Polymorphic and solvate structures of ethyl ester and carboxylic acid derivatives of WIN 61893 analogue and their stability in solution

Article abstract of DOI:10.1039/c4ce01152j

3-Ethyl ester- (1) and 3-carboxylic acid-isoxazole (2) derivatives of an antiviral drug analogue WIN 61893 were synthesized and characterized by X-ray crystallography and NMR spectroscopy. Crystallization experiments afforded two polymorphic structures fo

Full text of DOI:10.1039/c4ce01152j

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Polymorphic and solvate structures of ethyl ester
and carboxylic acid derivatives of WIN 61893
analogue and their stability in solution†
Cite this: DOI: 10.1039/c4ce01152j
Kirsi Salorinne, a Tanja Lahtinen,a Varpu Marjomäkib and Hannu Häkkinenac
*
3-Ethyl ester- (1) and 3-carboxylic acid-isoxazole (2) derivatives of an antiviral drug analogue WIN 61893
were synthesized and characterized by X-ray crystallography and NMR spectroscopy. Crystallization experi-
ments afforded two polymorphic structures for the ethyl ester derivative and two solvate structures for the
carboxylic acid derivative based on their ability to form intermolecular hydrogen bonding interactions with
the solvent molecules. The conformations of the derivatives depended greatly on the orientation of the
planar isoxazole and phenyl-oxadiazole ring systems with respect to one another and were found to take
up perpendicular, linear or tilted conformations. The carboxylic acid derivative was furthermore observed
to undergo isoxazole ring cleavage by decarboxylation in DMSO solution and transforming into a β-keto
nitrile ring-opening product over several days, whereas, the isoxazole ring of the ethyl ester derivative was
not affected.
Received 5th June 2014,
Accepted 12th August 2014
DOI: 10.1039/c4ce01152j
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Pleconaril, which showed a drastic decrease in the metabolic
degradation of the molecule and a broad range of antiviral
Introduction
Enteroviruses cause a variety of human illnesses related to
common respiratory infections, rash or mild fever to serious
or life-threatening infections including meningitis, myocardi-
tis, encephalitis, and paralytic poliomyelitis.1,2 Rhinoviruses
in the enterovirus genus, in particular, are the predominant
cause of the common cold in humans. Therefore, the develop-
ment of antiviral drugs designed to inhibit or interfere with
the viral replication process have been pursued over the past
decades and still continues to be a current topic.1,3,4
activity against enteroviruses in comparison to the other WIN
analogues.5,6 Structural studies,7–10 of several WIN analogues
including Pleconaril with human rhinoviruses (e.g. HRV14
and HRV16) showed the drug molecules to bind specifically
into an interior hydrophobic pocket within the virus capsid
causing a conformational change in the virus that eventually
One such family of potential antiviral drugs designed
to target the early events (attachment, entry and uncoating)
of viral replication are the “WIN compounds” named after
the developer Sterling-Winthrop (Scheme 1).1,3 The antiviral
drug candidate development – after a few promising lead
structures – led to WIN 63843 analogue or better known as
a Department of Chemistry, Nanoscience Center, University of Jyväskylä,
P.O. Box 35, 40014 JYU, Finland. E-mail: kirsi.salorinne@jyu.fi
b Departments of Biology and Environmental Science, Nanoscience Center,
University of Jyväskylä, P.O. Box 35, 40014 JYU, Finland
c Department of Physics, Nanoscience Center, University of Jyväskylä, P.O. Box 35,
40014 JYU, Finland
† Electronic supplementary information (ESI) available: Crystallographic data for
1-form I, 1-form II, 2-EtOH and 2-DMSO, respectively. Synthesis scheme, 1H and
13C NMR spectra of the intermediates (4–5) and products (1–2), crystallographic
data and details, 1H and 13C NMR spectra and details of the ring opening
product (3) are included in the ESI. CCDC 999254–999257. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4ce01152j
Scheme 1 Chemical structures of WIN 61893, WIN 63843 (Pleconaril)
and derivatives 1 and 2.
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blocks the replication and infectivity of the virus.1,3,5,11 Thus,
the structural and conformational properties of the WIN
analogues seem to be crucial for their mechanism of action
and, therefore, it is important to elucidate their structural
characteristics in detail.
Previously, three different polymorphic structures have
been reported for Pleconaril, two of which the single crystal
structure is known (forms I and III).12,13 In these two poly-
morphic structures the Pleconaril molecules were described
to pack either as a network of dimers (form I) or as a three-
dimensional weakly hydrogen-bonded network of monomers
(form III).13 In comparison to the structure of the prior WIN
61893 analogue, the oxadiazole ring methyl group was
substituted with a trifluoromethyl group in the Pleconaril
derivative (Scheme 1).14
Our aim was to functionalize the isoxazole ring 3-position
with an anchoring carboxylate group in order to produce a
potentially better analogue to the inherent palmitic acid that
is commonly found in the hydrophobic pocket of enterovirus
capsid. Here, we focused on the structural and conforma-
tional properties of the modified WIN 61893 compound.
Therefore, 3-(ethyl ester)- (1) and 3-(carboxylic acid)-isoxazole
(2) derivatives of WIN 61893 compound were prepared and
characterized (Scheme 1). Their structural and conforma-
tional properties were studied in detail by single crystal
X-ray diffraction analysis revealing the importance of the
hydrogen bonding ability of the carboxylate group on the
crystal packing. Solution stability was furthermore investi-
gated by NMR spectroscopy, which showed the isoxazole
ring of the carboxylic acid derivative (2) susceptible to
undergo ring opening by decarboxylation under certain reac-
tion conditions.
KI (0.12 g, 0.72 mmol) was suspended in dry DMF (15 mL)
with vigorous stirring under nitrogen atmosphere. 5-Chloro-
1-pentyne (1.05 mL, 9.91 mmol) was then added and the
reaction mixture was heated to 75 °C and allowed to stir
overnight. The resulting yellow suspension was filtered by
suction through a pad of Hyflo Super® and solvent was
removed by rotavapor. The yellow residue was dissolved in
ethyl acetate and the organic layer was washed once with
water and once with brine. The organic layer was separated,
dried with anhydrous Na2SO4 and solvent was removed by
rotavapor. Purification by flash column chromatography
on silica (mesh 0.063–0.200 mm) with ethyl acetate–hexane
2 : 8 provided the title compound as a white solid after
recrystallization from hexane. Yield 1.3 g (89%). m.p. 48–49 °C.
1H NMR (CDCl3, 400 MHz): δH = 7.32 (q, 2H, J = 0.59 Hz, ArH),
3.91 (t, 2H, J = 6.10 Hz, CH2O), 2.48 (td, 2H, J = 6.88 and
2.68 Hz, CH2), 2.30 (s, 6H, ArCH3), 2.02 (t, 2H, J = 6.52 Hz,
CH2–C), 1.99 (t, 1H, J = 2.66 Hz, CH) ppm. 13C NMR
(CDCl3, 100 MHz): δC = 159.7, 132.8, 132.6, 119.0, 107.4, 83.2,
70.4, 69.1, 29.0, 16.2, 15.10 ppm. HRMS (ESI-TOF) m/z:
236.1037 [M + Na+], required 236.1046.
3-(3,5-Dimethyl-4-(pent-4-yn-1-yloxy)phenyl)-5-methyl-1,2,4-
oxadiazole (5). A mixture of 4 (0.50 g, 2.35 mmol), anhydrous
and finely divided K2CO3 (1.6 g, 11.9 mmol) and
hydroxylamine hydrochloride (0.83 g, 11.9 mmol) in absolute
ethanol (10 mL) was refluxed with vigorous stirring for
17 hours. The hot reaction mixture was filtered by suction
and washed thoroughly with ethanol. Solvent was removed by
rotavapor yielding crude amidoxime intermediate, which was
redissolved in dry pyridine (2.5 mL) with stirring. Acetyl
chloride (0.335 mL, 4.71 mmol) was added carefully to
maintain a gentle reflux and the yellow reaction mixture was
then refluxed for 2 hours. The resulting dark brown mixture
was allowed to cool to RT, diluted with water and extracted
two times with dichloromethane. The combined organic
layers were washed successively with water and brine, and
the separated organic layer was dried with Na2SO4. Solvent
was removed by rotavapor and the crude mixture was passed
through a silica column (mesh 0.063–0.200 mm) with ethyl
acetate–hexane 3 : 7, which provided the title compound as
yellow oil. Yield 0.20 g (31%). 1H NMR (CDCl3, 300 MHz):
δH = 7.72 (q, 2H, J = 0.63 Hz, ArH), 3.91 (t, 2H, J = 6.09 Hz,
CH2O), 2.64 (s, 3H, CH3), 2.50 (td, 2H, J = 4.29 and 2.69 Hz,
CH2), 2.33 (s, 6H, ArCH3), 2.02 (t, 2H, J = 6.52 Hz, CH2–C),
1.99 (t, 1H, J = 2.66 Hz, CH) ppm. Product was used
directly without further analyses.
Experimental
Materials and methods
All materials were commercial unless otherwise mentioned.
Ethyl chlorooximidoacetate was prepared according to litera-
ture procedures.15 DMF was distilled over 4 Å molecular
sieves and stored under nitrogen over 3 Å molecular sieves.
K2CO3 was dried in an oven at 120 °C and stored in a desicca-
tor. 1H and 13C NMR spectra were recorded with Bruker
Avance 300, 400 or 500 MHz spectrometers and chemical
shifts were calibrated to the residual proton or carbon reso-
nance of the solvent. Accurate HRMS spectra were measured
with Micromass LCT ESI-TOF mass spectrometer using Leu-
cine Enkephalin as the internal calibration. Melting points
were determined with Stuart SMP3 melting point apparatus
in open capillaries. Synthesis scheme is shown in the ESI†
(Scheme S1).
Ethyl 5-(3-(2,6-dimethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)-
phenoxy)propyl)isoxazole-3-carboxylate (1). Oxadiazole
(0.39 g, 1.44 mmol) was dissolved in dry DMF (10 mL) at RT
under nitrogen atmosphere. solution of ethyl
5
A
chlorooximidoacetate15 (0.66 g, 4.33 mmol) in DMF (5 mL)
was added slowly over 20 minutes with stirring. The reaction
mixture was allowed to stir at RT for 30 minutes and then
heated to 90 °C. A solution of triethylamine (0.605 mL) in
DMF (5 mL) was then added slowly over 30 minutes and the
resulting yellow reaction mixture was allowed to stir at 90 °C
Synthesis of WIN 61893 derivatives16
3,5-Dimethyl-4-(pent-4-yn-1-yloxy)benzonitrile (4).
A
mixture of 3,5-dimethyl-4-hydroxy-benzonitrile (1.0 g, 6.93 mmol),
anhydrous and finely divided K2CO3 (1.9 g, 13.7 mmol) and
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for one day. After cooling to RT, the reaction mixture was
diluted with water and extracted three times with ethyl
acetate. The combined organic layers were washed with
water, aqueous 10% NaHSO4 solution and brine. The
separated organic layer was dried with Na2SO4 and solvent
was removed by rotavapor. Flash chromatographic
purification on silica (mesh 0.063–0.200 mm) with gradient
ethyl acetate–hexane 1 : 3 → 1 : 1 gave the title compound as a
white solid. Yield 0.26 g (46%). m.p. 95–96 °C. 1H NMR
(CDCl3, 400 MHz): δH = 7.73 (s, 2H, ArH), 6.49 (s, 1H, C = CH),
4.44 (q, 2H, J = 7.15 Hz, CH2OCO), 3.87 (t, 2H, J = 6.04 Hz,
CH2O), 3.13 (t, 2H, J = 15.1 Hz, CH2), 2.64 (s, 3H, CH3), 2.31
(s, 6H, ArCH3), 2.25 (q, 2H, J = 6.83 Hz, CH2), 1.42 (t, 3H,
J = 7.14 Hz, CH3) ppm. 13C NMR (CDCl3, 100 MHz): δC = 176.3,
174.6, 168.1, 160.1, 158.1, 156.5, 131.6, 128.1, 122.3, 101.8,
70.4, 62.1, 28.2, 23.6, 16.3, 14.1, 12.4 ppm. HRMS (ESI-TOF)
m/z: 408.1542 [M + Na+], required 408.1530.
Crystal data of structure 1-form I. C20H23N3O5, M = 385.41,
monoclinic, a = 7.8644(2), b = 23.7196(5), c = 10.4242(2) Å,
β = 96.213(3)°, V = 1933.11(7) Å3, T = 170(2) K, space group
P21/n, Z = 4, μ(Mo Kα) = 0.096 mm−1, 11 637 reflections
collected, 5318 unique (Rint = 0.0202) which were used in all
calculations. The final wR2 was 0.1328 (all data) and R1 was
0.0530 (>2σ(I)), CCDC number 999254.
Crystal data of structure 1-form II. C20H23N3O5, M =
385.41, monoclinic, a = 18.6194(5), b = 8.3392(2), c =
12.7669(3) Å, β = 100.331(3)°, V = 1950.19(8) Å3, T = 170(2) K,
space group P21/c, Z = 4, μ(Mo Kα) = 0.096 mm−1, 10 497
reflections collected, 5164 unique (Rint = 0.0420) which were
used in all calculations. The final wR2 was 0.1674 (all data)
and R1 was 0.0596 (>2σ(I)), CCDC number 999255.
Crystal data of structure 2-EtOH. C40H50N6O12, M =
806.86, monoclinic, a = 8.3029(3), b = 45.516(2), c =
11.0152(4) Å, β = 103.692(4)°, V = 4044.5(3) Å3, T = 170(2) K,
space group P21/c, Z = 4, μ(Mo Kα) = 0.099 mm−1, 18 378
reflections collected, 9215 unique (Rint = 0.0267) which were
used in all calculations. The final wR2 was 0.1408 (all data)
and R1 was 0.0593 (>2σ(I)), CCDC number 999256.
Crystal data of structure 2-DMSO. C20H25N3O6S, M =
435.49, monoclinic, a = 13.7775(3), b = 7.2131(1), c =
21.4071(3) Å, β = 90.490(2)°, V = 2127.32(6) Å3, T = 170(2) K,
space group P21/c, Z = 4, μ(Mo Kα) = 0.194 mm−1, 11 563
reflections collected, 5806 unique (Rint = 0.0182) which were
used in all calculations. The final wR2 was 0.1245 (all data)
and R1 was 0.0446 (>2σ(I)), CCDC number 999257.
5-(3-(2,6-Dimethyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)-
phenoxy)-propyl)isoxazole-3-carboxylic acid (2). A solution of
1 (80 mg, 0.21 mmol) and NaOH (14 mg, 0.35 mmol) in
ethanol–water mixture (1 : 1, 20 mL) was refluxed with
stirring for 1 hour. Ethanol was removed by rotavapor. The
remaining aqueous solution was washed with diethyl ether
and acidified by the addition of 6 M hydrochloric acid.
The precipitated white solid was extracted with ethyl acetate
and the combined organic layers were washed with water
and brine. The separated organic layer was then dried
with Na2SO4 and solvent was removed by rotavapor.
Recrystallization of the crude product from methanol
afforded the title compound as a white solid. Yield 62 mg
Solution stability experiments
(84%). m.p. 144–146 °C. 1H NMR (CDCl3, 400 MHz): δH
=
The stability of derivatives 1 and 2 in solution was investi-
gated by NMR spectroscopy. Samples were prepared in
DMSO-d6 and CDCl3 solutions. 1H NMR spectra were
recorded repeatedly with a Bruker 400 MHz spectrometer at
30 °C during several weeks. In case isoxazole ring opening
was observed, 13C NMR spectrum was also measured of the
final ring-opening product for a complete structure analysis.
7.73 (s, 2H, ArH), 6.55 (s, 1H, C = CH), 3.88 (t, 2H, J = 5.88 Hz,
CH2O), 3.16 (t, 2H, J = 15.0 Hz, CH2), 2.65 (s, 3H, CH3), 2.32
(s, 6H, ArCH3), 2.26 (q, 2H, J = 6.86 Hz, CH2) ppm. 13C NMR
(CDCl3, 100 MHz): δC = 176.6, 175.5, 168.2, 161.7, 158.3, 155.9,
131.7, 128.3, 122.4, 102.1, 70.5, 28.4, 23.8, 16.5, 12.5 ppm.
HRMS (ESI-TOF) m/z: 713.2590 [2 M − H−], required 713.2577.
Results and discussion
X-ray crystallography
Synthesis and crystallization
Data were recorded with AGILENT SuperNova diffractometer
using a micro-focus X-ray source and multilayer optics mono-
chromatized MoKα [λ(MoKα) = 0.71073 Å; 50 kV, 0.8 mA] radi-
ation. All data were collected at a temperature of 170(2) K.
Data reduction and analytical absorption correction by Clark
WIN 61893 analogues having either ethyl ester (1) or carbox-
ylic acid (2) functionality at the 3-position of the isoxazole
ring were synthesized according to previously published pro-
cedures using 3,5-dimethylphenol as the aromatic unit
(Scheme 1 and S1†).16 Crystallization experiments gave two
polymorphic structures for the ethyl ester derivative 1 by slow
evaporation at room temperature from CH2Cl2-hexane
(1-form I) or from hot methanol (1-form II) solutions. Form I
could also be obtained by slow vapor diffusion with water
from either methanol or ethanol solutions of derivative 1.
Carboxylic acid derivative 2, on the other hand, crystallized
as two different solvate structures (2-EtOH and 2-DMSO).
Ethanol-solvate (2-EtOH) was obtained by slow evaporation at
room temperature from hot ethanol solution, whereas,
DMSO-solvate (2-DMSO) crystallized by slow vapor diffusion
and Reid17 were made with CrysalisPRO 18
The structures were
.
solved by direct methods (SHELXS-97 (ref. 19)) or by charge
flipping (Superflip20) integrated in the program of Olex2,21
and refinements based on F2 were made by full-matrix least-
squares techniques (SHELXL-97 (ref. 19)). Hydrogen atoms
were calculated to their idealized positions with isotropic
temperature factors (1.2 or 1.5 times the C/O temperature
factor) and refined as riding atoms. See Table S1 (ESI†) for
complete crystallographic data and structural refinement
parameters. CCDC numbers 999254–999257 contain the
supplementary crystallographic data for this paper.
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form III – reveals similar conformational features in the main
WIN 61893 framework.13 Likewise, the propyloxy unit is
found in a gauche conformation in both of the Pleconaril
forms I and III (63.6–67.9°) structures. However, only linear
conformation between planes A and B is observed for both
of the Pleconaril polymorphs (form I: ϕ = 17.2° and form III:
ϕ = 9.9°).13
Fig.
1 A representative Ortep figure of WIN 61893 analogue
(derivative 1, structure 1-form II) with 50% ellipsoid probability and a
numbering scheme used for all structures.
Crystal packing interactions
Although the structural characteristics discussed above are
very similar for both derivatives 1 and 2, the differences
between the structures come into play when considering the
crystal packing interactions that the molecules are able to
form. The pivotal difference between the derivatives 1 and 2
is the ester or the carboxylic acid functionality of the
isoxazole ring, and namely the ability of this functional group
to form hydrogen-bonding interactions. Therefore, polymor-
phic structures (1-form I and 1-form II) are observed with the
ethyl ester derivative 1, while the carboxylic acid derivative 2
more readily forms solvate structures (2-EtOH and 2-DMSO)
by hydrogen bonding to the solvent molecules included in
the crystal lattice.
When looking at the packing interactions of the ethyl ester
derivative 1, the conformation of the molecule – perpendicu-
lar or linear – seems to have a major role in the crystal pack-
ing of the two polymorphs (Fig. 2, Table 2). A common
descriptor in both structures, however, is the formation of an
inversion dimer by two opposite facing molecules. In 1-form
I, the inversion dimer is held together by aromatic C13–H⋯π
(distance to centroid of 3.30 Å) interactions between the
parallel propyloxy and phenyl units, respectively (Fig. 2a, top).
The ethyl ester functionalized isoxazole unit (plane A),
which is in an almost perpendicular orientation to the
phenyl-oxadiazole unit (plane B), conveniently forms
N7⋯H–C11 (2.67 Å) and OC4⋯O14 (3.17 Å) interactions,
respectively, with the propyloxy unit of the neighboring dimer
in the diagonal direction. The carbonyl oxygen furthermore
forms O5⋯H–C13/22 (2.62 Å) interactions to the propyloxy
unit and the phenyl ring methyl group of the second neigh-
boring dimer, which altogether translates into a zigzag layer
assembly of infinite arrays of molecules in the crystal lattice
(Fig. 2a, middle and bottom).
with water from DMSO solution (Table S1†). All structures
crystallized in a monoclinic space group, P21/n or P21/c, with
one molecule in the asymmetric unit, except for structure
2-EtOH, which contained two molecules in the asymmetric
unit (2-EtOH-a and 2-EtOH-b).
Structural characteristics
All three rings (isoxazole, phenyl and oxadiazole) in the WIN
61893 derivatives 1 and 2 structures have a planar conforma-
tion as expected. The skeleton of the derivatives can be
viewed as consisting of three different structural units:
carboxylic acid or ester functionalized isoxazole ring [plane A,
defined as O3, C4, O5, C6, N7, O8, C9, C10 and C11] at one
end, which is connected by a propyloxy unit [C11–C12–C13–
O14] to a conjugated phenyl-oxadiazole ring system [plane B,
defined as O14, C15, C16, C17, C18, C19, C20, C21, C22, C23,
N24, O25, C26, N27 and C28] at the other end (Scheme 1,
Fig. 1). As such, the conformation of the molecule depends
to a great extent on the orientation of these structural units
with respect to one another (Table 1). In all of the structures
of 1 and 2 the connecting propyloxy unit is in a gauche
conformation with a torsion angle (C11–C12–C13–O14) in the
range of 59.7–67.6°. The set of five torsion angles determined
for the propyloxy unit (Table 1) shows the different conforma-
tional properties between the four structures, but the orienta-
tion of the structural units can also be simply viewed as the
angle (ϕ, Scheme 1) between planes A and B, which shows
the derivatives 1 and 2 to take up three different conforma-
tions: perpendicular (1-form I, ϕ = 60.8°), linear (1-form II
and 2-EtOH-b, ϕ = 14.2 and 5.9°) and tilted (2-EtOH-a and
2-DMSO, ϕ = 37.7 and ϕ = 33.7°) conformations.
Comparison of the derivatives 1 and 2 structures with the
known polymorphic structures of Pleconaril – form I and
In 1-form II, on the other hand, the inversion dimers are
connected by intermolecular C13–H⋯O3 (2.67 Å) interactions
Table 1 Geometrical parameters of the WIN 61893 derivatives 1 and 2 structures
Torsion angles (°)
1-Form I
1-Form II
2-EtOH-a/b
2-DMSO
O8–C9–C11–C12
73.1
177.0
171.6, 164.1
−168.5
−169.9
−59.7 (g)
164.3
80.4
33.7 (T)
C9–C11–C12–C13
−179.0
−67.4 (g)
176.0
82.7
60.8 (P)
−173.1
−63.3 (g)
174.3
82.3
14.2 (L)
173.8, −177.2
63.7 (g), −67.6 (g)
−166.7, 163.4
−95.7, 86.9
C11–C12–C13–O14a
C12–C13–O14–C15
C13–O14–C15–C16
Planes A and B angle, ϕ (°)b
37.7 (T), 5.9 (L)
a
b
Conformation of the propyloxy unit, gauche = g. Planes A and B angle: ϕ > 60° noted as perpendicular (P), ϕ > 20° noted as tilted (T)
and ϕ < 20° noted as linear (L) orientation with respect to one another.
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Fig. 2 Inversion dimers and crystal packing of the polymorphic structures of derivative 1: a) 1-form I and b) 1-form II. Dashed black or solid green
lines highlight the selected intermolecular interactions. Hydrogen atoms have been omitted for clarity when applicable.
between the parallel propyloxy unit and the isoxazole ester
group (Fig. 2b, top). In this case, derivative 1 is in a linear
conformation and the neighboring dimers interact most
favorably in the diagonal direction by forming intermolecular
counterparts by forming intermolecular C10a/b–H⋯N24a/b (dis-
tance of 2.67–2.73 Å) interactions between the acidic iso-
xazole hydrogen and the oxadiazole nitrogen, C12b–H⋯O25b
(2.69 Å) interactions between the propyloxy unit and
the oxadiazole oxygen, and O5a/b⋯H–C19a,21a/b (distance of
2.46–2.55 Å) interactions between the carbonyl oxygen and
the phenyl ring methyl groups (Fig. 3a, top). The conforma-
tional difference of the structural isomers is seen as the num-
ber of formed intermolecular interactions with the respective
inversion counterparts, which in the case of the linear isomer
is four interactions; whereas, the tilted isomer is only able to
make two of the above mentioned intermolecular interac-
tions. The assemblies of four molecules then layer diagonally
(highlighted with magenta color), which creates once again
a zigzag type of layer packing in the crystal lattice (Fig. 3a,
middle and bottom).
For the DMSO-solvate structure 2-DMSO, a different kind
of packing of derivative 2 molecules into the crystal lattice is
observed. Since DMSO can only function as a hydrogen bond
acceptor, only one type of hydrogen bonding interaction of
O3–H⋯ODMSO (1.73 Å) is formed between the carboxylic acid
group and the DMSO solvent molecule. Contrary to the other
structures, the inversion molecules stack in an alternating
fashion via intermolecular aromatic π⋯H–C21 (distance to
C
28–H⋯N24 (2.64 Å) interactions between two oxadiazole
groups, C22–H⋯O5 (2.34 Å) interactions between the phenyl
ring methyl and carbonyl groups, and C 22–H⋯C 11/22
(distance of 2.40–2.87 Å) interactions between the propyloxy
unit and the phenyl ring methyl groups, which lead into a
diagonal criss-cross type of packing in the crystal lattice
(Fig. 2b, middle and bottom).
As expected, in the solvate structures of derivative 2, the
hydrogen bonding interactions with the solvent molecules
play a major role in the crystal packing (Fig. 3). As seen in
the EtOH-solvate structure 2-EtOH, a solvent mediated dimer
is formed of two ethanol molecules and of two derivatives 2
molecules of different conformations – tilted (2-EtOH-a) and
linear (2-EtOH-b). The dimer is held together by inter-
molecular hydrogen bonded network of the R44(14) motif, in
which two types of hydrogen bonds are formed between the
solvent ethanol and isoxazole nitrogen (OEtOH–H⋯N7, dis-
tance of 2.05–2.06 Å) or carboxylic acid oxygen (O3–H⋯OEtOH
,
distance of 1.74–1.78 Å), respectively (Fig. 3a, top). The dimer
interacts with the respective conformational inversion
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Table 2 Selected hydrogen bond parameters of the WIN 61893 derivatives 1 and 2 structures
1-Form I
1-Form II
Interaction
D–H⋯A (Å)
υ (°)
Interaction
D–H⋯A (Å)
υ (°)
C
C
C
C
C
C
22–H⋯O5
13–H⋯O5
11–H⋯N7
13–H⋯Cta
13–H⋯C17
22–H⋯C22
2.62
2.62
2.67
3.30
2.73
2.86
3.17
150.0
150.3
167.6
149.2
125.2
113.4
—
C13–H⋯O3
C22–H⋯O5
C28–H⋯N24
C11–H⋯C22
C22–H⋯C22
2.67
2.34
2.66
2.87
2.40
160.4
164.9
169.2
164.9
130.0
OC4⋯O14
2-EtOH
2-DMSO
Interaction
D–H⋯A (Å)
υ (°)
Interaction
D–H⋯A (Å)
υ (°)
O3–H⋯OEtOH
1.78
1.74
2.05
2.06
2.46
2.50
2.55
2.70
2.73
2.67
172.7
171.7
177.3
168.7
153.2
153.5
150.5
134.5
163.8
178.8
O3–H⋯ODMSO
1.73
2.52
2.57
2.59
2.72
2.82
2.87
3.40
2.74
3.51
2.71
2.63
177.1
137.7
149.2
153.4
133.3
141.1
160.1
165.9
159.5
152.7
123.5
154.3
C
C
C
C
C
C
C
C
C
C
C
DMSO-103–H⋯O5
DMSO-103–H⋯O8
28–H⋯O3
OEtOH–H⋯N7
C21–H⋯O5
28–H⋯N7
21–H⋯Cta
C19B–H⋯O5B
C12B–H⋯O25B
C10–H⋯N24
11–H⋯C18
11–H⋯Cta
13–H⋯C19
13–H⋯Cta
DMSO-102–H⋯ODMSO
DMSO-103–H⋯ODMSO
a
Ct = aromatic ring centroid (C15–C16–C17–C18–C19–C20).
Fig. 3 Inversion dimer (a) and inversion stack (b) of the solvate structures of derivative 2 with crystal packing shown for: a) 2-EtOH and
b) 2-DMSO. Dashed black or solid green lines highlight the selected intermolecular interactions. Hydrogen atoms have been omitted for clarity
when applicable.
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centroid of 2.82 Å), C28–H⋯O3 (2.59 Å), C28–H⋯N7 (2.72 Å)
and C28–H⋯C4 (2.90 Å) interactions, rather than forming
separate inversion dimers as seen with structures 1-form I,
1-form II and 2-EtOH (Fig. 3b, top). The inversion stacks
assemble in the crystal lattice in a brick wall pattern, in
which the neighboring inversion stacks are slightly shifted
to one another (Fig. 3b, middle). The channels that are
formed between the neighboring stacks are filled with solvent
DMSO molecules that coordinate with one another via
and 2 in solution. As a conjugated heterocycle, the isoxazole
ring has typical properties of an aromatic system, but in addi-
tion it contains a weak nitrogen–oxygen bond, which is sus-
ceptible to undergo ring cleavage when exposed to certain
reaction conditions.22,23 The most typical ring cleavage prod-
ucts of the isoxazole ring include, for example, 1,3-
dicarbonyl, enaminoketone, γ-amino alcohol, α,β-unsaturated
oxime, β-hydroxy nitrile and β-hydroxy ketone compounds
depending on the nature and number of substituents in the
isoxazole ring.22 Therefore, the stability of the derivatives 1
C
102/103–H⋯ODMSO (distance of 2.63–2.71 Å) interactions
1
between the DMSO methyl and carbonyl groups, and by
and 2 were studied by H NMR spectroscopy in polar aprotic
C
103–H⋯O5/8 (distance of 2.52–2.57 Å) interactions between
DMSO-d6 and in non-polar CDCl3 solutions.
the DMSO methyl group and the carbonyl and isoxazole
oxygens of the neighboring derivatives 2 molecules (Fig. 3b,
bottom).
Coincidentally, it was observed that the carboxylic acid
derivative 2 underwent slow isoxazole ring cleavage within a
few weeks at room temperature in the polar aprotic DMSO-d6
solution, whereas, it was stable in the non-polar CDCl3 solu-
tion (Fig. 4 and S4†). The ethyl ester derivative 1, on the other
hand, did not show any ring cleavage and remained stable in
both solvents even after several weeks at room temperature
(Fig. S3 and S5†). In order to track down the ring-opening
product of derivative 2, the NMR spectra was carefully
analysed after the isoxazole ring opening had completely
While the conformational features of the main WIN 61893
framework of derivatives 1 and 2 structures were found to be
very similar to Pleconaril forms I and III structures, there are
differences seen in the crystal packing, which seem to greatly
depend on the characteristic intermolecular interactions
formed by the carboxylate or the trifluoromethyl functionali-
ties, as well as, on the preferred orientation of the planes A
and B with respect to one another.
1
taken place. From the H NMR spectra it was apparent that
In the case of Pleconaril forms I and III structures, only
linear conformation was observed, which allows the mole-
cules to pack in a highly parallel layers by C–H⋯O, C–H⋯N,
and C–H⋯F hydrogen bonding and π–π stacking interac-
tions13 in comparison to the more complex zigzag, diagonal
criss-cross and brick wall type of networks that were observed
with derivatives 1 and 2 structures having linear, tilted and
perpendicular conformations. Furthermore, the key feature
in the formation of hydrogen bonded structural motifs (inver-
sion dimer of form I and catemer synthon of form III) in the
Pleconaril structures was shown to be due to the C–H⋯O
and C–H⋯N hydrogen bonds formed primarily by the
oxadiazole ring with the neighboring oxadiazole (form I) or
isoxazole (form III) rings.13 Although similar motifs were also
seen in derivatives 1 and 2 structures, it should be empha-
sized that the carboxylate functionality additionally plays a
significant role in the formation of the structural motifs and
hydrogen bonding interactions as described above. There-
fore, the similarities in the structures of derivatives 1 and 2
and Pleconaril seem to arise from the interactions formed
by the shared WIN 61893 framework, whereas, the structural dif-
ferences are brought upon the specific hydrogen bond
forming abilities of the carboxylate or trifluoromethyl func-
tionalities, and consequently due to the multitude of confor-
mations of derivatives 1 and 2 in comparison to Pleconaril.
after one week in DMSO-d6 solution the isoxazole ring proton
H-10 of derivative 2 at 6.70 ppm began to disappear and a
new singlet peak was formed at 4.09 ppm, which became a
single H-10 resonance after several weeks (Fig. 4). Also, all
the propyloxy unit protons H-11 through H-13 shifted upfield
due to the isoxazole ring opening (Fig. 4, Table S2†). It was
considered that the carboxylic acid group was responsible
Solution stability
The isoxazole unit of WIN 61893 derivatives 1 and 2 struc-
tures not only convey the essential structural difference
between the two derivatives, but as a structural motif the
heterocyclic isoxazole ring has interesting properties on its
own, which play a key role in the stability of the derivatives 1
Fig. 4 Time dependent 1H NMR spectra of WIN 61893 carboxylic acid
derivative 2 in DMSO-d6 at 30 °C showing the isoxazole ring cleavage
into β-keto nitrile derivative 3 over several days: a) freshly prepared
solution, b) after 8 days and c) after 5 weeks. Molecular formula is
shown only up to carbon C-11 for clarity (see Scheme 2).
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of the WIN 61893 skeleton was mainly defined by the orienta-
tion of the isoxazole ring with respect to the planar phenyl-
oxadiazole unit taking either perpendicular, linear or tilted
conformations. The propyloxy unit between the planar ring
structures was found in a gauche orientation. The stability of
the ethyl ester and carboxylic acid derivatives were studied in
solution, which revealed that the carboxylic acid derivative
was susceptible to undergo isoxazole ring cleavage in DMSO
solution by decarboxylation yielding a ring opening product
β-keto nitrile derivative.
Acknowledgements
Elina Kalenius and Johanna Lind are thanked for their help
with ESI-HRMS analyses. Esa Haapaniemi is thanked for his
help with NMR measurements.
Notes and references
Scheme 2 Decarboxylation of 3-carboxyisoxazole group of derivative
2 into a β-keto nitrile or α-cyanoenol tautomer are shown for
derivative 3.
1 A. M. De Palma, I. Vliegen, E. D. Clercq and J. Neyts, Med.
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3 G. D. Diana, Curr. Med. Chem., 2003, 2, 1.
for the isoxazole ring cleavage since no ring opening
was observed with the ethyl ester derivative 1. Therefore,
subsequent decarboxylation and ring opening of the
3-carboxyisoxazole ring of derivate 2 into a β-keto nitrile
derivative 3 was suggested23,24 and supported by 13C NMR
spectrum (Fig. S6†) as the upfield shift of carbon C-6 from
158.3 to 115.5 ppm corresponding the resonance of the cyano
group, and as the downfield shift of carbon C-9 from 161.7 to
199.5 ppm associated with the carbonyl group formation
(Scheme 2, Table S2†). In addition, the loss of aromaticity
upon the isoxazole ring opening was seen as a drastic upfield
shift of carbon C-10 from 102.1 to 37.9 ppm.
The stability of the isoxazole ring of the WIN 61893 deriva-
tives 1 and 2 in solution is a key issue when considering
these compounds as possible antiviral agents and their distri-
bution and metabolism in biological media, as well as, mech-
anism of action. Isoxazole unit containing biologically active
compounds have been shown to undergo metabolic processes
resulting in the aforementioned isoxazole ring-opening prod-
ucts.25 3-Carboxyisoxazole and 3-unsubstituted isoxazole
rings in particular were shown to convert into either β-keto
nitrile or the tautomer α-cyanoenol ring-opening products
during metabolic biotransformation processes.24,25
4 For more recent examples, see: H. A. Basta, S. Ashraf,
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Conclusions
In conclusion, WIN 61893 analogues having either ethyl ester
or carboxylic acid functionality at the 3-position in the
isoxazole ring were prepared and crystallized. Two polymor-
phic structures of the ethyl ester derivative were obtained,
whereas, the carboxylic acid derivative crystallized as two
solvate structures. In all of the structures, the conformation
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24 This type of isoxazole ring cleavage is particularly known
and studied with 3-carboxybenzisoxazoles, see: D. S. Kemp
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