Polymorphism in the Anti-inflammatory Drug Flunixin and Its Relationship with Clonixin

Article abstract of DOI:10.1021/acs.cgd.0c00284

Crystallization of the nonsteroidal anti-inflammatory drug (NSAID) flunixin in acetone and in acetone-hexane produced two conformational polymorphs, as predicted by hydrogen-bond propensity analyses, which are similar to three of the polymorphs of the rel

Full text of DOI:10.1021/acs.cgd.0c00284

Subscriber access provided by Uppsala universitetsbibliotek
Article
Polymorphism in the anti-inflammatory drug
Flunixin and its relationship with Clonixin.
María Cecilia Dávila-Miliani, Analio Dugarte-Dugarte, Robert A. Toro, Jines E. Contreras,
Hernando A. Camargo, Jose Antonio Henao, José Miguel Delgado, and Graciela Díaz de Delgado
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.0c00284 • Publication Date (Web): 14 May 2020
Downloaded from pubs.acs.org on May 14, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
Polymorphism in the anti-inflammatory drug Flunixin and its relationship
with Clonixin.
1
1
2
1
María Cecilia Dávila-Miliani , Analio Dugarte-Dugarte , Robert A. Toro , Jines E. Contreras ,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
1
1
Hernando A. Camargo , José Antonio Henao , José Miguel Delgado , Graciela Díaz de Delgado
1Laboratorio de Cristalografía-LNDRX, Departamento de Química, Facultad de Ciencias,
Universidad de los Andes, Mérida 5101, Venezuela.
2Grupo de Investigación en Química Estructural (GIQUE), Escuela de Química, Universidad
Industrial de Santander, Bucaramanga, Colombia.
3Grupo de Investigación en Nuevos Materiales y Energías Alternativas, Facultad de Química
Ambiental, Universidad Santo Tomás, Florida Blanca, Colombia.
email: diaz@ula.ve / gdiazdedelgado@gmail.com
ABSTRACT
Crystallization of the NSAID flunixin in acetone and in acetone-hexane produced two
conformational polymorphs, as predicted by hydrogen bond propensity analyses, which are similar
to three of the polymorphs of the related NSAID clonixin. The two polymorphs of flunixin display
an intramolecular hydrogen bond between the amine N—H and the carbonyl oxygen of the
carboxylic group. However, the intermolecular hydrogen bond pattern of Form I (crystallized from
acetone) is characterized by the acid—pyridine heterosynthon while Form II (from acetone-
hexane) displays the acid—acid homosynthon. Form I of flunixin is similar to form I of clonixin
while form II of flunixin is related to forms III and IV of clonixin. Hirshfeld surface maps were
used to show the differences in the interactions present in the two flunixin polymorphs and the
similarities with the polymorphs of the related compound clonixin. Energy framework calculations
corroborate the similarity between Form I of flunixin and clonixin and indicate that Form II of
flunixin is more closely related to Form IV of clonixin than to Form III.
Keywords: Flunixin, Clonixin, NSAID, polymorphism, hydrogen bond propensity, Hirshfeld
surfaces, energy frameworks.
ACS Paragon Plus Environment
1

Crystal Growth & Design
Page 2 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Non-steroidal anti-inflammatory drugs (NSAIDs) comprise a heterogeneous family of compounds
which inhibit the prostaglandin G/H synthase enzymes (or cyclooxygenases) involved in painful
and inflammatory responses to a variety of stimuli. Selective pain and inflammation relief by
NSAIDs is thought to be related to inhibition of Cyclooxygenase-2 (COX-2) while COX-1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
inhibition is usually related to the adverse effects of such drugs. An important class of non-narcotic
NSAIDs are diarylamine derivatives containing a carboxylic acid group. They can also be regarded
as derivatives of phenylacetic and nicotinic acids. Diclofenac, mefenamic acid, tolfenamic acid,
1
niflumic acid, belong to this group of compounds. In particular diclofenac is among the top 200
2
most prescribed drugs, with more than 9.5 million prescriptions in 2015 in the US. Other
compounds of this group, such as clonixin (3, figure 1) in the form of its lysine salt, have shown
improved analgesic activity. The development and use of clonixin led to the discovery and study
of the closely related compound flunixin (1, figure 1) which is presented in this contribution.
3
Flunixin is usually formulated as its meglumine salt where meglumine (2, figure 1) can be
4
considered as an excipient which increases the solubility of flunixin. Some commonly used brand
names are Banamine®, Flumeglumine®, Suppressor®, Binixin®, and Finadyne®. It is frequently
used in veterinary medicine, including food-producing animals, in contrast to clonixin which is
prescribed only for human consumption. Flunixin is widely administered to horses to alleviate
muscle pain, colic pain, and in the treatment of joint diseases. It is considered a controlled substance
5
in equestrian sports and, therefore, its use is under strict regulation. The analgesic action of
flunixin involves the same mechanism as the other NSAIDs, blocking pain impulse generation by
inhibiting the enzyme cyclooxygenase and causing a decrease in the synthesis of prostaglandins.3,6
4
The crystal structure of the flunixin-meglumine salt has been previously reported, but searches in
7
8
the Cambridge Structural Database (CSD) and the PDF-4/Organics database indicated that the
structure of flunixin is not known.
Many diarylamines and drugs belonging to this family exhibit polymorphism.9-16 Given the close
relationship between flunixin and clonixin, a screening was carried out to explore the possibility
1
7
of polymorphism in flunixin. For clonixin four polymorphs have been identified. Forms I, III,
ACS Paragon Plus Environment
2

Page 3 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
and IV are neutral, while form II is zwitterionic (Figure 2). It is one of the few molecules in the
1
5
CSD that has zwitterionic polymorphs. In forms I and II the molecules form carboxylic-pyridine
hydrogen bonds (hetero-synthons) while in forms III and IV they display carboxylic acid dimers
(homo-synthons). The conformation of the molecules is different in each polymorph.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Molecular diagrams of flunixin (1), meglumine (2), and clonixin (3).
In view of the polymorphism exhibited by clonixin, the possible formation of polymorphs of
flunixin was examined experimentally, by performing crystallization experiments, and by using
the Hydrogen Bond Propensity tool included in the CSD-Materials suite implemented in
1
8
Mercury , comparing with the calculation for the polymorphs of clonixin. This tool, based on
statistical considerations, has proven to be of great value in the study, prediction, and
rationalization of polymorphism of many pharmaceutical compounds as well as to assess the
potential formation of multicomponent materials.19-21
As part of the work being carried out in our laboratories on the structural characterization of active
pharmaceutical ingredients (APIs) and their metal derivatives, the characterization by Attenuated
Total Reflectance Infrared Spectroscopy (ATR-FTIR), Thermogravimetric analysis (TGA),
Differential Scanning Calorimetry (DSC), and powder and single crystal X-ray diffraction (PXRD
and SCXRD), of two polymorphs of flunixin (obtained so far) is reported in the present work.
Hydrogen Bond Propensity studies, Hirshfeld surface analysis, and Energy frameworks
calculations are also presented.
ACS Paragon Plus Environment
3

Crystal Growth & Design
Page 4 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
I
II
III
IV
Figure 2. Hydrogen bonds present in the four polymorphs of clonixin. Figures drawn using the
data included in the corresponding CSD entries (I; BIXGIY, II: BIXGIY04, III: BIXGIY02, IV:
BIXGIY03).
RESULTS AND DISCUSSION
1
. Crystallization experiments
Flunixin was separated from flunixin-meglumine raw material (FM-RM) as described in the
Experimental section. Crystallization in acetone solution of the precipitated flunixin (FP) produced
the colorless prisms of Form I shown in Figure SI-1a of the Supporting Information file. On the
other hand, crystallization in acetone/hexane produced needles of Form II (Figure SI-1b) as well
as prismatic crystals of Form I.
2
. Infrared spectroscopy and thermal analysis
ACS Paragon Plus Environment
4

Page 5 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
The ATR-FTIR spectra of flunixin-meglumine raw material (FM-RM), flunixin precipitated (FP),
and Form I of flunixin (F-I) are shown in Figure SI-2. The characteristic bands of the functional
groups present in the molecules are observed as summarized in Table SI-1. The infrared spectrum
of FM-RM (Figure SI-2a) shows the N-H stretching vibration of the secondary amine group of the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
flunixin moiety at 3359 cm and the N-H stretching vibrations of the protonated amine of
-1
meglumine between 2842 and 2915 cm , overlapped with the C –H and C –H stretching
sp3
sp2
vibrations. The multiple hydrogen-bonded hydroxyl groups present in the meglumine moiety
-1
-1
-1
produce a broad band centered around 3150 cm . The two bands at 1584 cm and 1370 cm
correspond to the asymmetric and symmetric stretching vibrations of the carboxylate group of
-1
flunixin. The C=C stretching bands of flunixin are observed at 1601 cm . For FP and F-I (Figures
-1
SI-2b and SI-2c) the N-H stretch is observed at 3237 and 3236 cm , respectively. The stretching
band of the carboxylic O–H is very broad and weak. The C=O stretch appears between 1673 cm-1
-1
and 1672 cm . The absorption bands due to the φ–N and N–py stretches are observed at 1316 and
-1
1
239 cm , respectively, while the stretching vibrations of the C=N and C=C bonds appear as two
-1
overlapped bands at 1599-1580 cm . The overtones of the O–H bending are expected to overlap
with the asymmetric and symmetric vibrations of the methyl C–H bonds between 2900 and 2200
-1
-1
cm , while the O-H bending vibration is observed at 1452-1451 cm . In the two spectra, the
characteristic absorption bands of the trifluoromethyl group are observed in the range 1141-1120
-
1
cm .
The thermogram for flunixin recrystallized in acetone (Form I, Figure SI-3a) shows stability up to
approximately 200 °C when melting and decomposition start to occur. The total weight loss is 94.7
%. The DSC shows a sharp endotherm at 235.0 °C associated to melting. A broad endotherm at
277.1 °C and a sharp endotherm at 283.3 °C are associated to decomposition. The TGA/DSC of
Flunixin recrystallized in acetone/hexane, from which Form-II was identified, showed the same
thermal behavior observed for Form I (Figure SI-3b). This may be the result of the presence of an
important amount of Form I in the sample, as mentioned before. For comparison, the TGA/DSC of
flunixin-meglumine raw material (FM-RM) is presented in Figure SI-3c. FM-RM shows stability
up to 258 °C when a two-step weight loss of 85.52%, with peak temperatures 288 and 350 °C is
observed. The DSC shows an endothermic transition without weight loss with peak temperature of
1
42.0 °C that corresponds to melting. The TGA/DSC of Flunixin powder (Figure SI-3d) shows
ACS Paragon Plus Environment
5

Crystal Growth & Design
Page 6 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
melting at 230.9 °C and the endotherm associated with decomposition is observed at 267.9 °C. An
endothermic event at 171.4 °C is observed which has not been possible to correlate to known phases
in the system. It is being investigated and any results will be reported elsewhere.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
. X-ray diffraction
Structure determination and refinement from powder diffraction data of Form I.
The excellent quality of the powder diffraction pattern obtained for Flunixin precipitated from
flunixin-meglumine raw material (FM-RM) as described in the Experimental section, allowed us
to successfully determine the structure from powder data. The details of the structure determination
and refinement from powder data are presented in the Supporting Information file, Section SI-3,
Figure SI-4 and Tables SI-2a,b,c.
Structure determination and refinement from single crystal data
Table 1 summarizes the crystal data, experimental conditions, and final refinement parameters for
the studies at ambient temperature (RT) and low temperature (LT) of flunixin Form I, and at
ambient temperature of Form II. A low temperature study of the needles of Form II was not possible
due to the small amount obtained and their low crystal quality. Section SI-4 of the Supporting
Information contains tables of atomic coordinates, anisotropic displacement parameters for non-
hydrogen atoms, and isotropic displacement parameters for hydrogen atoms (Tables SI-3a,b,c) for
Form I-RT, Form I-LT, and Form II-RT. Corresponding bond lengths and angles are presented in
Tables SI-4a,b,c and Table SI-5 summarizes the calculated geometric parameters for hydrogen
bonds and intermolecular contacts for the two polymorphs of flunixin.
ACS Paragon Plus Environment
6

Page 7 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
Table 1. Crystal data, experimental conditions, and refinement results for flunixin Form I
(ambient and low temperature) and Form II (ambient temperature).
Form I
Amb. temperature) (Low temperature)
C H F N O C H F N O
Form I
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Form II
(
Formula
C H F N O
2
1
4
11
3
2
2
14 11
3
2
2
14 11
3
2
Formula Weight
Crystal System
Space group
296.25
monoclinic
P2 /c (No. 14)
296.25
monoclinic
P2 /c (No. 14)
296.25
triclinic
P1 (No. 2)
1
1
a = 7.6842(3) Å
b = 14.1394(5) Å
c = 12.5115(5) Å
a = 7.580(14) Å
b = 14.14(3) Å
c = 12.45(2) Å
a = 7.3684(5) Å
b = 7.6617(5) Å
c = 11.7865(8) Å
Unit cell parameters

= 79.684(5)°

 102.138(4)°
  102.32(2)°
 = 85.082(6)°

= 87.191(6)°
651.85(8)
2
V ( Å3)
Z
D(calc) (g cm )
1328.98(9)
4
1304(4)
4
1.509
-3
1.481
0.128
1.509
0.131
-1

(MoK) (mm )
0.131
Crystal Size (mm)
Temperature (K)
Radiation, λ (Å)
Min-Max (°)
Limiting indices
0.23 x 0.44 x 0.46
293
MoK 0.71073
3.6, 27.4
0.09 x 0.10 x 0.22
100
0.03 x 0.08 x 0.23
293
MoK 0.71073
2.8, 28.2
MoK 0.71073
2.2, 32.3
-4  h  10
-13  k  19
-18  l  3

-9  h  9
-18  k  18
-9  h  8
-9  k  10
-14  l  15
-16  l  15
Tot., Uniq. Data, Rint
Obs. Data (I > 2.0 (I))
Nref, Npar
11671, 2939, 0.023 3642, 2717, 0.057 10000, 2681, 0.062
2407
1441
1576
2939, 197
2717, 197
2681, 197
R, wR , S
0.0432, 0.1255, 1.03 0.0394, 0.0716, 0.75 0.0551, 0.1618, 0.96
-0.22, 0.19 -0.22, 0.26 -0.19, 0.18
2
Min. and Max. Resd.
-
-3
Dens. (e Å )
Crystal structure of Form I
During the course of the Rietveld refinement of the structure of flunixin (FP) obtained by separation
from the flunixin-meglumine raw material, colorless prismatic crystals of appropriate size and
quality for single crystal studies were obtained from an acetone solution. The analysis of the single
ACS Paragon Plus Environment
7

Crystal Growth & Design
Page 8 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
crystal data indicated that the structure of these crystals is the same as the structure determined
from powder data. This phase was assigned as Form I of flunixin, which crystallizes in a monoclinic
unit cell with space group P2 /c and unit cell parameters a = 12.484(2) Å, b = 14.126(3) Å, c =
1
3
7
.682(2) Å,  = 102.01(1)°, V = 1327.58(3) Å , Z = 4. It is worth noting that bond distances and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
angles from the powder diffraction study are in the range suggested in the statistical analysis
2
2
performed with Mogul Geometry Check and agree with the results from the single crystal data.
The following discussion of the structure of Form I will use the results from the single crystal data.
The asymmetric unit contains one flunixin molecule (Figure 3a) in which the pyridine ring forms
a 71.12(7)° angle with the plane of the substituted phenyl ring. The superposition of the molecules
of flunixin in Form I obtained in the powder diffraction study and in the single crystal studies at
ambient and low temperature shows that the conformations are the same (Figure 3b) and indicates
the good quality of the structure determination from powder diffraction data. The torsion angle C5-
N2-C6-C7 is 111.8(2)° at 293 K and 113.7(2)° at 100 K.
(a)
(b)
Figure 3. (a) Molecular structure of flunixin form I (from the low temperature study). Ellipsoids
drawn at the 50% level of probability. (b) Superposition of the flunixin molecules in Form I
obtained from powder diffraction (green), ambient temperature (orange), and low temperature
(purple) studies.
The packing of flunixin molecules in form I is shown in Figure 4. There is one intramolecular
hydrogen bond [N2—H2A···O2] represented by the graph set symbol S(6).23 The molecules
ACS Paragon Plus Environment
8

Page 9 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
interact by carboxylic-pyridine O1—H1A···N1 and C1—H1···O2 hydrogen bonds to form chains
that alternate directions along the +b and -b axis. This hetero-synthon produces a ring which can
2
be described by the symbol 푅 (7). The same C1—H1 donor participates in a bifurcated hydrogen
2
bond with one fluorine atom [C1—H1···F2] connecting the chains in a zigzag fashion. Additionally,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

··· interactions between the phenyl rings build up a bidimensional network approximately
parallel to the ab-plane (Figure 4a). These layers are further connected via weaker ··· interactions
between the pyridine rings along the c-axis (Figure 4b). Hydrogen bonds and ··· contacts,
2
4
calculated with PLATON, are summarized in Table SI-5 in the Supporting Information.
(a)
(b)
Figure 4. Two views of the structure of Form I of flunixin. (a) Hydrogen bonds and ···
interactions which produce layers parallel to the ab-plane. (b) Detail of the ··· interactions which
connect the layers along the c-axis.
Crystal structure of Form II
Flunixin Form II crystallizes in the triclinic system, space group 푃1, with parameters a = 7.3700(5)
Å, b = 7.6640(5) Å, c = 11.7894(7) Å,  = 79.687(5)°,  = 85.089(5)°,  = 87.190(5)°, V =
3
6
52.36(7) Å , Z = 2. The asymmetric unit also contains one flunixin molecule but, in contrast to
Form I, the molecules adopt a planar conformation (Figure 5a). The C5-N2-C6-C7 torsion angle is
ACS Paragon Plus Environment
9

Crystal Growth & Design
Page 10 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.2(4)°, in contrast with the torsions displayed by the flunixin molecule in Form I. The
superposition of the molecules in Forms I and II is shown in Figure 5b.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
Figure 5. (a) Molecular structure of flunixin form II. Ellipsoids drawn at the 30% level of
probability. (b) Superposition of the molecules in form I (red) and form II (blue).
The packing in form II is shown in Figure 6. There is one intramolecular hydrogen bond as in form
I, N2—H2A···O2, represented by the graph set symbol S(6). The molecules interact via the
carboxylic acid – carboxylic acid homosynthon O1—H1A···O2, which is represented by the
2
symbol 푅 (8), forming dimers which connect through C3—H3···F3 hydrogen bonds to produce
2
ribbons parallel to the bc plane (Figure 6a). The ribbons stack in an alternating fashion at an angle
of 18.82° from the a-axis, by means of C14—H14C···· interactions between the methyl group of
a phenyl ring and a pyridine ring related by the symmetry operation 1-x, 1-y, 1-z as depicted in
Figure 6b. Additionally, there are ···· interactions between these rings. As mentioned before, the
geometric parameters for these interactions are summarized in Table SI-5.
ACS Paragon Plus Environment
1
0

Page 11 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
Figure 6. Two views of the structure of form II of flunixin. (a) Cyclic O—H···O hydrogen bonds
and C—H···F interactions which produce ribbons parallel to the bc-plane. (b) C—H···· and ····
interactions which connect ribbons approximately along the a-axis.
For comparison purposes, the experimental powder diffraction pattern for flunixin Form I is
presented along with the calculated patterns from single crystal data for Form I and Form II (Figure
SI-5). As can be seen, the calculated diffraction pattern of Form I coincides with the recorded
pattern of the precipitated flunixin powder.
4
. Hydrogen bonding propensity analysis
Given the close relationship between flunixin (1) and clonixin (3), and the report of four
polymorphs for the latter, it was considered of interest to carry out a hydrogen bond propensity
(
HBP) analysis with Mercury.18,20-21 Section SI-5 of the Supporting Information summarizes de
details of the calculations. Figure SI-6 shows the selected hydrogen bond donors and acceptors.
The results of the calculations are presented in Table 2.
ACS Paragon Plus Environment
1
1

Crystal Growth & Design
Page 12 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Predicted and Realized Intermolecular Hydrogen Bonds for polymorphs I and II of
Flunixin and I, II, III, and IV of Clonixin
Flunixin Forms I and II
Obs.
Clonixin Forms I, III, IV
Clonixin Form II
D
A
P
D
A
P
Obs.
D
A
P
Obs.
O1—H N1 0.45 Form I O1—H
O1—H O2 0.41 Form II O1—H
N1
O2
N1
O2
O1
Cl1
O1
Cl1
N2
N2
0.42
Form I
N1—H O1
0.74 Form II
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.36 Forms III and IV N1—H O2
0.74
0.65
0.65
0.01
0.01
0.00
0.00
N2—H N1 0.25
N2—H O2 0.22
N2—H
N2—H
O1—H
O1—H
N2—H
N2—H
O1—H
N2—H
0.23
0.19
0.03
0.02
0.01
0.01
0.00
0.00
N2—H O1
N2—H O2
N1—H Cl1
N2—H Cl1
N1—H N2
N2—H N2
O1—H
O1—H
O1—H
F1 0.06
F2 0.06
F3 0.06
O1—H O1—H 0.06
N2—H
N2—H
N2—H
F1 0.03
F2 0.03
F3 0.03
D: Donor; A: Acceptor; P: Propensity.
For all polymorphs of flunixin and clonixin, the calculations indicate an intramolecular hydrogen
bond with the highest propensity involving the N2 from the secondary amine and a carboxylate
oxygen O2 (N2—H···O2). This intramolecular hydrogen bond is, indeed, present in the two
polymorphs of flunixin and the four polymorphs of clonixin.
Regarding the intermolecular hydrogen bonds, the highest propensities calculated involve the
carboxylic acid-pyridine hydrogen bond O1—H···N1, as shown in Table 2. This interaction is
present in forms I of flunixin and of clonixin. The second highest propensities are for the carboxylic
acid dimer O1—H···O2, which is present in flunixin form II and clonixin forms III and IV. For
polymorph II of clonixin (zwitterionic) the HBP analysis correctly predicts the intermolecular
hydrogen bond between the pyridine N1—H and the carboxylate oxygens present in this structure
(
N1—H···O1).
Figure SI-7 is the putative landscape for flunixin and shows that the two polymorphs obtained fall
in the highest propensity zone. Based on these calculations, it should also be possible to obtain
other polymorphs of flunixin, as is observed for clonixin and other related compounds. This
possibility is currently under investigation. Interestingly, for the related NSAID niflumic acid,
ACS Paragon Plus Environment
1
2

Page 13 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
which differs from flunixin in the absence of the methyl group next to the –CF in the phenyl group,
3
only one crystal form has been reported.25
5
.
Hirshfeld surface analysis.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hirshfeld surface analysis has recently gained attention for the description of intermolecular close
contacts since “fingerprint plots” of d /d interactions are particularly useful in assessing differences
e
i
26
in the contact environment of molecules in polymorphs. These fingerprint plots, calculated with
CrystalExplorer17,27 are shown in Figure 7 for polymorph I of flunixin and in Figure 8 for
polymorph II.

From Figure 7, the dominant interaction in polymorph I corresponds to the H N hydrogen bonds,




followed by H O, H F, and H C interactions. Weaker interactions such as   contacts are
present but do not contribute significantly to the fingerprint plot. The fingerprint plot for polymorph
II (Figure 8) is very different from the plot for polymorph I. In this case, the dominant interaction



is the O—H O cyclic hydrogen bond. H F and H C interactions are also important. The
contribution of ···· interactions is more important in polymorph II than in polymorph I. Figure
9 summarizes the percentage contribution of important intermolecular contacts to the Hirshfeld
surface area for the two polymorphs of flunixin.
ACS Paragon Plus Environment
1
3

Crystal Growth & Design
Page 14 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

Figure 7. Fingerprint plots for the molecule of flunixin Form I. (a) all contacts; (b) H N contacts;





(
c) H O contacts; (d) H F contacts; (e) H C contacts; (f) O F contacts; (g) C F contacts; (h)


C C contacts; (i) H H contacts. The percent of surface area included is shown for each plot.
Fingerprint plots were also calculated for the four polymorphs of clonixin and are included in
Figures SI-8 to SI-11 of the Supporting Information file. Figure 10 shows the fingerprint plots
including all contacts for the two polymorphs of flunixin and the four polymorphs of clonixin. It is
worth noting the resemblance of the plots for flunixin form I and clonixin form I and between form
II of flunixin and forms III and IV of clonixin. A summary of the contribution of intermolecular
interactions to the Hirshfeld surface in terms of percentage surface area is presented in Figure SI-
1
2.
ACS Paragon Plus Environment
1
4

Page 15 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

Figure 8. Fingerprint plots for the molecule of flunixin Form II. (a) all contacts; (b) H N contacts;





(
c) H O contacts; (d) H F contacts; (e) H C contacts; (f) O F contacts; (g) C F contacts; (h)


C C contacts; (i) H H contacts. The percent of surface area included is shown for each plot.
Flunixin Form I
Flunixin Form II
0%
10%
H-F
20%
30%
O-F
40%
H-N
50%
C-F
60%
C-C
70%
H-H
80%
90%
100%
H-C
H-O
Other
Figure 9. Contribution of important intermolecular contacts (%) to the Hirshfeld surface area for
the two polymorphs of flunixin.
Additionally, the dnorm parameter mapped onto the Hirshfeld surface allows to better visualize
contacts shorter than the sum of van der Waals radii. Figure 11 shows dnorm mapped for both
polymorphs of flunixin. In form I, the strongest red area (shortest contact) corresponds to the O—

H N hydrogen bond, shown on both sides of the molecule (the carboxylic group and the pyridine
ACS Paragon Plus Environment
1
5

Crystal Growth & Design
Page 16 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6


ring) while smaller red areas are related to weaker C—H O and C—H F interactions. In form II,

the cyclic O—H O hydrogen bond corresponds to the strongest red spots because it is the strongest

interaction in the structure, even stronger than the O—H N hydrogen bond in polymorph I. A

small red spot related to the C—H F interaction is also observed in the map of form II.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 10. Fingerprint plots calculated for all contacts in the two polymorphs of flunixin and the
four polymorphs of clonixin.
ACS Paragon Plus Environment
1
6

Page 17 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
Figure 11. Mapping of the dnorm parameter in the range -0.70 to 1.32 onto the Hirshfeld surface
for (a) Form I and (b) Form II of flunixin. The strongest close contacts with neighboring molecules
are shown for each polymorph.
6
.
Energy framework analysis.
Figures 12 and 13 contain a representation, in 2x2x2 unit cells, of the electrostatic, dispersion, and
total energy for the polymorphs of flunixin and clonixin. Table 3 presents a summary of the
contributions to the total energy of the individual energy components for these polymorphs. The
complete results of the calculations are presented in Tables SI-8 to SI-13 of the Supporting
Information.
As it was discussed in previous sections, polymorphs I of flunixin and of clonixin display the same
molecular configuration, hydrogen bonding pattern, and a similar distribution of intermolecular
interactions as seen in the calculated fingerprint plots. Similarly, polymorph II of flunixin shares
those same features with polymorphs III and IV of clonixin. The calculations of energy frameworks
for all polymorphs support those findings. A comparison with clonixin Form II is not appropriate
because of the zwitterionic nature of the clonixin fragment in this polymorph.
ACS Paragon Plus Environment
1
7

Crystal Growth & Design
Page 18 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As seen in Figure 12, forms I of flunixin and of clonixin show the same topology for the
contributions of the electrostatic and dispersive energies to the total energy of interactions. The
values for the individual contributions are also similar (Table 3) leading to similar values of Etot.
On the other hand, a comparison of the energy framework for polymorph II of flunixin (F-II) with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
clonixin polymorphs III and IV (C-III and C-IV) indicates a closest resemblance of F-II to C-IV
(Figure 13). Fingerprint plots pointed to similarities in intermolecular contacts between F-II, C-III,
and C-IV, but energy frameworks calculations provide a better insight into the contributions to the
total interaction energy which allows to identify a closer resemblance between F-II and C-IV.
Figure 12. Energy frameworks viewed down the a-axis for (a) Flunixin Form I and (b) Clonixin
-1
Form I. The cylinder radii was scaled to 75 arbitrary units and the energy cutoff is 5 kJ mol . E ,
ele
E , and E are represented in red, green, and blue, respectively.
disp
tot
ACS Paragon Plus Environment
1
8

Page 19 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 13. Energy frameworks for a) Flunixin Form II viewed down the c-axis, b) Clonixin Form
III down the a-axis, and Clonixin Form IV down the c-axis. The cylinder radii was scaled to 75
-1
arbitrary units and the energy cutoff is 5 kJ mol . E , E , and E are represented in red, green,
ele
disp
tot
and blue, respectively.
-1
Table 3. Summary of calculated energies (kJ mol ) for the polymorphs of Flunixin and Clonixin.
(3.80 Å radius).
Eele
Epol
Edisp
Erep
Etot
Flunixin
Clonixin
Form I
-92.6
-23.6
-187.5
165.6
-176.3
Form II
Form I
-142.2
-98.1
-32.3
-24.7
-61.9
-35.1
-33.8
-251.3
-192.3
-149.2
-210.9
-277.8
237.6
184.7
146.6
248.4
260.9
-246.4
-175.4
-154.7
-208.6
-256.5
Form II
Form III
Form IV
-65.9
-143.7
-142.8
ACS Paragon Plus Environment
1
9

Crystal Growth & Design
Page 20 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Flunixin:Meglumine
Energy Model
-42.8
-13.7
-52.7
75.5
kdisp
-54.7
krep
kele
kpol
0.740
CE-B3LYP ... B3LYP/6-31G(d,p) electron densities 1.057
0.871 0.618
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSIONS
Two polymorphs of the NSAID flunixin were prepared and characterized by ATR-FTIR and
TGA/DSC analysis. Their structures, determined from powder and single crystal X ray diffraction,
indicate very different conformations which lead to different hydrogen bonding patterns and
different intermolecular interactions. The Hydrogen Bond Propensity calculation predicts the two
polymorphs of flunixin and points to the possible existence of more polymorphs. The Hirshfeld
surface analysis and energy framework calculations show the differences between the two
polymorphs of flunixin and their respective similarities with polymorphs of the related NSAID
clonixin. In particular, energy framework calculations demonstrated to be useful in distinguishing
small differences in the crystalline packing that allowed to correctly classify the flunixin
polymorphs. Crystallization experiments are underway to optimize the yield of polymorph II and
to obtain other possible polymorphs of flunixin, in particular a zwitterionic form.
EXPERIMENTAL
1
. Materials
Flunixin-meglumine raw material (FM-RM) was supplied by Laboratorios Reveex, Maracay,
Venezuela. Concentrated HNO , acetone, ethanol, and hexane, purchased from different vendors,
3
were used without further purification.
2
. Separation and crystallization of flunixin
ACS Paragon Plus Environment
2
0

Page 21 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
Flunixin powder (FP) was separated from the flunixin-meglumine raw material (FM-RM) by
potentiometric titration of a 0.05 M solution of flunixin-meglumine (FM) (2.458 g of FM dissolved
in 100 mL of water, pH = 8.515) with a 0.05 M solution of HNO (0.35 mL of HNO 65% dissolved
3
3
in 100 mL of water). The solution was kept under constant stirring and the nitric acid solution was
added slowly from a burette while measuring the pH with a Crimson Basic 20 pH-meter calibrated
with pH = 7 and pH = 4 buffer solutions (Laboratorio de Análisis Instrumental, Universidad de Los
Andes, Mérida, Venezuela). The temperature in the laboratory was 22 °C. After adding about 1 mL
of the acid solution (pH = 7.05) a white precipitate formed. This precipitate was later filtered by
gravity and allowed to dry at room temperature for 12 hours. The remaining solution was allowed
to evaporate slowly at ambient temperature.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Crystallization experiments were carried out using 50 mg of the precipitated flunixin in 1.5 mL of
ethanol, acetone, and acetone-hexane 1:1 solution in 2 mL Eppendorf Safe-Lock microcentrifuge
tubes. Transparent prismatic crystals were obtained from ethanol and acetone solutions while
transparent needles (along with prisms similar to those obtained from ethanol and acetone) grew
in the acetone-hexane solution (see Figure SI-1). Based on powder and single crystal X ray
diffraction studies performed on the materials, the separated flunixin and the prismatic crystals
(from either acetone or ethanol) were labeled form I (F-I) and the needles (from acetone/hexane)
were labeled form II (F-II).
3
. Infrared Spectroscopy and thermal analysis
ATR-FTIR spectra for FM-RM, the separated flunixin (FP), and recrystallized flunixin (both
-1
forms, F-I and F-II) were recorded from 4000 to 500 cm , in 24 scans, collected in 30 seconds,
-1
with a resolution of 4 cm . The instrument used was a Bruker Alpha spectrophotometer with an
ATR eco ZnSe accessory, located at Laboratorio de Química Orgánica y Biomolecular (LQOBio),
Universidad Industrial de Santander, Bucaramanga, Colombia.
TGA and DSC analyses were carried out using a NETZSCH STA 449F5 instrument under dynamic
-1
nitrogen atmosphere at 50.0 mL min . The instrument was allowed to equilibrate at 28.00 °C and
ACS Paragon Plus Environment
2
1

Crystal Growth & Design
Page 22 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
a heating ramp of 10.00 °C min was applied up to 450.00 °C. The data was analyzed with the
NETZSCH Proteus Thermal Analysis Version 6.1.0 software.
4
. Characterization by X ray powder diffraction. Structure determination and refinement of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Flunixin Form I from Powder Diffraction data.
Powder diffraction patterns for FM-RM and for FP were recorded on a Siemens D5005
diffractometer operating at 40kV/30mA, with CuK radiation (= 1.5418 Å), and slits of 0.02 mm.
The materials were gently ground in an agate mortar and dusted on top of a zero-background holder.
Data were collected from 5° to 45° (2) in steps of 0.02° at 120 seconds per step. The high quality
of the pattern of the separated flunixin allowed the structure determination of this material, prior
2
8
29
30
to the single crystal study, using the programs DICVOL14, TALP, and GSAS-II, as detailed
in Section SI-3 of the Supporting Information. The powder diffraction data has been incorporated
as entry PDF 00-068-1378 (as a Star-Quality pattern) in the Powder Diffraction File of the
International Centre for Diffraction Data (ICDD).8
5. Single crystal X ray diffraction
Single crystal X ray diffraction data were collected at 293 K for Flunixin Forms I and II with a
Rigaku XtaLAB P200 diffractometer operating at 50 kV and 40 mA. The diffractometer is
equipped with a Pilatus 200K detector and SHINE (curved graphite monochromator) optics. MoK
31
32
radiation (=0.71075 Å) was used. CrystalClear and CrysAlis Pro were used for data collection,
unit cell determination and refinement, integration, and reduction. Low temperature (100 K) data
for Flunixin Form I was collected at Northwestern University using a Bruker APEX-II CCD Kappa
Duo diffractometer, with MoK radiation ( = 0.71075 Å). Unit cell determination and refinement
and data reduction were accomplished with Bruker APEX233 and Bruker SAINT34 software.
SHELXT35 and SHELXL36 implemented in shelXle37 were used, respectively, for structure
3
8
determination and refinement by full matrix least-squares. Diamond 3.0 was used for graphics.
2
4
39
PLATON was used for structure validation and geometrical calculations and enCIFer for CIF
editing. Additional details are provided in the Supporting Information, Section SI-4.
ACS Paragon Plus Environment
2
2

Page 23 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
6
. Hirshfeld Surface analysis and Energy Frameworks calculations.
2
7
Hirshfeld surface (HS) analysis was accomplished with CrystalExplorer17. “Fingerprint plots” of
d vs. d were obtained for the two polymorphs of flunixin and for the four polymorphs of clonixin.
e
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For flunixin, dnorm values26 were mapped onto the HS in the range -0.80 to 1.70 to aid the
visualization of intermolecular contacts. Details are provided in Section SI-6 of the Supporting
Information. Section SI-7 describes the calculation of Energy frameworks using
27
40-41
CrystalExplorer17 and the scaling procedures employed.
CRYSTALLOGRAPHIC DATA
Single crystal data for flunixin Form I (Ambient Temperature and Low Temperature) and Form II
have been deposited with the Cambridge Crystallographic Data Centre under deposition numbers
1
827219, 1884640, and 1884673, respectively. They can be accessed free of charge at
https://www.ccdc.cam.ac.uk/structures. Powder diffraction data for flunixin Form I has been
deposited with the International Centre for Diffraction Data (ICDD) as entry PDF 00-068-1378.
ASSOCIATED CONTENT
Supporting Information. Additional information noted in text is available free of charge via the
ACS publications site http://pubs.acs.org. Sections SI-1 to SI-7 contain ATR-FTIR spectra,
TGA/DSC curves, details of the structure determination and refinement by powder data, details of
single crystal data collection, details of Hydrogen Bond Propensity, Hirshfeld Surface analysis and
Energy Frameworks calculations. Figures SI-1 to SI-12 and Tables SI-1 to SI-13 as well as
References for the experimental procedures are included.
ACS Paragon Plus Environment
2
3

Crystal Growth & Design
Page 24 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AUTHOR INFORMATION
Corresponding Author
*
E-mail: gdiazdedelgado@gmail.com / diaz@ula.ve
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACKNOWLEDGEMENTS
We dedicate this contribution to Dr. William Jones on the occasion of his retirement, in
appreciation for his contributions to the understanding of the solid-state chemistry of
pharmaceutical compounds.
The authors thank Lic. M. A. Ramírez and Lic. A. G. Mora (ULA) and Qco. C. Alvarez (UIS) for
technical assistance, and Lic. J. J. Dávila of Laboratorios Reveex, Venezuela. Thanks are also due
to Charlotte Stern (Northwestern U., USA) for data collection at low temperature of Form I, during
the 2017 ACA Summer Course in Chemical Crystallography and to Dr. Amy Sarjeant (CCDC) for
fruitful discussions regarding Hydrogen Bond Propensities. This work was supported in part by
Vicerrectoría de Investigación y Extensión, Universidad Industrial de Santander (UIS), Colombia.
Access to the Cambridge Structural Database (CSD) for Universidad de Los Andes (Venezuela)
was possible through the Frank H. Allen International Research & Education Programme (FAIRE)
from the Cambridge Crystallographic Data Centre (CCDC).
REFERENCES
(
1) Brunton, L. L.; Lazo, J. S.; Parker, K. L., Goodman & Gilman's: The Pharmacological Basis
of Therapeutics. 11 ed.; McGraw-Hill Medical Publishing Division: New York, NY, 2006;
p 2021.
(2) http://clincalc.com/DrugStats/Drugs/Diclofenac Access date: December 19, 2018
(
3) Jaussaud, P. La Flunixine et son utilisation chez le cheval. Ann. Rech. Vét. 1986, 17, 353-
62.
3
(
4) Cao, X.-J.; Sun, C.-R.; Pan, Y.-J. The complex of flunixin and meglumine. Acta Crystallogr.,
Sect. E 2003, 59, o1471-o1473.
ACS Paragon Plus Environment
2
4

Page 25 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
(
5) Fédération Equestre Internationale, F., Equine Prohibited Substances Database. Fédération
Equestre
Internationale:
Lausanne,
Switzerland,
2010-2018.
http://prohibitedsubstancesdatabase.feicleansport.org/.
(
6) USP Veterinary Pharmaceutical Information Monographs - Anti-inflammatories. J. Vet.
Pharmacol. Therap. 2004, 27, 1-110.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(7) Groom, C. R.; Bruno, I. J.; Lightfoot, M. P.; Ward, S. C. The Cambridge Structural Database.
Acta Crystallogr., Sect. B 2016, 72, 171-179.
(
8) Kabekkodu, S. PDF-4/Organics Database. International Centre for Diffraction Data
(ICDD): Newtown Square, PA, USA, 2015.
(
9) Long, S.; Parkin, S.; Siegler, M.; Brock, C. P.; Cammers, A.; Li, T. Polymorphism of an
Organic System Effected by the Directionality of Hydrogen-Bonding Chains. Cryst. Growth
Des. 2008, 8, 3137-3140.
(10) Long, S.; Parkin, S.; Siegler, M. A.; Cammers, A.; Li, T. Polymorphism and Phase Behaviors
of 2-(Phenylamino)nicotinic Acid. Cryst. Growth Des. 2008, 8, 4006-4013.
(
(
(
11) Long, S.; Li, T. Controlled Formation of the Acid−Pyridine Heterosynthon over the
Acid−Acid Homosynthon in 2-Anilinonicotinic Acids. Cryst. Growth Des. 2009, 9, 4993-
4997.
12) Long, S.; Li, T. Enforcing Molecule’s π-Conjugation and Consequent Formation of the
Acid−Acid Homosynthon over the Acid−Pyridine Heterosynthon in 2-Anilinonicotinic
Acids. Cryst. Growth Des. 2010, 10, 2465-2469.
13) Long, S.; Siegler, M. A.; Mattei, A.; Li, T. Phase Transition from Two Z′ = 1 Forms to a Z′
=
2 Form of a Concomitant Conformational Polymorphic System. Cryst. Growth Des. 2011,
1
1, 414-421.
(14) Lee, E. H.; Boerrigter, S. X. M.; Rumondor, A. C. F.; Chamarthy, S. P.; Byrn, S. R.
Formation and Solid-State Characterization of a Salt-Induced Metastable Polymorph of
Flufenamic Acid. Cryst. Growth Des. 2008, 8, 91-97.
(
15) Nath, N. K.; Kumar, S. S.; Nangia, A. Neutral and Zwitterionic Polymorphs of 2-(p-
Tolylamino)nicotinic Acid. Cryst. Growth Des. 2011, 11, 4594-4605.
16) Kumar, S. S.; Nangia, A. A Solubility Comparison of Neutral and Zwitterionic Polymorphs.
Cryst. Growth Des. 2014, 14, 1865-1881.
(
(17) Takasuka, M.; Nakai, H.; Shiro, M. Infrared spectral and X-ray studies of polymorphic forms
ACS Paragon Plus Environment
2
5

Crystal Growth & Design
Page 26 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of 2-(2-methyl-3-chloroanilino)nicotinic acid. J. Chem. Soc., Perkin Trans. 2 1982, 1061-
1067.
(
18) Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.; McCabe, P.; Pidcock, E.;
Rodriguez-Monge, L.; Taylor, R.; van de Streek, J.; Wood, P. A. Mercury CSD 2.0– new
features for the visualization and investigation of crystal structures. J. Appl. Crystallogr.
2008, 41, 466-470.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
19) Nauha, E.; Bernstein, J. “Predicting” Crystal Forms of Pharmaceuticals Using Hydrogen
Bond Propensities: Two Test Cases. Cryst. Growth Des. 2014, 14, 4364-4370.
(20) Galek, P. T. A.; Fábián, L.; Motherwell, W. D. S.; Allen, F. H.; Feeder, N. Knowledge‐based
model of hydrogen‐bonding propensity in organic crystals. Acta Crystallogr. Sect. B 2007,
6
3, 768-782.
(
21) Galek, P. T. A.; Allen, F. H.; Fabian, L.; Feeder, N. Knowledge-based H-bond prediction to
aid experimental polymorph screening. CrystEngComm 2009, 11, 2634-2639.
(
22) Bruno, I. J.; Cole, J. C.; Kessler, M.; Luo, J.; Motherwell, W. D. S.; Purkis, L. H.; Smith, B.
R.; Taylor, R.; Cooper, R. I.; Harris, S. E.; Orpen, A. G. Retrieval of Crystallographically-
Derived Molecular Geometry Information. J. Chem. Inf. Comp. Sci. 2004, 44, 2133-2144.
(23) Etter, M. C.; MacDonald, J. C.; Bernstein, J. Graph-set analysis of hydrogen-bond patterns
in organic crystals. Acta Crystallogr., Sect. B 1990, 46, 256-262.
(
(
(
(
24) Spek, A. L. Structure validation in chemical crystallography. Acta Crystallogr., Sect. D 2009,
65, 148-55.
25) Krishna Murthy, H. M.; Vijayan, M., 2{[3-(Trifluoromethyl)phenyl]amino}-3-
pyridinecarboxylic acid (niflumic acid). Acta Crystallogr. Sect. B 1979, 35, 262-263.
26) Spackman, M. A.; Jayatilaka, D. Hirshfeld surface analysis. CrystEngComm 2009, 11, 19-
32.
27) Turner, M. J.; McKinnon, J. J.; Wolff, S. K.; Grimwood, D. J.; Spackman, P. R.; Jayatilaka,
D.; Spackman, M. A. CrystalExplorer17; University of Western Australia: Crawley,
Australia, 2017. http://hirshfeldsurface.net.
(
28) Boultif, A.; Louer, D. Powder pattern indexing with the dichotomy method. J. Appl.
Crystallogr. 2004, 37, 724-731.
(
29) Vallcorba, O.; Rius, J.; Frontera, C.; Miravitlles, C. TALP: a multisolution direct-space
strategy for solving molecular crystals from powder diffraction data based on restrained least
ACS Paragon Plus Environment
2
6

Page 27 of 28
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
squares. J. Appl. Crystallogr. 2012, 45, 1270-1277.
(30) Toby, B. H.; Von Dreele, R. B. GSAS-II: the genesis of a modern open-source all purpose
crystallography software package. J. Appl. Crystallogr. 2013, 46, 544-549.
(
31) Rigaku CrystalClear-SM Expert, 2.1 b45; Rigaku: Rigaku Americas, The Woodlands, TX,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
USA, and Rigaku Corporation, Tokyo, Japan., 2015.
(32) RigakuOxfordDiffraction CrysAlisPro Software system, 171.39.28e; Rigaku Corporation:
Oxford, UK, 2015.
(
33) Bruker APEX2, Bruker AXS Inc.: Madison, Wisconsin, USA, 2012.
(34) Bruker SAINT, Bruker AXS Inc.: Madison, Wisconsin, USA, 2012.
(
(
(
(
35) Sheldrick, G. M. SHELXT - Integrated space-group and crystal-structure determination. Acta
Crystallogr., Sect. A 2015, 71, 3-8.
36) Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C 2015,
71, 3-8.
37) Hubschle, C. B.; Sheldrick, G. M.; Dittrich, B. ShelXle: a Qt graphical user interface for
SHELXL. J. Appl. Crystallogr. 2011, 44, 1281-1284.
38) Brandenburg, K. DIAMOND, 3.0; Crystal Impact GbR: Bonn, Germany, 1999.
(39) Allen, F. H.; Johnson, O.; Shields, G. P.; Smith, B. R.; Towler, M. CIF applications. XV.
enCIFer: a program for viewing, editing and visualizing CIFs. J. Appl. Crystallogr. 2004, 37
(2), 335-338.
(40) Mackenzie, C. F.; Spackman, P. R.; Jayatilaka, D.; Spackman, M. A. CrystalExplorer model
energies and energy frameworks: extension to metal coordination compounds, organic salts,
solvates and open-shell systems. IUCrJ 2017, 4, 575–587.
(
41) Edwards, A. J.; Mackenzie, C. F.; Spackman, P. R.; Jayatilaka, D.; Spackman, M. A.
Intermolecular interactions in molecular crystals: what’s in a name?. Faraday
Discuss. 2017, 203, 93–112.
ACS Paragon Plus Environment
2
7

Crystal Growth & Design
Page 28 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For Table of Contents Use Only
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Polymorphism in the anti-inflammatory drug Flunixin and its
relationship with Clonixin.
1
1
2
1
María Cecilia Dávila-Miliani , Analio Dugarte-Dugarte , Robert A. Toro , Jines E. Contreras ,
3
2
1
1
Hernando A. Camargo , José Antonio Henao , José Miguel Delgado , Graciela Díaz de Delgado
1Laboratorio de Cristalografía-LNDRX, Departamento de Química, Facultad de Ciencias,
Universidad de los Andes, Mérida 5101, Venezuela.
2Grupo de Investigación en Química Estructural (GIQUE), Escuela de Química, Universidad
Industrial de Santander, Bucaramanga, Colombia.
3Grupo de Investigación en Nuevos Materiales y Energías Alternativas, Facultad de Química
Ambiental, Universidad Santo Tomás, Florida Blanca, Colombia.
Synopsis. Two conformational polymorphs of the non-steroidal anti-inflammatory drug flunixin
were grown and characterized by ATR-FTIR, TGA/DSC, and powder and single crystal X ray
diffraction. Form I exhibits intermolecular hydrogen bonds based on the acid—pyridine
heterosynthon while Form II displays the acid—acid homosynthon.
ACS Paragon Plus Environment
2
8