Tuning the molecular/electronic structure of new substituted pyrazoles: Synthesis, biological trials, theoretical approaches and Hammett correlations

Article abstract of DOI:10.1016/j.molstruc.2018.05.088

The high yield synthesis, characterization and biological trials of a family of compounds containing the pyrazole core (E)-3,5-dimethyl-1-(4-R-phenyl)-4-(phenyldiazenyl)-1H-pyrazoles (with R = 4-CH₃O (1), 4-CH₃ (2), 4-H (3), 4-F (4), 4-Cl (5), 4-CF₃ (6), 4-CN (7), 4-NO₂ (8), 3-Cl (9), 3-NO₂ (10), 2-Cl (11) and –C₆F₅ (12)) are reported. Hammett correlations were found for the experimental and theoretical computed data, showing the dependence of all the obtained results on the donor or acceptor nature of the R substituent. In this sense, the absorption wavelength does decrease as the electron-withdrawing character of the substituent increases. Furthermore, the wavelength intensity does follow a Hammett correlation with the sigma donor character of the substituents. The compounds were characterized using MP, EA, UV–vis, FT-IR, ¹H NMR, ¹³C NMR including bidimensional NMR experiments (HMBC and HMQC). The structures of 4, 5 and 8 were successfully elucidated by means of X-Ray diffraction. Pyrazole based dyes are known in pharmacological areas. Therefore, biological activity of all these structures were tested against cancer cell lines over a wide library of cell lines (60), including leukemia, colon and brain cancer, showing that the compounds have anticancer activity, which expands the knowledge of this kind of dyes as targets for this critical application. Finally, quantum calculations were carried out in order to give a rational explanation to the observed UV–vis absorption bands and FT-IR signals. As shown, it is possible to tune the molecular/electronic structure of the synthesized dyes by means of changing the –R substituent in the structure.

Full text of DOI:10.1016/j.molstruc.2018.05.088

Accepted Manuscript
Tuning the molecular/electronic structure of new substituted pyrazoles: Synthesis,
biological trials, theoretical approaches and Hammett correlations
Carlos Bustos, Luis Alvarez-Thon, Elies Molins, Ignacio Moreno-Villoslada, Gabriel
Vallejos-Contreras, Christian Sánchez, Ximena Zarate, Desmond Mac-Leod Carey,
Eduardo Schott
PII:
S0022-2860(18)30667-7
10.1016/j.molstruc.2018.05.088
MOLSTR 25261
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 29 March 2018
Revised Date: 25 May 2018
Accepted Date: 28 May 2018
Please cite this article as: C. Bustos, L. Alvarez-Thon, E. Molins, I. Moreno-Villoslada, G. Vallejos-
Contreras, C. Sánchez, X. Zarate, D. Mac-Leod Carey, E. Schott, Tuning the molecular/electronic
structure of new substituted pyrazoles: Synthesis, biological trials, theoretical approaches and Hammett
correlations, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.05.088.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Tuning the molecular/electronic structure of new
substituted pyrazoles: synthesis, biological trials,
theoretical approaches and Hammett correlations
Carlos Bustosa*, Luis Alvarez-Thonb, Elies Molinsc, Ignacio Moreno-Villosladaa, Gabriel Vallejos-
Contrerasa, Christian Sáncheza, Ximena Zarated*, Desmond Mac-Leod Careyd and Eduardo Schotte*
a
b
Institituto de Ciencias Químicas, Universidad Austral de Chile, Avda. Las Encinas 220, Campus Isla Teja,Casilla 456,
Valdivia, Chile.
Universidad Central de Chile, Toesca 1783, Santiago de Chile, Chile
c Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Bellaterra, Spain.
d Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Av. Pedro de Valdivia
425, Santiago, Chile
e Departamento de Química Inorgánica, Facultad de Química, Pontificia Universidad Católica de Chile, Vicuña Mackenna
4860, Macul, Santiago.
Keywords: Pyrazoles; Hammett correlation; Synthesis; DFT; Anticancer activity.
Abstract
The high yield synthesis, characterization and biological trials of a family of compounds containing the
pyrazole core (E)-3,5-dimethyl-1-(4-R-phenyl)-4-(phenyldiazenyl)-1H-pyrazoles (with R= 4-CH3O
(1), 4-CH3 (2), 4-H (3), 4-F (4), 4-Cl (5), 4-CF3 (6), 4-CN (7), 4-NO2 (8), 3-Cl (9), 3-NO2 (10), 2-Cl
(11) and -C6F5 (12)) are reported. Hammett correlations were found for the experimental and
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theoretical computed data, showing the dependence of all the obtained results on the donor or acceptor
nature of the R substituent. In this sense, the absorption wavelength does decrease as the electron-
withdrawing character of the substituent increases. Furthermore, the wavelength intensity does follow a
Hammett correlation with the sigma donor character of the substituents. The compounds were
1
characterized using MP, EA, UV-vis, FT-IR, H-NMR, 13C-NMR including bidimensional NMR
experiments (HMBC and HMQC). The structures of 4, 5 and 8 were successfully elucidated by means
of X-Ray diffraction. Pyrazole based dyes are known in pharmacological areas. Therefore, biological
activity of all these structures were tested against cancer cell lines over a wide library of cell lines (60),
including leukemia, colon and brain cancer, showing that the compounds have anticancer activity,
which expands the knowledge of this kind of dyes as targets for this critical application. Finally,
quantum calculations were carried out in order to give a rational explanation to the observed UV-vis
absorption bands and FT-IR signals. As shown, it is possible to tune the molecular/electronic structure
of the synthesized dyes by means of changing the -R substituent in the structure.
Introduction
Pyrazole based dyes are fascinating compounds as they have biological activity and several
applications in coordination chemistry. These dyes are also pharmacological intermediates and the
dyestuff industry uses them[1-7]. It has been reported that many pyrazoles show biological properties
such as anti-neoplastic, anti-microbial, anti-inflammatory, anti-diabetic, among others[2–4,8].
Pyrazole derivatives, as biomolecules, have attracted more attention, as they have fascinating
pharmacological properties. In the pharmacology industry, the pyrazole ring can be found in a variety
of commonly used drugs, that belong to different categories and show diverse therapeutic properties.
[9]
Moreover, pyrazoles have much importance in coordination chemistry as the pyrazolates conjugate
bases have the ability to generate a bond with metals in different modes and have the ability to generate
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strong bridges [1–3]. For instance, complexes containing the pyrazolate ring have been reported as
starting material for chemical vapor deposition. For this application they have fascinating luminescent
properties[4–7]. The pyrazole ring is possible to be obtained with a variety of previously reported
methods[2–4,8]. Those reported procedures are well known and let the synthesis of the pyrazole ring
with substituent groups in different positions, which induces changes in their electronic structure, and
therefore in their absorption profile[10–12]. Furthermore, pyrazole ring and it pyrazolate form can be
considered as polydentate ligands[13–15]. On this subject, the polydentate complex most commonly
used in coordination chemistry are polypyrazolylborates[16,17]. The pyrazolium cation, which
corresponds to the protonated form of the pyrazole ring, forms part of many salts and, in some cases,
their behavior plays an important role as supramolecular guest towards adequate hosts[18–20].
Complexes that have in their structure pyrazolato ligands (one or more) might be synthetized by
reacting a pyrazolate anions with a suitable metal salt or trying a mixture pyrazole/metallic salt with a
base[21–28].
In terms of acid-base, pyrazoles are amphoteric substances as they can donate or accept protons (H+).
In fact, the free electron pair placed on the N-atom can act as an acceptor in a hydrogen-bond, while the
N-H group can act as a donor in a hydrogen-bond. This property (acting as a hydrogen-bond donor)
explains the fact that pyrazoles are widely used in supramolecular chemistry. In this context, it has been
shown aggregation in solid-state structures of pyrazoles through the N-H group, which is influenced by
the character of the substitution in the pyrazole ring[9,28].
Some pyrazole derivatives have interesting applications in the development of new materials, also of
new brightening agents. It has been also shown that the pyrazole containing compounds have
solvatochromic and electroluminescence properties. Furthermore, pyrazoles can act as semiconductors,
liquid crystals, and organic light-emitting diodes. A more complex structure having pyrazole rings in
their structure are also of much interest, due to their synthetic utility as synthetic reagents in
multicomponent reactions, chiral auxiliaries and guanylating agents. [29]
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In order to gain more insight on a pyrazole family of dyes, in the herein article we assess the substituent
effect over the molecular, electronic and biological properties of new systems. We reported the study
on the synthesis, characterization, anticancer activity and theoretical calculations of a series of twelve
(E)-3,5-dimethyl-1-(4-R-phenyl)-4-(phenyldiazenyl)-1H-pyrazoles. The -R group has been modified
from electron-donor to electron-withdrawing, R= 4-CH3O (1), 4-CH3 (2), 4-H (3), 4-F (4), 4-Cl (5), 4-
CF3 (6), 4-CN (7), 4-NO2 (8), 3-Cl (9), 3-NO2 (10), 2-Cl (11) and -C6F5 (12). While changing the -R
substituent group, Hammett correlations were found for some of the studied properties (experimental
and theoretical data). Those Hammett correlations are dependent on the capacity of the substituent to
act as an electron-donor or electron-acceptor in the phenylpyrazole ring. Specifically, the measured
main absorption band follows a Hammett correlation (decreasing its value as the electron-withdrawing
capacity of the substituent increases). Furthermore, the wavelength intensity does also follow a
Hammett correlation with the sigma donor character of the substituents. DFT and TDDFT
computations were carried out to get the composition of the frontier molecular orbitals (FMO), to
compute the UV-Vis electron promotion and to assign their character. It is shown that is possible to
tune the molecular/electronic structure of the synthetized dyes by means of changing the -R substituent
in the structure.
Methods and Experimental Procedures
Chemicals. All compounds (aniline, acetic acid, acetylacetone, sodium nitrite, sodium hydroxide,
sodium acetate, hydrochloric acid, and substituted arylhydrazines, R-C6H4-NH-NH2 {R=4-CH3O (1),
4-CH3 (2), 4-H (3), 4-F (4), 4-Cl (5), 4-CF3 (6), 4-CN (7), 4-NO2 (8), 3-Cl (9), 3-NO2 (10), 2-Cl (11)
and (Perfluorophenyl)hydrazine, NH2NH-C6F5 (12)} and solvents (ethanol and CHCl3, CDCl3) were
obtained in the common commercial sources (Merck, Fisher and T. J. Baker) and used without
purification. The precursor 3-(2-phenylhydrazinylidene)pentane-2,4-dione was synthesized as was
previously reported [30,31] and its obtention was checked using FT-IR.
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Physical measurements. Melting points were registered on Digital STUARD SMP10 equipment.
Elemental analysis was performed on a Fisons EA 1108 micro analyzer. The UV-Visible spectra were
recorded in the 1000-250 nm range in a 10 mm quartz cells, using Perkin Elmer Lambda 35 equipment,
diluting a concentrated solution ~1.0×10-5 mol/L in CHCl3 at ~1.0×10-3 mol/L. The IR spectra in solid
state were obtained on ATR Jasco, PRO450-S, mounted on a Jasco, FT/IR-4200 instrument. 1H-NMR,
13C-NMR and bidimensional experiments (HMBC and HMQC) were registered in a 5 mm glass tube at
298 K in CDCl3 solutions using the residual solvent as internal standard, using conventional procedures
on a Bruker, AVANCE AM 400 equipment.
Data collection. Single crystals X-Ray diffraction data sets were collected at 293 K, 4 and 5 and 298 K
8 in two different equipment. Compounds 4 and 5 up a max 2θ of ca. 52º on a Enraf-Nonius CAD
diffractometer and compound 8 up to max 2θ of ca. 61º on a Bruker-AXS Smart Apex II
diffractometer, using monochromatic MoKα radiation, λ= 0.71069 Å and a 0.3º separation between
frames. Data integration was performed using WinGX program in the diffractometer package[32].
Figures 1 exhibit an ORTEP of 4, 5 and 8, respectively. Table 1, in the “Description of structures”
section, shows selected bonds length and torsion angles. The data collection and structural refinement
parameters for each compound are given in Table S1. The structures were solved by direct methods and
Fourier’s difference, and refined by least squares on F2 using the SIR-2004 program[33] and the
anisotropic displacement parameters for non-H atoms using the SIR-2004 program[33]. All
calculations to solve the structures to refine the model proposed and to obtain results were carried out
with the computer programs SHELXL97[34]. Crystallographic data (excluding structure factors) for
the structures reported in this paper have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication, CCDC No. 295833 for compound (4), CCDC No. 295834 for
compound (5) and CCDC No. 295835 for compound (8). Copies of this information may be obtained
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free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223
336 033. E-mail: data_request@ccdc.cam.ac.uk. Web page: http://www.ccdc.cam.ac.uk.
Figure 1. ORTEP view of compounds 4, 5 and 8 with a probability level of 50%.
Pyrazole Synthesis.
Procedure. In the round-bottomed flask were added 4.9 mmol (1.0 g) of 3-(2-
phenylhydrazinylidene)pentane-2,4-dione, 4.9 mmol of any arylhydrazine R-C6H4-NHNH2 {R= 4-
CH3O (1) 98%, 0.87 g; 4-CH3 (2) 98 %, 0.79 g; 4-H (3) 97 %, d= 1.099 g/mL, 0.50 mL; 4-F (4), 97 %
0.82 g; 4-Cl (5) 98 %, 0.89 g; 4-CF3 (6), 96%, 0.90 g; 4-CN (7), 97 %, 0.86 g; 4-NO2 (8), 97%, 0.77 g;
3-Cl (9), 97 %, 0.90 g; 3-NO2 (10), 98%, 0.95g; 2-Cl (11), 97 %, 0.90 g and -C6F5 (12), 97%, 0.53 g},
5 mL of glacial acetic acid and 30 mL of ethanol. The reaction mixture was stirred and heated during
36 h near the boiling point. Then, after cool at room temperature, the yellow/orange solid was filtered,
washed with water (300 mL) and dried under vacuum for 24 h. All compounds can be recrystallized in
ethanol or ethanol/H2O mixtures of variable composition. Slow evaporation of the ethanol from
solutions that contain compounds 4, 5 and 8 yielded suitable crystals for X-ray diffraction.
(E)-1-(4-methoxyphenyl)-3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazole
(1).
Yield:
72%.
Recrystallized in EtOH/H2O 9:1, final yield: 58%. MP (ºC): 96-97. EA for C18H18N4O (Mτ: 306.36
g/mol), Calc.(%) C, 70.57; H, 5.92; N, 18.29; found(%) C, 70.78; H, 6.03; N, 18.33. UV-Visible
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spectrum, CHCl3 1.96×10-5 mol/L, λmax, nm(logε): λ1: 429(3.59), λ2: 339(4.72). IR spectrum, ν(cm-1):
ν(C-H, Ar.): 3051w, 3005w; ν(C-H, Aliph.): 2961w, 2935w, 2921w, 2837w; ν(C=N), ν(C=C) or
ν(N=N): 1609w, 1589w, 1552m, 1523s, ν(N-N): 1416s. 1H NMR spectrum (400 MHz, CDCl3) δ ppm:
7.82 (d, J = 7.9 Hz, 2H), 7.48 (t, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 3H), 7.01 (d, J = 8.7 Hz, 2H),
3.86 (s, 3H), 2.61 (s, 3H), 2.60 (s, 3H). 13C NMR spectrum (101 MHz in CDCl3) δ ppm: 159.46,
153.75, 143.75, 139.10, 136.04, 132.27, 129.57, 129.03, 126.54, 121.96, 114.48, 55.68, 14.11, 11.35.
(E)-3,5-dimethyl-4-(phenyldiazenyl)-1-p-tolyl-1H-pyrazole (2): Crude yield: 92%. Recrystallized in
EtOH, final yield 78%. MP (ºC): 84-85. EA for C18H18N4 (Mτ: 290.36 g/mol): Calc.(%): C, 74.46; H,
6.25; N , 19.30; found(%): C, 74.31; H, 6.30; N, 19.40. UV-Visible spectrum, CHCl3 1.03×10-5 mol/L,
λmax, nm(logε): λ1: 430(3.81), λ2: 339(4.94). IR spectrum, ν(cm-1): ν(C-H, Ar.): 3060w, 3034w; ν(C-H,
Aliph.): 2965w, 2924w, 2857w; ν(C=N), ν(N=N) or ν(C=C): 1610w, 1587w, 1553m, 1520s, ν(N-N):
1410s. 1H NMR spectrum (400 MHz, CDCl3) δ ppm: 7.83 (d, J = 7.8 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H),
7.43 -7.34 (m, 3H), 7.30 (d, J = 8.1 Hz, 2H), 2.64 (s, 3H), 2.61 (s, 3H), 2.43 (s, 3H). 13C NMR
spectrum (101 MHz, CDCl3) δ ppm: 153.76, 143.82, 139.11, 138.19, 136.76, 136.21, 129.89, 129.59,
129.03, 124.89, 121.97, 21.25, 14.17, 11.43.
(E)-3,5-dimethyl-1-phenyl-4-(phenyldiazenyl)-1H-pyrazole (3): Crude yield: 97%. Recrystallized in
EtOH, final yield: 81%. MP(ºC): 59-60. EA for C17H16N4 (Mτ: 276.34 g/mol): Calc.(%): C, 73.89; H,
5.84; N, 20.27; found(%):C, 74.02; H, 5.94; N, 20.04. UV-Visible spectrum, CHCl3 1.00×10-5 mol/L,
λmax, nm(logε): λ1: 430(3.84), λ2: 337(4.99). IR spectrum, ν(cm-1): ν(C-H, Ar.): 3060w, 3035w; ν(C-H,
Aliph.): 2962w, 2918w; ν(C=N ), ν(N=N) or ν(C=C): 1599m, 1554m, 1509s; ν(N-N): 1411s. 1H NMR
spectrum (400 MHz, CDCl3) δ ppm: 7.83 (d, J = 7.8 Hz, 2H), 7.56-7.36 (m, 8H), 2.67 (s, 3H), 2.60 (s,
3H). 13C NMR spectrum (101 MHz, CDCl3) δ ppm: 153.77, 144.04, 139.29, 139.22, 136.37, 129.70,
129.38, 129.08, 128.21, 125.06, 122.03, 14.21, 11.51.
(E)-1-(4-fluorophenyl)-3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazole (4): Crude yield: 75%.
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Recrystallized in EtOH, final yield 66.0%. MP(ºC): 125-126. EA for C17H15FN4 (Mτ: 294,33 g/mol):
Calc.(%): C, 69.37; H, 5.14; N, 19.04; found(%): C, 69.36; H, 5.43; N, 19.12. UV-Visible spectrum,
CHCl3 9.85×10-6 mol/L, λmax, nm(logε): λ1: 435(3.98); λ2: 337 (5.02). IR spectrum, ν(cm-1): ν(C-H,
Ar.): 3065w, 3034w; ν(C-H, Aliph.): 2988w, 2967w, 2927w; ν(C=N ), ν(N=N) y/o ν(C=C): 1604w,
1
1557w, 1542w, 1514s; ν(N-N): 1409m. H NMR (400 MHz, CDCl3) δ ppm: 7.82 (d, J = 7.9 Hz, 2H),
7.47 (t, J = 7.1 Hz, 4H), 7.39 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 8.0 Hz, 2H), 2.62 (s, 3H), 2.58 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ ppm: 162.18 (d, J = 248.4 Hz), 153.68 (s), 144.13 (s), 139.16 (s), 136.28
(s), 135.38 (d, J = 3.0 Hz), 129.77 (s), 129.08 (s), 126.92 (d, J = 8.7 Hz), 122.03 (s), 116.32 (d, J = 23.0
Hz), 14.13 (s), 11.38 (s).
(E)-1-(4-chlorophenyl)-3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazole (5): Crude yield: 60%.
Recrystallized in EtOH, final yield: 43%. MP(ºC): 141-142. EA for C17H15ClN4 (Mτ: 310.78 g/mol),
Calc.(%): C, 65.70; H, 4.86; N, 18.03; found(%): C, 65.75; H, 4.89; N, 18.02. UV-Visible spectrum,
CHCl3 1.0×10-6 mol/L, λmax, nm(logε): λ1: 435(3.84); λ2: 336(4.93). IR spectrum, ν(cm-1): ν(C-H, Ar):
3064w, 3033w; ν(C-H, Aliph.): 2988w, 2963w, 2926w, 2853w; ν(C=N), ν(N=N) y/o ν(C=C):1594w,
1585w, 1556m, 1542w, 1504s; ν(N-N): 1406s. 1H NMR spectrum (400 MHz, CDCl3) δ ppm: 7.82 (d, J
= 7.5 Hz, 2H), 7.52-7.43 (m, 6H), 7.40 (t, J = 7.3 Hz, 1H), 2.66 (s, 3H), 2.59 (s, 3H). 13C NMR
spectrum (101 MHz, CDCl3) δ ppm: 153.67, 144.34, 139.19, 137.77, 136.49, 133.98, 129.86, 129.57,
129.10, 126.11, 122.06, 14.18, 11.51.
(E)-3,5-dimethyl-4-(phenyldiazenyl)-1-(4-(trifluoromethyl)phenyl)-1H-pyrazole (6): crude yield:
90%. Recrystallized in EtOH/H2O 1:8, final yield: 73.5%. MP (ºC): 109-110. EA for para C18H15F3N4
(Mτ: 344,33 g/mol): Calc.(%): C, 62.79; H, 4.39; N, 16.27; found(%): C, 62.72; H, 4.45; N, 16.63. UV-
Visible spectrum, CHCl3 9.87×10-6 mol/L, λmax, nm(logε): λ1: 430(3.80), λ2: 335(4.92). IR spectrum,
ν(cm-1): ν(C-H, Ar.): 3083d, 3057d; ν(C-H, Aliph.): 2969w, 2926w; ν(C=N), ν(N=N) y/o ν(C=C):
1
1613m, 1593w, 1560m, 1523m; ν(N-N): 1410s. H NMR (400 MHz, CDCl3) δ ppm: 7.84 (d, J = 7.5
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Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.2 Hz,
1H), 2.72 (s, 3H), 2.61 (s, 3H). 13C NMR spectrum (101 MHz, CDCl3) δ ppm: 153.60, 144.70, 142.11,
139.42, 136.82, 129.99, 129.11, 129.85 (q, J = 33.1 Hz), 126.58 (q, J = 3.7 Hz), 124.64, 123.92 (q, J =
272.2 Hz), 122.10, 14.27, 11.63.
(E)-4-(3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazol-1-yl)benzonitrile (7). Crude yield: 91%,
recrystallized in EtOH, final yield: 80.5%. MP(ºC): 197-198. EA for C18H15N5 (Mτ: 301.35 g/mol):
Calc.(%): C, 71.74; H, 5.02; N, 23.24; found(%): C, 71.99; H, 4.98; N, 23.04. UV-Visible spectrum,
CHCl3 1.00×10-6 mol/L, λmax, nm(logε): λ1: 437(3.60); λ2: 335(4.70). IR spectrum, ν(cm-1): ν(C-H,
Ar): 3081w, 3057w, 3031w; ν(C-H, Aliph.): 2994w, 2963w, 2925w, 2854w; ν(C≡N): 2221m; ν(C=N ),
1
ν(N=N) or ν(C=C): 1601m, 1580w, 1556m, 1541s; ν(N-N): 1404s. H NMR spectrum (400 MHz,
CDCl3) δ ppm: 7.82 (dd, J = 12.0, 8.1 Hz, 4H), 7.70 (d, J = 8.1 Hz, 2H), 7.49 (t, J = 7.5 Hz, 2H), 7.41
(dd, J = 16.6, 9.5 Hz, 1H), 2.75 (s, 3H), 2.59 (s, 3H). 13C NMR spectrum (101 MHz, CDCl3) δ ppm
153.52, 145.10, 142.83, 139.54, 137.12, 133.40, 130.15, 129.13, 124.49, 122.13, 118.27, 111.22, 14.35,
11.80.
(E)-3,5-dimethyl-1-(4-nitrophenyl)-4-(phenyldiazenyl)-1H-pyrazole (8): Crude yield: 89%.
Recrystallized in EtOH, final yield 72.0%. MP (ºC): 196-197. EA for C17H15N5O2 (Mτ: 321,33 g/mol):
Calc.(%): C, 63.54; H, 4.71; N, 21.79; found(%): C, 63,52 ; H, 4.81; N, 22.03. UV-Visible spectrum,
CHCl3 1.00×10-6 mol/L, λmax, nm(logε): λ1: 431(3.86); λ2: 334(5.08). IR spectrum, ν(cm-1): ν(C-H,
Ar.): 3080w; ν(C-H, Aliph.): 2967w, 2928w, 2844w; ν(C=N), ν(N=N) y/o ν(C=C): 1609w. 1595s,
1
1561s, 1522s, 1507s; ν(N-N): 1407s. H NMR (400 MHz, CDCl3) δ ppm: 8.34 (t, J = 10.8 Hz, 2H),
7.78 (dd, J = 29.1, 8.2 Hz, 4H), 7.44 (dt, J = 14.4, 7.4 Hz, 3H), 2.77 (s, 3H), 2.58 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ ppm: 153.51, 146.39, 145.32, 144.38, 139.75, 137.30, 130.23, 129.15, 124.99,
124.17, 122.17, 14.40, 11.92.
(E)-1-(3-chlorophenyl)-3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazole (9). Crude yield: 77%.
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Recrystallized in EtOH, final yield: 62%. MP(ºC): 84-85. EA for C17H15ClN4 (Mτ: 310.78 g/mol):
Calc.(%): C, 65.70; H, 4.86; N, 18.03; found(%): C, 65.78; H, 4.82; N, 18.34. UV-visible spectrum,
CHCl3 1.0×10-6 mol/L, λmax, nm(logε): λ1: 430(3.28); λ2: 336(4.49); 257sh. IR spectrum, ν(cm-1): ν(C-
H, Ar.): 3079w, 3062w, 3033w; ν(C-H, Aliph.): 2986w, 2955w, 2924w; ν(C=N), ν(N=N) /or ν(C=C):
1
1596s, 1553s, 1497s; ν(N-N): 1403s. H NMR spectrum (400 MHz, CDCl3) δ ppm: 7.83 (d, J = 7.9 Hz,
2H), 7.57 (s, 1H), 7.52-7.35 (m, 6H), 2.68 (s, 3H), 2.60 (s, 3H). 13C NMR spectrum (101 MHz, CDCl3)
δ ppm: 153.60, 144.34, 140.28, 139.30, 136.52, 135.07, 130.26, 129.84, 129.05, 128.16, 125.07,
122.74, 122.04, 14.22, 11.50.
(E)-3,5-dimethyl-1-(3-nitrophenyl)-4-(phenyldiazenyl)-1H-pyrazole (10): Crude yield: 85%.
Recrystallized in EtOH, final yield: 72%. MP(ºC): 146-147. EA for C17H15N5O2 (Mτ: 321,33 Calc.(%):
C, 63.54; H, 4.71; N, 21.79; found(%): C, 63.51; H, 4.90; N, 22.02. UV-Visible, CHCl3 1.00×10-6
mol/L, λmax, nm(logε): λ1: 434(3.28); λ2: 334(4.43). IR spectrum, ν(cm-1): ν(C-H, Ar.): 3109w, 3071w;
ν(C-H, Alif.): 2986w, 2971w, 2925w; ν(C=N), ν(N=N) y/o ν(C=C): 1614w, 1559m, 1526s; ν(N-N):
1410m. 1H NMR (400 MHz, CDCl3) δ ppm: 8.44 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.0 Hz,
1H), 7.84 (d, J = 7.9 Hz, 2H), 7.70 (t, J = 8.1 Hz, 1H), 7.49 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.1 Hz, 1H),
2.76 (s, 3H), 2.60 (s, 3H). 13C NMR (101 MHz, CDCl3) δ ppm: 153.55, 148.79, 145.01, 140.38, 139.47,
136.92, 130.35, 130.13, 130.04, 129.14, 122.41, 122.15, 119.37, 14.30, 11.65.
(E)-1-(2-chlorophenyl)-3,5-dimethyl-4-(phenyldiazenyl)-1H-pyrazole (11): Crude yield: 93%.
Recrystallized in EtOH, final yield: 72%. MP(ºC): 78-79. EA for C17H15ClN4 (Mτ: 310.78 g/mol):
Calc.(%): C, 65.70; H, 4.86; N, 18.03; found(%): C, 65.81; H, 4.90; N, 18.05. UV-Visible spectrum,
CDCl31.00×10-6 mol/L, λmax, nm(logε): λ1: 428(3.49); λ2: 333(4.73). IR spectrum, ν(cm-1): ν(C-H, Ar):
3080w, 3066w, 3034w; ν(C-H, Aliph.): 2962dw, 2923w, 2853w; ν(C=N), ν(N=N) or ν(C=C): 1590w,
1580w, 1552m, 1536s, 1510m; ν(N-N): 1416s. 1H NMR spectrum (400 MHz, CDCl3) δ ppm: 7.83 (d, J
= 8.0 Hz, 2H), 7.62-7.33 (m, 7H), 2.61 (s, 3H), 2.48 (s, 3H). 13C NMR (101 MHz, CDCl3) δ ppm:
10

ACCEPTED MANUSCRIPT
153.72, 144.61, 140.70, 136.81, 135.59, 132.52, 130.85, 130.49, 129.87, 129.73, 129.06, 127.90,
122.03, 14.14, 10.72.
(E)-3,5-dimethyl-1-(perfluorophenyl)-4-(phenyldiazenyl)-1H-pyrazole (12): Crude yield: 96%.
Recrystallized in EtOH, final yield 86%. MP(ºC): 79-80. EA for C17H11F5N4 (Mτ: 366.29. Calc.(%): C,
55.74; H, 3.03; N, 15.30; found(%): C, 55.65; H, 3.21; N,15.60. UV-Visible spectrum, CHCl3 1.00×10-
5 mol/L, λmax, nm(logε): λ1: 432(3.46); λ2: 327(4.53). IR spectrum, ν(cm-1): ν(C-H, Ar.): 3076w; ν(C-H,
1
Alif.): 2967w, 2928w; ν(C=N ), ν(N=N) y/o ν(C=C): 1562w, 1534s, 1512s; ν(N-N): 1411m. H NMR
(400 MHz, CDCl3) δ ppm: 7.84 (d, J = 7.9 Hz, 2H), 7.49 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.1 Hz, 1H),
13
2.60 (s, 3H), 2.53 (s, 3H). C NMR (101 MHz, CDCl3) δ ppm: 153.46 (s), 146.66 (s), 145.53-145.23
(m), 143.83-143.34 (m), 142.83 (ddd, J = 11.8, 7.8, 3.9 Hz), 141.54 (s), 141.21-140.80 (m), 139.57 –
139.09 (m), 137.20-136.57 (m), 136.00 (s), 130.25 (s), 129.12 (s), 122.16 (s), 114.46 (td, J = 14.2, 4.2
Hz), 14.14 (s), 10.19 (s).
In vitro anti-tumor assays. The NCI’s in vitro anti-tumor screening protocol consists of 60 human
tumor cell lines against which compounds 1-8 and 12 were tested with 3-4,5-dimethylthiazol-2-yl-2,5-
diphenyl-tetrazolium bromide (MTT) assay (11). Cancer cells were treated with 1 ꢀM of test
compounds. Dimethyl sulfoxide (DMSO) was used as a vehicle control and six wells were prepared for
each compound. After treatment, the supernatant was carefully aspirated and 150 ꢀl of DMSO was
added to each well. The absorbance was measured at 590 nm.
Computational details. All the computations were performed in Gaussian 09 [35], performing ground-
state geometry optimization with Becke's three-parameter hybrid exchange functional and the Lee-
Yang-Parr nonlocal correlation functional including the long-range interaction correction CAM-
B3LYP[36–40]. The basis set 6-31G* was used for C, N, O, Cl, Br, and H atoms[41]. The Hessian for
all compounds was also calculated to assure them as local minimum (no imaginary values were found).
Time-dependent DFT (TDDFT) calculations were also performed using the same theoretical level. The
first 60 singlet excited states were computed. Calculations by the first-principles method were used to
11

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obtain accurate excitation energies and oscillator strengths. The effect of the solvent was included with
the polarizable continuum model (PCM) using as solvent ethanol[42] for the optimization and TDDFT
calculations. See Figure S1 for all the FMO involved in the transitions.
Results and discussion
Synthesis. The compound 3-(2-phenylhydrazinylidene)pentane-2,4-dione, of formula C6H5-
NHN=C(COCH3)2, reacts with substituted arylhydrazines, R-C6H4-NH-NH2, in 1:1 molar ratio, to give
in good yields a series of 1,3,4,5-tetrasubstituted pyrazoles 1-12 (See Scheme 1). These reactions were
carried out refluxing the reactants during 36 h in EtOH and glacial HOAc as catalyst. The crude
products were purified by recrystallization in EtOH or mixtures of EtOH/H2O of variable composition,
under these conditions single crystals of three members of this series were obtained.
Scheme 1. Proposed reaction mechanism. (R=4-CH3O (1), 4-CH3 (2), 4-H (3), 4-F (4), 4-Cl (5), 4-CF3
12

ACCEPTED MANUSCRIPT
(6), 4-CN (7), 4-NO2 (8), 3-Cl (9), 3-NO2 (10), 2-Cl (11) and (Perfluorophenyl)hydrazine, NH2NH-
C6F5 (12)).
In concordance with Scheme 1, it is proposed that the reaction produces the β-dihydrazone as
intermediate that, in the course of the heating in acid medium, suffers an addition reaction followed by
displacement of one H2O molecule, yielding the respective pyrazoles[9]. The pure compounds were
characterized using analytical techniques (MP and EA), spectroscopic methods (UV-Visible, FT-IR
and NMR) and by X-ray diffraction methods the crystalline structures of 4, 5 and 8 were obtained.
Description of structures. Figure 1 shows the molecular views of compounds 4, 5 and 8. In the
Supplementary Material, in Table S1 are provided a survey of crystallographic and refinement data for
the three compounds. Moreover, Table 1 shows some selected bond distances, angles, torsion angles
and the DFT results for the geometry optimizations. The discussion of the DFT data will be further
mentioned in the theoretical calculation section.
These structures have been well refined with final R indices with [I>2σ(I)] of R1= 0.0752, wR2
=0.1202 4, R1= 0.0751, wR2= 0.1302 5 and R1=0.071, wR2=0.1565 8, see Table S1. Further
information concerning the resolution of these structures can be seen in the Supplementary Material.
Compounds 4, 5 and 8 are isostructural and the presence of the pyrazole ring confirms the cyclization
reaction proposed in the mechanism of Scheme 1. Furthermore, the azo bridge adopts E configuration
(see Figure 1). The torsion angle measured between the planes generated by each ring show that these
structures are not planar and therefore no electronic communication in the structure might be expected.
The measured distances in the three compounds for N1-N2 are consistent with the presence of a double
bond and the distances N1-C1 and N2-C9 are consistent with single bonds, see Table 1. Furthermore, the
proximity to 120º of the angles C1-N1-N2 indicate that both nitrogen atoms, N1 and N2, have a marked
sp2 character. In case of 4, a certain degree of disorder is observed. Similar previously reported
13

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structures were found in literature and a comparison between the herein reported and the previously
reported results[43] are also shown in Table 1. The previous reported structure showed a cyanide group
in the azophenyl ring. All other reported compounds show a good correlation between bond distances,
angles and the torsion angle involved in the molecules. Finally, there are no conventional
intermolecular and intramolecular hydrogen bonds in 4, 5 and 8 and the whole supramolecular structure
is built by weak interactions. However, the nitro group plays a crucial role in the crystal packing of 8
since two NO2 groups of neighboring molecules are linked via a dipole-dipole interaction. This special
interaction involving to the nitro-group has been described as type V interaction in the literature[44].
Details on the supramolecular structure of each compound are shown in the SI.
Table 1. Geometrical parameters (in Å) at DFT theoretical level are reported for compounds 1-12. For
4, 5 and 8, the experimental data obtained from the X-Ray structures are labelled with (exp).
-C1-N2=N1-
Distance -N1=N2- -C9-N2= =N1-C1- -N3-N4- -N4-C12-
-N2=N1-C9
1.252
1.252
1.252
1.252
1.077
1.252
1.222
1.422
1.422
1.422
1.422
1.667
1.422
1.470
1.386
1.386
1.386
1.387
1.675
1.387
1.440
1.365
1.366
1.366
1.366
1.386
1.366
1.379
1.421
1.420
1.420
1.419
1.426
1.418
1.426
114.8
114.8
114.8
114.8
91.9
115.8
115.8
1.4
115.7
90.4
1
2
3
4
4EXP
114.9
111.8
115.7
112.6
5
5EXP
1.251
1.251
1.251
1.250
1.421
1.421
1.421
1.432
1.388
1.388
1.389
1.404
1.367
1.368
1.368
1.381
1.415
1.414
1.412
1.423
114.9
114.9
115.0
113.0
115.6
115.6
115.5
114.8
6
7
8
8EXP
1.252
1.251
1.252
1.251
1.188
1.422
1.421
1.422
1.422
1.440
1.387
1.388
1.387
1.389
1.567
1.366
1.367
1.366
1.368
1.372
1.417
1.414
1.418
1.405
1.408
114.9
114.9
114.8
114.9
102.4
115.7
115.5
115.7
115.5
106.2
9
10
11
12
[43]*
*(E)-4-(3,5-dimethyl-1-(perfluorophenyl)-1H-pyrazol-4-yl)diazenyl)benzonitrile
Spectroscopic studies. The UV-vis spectra of compounds 1-12 exhibit two main absorption bands.
Both bands character was assigned using TDDFT calculation (vide infra). The most intense, λ1, is
14

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located in the range 327-339 nm (logε=4.43-5.08) and is attributed to the π*
←π transition centered in
the group -C-N=N-C-, which is conjugated with the pyrazolyl and phenyl rings in each compound. On
the other hand, the absorption band placed at lower energy, λ2, has lower intensity and it is centered in
the range 428-437 nm. In all compounds this absorption appears as a shoulder besides λ1 or has some
contribution of λ1, which increases the absorption coefficient of λ2 at values within the range
logε=3.28-3.98, see Figure 2 and the SI. According with the above observation, it is proposed that λ2 is
a π*←n transition that emerges from the azo group, -N=N-, present in each molecule. The UV-vis
spectra of each compound may be found in Figure 2. The absorption maxima are summarized in Table
2. Further discussion might be found in the theoretical calculation section.
Figure 2. Experimental UV–vis absorption spectrum in CHCl3.
15

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Table 2. Hammett constant value (σ), UV-Vis (exp) and TDDFT calculations (Th) for all the studied
compounds, oscillator strength (f), involved molecular orbitals and contribution (%).
Comp.
1
σt
λexp logε eV
λTh
f
Transition
HOMO
%
-0.27 429 3.59 2.97 418 0.0001
LUMO
LUMO
LUMO
LUMO
LUMO
LUMO
85
15
70
13
96
80
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
HOMO-1
HOMO-1
HOMO-4
HOMO
339 4.72 3.81 325 1.1464
2
-0.17 430 3.81 2.96 418 0.0000
339 4.94 3.82 324 1.1490
HOMO-3
HOMO
LUMO+3 12
LUMO 100
3
4
5
6
7
0
430 3.84 2.96 419 0.0000
337 4.99 3.84 323 1.1253
435 3.98 2.96 419 0.0000
337 4.89 3.85 322 1.1153
435 3.84 2.96 419 0.0000
336 4.93 3.84 323 1.1891
430 3.8 2.95 420 0.0001
335 4.92 3.85 322 1.1958
437 3.6 2.95 420 0.0001
335 4.7 3.83 323 1.3382
HOMO
HOMO-1
HOMO
LUMO
97
0.06
0.23
0.54
0.66
LUMO 100
HOMO-1
HOMO
LUMO
LUMO
LUMO
97
98
91
HOMO-1
HOMO
LUMO 100
HOMO-1
HOMO
LUMO
LUMO
LUMO
94
96
87
HOMO-1
HOMO-1
HOMO
LUMO+1 13
LUMO+1 52
8
0.78
431 3.86 2.94 421 0.0002
334 5.08 3.77 329 1.4864
HOMO
LUMO
LUMO+1 87
LUMO 10
LUMO 100
42
HOMO-1
HOMO-1
HOMO
9
0.37
0.71
430 3.28 2.96 419 0.0003
336 4.49 3.85 322 1.1611
434 3.28 2.95 420 0.0000
334 4.43 3.86 321 1.1763
428 3.49 2.97 417 0.0002
333 4.73 3.91 317 1.0561
432 3.46 2.96 418 0.0000
327 4.53 3.95 314 1.0800
HOMO-1
HOMO-1
HOMO-2
HOMO
LUMO
LUMO
LUMO
98
96
98
10
11
12
LUMO 100
LUMO 96
LUMO 100
LUMO 97
HOMO-1
HOMO
HOMO-1
The main characteristics of the IR spectra of 1-12 show the weak stretching modes ν(C-H), attributed to
the aromatic and aliphatic groups over and under 3000 cm-1, respectively. Besides, all compounds
display a set of absorptions of variable intensity in the range 1614-1497 cm-1, assigned to the stretching
modes ν(C=N), ν(N=N) or ν(C=C). The absorptions located in the range 1416-1403 cm-1 have been
16

ACCEPTED MANUSCRIPT
assigned to the single ν(N-N) of the pyrazole ring and compound 7 displays the respective stretching
mode ν(C≡N) at 2221 cm-1. All the set of IR spectra can be found in the SI.
1
The H-NMR and 13C-NMR spectra of compounds 1-12 were labeled using the numeration shown
according to Figure 3, the full summary of the observed signals for each compound and each atom is
shown in the SI. The 1H-NMR spectra display the resonances in the range 8.34-7.01 ppm attributed to
the aromatic protons. All compounds display two singlets in the ranges 2.77-2.48 ppm and 2.61-2.58
ppm that reveal the presence of the protons H1 and H5 assigned to CH3 groups linked to the pyrazole
ring. Besides, compounds 1 and 2 show the typical resonances of the substituent -R, 4-OCH3 and 4-
CH3 at 3.86 ppm and 2.43 ppm, respectively. Finally, the relative area of these resonances shows a
good agreement with the total number of expected protons for each molecule. On the other hand, the
total number of carbon resonances observed in the 13C-NMR spectra of 1-12 are consistent with the
expected number for each molecule and, the most general features are associated with the pyrazole ring
and with the -R groups of the molecules. In fact, we have found that C1, C2, C3, C4 and C5 are located
in ranges of chemical shift almost invariable around 14.11-14.40 ppm, 143.75-146.66 ppm, 135.59-
137.30 ppm, 139.10-141.54 ppm y 10.19-11.92 ppm, respectively. Also, the substituent -R of 1, 4-
OCH3, 2, 4-CH3 and 7, 4-C≡N, are placed at 55.68 ppm, 21.25 ppm and 111.22 ppm, respectively.
17

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Figure 3. Used label of hydrogen and carbon atoms for the assignments of the 1H-NMR and 13C-NMR
signals of compounds 1-12.
C-F coupling was observed in the carbon resonances of compounds 4, 6 and 12. In fact, in case of the
4-F-C6H4- group for 4 the four expected resonance signals for carbons C13, C12, C11 and C10, appear as
doublets in 162.18 ppm (J13-F=248.4 Hz), 116.32 ppm (J12-F=23.0 Hz), 126.92 ppm (J11-F=8.7 Hz) and
135.38 ppm (J10-F=3.0 Hz), respectively. The 4-CF3-C6H4- group of 6 exhibits three quartet attributed to
CF3-, C13 and C12 in 123.92 ppm (JCF3-F=272.2 Hz), 129.85 ppm (J13-F=33.1 Hz) and 126.58 ppm (J12-
F=3.7 Hz), respectively. In this case the signal of C10 remain as a singlet. Finally, the C6F5- ring of 12
displays a multiplet for C10 at 114.33 ppm and, the resonances of the carbons C11, C12 and C13 emerge
as a pair of symmetrical multiplets at 137.97 ppm (J=258.7 Hz), 143.97 ppm (J=255.7 Hz) and 142.17
ppm (J=258.4 Hz), respectively. The JC-F values observed between each pair of multiplets are in
concordance with the geminal C-F coupling; while each multiplet is produced by the C-F coupling at
two or more bonds. Figure 4 shows an approximated design of the chemical shift of the signals
observed for the C6F5- group of 12.
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Figure 4. Resonances of carbons C10, C11, C12 and C13 with C-F coupling displayed by the C6F5- group
of 12.
Additional structural information of systems 1-12 has been obtained from the bidimensional NMR
experiments (HMQC and HBMC). All the HMBC spectra can be found in the SI. In fact, all HMQC
and HMBC spectra show the same interaction pattern for the methyl protons with their neighbor
carbons located on the pyrazole rings. This pattern is in agreement with the complementary information
obtained from the HMQC spectra. To illustrate the common patterns observed in the bidimensional
spectra, Figures 5 shows the HMQC spectrum for 12, where it is possible to observe the main C-H
interactions observed in this studied compound.
19

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Figure 5. HMQC spectrum of compound 12.
In vitro anti-tumor assays. Primary in vitro one dose anticancer assay was performed over the full
NCI 60 cell panel which include 6 leukemia, 8 melanoma, 9 non-small lung cancer, 7 colon cancer, 6
brain cancer, 8 breast cancer, 6 ovarian cancer, 8 kidney cancer and 2 prostate cancer cell lines. All the
experiments were performed in accordance with the protocol of the NCI, USA. One-dose data of all the
compounds are reported as a mean graph of the percent growth of treated cells, see Figure 6, 7 and SI.
The value obtained for the One-dose assay is referred as growth relative to the control (no-drug), and
relative to the time zero number of cells. This allows detection of growth inhibition (values between 0
and 100) and lethality (negative values). For example, a value of 100 means no growth inhibition. A
value of 20 would mean 80% growth inhibition. A value of 0 means no net growth during the
experiment time. Finally, a value of -50 would mean 50% lethality. A value of -100 means all cells are
20

ACCEPTED MANUSCRIPT
dead. The one-dose data of all the compounds is given in the SI.
Figure 6. Percentage of cell growth of NCI 60 cancer cell lines displayed by the compounds 1-8 and
12.
As can be observed in Figure 6, all compounds show certain degree of growth inhibition against
different cell lines. Furthermore, all compounds also show certain degree of lethality against different
cell lines. In this sense, there is no correlation between the activity and the substituent. It is important to
notice that highly selective growth inhibition activity was observed against NCI-H522 (lung cancer cell
line), UO-31 (kidney cancer cell line) and SR (leukemia cell line) compared to the remaining cell lines,
which suggested a certain degree of tumor-tissue-type (disease-oriented) selectivity. Previous reports
have shown that the mentioned cell lines (against which the studied compounds were active) commonly
21

ACCEPTED MANUSCRIPT
have an over-expression of growth factor kinases (EGFR, HER2/Neu, PDGFR, VEGFR, in between
others).[45] On the other hand in terms of lethality, it is observed a clear activity against SK-MEL-28,
SK-MEL-28, NCI-H460, DU-145, SN12C, SF-295, SF539 and LOX IMVI. Although, in each of these
mentioned cell lines, every compound shows activity, it is only modest. Furthermore, there is no
connection of any metabolism in those cell lines, which might be a target of activity of the studied
compounds.
22

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Figure 7. Percentage of cell growth of NCI 60 cancer cell lines displayed by the compounds 1-8 and 12
for each cell line.
Theoretical Calculations. To get further understanding of compounds 1-12, theoretical calculations
were performed. It is observed that the geometrical parameters of the optimized molecular structures,
see Table 1, are in good agreement with the herein X-Ray reported values. Moreover, the bond lengths
and angles of the azo group and pyrazole ring are mostly unaffected by the modification of the -R
substituent. However, the distances between the phenyl substituted rings and the pyrazole ring get
shorter as the substituent over the phenyl ring turns more electron-attracting. This fact is observed
when the same substitution positions are compared.
The vibrational frequencies of the optimized structure of each compound were calculated to probe that
the obtained structure correspond to an energy minimum. Only positive values were obtained and a
good agreement was observed among the experimental and theoretical FT-IR spectra. The
superposition of the experimental and theoretical spectra can be found in the SI in figures S2-S13.
23

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Furthermore, in the FT-IR is observed that the vibrations positioned between 1595-1619 cm-1 of the
experimental FT-IR are assigned to the bonds -C=N- and -N=N- of the chromophores.
To carry out a description of the UV-Vis absorption spectra of 1-12, TDDFT simulations were done.
All the involved MOs of the calculated transitions are depicted in the SI (Figure S1). The calculated
transition energies show a good agreement with the experimental results. In every case the first
calculated transitions located between 421 and 417 nm goes from the HOMO, which is an orbital
delocalized over the complete structure, towards the LUMO, which is an orbital located mainly over
one side of the molecule. Specifically, in most of the cases, the LUMO is localized over the azo region
of the molecule, however in case of the two nitro substituted compounds (8 and 10), the LUMO is
localized over the phenyl ring that is connected to the pyrazole. The second and most intense calculated
transition located in the 329-314nm range, goes from the HOMO-1, which is an orbital located over the
azo group, towards the LUMO.
On the other hand, the reactivity indexes are reported in Table 3 (electronic chemical potential (µ),
chemical hardness (η), and electrophilicity (ω))[46–48]. η represents the resistance of a reagent to
modify its electronic structure[46]. It can be related, such as electronegativity, to the chemical
reactivity and stability of a system. In this family of compounds, the biggest value for η is observed for
12, which has the perfluorinated substituent (being the most electron-attracting substituent). While the
smallest value is observed for 1 which has the most electron-donor substituent. µ is related to the
direction of the electronic flux during a chemical interaction[46]. This index behaves opposite than the
tendency shown by η. In this sense the highest value is shown by 1, which has the most electron donor
substituent. On the other hand, the smallest value is observed for 8, 10 and 12, which are the three most
electron-attracting substituent. Electrophilicity index, ω, can be defined as the stabilization in energy
that an electron acceptor undergoes, when is surrounded by an electron bath at constant electronic
chemical potential. The observed values of ω give information whereas in between two molecules, one
24

ACCEPTED MANUSCRIPT
behaves as an electrophile (or nucleophile), indicated by a higher (or lower) ω value[47,48]. In this
case the highest value of ω was found for compound 10, which has one of the most electron-attracting
substituent. The smallest value is shown by 1. In this sense, low values of ω indicate an antioxidant
behavior, thus the antioxidant potential of these compounds increase as the donor capability of the R
substituent increases. Therefore, the best antioxidant would be 1.
Table 3. HOMO and LUMO orbital energies, HOMO-LUMO gap (GAP) and Reactivity indexes,
electronic chemical potential (µ), chemical hardness (η) and electrophilicity (ω), all in eV
σHammett
µ
η
ω
Comp.
HOMO LUMO GAP
-0.27 -7.31 -1.01 6.30 -4.16 3.15 2.75
-0.17 -7.41 -1.03 6.38 -4.22 3.19 2.79
1
2
0
-7.47 -1.05 6.42 -4.26 3.21 2.82
-7.50 -1.07 6.43 -4.28 3.22 2.85
-7.51 -1.09 6.42 -4.30 3.21 2.88
-7.59 -1.15 6.44 -4.37 3.22 2.96
-7.62 -1.23 6.39 -4.42 3.20 3.06
-7.71 -1.25 6.46 -4.48 3.23 3.11
-7.54 -1.10 6.45 -4.32 3.22 2.90
-7.64 -1.24 6.39 -4.44 3.20 3.08
-7.57 -1.01 6.56 -4.29 3.28 2.81
-7.73 -1.14 6.59 -4.44 3.30 2.99
3
4
5
6
7
8
9
10
11
12
0.06
0.23
0.54
0.66
0.78
0.37
0.71
-
-
Hammett Correlations. To have a clearer description observed for the overall trends due to the
modification of the substituent -R on the UV-vis absorption wavelengths, geometrical parameters,
EHOMO, ELUMO, HL GAP and reactivity indexes, taking into account the substituent electron
donor/accepting strength, Hammett constants can be considered a numerical measure of this
strength[49]. Contrasts between the two sets of compounds (electron-withdrawing and electron-donor)
confirmed a correlation between Hammett parameters and the EHOMO and ELUMO relative to R=H
(compound 3). In case of the geometrical parameters, all measured distances show a Hammett
correlation, see SI. As it can be observed, the variation in the calculated geometrical parameters, bond
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distances and angles, is small (the variation is observed in the fourth decimal place), however this result
shows that the geometrical parameters are only influenced to some extent due to the electron
donor/acceptor character of the substituent over the phenyl ring in the pyrazole ring. This is shown by
the slope near to zero in each case. The small variations measured for the interatomic distances situated
several bonds away from the -R group, can be related to the fact that there is small electronic
delocalization in the whole molecule.
In case of the energy of the system, as shown in Figure 8 the EHOMO and ELUMO, as the electron-
acceptor character of the -R group increases, the value of the HL GAP decreases, which is verified by
the confluent slopes of the trend lines.
Figure 8. Hammett correlation plot of HOMO (R2=0.94) and LUMO (R2=0.93) energies for
compounds 1-12.
In case of the measured UV-vis spectra and TDDFT vertical transitions, a Hammett correlation was
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shown in case of the lower wavelength excitation, which has the highest extinction coefficient. It was
shown that, as the electron-withdrawing character of the -R substituent increase, the absorption
wavelength decreases, see Figure 9a. Interestingly, the molar extinction coefficient also follows a
Hammett correlation with the sigma donation character of the -R substituent. As shown in Figure 9b, as
the sigma donation character of the -R substituent decreases, the molar extinction coefficient increases.
On the other hand, for the -R substituents with electron-withdrawing sigma donation, the molar
extinction coefficient does not follow any trend. finally, the second observed transition involves lower
energy MOs and no Hammett correlation was observed.
a)
b)
Figure 9. a) Hammett correlation plot of λ2 absorption wavelength (R2=0.95). b) Hammett correlation
plot of ε (R2=0.97).
In terms of the reactivity indexes, µ and ω show a Hammett correlation with similar trends, as observed
before. This shows that the general reactivity of this type of compounds can be tuned, by means of
changing the peripheral substituent. See the SI for all the mentioned trends. As shown by the Hammett
correlation results, as the electron donor/acceptor character of the substituent changes, the
molecular/electronic structure of the studied dyes changes, showing a linear tendency as the substituent
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is modified. This allows to tune the measured properties in this family of dyes, by means of changing
the -R peripheral substituent.
Conclusions
The synthesis of a new family of 12 non-previously reported pyrazoles dyes of the type 3,5-dimethyl-1-
(R-phenyl)-4-[(E)-2-phenyldiazen-1-yl]-1H-pyrazole is reported, with high yields. The substituent over
the phenyl ring was modified from electron-withdrawing to electron-donor. The nature and type of the
substituent induced changes in the geometrical parameters and significant changes over the electronic
structure and the prominent reactivity that the compounds might show. These observed effects were
studied by means of Hammett correlations. Those results allowed us to propose the tuning in the
properties depending on the donor/acceptor character of the -R substituent, due to the observed linear
tendency as the substituent is modified.
The crystal and molecular structure of three compounds (4, 5 and 8) were determined by single crystal
X-ray diffraction. In case of 4, disorder for the nitrogen atoms was observed in the fragment -C-N=N-
C-. This disorder is related to the vibrational coupled mode of this group. The molecule shows a
deviation from planarity in the angle generated by the plane of each ring that constitutes the molecule,
this fact might be a result of the lack of electronic delocalization. Variable degree of growth inhibition
against different cell lines was observed for every compound. Also, all compounds also show certain
degree of lethality against different cell lines.
DFT calculations were performed over the studied compounds, and a good correlation between the
experimental and theoretical geometrical parameters was observed. Furthermore, no big variations
were observed between the studied compounds, which is related to the robustness of the system. Also,
the IR and the frequency calculations show a good correlation. Furthermore, TDDFT calculations were
performed to obtain the vertical transitions involved in the UV-Vis absorption spectra, showing that the
first calculated excitations have involved MOs that are described as π*←π and the second calculated
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