Design and synthesis of 2,4-difluorophenylpyruvic acid and of its azlactone precursor for macrophage migration inhibitory factor (MIF) tautomerase activity

Article abstract of DOI:10.1016/j.molstruc.2003.11.022

The macrophage migration inhibitory factor (MIF) is an important cytokine implicated in several diseases and currently a target for drug development. This study aimed to synthesize phenylpyruvic acid like structures that fit the molecular requirements of

Full text of DOI:10.1016/j.molstruc.2003.11.022

Journal of Molecular Structure 690 (2004) 89–94
www.elsevier.com/locate/molstruc
Design and synthesis of 2,4-difluorophenylpyruvic acid and of its
azlactone precursor for macrophage migration inhibitory factor (MIF)
tautomerase activity
a,
P.P. Haasbroek , D.W. Oliver , A.J.M. Carpy
b
c
*
aDepartment of Pharmacy and Pharmacology, University of the Witwatersrand, Johannesburg, South Africa
bPharmacology, Faculty of Health Sciences, Potchefstroom University for C.H.E., Potchefstroom, South Africa
c
` ´
Laboratoire de Physico- and Toxico-Chimie des Systemes Naturels (LPTC), UMR 5472 CNRS, Universite de Bordeaux I,
´
351 Cours de la Liberation, Talence Cedex 33405, France
Received 11 July 2003; revised 10 November 2003; accepted 17 November 2003
Abstract
The macrophage migration inhibitory factor (MIF) is an important cytokine implicated in several diseases and currently a target for drug
development. This study aimed to synthesize phenylpyruvic acid like structures that fit the molecular requirements of MIF with respect to
keto/enol tautomerism and E/Z isomerism of the enol forms. The synthesis of 2,4-difluorophenylpyruvic acid and its azlactone precursor as
potential ligands to interact with the active site of MIF is reported here. Both the E and Z isomers of the azlactone of 2,4-
difluorobenzaldehyde were synthesized using different synthetic methods. Hydrolysis of these isomeric azlactones yielded similar enol/keto
tautomeric mixtures of 2,4-difluorophenylpyruvic acid, with the enol form predominating. NMR spectroscopy was used to confirm the
structures of these compounds, while an X-ray crystallographic study also confirmed the configuration of the E isomer of the azlactone.
q 2003 Elsevier B.V. All rights reserved.
Keywords: Macrophage migration inhibitory factor; NMR; X-ray; Azlactones; 2,4-Difluorophenylpyruvic acid
1. Introduction
indicated that the configuration of the E isomer of the enol
form of 2,4-difluorophenylpyruvic acid shows potential in
fitting the structural requirements for MIF interaction.
Spectral data of phenylpyruvic acid and derivatives
obtained by us [17–22] and others [23–27] revealed some
interesting structural features of these compounds. While
our previous studies focussed on the Z isomers [17–22] and
their synthesis from the Z azlactones, this study concen-
trated on the synthesis of the E isomers of these derivatives.
The E isomers of the azlactones are more difficult to
synthesize and require specific methods [28]. From our
previous efforts to synthesize the E azlactones, it appears
that the 2-phenyl azlactones (using hippuric acid)
give better results than the 2-methyl azlactones (using
N-acetylglycine). Both isomers of the azlactone of 2,4-
difluorobenzaldehyde were synthesized in this study in
order to make a comparison and identify differences in their
NMR spectra. The azlactones were then converted to their
In continuation of our cooperative program1 dealing with
the chemistry, synthesis and structural features of phenyl-
pyruvic acid derivatives, we recently reported our interest in
the synthesis of phenylpyruvic acid derivatives as potential
inhibitors on the phenylpyruvate tautomerase activity
catalysed by Macrophage Migration Inhibitory Factor
(MIF) [1,2]. There is an increasing interest in MIF as a
target for drug development in view of its biological
functions amongst others macrophage activation, tumorcidil
activity and induction of nitric oxide production as well as
its role in pathology such as sepsis, acute respiratory distress
syndrome, nephropathy and hypersensitivity reactions
[3–16]. A preliminary molecular modelling investigation
was used to determine the molecular requirements of novel
ligands to fit in the active site for MIF interaction. The data
*
1
Corresponding author. Tel.: þ27-11-742-2342; fax: þ27-11-642-4355.
1
corresponding phenylpyruvic acids by acid hydrolysis. H
NMR and 13C NMR spectral data were obtained for these
The financial support from the South African and French Governments
is gratefully acknowledged.
0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2003.11.022

90
P.P. Haasbroek et al. / Journal of Molecular Structure 690 (2004) 89–94
compounds while an X-ray crystallographic study was also
performed to confirm the configuration of the E isomer of
the azlactone.
(20 ml) was stirred for 2 h at 95–100 8C. The mixture was
poured into a mixture of water and ethanol (2:1), cooled,
filtered and dried (5.55 g; 0.019 mol). Recrystallization
from carbon tetrachloride gave fine light yellow crystals of
3, mp 187–189 8C.
2. Experimental
2.1.3. Synthesis of 2,4-difluorophenylpyruvic acid, 4
2.1. Synthesis
The azlactone of 2,4-difluorobenzaldehyde 2(E), (1.0 g;
0.0035 mol) was boiled under reflux in a mixture (20 ml) of
glacial acetic acid and concentrated hydrochloric acid (3:7).
After 3 h refluxing, water (20 ml) was added and the
mixture cooled. The precipitate that formed, was filtered-
off, washed with water and dried (0.70 g; 0.0035 mol).
Recrystallization from a mixture of n-hexane and benzene
(3:10) gave white crystals of 4, mp 153–158 8C.
The E isomer of the azlactone, 2, was synthesized from
2,4-difluorobenzaldehyde, hippuric acid and polyphospho-
ric acid, PPA (Scheme 1) [28]. The synthesis of the Z isomer
3, involved the Erlenmeyer method [1,17,18,20–22,29] for
the preparation of aromatic azlactones starting from 2,4-
difluorobenzaldehyde, hippuric acid and acetic anhydride.
Acid hydrolysis of 2 and 3, respectively, yielded the same
tautomeric phenylpyruvic acid mixture 4 (Scheme 2).
Hydrolysis of the azlactone 3(Z) under the same
conditions, gave the same product as the E isomer (same
melting points and same NMR spectra).
2.1.1. Synthesis of 4-(2,4-difluorobenzylidene)-2-phenyl-5-
oxazolone (E), 2
2.2. Spectroscopic analysis
A mixture of 2,4-difluorobenzaldehyde 1 (7.1 g;
0.05 mol), hippuric acid (8.95 g; 0.05 mol) and polypho-
sphoric acid (60 g) was heated at 90 8C for 2 h with
occasional stirring. The mixture was poured into water
(250 ml), cooled, filtered, washed several times with water,
and dried (12.9 g; 0.045 mol). Melting point and Thin Layer
Chromatography (TLC) showed a mixture of the E and Z
isomers. The two isomers were separated by fractional
crystallization using carbon tetrachloride as solvent. The Z
isomer 3, crystallized out first, forming fine yellow crystals,
mp 187–189 8C (4.3 g; 0.015 mol). Concentration of the
filtrate, followed by cooling in a refrigerator, resulted in the
crystallization of the E isomer, which was further purified
by recrystallization from a benzene/n-hexane (1:2) mixture.
Yellow needle-like crystals of 2 formed, mp 127–128 8C,
(2.1 g; 0.0074 mol). The purity of the E isomer was
confirmed by TLC, using silica gel 60 and benzene/n-
hexane (1:1) as eluent. Rf values of 0.56 and 0.35 were
obtained for Z and E, respectively.
NMR analyses of 2, 3 and 4 were performed on a Bruker
Avance 300 spectrometer, using CDCl3 as solvent for 2 and
3 and DMSO-d6 as solvent for 4 while TMS was used as
internal standard (Tables 1 and 2). The melting points
(uncorrected) were determined on an electrothermal melting
point apparatus.
2.3. X-ray analysis
Yellow prismatic crystals of 2 were obtained from a
mixture of benzene and n-hexane. All diffraction measure-
ments were performed at room temperature and data
collected with an Enraf-Nonius CAD-4 diffractometer
using graphite-monochromated Cu Ka radiation. The struc-
ture was solved by direct methods using MolEN [30].
Difference Fourier maps showed the presence of water
molecules. Atoms were refined anisotropically, except H-
atoms placed in theoretical positions (Table 3). The final
atomic coordinates are given in Table 4 and the bond
lengths and angles in Tables 5 and 6. Some relevant torsion
angles are shown in Table 7 and the molecular structure
with its atomic numbering scheme in Fig. 1.
2.1.2. Synthesis of 4-(2,4-difluorobenzylidine)-2-phenyl-5-
oxazolone (Z), 3
A
mixture of 2,4-difluorobenzaldehyde (3.55 g;
0.025 mol), hippuric acid (4.475 g; 0.025 mol), anhydrous
sodium acetate (4.1 g; 0.05 mol) and acetic anhydride

P.P. Haasbroek et al. / Journal of Molecular Structure 690 (2004) 89–94
91
Scheme 2.
3. Results
agreement with values reported for similar 2-phenyl
azlactones [31], where the E isomer also absorbs further
downfield from that of the Z isomer. The 13C NMR data of 2
and 3 appear in Table 2. The 13C NMR spectra of the two
isomers are also very similar, with the major difference in
the absorption of the benzylic carbon of the two isomers
with E at d 129.1 and Z at d 121.5.
3.1. Spectroscopic analysis
The 1NMR data of 2(E) and 3(Z) appear in Table 1 The
1H NMR spectra of the aromatic protons of 2 and 3 are very
similar with the signals of 2 shifted slightly upfield
compared to those of 3 except for H-3 which has exactly
the same chemical shift values for both isomers. There is,
however, a marked difference in the absorption of the
benzylic protons (Ar–CHyC) of the two isomers, with the
benzylic proton of 3 giving a singlet at d 7.42 and the same
proton of 2, a singlet at d 7.69. These values are in
1
The H NMR spectrum for the product 4 (d 8.28–8.35
(m), H-6; d 7.10–7.26 (m), H-3, H-5; d 6.53 (s), CHyC; d
4.26 (s), CH2), obtained from the hydrolysis of the
azlactones 2 and 3, clearly shows a tautomeric mixture of
the enol (one proton singlet at d 6.53 for benzylic proton)
and the keto (two proton singlet at d 4.26 for the CH2
protons) forms, with the enol form predominating (,88%)
in solution. Similar values were reported for phenylpyruvic
acid keto/enol (Z) tautomeric mixtures [18,22]. The 13C
NMR spectrum for 4 also confirms a tautomeric mixture,
with the enol form giving characteristic peaks at d 143.1 for
the enolic carbon (C HyC–OH) and at d 98.7 for the
benzylic carbon (CHyC–OH), while the keto form gives
Table 1
1H NMR data (d; ppm) of 2 and 3
Hydogen atom
2
3
H-6
8.68–8.76 (m)a
6.93–6.99 (m)
6.82–6.89 (m)
7.69 (s)
8.87–8.95 (m)
6.99–7.05 (m)
6.82–6.89 (m)
7.42 (s)
H-5
H-3
Ar–CHyC
b
H-20 , H-60
8.03–8.06 (m)
7.54–7.59 (m)
7.46–7.50 (m)
8.11–8.14 (m)
7.59–7.64 (m)
7.49–7.54 (m)
Table 3
H-40
Crystal data and structure refinement for 2
H-30, H-50
Chemical formula
Formula weight
Temperature (K)
C16H9F2NO2
a
m, Multiplet; s, singlet.
285.26
b
Prime numbers represent protons of the 2-phenyl ring.
296(2)
˚
Wavelength (A)
1.54180
Monoclinic
P21=c
Crystal system
Space group
Table 2
13C NMR data (d; ppm) of 2 and 3
˚
˚
Unit cell dimensions
a ¼ 3:804ð1Þ A, b ¼ 27:947ð1Þ A,
˚
c ¼ 13:376ð5Þ A, b ¼ 98:04ð5Þ8
3
˚
Carbon atom
2
3
Volume (A )
1408.0(6)
Z
4
Calculated density (Mg m23
)
1.346
CyO
C-4
166.5
166.3
Absorption coefficient (mm21
)
1.004
163.8
164.1
Fð000Þ
664
CyN
C-2
163.1
162.8
Crystal size (mm3)
0.40 £ 0.25 £ 0.10
3.16–55.00
160.4
132.9 (d)a
160.7
Theta range (8)
Index ranges
C-6
134.1 (d)
133.7
24 # h # 4; 0 # k # 29; 0 # l # 14
1637/1637 ½RðintÞ ¼ 0:0000ꢀ
93.5%
CHyC
C HyC
135.0
Reflections collected/unique
Completeness to u ¼ 558
Max. and min. transmission
Refinement method
129.1 (d)
133.1 (d)
128.9 (d)
127.9 (d)
125.2
121.5 (d)
133.6 (d)
129.0 (d)
128.4 (d)
125.3
b
C-40
C-30, C-50
C-20, C-60
C-10
0.9063 and 0.6896
Full-matrix least-square on F2
1637/0/218
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices ½I . 2sðIÞꢀ
R indices (all data)
1.151
C-1
117.3
118.3
R ¼ 0:1105; R2w ¼ 0:2885
R ¼ 0:1188; R2w ¼ 0:2939
0.046(6)
C-5
111.9 (d)
104.0 (d)
112.4 (d)
104.1 (d)
C-3
Extinction coefficient
Largest diff. peak and hole
a
23
d, Doublet.
˚
0.363 and 20.306 e A
b
Prime numbers represent carbons of the 2-phenyl ring.

92
P.P. Haasbroek et al. / Journal of Molecular Structure 690 (2004) 89–94
Table 4
Atomic coordinates ( £ 104) and equivalent isotropic displacement
Table 6
Bond angles (8) for 2
2
3
˚
parameters (A £ 10 ) for 2
C(6)–C(1)–C(2)
121.0(7)
119.0(7)
120.0(7)
118.4(8)
120.4(8)
118.8(8)
121.1(8)
120.3(8)
114.4(6)
128.2(7)
117.4(6)
107.9(6)
120.8(7)
133.5(7)
105.6(6)
120.0(7)
133.4(8)
106.5(6)
105.6(5)
132.5(7)
115.3(7)
126.6(7)
118.1(7)
116.7(7)
118.6(7)
124.7(8)
116.1(8)
119.7(8)
123.6(8)
116.7(8)
118.1(8)
122.2(8)
x
y
z
UðeqÞ
C(6)–C(1)–C(7)
C(2)–C(1)–C(7)
C(1)–C(2)–C(3)
C(1)
805(19)
960(20)
5963(3)
4316(6)
4004(6)
3050(7)
2418(6)
2770(6)
3694(6)
5329(6)
5780(5)
6736(6)
6785(6)
5882(4)
7414(5)
7384(6)
8435(6)
8912(6)
9895(7)
53(2)
57(2)
65(2)
69(3)
62(2)
57(2)
47(2)
57(2)
50(2)
59(2)
58(2)
84(2)
52(2)
49(2)
57(2)
75(3)
71(3)
65(2)
62(2)
80(2)
100(2)
104(11)
114(11)
64(9)
C(2)–C(3)–C(4)
C(2)
5490(3)
5367(3)
5716(3)
6186(3)
6306(3)
6103(2)
6501(2)
6466(3)
5988(3)
5769(2)
5781(2)
6832(3)
6883(2)
7323(3)
7421(3)
7050(3)
6608(3)
6527(3)
7688(2)
7139(2)
4863(10)
5204(10)
5326(12)
C(5)–C(4)–C(3)
C(3)
2740(20)
22520(20)
22590(20)
2980(20)
2471(18)
2232(17)
4212(19)
5770(20)
4551(14)
7821(18)
4390(20)
5981(19)
5620(20)
6930(30)
8680(30)
9070(20)
7720(20)
4010(15)
9932(18)
21490(110)
7620(120)
4110(110)
C(6)–C(5)–C(4)
C(4)
C(5)–C(6)–C(1)
C(5)
N(8)–C(7)–O(11)
N(8)–C(7)–C(1)
C(6)
C(7)
O(11)–C(7)–C(1)
C(7)–N(8)–C(9)
N(8)
C(9)
C(13)–C(9)–N(8)
C(13)–C(9)–C(10)
N(8)–C(9)–C(10)
O(12)–C(10)–O(11)
O(12)–C(10)–C(9)
O(11)–C(10)–C(9)
C(7)–O(11)–C(10)
C(9)–C(13)–C(14)
C(19)–C(14)–C(15)
C(19)–C(14)–C(13)
C(15)–C(14)–C(13)
F(20)–C(15)–C(16)
F(20)–C(15)–C(14)
C(16)–C(15)–C(14)
C(15)–C(16)–C(17)
C(18)–C(17)–F(21)
C(18)–C(17)–C(16)
F(21)–C(17)–C(16)
C(17)–C(18)–C(19)
C(14)–C(19)–C(18)
C(10)
O(11)
O(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
F(20)
F(21)
O(101)
O(102)
O(103)
10442(6)
10058(6)
9047(6)
8361(4)
11426(4)
240(20)
9590(20)
9249(20)
UðeqÞ is defined as one-third of the trace of the orthogonalized Uij
tensor.
peaks at d 43.3 for the CH2 carbon and at d 192.3 for the
carbonyl carbon (CO–COOH) [18,22]. The remainder of
the carbon spectrum shows a near duplication of the carbon
signals due to the small chemical shift differences for the
carbons of this mixture of two geometric isomers.
3.2. X-ray analysis
The poor quality of the crystals, the low data/parameters
ratio and inclusion of disordered water can explain the
relatively poor final R factor ðR ¼ 0:11Þ (Table 3).
However, as the aim of this study was to confirm the
configuration of 2, the conclusions that can be drawn do not
suffer any suspicion.
Table 5
˚
Bond lengths (A) for 2
C(1)–C(6)
1.383(10)
1.391(11)
1.466(11)
1.391(12)
1.401(12)
1.397(12)
1.341(11)
1.276(9)
C(1)–C(2)
C(1)–C(7)
C(2)–C(3)
Torsion angles (Table 7) show that the three planar rings
almost lie in the same plane. The E configuration of 2 was
confirmed by the relative positions of the nitrogen N(8) and
the benzylic H on the same side of the C(9)–C(13) double
bond. Another important feature to be noticed is the
orientation of the difluoro aromatic moiety. The o-fluoro
atom F(20) of the disubstituted benzyl moiety is oriented
anti with respect to the ketone functionality or syn with
respect to the benzylic hydrogen atom.
C(3)–C(4)
C(4)–C(5)
C(5)–C(6)
C(7)–N(8)
C(7)–O(11)
N(8)–C(9)
1.371(8)
1.394(10)
1.337(10)
1.459(11)
1.212(9)
C(9)–C(13)
C(9)–C(10)
C(10)–O(12)
C(10)–O(11)
C(13)–C(14)
C(14)–C(19)
C(14)–C(15)
C(15)–F(20)
C(15)–C(16)
C(16)–C(17)
C(17)–C(18)
C(17)–F(21)
C(18)–C(19)
1.376(9)
1.458(11)
1.395(11)
1.400(11)
1.353(9)
This result was confirmed by a semi-empirical quantum
mechanics calculation using AMPAC [32]. This was done
Table 7
1.367(11)
1.386(13)
1.352(12)
1.358(9)
Selected torsion angles (8) for 2
C(2)–C(1)–C(7)–N(8)
N(8)–C(9)–C(13)–C(14)
C(9)–C(13)–C(14)–C(15)
2 170.9(8)
174.5(8)
1.396(12)
2178.8(8)

P.P. Haasbroek et al. / Journal of Molecular Structure 690 (2004) 89–94
93
Fig. 1. ORTEP view of 2. Thermal ellipsoid percentage 50%.
on the E isomer of the o-chlorophenyl azlactone. The
AMPAC results indicated that the orientation of the
aromatic moiety was so that the o-chloro substituent was
always syn vs. the benzylidene hydrogen (and the nitrogen
atom of the oxazolone moiety) independently of the starting
geometry2.
position of the ring [18,22]. Although the E isomer of the
azlactone could be successfully synthesized, this configur-
ation was not retained for the enol form of the phenylpyru-
vic acid on hydrolysis of this azlactone, as is the case with
the Z isomers. The total absence of the E isomer is an
indication that it isomerises via the keto form to the Z
configuration during the hydrolysis reaction. Although, our
molecular modelling investigation favoured the configur-
ation of the E isomer of 2,4-difluorophenylpyruvic acid for
interaction with MIF, we were unsuccessful in synthesizing
the E isomer, as transformation to the Z isomer appears to
take place under these chemical conditions. Biological
evaluation of these compounds is currently under
investigation.
Although not very precise, bond lengths and angles of 2
have expected values.
4. Discussion
Even though the method used for the synthesis of the E
isomer of the azlactone 2, formed a mixture of E and Z, the E
isomer could be successfully isolated by fractional crystal-
lization. The Z fraction was identified by direct preparation
using the conventional method for the preparation of the Z
isomers. Interestingly, the E isomer forms large needle-like
crystals and the Z isomer small fine crystals with a melting
point difference of approximately 60 8C. The purity of E
was confirmed by TLC, which can also be successfully
employed to distinguish between the two isomers. NMR
spectral data was also used to distinguish between the two
isomers, as there is a clear difference in the chemical shift
values of their benzylic protons and carbons, with the E
isomer absorbing further downfield. The X-ray data of the
crystals of 2 confirmed its E configuration.
Acknowledgements
The authors wish to extend their sincere gratitude to Prof.
´
J.M. Leger (Laboratoire de Chimie Analytique, UFR des
´
Sciences Pharmaceutiques, Universite de Bordeaux II) for
participating to the X-ray crystal analysis of 2.
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