 117.1
C3–C4–N6 119.6 (1) 120.8 120.5
C1–N6–C4 122.9 (1) 123.3 123.7
C1–N9–C2 126.7 (1) 127.6 128.1
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HF/6- DFT/6-
Atoms connected Experimental
311++(d,p) 311++(d,p)
C14#1–C23–C13 119.9 (1) 119.4 119.0
O18–C13–C14 120.3 (1) 118.2 118.1
O18–C13–C23 119.6 (1) 122.4 122.9
C14–C13–C23 120.1 (1) 119.4 119.0
Symmetry transformations used to generate equivalent atoms:
#1 -x,-y,-z+1

Table 3

Hydrogen bonding geometry of 5FUHYQ

D-H···A (D-H) (H···A) (D···A) (D–H···A)

(Å, ) (Å) (Å) (Å) ()
O18–H3O∙∙∙O1W 1.18 (4) 1.53 (4) 2.701 (2) 174.0 (3)
N6–H5∙∙∙O10#2 0.86 1.96 2.820 (2) 173.4
C23–H24∙∙∙F8#3 0.93 2.49 3.267 (2) 141.1
N9–H12∙∙∙O10#4 0.87 (2) 1.99 (2) 2.861 (2) 179.6 (2)
O1W–H2W∙∙∙O11#3 0.91 (4) 1.90 (4) 2.782 (2) 163.0 (3)
O1W–H2W∙∙∙O1W#5 0.74(3) 2.33(3) 2.788(5) 121(3)
Symmetry transformations used to generate equivalent atoms:
#2 -x+1,-y+1,-z+1 #3 -x,-y+1,-z+2 #4 -x,-y+1,-z+1 #5 -x+1,-y,-z+2
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Table 4

Atomic charges for optimized geometry of 5FUHYQ

HF/6- DFT/6-
Atoms
311++(d,p) 311++(d,p)
C1 0.442 0.200
C2 0.580 0.366
C3 -0.520 -0.482
C4 0.431 0.293
H5 0.264 0.224
N6 -0.594 -0.358
H7 0.393 0.349
F8 -0.235 -0.175
N9 -0.531 -0.291
O10 -0.451 -0.367
O11 -0.375 -0.268
H12 0.605 0.506
C13 -0.396 -0.515
C14 -0.070 -0.067
H15 0.211 0.190
C16 0.139 0.064
H17 0.184 0.140
O18 -0.345 -0.251
H19 0.356 0.330
C20 -0.439 -0.483
C21 -0.105 -0.003
H22 0.233 0.195
C23 0.083 0.198
H24 0.189 0.194
O25 -0.320 -0.243
H26 0.270 0.254
 Table 5
Experimental and calculated vibrational wave numbers of 5FUHYQ
Mode Experimental HF/6-311++G(d,p) B3LYP/6-311++G(d,p)
Assignment
Number FT-IR Raman νcal aI IR bI Raman νcal aI IR bI Raman

1 5 0.237 1.544 15 0.596 1.501 γ (C–H + O–H)

2 20 0.529 1.511 19 2.635 5.384 γ (C–H + N–H)
3 24 4.97 0.841 24 1.947 4.214 γ (C–H + N–H)
4 43 0.384 1.897 57 0.111 3.479 β (C–H + N–H)
5 74 0.426 0.903 65 1.786 2.724 γ C–H
6 113 1.881 0.314 105 0.942 1.663 β (C–H+ N–H)
7 123 0.186 0.479 129 1.252 0.607 γ (C=O + O–H +C–F)
8 158 0.106 0.016 156 0.262 0.134 γ (N–H+ O–H)
9 176 3.901 0.03 167 2.731 0.15 γ (C=O + O–H)
10 198 123.968 1.394 246 106.891 1.479 γ O–H
11 331 0.889 0.256 305 0.882 0.198 β (C=O + C–F)
12 368 25.888 0.354 345 9.415 0.452 β C–C + C–H+ N–H)
13 384 23.408 0.03 353 12.664 0.326 β (O–H)
14 407 0.155 2.895 366 0.278 3.501 β (C–C + C–H+ O–H)
15 412 5.063 2.356 381 8.384 1.129 β (C–H + N–H)
16 444 64.205 1.511 401 41.42 1.894 β C=O
17 463 0.083 0.089 428 0.486 0.095 γ C–H
18 441 w 470 w 495 6.376 0.529 455 4.621 1.325 β (C–H + O–H)
19 496 10.413 1.878 460 4.94 2.547 ring distortion
 Mode Experimental HF/6-311++G(d,p) B3LYP/6-311++G(d,p)
Assignment
Number FT-IR Raman νcal aI IR bI Raman νcal aI IR bI Raman

20 510 0.557 11.336 472 0.788 7.813 β (C–H + O–H)

21 543 76.205 0.037 521 20.262 0.321 γ C–H
22 552 64.478 1.468 539 11.856 4.198 ring distortion
23 548 w 564 w 578 66.761 0.26 542 65.152 0.206 γ N–H
24 587 10.875 3.344 619 202.128 11.52 γ O–H
25 693 3.338 5.639 634 5.884 5.807 β (C–H + N–H)
26 646 w 707 0.181 5.907 659 2.911 5.502 ring distortion
27 777 1.006 1.713 702 26.913 1.36 γ N–H
28 788 59.378 0.784 706 10.663 1.111 γ (C–C + N–H)
29 795 14.465 14.708 742 16.261 22.884 ring distortion
30 758 w 751 w 820 73.023 0.226 751 46.416 0.623 γ (C–C + N–H)
31 763 w 842 112.031 2.364 763 1.51 0.853 γ (C–C + N–H)
32 851 23.195 1.394 767 81.406 0.359 β C–H + γ N–H
33 893 74.709 1.621 811 1.972 0.256 γ C–H
34 815 w 832 w 906 0.618 0.79 819 65.914 3.943 β (C–H + O–H)
35 921 0.046 51.132 841 63.133 0.252 γ C–H
36 936 91.712 0.044 861 0.459 47.585 ring brething
37 1023 34.465 3.861 894 29.935 1.831 γ C–H
38 1037 0.301 0.039 924 1.642 0.363 γ (C–H + N–H)
39 1067 8.038 1.431 952 2.062 0.429 γ (C–H + N–H)
40 1068 0.137 0.024 981 12.503 1.531 β (C–H + N–H)
 Mode Experimental HF/6-311++G(d,p) B3LYP/6-311++G(d,p)
Assignment
Number FT-IR Raman νcal aI IR bI Raman νcal aI IR bI Raman

41 1098 0.956 0.028 1023 0.215 0.168 β C–H

42 1159 78.464 0.112 1114 26.198 0.501 β C–H
43 1209 116.317 0.027 1148 25 3.064 ν C–N + β N–H
44 1178 w 1173 w 1240 33.501 1.085 1180 1.625 4.792 β C–H
45 1271 0.78 4.226 1185 59.105 0.414 β (C–H+ N–H + O–H)
46 1315 12.466 3.417 1187 207.139 6.621 β (C–H+ N–H)
47 1261 w 1243 w 1319 177.903 3.96 1246 75.961 5.334 β (C–H+ O–H)
48 1366 722.325 1.506 1259 220.509 3.775 ν C–F + C–N
49 1372 83.868 1.221 1262 204.848 1.813 β (C–H+ O–H)
50 1386 64.375 33.128 1289 4.39 30.779 ν C–O + β C–H
51 1335 w 1336 m 1391 133.833 7.08 1353 4.926 27.287 ν C–C +β C–H
52 1479 28.107 2.476 1356 43.482 1.216 β C–H+ O–H
53 1484 18.1 37.257 1383 14.884 1.649 β C–H+ O–H
54 1566 146.064 1.791 1423 73.197 8.213 β N–H
55 1448 m 1594 6.208 8.749 1435 28.662 7.905 β N–H
56 1606 158.729 3.272 1487 119.097 0.258 β (C–H+ O–H)
57 1645 69.126 5.465 1498 27.985 7.842 β C–H + ν C–N
58 1522 w 1678 267.325 0.055 1545 174.021 1.568 β C–H
59 1792 7.808 12.025 1636 0.678 11.422 ν C=C + β C–H
60 1658 m 1686 s 1817 0.516 15.906 1664 4.56 13.814 β C–H
61 1901 75.289 73.03 1713 63.04 46.247 ν C=C + β (N–H +C–H)
 Mode Experimental HF/6-311++G(d,p) B3LYP/6-311++G(d,p)
Assignment
Number FT-IR Raman νcal aI IR bI Raman νcal aI IR bI Raman

62 1939 1149.931 3.174 1767 999.687 8.786 ν C=O + β N–H

63 1894 w 1987 616.6 65.177 1793 314.212 69.112 ν C=O
64 3012 w 3060 w 3310 22.309 69.516 3151 17.668 97.795 νs C–H
65 3113 w 3100 w 3319 8.948 76.252 3171 5.455 60.964 νs C–H
66 3350 8.248 45.233 3191 7.085 1.639 νas C–H
67 3354 2.879 164.551 3193 0.112 239.94 νas C–H
68 3387 3.354 99.298 3223 2.84 131.85 ν C–H
69 3392 s 3752 361.878 119.679 3517 259.209 331.48 ν N–H
70 3882 143.578 73.887 3637 153.066 112.1 ν N–H
71 4082 373.374 95.817 3659 603.209 184.72 ν O–H
72 4193 96.953 78.494 3843 63.879 116.03 ν O–H

w-weak; s-strong; ν-stretching; νas- asymmetric stretching: νs- symmetric stretching; β-in-plane bending; γ-out of plane bending; I IR -
calculated IR intensity; I Raman - calculated Raman intensity
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Table 6 Energy values of 5FUHYQ

HF / B3LYP/
Parameters (au)
6-311++G(d,p) 6-311++G(d,p)
ELUMO 0.03 -0.079
EHOMO -0.297 -0.211
Δ(ELUMO - EHOMO) 0.267 0.132
Electron Affinity (A) -0.03 0.079
Ionization Potential (I) 0.297 0.211
Chemical Hardness (η) 0.134 0.066
Electronegativity (χ) 0.164 0.145
Chemical Potential (μ) -0.164 -0.145