Physical and crystallographic characterisation of the mGlu5 antagonist MTEP and its monohydrochloride

Article abstract of DOI:10.1002/jps.21823

An improved medium scale synthesis of 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl]pyridine (MTEP), a selective and potent metabotropic glutamate subtype 5 (mGlu5) antagonist, has allowed thorough characterisation of the crystal structures of the free base and the previously unreported hydrochloride (MTEP.HCl). Hirshfeld surface analysis has revealed that molecules in crystalline MTEP are weakly polar, and aggregate through nonclassical C-H...N hydrogen bonds. A strong ionic N-H⁺...Cl ^- hydrogen bond dominates the crystal packing in MTEP.HCl. Despite significant differences in the crystal packing, the molecular structures of MTEP and MTEP.HCl are very similar. The acid dissociation constants for MTEP were investigated using ¹H NMR spectroscopy. The second acid dissociation constant (pK_a2), associated with the pyridine nitrogen, was determined to be 3.40±0.01, whilst pK_a1, associated with the thiazole nitrogen, was estimated to be 0.2. The low pK_a values make it unlikely that MTEP is protonated in its biologically active form.

Full text of DOI:10.1002/jps.21823

Physical and Crystallographic Characterisation of the
mGlu5 Antagonist MTEP and Its Monohydrochloride
1
1
1
2
MATTHEW J. McILDOWIE, MICHAEL N. GANDY, BRIAN W. SKELTON, JONATHAN M. BROTCHIE,
1
1
1
GEORGE A. KOUTSANTONIS, MARK A. SPACKMAN, MATTHEW J. PIGGOTT
1
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, WA 6009,
Australia
2
Toronto Western Research Institute, University Health Network, Toronto Western Hospital, 399 Bathurst Street,
MC 11-419, Toronto, Ontario, Canada M5T 2S8
Received 25 February 2009; revised 19 April 2009; accepted 22 April 2009
Published online 4 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21823
ABSTRACT: An improved medium scale synthesis of 3-[(2-methyl-1,3-thiazol-4-yl)ethy-
nyl]pyridine (MTEP), a selective and potent metabotropic glutamate subtype 5 (mGlu5)
antagonist, has allowed thorough characterisation of the crystal structures of the free
base and the previously unreported hydrochloride (MTEP.HCl). Hirshfeld surface
analysis has revealed that molecules in crystalline MTEP are weakly polar, and
þ
À
aggregate through nonclassical C–Hꢀ ꢀ ꢀN hydrogen bonds. A strong ionic N–H ꢀ ꢀ ꢀCl
hydrogen bond dominates the crystal packing in MTEP.HCl. Despite significant differ-
ences in the crystal packing, the molecular structures of MTEP and MTEP.HCl are very
1
similar. The acid dissociation constants for MTEP were investigated using H NMR
spectroscopy. The second acid dissociation constant (pKa2), associated with the pyridine
nitrogen, was determined to be 3.40 Æ 0.01, whilst pKa1, associated with the thiazole
a
nitrogen, was estimated to be 0.2. The low pK values make it unlikely that MTEP is
protonated in its biologically active form. ß 2009 Wiley-Liss, Inc. and the American Pharma-
cists Association J Pharm Sci 99:234–245, 2010
Keywords: 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine; MTEP; MTEP hydro-
chloride; crystal structure; Hirshfeld surface analysis; synthesis; acid dissociation
constant; solubility; physicochemical properties; NMR spectroscopy
2
–10
6,7,11–16
2,17,18
27,28
INTRODUCTION
anxiety,
depression,
pain,
drug
1
9–26
addiction,
appetite suppression,
acid
2
9
30,31
32–34
3
-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]pyridine
MTEP) (3) (Scheme 1) is a selective and potent
antagonist of metabotropic glutamate subtype 5
mGlu5) receptors that has received considerable
reflux,
and other neurodegenerative diseases.
In connection with our Parkinson’s disease drug
stroke,
Parkinson’s disease,
3
5,36
(
3
7
(
discovery program, we
required significant
1
attention in recent times. Several psychiatric,
neurological and neurodegenerative diseases are
associated with excessive mGlu5 receptor activa-
tion, and thus MTEP is a promising drug lead for
quantities of the previously unreported
MTEP.HCl, which was favoured over the free
base because of its improved water solubility.
Herein we report an improved medium-scale
synthesis of MTEP and the corresponding hydro-
chloride, along with X-ray crystal structures of
both compounds, which may be of use in modelling
studies. In addition we present a Hirshfeld
surface analysis of the crystal structures using
CrystalExplorer. The physiologically relevant
Correspondence to: Matthew J. Piggott (Telephone: þ61-8-
6
488-3170; Fax: þ61-8-6488-1005;
E-mail: piggott@cyllene.uwa.edu.au)
Journal of Pharmaceutical Sciences, Vol. 99, 234–245 (2010)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
234
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CRYSTALLOGRAPHIC CHARACTERISATION OF MTEP AND MTEP.HCL
235
Elution with 1:5 ethyl acetate–hexanes afforded
1
MTEP (3) as a tan solid (4.78 g, 78%). H NMR
(
500 MHz, CDCl ) d: 8.79 (m, 1H, H2), 8.57 (dd,
3
J ¼ 4.9, 1.6 Hz, 1H, H6), 7.85–7.82 (m, 1H, H4),
0
0
7
.44 (s, 1H, H5 ), 7.31–7.27 (m, 1H, H5), 2.76 (s,
3
H, CH ). The spectrum agrees closely with that
3
9
reported. A sample crystallised from ether at
9
Scheme 1. Synthesis of MTEP (3).
À188C as colourless plates, mp 77–798C [lit. 78–
7
98C].
acid dissociation constant has also been deter-
mined by H NMR spectroscopic titration.
MTEP.HCl
1
MTEP (3) was dissolved in one equivalent of
methanolic HCl and the resulting solution was
evaporated to give a tan solid, which crystallised
MATERIALS AND METHODS
General Experimental Details
1
from isopropanol as tan plates, mp 154–1568C. H
NMR (600 MHz, D O) d: 8.95 (s, 1H, H2), 8.74 (d,
2
J ¼ 6.3 Hz, 1H, H6), 8.62 (m [apparent dt], 1H,
H4), 8.01 (dd, J ¼ 8.4, 6.0 Hz, 1H, H5), 7.84 (s, 1H,
2
-Methyl-4-[(trimethylsilyl)ethynyl]-1,3-thiazole
9
00
H5 ), 2.73 (s, 3H, CH ). (500 MHz, DMSO-d ) d:
3 6
(
1) was prepared as described previously. Tetra-
hydrofuran and methanol were distilled. Metha-
nolic hydrogen chloride was prepared by bubbling
HCl gas through methanol; concentration was
determined by titration with standardised sodium
hydroxide. All other reagents were purchased
from commercial suppliers and used without
purification. NMR spectra were acquired using
8.89 (m, 1H, H2), 8.70 (dd, J ¼ 5.1, 1.6 Hz, 1H, H6),
0
0
8.21 (m [apparent dt], 1H, H4), 8.02 (s, 1H, H5 ),
7.66 (ddd, J ¼ 8.0, 5.1, 0.8 Hz, 1H, H5) 6.8, br s,
1
3
2.4H, NH þ H O), 2.69 (s, 3H, CH ). C NMR
2
3
(500 MHz, DMSO-d ) d: 166.1, 149.5, 147.1, 141.2,
134.5, 125.9, 124.7, 119.7, 87.9 (C–), 84.0 (C–),
6
–
–
–
–
18.7 (CH ). Elemental analysis: C, 55.9; H, 3.8; N,
3
1
13
Bruker Avance 500 (500 MHz, H; 125 MHz, C)
11.7. C H ClN S requires C, 55.8; H, 3.8; N,
1
1
9
2
1
or 600 (600 MHz, H) spectrometers. Chemical
11.8%. Slow crystallisation of a sample from
isopropanol gave clusters of tan plates, a fragment
of which was suitable for X-ray diffraction.
shift values are expressed in ppm, relative to
1
CHCl3 ( H, 7.26 ppm), sodium 2,2-dimethyl-2-
1
silapentane-5-sulfonate (DSS, H, 0.00 ppm),
1
CD SOCD H ( H, 3.50 ppm) and SO(CD3)2
(
3
2
1
3
C, 39.5 ppm), for spectra acquired in
Crystallography
CDCl , D O or DMSO-d , respectively. Elemental
analysis was performed by Robertson Microlit
Laboratories Inc. (Madison, NJ).
3
2
6
Oxford Diffraction Xcalibur (MTEP) or Gemini
(MTEP.HCl) CCD (charge-coupled device) dif-
fractometers fitted with Oxford Instrument Cryo-
jet cold nitrogen gas flow systems were used
by way of v-scans and monochromatic Mo
˚
˚
Synthesis
Ka ¼ 0.71073 A (MTEP) or Cu Ka ¼ 1.54184 A
(
for the small crystals of MTEP.HCl) radiation,
MTEP (3)
yielding N reflections, these merging to N unique
t
3
-Bromopyridine (2) (5.82 g, 36.8 mmol) and 2-
(Rint cited) after multiscan absorption correction
9
methyl-4-[(trimethylsilyl)ethynyl]-1,3-thiazole (1)
6.00 g, 30.7 mmol) were added to a stirred
(using the proprietary software), with N having
o
(
I > 2s(I). Minimum and maximum transmission
coefficients ‘T’min/max are quoted. All reflections
were used in the full matrix least squares
mixture of copper iodide (0.35 g, 6 mol%) and
palladium tetrakis(triphenylphosphine) (1.07 g,
2
3
(
mol%) in deoxygenated diisopropylamine
400 mL). The mixture was heated under reflux
and tetrabutylammonium fluoride solution
39.9 mL, 1 M in tetrahydrofuran) was added by
refinement on F , refining anisotropic displace-
ment parameter forms for the nonhydrogen
atoms, hydrogen atom treatment following a
riding model with isotropic displacement para-
meters based on those of the parent atom. Neutral
atom complex scattering factors were employed
(
syringe pump over 30 h. After heating for a
further 18 h, the solvent was evaporated and
the residue was subjected to rapid silica filtration.
38
within the SHELXL 97 program.
Pertinent
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236
McILDOWIE ET AL.
results are given in the text, table and figures, the
latter showing nonhydrogen atoms with 50%
probability amplitude displacement envelopes,
using nonlinear least squares regression, from
which pK values were determined.
a
˚
hydrogen atoms having arbitrary radii of 0.1 A.
CCDC 720986 and 720987 contain the supple-
mentary crystallographic data for this paper.
These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk.
RESULTS AND DISCUSSION
Synthesis
MTEP (3) was synthesised as described pre-
9
viously, with minor modifications to the key
Sonogashira coupling between the terminal
alkyne generated in situ from the silane 1 and
3-bromopyridine (2) (Scheme 1). Specifically, the
use of diisopropylamine in place of triethylamine/
Hirshfeld Surface Analysis
Intermolecular interactions and crystal packing
1
,2-dimethoxyethane, and slower generation of
39
were investigated using Mercury 2.0
and
the terminal alkyne by extended syringe-pump
addition of tetrabutylammonium fluoride, led to a
4
0
CrystalExplorer 2.1, with electrostatic poten-
tials obtained from ab initio wavefunctions
computed at the HF/Midi! level of theory using
1
3% improvement in yield. An ether solution of
MTEP at À188C produced a crystal suitable for
4
1
Gaussian03.
X-ray diffraction.
The (pyridinium) monohydrochloride was pre-
pared by dissolution of the free base in one
equivalent of methanolic hydrogen chloride,
followed by evaporation. Slow cooling of an
isopropanol solution of the hydrochloride gave a
crystal suitable for X-ray diffraction.
Measurement of pK values
a
A solution of MTEP.HCl (1.2 mg, 5.1 mmol) in
9
:1 H O/D O (1.0 mL), containing acetone
2 2
(0.32 mL, 4.3 mmol) as an internal standard, was
basified to pH 11.4 using 1 M aqueous sodium
hydroxide (10 mL). An initial aliquot of 0.1 M
aqueous hydrochloric acid (5 mL) was added
directly into the NMR tube, the resultant pH
Crystal Structures
The results of the single-crystal X-ray structure
determinations of MTEP and MTEP.HCl are
consistent in terms of stoichiometry and connec-
tivity with the proposed formulations. Crystal and
refinement data for both structures are collated in
Table 1.
1
was measured, and the H NMR spectrum was
acquired. This process was repeated using hydro-
chloric acid of progressively increasing concentra-
tion up to 10 M, until the solution reached a pH
of 0.
All pH values were determined using a
combination pH electrode (Wilmad LabGlass,
Buena, NJ) by direct insertion into the NMR
tube. The probe was calibrated using buffers 0.00,
The crystal structure of MTEP (Fig. 1) revealed
two molecules per asymmetric unit in space group
P2 /c. There are apparently very few structural
1
differences between the two molecules, with the
atoms of each being almost coplanar, but with the
two ring systems being rotated slightly; the angles
between the pyridine and thiazole rings being
7.36(7)8 and 4.79(7)8 for molecules 1 and 2,
respectively. The two molecules themselves also
approximate coplanarity, the angle between them
being 2.76(4)8.
1.00, 4.01 for the pH range 0–4, buffers 4.01 and
7.01 for the pH range 4–7, and buffers 7.01 and
10.00 for pH readings above 7. Values of pH were
not corrected for isotope effects.
1
H NMR spectra were acquired using the
WATERGATE (water suppression through gra-
dient tailored excitation) pulse sequence on a
Bruker Avance 600 MHz spectrometer. All spectra
were calibrated to the internal acetone signal
A search of the Cambridge Crystallographic
Database revealed no examples of a simple
internal alkyne with five- and six-membered ring
substituents. A pertinent example to both MTEP
and MTEP.HCl is the structure of (phenylethy-
(
d 2.12). A solution temperature of 238C was
maintained during all pH determinations and
1
H NMR experiments.
42
SigmaPlot 10.0 (Systat Software, Inc., San Jose,
CA) was used to fit an empirical sigmoidal
function to plots of pH versus chemical shift,
nyl)ferrocene, although the five and six-mem-
bered rings are not heterocyclic in this case. In
–
˚
this molecule the C–C bond length (1.192(3) A) is
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CRYSTALLOGRAPHIC CHARACTERISATION OF MTEP AND MTEP.HCL
237
00
the nitrogen of the thiazole (N3 ) and the thiazole
proton (H5 ), and are characteristic of weak
Table 1. Crystal Data and Refinement Details
MTEP MTEP.HCl
2
ClN S
0
0
˚
00
00
hydrogen bonds (2.48 A for H5 ꢀ ꢀ ꢀN3 a (x, y,
˚
00
00
z À 1), and 2.62 A for H5 aꢀ ꢀ ꢀN3 , where the
Formula
C
11
H
8
N
2
S
C
11
H
9
0
0
00
M (Da)
200.25
Monoclinic
P2 /c
10.5131(4)
17.9060(5)
11.2329(3)
111.660(4)
1965.3(1)
1.354
236.71
Monoclinic
P2 /c
7.4031(1)
9.2421(2)
15.6131(4)
92.188(2)
1067.47(4)
1.473
notation H5 a refers to atoms H5 in the second
r
Crystal system
Space group
molecule, etc., and for a standard C–H distance
˚
1
1
of 0.95 A).
˚
a (A)
In the crystal structure of the hydrochloride
˚
b (A)
(
Fig. 3) a strong hydrogen bond between the
˚
c (A)
À
pyridinium NH and the Cl is apparent. Relevant
˚
b (A)
˚
geometries are: N1–H1 0.88 A, H1ꢀ ꢀ ꢀCl1
˚
3
V (A )
˚
˚
2.09 A, N1ꢀ ꢀ ꢀCl1 2.956(3) A, N1–H1ꢀ ꢀ ꢀCl1 1688.
À3
D
Z
c
(g cm )
–
˚
The C–C bond length is 1.197(6) A with the
8
0.29
Mo Ka
4
4.70
Cu Ka
0
0
00
À1
adjacent bonds, C1 –C3, C2 –C4 shortened
m (mm
Radiation
Specimen (mm )
T’min/max
umax (8)
)
˚
(1.422(6), 1.429(6) A) compared to usual values
3
0.26, 0.25, 0.03
0.98, 1.00
60.6
0.11, 0.09, 0.03
0.78, 1.00
134.5
for single bonds, as expected in this conjugated
À
‘
2
system. The molecule and the Cl ion are almost
coplanar; the maximum deviation from a plane of
N
t
22,216
14,501
˚
best fit through all nonhydrogen atoms is 0.073 A
N (Rint
No
)
5410 (0.058)
3265
1878 (0.042)
1681
00
for C5 .
The two rings are essentially coplanar, the
dihedral angle being 175.6(2)8. Planes of mole-
cules pack in columns in the unit cell (Fig. 4),
closest contacts between atoms of these stacked
R
1
(I > 2s(I))
wR (all data)
T (K)
0.049
0.088
100
0.059
0.184
100
2
2
o
2
c
2
o
1/2
00
R
1
¼ (SjjF
o
j À jF
c
jj)/SjF
o
j, wR
2
¼ (SwðF À F Þ/SwðF Þ)
.
molecules being C6ꢀ ꢀ ꢀC2 (Àx, 1 À y, 1 À z)
˚
00
3
3
.312(6) A, and C2ꢀ ꢀ ꢀC4 (1 À x, 1 À y, 1 À z)
˚
.386(6) A.
comparable to the analogous distances in both
MTEP and MTEP.HCl, as are the C–C–C angles
The individual molecules differ very little in the
–
structures of MTEP and MTEP.HCl. This is
confirmed in Figure 5 where the molecular
structures are overlaid to minimize the separation
between the eight central atoms in each molecule.
The figure clearly shows there is a greater
deviation from planarity for MTEPa (i.e. molecule
2) compared to MTEP (molecule 1), as well as
highlighting the fact that the protonated mole-
(
178.5(3) and 179.0(2)8).
The extended crystal structure of MTEP is
depicted in Figure 2. The two independent
molecules alternate to form strips along c, the
strips packing in a herringbone fashion with the
angle between them being 86.28. Close contacts
between the two molecules within strips involve
Figure 1. Molecular projection of MTEP molecule 1 (MTEP), molecule 2 (MTEPa)
˚
0
0
being similar. Selected bond lengths (A) and angles (8) for molecule 1, 2: C1 –C2 1.196(3),
0
0
0
0
0
0
1
.196(3); C1 –C3 1.434(3), 1.436(3); C2 –C4 1.432(3), 1.434(3); C3–C1 –C2 178.9(2),
0
0
00
1
77.3(2); C1 –C2 –C4 178.5(2), 179.2(2).
DOI 10.1002/jps
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238
McILDOWIE ET AL.
Figure 2. Unit cell for MTEP (molecule 1 in the cif), viewed down c (a), and down a (b);
H atoms have been omitted for clarity. The second independent MTEP molecule is
denoted by the ‘‘a’’ (molecule 2 in the cif).
cules in MTEP.HCl differ from those in MTEP by a
molecules into the lattice. In the neutral MTEP
the lattice packing appears to be facilitated by the
intermolecular hydrogen bonding, resulting in a
very different three-dimensional structure.
–
1
808 rotation about the central C–C–C–C axis. In
MTEP.HCl, the protonated nitrogen atom plays a
major role in distinguishing the packing of the
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CRYSTALLOGRAPHIC CHARACTERISATION OF MTEP AND MTEP.HCL
239
Figure 5. Aligned structures of the two molecules of
MTEP and MTEP.HCl, indicating similar structures
but flipped conformation of the hydrochloride relative
to the neutral molecules.
Figure 3. Molecular projection of MTEP.HCl.
˚
0
0
Selected bond lengths (A) and angles (8): C1 –C2
0 0 00 0
1
.197(6); C1 –C3 1.422(6); C2 –C4 1.429(6); C3–C1 –
0
0
0
00
C2 177.9(5); C1 –C2 –C4 179.4(5).
4
9
Hirshfeld Surface Analysis
feld surfaces to highlight the differing electro-
static nature of the intermolecular interactions in
MTEP and MTEP.HCl that arise as a consequence
of protonation of the pyridine nitrogen atom.
Figures 6 and 7 display clusters of molecules in
MTEP and MTEP.HCl, respectively, with the
electrostatic potential mapped on molecular
Hirshfeld surfaces for selected molecules. The
range of the electrostatic potential is identical in
both figures, the much more intense blue and red
colourings for MTEP.HCl highlighting the fact
The analysis of molecular crystal structures using
tools based on Hirshfeld surfaces is becoming
increasingly common, and is gaining acceptance
as a complementary tool in the solid state
43–47
characterization of pharmaceutical materials.
The present suite of tools and techniques incor-
porated in Hirshfeld surface analysis are con-
4
8
veniently summarized in a recent review article.
For the present analysis we employ the mapping
of the electrostatic potential on molecular Hirsh-
Figure 4. Unit cell for MTEP.HCl, viewed down a.
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240
McILDOWIE ET AL.
Figure 6. The molecular electrostatic potential mapped on Hirshfeld surfaces for a
cluster of molecules in MTEP. The electrostatic potential is mapped over the range
À0.05 au (red) to þ0.05 au (blue), and hydrogen bonds are indicated by dashed lines.
Atoms involved in interactions within a strip have black labels, while those between
strips have blue labels.
that the interactions in that structure comprise a
greater ionic component than in MTEP. The
electrostatic potential for MTEP (Fig. 6) shows
the expected electronegative (red) regions around
the pyridine and thiazole nitrogen atoms and a
weakly electronegative region around the thiazole
sulfur atom, while the surface in the vicinity of the
hydrogen atoms is mostly electropositive (blue).
Both MTEP and MTEPa display near identical
features, and the HF/Midi! dipole moments are
around 3.1 D for both molecules. The cluster of
molecules in Figure 6 is chosen to illustrate the
important patterns of hydrogen bonding interac-
tions between molecules in the MTEP crystal
structure. As observed above, the crystal struc-
ture of MTEP consists of strips of molecules
aligned side by side, MTEP and MTEPa alternat-
ing, and running along the c axis (see Fig. 2).
Molecules in adjacent strips interdigitate and
are oriented at almost 908 to one another.
The important interaction within strips involves
atoms H5 and N3 on the thiazole, and can be
summarised as an infinite sequence of the type
0
0
00
0
0
00
00
00
ꢀ ꢀ ꢀH5 a–N3 aꢀ ꢀ ꢀH5 –N3 ꢀ ꢀ ꢀ. There are two simi-
lar sets of interactions between strips that involve
only atoms on the pyridine; that on one side of the
strip involves only MTEP, with a sequence ꢀ ꢀ ꢀH5–
˚
N1ꢀ ꢀ ꢀH5–N1ꢀ ꢀ ꢀ (N1ꢀ ꢀ ꢀH5 ¼ 2.55 A), while on the
other side of the strip only MTEPa molecules
interact with one another in a similar fashion,
˚
ꢀ ꢀ ꢀH5a–N1aꢀ ꢀ ꢀH5a–N1aꢀ ꢀ ꢀ (N1aꢀ ꢀ ꢀH5a ¼ 2.62 A).
All of these hydrogen bond interactions show a
characteristic feature, namely the complementary
nature of the electrostatic potentials where
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CRYSTALLOGRAPHIC CHARACTERISATION OF MTEP AND MTEP.HCL
241
of the complex is 14.4 D. Figure 7a shows how
these moieties pack in a sheet in a way that
À
completely surrounds the Cl ion with electro-
positive regions of neighbouring molecules.
Figure 7b illustrates how the columnwise
þ
À
stacking of MTEPH .Cl complexes in MTEP.HCl
is the result of an energetically favourable
arrangement where large dipoles alternate in
an antiparallel fashion. Protonation of the pyr-
idine also enhances the electropositive nature of
the methyl group attached to the thiazole (the
surface around the methyl group in Figure 7 is
considerably darker blue than that for MTEP in
Fig. 6). As a consequence, a secondary interaction
between the methyl group and the nitrogen in
the thiazole is also apparent in Figure 7
0
0
00
˚
(
H6 ꢀ ꢀ ꢀN3 ¼ 2.60 A).
pK Determination
a
The dissociation constants for MTEP were inves-
tigated by titration of hydrochloric acid against a
1
solution of MTEP in 9:1 H O/D O using H NMR
2
2
50
spectroscopy. Figure 8 contains overlaid plots of
the H NMR spectra with varying pH, indicating
1
the resonances used in this study, and easily
observable chemical shift changes. It should be
noted that these data only allow an estimation
Figure 7. The molecular electrostatic potential
mapped on Hirshfeld surfaces for (a) a layer of mole-
cules in MTEP.HCl; and (b) a stack of three molecules in
MTEP.HCl. The electrostatic potential is mapped over
the same range as in Figure 6, and hydrogen bonds are
indicated by dashed lines.
of pK , as the solvent is not pure water, however,
a
the presence of D O has a minimal impact on
2
51
the pK values determined.
a
The second dissociation constant, correspond-
ing to protonation/deprotonation of the pyridyl
nitrogen, was determined by analysis of plots of
the chemical shifts of the four pyridyl protons
versus pH (Fig. 9). Each dataset was fitted to an
empirical sigmoidal function (Eq. 1) using non-
linear least squares regression analysis (Sigma-
Plot 10.0),
adjacent Hirshfeld surfaces touch one another (i.e.
the red electronegative region around the acceptor
atom contacts a blue region around the donor
atom). These nonclassical hydrogen bonds are
favoured in MTEP by the presence of electro-
negative atoms in both heterocycle which enhance
the positive nature of the hydrogen atoms
attached to the rings, and this can be seen quite
clearly from the electrostatic potential maps in
Figure 6.
a
d ¼ d0 þ
(1)
þ eÀððpHÀpKaÞ=bÞ
1
where d is the chemical shift observed at
For MTEP.HCl we have chosen to compute the
electrostatic potential for the neutral complex
various pH values, d is the shift for the fully
0
deprotonated base, and a and b are substance and
þ
À
49
MTEPH .Cl , and map it on the Hirshfeld surface
for that moiety. The strongly dipolar nature of
the complex is immediately evident by the
extensive electronegative (red) region completely
surrounding the chloride ion, and the surface
around the pyridine is now completely electro-
positive (blue), a direct result of the protonation of
the pyridine N atom; the HF/Midi! dipole moment
condition dependant constants.
The value
of pKa2 corresponds to the point of inflection of
the fitted curve, which in this case was found to be
3.40 Æ 0.01. This result was given consistently by
each of the four curves. The pK of 3.42 Æ 0.12
a
predicted by Advanced Chemistry Development
52
(ACD/Labs) Software is impressively close to our
experimentally determined result. It is unlikely
DOI 10.1002/jps
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242
McILDOWIE ET AL.
1
2 2
Figure 8. Stack plot of the H NMR spectra of solutions of MTEP in 9:1 H O/D O with
varying pH.
that MTEP is protonated in its biologically active
gen, could not be determined accurately, as pH
measurement below pH ꢁ 0 using our equipment
is subject to considerable error. Nevertheless, part
of the relevant buffer region (below pH 1.5) can be
form given this low pK value.
a
The first dissociation constant, corresponding to
protonation/deprotonation of the thiazole nitro-
Figure 9. Plot of pH versus the chemical shift of
pyridyl protons H2 (*), H6 (4), H4 (&) and H5 (^).
Chemical shift values recorded for pH values below 1.50
(
shaded points) were omitted when fitting each curve
owing to an effect of protonation at the thiazolyl nitro-
gen on the chemical shift of the pyridyl protons.
Figure 10. Plot of pH versus the chemical shift of
thiazolyl protons H5 (*) and CH (~).
3
0
0
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DOI 10.1002/jps

CRYSTALLOGRAPHIC CHARACTERISATION OF MTEP AND MTEP.HCL
243
seen in a the plot of pH versus the chemical shift of
the methyl and thiazolyl protons (Fig. 10), and
from this we estimate pKa1 to be approximately
Reggiani A, Bertorelli R. 2005. The antinociceptive
and anxiolytic-like effects of the metabotropic glu-
tamate receptor 5 (mGluR5) antagonists, MPEP
and MTEP, and the mGluR1 antagonist,
LY456236, in rodents: A comparison of efficacy
and side-effect profiles. Psychopharmacology (Ber-
lin, Germany) 179:207–217.
0
.2. As the exact value is not physiologically
relevant, we did not pursue an accurate determi-
nation any further.
3
4
. Stachowicz K, Golembiowska K, Sowa M, Nowak G,
Chojnacka-W o´ jcik E, Pilc A. 2007. Anxiolytic-like
action of MTEP expressed in the conflict drinking
Vogel test in rats is serotonin dependent. Neuro-
pharmacology 53:741–748.
. Pietraszek M, Sukhanov I, Maciejak P, Szyndler J,
Gravius A, Wislowska A, Pla z´ nik A, Bespalov AY,
Danysz W. 2005. Anxiolytic-like effects of mGlu1
and mGlu5 receptor antagonists in rats. Eur J
Pharmacol 514:25–34.
CONCLUSIONS
An improved medium scale synthesis has allowed
thorough characterisation of the crystal struc-
tures of MTEP and the previously unreported
hydrochloride MTEP.HCl, which is more amen-
able to biological testing due to its enhanced water
solubility. Molecules in crystalline MTEP are
weakly polar, and aggregate through weak
C–Hꢀ ꢀ ꢀN hydrogen bonds. The structure of the
5. Page ME, Szeliga P, Gasparini F, Cryan JF. 2005.
Blockade of the mGlu5 receptor decreases basal and
stress-induced cortical norepinephrine in rodents.
Psychopharmacology (Berlin, Germany) 179:240–
protonated molecule in MTEP.HCl differs from
that in MTEP by a 1808 rotation of the pyridine
ring relative to the thiazole ring, and the
dominant interaction in the crystal involves a
2
46.
6
. Nicolas LB, Kolb Y, Prinssen EPM. 2006.
A combined marble burying-locomotor activity test
in mice: A practical screening test with sensitivity
to different classes of anxiolytics and antidepres-
sants. Eur J Pharmacol 547:106–115.
þ
À
strong ionic N–H ꢀ ꢀ ꢀCl hydrogen bond. Electro-
static potentials mapped on molecular surfaces
illustrate the way in which the electrostatic
nature of the various regions of MTEP changes
as a result of protonation of the pyridine nitrogen,
and highlight the much greater importance of
electrostatic interactions in the crystal structure
of MTEP.HCl compared with MTEP. Despite
these differences in the crystal packing, the
molecular structures of MTEP and MTEP.HCl
7
. Molina-Hern a´ ndez M, Tellez-Alc a´ ntara NP, P e´ rez-
Garc ´ı a J, Olivera-Lopez JI, Jaramillo MT. 2006.
Antidepressant-like and anxiolytic-like actions of
the mGlu5 receptor antagonist MTEP, microin-
jected into lateral septal nuclei of male Wistar rats.
Prog Neuro-Psychopharmacol Biol Psychiatry
3
0:1129–1135.
8
. Klodzinska A, Tatarczy n´ ska E, Chojnacka-W o´ jcik
E, Nowak G, Cosford NDP, Pilc A. 2004. Anxiolytic-
like effects of MTEP, a potent and selective mGlu5
receptor agonist does not involve GABAA signaling.
Neuropharmacology 47:342–350.
are very similar. The low pK values determined
in this study make it unlikely that MTEP is
protonated in its biologically active form.
a
9
. Cosford NDP, Tehrani L, Roppe J, Schweiger E,
Smith ND, Anderson J, Bristow L, Brodkin J, Jiang
X, McDonald I, Rao S, Washburn M, Varney MA.
ACKNOWLEDGMENTS
The authors thank Atuka Ltd. and the University
of Western Australia for financial support, and
Dr. Lindsay Byrne for expert NMR spectroscopy
assistance. M.N.G. is the recipient of a UWA
postgraduate scholarship. M.A.S. has been sup-
ported by the Australian Research Council.
2
003. 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]- pyri-
dine: A potent and highly selective metabotropic
glutamate subtype 5 receptor antagonist with
anxiolytic activity. J Med Chem 46:204–206.
10. Busse CS, Brodkin J, Tattersall D, Anderson JJ,
Warren N, Tehrani L, Bristow LJ, Varney MA,
Cosford NDP. 2004. The behavioral profile of the
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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010