Geometrical Isomerism in Guanabenz Free Base: Synthesis, Characterization, Crystal Structure, and Theoretical Studies

Article abstract of DOI:10.1021/acs.cgd.9b00026

Guanabenz is a drug used for the treatment of hypertension, and it exhibits isomerism and tautomerism. In this article, X-ray diffraction studies (single crystal and powder) as well as thermochemical analysis on the guanabenz have been reported. The resul

Full text of DOI:10.1021/acs.cgd.9b00026

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Article
Geometrical Isomerism in Guanabenz Free Base: Synthesis,
Characterization, Crystal Structure and Theoretical Studies
Deepika Kathuria, Sumit S. Chourasiya, Aabid Abdullah Wani,
Manjeet Singh, Subash C. Sahoo, and Prasad V. Bharatam
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00026 • Publication Date (Web): 02 May 2019
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Geometrical Isomerism in Guanabenz Free Base:
Synthesis, Characterization, Crystal Structure and
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Theoretical Studies
Deepika Kathuria,† Sumit S. Chourasiya,† Aabid Abdullah Wani,† Manjeet Singh, §
Subash C. Sahoo*§ and Prasad V. Bharatam*†
†Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S. A. S. Nagar – 160062, Punjab, India
§Department of Chemistry, Panjab University Chandigarh, Sector-14, Chandigarh, 160014, India
KEYWORDS: Guanabenz, Isomerism, Crystal Structure, Polymorphism, DFT
ABSTRACT: Guanabenz is a drug used for the treatment of hypertension, it exhibits isomerism
and tautomerism. In this article, X-Ray diffraction studies (single crystal and powder), as well as
thermochemical analysis on the guanabenz have been reported. The results indicated that E/Z
isomerism in guanabenz is responsible for the identification of two solid forms, Form I (E-isomer)
and Form II (Z-isomer). These two forms may be treated as geometrical polymorphs. Form I of
guanabenz was already known. Identification of Form II and its X-Ray structure are reported first
time in this article. Quantum chemical analysis and 2D fingerprint plots as Hirshfeld surfaces of
these two forms highlight the differences in intermolecular hydrogen bonding, energy differences
and π- stacking interactions. The quantum chemical studies indicate that Form-I (E isomer) is more
stable than Form-II (Z isomer) by 2.13 kcal/mol. The presence of Form II in the crystal structure
has been rationalized by quantum chemical calculations on the basis of dimerization energies
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which suggest that the dimer of Z isomer is more stable than the dimer of the E isomer by ~7
kcal/mol.
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Introduction
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In a search for new and better antihypertension drugs during 1960’s, a series of benzylidene
aminoguanidines were synthesized by Wyeth Laboratories, Inc., Philadelphia. One of the
compounds of this series, Wy-8678 (E-2,6-dichlorobenzylidene aminoguanidine acetate, 1, Figure
1) was identified as a new potent hypotensive agent in 1969.1 The common name given to this
compound was guanabenz due to the presence of guanidine and benzylidene moieties. Its
antihypertensive action is attributed towards its high affinity to α2-adrenergic receptor.2
Subsequently, the clinical trials were carried out and guanabenz stands out the most potent
compound of the series.3,4 Guanabenz acetate (1) is now in use for the treatment of hypertension
and is sold under the brand names Wytensin, Rexitene, Hipten, etc.4 Apart from the anti-
hypertensive activity of guanabenz, it has also been reported for the treatment of various diseases
such as cutaneous cystic fibriosis,5,6 amyloidosis,7 prion-based diseases,8-10 inflammation etc.11,12
Cl
Cl
NH2
NH2
NH2
H
N
N
N
H
N
H
N
Cl
Cl
O
O
1
2
Wy-8678
(Guanabenz acetate)
(Guanabenz free base)
Figure 1. The structure of guanabenz acetate (1) and guanabenz free base (2).
Due to the presence of highly basic guanidine moiety in guanabenz, it is marketed as an
acetate salt for oral administration (Figure 1). The presence of imine (–CH=N) and guanidine
structural moieties in guanabenz attracted researchers to study its structural properties. The (E/Z)
isomerization study on guanabenz acetate and its derivatives was carried out in 1976 by Tsuijkawa
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et al.13 The authors suggested that the E-isomer of guanabenz acetate (colourless needle) can easily
isomerize to Z-isomer (pale yellow powder) by irradiation of U.V. rays on aqueous and methanol
solution. The structural differences between E/Z isomers were established by Nuclear Overhauser
Effect (NOE) experiments.13 These observations were also supported by Holzer and Gyorgydeak
in 1992 in their study on the structure of various guanylhydrazones obtained from aromatic
aldehydes.14 Recently, in 2008, a crystal structure of acetate salt of guanabenz (1) has been reported
by Tanaka and Hirayama.15 The single crystal X-ray structure of guanabenz acetate indicates that
guanabenz (base) and acetic acid are connected by two hydrogen bonds (Figure 1) and the entire
salt lies substantially within a single plane.15
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Although, guanabenz is marketed in its monoprotonated form but its free base (2) cannot be
ignored, since the available literature on the biological activity of guanabenz and other
guanylhydrazone hydrochloride suggested the existence of considerable portion (~16 %) of free
base at physiological temperature (~37 °C) and pH (7.4).16,17 A few studies were reported on the
structural aspects of the free base of guanabenz which includes (i) conformational analysis of both
monoprotonated as well as free base of guanabenz,16 the single crystal X-Ray diffraction analysis
of E-isomer of Guanabenz (its details are not available in CCDC)16 (ii) the experimental and
quantum chemical studies on guanabenz to understand prototropic tautomerism.18,19 Recently, we
have investigated the tautomerism,18,19 isomerism20 and medicinal applications21 of azines and
concluded that guanylhydrazones exist preferentially in their azine tautomeric state with E-
geometry across -C=N, which were evidenced by analyzing the crystal structures.
The quantum chemical studies on guanabenz also suggested that, it prefers to exist in the
azine tautomeric state (∆G = 5.96 kcal/mol) in comparison to its hydrazone tautomeric state. It
was found that the azine tautomer of guanabenz carries very interesting properties as it possess
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two imine bonds linked by N-N single bond. The conjugation22 vs. conjugation stopper property23
vs. conjugation switch property24 across azines was explored using structural studies (X-rays and
quantum chemical). In addition, polymorphism and conformation isomerism in azines was also
reported earlier by Glaser and co-workers.25 Taking these facts together it can be considered that
guanabenz can also show E/Z isomerisation or/and polymorphism (Figure 2). This proposition
prompted us to carry out the solid state and computational studies on guanabenz free base.
Previous Study: Prototropic tautomerism18,19
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Cl
Cl
NH2
NH
NH2
NH2
G = 5.96 kcal/mol
N
N
N
N
H
Cl
H
Cl
H
Hydrazone form
E-Azine form
Form I
This Study: Geometrical Isomerism
Cl
H
NH2
NH2
NH2
Isomerism
H
N
N
N
N
NH2
Cl
Cl
Cl
Z-Azine form
Form II (Minor)
E-Azine form
Form I (Major)
Figure 2. Tautomerism and Geometrical Isomerism in guanabenz free base.
Experimental Section
Materials and Methods
The reagents and chemicals required for the study were procured from Sigma Aldrich and
were used as such without further purification unless otherwise mentioned. The progress of the
reaction was monitored by Thin Layer Chromatography (TLC) performed on silica gel aluminium
1
13
plates and visualization was done by UV light. H NMR (400 MHz) and C NMR (101 MHz)
spectra were recorded on a Bruker Advance spectrometer with TMS as an internal standard. The
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1H NMR and 13C NMR spectra were recorded for DMSO-d6 at 2.50 ppm and 39.51 ppm
respectively. Chemical shift (δ) values are reported in part per million (ppm). Coupling constants
(J) were reported in Hertz (Hz). The abbreviations used to characterize the signals are as follows:
s = singlet, d =doublet, t = triplet, br s = broad singlet. High resolution mass spectra were taken
using ESI-TOF method. Mass spectra were recorded using ESI mode. The IR spectra were
recorded on Thermo Scientific (NICOLET IS50) FT-IR spectrophotometer in the range 4000-400
cm-1.
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X-ray Crystallography
Crystals were mounted on Hampton cryoloops. All geometric and intensity data for the
crystals were collected using a Super-Nova (Mo) X-ray diffractometer equipped with a micro-
focus sealed X-ray tube Mo-Kα (λ = 0.71073 Å) X-ray source, and HyPix3000 (CCD plate)
detector of with increasing ω (width of 0.3_ per frame) at a scan speed of either 5 or 10 s/frame.
The CrysAlisPro software was used for data acquisition, and data extraction. Using Olex2,26 the
structure was solved with the SIR200427 structure solution program using direct methods and
refined with the ShelXL28 refinement package using Least Squares minimization. All non-
hydrogen atoms were refined with anisotropic thermal parameters. Details of crystallographic data
and structural refinement parameters are summarized in Table S1. Bond angle and bond length
geometric parameters are listed in Table S2.
Powder X-ray Diffraction
Powder X-ray Diffraction (PXRD) patterns were recorded on a Phillips PAN analytical
diffractometer for Cu Kα radiation (λ = 1.5406 Å), with a scan speed of 2° min−1 and a step size
of 0.02° in 2θ.
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Thermal Analysis
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Thermal analysis methods were used in this study which includes thermogravimetric
analysis (TGA) and differential scanning calorimetry (DSC). Thermal gravimetric analysis was
carried out using Mettler Toledo instrument with an SDTA sensor by loading over an alumina pan
and heating from 30 °C to 1000 °C at a heating rate of 10 °C min-1 under N2 atmosphere. DSC was
performed using a Mettler-Toledo DSC-821 differential scanning calorimeter). Indium was used
for calibration. Accurately weighed samples (5-8 mg) were placed in hermetically sealed
aluminum pans and scanned at 10 °C/min under a nitrogen purge. The powders of the Form I and
Form II were heated at 10 °C/min. 2D fingerprint plots as Hirshfeld surfaces were calculated using
crystalexplor software.29
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Computational Methods
The quantum chemical calculations were carried out using the Gaussian09 package.30 Geometry
optimizations were carried out by DFT31 using Becke−Lee−Yang−Parr (B3LYP)32/X-D33
level of theory with 6-311++G(d,p) basis set. Frequencies were computed analytically for all the
optimized species to characterize each stationary point minima. Gibbs free energy was employed
in estimating relative energies.
Results and Discussion
Synthesis and Characterization
Aminoguanidine hydrochloride (1 mmol) was dissolved in water (2 mL) and to the stirred
solution, NaOH pellets were added (5 mmol). The reaction mass was stirred to get clear solution,
subsequently 2,6-dichlorobenzaldehyde (1 mmol) was added. The reaction mass was stirred at
room temperature which resulted in the formation of precipitate of product in 1h. (Scheme 1).19
The resultant precipitates were filtered and washed with water (2×2 mL) to remove the excess of
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NaOH. The precipitates were dried under reduced pressure to afford guanabenz free base in 96 %
yield. This synthetic procedure is different from the standard method in which protonated
guanabenz is isolated.34
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Cl
Cl
NH.HCl
NH2
H2O
NH2
NH2
H2N
N
N
NaOH, rt, 1h
CHO
N
H
Cl
2,6-dichlorobenzaldehyde
Cl
2
Aminoguanidine
hydrochloride
Guanabenz ( )
(Azine tautomer, E isomer)
Scheme 1. Synthesis of guanabenz free base (2).
1
The characterization of the product by H NMR spectroscopy in DMSO-d6 confirms the
formation of guanabenz in its preferred azine tautomeric state as it is evidenced from the presence
of two broad singlets of two protons each at 5.81 ppm and 5.84 ppm which are the characteristic
probe for the presence of azine tautomer.19 Further, the confirmation for the presence of azine
tautomer was provided by 13C NMR spectroscopy where a signal at δ 161.98 ppm was observed
which is attributed to the C(NH2)2 carbon, a characteristic probe of azine tautomer. From the 1H
NMR study, we observed the presence of only one isomer and it is assumed to be belong to the E-
isomer of guanabenz.
When the compound was dissolved in pure methanol, suitable block shape crystals (Form I)
started crystallizing out from pure methanol via slow evaporation at room temperature within 2
days and the yield kept on increasing subsequent days but there is no sign of Form II crystals in
the same pot. The same observation occurs in pure water as solvent medium but the time duration
is slightly longer as compared to pure methanol. Interestingly, a 5-10 % of water in methanol gave
the first indication of existence of Form II, which appeared towards the end of the crystallization.
However, the quality of the Form II crystals were very poor as well as brittle in nature. After
several combination of mixture, it was found that the 3:1 mixture of methanol-water was the best
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choice to improve the quality and yield of crystals of Form II along with Form I, however, the final
yield ratio achieved is of 95:5 (Form I : Form II).
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From the crystal data, it is clear that Form I has slightly higher density than Form II (Table
1) and the crystallization behavior is found to be very interesting. Either pure water or methanol
yields Form I exclusively, while the mixture of solvents (MeOH: Water = 3:1) gives Form I and
Form II with final yield ratio of 95:5. The above results are in well agreement with the observation
by Diamant et al., where Form I was obtained while crystallizing from pure water.16 Again, it is
found that Form I always crystalizes faster with good yields than Form II in methanol-water
mixture with a time gap of more than two weeks. Keeping the crystallization till dryness of the
solvent, results in lots of cracks on the surface of the Form I crystals. Both the high yield and fast
crystalizing properties of Form I over Form II, might be the possible reasons because of which the
existence of Form II was overlooked easily even though Form I has been known since 1985.
Attempts to grow only Form II, was not successful in either of the pure solvent (methanol and
water) and at the same time the screening of crystallization in other solvents like ethanol,
acetonitrile, acetone, chloroform, ethyl acetate, and acetone gave no fruitful results mainly due to
poor solubility. The block shaped Form I crystals are relatively harder while Form II crystals are
rod shaped and fragile in nature. The quality of diffractions for Form II crystals are found to be
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very poor (diffracting to only low resolution) as compared to Form I, and as a result of, data
quality could not be improved even after several attempts.
It is well known that seeding facilitates the crystallization process via heterogeneous or
secondary nucleation. Attempts to obtain more crystals of Form II in the presence of few crystals
of Form II to facilitate secondary nucleation, resulted in no further success and crystallizing
properties remain same. It is again confirmed that Form I grows faster than the seed crystals and
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crystallize out. As per the cross nucleation phenomena35 several different solid state forms nucleate
on each other and the state with a fastest growth rate will be eventually observed regardless of the
rate of nucleation, in the present case this concept holds good for Form I.
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Crystallographic and Structural Details
The two types of crystals can be separated mechanically from the same crystallization pot
due to their distinct crystal morphology – Form I exclusively adopts block shape and Form II
adopts rod shape. The optical photographs of the two forms show distinct crystal morphologies of
the two isomers (Figure 3). The two forms show prominent dissimilarities in many aspects like
crystal habits, molecular conformations and crystallization towards different solvent environment.
In this contribution, we were thus presented with an opportunity for the comparison of the
molecular assembly in the crystals of both the Forms, thermal studies and theoretical calculations.
Figure 3. Ball and stick models of (a) Form I and (b) Form II; optical pictures of the crystals (c)
Form I and (d) Form II; overlap diagrams of Form I and Form II at each ends showing level of
mismatches (e-f, red: Form I, green: Form II).
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Table 1 lists the cell parameters of Form I and Form II obtained in this study. A comparison
of cell parameters of Form I has also been made with that of reported by Diamant et al. in 1985.16
The cell parameters of the two forms are quite different with Z′ values (Form I, Z′ = 1; Form II, Z′
= 2; Table 1). The ORTEP plots of the two forms are provided in Figure 4.
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Table 1. Comparison of cell parameters of the reported crystal structures of guanabenz Form I
with Form II. Structural details of the reported structure (Form I) were not available in CCDC and
hence a new data set collected to highlight the differences.
Compound
CCDC No.
Reference
space group
T/K
Form I (E) Form I (E)
not found 1866599
Reference-16 This work
Form II (Z)
1866600
This work
P-1
P21/c
unknown
16.290
8.309
7.490
90
P21/c
293
293
a/Å
16.3082(4)
8.2933(3)
7.4489(3)
90
7.9018(3)
10.8460(7)
12.9559(8)
72.515(4)
78.313(5)
83.119(4)
2/2
b/Å
c/Å
α/deg
β/deg
98.34
90
98.201(3)
90
γ/deg
Z′/Z
1/4
1/4
V/Å3
unknown
-do-
997.15(6)
1.539
1035.03(10)
1.483
Dcal/(gcm-3)
R1[I>2σ(I)]
Reflns collected
Unique reflns
Observed reflns
0.035
unknown
-do-
0.0426
7796
0.0712
14259
2112
4384
-do-
1768
2410
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WR2[all]
Rint
-do-
0.0988
0.0324
1.054
0.2422
0.0916
1.114
-do-
-do-
GOOF
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Figure 4. ORTEP diagram for (a) Form I and (b) Form II at 35% and 50 % probability ellipsoids
respectively; (c) and (d) are the overlap diagram of the two independent molecules of Form II in
different orientations.
There are clear differences between the crystal structures of Form I (zigzag chain of
molecules) and Form II (planar and lamellar packing), apart from the fact that their unit cell
parameters, space groups, and Z′ values are different. Form I is crystallized in the P21/c symmetry
and Form II is crystallized in P1 symmetry. Form I has only one molecule in asymmetric unit cell,
in contrast, Form II has two molecules in the asymmetric unit cell. These two molecules of Form
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II have difference in their geometry as it is evident by overlapping them in different orientation
(Figure 4c, 4d). Although both Form I and Form II possess equal number of donor-acceptor sites
(amino group-donor; imino group-acceptor), due to different flipping arrangement, in Form I, only
one amino group of aminoguanidine group participates in H-bonding while in Form II both amino
groups actively involved in H-bonding. As a result, in Form I there are two sets of H-bonds in total
while three sets of H-bonds are present in Form II. It is well known that there is always a
competition between directional interactions versus better packing in crystal engineering, which
leads to different crystallization. There are reports which suggest that the solid state forms with
looser packing and better H-bonding can lead to higher Z′ value and similar observation is found
in Form II.36,37
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From the structural study, it became clear that the differences in the two forms are due to
E/Z isomerism across the C7-N1 bond. Further, the differences due to the rotation across C1-C7
single bond are also noticeable. The aromatic 2,6-dichlorophenyl ring and the methylene
aminoguanidine moieties are planar individually and are arranged orthogonally to each other in
Form II whereas they tend to be coplanar in Form I. The angle ɸ between the two planes is 82.34°
and the corresponding angle for Form I is 40.32°. The overlap structures of the Form I and Form
II reveal the level of mismatch of the forms after making a suitable alignment at any one end
(aromatic group or guanidine moiety) creates the corresponding mismatch automatically in the
other end (Figure 3e and 3f).
It is intriguing to note that only one isomer was noticed in the solution state (1H NMR, Figure
S11), whereas two forms observed in solid state (which are clearly distinguishable). To verify,
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both solid forms were dissolved in DMSO-d6 and subjected to H NMR studies. The H NMR
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spectra of Form I and Form II are provided in Figure 5. The H NMR of the solid compound
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obtained after synthesis (as synthesized, Figure S11) is identical as that of Form I confirming the
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presence of E-isomer in solution phase. The H NMR spectrum of Form II exhibits distinct
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characteristic peaks. The chemical shift () value of –CH=N proton in Form I and Form II appear
at 8.20 ppm and 7.10 ppm respectively. A shifting of signal due –CH=N proton from 8.20 ppm
(Form I) to 7.10 ppm (Form II) is due to the presence of nitrogen lone pair on the same side of
proton nucleus in Form II causing its shielding, resulting into a shift of signal in upfield region.
Thus, the two isomers can be easily distinguishable in solution state also.
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Figure 5. An overlay of 1H NMR of Form I and Form II of guanabenz in DMSO-d6.
Figures 6a and 6b show the interactions within the dimers of Form I and Form II
respectively. In both cases the dimers are characterized by two intermolecular hydrogen bonds (
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2.27 Å), leading to R
(8) supramolecular synthon. The differences between the two crystal
structures are very clear in the dimers of Form I and Form II – In Form I, the phenyl groups are
coplanar with the molecules and are away from the central synthon whereas in Form II, the phenyl
groups flank the central synthon unit. In the crystal structures of Form I and Form II, repeated units
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of tetramers exist (Figure 6c and 6d respectively) which are generally connected by two sets of H-
bonds of N H···N type (Form I: 2.27, 2.74 Å; Form II: 2.27, 2.07 Å). Again for Form I, each
dimer unit is connected via π···π interactions (3.61Å, centroid-centroid) (Figure 6e) and extends
the chain along bc-plane. Similarly,  stacking interactions between molecules are clear in Form
II also, which become apparent only when the molecular assembly is considered beyond tetrameric
state (Figure 6d vs. 6f)
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Figure 6. Hydrogen bonding and molecular packing in the crystal structures of guanabenz Form I
(left panel) and Form II (right panel).
The 2D fingerprint plots as a Hirshfeld surface38-40 for the two forms display various
interactions as the main structure governing the N4 H···N2 hydrogen bond is the same in both the
structures, confirmed by a pair of sharp spikes in the plots (Figure 7). The relative contributions
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of each interaction to the Hirshfeld surface are shown in Figure-S7. Both the forms have almost
similar N···H, Cl···H and H···H interactions, however Form II has less C···C contacts compared
to Form I (Figure 7). The presence of π-π stacking interaction is more prominent in case of Form
I which is evidenced by comparing the crystal structures and the Hirshfeld finger print plots.
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Figure 7. 2D fingerprint Hirshfeld surfaces plots of Form I and Form II showing the various
interactions and their percentage.
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FTIR and PXRD Analyses
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The IR spectra of two forms are shown in Figures S4-S6. There are several strong and distinct
transmittance bands for Form I and II and can be easily comparable. As shown in Figure S4, for
Form I the peaks are in the region 2917 cm-1 (N=C-H), 1594 cm-1 (C=N) and 840 cm-1(C-Cl), 660
cm-1 (N=C-H, Bending). Form II does not show any peak near 2917 cm-1, whereas the other two
signals are partly shifted at 1592 cm-1, 862 cm-1 and 671 cm-1. One significant difference is due to
the iminic C-H stretch. In Form I, the C-H bond of the imine group shows distinct signals (2917
cm-1) as it is not involved in any kind of interactions in the crystal structure. On the other hand, in
Form II, the iminic C-H bond is involved in a C-HCl type interaction, thus the vibrational
frequency is not sufficiently free to exhibit peak in IR spectrum (Figure S5). This observation is
in good agreement with the Hirshfield analysis also (Figure S7).
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A comparison of experimental PXRD pattern of Form I, Form II and mixture reveals the
differences in the crystal arrangement and phase of the two Forms (Figure 8). After physically
separating the two forms and/or exclusive isolation of Form I, the experimental and simulated
PXRD patterns were compared (Figure S1 and S2). The experimental patterns match well with the
simulated ones, confirming the phase purity of the physically separated forms. After successfully
isolating the two forms in the same pot, the mixture was subjected to PXRD analysis (Figure S3).
The PXRD pattern shows distinctive peaks coming from each form as well as percentage of each
isomer. At least in three different instances of isolation of mixture, we observed that the percentage
of Form I isomer varies between 80-95 % and can be clearly observed both under optical
microscopy (Figure S10) as well as in PXRD analysis.
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Figure 8. Experimental PXRD pattern of Form I (red), Form II (blue) and mixture (green).
Thermal Analysis of Form I and II
The thermograms of Form I and II give almost identical thermal signatures (Figure 9). There
are horizontal plateau without any weight loss which confirms the stability of the two forms till
250 ºC. However, after 250 ºC both the forms start decomposing, this process is faster for Form II
in comparison to Form I. At around 550 ºC, both forms of the guanabenz decomposed completely.
From the thermogram plot, it is clear that the both the isomers of guanabenz melting starts nearly
at ~230 ºC and it is matching exactly with the value of 227 ºC for Form I isomer reported by
Diamant et al.16 A good correlation can be observed from the crystal structure of both the Forms.
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In Form I, the molecules are held tightly by strong H-bonds (2.27 Å) as compared to Form II (2.28,
2.0 Å) with an additional π···π stacking interactions (Figure 6c) as a result more energy may
require to make the molecules free.
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Figure 9. Thermogravimetric analysis (TGA) plots of Form I (blue) and Form II (red).
DSC Analysis
The DSC profile for Form I shows weak endotherm around 61.7 °C followed by a weak
exotherm at 215 °C and finally the compound decomposed at 318 °C (Figure S8). Apart from that,
Form I also shows a few unresolved multiple weak peaks between 110-160 °C which may be due
to the association of martensitic phase transition.41 On the other hand, the DSC profile of Form II
is rather smooth till 215 °C and the compound shows a sharp endotherm (217 °C) related to melting
of the compound followed by an immediate exotherm (225 °C) with just a gap of 8 °C correspond
to the oxidative degradation (Figure S9). Comparing the TGA and DSC profiles of Form II, it is
clear that the first endotherm occurs at 217 C which is very much consistent with the
decomposition temperature of ~220
̊C obtained from TGA data. Although melting of both Form I
and Form II starts nearly at the same temperature, the TGA profile of both the forms are quite
distinguishable. The Form II shows a sharp fall in weight loss (200-350 ̊C) as compared to Form I
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which shows a gradual rate of decomposition at temperature 200-500 ̊C. Comparing the whole
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range of decomposition, we assume that Form I is little more thermodynamically stable than Form
II.
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Quantum Chemical Analysis
To understand the driving factors for the observed isomers in guanabenz, quantum
chemical calculations were performed. Initially, the calculations were performed on the monomers
of guanabenz (E- and Z- isomers). The results from B3LYP/6-311+G(d,p) analysis suggested that
the E-isomer (Form I) is more stable than the Z-isomer (Form II) by 2.13 kcal/mol suggesting that
the E-isomer is thermodynamically more stable than Z-isomer. GIAO (Gauge-Independent Atomic
Orbital) method was employed to estimate the chemical shift values. The chemical shift value for
the iminic proton of E-isomer and Z-isomer is 8.81 and 7.57 ppm respectively which is consistent
with the experimentally obtained chemical shift values. The N1-inversion barrier (also the –C7=N1
rotational barrier) for the E/Z isomerization is ~37 kcal/mol. The N1-inversion barrier after
protonation is ~39 kcal/mol, suggesting that the inversion process is thermodynamically prohibited
between the E and Z isomers in guanabenz. Since guanabenz is highly basic, it prefers to exist in
protonated state. Quantum chemical analysis showed that the proton affinity (PA) values for the E
and Z isomers are 237 and 238 kcal/mol respectively showing the highly basic character for both
the isomers. The intrinsic energy difference (2.13 kcal/mol) may be the driving force for observing
the Z-isomer as the minor isomer in the mixture. Considering that only one isomer is found in the
reaction product and also considering that the E/Z conversion is an energetically prohibited
process, it is intriguing that Form II is found in solid state. To explore this, the dimeric states of
the E- and Z-isomers of guanabenz are also studied using quantum chemical methods. Since, the
DFT-B3LYP functional cannot adequately account for the dispersion interactions (non-bonding)
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the dispersion corrected DFT functional i.e. B97X-D33 was used to calculate the energy
differences between both the isomers in their dimeric state.
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The 3D structures of dimers according to B97X-D based geometry optimization are
given in Figures 10a and 10b. According to the crystal structure data (Figure 6a and 6b), the
intermolecular hydrogen bonds in E and Z dimers are of the order of approximately 2.28 Å.
According to the gas phase calculations, these intermolecular hydrogen bond lengths are much
shorter (1.93 Å in the E-isomer and 1.92 Å in the Z-isomers). In the crystal structure, an 2 type of
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cation-π interaction has been noticed for Z-dimer. In the optimized Z-dimer, an 6 type of cation-
π interaction (~3.2 Å) has been noticed. The stabilization energies due to the dimer formation in
the E-isomer is 2.21 kcal/mol. The stabilization energy due to the dimer formation in Z-isomer is
4.89 kcal/mol. This greater stabilization in Z-isomer (2.68 kcal/mol) can be attributed to the strong
cation-π interaction. This is also reflected in the greater stability of the Z-dimer (by 1.45 kcal/mol).
a) E-dimer
c) E-pair with H-bonding
e) E-pair with  stacking
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d) Z-pair
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Figure 10. The optimized 3D structures of E, Z-dimers (a,b) and E, Z-pairs (c-e). The dotted lines
represents the hydrogen bond interactions and the distances are in Å.
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The tetrameric states of Form I and Form II (Figure 6c and 6d) clearly indicate the presence
of additional set of intermolecular hydrogen bond interactions between the pairs of monomers. To
estimate the strength of these additional interactions the 3D geometries of these pairs are extracted
from the crystal structure and further subjected to geometry optimization. The optimized 3D
structures of these pairs (E-pair and Z-pair) are given in Figures 10c and 10d respectively. An
additional pair of E-isomer was also considered i.e. E-pair with π- π stacking interaction due to
arene moieties (Figure 10e). In the E-pair (Figure 10c), two intermolecular hydrogen bonds of
lengths 1.99 Å and 2.09 Å are found according to the quantum chemical study. However, in the Z-
pair (Figure 10d), bifurcated hydrogen bonds of the order of 2.08 Å and 2.19 Å have been noticed
along with cation-π interaction. The stabilization energy due to the E- pair formation with H-
bonding is 1.49 kcal/mol. In case of E-pair with π- π stacking interaction, the energy gain due to
pair formation is only 0.30 kcal/mol suggesting that π-π stacking interaction is playing a minor
role in the stabilization of E pair. The stabilization energy due to pair formation in Z-isomer is 5.79
kcal/mol, this stabilization energy is relatively larger compared to E-pair with H-bonding and π- π
stacking interaction, this result further suggests that the cation-π interaction present in Z-dimer and
Z-pair is playing crucial role in their stabilizing them in the solid state. Hence, the overall extra
stabilization due to dimer (2.68 kcal/mol) and pair formation (4.00 kcal/mol) in the Z-isomer is of
the order of the ~7 kcal/mol for each molecule of guanabenz. Though, the Z-isomer is less stable
than E-isomer in monomeric state, it experiences extra stability (hydrogen bonding and
hydrophobic interactions) in the solid state due to intermolecular interactions. In addition, added
advantages, presumably due to hydrophobic contacts, crystal packing forces might be contributing
to the observation of Form II of guanabenz.
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Isomerism vs. Polymorphism
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Different crystalline states of a chemical substance are known as polymorphs.
Polymorphism observed due to conformational differences in the structure in solid state leads to
conformational polymorphism. Tautomeric difference observed in solid state leads to tautomeric
polymorphism. Though there is “a need for clear definition of polymorphism there is an implied
correspondence between different molecular arrangements and different crystal forms”.42,43
Especially, when the information from solid state studies leads to the identification of two different
arrangements, the use of the term polymorphism is quite justified.
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In the current work, the two different molecular arrangements of the drug guanabenz were
identified “from the solid state information to the structure information” rather than “from the
structural information to the solid state information” i.e. From I and Form II of guanabenz free
base were first identified which further lead to the identification of E/Z isomerism in guanabenz.
Thus, Form I and Form II may be considered as two polymorphs of same molecule and the origin
of the observed polymorphism has been attributed to the differences in the geometrical
arrangements of atoms. In such context it may be worth considering the two polymorphs as
configurational polymorphs,44,45 on the same lines as that of “conformational polymorphs” and
that of “tautomeric polymorphism”.46 The observation of polymorphism due to geometrical
isomers was earlier reported by a few scientific groups – for ex. Configurational isomerism and
polymorphism in chalcones reported by Xu and coworkers.47
Conclusions
In summary, it was demonstrated that guanabenz can exist in two forms (Form I and Form
II), the two forms differ in terms of their geometry across –C=N. The E isomer is
thermodynamically more stable and lead to the major polymorph i.e. Form I. The Z isomer, which
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is 2.13 kcal/mol less stable leads to the formation of the minor polymorph (Form II). In both the
forms, guanabenz exists exclusively in an azine tautomeric state, in contrast to the general
perception i.e. it is a hydrazone derivative. In solution state (NMR analysis in DMSO-d6), only
one form was noticed with E geometry, however, after crystallization the 1H NMR of both isomers
could be recorded. Both the isomers can be separated by mechanical means due to their distinct
crystal morphology – (i) Form I exclusively adopts a block shaped arrangement, (ii) Form II adopts
a rod shaped arrangement. Both the forms were characterized by single crystal XRD. The single
crystal and powder XRD studies clearly show the differences between the two forms. The quantum
chemical analysis was carried out on the dimers of the Form I and Form II to rationalize their
existence in the solid state, to explore the energy as well as electronic factors associated with the
two forms.
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Experimental
(E)-4-(2,6-dichlorophenyl)-1,1-diamino-2,3-diazabuta-1,3-diene (2)
1
White solid (315 mg, 96%). mp 220-222 °C; IR (KBr, cm-1): 3358, 1663, 1538, 1148; H NMR
(400 MHz, DMSO-d6)  8.20 (s, 1H), 7.49-7.55 (d, J = 8.2 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H); 5.81
13
(br s, 2H), 5.84 (br s, 2H); C NMR (100 MHz, DMSO-d6)  161.98, 137.86, 133.72, 132.22,
129.68, 129.25; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C8H9Cl2N4 231.0198; Found 231.0195.
ASSOCIATED CONTENT
Supporting information
Experimental details, X-ray crystallography, crystal structure analysis, thermal analysis,
and CIF files. The coordinates of the quantum chemically optimized geometries of monomers and
dimers of the Form I and Form II of guanabenz are also included along with their absolute energies.
CCDC 1557940 and 1870736, contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.cam.ac.uk.
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AUTHOR INFORMATION
Corresponding Author
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*E-mail: pvbharatam@niper.ac.in; subash.chem@gmail.com
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
Authors acknowledge DST-INSPIRE, New Delhi and CSIR, New Delhi for financial support.
SCS thanks DST-FIST for single crystal facility at Panjab University.
REFERENCES
(1) Baum, T.; Eckfeld, D. K.; Metz, N.; Dinish, J. L.; Rowles, G.; van Pelt, R.; Shropshire, A.
T.; Fernandez, S. P.; Gluckman, M. I.; Bruce, W. F. 2, 6-Dichlorobenzylidene amino
guanidine acetate (Wy-8678). A new hypotensive agent. Experientia 1969, 25, 1066-1067.
(2) Baum, T.; Shropshire, A. T. Studies on the centrally mediated hypotensive activity of
guanabenz. Eur. J. Pharmacol. 1976, 37, 31-44.
(3) Nash, D. T. Clinical trial with guanabenz, a new antihypertensive agent. J. Clin. Pharmacol.
New Drugs, 1973, 13, 416-421.
(4) Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.;
Johnson, D.; Li, C.; Sayeeda, Z. DrugBank 5.0: a major update to the DrugBank database
for 2018. Nucleic Acids Res. 2017, 46, D1074-D1082.
(5) Norez, C.; Vandebrouck, C.; Antigny, F.; Dannhoffer, L.; Blondel, M.; Becq, F. Guanabenz,
an α2-selective adrenergic agonist, activates Ca2+-dependent chloride currents in cystic
fibrosis human airway epithelial cells. Eur. J. Pharmacol. 2008, 592, 33-40.
(6) Neuber, C.; Uebeler, J.; Schulze, T.; Sotoud, H.; El-Armouche, A.; Eschenhagen, T.
Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes. PLoS
One 2014, 9, e98893.
(7) Da, C. L. E.; da Rosa Santos, O. Silvestre Labs Químia E Farmaceutica Ltda.,
2012. Guanabenz-containing compound for the treatment of primary cutaneous amyloidosis.
(8) Gug, F.; Oumata, N.; Tribouillard-Tanvier, D.; Voisset, C.; Desban, N.; Bach, S.; Blondel,
M.; Galons, H. Synthesis of conjugates of 6-Aminophenanthridine and guanabenz, two
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