Chloramphenicol·cyclodextrin inclusion compounds: Co-dissolution and mechanochemical preparations and antibacterial action

Article abstract of DOI:10.1039/c3ce26414a

β-Cyclodextrin (βCD) inclusion compounds of chloramphenicol in the solid-state were prepared via two different procedures, co-crystallisation from aqueous solution and the solvent-free method of co-grinding. Chloramphenicol inclusion into permethylated βCD (TRIMEB) was also performed by co-dissolution in ethanol. The co-crystallisation procedure was the best for preparing chloramphenicol·βCD in 1:1 stoichiometry in high purity, while with the co-grinding treatment and the same starting proportion, a complete inclusion was not achieved. Microcrystals of chloramphenicol· βCD inclusion compound presented polymorphism, crystallising simultaneously in the triclinic P1 or monoclinic C2 space groups. Only crystals of the latter were suitable for single-crystal diffraction and data for the guest atoms was comprised of a smeared-out electron cloud, so theoretical calculations were used to propose their plausible geometry and location inside the host molecules. The co-grinding procedure curbed polymorphism and allowed the preparation of chloramphenicol·βCD in the amorphous state; chloramphenicol· TRIMEB prepared by co-dissolution of the components in a 1:1 proportion was also an amorphous material. The influence of inclusion with βCD and TRIMEB on the antimicrobial performance of chloramphenicol was evaluated, and both inclusion compounds demonstrated selective action against Enterococcus faecalis strains ATCC 29212 and A33562 and Listeria monocytogenes ATCC 7644. Chloramphenicol·TRIMEB also had higher activity against the E. faecalis strains A35906, 9308 and E4856 and against Listeria inocua.

Full text of DOI:10.1039/c3ce26414a

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Chloramphenicol?cyclodextrin inclusion compounds:
co-dissolution and mechanochemical preparations and
antibacterial action3
Cite this: CrystEngComm, 2013, 15,
822
2
a
b
a
a
Ana I. Ramos, Teresa M. Braga, Patr ´ı cia Silva, Jos e´ A. Fernandes, Paulo Ribeiro-
a
bc
a
ad
Claro, Maria de F a´ tima Silva Lopes, Filipe A. Almeida Paz* and Susana S. Braga*
b-Cyclodextrin (bCD) inclusion compounds of chloramphenicol in the solid-state were prepared via two
different procedures, co-crystallisation from aqueous solution and the solvent-free method of co-grinding.
Chloramphenicol inclusion into permethylated bCD (TRIMEB) was also performed by co-dissolution in
ethanol. The co-crystallisation procedure was the best for preparing chloramphenicol?bCD in 1 : 1
stoichiometry in high purity, while with the co-grinding treatment and the same starting proportion, a
complete inclusion was not achieved. Microcrystals of chloramphenicol?bCD inclusion compound
presented polymorphism, crystallising simultaneously in the triclinic P1 or monoclinic C2 space groups.
Only crystals of the latter were suitable for single-crystal diffraction and data for the guest atoms was
comprised of a smeared-out electron cloud, so theoretical calculations were used to propose their
plausible geometry and location inside the host molecules. The co-grinding procedure curbed
polymorphism and allowed the preparation of chloramphenicol?bCD in the amorphous state;
chloramphenicol?TRIMEB prepared by co-dissolution of the components in a 1 : 1 proportion was also
an amorphous material. The influence of inclusion with bCD and TRIMEB on the antimicrobial
performance of chloramphenicol was evaluated, and both inclusion compounds demonstrated selective
action against Enterococcus faecalis strains ATCC 29212 and A33562 and Listeria monocytogenes ATCC
Received 31st August 2012,
Accepted 23rd January 2013
DOI: 10.1039/c3ce26414a
7
644. Chloramphenicol?TRIMEB also had higher activity against the E. faecalis strains A35906, 9308 and
www.rsc.org/crystengcomm
E4856 and against Listeria inocua.
The poor water solubility of chloramphenicol requires
formulation with the help of solubilising agents, one common
approach being molecular encapsulation with cyclodextrins
Introduction
Chloramphenicol (drawn in Scheme 1) is a naturally occurring
antibiotic resulting from the metabolism of chorismic acid in
Streptomyces venezuelae. It has a broad spectrum of activity
(CDs) to form inclusion compounds (ICs). The best procedure
1
to obtain a pure IC is co-crystallisation from aqueous solution.
For the IC chloramphenicol?bCD, studies in the aqueous
(
Gram-positive and Gram-negative cocci and bacilli, including
2
anaerobes) and remains the most prescribed active pharma-
ceutical ingredient (API) in Europe for acute infection
conjunctivitis. Its good ability to pass the blood brain barrier
phase are already available: stoichiometry was determined as
6
1
: 1, with a tight fit of chloramphenicol in the cavity, and the
3
affinity constant, assessed by different methods, was esti-
4
makes it also of value in uncomplicated meningitis, being the
recommended API by the World Health Organization to treat
epidemics of meningococcal meningitis in Africa.
3
21
7
mated to round 2 6 10
M
at 298 K. Chloramphenicol
encapsulation by methylated CDs is also reported, but only in
5
8
patents. One of these (developed in Portugal by Oftalder
a
Department of Chemistry, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal.
E-mail: sbraga@ua.pt; Fax: +351 234 401470; Tel: +351 234 401418
b
Antibiotic Stress and Virulence of Enterococci Laboratory, Instituto de Tecnologia
Qu ´ı mica e Biol o´ gica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras,
Portugal
c
IBET, Apartado 12, 2781-901 Oeiras, Portugal
d
QOPNA Research Unit (Qu ´ı mica Org aˆ nica, Produtos Naturais e Agro-alimentares),
Aveiro, Portugal
3
CCDC 762697. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3ce26414a
Scheme 1 Molecular diagram of chloramphenicol.
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822 | CrystEngComm, 2013, 15, 2822–2834
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Laboratories) comprises an eye drop formulation with the
trade name Clorocil1. It used 2,6-di-O-methyl-bCD (DIMEB)
as the molecular capsule, which is claimed to increase both
Hungary, and chloramphenicol from Sigma-Aldrich (Iberian
distribution, manufactured in China).
Microanalyses for CHN were performed at the University of
Cambridge (Department of Chemistry) on an Exeter Analytical
CE 440 Elemental Analyser. Samples were combusted under an
oxygen atmosphere at 975 uC for 1 minute, and helium used as
a purge gas.
9
solubility and stability.
In solid formulations, CDs not only enhance API solubility
but also its dissolution and rate of absorption, and may help
reduce polymorphism. The hydroxypropylated derivative of
1
0
Conventional powder X-ray diffraction data were collected at
ambient temperature on an X9Pert MPD Philips diffractometer
bCD was shown to have these effects on chloramphenicol,
thus enhancing the bioavailability. We showed recently that
heptakis-2,3,6-tris-O-methyl-bCD (TRIMEB) inclusion reduces
1 2
(Cu Ka1,2 X-radiation, l = 1.540598 Å and l = 1.544426 Å),
equipped with an X9Celerator detector and a flat-plate sample
holder in a Bragg–Brentano para-focussing optics configura-
tion (40 kV, 50 mA). Intensity data were collected by the step
counting method (step 0.04u), in continuous mode, in the ca. 4
the
RuCl([9]aneS
of cytotoxic organometallic guests with bCD and TRIMEB,
polymorphism
of
the
inorganic
complex
1
1
[
3
)(dmso)]Cl. We have also prepared solid ICs
1
2
1
2a
namely
a
ferrocene derivative
and two metallocenes
¡ 2h ¡ 50u range.
TGA studies were performed on a Shimadzu TGA-50
(
Cp MCl with Cp = cyclopentadienyl, M = Nb, Mo) and shown
2
2
how IC formation helps enhance antitumoral action; best
thermogravimetric analyser, using a heating rate of 5 uC
1
2d
21
21
results were obtained with Cp
2
MoCl
2
?TRIMEB.
min , under air atmosphere, with a flow rate of 20 mL min
.
In opposition to co-crystallisation techniques that afford
pure ICs in the crystalline state, procedures with little or even
no solvent are of higher interest to the pharmaceutical
The sample holder was a 5 mm Ø platinum plate and the
sample mass was typically 1 to 5 mg.
FT-IR spectra (range 4000–400 cm ) were collected as KBr
pellets using a Bruker Tensor 27 spectrometer by averaging
2
1
1
3
industry; cutting solvent recovery or disposal brings obvious
economic and environmental advantages. In this context,
2
1
2
00 scans at a maximum resolution of 2 cm
.
1
3
1
1
3a,14
C{ H} CP/MAS NMR spectra were recorded at 125.72 MHz
mechanochemistry
has become a powerful tool in crystal
on a (11.7 T) Bruker AVANCE 500 spectrometer, with an
engineering of API molecules. CDs are also capable of
including guest APIs by these solid–solid reactions, which
has rekindled their industrial interest. The inclusion can be
driven by different forces, such as shear (used in extrusion
1
optimized p/2 pulse for H of 4.5 ms, 2 ms contact time, a
spinning rate of 7 kHz and 12 s recycle delays. Chemical shifts
are quoted in ppm from TMS.
SEM images were acquired using a scanning electron
microscope Hitachi SU-70 Schottky emission instrument
working at 15 kV or 4 kV. Energy-dispersive X-ray spectroscopy
1
5
16
processes), heat and/or pressure, grinding or ball milling.
Some reports describe the use of manual of co-grinding in the
1
7
formation of ICs and rotaxanes, but for convenience, it is
commonly replaced by the electromechanical method of ball
milling (BM), which is already patented for bCD inclusion of
(EDS) data and maps were acquired using a Bruker QUANTAX
400 microanalysis system. Samples were prepared by deposi-
tion on aluminum sample holders followed by carbon coating
in an Emitech K950X carbon evaporator.
1
8
menthol and cyclohexane. BM was also used to promote
1
9–22
dispersion of APIs on CD carriers,
yielding blends with
Preparation of the compounds
better solubility profiles. IC formation by BM was achieved
with a few molecules of larger dimensions including para-
Chloramphenicol?bCD by co-dissolution (1). To a solution of
bCD (197 mg, 0.15 mmol) in water (2.0 mL) at 80 uC was added
solid chloramphenicol (40 mg, 0.15 mmol) step by step, always
allowing each fraction to dissolve before adding the next. The
resulting pristine solution was stirred for 40 minutes and
slowly cooled to ambient temperature in an isothermic
container with a water bath. After 48 h, clear prismatic crystals
of chloramphenicol?bCD had formed.
2
3
24
25
cetamol, loratadine, and fentanyl.
The present work presents the preparation of bCD and
TRIMEB solid inclusion compounds with chloramphenicol by
the classic co-dissolution method and of chloramphenicol?bCD
by mechanochemical grinding. The products are characterised
in depth by a number of solid-state techniques: FT-IR,
thermogravimetric analysis, X-ray diffraction, theoretical calcu-
lations and solid-state NMR studies. In addition, the influence
of CDs on the biological activity of chloramphenicol is
evaluated, by determining the minimum inhibitory concentra-
tions (MICs) of the inclusion compounds (prepared by the co-
dissolution method) against Listeria, Enterococcus, Bacillus,
Staphylococcus and Salmonella strains.
Elemental Composition (in%). Anal. Calc. for (C11
35)?6H O (MW 1584.2): C 40.6; N 1.79; H 6.05. Found: C
0.2; N 1.93; H 6.02. TGA revealed a dehydration step of 7%,
corresponding to 6.08 water molecules.
12 2 5 2
H N O Cl )?
42 70
(C H O
2
4
2
1
FT-IR (KBr pellet; in cm ): 3358 br, 2926 m, 1698 m, 1641
m, 1523 m, 1413 m, 1370 m, 1348 m, 1244 m, 1205 m, 1158 s,
1
7
102 m, 1079 s, 1056 vs, 1028 vs, 1002 s, 945 m, 863 m, 814 w,
54 m, 704 m, 648 w, 576 m, 530 w, 478 w, 360 m.
1
3
1
C{ H} CP/MAS NMR (see Scheme 2): 162.8 (sh), 161.0
(guest, CO), 148.4 (guest, C–NO ), 146.3 (guest, C ), 126.0
guest, C ), 123.2 (guest, C ), 103.6 (bCD, C1), 80.4 (bCD, C4),
2.7 (bCD, C2,3,5), 60.3 (bCD, C6) ppm.
Chloramphenicol?bCD by mechanochemistry. A mixture
Experimental section
2
d
(
7
f
e
Materials and methods
bCD was kindly donated by Laboratoires Roquette, Lestrem,
France. 2,3,6-Tris-O-methyl-bCD was purchased from Cyclolab,
containing solid bCD (1.00 g, 0.76 mmol) and chlorampheni-
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FT-IR (KBr pellet; in cm ): 3431 s, 2981 s, 2931 s, 2900 sh,
836 s, 2636 w, 2600 w, 2068 m, 1747 m, 1700 s, 1641 m, 1607
1
2
m, 1526 s, 1459 s, 1349 s, 1305 m, 1252 m, 1196 s, 1161 vs,
1
8
3
142 vs, 1105 vs, 1089 vs, 1068 vs, 1039 vs, 969 s, 911 m, 858 m,
12 m, 754 m, 706 m, 655 w, 551 s, 472 w, 350 m, 327 w, 308 w,
04 w.
1
3
1
C{ H} CP/MAS NMR (see Scheme 2): 163.7 (sh), 162.8
guest, CO), 149.5 (guest, C–NO ), 147.0 (guest, C ), 126.4
(
(
2
d
Scheme 2 Carbon labelling for the guest chloramphenicol, on the left, and the
guest, C ), 122.8 (guest, C ), 98.3 (TRIMEB, C1), 82.0 (TRIMEB,
f
e
3
hosts bCD (R = H) and TRIMEB (R = CH ), on the right.
C2,3,4), 70.8 (TRIMEB, C5,6), 58.3 (TRIMEB, O–CH ) ppm.
3
Single-crystal X-ray diffraction
col (203 mg, 0.76 mmol) was manually ground in a granite
mortar for 120 minutes. Fractions of this mixture were
collected at 2, 15, 30, 60, 90 and 120 minutes of grinding for
further analyses; aliquots were also collected at 5 and 10 min,
for which only powder XRD data is shown.
Crystal solution and refinement of the bCD network in 1. A
single-crystal of 1 (as obtained from the co-dissolution
method) was mounted on a Hampton Research CryoLoop
using FOMBLIN Y perfluoropolyether vacuum oil (LVAC 25/6)
27
purchased from Aldrich, with the help of a Stemi 2000
stereomicroscope equipped with Carl Zeiss lenses. Data were
collected at 150(2) K on a Bruker X8 Kappa APEX II charge-
coupled device (CCD) area-detector diffractometer (Mo Ka
graphite-monochromated radiation, l = 0.71073 Å) controlled
Estimation of the inclusion rate by peak fitting of FT-IR data.
The carbonyl stretching band in pure chloramphenicol is
2
1
observed at 1688 cm
roughly 1700 cm
and shifts upon bCD inclusion to
2
1
2
8
(as observed in chloramphenicol?bCD
by the APEX2 software package, and equipped with an
Oxford Cryosystems Series 700 cryostream monitored remotely
prepared by co-dissolution). Thus, inclusion rate was esti-
mated by averaging the relative contribution of each band in
the spectra of the mixture over the time of the grinding.
Identification and deconvolution of the peaks was per-
2
9
using the software interface Cryopad. Images were processed
3
0
using the software package SAINT+, and data were corrected
for absorption by the multi-scan semi-empirical method
TM
31
formed using the software package PeakFit v4.05 (baseline:
implemented in SADABS. The structure was solved by the
3
2
exponential D2, maximum smoothing: 2%, peak detection:
second derivative method, peak type: gaussian). The areas
direct methods of SHELXS-97, and refined by full-matrix
2
32b,33
least squares on F using SHELXL-97.
All non-hydrogen
2
1
under the peaks 1688 and 1700 cm
(Scheme 3) give the
atoms were directly located from difference Fourier maps and
refined with independent isotropic displacement parameters
because the smeared-out electron density surrounding the CD
chemical moieties (see below) prevented a sensible refinement
of the atoms using anisotropic displacement parameters.
Hydrogen atoms attached to carbon and to the terminal
hydroxyl groups were located at their idealised positions and
included in the structural model in subsequent refinement
percentages of free and included chloramphenicol, respec-
tively.
Chloramphenicol?TRIMEB by co-dissolution (2). To a solu-
tion of chloramphenicol (40.0 mg, 124 mmol) in ethanol (3
mL) was added solid TRIMEB (177 mg, 124 mmol) step by step,
always allowing each fraction to dissolve before adding the
next. The resulting mixture was kept in a thermostated bath at
cycles in riding-motion approximation with Uiso
= 1.2
2
5 uC and stirred for 120 min. The solution was then heated to
(aromatic carbon) or 1.5 (–CH3 and –OH groups) of Ueq of
achieve oversaturation (TRIMEB solubility lowers with heating)
2
6
the parent atom.
and attempt crystallisation, but only a viscous material was
obtained, so solvent removal (under vacuum) was performed
to isolate a white vitreous solid.
A considerable smeared-out electron density was found in
the empty spaces available in the structure, which prevented a
sensible location and refinement of all guest and the vast
majority of the solvent molecules. Searches for the total
Elemental composition (in %). Anal. Calcd for
(
1
C H N O Cl )?(C H O )?4H O (MW 1824.7): C, 48.7; N,
11 12 2 5 2 63 112 35 2
3
4
potential solvent area using the software package PLATON
.54; H, 7.29. Found: C, 48.6; N, 1.85; H, 6.79.
revealed the presence of large cavities in the unit cell, with a
3
total internal volume of ca. 2406 Å (ca. 35% of the total
volume of the unit cell). The original data set was then
3
5
mathematically treated using the SQUEEZE subroutines in
order to remove the contribution of these highly disordered
molecules in the solvent-accessible volume. It was estimated
that the ca. 35% of empty internal volume would contain ca.
975 electrons. Since a value of 0(4) for the Flack parameter is
meaningless, the absolute configuration was established by
comparison with the expected configuration of a bCD
molecule.
The calculated solvent-free reflection list was then used for
further structural refinement of the model composed solely by
the bCD molecule.
Scheme 3 Examples of the peaks and calculated areas for the CLO band of free
and included chloramphenicol. Dotted lines represent the collected data and
block lines the calculated fitting.
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Crystal data for the squeezed structure. C42
135.0), monoclinic, space group C2, Z = 4, a = 19.056(5) Å, b
H
70
O
35 (MW
All incubations were performed at 37 uC. For each compound
tested with a particular strain, five replicas were made.
Results, unless otherwise specified, correspond to, at least,
three concordant values, with others falling within ¡1 log2
1
=
3
24.415(6) Å, c = 15.698(4) Å, b = 109.463(13), V = 6886(3) Å ,
2
1
m(Mo Ka) = 0.097 mm , colourless blocks with crystal size of
3
40
0
.10 6 0.08 6 0.02 mm . Of a total of 16 078 reflections
collected, 9408 were independent (Rint = 0.0873). Final R1
.1366 [I . 2s(I)] and wR = 0.3942 (all data). Data
dilutions, which is acceptable for these tests. For the E. coli
=
ATCC 8739 strain and the E. faecium 29379 strain, obtained
values for 1 and 2 were non-concordant and, therefore, were
omitted.
0
2
completeness to theta = 25.35u, 96.1%. CCDC 762697.
Theoretical studies
Ab initio calculations were performed using the G03w
Results and discussion
3
6
package on the multiprocessor cluster ‘‘Flamingo’’ belong-
ing to the Associated Laboratory CICECO, University of Aveiro.
The B3LYP method at the 6-31G level was used. The initial
molecular geometry of chloramphenicol (guest) was calculated
from crystallographic data taken from its published crystal
Inclusion compounds prepared from co-solutions
Inclusion of chloramphenicol by co-dissolution followed our
1
1,41
previously published strategy
which is based on the
3
7
relative solubilities of the CDs. The reaction with TRIMEB,
structure and deposited with the Cambridge Structural
4
2
3
8
soluble in most organic solvents, was carried out in ethanol.
bCD requires water to dissolve, so its mildly-heated aqueous
solution was added with solid chloramphenicol and slowly
cooled to ambient temperature to isolate single crystals of
chloramphenicol?bCD (1) used in the X-ray studies.
Initial assessment of the inclusion of chloramphenicol into
bCD and TRIMEB was carried by elemental analysis and FT-IR
spectroscopy studies. The CLO stretching mode of chloram-
phenicol is sensitive to weak interactions provided these
change the relative contributions of the non-polarized and
Database (CSD, Version 5.33, February 2012). The molecular
geometry of bCD (host) was that of the crystal structure
determined from single-crystal X-ray diffraction (see the
previous sub-section for additional details). The structures of
the inclusion compounds were simulated by iterative geome-
trical inclusion of the guest inside the fixed host (as
determined from X-ray diffraction). The first step involved
the partial optimization of two distinct dimeric structures,
both with the guests included in a head-to-head cavity of a
i
bCD dimer and presenting C
2
symmetry: A , approaching
i
polarized resonance structures of the CLO bond (.CLO and
through the aliphatic part; and B , approaching through the
aromatic fragments. When the final calculated structures (A
and B) were transposed into the crystal packing, it was found
that it was not possible to adjust B to the real packing
geometry. A 2 : 2 bCD dimer with tail-to-tail cavity structure, C,
was ultimately derived using structure A as starting geome-
trical premise: the aliphatic section of the guest was fixed
while the aromatic one was allowed to optimise. This new
geometry was found to be chemically feasible in a hypothetical
crystal packing.
2
.
C–O , respectively). Such cases are common in the host–
guest interactions of ICs and are also observed for the
compounds prepared in this work, 1 and 2: the band for the
CLO mode occurs at 1688 cm
2
1
in the spectrum of pure
chloramphenicol and appears blue-shifted to 1698 and 1700
2
1
cm in the spectra of 1 and 2, respectively, a clear indication
of IC formation. In addition, elemental analysis data (details
in the Experimental section) show 1 : 1 host-to-guest stoichio-
metry in both 1 and 2.
Structural model of 1 from combined single-crystal data
refinement and theoretical calculations. Although a large
number of single-crystals could be directly isolated from the
reaction media of distinct batches, all of them systematically
exhibited very poor diffraction above ca. 1 Å. Tested crystals
were all classified as pertaining to the monoclinic C2 space
group and had unit cell parameters characteristic of ICs with
dimeric supramolecular arrangement of bCD molecules,
Determination of minimum inhibitory concentration (MIC)
The MIC studies of pure chloramphenicol and compounds 1
and 2 were performed on 34 bacterial strains: two Gram-
negative strains, Escherichia coli ATCC 8739 and Salmonella
poona NCTC 4840, and 32 Gram-positive strains,
Staphylococcus aureus ATCC 6538, Listeria monocytogenes
ATCC 7644, Listeria innocua 4202 (from the culture collection
of National Food Biotechnology Center, University College
Cork, Ireland), Bacillus cereus ATCC 11778, Enterococcus
faecalis ATCC 29212, Enterococcus faecalis V583 and a total of
4
3
referenced as isostructural series number 11 by Caira.
A
reasonable crystal structure solution, with a stable overall
refinement, could only be achieved for the crystallographically
independent CD host: the electron density arising from the
guest species and water molecules of crystallisation was
considerably smeared-out (seemingly due to strong thermal
and positional disorder) and therefore stripped out from the
original data set. The determined C2 space group packing of 1
26 clinical isolates of the genus Enterococcus (from the culture
collection of Stress Antibiotic and Virulence Enterococci
laboratory, IBET/ITQB Universidade Nova de Lisboa, Oeiras).
Strains were grown overnight in Brain Heart Infusion (BHI)
broth from Oxoid and plated in BHI agar. Colonies were
picked and incubated in Muller–Hinton (MH) broth, also from
Oxoid. The inoculum density was adjusted to 0.5 McFarland in
fresh MH broth and diluted to 1 : 100. On a 96 well
microplate, 100 mL of each bacterial culture diluted to
2
implies a C rotation axis at the centre of the dimer and
another between adjacent dimers. The bCD head rims are
about 2.6 Å apart, while for the tail rims the analogous
distance is ca. 3.2 Å. We must stress that, though the guest
molecules could not be located, the observed crystal packing
1
: 100 were added to 100 mL solution of each compound
2
1
39
43
(range from 1–2048 mg mL ). Results were read after 24 h.
features are clearly typical of ICs. Note also that according to
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solution NMR studies, 1 : 1 ICs of bCD with chloramphenicol
are chemically feasible for its two tautomeric forms, so
crystallisation of these would ultimately lead to structural
disorder. Efforts to locate or model guest disorder (with
heavily constrained models) did not afford stable or realistic
initial). In this structure, two parts with polar atoms are
interacting with each other. Another arrangement, B , was set
with an interaction of two aromatic rings.
i
Partial optimisation of the structure (level B3LYP/6-31G, see
Experimental section), with the bCD hosts fixed and guests
allowed to run freely, afforded the inclusion geometries A and
B, represented in Fig. 1. Structure B has lower energy than A
4
4
molecular geometries, and the overall fitting quality also did
not improve significantly.
2
1
The geometry of 1 was further studied using in silico
methods. These followed the methodology presented by some
(by 314 kJ mol ) but, when applied to fit the crystal lattice, it
gives rise to a strong superimposition of atoms thus being an
unrealistic model (Fig. 1b). Structure A, presenting the
aromatic rings of the guest inside the hydrophobic cavity of
bCD, generates a significantly less severe superimposition
when fitted into the crystal (Fig. 1a), and it can be further
minimised by modifying two of the chloramphenicol dihedral
angles. Also, it is in good agreement with the reported results
1
2a
of us in a previous work, in which the structural features of
a ferrocene derivative included in bCD were investigated by
using ab initio calculations to optimise the structural features
of the guest inside the known host geometry (from crystal-
lography refinement). In the present case, the initial structure
for optimisation comprised the bCD dimer (coordinates from
the crystallography data) which must have, according to the
6
from solution NMR, thus making it a very plausible model for
1
: 1 stoichiometry evidenced by elemental analysis, two
chloramphenicol molecules inside. Note that these, in spite
of their disorder, should also be related by the same C
1.
Further optimisation of structure A to minimise the atomic
collisions followed the reasoning represented in Scheme 4.
A new structure, C, was considered, which belongs to the
same hypothetical crystal model and merely changed the type
of dimeric unit chosen for optimisation. In opposition to
structure A, structure C comprises two bCD molecules which
do not belong to the same dimer.
2
rotation axis as bCD molecules. Their initial geometries were
taken from the published structure of chloramphenicol (space
3
7
group C222
1
), which also presents a dimeric arrangement
formed by a C rotation axis. This axis was very conveniently
2
2
superimposed into the C axis of the crystal packing of the
bCD dimers from the crystal structure of 1 to produce the
initial structure for optimisation coined as A (the i stands for
If C were constructed only by symmetry operations from A, a
superimposition of the guest molecules would ultimately
i
i
i
Fig. 1 Schematics of two hypothetical crystal packing arrangements (in monoclinic C2) of chloramphenicol?bCD (1). Structures A and B were optimised from A and B ,
with two different supramolecular arrangements of chloramphenicol dimers taken from its native crystal structure. The locations with strong superimposition (i.e.,
collision) of atoms from symmetry-related guest molecules are highlighted.
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planar group; the three angles of the NH group range from
114.6u to 123.0u, with a sum of 356.0u. Nevertheless, a number
of short distance contacts appear in this structure (the sum of
van der Waals radii are given inside parentheses for
comparative purposes): one of the chlorine atoms is at
3.02 Å (3.45 Å) from the Cnitro atom of the aromatic ring
belonging to the adjacent molecule; another chlorine atom
stays at 2.31 Å (2.95 Å) from a CH O proton of the
2
neighbouring guest; the inter-nitrogen distance between two
neighbouring nitro groups is 2.61 Å (3.10 Å). Additionally,
some short contacts also occur between guests and their hosts,
…
with the shortest of each type being Cl H at 2.50 Å (three
…
distances smaller than 2.95 Å) and O O at 2.13 Å (three
Scheme 4 Schematic representation of the transformation of structure A into C.
bCD molecules are represented in black and chloramphenicol in blue. Arrows
indicate the conformational rotations of the guest molecule.
distances smaller than 3.04 Å). The shortest distance between
hydrogen atoms is ca. 1.68 Å: while one pair has an
internuclear distance smaller than 2 Å, seven are between
2.0 Å and 2.4 Å, which is the sum of the van der Waals radii.
We note that a distance between hydrogen atoms smaller
happen (gray trace in Scheme 4). To avoid this, two dihedral
angles of chloramphenicol were modified, namely the dihedral
Ph–C–C–N angle and the rotation of the aromatic ring plane.
This empirical manipulation required a further optimisation
procedure while keeping the HNCOCHCl guest moiety fixed
2
2
to maintain its geometry around the central C axis. This
than the sum of van der Waals radii is not usual in geometry
optimisations, and even a distance shorter than 2 Å is not
4
5
completely artificial. The same applies to all other small
distances, which may be envisaged as particularly strong
interactions. On the other hand, it should be recognised that
structure C is not a product of full optimisation, many of the
short distances appear from the imposed rigidity of the bCD
molecule. A full optimisation could enlarge these small
distances but it arises as very time-consuming and compu-
ter-expensive and falls beyond the scope of the present study.
Crystal morphology and powder X-ray diffraction. Crystals of
compound 1 were studied by electron microscopy using SEM
and EDS mapping to evaluate: i) crystal morphology, ii) crystal
constraint allowed convenient crystal packing.
Fig. 2 depicts the partially optimised structure C and the
corresponding crystal packing (and slices of the structure). The
guest has a strongly twisted geometry (described by Ali and
6
collaborators as a ‘‘tight fit’’), yet all internal angles seem to
3
be realistic. The H–O–C angles are 106.4u and 115.03u; Csp
angles range from 103.7u to 115.3u; the three angles involving
the carbonyl moiety range from 109.3u to 125.6u comprising a
Fig. 2 Schematic representation of the structure C for the IC of chloramphenicol?bCD (1), determined from a combination of X-ray diffraction data (all bCD molecules)
and theoretical modelling (all guest molecules): (a) and (b) side and top views of the 1 : 1 chloramphenicol?bCD units; (c) columnar arrangement of 1 : 1 ICs
emphasising the absence of superimposing atoms belonging to adjacent guest molecules (represented in different colours); (d) idealised crystal packing of 1 viewed
in perspective along the [001] direction of the unit cell.
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Fig. 3 EDS mapping of chloramphenicol?bCD (1).
integrity, and iii) material homogeneity. Parallelepipeds with a
typical shape for CD adducts were observed throughout the
bulk material.
EDS mapping studies (Fig. 3) reveal a fairly homogenous
distribution of the elements composing the chloramphenicol?bCD
inclusion compound, in particular N and Cl. We note the presence
of green spots (corresponding to Cl) located in the background
mentation. The collection of patterns for the first batch and
the five replicas is shown in Fig. 4.
The traces were grouped into two families according to their
observed reflection envelopes. Again in reference to Caira’s
series, we can conclude that: i) in three of our batches the
monoclinic C2 structure is predominant; ii) in the remaining
three, there is no dominant phase. Electron microscopy and
powder X-ray diffraction studies were also conducted on
samples of chloramphenicol?TRIMEB (2) and have revealed a
glassy amorphous material with a homogenous distribution of
elements (namely N and Cl, data not shown). The results have
nonetheless allowed us to rule out the presence of a mere
physical mixture of TRIMEB and chloramphenicol crystallites
in compound 2.
(i.e., sample holder) which are attributed to artifacts arising from
the fact that the sample in study is not completely flat. In addition,
we have also observed that no guest excess can be derived from
elemental analysis data (see the Experimental section).
The experimental powder X-ray diffraction pattern of 1 was
compared with a simulation based on the crystallographic
information gathered from the single-crystal studies
1
3
1
(described in the previous subsection). There was not a perfect
Solid-state NMR. The C{ H} CP/MAS NMR spectra of the
ICs 1 and 2 are shown in Fig. 5 along with those of their
components: chloramphenicol and bCD or TRIMEB.
match due to the appearance of some extra reflections, thus
indicating the presence of, at least, another crystalline phase
in the bulk material. Indeed, according to Caira’s system-
In agreement with the literature, the spectra of bCD
4
3
46,47
48
atisation of crystalline CD inclusion compunds, the same
channel packing geometry may also appear in crystals with P1
symmetry (Caira’s series 10). The two overall symmetries of
this same channel packing, defined by the same author as
polymorphism, produce almost identical powder X-ray diffrac-
tion patterns (i.e., series 10 and 11). Nevertheless, the overall
envelopes are slightly different and can be used for qualitative
differentiation.
hydrate
and TRIMEB (Fig. 5b and d, respectively), are
crowded with multiple sharp resonances for each type of
carbon atom. In the ICs 1 and 2 this multiplicity is lost: the
spectrum of chloramphenicol?bCD presents single peaks for
the host carbon atoms, with fwhms not larger than those
observed for pure bCD hydrate resonances (Fig. 5c) and the
spectrum of chloramphenicol?TRIMEB exhibits broad peaks
for the TRIMEB carbons. It must be noted that these slight
differences in the spectral features of the ICs also imply that
the geometrical features in 1 and 2 should differ. Indeed, in
the spectrum of 1, the singularisation and narrowing of host
carbon resonances (with maxima at d = 103.6, 80.4, 72.7 and
60.3 ppm for C1, C4, C2,3,5 and C6 as labeled in Scheme 2,
respectively) are associated with symmetrisation of the bCD
To attempt a confirmation of the existence of this structural
polymorphism (and thus explain the extra reflections), a set of
five replicas were synthesised. The preparations followed the
procedure of the first batch (see the Experimental section) and
were carried out simultaneously to minimise errors associated
with human intervention, ambient temperature and instru-
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Fig. 4 A comparison between the experimental powder X-ray diffraction patterns for chloramphenicol?bCD from the first batch (represented as 1) and a set of five replicas (i–v).
1
2d,49
macrocycle in order to better accommodate the guest,
while the spectral features observed for 2 are attributed to the
loss of overall crystallinity, to conformational modification of
4
2
the host upon inclusion, or to a combination of both.
Resonances arising from the chloramphenicol carbons are
also observed in the spectra of 1 and 2. We note that the CLO
resonances seem to be duplicated. This is particularly evident
in the spectrum of 1, with two resonances peaking at 161.0 and
162.0 ppm (Fig. 5c), thus evidencing that the guest is
disordered over at least two positions inside the host
5
0
cavities. In the case of chloramphenicol?bCD, this feature
is in good agreement with the data collected from X-ray
diffraction.
Thermogravimetric analysis (TGA). Fig. 6 shows the thermo-
gravimetric (TG) traces for chloramphenicol, the ICs 1 and 2
and, for comparison, the physical mixtures (mixes) of their
components ground for a few seconds. Chloramphenicol starts
to decompose around 200 uC and leaves a residual mass of
1.7% at 600 uC. The same step, expected to appear in the TG
traces of the mixes, is absent in the TG trace of the bCD mix,
which follows closely that of the IC 1. These two samples differ
only slightly in the initial dehydration steps (7% for 1 and 11–
12% for the bCD mix), then present no further mass changes
until the decomposition step which is observed for both at 240
uC, a temperature lower than that of pure bCD hydrate (ca. 270
uC, trace not shown).
These almost superimposing TG traces, uncommon among
ICs, may be evidence that the bCD chloramphenicol host–
guest system is suitable for fast mechanochemical formation.
This alternative preparation method is further studied in the
following sub-section.
13
1
Fig. 5 C{ H} CP/MAS spectra for (a) pure chloramphenicol, (b) bCD hydrate, (c)
chloramphenicol?bCD (1), (d) TRIMEB and (e) chloramphenicol?TRIMEB (2).
Spinning sidebands are denoted by asterisks. The carbon labeling scheme for
chloramphenicol is detailed in Scheme 1, with carbon resonances attributed by
51
6
reference to the reported DMSO-d solution spectrum.
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The TG trace for chloramphenicol?TRIMEB (2) differs from
CrystEngComm
those commonly observed for ICs of TRIMEB with organic
guests, for which it is common to observe decomposition
5
2
starting at lower temperatures than the host.
Indeed,
compound 2 has a higher thermal stability (onset of decom-
position around 230 uC) than TRIMEB (onset of decomposition
at 180 uC), a feature which also occurs, though with less extent,
in the 1 : 1 mix with chloramphenicol (decomposing around
205 uC). Such an unusual behaviour, albeit unprecedented for
organic guests, has been observed in some of our previous
reports for ICs of TRIMEB with organometallic guests and
1
1,12d,26
metal coordination complexes.
Chloramphenicol?bCD by co-grinding
As evidenced by the thermogravimetry data, a brief co-grinding
of chloramphenicol and bCD to prepare the physical mixture
afforded a blend with thermal behavior very close to that of the
IC obtained from the co-dissolution. Therefore, in the present
work it was considered worthy of interest to extend our
investigations and study the utility of co-grinding as a suitable
preparation method for chloramphenicol?bCD.
Fig. 7 Time-dependent chloramphenicol inclusion into bCD in a 1 : 1 powder
mixture subject to manual grinding in mortar, monitored by following the guest
CLO stretching bands (included and non-included) in FT-IR spectra.
details in the Experimental section). The evolution of this rate
over the grinding time is presented in Fig. 7; note how the
inclusion rate mounts strongly in the first minutes and then
proceeds asymptotically to a maximum of about 32%.
The powder diffractograms show that the crystallinity of the
starting materials is strongly reduced from early time-points of
grinding; diffractograms collected for aliquots taken at 5 and
A 1 : 1 (molar ratio) mixture of these starting materials was
manually ground in a mortar for 120 minutes. Fractions of the
ground mixture were taken at prescribed times and thoroughly
1
3
1
analysed using FT-IR, powder X-ray diffraction, C{ H} CP/
MAS NMR and EDS mapping for the distribution of elements.
In a recent literature report, deconvolution and curve-fitting
of FT-IR spectra were used to evaluate and monitor the rate of
inclusion of loratadine into HPbCD over time (co-ground in a
10 minutes of grinding were included in Fig. 8 for a better
visualisation of crystallinity loss.
After only 30 minutes of grinding, reflections are practically
absent and from this time-point onward there are negligible
changes in the diffractograms of the mixture (traces of
fractions collected at 60 and 90 minutes were omitted).
The grinding action on homogenisation and amorphisation
of the two phases is further evidenced by EDS mapping of the
most significant elements, depicted in Fig. 9. At 2 minutes, it
is still possible to observe crystallites of the guest, while after
5
3
ball mill). In our samples we monitored the CLO stretching
band of chloramphenicol as a probe for inclusion; it is known
from the studies on compound 1 (from co-dissolution, see
previous sections) that inclusion blueshifts the band (from
2
1
1
688 to around 1700 cm ). The inclusion rate was determined
using the areas under the peaks for included and free
chloramphenicol (calculated with the PeakFit software, see
Fig. 6 Thermogravimetric traces of pure chloramphenicol, the ICs
chloramphenicol?bCD (1) and chloramphenicol?TRIMEB (2), and of the briefly
co-ground 1 : 1 ground physical mixtures of bCD/TRIMEB with chlorampheni-
col.
Fig. 8 Powder X-ray diffraction patterns for aliquots taken at different times of
the ground mixture of bCD and chloramphenicol (1 : 1 ratio).
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Fig. 9 SEM imagery with EDS mapping for aliquots of the co-ground mixture of
bCD and chloramphenicol (1 : 1 ratio) collected at 2 (left) and 30 minutes
(
right).
30 minutes chloramphenicol seems to be completely distrib-
uted throughout the bulk material.
1
3
1
Further studies using solid-state C{ H} CP/MAS NMR show
the progressive transformation of the collected spectra from
that of a physical mixture into a spectrum (Fig. 10, 120 min)
sharing similarities with that of compound 1 (from co-
crystallisation), in particular, the loss of multiplicity for the
host carbon resonances and lower intensities for the chlor-
amphenicol signals.
In summary, the use in tandem of NMR, FT-IR, powder XRD
and EDS mapping shows that co-grinding does promote
inclusion of chloramphenicol into bCD. However, the esti-
5
3
mated inclusion rate (by fitting selected FT-IR peaks) was
only ca. 32% after 120 min of grinding.
13
1
Fig. 10 C{ H} CP/MAS spectra of a solid 1 : 1 physical mixture of chloram-
phenicol and bCD subject to co-grinding over 120 minutes; sample aliquots are
labelled according to grinding time in minutes. For comparison the spectrum of
pure bCD (below), as well as that of chloramphenicol (on top), are shown.
Antimicrobial activity studies
MIC determination was employed to assess the influence of
CD encapsulation on the antimicrobial activity of chloram-
phenicol. The compounds 1 and 2 (prepared by co-dissolution)
were tested against a selection of Gram-negative and Gram-
positive strains, including some vancomycin- and multidrug-
resistant Enterococcus. Pure chloramphenicol was used as a
standard, and a comparison with the results for 1 and 2 is
shown in Table 1.
host is a known membrane permeability enhancer). Given that
changes in permeability of the membrane are a common
mechanism of resistance of bacteria to chloramphenicol, the
present results show that TRIMEB may be helpful in curbing
bacterial resistance to this agent.
5
4
In the majority of the tested Enterococcus, encapsulation into
bCD and TRIMEB was unsuccessful in improving activity as it
afforded MIC values similar or even higher than those of pure
chloramphenicol. A few noteworthy exceptions are the E.
faecalis strains ATCC 29212 and A33562 and the bacterium L.
monocytogenes ATCC 7644, for which inclusion yielded a useful
enhancement of activity, with MIC reductions of two to eight
times. A similar response of E. faecalis ATCC 29212 to bCD and
TRIMEB inclusion of the antimicrobial metal complex
Concluding remarks
Literature reports on the inclusion of chloramphenicol into
CDs have thoroughly examined the effects of these hosts on
solubility improvement, but no data on the modifications
exerted on the biological activity of this API were available to
date. We have thus used the ICs prepared as pure solids with
known 1 : 1 stoichiometry (i.e. those from co-dissolution) to
evaluate alterations to chloramphenicol MICs upon IC forma-
tion with bCD and TRIMEB. Our results show, for both ICs 1
and 2, reduced MIC values against the A33562 and ATCC
29212 strains of E. faecalis and a remarkable ten-fold MIC
reduction on L. monocytogenes ATCC 7644. Of note was also the
1
1
[Ru([9]aneS
3
)phenCl]Cl had been previously observed by us.
TRIMEB was also able to reduce the MICs of chloramphe-
nicol against E. faecalis strains A35906, 9308 and E4856, and
against L. inocua, whereas bCD inclusion was not equally
effective. These microorganisms may, thus, present a higher
susceptibility to the TRIMEB action on cell membrane (this
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Table 1 MIC values for compounds 1, 2 and chloramphenicol against selected
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MIC values of 1 and 2 are expressed in mass equivalents (mg mL
of chloramphenicol, calculated based on the purity values derived
from their molecular formulae (see Experimental section), which
)
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The FCT and the European Social Fund are acknowledged for
grants SFRH/BPD/63736/2009 (to JAF), 78020/2011 (to SSB) and
SFRH/BD/46601/2008 (to PS) and BD/41882/2007 (to TMB). We
thank CICECO for granting access to the Flamingo computer
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