Crystal structures and Hirshfeld surface analyses of the di- and tri-hydrates of (5α,17E)-17-hydrazonoandrostan-3-ol: Significant differences in the hydrogen bonding patterns and supramolecular arrangements

Article abstract of DOI:10.1016/j.steroids.2018.09.010

The crystal structures, Hirshfeld surface analyses and electrostatic potential surfaces of the di- and tri-hydrates of (5α,17E)-17-hydrazonoandrostan-3-ol, 3, namely [3·(H₂O)₂] and [3·(H₂O)₃], are reported. The trihydrate, isolated from a solution of 3 in moist methanol, recrystallizes in the orthorhombic space group, P2₁2₁2₁, while that of the dihydrate, isolated from a 1:1 aqueous methanol solution, recrystallizes in the monoclinic space group, P2₁. The asymmetric unit of the trihydrate involves one steroid and three water molecules, while that of the dihydrate has two similar but independent steroid molecules and four hydrate molecules. Very similar conformations are found for the steroid molecules in both hydrates. As expected, the different mole ratios of water: steroid have major influences on the structures. In both cases, complex crystal structures are constructed from various classical hydrogen bonds, involving the hydrate molecules and the hydroxy and hydrazonyl moieties of the steroid. In the trihydrate, there are no direct connections between the steroid molecules, instead the water molecules link the steroid molecules, with only weak van der Waals forces between the steroid molecules. There are some direct links between the steroid molecules in the dihydrate, involving O–H(steroid hydroxyl)?O(steroid oxo) hydrogen bonds, in a head to head fashion, and O–H?N(hydrazonyl) hydrogen bonds, in a head to tail fashion. However, the major occurrence throughout the structure is of steroid molecules linked by water molecules.

Full text of DOI:10.1016/j.steroids.2018.09.010

Steroids140(2018)92–103
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Crystal structures and Hirshfeld surface analyses of the di- and tri-hydrates
of (5α,17E)-17-hydrazonoandrostan-3-ol: Significant differences in the
hydrogen bonding patterns and supramolecular arrangements
⁎
Ligia R. Gomesa,b, John N. Lowc, Alan B. Turnerc,1, Thomas C. Baddeleyc, James L. Wardellc,d,
a
FP-ENAS-Faculdade de Ciências de Saúde, Escola Superior de Saúde da UFP, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal
b
c
REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 687, P-4169-007 Porto, Portugal
Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, Scotland
d
Instituto de Tecnologia em Fármacos e Farmanguinhos, Fundação Oswaldo Cruz, 21041-250 Rio de Janeiro, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
The crystal structures, Hirshfeld surface analyses and electrostatic potential surfaces of the di- and tri-hydrates of
(5α,17E)-17-hydrazonoandrostan-3-ol, 3, namely [3·(H2O)2] and [3·(H2O)3], are reported. The trihydrate, iso-
lated from a solution of 3 in moist methanol, recrystallizes in the orthorhombic space group, P212121, while that
of the dihydrate, isolated from a 1:1 aqueous methanol solution, recrystallizes in the monoclinic space group, P21.
The asymmetric unit of the trihydrate involves one steroid and three water molecules, while that of the dihydrate
has two similar but independent steroid molecules and four hydrate molecules. Very similar conformations are
found for the steroid molecules in both hydrates. As expected, the different mole ratios of water: steroid have
major influences on the structures. In both cases, complex crystal structures are constructed from various
classical hydrogen bonds, involving the hydrate molecules and the hydroxy and hydrazonyl moieties of the
steroid. In the trihydrate, there are no direct connections between the steroid molecules, instead the water
molecules link the steroid molecules, with only weak van der Waals forces between the steroid molecules. There
are some direct links between the steroid molecules in the dihydrate, involving OeH(steroid hydroxyl)⋯O
(steroid oxo) hydrogen bonds, in a head to head fashion, and OeH⋯N(hydrazonyl) hydrogen bonds, in a head to
tail fashion. However, the major occurrence throughout the structure is of steroid molecules linked by water
molecules.
Hydrogen bonds
Crystal structures
Hirshfeld surface analysis
Hydrazones
Androsterones
Molecular electrostatic potential
1. Introduction
to form head-to-tail linked molecular chains utilizing OeH⋯O hy-
drogen bonds. A strong preference is to use a hydroxy group on C-3 in
The functions of 5α-androstane-3α-ol-17-one, 1, epi-androsterone
(3α-androstane-3β-ol-17-one, 2), their derivatives and related steroids,
in both plant and animal domains, have attracted much interest [1–5].
As well as the study of synthetic procedures and biological activities,
study of the crystal structures of these compounds has been of interest.
Among the crystal structures reported include those of 1 and its hemi-
hydrate, both recently confirmed, and that of 2 [6 and references there
in], see Scheme 1.
the OeH⋯O hydrogen bonding even in the presence of other hydroxyl
groups in the steroid. However, in the absence of a 3-hydroxyl group,
other hydroxyl groups can be used in the hydrogen bonding with the C-
17 carbonyl [16–19].
In hydrates and other solvates, the solvate molecules can play
dominate and varying roles in the crystal packing. Thus in the hemi-
hydrate of 5α-androstane-3β-ol-17-one, 6, the water molecule acts as a
link between the androsterone molecules, forming three strong hy-
drogen bonds, each to a separate androsterone molecule by acting as a
donor to two androsterone molecules and an acceptor to a third [6].
The HO unit on C-3 hydroxyl group in androsterone-3β,5α,6β,17β-te-
trol·trihydrate [20] acts as the donor in two hydrogen bonds with water
molecules as well as acting as an acceptor in O3eH3⋯O(hydroxyl)
An area of interest in the crystallographic studies has been the
linkage of the steroidal molecules via hydrogen bonds. With a donor
group, such as a hydroxyl group, at one end, e.g., on C-3, and an ac-
ceptor group such as a carbonyl group at the other end of the steroid
molecule, e.g., on C-17 [6–14], or vice versa [15], a strong tendency is
⁎
Corresponding author.
E-mail address: j.wardell@abdn.ac.uk (J.L. Wardell).
1
Died: 16th February 2014.
https://doi.org/10.1016/j.steroids.2018.09.010
Received 19 July 2018; Received in revised form 19 September 2018; Accepted 24 September 2018
Availableonline29September2018
0039-128X/©2018ElsevierInc.Allrightsreserved.

L.R. Gomes et al.
Steroids140(2018)92–103
molecules, we undertook crystal structure determinations and Hirshfeld
surface analyses of these hydrates of 3 to ascertain which of the po-
tential intermolecular interactions were being utilized and how the
molecules of 3 are linked.
2. Experimental
2.1. General
Melting points were determined using an MQAPF-302 (MicroQuímica
Ltd, Santa Catarina, Brazil) apparatus and are uncorrected. High-resolution
mass spectra were determined using a Water Mass Spec. Model Xevo G2 QT
of instrument and MassLynx version 4.1 software.
Scheme 1. Formation of 3.
hydrogen bonds: these link the steroid molecules in a head-to-tail
manner. In the methanol solvate of 3β-hydroxyandrost-5-en-17-one, the
disordered solvate molecules link pairs of steroid molecules in a head-
to-head manner [21]. In contrast, the water molecules in 17α-hydroxy-
17-methylandrost-4-ene-3-one·hemi-hydrate [15] appear not to be in-
volved in linking the steroid molecules.
Differential scanning calorimetry. A Mettler Toledo STARe System
DSC1, cooled by liquid nitrogen and calibrated using indium and zinc as
standards, was used with a time–temperature programme of heating
from 25 to 250 °C at 10 °C/min.
Thermal gravimetric analysis. A Mettler Toledo STARe System TGA2
was used with a time–temperature programme of heating from 25 to
250 °C at 10 °C/min.
An interesting derivative of 5α-androstane-3β-ol-17-one is the hy-
drazonylandrostane compound (5α,17E)-17-hydrazonoandrostan-3-ol,
3, prepared from the reaction of epi-androsterone, 2 with hydrazine
hydrate 80% under reflux [22], see Scheme 1]. Among other things,
compound 3 has proved to be a useful precursor of nitrogen containing
steroidal heterocycles [23].
2.2. Synthesis
Our studies with compound 3 have revealed that hydrates can be
obtained on recrystallisation from organic solvents in air. For example
slow evaporation of solutions of 3 in moist methanol at room tem-
perature led to the isolation of crystals of a trihydrate, and from a 1:1
(v:v) aqueous MeOH solution, surprisingly only a dihydrate. With hy-
droxyl and hydrazonyl substituents, in addition to the hydrate
(5α,17E)-17-Hydrazonoandrostan-3-ol, 3, was prepared from the
reaction of epi-androsterone, 1 with hydrazine hydrate 80% under re-
flux, as previously reported [22]. The trihydrate and dihydrate were
isolated, respectively, from solutions of 3 in moist methanol and 1:1
aqueous methanol and used without further treatment in the structure
determinations.
Table 1
Crystallographic experimental details.
[3·(H2O)3]
[3·(H2O)2]
Crystal data
Chemical formula
C
19H38N2O4·
C19H36N2O3
Mr
358.51
340.50
Crystal system, space group
Temperature (K)
a, b, c (Å)
Orthorhombic, P212121
Monoclinic, P21
200
100
5.9299 (2), 12.3916 (4), 28.1777 (9)
7.2600 (2), 9.9010 (2), 26.7004 (6)
α, β, γ (°)
90, 90, 90
2070.52 (12)
4
90, 94.024 (2), 90
V (Å3)
1914.53 (8)
Z
4
Radiation type
Mo Kα
Mo Kα
μ (mm−1
)
0.08
0.08
Crystal size (mm)
0.30 × 0.27 × 0.20
0.20 × 0.20 × 0.02
Data collection
Diffractometer
Rigaku AFC12 (Right) diffractometer
XtaLAB AFC12 (RCD3): Kappa single diffractometer
Absorption correction
Multi-scan: CrysAlis PRO, Agilent Technologies, Version 1.171.37.35
Multi-scan: CrysAlis PRO 1.171.39.9g (Rigaku Oxford Diffraction,
2015) Empirical absorption correction using spherical harmonics,
implemented in SCALE3 ABSPACK scaling algorithm
0.368, 1.000
(release 13-08-2014 CrysAlis171 .NET) (compiled Aug 13, 2014,
18:06:01)
T
min, Tmax
0.583, 1.000
No. of measured, independent and
observed [I > 2σ(I)] reflections
Rint
15,056, 5210, 4032
24,152, 8680, 8105
0.032
0.695
0.033
0.649
(sin θ/λ)max (Å−1
Refinement
)
R[F2 > 2σ(F2)], wR(F2), S
No. of reflections
0.049, 0.114, 1.02
0.044, 0.101, 1.07
5210
8680
No. of parameters
No. of restraints
264
494
0
1
H-atom treatment
H atoms treated by a mixture of independent and constrained
H atoms treated by a mixture of independent and constrained
refinement
refinement
Δρmax, Δρσmin (e Å−3
Absolute structure
)
0.15, −0.25
0.33, −0.19
Flack x determined using 1322 quotients
Refined as an inversion twin
[(I+) − (I−)]/[(I+) + (I−)] [24]
Absolute structure parameter
CCDC deposit numbers
0.3 (5)
0.6 (10)
1817743
1538473
Programs used: CrystalClear-SM Expert 3.1 b27 [25], CrysAlis PRO, Agilent Technologies, Version CrysAlis PRO 1.171.39.9g [26], CrysAlis PRO, Agilent Technologies,
Version 1.171.37.35, ORTEP [28], SHELXT [29], SHelXle [30], SHELXL7 [31], MERCURY [32], PLATON [33].
93

L.R. Gomes et al.
Steroids140(2018)92–103
Fig. 1. Atom arrangements and the numbering scheme and side-on views of the molecular conformations of 3 in the two hydrates: ellipsoid probabilities are drawn at
the 50% level. Hydrogen atoms have been drawn as spheres of arbitrary radius. Hydrogen atoms have been omitted in the drawings of the conformations.
Table 2
Pucker parameters.
Compound
Q(2), Å
Phi (2), °
Ring D: 5-membered ring
[3·(H2O)3]
0.460(2)
0.460(2)
0.435(2)
336.5(3)
203.5(3)
208.5(3)
[3·(H2O)2]·Mol. A
[3·(H2O)2]·Mol. B
Q
Theta
Phi
Six membered rings
[3·(H2O)3]
A-ring
0.577(2)
0.570(2)
0.567(2)
0.4(2)
3.0(2)
5.9(2)
258(7)
349(3)
292(2)
B-ring
C-ring
[3·(H2O)2]·Mol. A
A-ring
0.570(2)
0.573(2)
0.578(2)
3.3(2)
3.9(2)
0.0(2)
298(4)
297(3)
213(10)
B-ring
C-ring
[3·(H2O)2]·Mol. B
A-ring
0.580(2)
0.584(2)
0.579(2)
4.4(2)
6.9(2)
3.1(2)
136(3)
B-ring
313.2(19)
267(3)
C-ring
94

L.R. Gomes et al.
Steroids140(2018)92–103
Table 3
Geometric parameters and symmetry operations for hydrogen bonds (Å, °).
DeH⋯A
DeH
H⋯A
D⋯A
DeH⋯A
Symmetry code
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)3]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
[3·(H2O)2]
O1WeH1W1⋯O22i
O1WeH2W1⋯O3Wii
O2WeH1W2⋯N20iii
O2WeH2W2⋯N21iv
O3WeH1W3⋯O22v
O3WeH2W3⋯O1W
N21eH21B⋯O3W
O22eH22⋯O2W
O3eH3X⋯N120
O3eH3Y⋯O122
O4eH4X⋯N220
O4eH4Y⋯N121
O5eH5X⋯O222
O5eH5Y⋯O4
0.88(4)
0.92(3)
0.89(4)
0.85(4)
0.83(4)
1.00(4)
0.89(4)
0.79(4)
0.83(3)
0.88(5)
0.87(4)
0.89(4)
0.78(5)
0.90(4)
0.86(5)
0.86(4)
0.86(4)
0.87(4)
0.82(3)
0.89(4)
1.97(4)
1.85(4)
1.93(4)
2.08(4)
1.97(4)
1.73(4)
2.15(4)
1.89(4)
2.18(3)
2.11(5)
2.04(4)
2.00(3)
2.13(5)
1.96(4)
2.60(4)
1.91(4)
1.77(4)
2.24(4)
1.90(3)
2.19(3)
2.851(3)
2.760(3)
2.809(3)
2.903(4)
2.801(3)
2.733(3)
2.995(3)
2.677(3)
2.988(3)
2.963(3)
2.903(3)
2.873(3)
2.904(3)
2.826(3)
3.243(3)
2.751(4)
2.626(3)
3.011(3)
2.724(2)
3.050(3)
177(3)
172(3)
172(3)
166(3)
178(5)
176(4)
159(3)
174(3)
164(3)
165(4)
169(3)
170(3)
171(4)
161(3)
133(3)
178(5)
176(3)
149(3)
175(3)
164(3)
3/2 − x, −y, 1/2 + z
−1/2 + x, −1/2 − y, 1 − z
2 − x, −1/2 + y, 1/2 − z
3/2 − x, −y, −1/2 + z
5/2 − x, −y, 1/2 + z
1 − x, −1/2 + y, 1 − z
1 − x, 1/2 + y, −z
1 − x, 1/2 + y, 1 − z
−1 + x, y, z
O6eH6X⋯N220
O6eH6Y⋯O5
1 − x, 1/2 + y, −z
O122eH122⋯O6
N121eH124⋯O222
O222eH222⋯O122
N221eH224⋯O3
2 − x, 1/2 + y, 1 − z
1 − x, 1/2 + y, −z
3 and three water molecules, while that of [(3)·(H2O)2] consists of two
independent molecules, Mol. A & Mol. B, of 3 and four water molecules.
The trihydrate, [3·(H2O)3], recrystallises in the orthorhombic space
group, P212121 and the dihydrate in the monoclinic space group, P21.
The atom arrangements, numbering schemes and molecular con-
formations of the steroidal molecules in both hydrates are shown in
Fig. 1.
Table 4
Percentages of the most important atom–atom contacts involving 3.
H⋯H
H⋯O
O⋯H
H⋯N
N⋯H
H⋯C/C⋯H
[3·(H2O)3]
85.4
84.8
85.2
6.6
6.9
5.3
2.9
2.8
3.4
0.8
0.6
0.8
4.1
4.7
4.8
0.2
0.1
0.4
[3·(H2O)2]·Mol. A
[3·(H2O)2]·Mol. B
The three six-membered rings in each molecule of 3 in both hy-
drates have ideal or near ideal chair shapes, while the five membered
rings in [(3)·(H2O)3] and Mol. B of [(3)·(H2O)2] have envelope shapes
with the flaps at C14 and C24, respectively, and that of Mol. A of
[(3)·(H2O)2] has a twist on C113eC114, see Table 2. Thus there are
only minor differences between the conformations of molecules of 3 in
both hydrates. The bond lengths and angles are all in the expected re-
gions and are not further discussed.
2.3. Compound [3·(H2O)3]
M.p. 187–192 °C.
HR-MS (ESI): Calcd for C19H33N2O+ (3+H+): 305.2587; Found:
305.2596.
2.4. Compound [3·(H2O)2]
3.1. Intermolecular interactions
M.p. 195–199 °C.
HR-MS (ESI): Calcd.
305.2590.
C
19H33N2O+ (3+H+): 305.2587; Found:
A large number of classical hydrogen bonds in both hydrates, as
determined using PLATON [33], and confirmed by the Hirshfeld surface
analyses [34,35], link the steroid and the water molecules into complex
three-dimensional arrays, see Table 3 for the intermolecular interac-
tions and symmetry operations. As a consequence of the steroid nature
of the molecule, the most abundant contacts involving the steroid
molecules are H⋯H contacts, see Table 4. Table 4 lists the contact
percentages, selected by partial analyses of the FP plots. As well as the
hydrate molecules, the two functional groups, H2NeN and HO, at op-
posite ends of the steroid molecule, 3, are involved in classical hy-
drogen bonds. These result in the N/OeH⋯O interactions listed in
Table 3. Only small percentages of H⋯C/C⋯H are noted. As shown in
Table 3 there are only small differences in the percentages of contacts
involving the steroid molecules.
2.5. Experimental crystallography
All details are listed in Table 1 [24–33].
2.6. Hirshfeld surface analyses
The Hirshfeld surfaces and two-dimensional Fingerprint (FP) plots
[34] were generated using Crystal Explorer 3.1 [35]. The Hirshfeld
surface mapped over dnorm was scaled between −0.750 and 1.560. The
electrostatic potential (ESP) was calculated with TONTO [36] as im-
plemented in Crystal Explorer 3. The surfaces computed refer only to
the steroid part of the compound. The same applies to the percentages
of the various contacts, which were selected by the partial analysis of
the FP plots.
3.1.1. Supramolecular arrangements in [(3)·(H2O)3]
As indicated in Table 3, each hydrate molecule is involved as a
donor in two hydrogen bonds and as acceptor in another. Both nitrogen
atoms of the hydrazonyl group also act as acceptor atoms, but only one
of the hydrogens in each ]N-NH2 moiety is utilized in hydrogen
bonding in [(3)·(H2O)3]. Each of the three independent water mole-
cules is involved in different sets of hydrogen bonds, see Table 3. Thus
(i) water molecule, W*1 (with O1W), acts a donor to a HO group of a
steroid molecule, and as an acceptor and a donor in two hydrogen
bonds with two molecules of W*3, (ii) W*2 (with O2W), acts as donor
3. Results and discussion
Crystals of the trihydrate of 3 were isolated by slow evaporation of a
solution of 3 in moist methanol maintained at room temperature, over
5 days while those for the dihydrate were harvested after 3 days from a
1:1(v:v) aqueous methanol solution maintained at room temperature in
air.
The asymmetric unit of [(3)·(H2O)3] consists of a single molecule of
95

L.R. Gomes et al.
Steroids140(2018)92–103
Fig. 2. [3·(H2O)3]. (a) Part of a slice through the 3-dimensional structure showing a puckered sheet of molecules, viewed down the a axis, (b) part of another slice
through the three dimensional hydrogen-bonded structure, this time looking down the b axis: this slice shows a regular stacking arrangement of the steroid molecules.
The various hydrogen bonds are drawn as thin dashed lines. Symmetry operations are listed in Table 2.
to two molecules of 3, via the N20 and N21 atoms, and as an acceptor to
the HO group of a steroid molecule, and (iii) water molecule, W*3 (with
O3W) acts as a donor to the HO group of a steroid molecule and to W*1,
and as acceptor to another W*1 and to the HN moiety of another steroid
molecule. The hydroxyl group of the steroid acts as an acceptor to both
W*1 and W*3 and a donor to W*2, while the ]N-NH2 moiety acts as
acceptor to two W*2 and as a donor to W*3 molecules. There are no
direct steroid to steroid links.
Fig. 2, different combinations of hydrogen bonds, generated from the
water molecules and the HO and H2N-N] moieties of molecule 3,
create different complex chains within the slices, which effectively
segregate the steroid molecules within columns. These hydrogen
bonded chains are further linked into a more elaborate hydrogen
bonded framework throughout the structure as shown in Fig. 3: only the
atoms in 3 which participate in the hydrogen bonding network have
been drawn for reasons of clarity. The arrangement shown in Fig. 3
contains a network of various rings, including R55(10), R55(12), and
R66(14) rings [37].
Involvement of all the hydrogen bonds results in a overall complex
three-dimensional structure. Different slices through the three dimen-
sional hydrogen bonded structure reveal different molecular arrange-
ments generated from different combinations of hydrogen bond.
Examples of such slices are illustrated in Fig. 2. It is also apparent that
spiral chains of molecules, formed from hydrogen bonds, occur
throughout the structure. As can be observed in the two slices shown in
Three views of the Hirshfeld surface of the steroid molecule in
[3·(H2O)3] are illustrated in Fig. 4, which also show the connections of
3 with the three water molecules W*1, W*2 and W*3. The red areas
indicate contact points at the hydroxyl and ]NNH2 groups of the
steroid, which result from O3WeH1W3⋯O22, O1WeH1W1⋯O22, and
96

L.R. Gomes et al.
Steroids140(2018)92–103
Fig. 3. [3·(H2O)3]. (a) Part of the hydrogen bond framework, formed from the water molecules, and the HO and H2N-N] moieties of molecule 3. Only the
participating atoms of 3 in the hydrogen bonding network are drawn, (b) another view of the hydrogen bonded network showing the cell axes. As can be seen, each of
the three independent water molecules are involved in different sets of hydrogen bonds, see also Table 2.
Fig. 4. Three views of the Hirshfeld surface of 3 in [3·(H2O)3], mapped over dnorm, (a) Connections of 3 with W*2, via O22, and with W*3, via ]NNH2, also showing
is a further link to W*1, (b) the highlighted red spots indicate contact points with the hydroxyl atoms of the steroid, due to O3WeH1W3⋯O22, O1WeH1W1⋯O22,
and O22eH22⋯O2W hydrogen bonds, (c) the three highlighted red spots indicate contact points with the ]NNH2 atoms of the steroid, (N21eH21B⋯O3W,
N21⋯H2W2eO2W and N20⋯H1W2eO2W, as designated, also a small red spot area on the top face has a complementary spot on the hidden face and is due to a
C⋯H contact.
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L.R. Gomes et al.
Steroids140(2018)92–103
Fig. 5. Views of the Hirshfeld surfaces mapped over dnorm of the three water molecules, W#1, W#2 and W#3, in [3·(H2O)3]: also indicated are their immediate
connections. The highlighted red spots on the surfaces indicate contact points with the N/O⋯H atoms (dashed lines: red for O⋯H and yellow for N⋯H). The positions
of additional water molecules W#1, W#2 and W#3 with contacts to the identified hydrate molecules are also indicated.
O22eH22⋯O2W,
N21eH21B⋯O3W,
N21⋯H2W2eO2W
and
The combination of all the intermolecular interactions generates a
complex three dimensional supramolecular array. As in the trihydrate,
different slices through the three dimensional structure of the dihydrate
reveal different molecular arrangements formed from different combi-
nations of hydrogen bonds. Two such slices are illustrated in Fig. 6. In
each of these slices, complex chains, formed from the combinations of
hydrogen bonds, effectively separate the steroid molecules into col-
umns. It is also apparent that spiral chains of molecules, linked by
hydrogen bonds, occur throughout the structure. These complex hy-
drogen bonded chains, observed in the slices, are further linked into a
more elaborate hydrogen bonded framework throughout the structure
as shown in Fig. 7: only the atoms in 3 which participate in the hy-
drogen bonding network have been drawn for reasons of clarity. Each of
the four independent water molecules is involved in different sets of
hydrogen bonds, see Table 2 and Fig. 8. Thus, (i) water molecule, W*3,
is hydrogen bonded to a Mol. B of 3, via its H2N-N] group, a Mol. A of
3, via its OH group and to W*4, (ii) water molecule W* 4, to the H2N-
N] groups of both Mol. A and Mol. B and to W*5, (iii) water molecule,
W*5, to HO of Mol. B, W*6 and W*4, and (iv) and water molecule, W*6,
to HO of Mol. A, H2N-N] group of Mol. B and to W*5. The network
shown in Fig. 7 contains a set of R55(10), R55(12), and R66(14) rings
[37].
N20⋯H1W2eO2W hydrogen bonds. There is also a small red area on
the top face, with a complementary spot on the hidden face, these arise
from C⋯H contacts. Fig. 5 illustrates the Hirshfeld surfaces of the three
hydrate molecules. The immediate connections to the water molecules
are also highlighted as are the red areas indicating contact points with
N/O⋯H atoms.
3.1.2. Supramolecular arrangements in [(3)·(H2O)2]
A consequence of having two steroid molecules and four water
molecules in the asymmetric unit of the dihydrate is that a larger
number of classical hydrogen bonds have to be considered than those
involved in the trihydrate, with just one steroid and three hydrate
molecules in the asymmetric unit. The different steroid to hydrate mol
ratio results in significant differences in the supramolecular arrays of
the two hydrates. With a reduction in the relative amount of water
molecules, there are now some direct links between the steroidal mo-
lecules in the dihydrate: molecules, A and B, are directly linked by
N121eH124⋯O222 and O222eH222⋯O122 hydrogen bonds. Of in-
terest, these direct links involve hydroxyl–hydroxy and hy-
drazonyl–hydroxy interactions but not hydrazonyl–hydrazonyl inter-
actions.
Views of the Hirshfeld surfaces for both Mol. A and Mol. B are illustrated
in Fig. 8, which also indicates the direct contacts between the steroid
molecules, O222eH222⋯O122, and N121eH124⋯O222, and connections
to the four water molecules, W*3–W*6, i.e., O3eH3Y⋯O122,
O3eH3X⋯N120, N221eH224⋯O3, O4eH4X⋯N220, O4eH4Y⋯N121,
O5eH5X⋯O222, O122eH122⋯O6 and O6eH6X⋯N221. There is also a
small red area on the top face, which has a complementary spot on the
hidden face and is due to a C⋯H contact. Fig. 9 illustrates the Hirshfeld
surfaces of the four hydrate molecules. The immediate connections to other
water molecules are also highlighted as are the contact points with the N/
O⋯H atoms. The FP plots for both hydrates are shown in Fig. 10.
Both hydrogen atoms and the oxygen atom of each hydrate mole-
cule, termed W*3 to W*6, are involved in the hydrogen bonding
scheme in the trihydrate, see Table 2. However it is noticeable that the
proportion of nitrogen to oxygen atoms involved as acceptors towards
the hydrate molecules changes from 2:4 in the trihydrate to 5:4 in the
dihydrate. These changes are still significantly different to the ratios of
nitrogen to oxygen atoms in the trihydrate and dihydrate of 2:4 and 4:6,
respectively. Both nitrogen atoms of each hydrazonyl group in
[(3)·(H2O)2] act as acceptor atoms, but only one of the hydrogens in
each ]N-NH2 moiety is utilized in hydrogen bonding in [(3)·(H2O)2].
The steroidal hydroxyl groups in Mol. A (with O122) and Mol. B (with
O222) are involved in different sets of hydrogen bonds, thus the OH
group in Mol. A acts as an acceptor to both W*3 and the hydroxyl group
of Mol. B, and as a donor to W*6, while the OH group of Mol. B acts as
an acceptor to W*5 and to the ]N-NH2 unit of Mol. A and as a donor to
the hydroxyl group of Mol. A.
3.2. Molecular electrostatic potential (ESP)
Because Hirshfeld surface partitions of the crystal space give non-
overlapping volumes associated with each molecule, these surfaces
98

L.R. Gomes et al.
Steroids140(2018)92–103
Fig. 6. [3·(H2O)2]. (a) Part of a slice through the 3-dimensional structure showing a puckered sheet of molecules, viewing down the a axis, (b) part of another slice
through the three dimensional hydrogen-bonded structure, this time looking down the b axis. The various hydrogen bonds are drawn as thin dashed lines. Symmetry
operations are listed in Table 2.
provide a kind of “electrostatic complementarity”. Negative electro-
static potential regions are represented by red areas and those of elec-
tropositive domains by blue areas. The grey areas on the remaining
mapped Hirshfeld surfaces indicate regions close to electroneutrality.
Thus as shown in Fig. 11a and b, for the trihydrate, regions close to the
lone pairs on the oxygen atom of the hydroxyl group and the nitrogen
atoms of the hydrazonyl group are shown as red areas. The blue re-
gions, the electropositive domains, are located near the H atoms of the
hydroxyl and hydrazonyl groups, Fig. 11c. In Fig. 11c, the electrostatic
complementarity of the N/OeH⋯O (water) contacts of the steroid with
hydrate molecules is highlighted: here the hydrogen atoms of the hy-
droxyl and hydrazonyl groups involved in the N/OeH⋯O (water)
contacts are indicated by the blue regions just above the surface of the
steroid molecule.
The Hirshfeld surfaces mapped with electrostatic potential of both
independent molecules of [3·(H2O)2], are provided in Fig. 12. Fig. 12a
illustrates the electropositive area of Mol. B involved in the direct
contact [via the hydrogen atom of its hydroxyl group] with the lone
pair of the oxygen atom of the eOH group of Mol. A, thereby creating
the O222eH222⋯O122 hydrogen bond. In Fig. 12b is illustrated the
other direct contact between Mol. A with Mol. B via the electropositive
area of Mol. A [hydrogen atom of its hydroxyl group] with the forma-
tion of the N121eH124⋯O222 hydrogen bond. In Fig. 12c, Hirshfeld
surfaces of both molecules of [3·(H2O)2] are shown. The remaining blue
99

L.R. Gomes et al.
Steroids140(2018)92–103
peak at 115.2 °C, corresponding to an energy of 8.9 kJ/mol. The onset
of melting occurred at 195 °C, with the peak at 199 °C; the corre-
sponding energy is 24.2 kJ/mol.
Thermal gravimetric analysis. A Mettler Toledo STARe System TGA2
was used. A sample of the dihydrate [(3)·(H2O)2], 15.69 mg, was used
with a time–temperature program of heating from 25 to 250 °C at 10 °C/
min. Two significant mass loss regions (i) 4.98% mass loss between 39
and 106 °C and (ii) 5.46% between 106 and 146 °C, see Supplementary
Fig. 2. The molecular weight of [(3)·(H2O)2] is 340.50, and hence the
calculated percentage loss of one hydrate molecule is 5.28%. The
combined weight loss is that of the two hydrates [total 10.56%, com-
pared to the found loss of 10.44%]. Thus the dehydration of
[(3)·(H2O)2], occurs essentially stage by stage, with just a small
overlap.
Successive loss of the three hydrate molecules from the trihydrate,
[(3)·(H2O)3], are expected too.
Fig. 7. [3·(H2O)2]. (a) Part of the hydrogen bond framework, formed from the
water molecules, and the HO and H2N-N] moieties of molecule 3. Only the
participating moieties within 3 in the hydrogen bonding network are drawn.
Each of the four independent water molecules are involved in different sets of
hydrogen bonds, see also Table 2 for symmetry operations. The hydrogen bonds
are drawn as thin dashed lines.
4. Conclusions
The combination of the Hirshfeld pixel approach and the PLATON
geometric approach provides complimentary insights into the supra-
molecular structure of [(3)·(H2O)2] and [(3)·(H2O)3]. Strong classical
hydrogen bonds in both hydrates, utilizing the hydrate molecules and
the hydroxyl and hydrazono moieties of the steroid, generate different
complex three dimensional structures. There are no direct steroid–-
steroid contacts in the trihydrate, but there are some head-to-head and
head to tail direct contacts in the dihydrate, although hydrate linked
steroid–steroid contacts still dominate. The influence of the number of
hydrate molecules on the overall structure, in particular on the contact
between the steroid molecules, is clearly apparent.
and red areas are due to interactions with water molecules.
3.2.1. DSC and TGA studies of [(3)·(H2O)2]
Differential scanning calorimetry. A Mettler Toledo STARe System
DSC1, cooled by liquid nitrogen and calibrated using indium and zinc as
standards, was used. A sample of the dihydrate, [(3)·(H2O)2] (8.63 mg),
was used with a time–temperature program of heating from 25 to
250 °C at 10 °C/min. The DSC trace, see Supplementary Fig. 1, indicates
a glass transition occurring at an initial temperature of 101.65 °C with a
Fig. 8. [3·(H2O)2]. Views of the Hirshfeld surfaces mapped over dnorm of (a) and (b) Mol. A, (c) and (d) Mol. B. The highlighted red spots on the surfaces indicate
contact points with the OeH and the N-NH2 atoms of the steroid as identified. The small red area on the top face of c arises from C⋯H contacts.
100

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Steroids140(2018)92–103
Fig. 9. [3·(H2O)2]. Views of the Hirshfeld surface mapped over dnorm for the hydrate molecules, W#3, W#4, W#5 and W#6. The highlighted red spots on the
surfaces indicate contact points with the N/O⋯H atoms (dashed lines: red for O⋯H and yellow for N⋯H). Contacts with the highlighted water molecules by
molecules of 3 and other water molecules are indicated.
Fig. 10. The FP plots for the molecule 3 in (a) [3·(H2O)3], (b) Mol. A of [3·(H2O)2] and (c) Mol. B of [3·(H2O)2]. The light blue areas in the middle of the FP plots at
de/di ≅ 1.6 Å show high pixel frequencies due to H⋯H contacts. The sharp spikes pointing to southwest are due to N/O⋯H contacts: the inner ones on the right are
due to O⋯H contacts; the outer ones on the right are related to N⋯H contacts. The left spikes are due to the H⋯O contacts.
Fig. 11. The electrostatic potential surfaces for mo-
lecule 3 in [3·(H2O)3]. The surfaces show the com-
plementary electropositive areas (blue) with the
water molecules. The ESP (ranging from −0.077 to
0.066) is electronegative in the vicinity of the lone
pairs of the oxygen atom and of the lone pairs of the
nitrogen atoms of the hydrazide fragment.
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Steroids140(2018)92–103
Fig. 12. Hirshfeld surfaces for 3 in [(3)·(H2O)2] 2 mapped with electrostatic potential. (a) The electropositive area of Mol. B indicating the interaction site with the
lone pair of the oxygen atom of the HO group of Mol. A, (b) the electronegative area of Mol. B indicating interaction with another steroid involving a NH⋯O
hydrogen bond, (c) Hirshfeld surfaces of Mol. A and Mol. B. The remaining blue and red areas are due to interactions with water molecules.
Acknowledgements
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The authors thank the National Crystallographic Service, University
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This work was supported by the Portuguese Foundation for Science and
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