Structural influence on the intermolecular/intramolecular hydrogen bonding in solid state of substituted leflunomides: Evidence by X-ray crystal structure

Article abstract of DOI:10.1016/j.molstruc.2005.02.024

We report the results of an X-ray crystal structure study of nine substituted leflunomide metabolite analogs (LFM). Comparison of the hydrogen bonding characteristics exhibited by these structurally distinct LFM analogs was especially informative about the inter- and intra-molecular hydrogen bonding patterns that exist in the crystal structure of individual compounds. All compounds had the strong intramolecular hydrogen bonds. In addition, with the exception of the 2,5-difluorophenyl substituted LFM analog, all other compounds formed inter- or intra-molecular hydrogen bonds with the halogen atom and the NH group. However, we found that the presence of a fluorine atom at the 2-position on the phenyl ring of the 2,5-difluoro and 2-fluoro derivatives resulted in only one intramolecular hydrogen bond in the structural framework. Conversely, the 3,5-difluoro substituted LFM analog had an intramolecular hydrogen bond common to the other halide substituted derivatives. The anomaly exhibited by the 2,5-difluoro and the 2-fluoro substituted compounds may be owing to the smaller size of fluorine atom in comparison with the chlorine and bromine atoms in the structures of the other analogs. The presence of a fluorine at the 2-position of the phenyl ring may disrupt the intermolecular hydrogen bonding that was observed for the other derivatives due to differences in the crystal packing for these molecules.

Full text of DOI:10.1016/j.molstruc.2005.02.024

Journal of Molecular Structure 743 (2005) 103–115
www.elsevier.com/locate/molstruc
Structural influence on the intermolecular/intramolecular hydrogen
bonding in solid state of substituted leflunomides:
evidence by X-ray crystal structure
T.K. Venkatachalama, Y. Zhenga, S. Ghoshb, F.M. Uckunc,
*
aDepartment of Chemistry, Parker Hughes Institute, 2685 Patton Road, Roseville, MN 55113, USA
bDepartment of Structural Biology, Parker Hughes Institute, 2685 Patton Road, Roseville, MN 55113, USA
cDepartment of Virology, Parker Hughes Institute, 2685 Patton Road, Roseville, MN 55113, USA
Received 15 November 2004; revised 1 February 2005; accepted 14 February 2005
Abstract
We report the results of an X-ray crystal structure study of nine substituted leflunomide metabolite analogs (LFM). Comparison of the
hydrogen bonding characteristics exhibited by these structurally distinct LFM analogs was especially informative about the inter- and
intra-molecular hydrogen bonding patterns that exist in the crystal structure of individual compounds. All compounds had the strong
intramolecular hydrogen bonds. In addition, with the exception of the 2,5-difluorophenyl substituted LFM analog, all other compounds
formed inter- or intra-molecular hydrogen bonds with the halogen atom and the NH group. However, we found that the presence of a fluorine
atom at the 2-position on the phenyl ring of the 2,5-difluoro and 2-fluoro derivatives resulted in only one intramolecular hydrogen bond in the
structural framework. Conversely, the 3,5-difluoro substituted LFM analog had an intramolecular hydrogen bond common to the other halide
substituted derivatives. The anomaly exhibited by the 2,5-difluoro and the 2-fluoro substituted compounds may be owing to the smaller size
of fluorine atom in comparison with the chlorine and bromine atoms in the structures of the other analogs. The presence of a fluorine at the
2-position of the phenyl ring may disrupt the intermolecular hydrogen bonding that was observed for the other derivatives due to differences
in the crystal packing for these molecules.
q 2005 Elsevier B.V. All rights reserved.
Keywords: Leflunomides; Crystal structures; Fluorine atom; Hydrogen bonding
1. Introduction
agents with apoptosis promoting and chemosensitizing
properties. The epidermal growth factor receptor (EGFR)
is a membrane-associated tyrosine kinase that serves as an
endogenous negative regulator of apoptosis in breast cancer
cells [5]. Consequently, the development of new potent anti-
breast cancer agents has emerged as an important focal point
for translational research in the treatment of breast cancer
[6]. Recently, compound A77 1726, which is the primary
metabolite of the isoxazole leflunomide [N-(4-trifluoro-
methylphenyl)-5-methylisoxazol-4-carboxamide), was
shown to possess anti-inflammatory activity with pleio-
tropic effects [7–10]. Mattar et al. [11] also provided
experimental evidence that the above compound inhibited
EGFR kinase in micromolar concentrations.
Apoptosis is a common mode of eukaryotic cell death
that is triggered by an inducible cascade of biochemical
events leading to the activation of endonucleases that cleave
the nuclear DNA into oligonucleosome length fragments
[1–4]. Several biochemical events contribute to apoptotic
cell death. An improved understanding of the molecular
basis of apoptosis and the pro-apoptotic versus anti-
apoptotic regulatory signals may provide further insight
into the pathogenesis of human lymphoid malignancies.
Selective inhibitors of the anti-apoptotic tyrosine kinase,
BTK hold promise as a new generation of anti-leukemic
In a systematic search for potent anti-cancer agents, we
have identified several structurally distinct LFM analogs as
potent inhibitors of tyrosine kinases. In the previous studies,
we have examined the X-ray crystal structures of a few LFM
analogs [12–16]. In this paper we report a detailed study on
*
Corresponding author. Tel.: C1 612 697 9228; fax: C1 612 697 1042.
E-mail address: fatih_uckun@ih.org (F.M. Uckun).
0022-2860/$ - see front matter q 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2005.02.024

104
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
spectra were obtained on a Varian Mercury 300 instrument at
ambient temperature in DMSO-d6. FT-IR spectra were
X
X
O
O
+
NC
NC
OH
O
DIC
´ ´
N
H
recorded on a Nicolet Protege 460 spectrometer between
H2N
600and4000 cmK1 asKBrpellets. UVspectrawererecorded
from a Beckmann DU 7400 UV/Vis spectrometer using a cell
path length of 1 cm in methanol. Mass spectra were
performed on a Hewlett Packard MALDI-TOF spectrometer
(Model G2025A LD-TOF) using dithranol as the supporting
matrix. Melting points were determined using a Melt John’s
apparatus and are uncorrected. HPLC data was collected
using a Hewlett Packard 1100 series instrument consisting of
an automatic sampler, an electronic degasser, a thermostatic
control unit, and a diode array detector in conjunction with a
Chemstation software assembly. The column was an
analytical RP-18 Lichrospher column (4.6!250 mm2,
5 mm particle size) and eluent was H2O (0.1% ACOH):ace-
tonitrile (35:65). The flow rate was maintained at 1.0 ml/min
and the detection wavelength was set at 275 nm. The column
was maintained at room temperature throughout the analysis.
A detailed procedure for the synthesis of 8 is provided
below, and should be considered representative for the
synthesis of the lefunomide derivatives presented in this
paper.
X
O
OH
NC
N
NaH, CH3COCl
N
H
Me
H
CN
Scheme 1. General synthetic scheme for leflunomide derivatives.
the X-ray crystal structures of several of these LFM
analogs and the influence of their structural differences
on the anisotropy in intermolecular hydrogen bonding
characteristics.
2. Experimental
All chemicals were purchased from Aldrich (Milwaukee,
WI) and were used without further purification. Unless
otherwise noted, each reaction vessel was secured with a
rubber septum, and the reaction was performed under a
nitrogen atmosphere. The leflunomide derivatives were
prepared using previously published data [13]. The prepared
compounds were purified using column chromatography on
silica gel, followed by recrystallization. Samples were
identified by NMR, IR and UV spectroscopy, mass
Synthesis of (2Z)-N-(2,4-dibromophenyl)-2-cyano-3-
hydroxybut-2-enamide (8). In a 250 ml RB flask, under a
nitrogen atmosphere, cyanoacetic acid (3.4 g, 40 mmol)
and 2,4-dibromoaniline (11.0 g, 44 mmol) were fully
dissolved in anhydrous tetrahydrofuran (80 ml). To this
spectrometry, and analytical HPLC. The H and 13C NMR
1
Table 1
Structures of LEF compounds
Compound
Structure
Compound
Structure
OH
1
6
Cl
O
O
OH
Me
O
O
N
H
Me
N
H
CN
OH
F
CN
Cl
Cl
F
2
3
7
8
Cl
Cl
OH
N
H
F
Me
Me
N
H
Me
Me
Me
CN
OH
CN
Br
O
O
OH
O
N
H
CN
Br
N
H
Cl
CN
4
5
9
OCF
Br
3
OH
O
O
OH
Me
Me
N
H
CN
N
H
CN
F
F
10
Br
Br
OH
O
OH
N
H
N
H
F
Me
CN
CN

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
105
Table 2
˚
Selective crystal data for compounds (A, deg)
Compound
Space group
a
b
c
a
b
g
1
2
3
4
5
6
7
8
9
P-1
5.5089
4.4845
4.4493
8.598
9.9183
9.5145
9.5719
5.595
11.8075
13.5388
14.378
68.40
91.22
77.10
90.0
82.59
87.72
94.00
86.07
90.0
P-1
95.40
83.75
92.31
81.71
78.59
95.94
108.59
81.09
P-1
P2(1)/n
P1
21.407
4.6397
5.2782
4.7724
9.2124
13.0502
11.5754
9.1565
75.69
69.79
90.0
85.54
75.84
90.0
P1
10.2335
24.1536
12.584
P21/c
C2/c
P-1
15.289
5.2834
13.714
90.0
90.0
10.8265
13.1634
69.01
83
solution was added 1,3-diisopropylcarbodiimide (6.3 ml,
40 mmol), and the mixture was allowed to stir overnight at
room temperature, during which a precipitate was formed.
The precipitated urea was removed by filtration and solution
partitioned between ethyl acetate and 0.5 N HCl [1:1]. The
organic layer was separated from the aqueous layer, washed
with brine, and dried over anhydrous sodium sulfate. After
filtration and removal of the solvent under vacuum the
residue was then crystallized from ethyl alcohol to obtain
12.1 g of N-(2,4-dibromophenyl)-2-cyanoacetamide.
precipitate the crude product. The precipitated product was
collected by filtration, washed with water, and dried under
vacuum. The product was recrystallized from ethanol to
furnish the analytically pure compound (9.8 g, 78% yield).
Crystals of the leflunomide derivatives were grown by
dissolving a compound in an organic solvent, which was then
allowed to evaporate slowly. The crystals were then mounted
carefully using an epoxy glue on a Bruker SMART CCD area
detector diffractometer. The following programs were
utilized for cell refinement, data reduction, structure solving,
andrefinementofthestructure:SAINT (Bruker1998a),SHELXTL
(Bruker, 1998b) and SHELXS97 [17,18]. The program used to
refine the structure was SHELXL [19]. Molecular graphics and
publication materials were prepared using SHELXTL.
In the subsequent step, N-(2,4-dibromophenyl)-2-cya-
noacetamide (11.1 g, 35 mmol) was dissolved in anhydrous
tetrahydrofuran (250 ml) under a nitrogen atmosphere and
sodium hydride (3.1 g, 77 mmol) was added carefully to the
solution. The mixture was allowed to stir atroom temperature
for 1 h. After this period, acetyl chloride (3.0 ml, 42 mmol)
was slowly added to the reaction mixture. The solution was
allowed to stir at room temperature for 1 h, then quenched by
adding acetic acid (5 ml). The mixture was poured into ice
water (500 ml) containing hydrochloric acid (12.5 ml) to
3. Results and discussion
The target LFM analogs were prepared in a two-step
procedure following Scheme 1. In brief, cyanoacetic acid
Table 3
˚
Selected bond and hydrogen bond lengths (A)
Compound
D–H/A
D–H
H/A
D/A
D–H/A
1
O(9)–H(9)/O(7)
O(9)–H(9)/O(7)#1
N(1)–H(1)/Cl(6)
N(1)–H(1)/N(11)#1
O(9)–H(9)/O(7)
O(9)–H(9)/O(7)#2
O(9)–H(9)/O(7)
N(1)–H(1)/N(11)#1
O(9)–H(9)/O(7)
O(9)–H(9)/O(7)
N(1)–H(1)/N(11)#1
O(9)–H(9)/O(7)
O(9)–H(9)/O(7)
N1–H1/N(11)#1
O(9)–H(9)/O(7)
N(1)–H(1)/Br(2)
O(9)–H(9)/O(7)
O(9)–H(9)/O(7)#1
N(1)–H(1)/N(11)#2
0.81(3)
0.81(3)
0.80(2)
0.87(2)
0.92(3)
0.92(3)
0.96(3)
0.86
1.78(3)
2.56(3)
2.50(2)
2.36(2)
1.70(3)
2.46(2)
1.62(3)
2.34
2.5329(17)
3.0477(18)
2.9434(13)
3.204(2)
2.5181(18)
2.9514(19)
2.512(3)
3.164(3)
2.500(5)
2.4802(19)
3.128(2)
2.540(2)
2.483(2)
3.134(3)
2.509(3)
3.056(3)
2.508(4)
3.027(4)
3.218(5)
153(3)
120(2)
116.8(19)
162.0(19)
147(2)
113.7(18)
153(3)
161.3
2
3
4
5
0.88(5)
0.82
1.70(5)
1.74
150(5)
148.5
0.83(2)
0.82
2.33(2)
1.81
161.9(18)
147
6
7
0.98(3)
0.87(2)
0.84(4)
0.77(3)
0.89(6)
0.89(6)
0.82(3)
1.56(3)
2.30(2)
1.70(5)
2.54(3)
1.70(6)
2.52(5)
2.44(4)
156(2)
163(2)
161(5)
126(3)
150(5)
117(4)
161(3)
8
9
Compound 1, symmetry code: KxK1, Ky, KzC1. Compound 2, symmetry code #1: KxC3, KyC1, KzC1; symmetry code #2:KxC1, Ky, KzC1.
Compound 3, symmetry code #1: KxC2, Ky, KzC1. Compound 5, symmetry code: KxC2, Ky, KzC2. Compound 7, symmetry code: K1Kx, 1Ky, 1Kz.
Compound 9, symmetry code#1: KxC1, Ky, Kz; symmetry code #2: KxC2, KyC1, Kz.

106
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
was condensed with substituted aniline in presence of
diisopropylcabodiimide to furnish the substituted amide.
This amide was further treated with sodium hydride and
acylated using acylchloride to obtain the desired
leflunomide.
these compounds is provided in Table 2. In addition, Table 3
provides the hydrogen bonds and their length, and Tables 4
and 5 show selected bond lengths and angles for all the
derivatives. Figs. 1–3 show the X-ray crystal structures of
these three compounds along with their respective crystal
packing diagram.
The structures of the compounds analyzed in this study
are provided in Table 1. Halide substituents on the phenyl
group were focused on as these compounds were found to be
biologically active [13]. We first compared the crystal
structures of similarly substituted LFM analogs for any
changes in the crystal structure characteristics.
Accordingly, compounds 1–3 are the compounds possessing
2,4-dichloro, 3,4-dichloro and 3,5-dichloro substituents,
respectively, on the phenyl ring. All three were triclinic with
a P-1 space group. Selected crystal data determined for
The crystal structure of 1 revealed two intramolecular
hydrogen bonds, one between the C]O and O–H group
and one for the N–H near the phenyl ring and the
chloride at the 2-position of the phenyl ring,
[O(9)–H(9)/O(7)Z2.5329(17) and N(1)–H(1)/Cl(6)Z
2.9434(13)] (Table 3). The hydrogen bonds can be
expressed in graph set motif form as S11ð6Þ and S11ð5Þ
Table 5
Selected bond angles
Table 4
Compound
Bond designation
Angles (deg)
Selected bond lengths
1
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
C(7)–N(1)–C(1)
O(7)–C(7)–N(1)
C(11)–C(8)–C(7)
O(9)–C(9)–C(8)
N(11)–C(11)–C(8)
128.81(13)
123.40(14)
119.16(13)
121.66(15)
175.96(18)
128.17(14)
122.53(15)
121.06(14)
121.87(15)
179.40(2)
128.4(2)
122.1(2)
121.2(2)
121.5(2)
179.6(3)
128.3(4)
121.7(4)
120.7(4)
121.5(4)
178.4(5)
127.69(15)
122.09(17)
121.39(17)
121.55(17)
179.4(2)
128.8
˚
Bond length (A)
Compound
Atom designation
1
Cl(4)–C(4)
Cl(6)–C(6)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
Cl(3)–C(3)
Cl(4)–C(4)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
Cl(3)–C(3)
Cl(5)–C(5)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
F(3)–C(3)
F(6)–C(6)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
F(3)–C(3)
F(5)–C(5)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
F(2)–C(2)
O(9)–C(9)
O(7)–C(7)
F(5)–C(5)
O(7)–C(7)
O(9)–C(9)
Br(2)–C(2)
Br(4)–C(4)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
Br(1)–C(5)
F(1)–C(12)
F(2)–C(12)
F(3)–C(12)
O(7)–C(7)
O(9)–C(9)
N(11)–C(11)
1.7425(17)
1.7360(15)
1.2376(17)
1.313(2)
1.138(2)
1.7294(17)
1.7342(17)
1.249(2)
1.319(2)
1.141(2)
1.744(3)
1.743(3)
1.254(3)
1.325(3)
1.141(3)
1.355(5)
1.345(5)
1.230(5)
1.304(5)
1.132(6)
1.352(2)
1.362(2)
1.249(2)
1.310(2)
1.138(2)
1.359(30
1.319(3)
1.246(3)
1.355(3)
1.249(2)
1.315(2)
1.900(3)
1.907(3)
1.245(3)
1.316(4)
1.130(4)
1.884(4)
1.288(7)
1.305(6)
1.286(7)
1.241(5)
1.320(5)
1.134(5)
2
3
4
5
6
7
8
9
2
3
4
5
121.8
6
7
8
119.8
122.2(2)
178.0(3)
127.5(17)
122.3(19)
121.7(17)
121.6(19)
179.6(2)
130.1(3)
122.40(3)
119.0(3)
121.2(3)
176.1(4)
128.8(4)
122.7(4)
120.2(3)
121.0(4)
177.9(4)
9

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
107
Fig. 1. X-ray crystal structure of 1 and its crystal packing diagram.
that imparts a more rigid conformation to the molecule.
These intramolecular hydrogen bonds are present forming
a six-member ring and a five-member ring. The
molecule is represented below in the graph set motif form
in Scheme 2.
No intermolecular hydrogen bonding was observed between
the second chloride at the 4-position with another molecule.
Compound 2, a 3,4-dichlorophenyl substituted LFM
analog displayed an intramolecular hydrogen bond between
the C]O and O–H group similar to that observed for 1.
There was no intramolecular hydrogen bond with chlorine
atom likely due to the positional change of chlorine atoms in
the structure of the molecule. Of interest, this compound did
show an intermolecular hydrogen bond between CN and
N–H as shown in the following graph set motif form R22ð12Þ
with a 12 member ring formation (Scheme 3).
Formation of an intramolecular hydrogen bond between
C]O and O–H was expected due to the higher electro-
negative character of the oxygen atom. Furthermore, the
formation of intramolecular hydrogen bond between
the chlorine and the N–H would eliminate the availability
of the N–H hydrogen for additional hydrogen bonding. The
formation of an N–H–Cl hydrogen bond would allow the
molecule to be highly planar and it would also provide a
balance between two rings, one at the top of the plane and
the other at the bottom. This hydrogen bond stabilizes the
molecule to a rigid structure due to the presence of five and
six membered rings. In other words, the free rotation
around C–NH bond attached to the phenyl ring appeared to
be curtailed due to this hydrogen bond. The compound
exhibited an intermolecular hydrogen bonding between
O–H and C]O in the crystal structure (Table 3).
This compound also formed a second intermolecular
hydrogen bond with C]O and O–H as seen in 1.
Compound 3, which has a 3,5-dichloro substituent on the
phenyl ring, formed an intramolecular hydrogen bond
between C]O and O–H very similar to that observed for
compounds 1 and 2. Once again, in the absence of a chlorine
atom at the 2-position on the phenyl ring, the N–H formed
a hydrogen bond with the CN intermolecularly. In addition a
third hydrogen bond with intermolecular hydrogen
bonding was observed with C]O and O–H as expected

108
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
Fig. 2. X-ray crystal structure of 2 and its crystal packing diagram.
(Fig. 3). The graph set motif form of this compound is
represented in Scheme 4.
no other hydrogen bonds present in the crystal structure of
the compound. This is of note since one would have
expected an intramolecular hydrogen bond between a
2-position fluoride and the N–H group similar to that
observed with the 2,4-dichloro substituted compound (1).
The absence of this intramolecular hydrogen bond for this
compound could probably be best explained by considering
the size of the fluoride in relation to that of chloride, since
both of them are highly electronegative in character. Further
comparison of the torsion angle data for these compounds
show that a twisting structure is also not the cause for this
behavior. The torsion angle only varied within K2.0–7.98
for all the compounds (Table 6). Recently, Yogavel et al.
[20] examined the crystal structure of 2-cyano-3-dimethy-
lamino-N-(4-methylphenyl)acrylamide and 2-cyano-3-
Compounds 1–3 each formed intermolecular hydrogen
bonds between the N–H and CN groups. In addition, the
O–H and C]O of individual molecules also formed
intermolecular hydrogen bonding in their solid state.
However, only 1 had an intramolecular hydrogen bond
with the chlorine atom likely due to its location at the
2-position on the phenyl ring. This aspect will be clear when
we consider other substituents at the 2-position of the
phenyl ring in the following discussion.
We extended our work by examining the crystal structure
of structurally similar compounds 4 and 5 (Figs. 4 and 5).
In both compounds there is a fluorine substitution on
the phenyl ring. Compound 4 formed a monoclinic crystal
and had only one intramolecular hydrogen bond, which was
between the C]O and the O–H groups. Notably, there were
dimethylamino-N-(2-methoxyphenyl)acrylamide and
found that the hydrogen bond formation occurs between

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
109
Fig. 3. X-ray crystal structure of 3 and its crystal packing diagram.
the methoxy group and N–H. This was further explained
1
S
(6)
1
due to the difference in torsion angles associated with
certain bonds of these two compounds [K3.7(3) and
K20.2(2)8]. In our example, the torsion angles for
individual compounds for C(7)–N(1)–C(1)–C(2) bond
were 1, K7.7(3); 2, K6.1(3); 3, 7.9(4); 4, K2.5(7); 5,
4.2; 8, 2.7(5); 9, K2.0(7). There was also no evidence of an
intermolecular hydrogen bond between N–H and CN of 4,
again in contrast to what was observed for the chloro
H
Cl
O
O
Me
N
H
CN
Cl
(5)
1
S
1
Scheme 2. Graph set motif of 1.

110
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
compound. Additionally, we found that there was no
intermolecular C]O and O–H hydrogen bond formation
either. Scheme 5 shows the graph set motif representation of
the molecule.
Having established that a 2,5-difluoro substituted LFM
analog does not form inter- and intramolecular hydrogen
bonds, we next examined the crystal structure of
3,5-difluoro substituted compound (5) (Fig. 5) to compare
its characteristics with those of the corresponding 3,5-
dichloro compound. Examining the crystal structure we
found one intramolecular hydrogen bond between the C]O
and O–H group. In contrast to compound 4, compound 5 had
an intermolecular hydrogen bond between the CN and N–H
groups of the molecule as represented below in the graphic
set motif form (Scheme 6). However, we did not observe
additional intermolecular hydrogen bonding involving
C]O and O–H, which was present in the chloro substituted
compounds as discussed before.
Scheme 3. Graph set motif of 2.
1
Cl
S
(6)
O
1
H
O
N
H
Cl
Me
It appears that the presence of a fluorine atom at the
2-position of the phenyl ring may disrupt the intermolecular
hydrogen bonds between the CN and N–H groups, as well as
the intermolecular hydrogen bonds between the C]O and
O–H groups in the crystal lattice. On the other hand, when
the fluoro atom is present in a different position of the
phenyl ring the usual inter- and intra-molecular hydrogen
bonds typical of these compounds are observed in
the crystal structure. Another example of this structural
relationship is found in the previously reported 2-fluoro
phenyl substituted LFM analog (6) which has only a C]O
C
N
2
R
(12)
2
N
C
H
N
Cl
Me
O
O
H
1
Cl
S
(6)
1
Scheme 4. Graph set motif of 3.
Fig. 4. X-ray crystal structure of 4 and its crystal packing diagram.

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
111
Fig. 5. X-ray crystal structure of 5 and its crystal packing diagram.
and O–H intramolecular hydrogen bond. As with compound
4, all other hydrogen bonds were absent (Scheme 7) [16].
Hence, the 2-fluoro compounds differed from the 2-chloro
substituted analogs.
torsion angle does not influence either the absence or
presence of the intramolecular hydrogen bond in these
derivatives.
From the previous discussion, we have established that
the chloro and fluoro compounds can behave differently in
their capacity to form inter- and intra-molecular hydrogen
bonds in their solid state. We sought to determine whether
the corresponding bromo compound would behave similar
to that of the chloro or the fluoro derivative. The
2,4-dibromo substituted leflunomide (8) showed two
intramolecular hydrogen bonds between C]O and O–H
as well as N–H and Br groups (Fig. 6). This trend is similar
to that exhibited by the chloro compound and is represented
below in the graphic motif form (Scheme 9).
We also examined the X-ray crystal structure of a
3-fluoro substituted leflunomide analog (7) for comparison.
This compound had an intramolecular hydrogen bond
between C]O and O–H and also an intermolecular
hydrogen bond with CN and N–H groups as shown in the
graph set motif form (Scheme 8). This provides evidence
that the fluorine atom at the 2-position and not merely the
presence of a fluorine on the phenyl ring has a remarkable
tendency to disrupt these intermolecular hydrogen bonds in
the crystal lattice.
To explain the influence of a 2-positioned fluorine on the
hydrogen bonding in the crystal lattice we compared the
torsion angles of all LFM analogs. Table 6 provides
the torsion angles determined for the bonds of interest in
the crystal structures of the compounds. From the values it is
evident that there was no significant differences in the
torsion angles observed in most of the halo-substituted
leflunomide derivatives. This result demonstrates that the
However, the compound did not show any intermolecular
hydrogen bonding with CN group and N–H as expected.
Both the chloro and bromo compounds indicated identical
hydrogen bonding characteristics in their solid state. Also
we can rationalize that the size of the bromide is larger
and hence it forms the intramolecular hydrogen bond in the
crystal lattice. Additionally, it shows the absence of
intermolecular hydrogen bond between the CN and N–H

112
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
Table 6
1
S
(6)
F
1
Selected torsion angles
H
Compound
Bond designation
Torsion angle (deg)
O
O
1
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
N(1)–C(1)–C(6)–Cl(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
N(1)–C(1)–C(6)–F(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
N(1)–C(1)–C(2)–Br(2)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
C(7)–N(1)–C(1)–C(2)
C(7)–N(1)–C(1)–C(6)
C(1)–N(1)–C(7)–C(8)
N(1)–C(7)–C(8)–C(11)
K7.7(3)
173.81(15)
0.8(2)
N
F
Me
C
N
H
179.06(14)
3.12(2)
2
R
O
(12)
2
N
C
2
3
4
K6.1(3)
174.9(17)
K178.32(15)
0.7(2)
H
N
F
Me
7.9(4)
O
K172.6(3)
178.6(3)
K1.6(4)
-2.5(7)
H
1
F
S
(6)
1
178.5(4)
0.0(6)
Scheme 6. Graph set motif of 5.
179.7(4)
0.8(6)
evidence of hydrogen bonding through the OCF3 of the
molecule.
5
4.2(3)
Overall, our results demonstrates that 2-chloro or
2-bromo substituted leflunomide derivatives have the
tendency to form intramolecular hydrogen bonds with
N–H group, where as the 2-fluoro group disrupts the
formation of the intramolecular hydrogen bond.
K176.22(18)
177.64(17)
1.1(3)
8a
K177.4(3))
2.7(5)
K1.8(4)
178.9(3)
0.1(4)
In previous discussion, we have demonstrated that
chloride and bromide groups will form intramolecular
hydrogen bonds with the NH group, while fluoride does not.
We propose that this is due to the smaller size of the fluorine
in comparison with the other halogens. The observed atomic
volume for all the three halogen are as follows: F 17.1 cm3;
Cl 22.7 cm3; Br 23.5 cm3 [21]. Comparing the bond lengths
of carbon–halogen bonds, it is known that in general
carbon–halogen bond increases as we proceed from fluorine
9
K2.0(7)
178.1(4)
179.7(4)
K0.8(6)
a
The carbon on the phenyl ring is numbered different in the X-ray crystal
structure.
groups for the similar reason that the hydrogen atom is not
available. In summary, our results demonstrates that
2-chloro or 2-bromo substituted leflunomide derivatives
have the tendency to form intramolecular hydrogen bonds
with N–H group, where as the 2-fluoro group disrupts the
formation of the intramolecular hydrogen bond.
1
S
(6)
1
H
O
O
N
H
Me
We completed the study by examining the crystal
structure of another bromo-substituted compound (9). The
reason for choosing this compound was to observe whether
introduction of additional group such as OCF3 on the phenyl
ring has any influence on the hydrogen bond formation. The
X-ray crystal structure of the compound is shown in Fig. 7.
Compound 9 had two hydrogen bonds similar to those
exhibited by the other chloro and bromo compounds
discussed previously. This compound also formed the
intermolecular hydrogen bonding, however, there was no
F
CN
Scheme 7. Graph set motif of 6.
1
S
(6)
1
H
O
O
N
H
F
Me
C
N
2
R
(12)
2
N
C
S11(6)
F
H
N
F
Me
H
O
O
O
O
H
Me
N
H
1
S
(6)
CN
F
1
Scheme 5. Graph set motif of 4.
Scheme 8. Graph set motif of 7.

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
113
Fig. 6. X-ray crystal structure of 8 and its crystal packing diagram.
to iodine and the values are as follows: C–F 1.39 A, C–Cl:
1.78, C–Br: 1.93, C–I: 2.14. The carbon–halogen bond
lengths of lefulonamide analogs are presented in Table 4.
The observed values of the bond lengths of carbon–halogen
bonds are consistent with the values described above. Based
on these bond lengths, we conclude that there is less
opportunity for the fluoride to form a hydrogen bond with
the NH group due to its relatively shorter bond length. In
summary we propose that all the above factors may
influence hydrogen bond formation of fluorine with NH,
in comparison with other halogens.
crystal structure of the BTK kinase domain (Fig. 8).
The molecule was docked inside the binding site of
BTK. Fig. 8 shows a ball and stick model of the BTK
inhibitor LFM-A13 and illustrates the favorable orien-
tation of this molecule in the kinase active site of BTK.
The figure was first published by our group and the
details of the interaction with various amino acid
residues in the BTK site have been discussed previously
[13]. The initial lead compound was the 2,5-dibromo
compound (LFM-A13, 10) for which we estimated
1
S
(6)
1
3.1. Biological activity of leflunomide analogs
H
Br
O
O
As part of our on-going program in structure-based
design of Bruton tyrosine kinase (BTK) inhibitors, we
have designed and synthesized several leflunomide
metabolite (LFM) analogs, targeting the adenosine
triphosphate (ATP) binding site of BTK. The com-
pounds were rationally designed based on the X-ray
N
H
Me
CN
Br
(5)
1
S
1
Scheme 9. Graph set motif of 8.

114
T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
Fig. 7. X-ray crystal structure of 9 and its crystal packing diagram.
a binding constant (Ki) value of 1.4 mM. It inhibited
human BTK in vitro with an inhibition (IC50) value of
17.1G0.8 mM and recombinant BTK expressed in a
baculovirus expression vector system with an IC50 value
of 2.5 mM. We have also examined the biological
activity of 6 and 7 against EGFR. Compound 6 showed
4. Conclusion
In conclusion, we have shown that the structural
variation in the leflunomide imparts molecular anisotropy
in solid state by preferentially forming inter- and intra-
molecular hydrogen bonds. We have identified that 2-fluoro
substituted compounds have significantly different hydro-
gen bonding schemes than other leflunomide derivatives
with different substitutions on the phenyl group. Addition-
ally, we have shown that there is no significant influence of
torsion angles towards this difference in hydrogen bonding
inhibitory
activity
against
EGFR
with
an
IC50Z74.5 mM. The overall structure–activity studies
revealed that ortho substitutions in the phenyl ring
would prevent the leflunomide metabolite analog from
having a close contact with the hinge region of the EGF
receptor kinase domain. Docking studies indicated that
meta substitutions are likely sandwiched between
residues Thr766 and Asp831. Finally, the para substituted
compounds maintain a close contact with the hinge
region of the EGFR kinase domain and are stabilized by
additional contact area through the para substituents and
the residues at the inner core of the protein [22].
Most of the leflunomides formed intramolecular
hydrogen bonds between the hydroxyl group and the
amide carbonyl group. In addition, the crystal packing
showed an additional weak intermolecular hydrogen
bond between the hydroxyl group and the amide
carbonyl-O atom of the centrosymmetrically related
molecule. Our study revealed the above compound
showed that its molecular conformation is very similar
to its corresponding energy-minimized molecular coor-
dinates which were generated and used for docking
studies with BTK.
Fig. 8. BTK inhibitor compound 10 docked in the active kinase domain.

T.K. Venkatachalam et al. / Journal of Molecular Structure 743 (2005) 103–115
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115
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bond with the NH group compared to other halogen
substituted compounds. However, the absence of the CN
and N–H intermolecular bonding in the case of 2-fluoro
substituted leflunomide may be due to the crystal packing
characteristic in solid state.
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