Structural and spectroscopic investigation on a new potentially bioactive di-hydrazone containing thiophene heterocyclic rings

Article abstract of DOI:10.1016/j.molstruc.2015.10.081

Hydrazones and several substituted hydrazones are associated with a broad spectrum of biological activities, as well as compounds containing the thiophene ring. In this context, a novel di-hydrazone derived from 2-thiophenecarboxylic acid hydrazide was sy

Full text of DOI:10.1016/j.molstruc.2015.10.081

Accepted Manuscript
Structural and spectroscopic investigation on a new potentially bioactive di-hydrazone
containing thiophene heterocyclic rings
Vanessa de S. Nogueira, Maria Clara Ramalho Freitas, Wellington S. Cruz, Tatiana
S. Ribeiro, Jackson A.L.C. Resende, Nicolás A. Rey
PII:
S0022-2860(15)30381-1
10.1016/j.molstruc.2015.10.081
MOLSTR 21926
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 July 2015
Revised Date: 21 October 2015
Accepted Date: 26 October 2015
Please cite this article as: V.d.S. Nogueira, M.C. Ramalho Freitas, W.S. Cruz, T.S. Ribeiro, J.A.L.C.
Resende, N.A. Rey, Structural and spectroscopic investigation on a new potentially bioactive di-
hydrazone containing thiophene heterocyclic rings, Journal of Molecular Structure (2015), doi: 10.1016/
j.molstruc.2015.10.081.
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Graphical abstract

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Structural and spectroscopic investigation on a new potentially bioactive di-hydrazone
containing thiophene heterocyclic rings
Vanessa de S. Nogueiraa, Maria Clara Ramalho Freitasb, Wellington S. Cruza,
Tatiana S. Ribeiroc, Jackson A. L. C. Resendeb, Nicolás A. Reya,*
*nicoarey@puc-rio.br
a
b
c
Department of Chemistry, PUC-Rio, Rua Marquês de São Vicente, 225
Institute of Chemistry, UFF, Outeiro de São João Batista, s/n 24020-141, Niterói, RJ, Brazil
DCNME, UFSCar, Rodovia Anhanguera, km 174 13600-970, Araras, SP, Brazil
− 22453-900, Rio de Janeiro, RJ, Brazil
−
−
Abstract
Hydrazones and several substituted hydrazones are associated with a broad spectrum of biological
activities, as well as compounds containing the thiophene ring. In this context, a novel di-hydrazone
derived from 2-thiophenecarboxylic acid hydrazide was synthesized and completely characterized
1
by elemental analysis, XRD, FT-IR, Raman and UV-Vis spectroscopies, thermogravimetry, H
1
1
NMR, H-1H COSY and H-1H ROESY. A preliminary in silico pharmacological evaluation was
also performed in order to assess the performance of the new compound regarding some molecular
properties relevant for a drug’s pharmacokinetics in the human body.
Keywords: Di-hydrazone; Thiophene; Synthesis; X-ray diffraction; Spectroscopy
1.
Introduction
Hydrazones are a class of organic compounds defined by the presence of the functional group
R1R2C=N−NR3R4 [1]. Literature studies reveal that hydrazones and several substituted hydrazones
are associated with a broad spectrum of biological activities, such as analgesic [2], antihypertensive
[3], anticonvulsant [4], anti-inflammatory [5], anti-TB [6, 7], antitumor [8, 9], anti-HIV [10, 11],
antimalarial [12], antidepressant [13], and vasodilatory [14]. Very recently, anti-Alzheimer activity
was also reported for hydrazones derived from 8-hydroxyquinoline-2-carboxaldehyde [15, 16]. It is
known that the interesting biological properties of carbonyl-containing hydrazones are related to the
presence of the R1R2C=N−NH−CO− pharmacophore [1], which also allows for those compounds to
act as bidentate ligands, coordinating biometals through the azomethine nitrogen and the carbonyl

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oxygen. Our research group at the Pontifical Catholic University of Rio de Janeiro (PUC-Rio) has
some experience in the synthesis of heterocyclic hydrazones [17, 18].
On the other hand, thiophene belongs to a class of heterocyclic sulfur compounds containing a five-
membered ring [19]. Its structure is similar to that of pyrrole and constitutes one of the most
important classes of heterocycles, presenting a variety of actions [20], such as antimicrobial [21,
22], antitumoral [23], analgesic and anti-inflammatory [24], antihypertensive [25], anti-diabetes
[26], inhibitory activity of cholesterol [27], anti-allergic [28], insecticide [29] and antioxidant [30].
Thiophene can be attached to various heterocyclic systems giving rise to new compounds, which
exhibit a broad range of biological effects, and reduced toxicity [31].
In view of the intrinsic biological relevance of both hydrazones and tiophene, and based on the
well-known concept of hybrid drugs, this paper reports on the synthesis of a novel di-hydrazone,
obtained by condensation between 2,5-dimethoxyterephthalaldehyde and 2-thiophenecarboxylic
acid hidrazide. This compound was characterized by elemental analysis, X-ray crystallography, FT-
1
1
1
IR, Raman and UV-Vis spectroscopies, thermogravimetry, H NMR, H-1H COSY and H-1H
ROESY experiments. A preliminary in silico (computational) pharmacological evaluation was also
performed in order to assess the performance of the synthesized compound regarding some
molecular properties relevant for a drug’s pharmacokinetics in the human body.
2.
Experimental methods
2.1. Syntheses of the target compound and its precursors
For the preparation of the desired di-hydrazone, we chose a protocol that involves the synthesis of a
symmetrical precursor center, namely, 2,5-dimethoxyterephthalaldehyde, which is obtained from
2,5-bis(chloromethyl)-1,4-dimethoxybenzene. This compound was, in turn, prepared from the
starting material 1,4-dimethoxybenzene (Scheme 1, top). All chemicals were purchased from
commercial sources and used without further purification.

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II
I
O
O
O
O
1) chloroform / urotropine,
reflux overnight
2) H3CCOOH, reflux for 12 h
1) 1,4-dioxane / HCl
O
Cl
Cl
O
2) H2CO / HCl(g)
stirring for 3 h
3) HClconc.
,
3) HClconc., reflux for 8 h
O
O
O
O
O
O
NH2
S
N N
S
methanol, reflux for 2 h
N
O
+ 2
H
N N
H
H
O
S
O
O
O
II
Scheme 1. Chemical synthesis of 2,5-dimethoxyterephthalaldehyde bis(thiophene-2-carbonyl hydrazone).
2.1.1. 2,5-bis(chloromethyl)-1,4-dimethoxybenzene (I)
This compound was prepared in the way described in literature [32]. Yield: 39.3g (23%) of a white
powder, m.p. 164 ºC (literature m.p.: 165 ºC). Main IR bands (KBr): 3050, 3019, 2966, 2938,
2837, 1512, 1464, 1432, 1409, 1319, 1259, 1224, 1179, 1136, 1041, 913, 877, 736, 680, 609, 475
cm-1. 1H NMR (DMSO-d6, 300 MHz): 3,78 ppm (s, 6H, −OCH3); 4,67 ppm (s, 4H, −CH2Cl); 7,12
ppm (s, 2H, aromatic ring).
2.1.2. 2,5-dimethoxyterephthalaldehyde (II)
This precursor center was prepared in three synthetic steps, from a procedure involving small
modifications of the method already reported [33]. Initially, we dissolved 24.1 g (0.10 mol) of I and
30.0 g (0.21 mol) of urotropine (hexamethylenetetramine) in 200 mL of chloroform. The mixture
was refluxed overnight. At the end of this first stage, after leaving the mixture cool to room
temperature, the solvent was removed under reduced pressure and the solid residue obtained was
dissolved in 320 mL of a 50% acetic acid aqueous solution, being refluxed subsequently for 12
hours. In the last part of the process, 25 mL of concentrated HCl was dropwise added to the
solution, which was then refluxed for 8 hours. After cooling, we observed the formation of a yellow
precipitate, which was filtered off, washed with cold water and ethanol and dried under vacuum.
Yield: 2.1 g ( 10%), m.p. 207 ºC (literature m.p.: 207 ºC). From the filtrate, an additional 0.8 g of
the product could be obtained; its m.p. was 200 ºC. The substance was used in the next step without

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further purification. Elemental analysis - Percentages found: C, 61.9; H, 5.2. Calcd. for C10H10O4:
C, 61.9;H, 5.2. Main IR bands (KBr): 3435, 3069, 3048, 2992, 2953, 2932, 2870, 2833, 1679,
1503, 1483, 1466, 1408, 1398, 1302, 1131, 1042, 878, 660 cm-1. 1H NMR (CDCl3, 300 MHz): 3,93
ppm (s, 6H, −OCH3); 7,44 ppm (s, 2H, aromatic ring); 10,48 ppm (s, 2H, −CHO).
2.1.3. 2,5-dimethoxyterephthalaldehyde bis(thiophene-2-carbonyl hydrazone)
A 1,0 mmol (0,142 g) 2-thiophenecarboxylic acid hydrazide methanolic solution was added, under
constant stirring, to another solution containing 0.5 mmol (0.097 g) of II in 10 mL of methanol
(Scheme 1, bottom). The mixture was refluxed for 2 hours and a yellow solid was formed during
this time. The precipitated was filtered off and washed with cold methanol. The resulting yellow
solution was maintained at room temperature and, after 7 days, pale orange crystals suitable for X-
ray diffraction were separated by filtration. Yield: 0.17 g (76%), m.p. 357-358 ºC. Elemental
analysis - Percentages found: C,54.3%; H, 4.1%; N, 12.8%; S, 14.2%. Calcd. for C20H18O4N4S2: C,
54.3%; H, 4.1%; N, 12.7%; S, 14.5%. The infrared spectrum of this compound, as well as the 1D
and 2D NMR assignments, will be discussed in the Results and Discussion section.
2.2. Instruments and methodology
The determination of carbon, hydrogen, nitrogen and sulfur were performed on a Thermo Electron
Corporation CHNS analyzer, model Flash EA 1112. Melting point was determined with a MS
TECNOPON Additional equipment LTDA, PMF-11 model, and series 8276.
2.2.1. X-ray crystallography
Single crystal X-ray diffraction (XRD) data for the di-hydrazone were collected on a Bruker D8
Venture diffractometer at room temperature, using Incoatec IµS microfocus X-ray source, MoKα
radiation (λ=0.71069 Å). The crystal was mounted on a Kappa Goniometer, and reflections
collected using a PHOTON 100 detector. Data collection and cell refinement were performed with
Bruker Instrument Service APEX2 v4.2.2 [34]. Data integration was carried out using SAINT [35].
Empirical multiscan absorption correction employing equivalent reflections was achieved with the
SADABS program [36]. The structure solutions and full-matrix least-squares refinements based on
F2 were performed with the SHELXS and SHELXL program packages [37]. Anisotropic parameters
were refined to all non-hydrogen atoms. Positions concerning the hydrogen atoms were constrained
to neutral diffraction distances values [38].

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2.2.2. Spectroscopic analyses (FT-IR, Raman, UV-Vis and NMR)
Infrared spectra were recorded on a Perkin-Elmer FT-IR 2000 apparatus. Samples were measured
from 4000 to 450 cm-1 as KBr pellets. Raman spectra of the solid sample of the di-hydrazone were
performed on a Perkin-Elmer Raman Station 400, using the 785 nm line for excitation. Electronic
spectra were obtained on a Varian spectrophotometer, model Cary 50 Scan. These analyses were
carried on in DMSO solution (10-4 mol L-1) at room temperature, by using a 1.0 cm quartz cells. 1H
NMR spectrum and the 1H-1H COSY and 1H-1H ROESY (rotating-frame nuclear Overhauser effect
spectroscopy, with a mixing time of 200 ms) contour plots of the di-hydrazone were obtained at
room temperature in a Bruker Avance III HD-400 spectrometer (9.4 T, 400 MHz for the 1H nuclei)
using a 5 mm probe and d6-DMSO as solvent. Calibration of the spectrum was made with the
solvent residual peaks as references (2.50 ppm) [39].
2.2.3. Thermogravimetry
Thermogravimetric analysis of the target compound was performed in a Perkin-Elmer analyzer;
model Pyris 1 TGA, under an atmosphere of flowing nitrogen. Temperature was varied from 20 to
900 ºC at a heating rate of 10 ºC min-1.
2.3. In silico pharmacological evaluation
Theoretical pharmacological analyses include, as a key process, the investigation of a compound’s
absorption by the organism. For the target di-hydrazone, this was performed by calculating certain
parameters by a 1D-QSAR methodology and then applying the Lipinski rule of five [40]. The
calculations involving log P, drug score and theoretical solubility were conducted using the Osiris®
Property Explorer software. The Wavefunction Spartan 10 v. 1.1.0 package was used for the
calculation of the surface electrostatic potential, and structural and QSAR analyses.
3.
Results and discussion
3.1. X-ray crystal analysis
Table 1 presents the crystallographic data for 2,5-dimethoxyterephthalaldehyde bis(thiophene-2-
carbonyl hydrazone). The crystalline structure belongs to the triclinic system, space group P -1 and
Z=2. This hydrazonic compound crystallizes in two different conformations, namely, (a) and (b),

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whose ORTEP representations are displayed in Figure 1. The asymmetric unit corresponds to two
half-molecules, each of them related to one of the conformers (depicted in yellow in the figure). The
symmetrical nature of the molecule is revealed in the crystal structure, as the conformations are
located around an inversion center of the space group. Table 2 shows the bond distances and Table
3, some selected angles for both conformers. Simple bonds, such as C5−N1 and C4−C5, allow for
free rotation, providing the cited individual conformations. Conformer (a) is more linear, while (b)
is bent around both C5−N1 and C52−N12 linkages. This makes (b) much more compact, displaying
a S1B···S1B2 distance equal to 10.709 Å. In (a), the distance between S1A and S1A1 is 17.585 Å.
The carbonyl-tiophene rotation reveals another important difference: (a) adopts a syn configuration,
while the conformation is anti in conformer (b). This can clearly be observed in the torsion angles
presented in Table 4. Noteworthy, conformer (b) shows an interesting S1···N2 interaction. The key
evidence for this is the atoms distance, equal to 2.883(2) Å, which is smaller than the van der Waals
radii of both atoms involved (3.35 Å). In order to validate this possibility, we conducted a search in
the database center, using IsoStar [41] and defining a S-tiophene interaction with an aromatic sp2
nitrogen atom. The results pointed out that, from 1,219 interactions, just 418 (34%) display a S···N
distance lower than the van der Waals radii. This can be considered as an indicative of nonbonding
S···N interaction in the synthesized di-hydrazone.
Despite the differences discussed above, there are some common aspects to both conformations:
C5−O1 and C5−N1 distances suggest that conformers (a) and (b) are in the keto (amide) form.
Moreover, all the hydrazonic N2=C6 bonds correspond to the (E) isomer. Both conformations are
almost planar, since the angle deviation of the thiophene heterocycles in relation to the aromatic
central ring is equal to 9.3(3) and 26.3(3)º for (a) and (b), respectively.
An intricate network of conventional and unconventional moderate to weak intermolecular H-bonds
(Table 5) gives rise to a 3D supramolecular structure. The Hirshfeld surface analysis (Figure 2)
indicates that the foremost contributions to the lattice stabilization come from H...H, O…H and
C...H interactions. Conformers (a) and (b) are perpendicularly oriented in the structure (Figure 3)
and there are π-π stacking interactions involving the central and the thiophene rings of (a), with a
centroid-centroid distance equal to 3.798(1) Å.

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Table 1. Crystallographic data for the target di-hydrazone
Formula
Weight (g mol-1)
Crystal System
Space Group
a (Å)
C20H18O4N4S2
442.5
Triclinic
P -1
8.8631(6)
9.6956(7)
12.6422(8)
79.850(2)
80.813(2)
71.011(2)
1005.06(12)
2
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
Z
dcalc. (g cm-3)
ꢀ (MoKα = 0.71073)
(mm-1)
1.462
0.301
3950 / 3104
271
Iobs / Iobs>2σ(Iobs)
Nº. of refined
parameters
2
2
2
R (Fo / Fo >2σFo )
0.0708 / 0.0531
0.1442
wR
S
1.056
RMSpeak (e- Å-3)
0.072

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Figure 1. ORTEP representations of conformers (a) and (b) in the structure of 2,5-dimethoxyterephthalaldehyde
bis(thiophene-2-carbonyl hydrazone). Thermal ellipsoids are shown at 50% probability level. Asymmetric units, with
the hydrogen atoms omitted for the sake of clarity, are highlighted in yellow (left side).

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Table 2. Bond distances (Å) for conformations (a) and (b) of the target di-hydrazone
Conformer (a)
Conformer (b)
Bond
Distance
Bond
Distance
S1A−C1A
S1A−C4A
C1A−C2A
C2A−C3A
C3A−C4A
C4A−C5A
C5A−O1A
C5A−N1A
N1A−N2A
N2A−C6A
C6A−C7A
C7A−C8A
C7A−C9A1
C8A−C9A
C8A−O2A
C9A−C7A1
O2A−C10A
1.689(4)
S1B−C1B
S1B−C4B
C1B−C2B
C2B−C3B
C3B−C4B
C4B−C5B
C5B−O1B
C5B−N1B
N1B−N2B
N2B−C6B
C6B−C7B
C7B−C8B
C7B−C9B2
C8B−C9B
C8B−O2B
C9B2−C8B2
O2B−C10B
1.700(4)
1.707(3)
1.338(5)
1.424(4)
1.383(4)
1.477(3)
1.221(3)
1.354(3)
1.380(3)
1.271(3)
1.463(3)
1.402(3)
1.395(3)
1.378(3)
1.371(3)
1.395(3)
1.406(4)
1.712(3)
1.347(5)
1.386(5)
1.392(4)
1.474(4)
1.232(3)
1.346(3)
1.374(3)
1.275(3)
1.467(4)
1.402(4)
1.391(4)
1.382(4)
1.365(3)
1.382(4)
1.419(4)
Symmetry codes: 1 1 − x, 1 − y, −z; 2 1 − x, −y, 1 − z

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Table 3. Bond angles (º) for conformations (a) and (b) of the target di-hydrazone
Conformer (a)
Conformer (b)
Atoms
Angle
Atoms
Angle
C1A−S1A−C4A
C2A−C1A−S1A
C1A−C2A−C3A
C2A−C3A−C4A
C3A−C4A−S1A
C3A−C4A−C5A
C5A−C4A−S1A
O1A−C5A−C4A
O1A−C5A−N1A
N1A−C5A−C4A
C5A−N1A−N2A
C6A−N2A−N1A
N2A−C6A−C7A
C8A−C7A−C6A
C9A1−C7A−C6A
C9A1−C7A−C8A
C9A−C8A−C7A
O2A−C8A−C7A
O2A−C8A−C9A
C8A−C9A−C7A1
C8A−O2A−C10A
91.81(15)
C1B−S1B−C4B
C2B−C1B−S1B
C1B−C2B−C3B
C2B−C3B−C4B
C3B−C4B−S1B
C3B−C4B−C5B
C5B−C4B−S1B
O1B−C5B−C4B
O1B−C5B−N1B
N1B−C5B−C4B
C5B−N1B−N2B
C6B−N2B−N1B
N2B−C6B−C7B
C8B−C7B−C6B
C9B2−C7B−C6B
C9B2−C7B−C8B
C9B−C8B−C7B
O2B−C8B−C7B
O2B−C8B−C9B
C8B2−C9B2−C7B
C8B−O2B−C10B
91.67(16)
112.7(2)
113.1(3)
110.7(3)
111.61(19)
130.2(2)
118.1(2)
122.2(2)
123.3(2)
114.5(2)
119.6(2)
114.4(2)
121.9(2)
118.7(2)
122.2(2)
119.1(2)
120.5(2)
115.1(2)
124.4(2)
120.4(2)
118.4(2)
112.6(3)
112.6(3)
113.0(3)
110.1(2)
121.1(3)
128.8(2)
119.5(2)
118.8(2)
121.7(2)
122.6(2)
115.4(2)
120.2(2)
120.4(2)
120.6(2)
119.0(2)
119.8(2)
116.5(2)
123.7(2)
121.2(2)
117.3(2)
Symmetry codes: 1 1 − x, 1 − y, −z; 2 1 − x, −y, 1 − z

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Table 4. Selected torsion angles (º) for conformations (a) and (b) of the target di-hydrazone
Atoms
Conformer (a)
Conformer (b)
C4−C5−N1−N2
−174.0(2)
−7.1(4)
5.3(4)
0.0(4)
172.6(2)
172.8(2)
O1−C5−N1−N2
S1−C4−C5−O1
S1−C4−C5−N1
C3−C4−C5−O1
C3−C4−C5−N1
C5−N1−N2−C6
N1−N2−C6−C7
179.3(2)
−7.4(4)
−5.9(4)
173.8(3)
−177.4(3)
1.9(4)
178.4(2)
−167.9(2)
178.5(2)
−179.7(2)
Table 5. Intermolecular H-bonding parameters for conformations (a) and (b) of the target di-hydrazone
˂DHA/º
Acceptor
d(H···A)/Å
Donor−H
N1B−H
d(D−H)/Å
d(D−A)/Å
3.017(3)
O1A
0.86
2.19
160.4
N1B−H
C6B−H6B
N2A
O1A
0.86
0.93
0.86
0.93
0.93
0.96
0.96
0.93
2.53
2.63
2.17
2.59
2.44
2.91
2.98
2.87
3.115(3)
3.381(3)
3.003(3)
3.463(4)
3.281(3)
3.691(4)
3.818(4)
3.741(3)
126.4
138.1
162.1
157.4
149.7
139.0
146.6
155.6
O1B*
O1B*
O1B*
S1A*
S1B**
S1A***
N1A−H
C3A−H3A
C6A−H6A
C10A−H10A’’
C10A−H10A’’’
C3B−H3B
Symmetry codes: * −x, 1 − y, −z; ** 1 − x, 1 − y, −z; *** x, 1 + y, z

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Figure 2. Hirshfeld surfaces, with the corresponding fingerprint plots broken down into contributions (%) from specific
pairs of atom-types, for conformations (a), top, and (b), bottom, of the target di-hydrazone.
Figure 3. Representation of the perpendicular disposition that conformations (a) and (b) adopt in the lattice.

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3.2. Vibrational spectroscopy
Figure 4 displays the infrared (top) and Raman (bottom) spectra of the di-hydrazone, while Table 6
lists some selected (stretching) vibrational absorptions, along with their respective assignments.
A comparative analysis of the infrared spectra of 2,5-dimethoxyterephthalaldehyde bis(thiophene-2-
carbonyl hydrazone) and that of its di-aldehyde precursor II (Figure S1, Supplementary material)
provides important information concerning the obtention of the target compound. For example, the
frequencies related to the aldehydes’ ν(C−H) and ν(C=O) modes, appearing at, respectively, 2870
and 1679 cm-1 in the spectrum of II are not observed in the FT-IR spectrum of the di-hydrazone
indicating the absence of such groups in the final product. Instead, a rather broad and intense IR
band at 1640 cm-1 (observed as a weak peak in the Raman spectrum) associated to the ν(C=O) and
ν(C=N) modes of carbonyl hydrazones, which are in this case overlapped, prove the attachment of
the 2-thiophenecarbonyl-derived arms to the precursor center II through a condensation reaction. In
fact, the C=N stretching modes of azomethine groups show absorptions close to that of carbonyl
stretchings [42]. This fact can sometimes difficult an accurate assignment. Also, the IR spectrum of
the target compound shows characteristic hydrazone bands at 3162 and 1135 (1124 cm-1 in Raman)
cm-1, which can be ascribed, correspondingly, to the ν(N−H) and ν(N−N) frequencies. These values
are in agreement with those observed for similar compounds [43, 44].

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Figure 4. Infrared (top) and Raman (bottom) spectra of the target di-hydrazone in the range 3250-500 cm-1.

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Table 6. Selected infrared and Raman absorptions, with their respective assignments
FT-IR (cm-1)
Raman (cm-1)
Assignment
3162
-
ν(N−H)
3078, 3045
2999
-
-
-
ν(C−H)arom.
νas(C−H)methyl
νs(C−H)methyl
2953, 2937, 2903
ν(C=O) +
1640
1640
ν(C=N)azomethine
1591, 1554, 1518
1594, 1560, 1520
ν(C=C)thiophene
ν(C=C)central ring
νass(C−O−C)
ν(N−N)
1465, 1454, 1416
- , 1448 , -
1226
1124
1037
860
1219
1135
1043
858
νs(C−O−C)
νass(C−S−C)thiophene
νs(C−S−C)thiophene
ν(C−S)
845
-
757
756
The carbon-carbon stretching modes of the thiophene rings and the benzene central ring are very
well separated in the vibrational spectra of the di-hydrazone, with those related to the heterocycles
being observed at higher wavenumbers. The parallel analysis of the IR spectra of the precursors II
and 2-thiophenecarboxylic acid hydrazide (Figure S2, Supplementary material) assisted us in the
assignment of these bands. Whereas the thiophene rings originate absorptions at 1591, 1554 and
1518 cm-1 (1594, 1560 and 1520 cm-1 in the Raman spectrum), the central ring is associated to a
series of bands at 1465, 1454 and 1416 cm-1. Hydrogen-containing aromatic rings usually exhibit
multiple weak bands in the region 3100-3000 cm-1 due to C–H stretching vibrations. In the present
case, these vibrations are registered at 3078 and 3045 cm-1 in the IR spectrum [45].

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The presence of the thiophene ring was confirmed by its characteristic stretching bands involving
the sulfur atom. The IR absorptions at 858 [νass(C−S−C)], 845 [νs(C−S−C)] and 757 cm-1 [ν(C−S)]
were, in this context, fundamental in order to identify the heterocycle in the target compound [46].
On the other hand, the asymmetric and symmetric stretching modes of the methoxyl substituents in
the central ring are observed, respectively, at 1219 and 1043 cm-1 in FT-IR. These modes appear,
correspondingly, at 1226 and 1037 cm-1 in the Raman spectrum. The C−H stretching vibrations of
the methoxyl groups occur at 2999, 2953, 2937, and 2903 cm-1 [47].
3.3. Electronic spectroscopy
The UV-Vis spectrum of 2,5-dimethoxyterephthalaldehyde bis(thiophene-2-carbonyl hydrazone) is
shown in Figure 5, along with those of its synthetic precursors II and 2-thiophenecarboxylic acid
hydrazide. In a similar way to that observed for the previously published isonicotinoyl hydrazone of
o-vanillin [17], there are two well-defined groups of bands, being the more energetic absorptions
related to π → π* transitions. These bands, detected at 329 (ε = 5600 L mol-1 cm-1) and 347 nm
(5300 L mol-1 cm-1), are red-shifted when compared to those of the compound in the cited reference
due to the more delocalized nature of a di-hydrazone.
On the other hand, n → π* transitions (responsible for the color of the di-hydrazone) appear as an
intense band at 395 nm (ε = 7500 L mol-1 cm-1) with a shoulder at 414 nm (6400 L mol-1 cm-1). The
band is closely related to that observed in the spectrum of precursor II (395 nm, 2100 L mol-1 cm-1).
1,0
0,9
n → π*
0,8
0,7
π → π*
0,6
0,5
0,4
0,3
0,2
0,1
300
350
400
450
500
550
Wavelength (nm)

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Figure 5. Electronic absorption spectra of the precursors II (green line) and 2-thiophenecarboxylic acid hydrazide
(black line), as well as that of the resulting di-hydrazone (red line) in the range 280-550 nm. [ ] = 10-4 mol L-1.
3.4. NMR spectral studies
For a better allocation of the di-hydrazone 1H NMR spectrum, a 2D COSY contour plot (Figure 6)
was used in order to correlate vicinal hydrogens. All the hydrogens present in the molecule were
found and Table 7 shows the proposed assignments, along with other useful information.
Despite the symmetric nature of the compound, two well-defined sets of associated signals are
observed in the 1D spectrum of the di-hydrazone, indicating that the symmetry of the molecule is
broken by some kind of intra- or intermolecular interactions. This effect probably involves rotation
around single bonds, which, as seen from the crystal structure of the compound, lead to conformers
with small energy differences between them. The most affected, i.e. more separated, signals are
those related to the azomethine (H6/H6*) hydrogens, followed by the central ring (H9/H9*), some
of the thiophene rings (H1/H1* and H3/H3*) and, finally, the −NH hydrogens. On the other hand,
the methoxyl (H10/H10*) and, mainly, the thiophene-related H2/H2* hydrogen atoms are virtually
1
equivalent. With the help of a H-1H ROESY contour plot, we were able to observe through-space
correlations between H6/H6* and −NH hydrogens, as well as between −NH and H3/H3*. The latter
implies that, in solution, the hydrazonic lateral chains of the molecule are in a conformation similar
to the one in conformer (a), i.e. a syn configuration. Another hypothesis is that free-rotation around
single bonds exceed the NMR chemical shift timescale. However, the reason behind the non-
equivalency of some hydrogens in this symmetric di-hydrazone remains unclear.
Table 7. 1H NMR chemical shifts, in ppm, for the target di-hydrazone (d6-DMSO) at room temperature. See Figure 6,
inset, for a complete atom labeling scheme, which is in agreement with the crystallographic number system
Atom
δ
N1H / N1H*
11.99 (s, 1H) / 11.94 (s, 1H)
H6 / H6*
H3 / H3*
H1 / H1*
H9 / H9*
8.79 (s, 1H) / 8.43 (s, 1H)
8.06 (d, 1H, 3JHH ~ 4.0) / 7.95 (d, 1H, 3JHH ~ 4.0)
7.98 (d, 1H, 3JHH ~ 4.0) / 7.88 (d, 1H, 3JHH ~ 4.0)
7.69 (s, 1H) / 7.52 (s, 1H)

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H2 / H2*
7.23 (t, 2H, 3JHH ~ 4.0)
H10 / H10*
3.93 (s, 3H) / 3.89 (s, 3H)
The signal multiplicities, as well as their respective integration and coupling constants (J, Hz) are in parentheses
Figure 6. Section of the 1H-1H COSY contour plot of the target di-hydrazone, displaying the aromatic region.
3.5. Thermogravimetric analysis
From the thermal decomposition curve of the compound, we observed the absence of crystallization
solvent in the structure, because there is no significant mass loss below 300 ºC. This information is

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confirmed by the XRD analysis, as discussed above. Between 350 and 390 ºC, with a maximum
decomposition rate at 372 ºC, an important and well-defined weight loss, corresponding to 72.3% of
the initial mass, takes place. As the observed melting point for the compound is around 357-358 ºC,
this phase transition is certainly associated to chemical transformations of the di-hydrazone.
3.6. Computational pharmacological evaluation
Table 8 presents some important theoretical parameters calculated for the di-hydrazone, along with
some reference values. Most parameters are within the desired ranges for good oral bioavailability,
with the polar surface area (PSA) being a little higher than the upper limit. On the other hand, the
calculated aqueous solubility of the compound is too low, which can be overcome through the use
of an adequate delivery system or by a salt (for example, hydrochloride) formation strategy. Based
on these parameters, the drug score of the compound, an estimate of the probability the substance
has to become an actual drug, can be calculated. This di-hydrazone displays a drug score of 45%.
Table 8. Performance of the target di-hydrazone concerning some molecular properties relevant for a drug’s
pharmacokinetics in the human body, along with reference values
di-hydrazone
Reference values
MW (g mol-1)
HBD
442
≤ 500
2
≤ 5
≤ 10
≤ 140
≤ 5
HBA
10
PSA (Å2)
157.8
4.34
−5.88
10
Calcd. log P
Calcd. log S
Rotatable bonds
Drug score
≥ −4
≤ 10
-
0.45
MW – molecular weight; HBD – H-bond donors; HBA – H-bond acceptors; PSA – polar surface area
Altogether, the in silico results indicate that the synthesized compound possesses some molecular
properties important for a drug’s pharmacokinetics in the human body. However, the Lipinski rule
does not predict if a specific compound is pharmacologically active.

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4. Conclusions
A novel di-hydrazone derived from 2-thiophenecarboxylic acid hydrazide was synthesized in good
yields through a protocol involving a condensation reaction with a terephthalaldehyde derivative.
The compound crystallizes in two different conformations, one more linear and the other, bent.
Interesting intramolecular S···N interactions seem to be involved in the stabilization of the latter.
All the hydrazonic bonds correspond, however, to (E) isomers. There is no crystallization solvent.
Distinctive IR and Raman bands denote hydrazone formation, as well as the presence of the
thiophene rings. This compound melts at 357-358 ºC in a process that comprises an important loss
1
of weight (thermal decomposition). The solution H NMR spectrum of the substance exhibits two
well-defined sets of signals, which is, a priori, unexpected for such a symmetric molecule. The 1H-
1H ROESY experiment suggests a syn configuration for the hydrazonic lateral chains. Finally, this
di-hydrazone performs quite well concerning the Lipinski rule of five, presenting a drug score of
45%. The compound is being screened towards a series of biological activities and the results will
be the subject of future reports.
Supplementary information
IR spectra of the synthetic precursors II and 2-thiophenecarboxylic acid hydrazide in the range
4000-450 cm-1. CCDC 1413069 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or
from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033).
Acknowledgments
Nicolás A. Rey wishes to thank FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do
Estado do Rio de Janeiro, Brazil) and CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico, Brazil) for the research fellowships awarded. The authors are indebted to Rosana
Garrido Gomes for her invaluable help in the NMR analyses, performed in the Central Analytical
Facility of the Department of Chemistry (PUC-Rio).
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Highlights
•
•
•
•
A novel di-hydrazone containing thiophene heterocyclic rings was synthesized.
This symmetrical compound crystallizes in two different conformations.
Its solution 1H NMR spectrum exhibits some unexpected non-symmetric features.
This di-hydrazone performs quite well concerning the Lipinski rule of five.