Synthesis of Imidazo[2,1-a]phthalazines, Potential Inhibitors of p38 MAP Kinase. Prediction of Binding Affinities of Protein Ligands

Article abstract of DOI:10.1002/1521-4184(200201)335:1<7::AID-ARDP7>3.0.CO;2-L

Based upon molecular modeling, the pharmacophore of potential inhibitors of p38 MAPK (mitogen-activated protein kinases) is discussed and the predictive binding affinities are calculated. Synthesis of original diarylimidazo[2,1-a]phthalazines obtained by

Full text of DOI:10.1002/1521-4184(200201)335:1<7::AID-ARDP7>3.0.CO;2-L

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Synthesis of Imidazo[1,2-a]phthalazines 7
Sylvie Mavel,
Isabelle Thery,
Alain Gueiffier
Synthesis of Imidazo[2,1-a]phthalazines, Potential
Inhibitors of p38 MAP Kinase. Prediction of Binding
Affinities of Protein Ligands
Laboratoire de Chimie
Thérapeutique, Faculté de
Pharmacie, Tours, France
Based upon molecular modeling, the pharmacophore of potential inhibitors of p38
MAPK (mitogen-activated protein kinases) is discussed and the predictive binding
affinities are calculated. Syntheses of original diarylimidazo[2,1-a]phthalazines ob-
tained by Suzuki coupling are described.
Keywords: Imidazophthalazine; Suzuki coupling; Molecular modeling; p38 MAPK
Received: July 16, 2001 [FP 611]
showed that pyridinylimidazole interacts with the ATP
binding pocket.The pyridyl nitrogen mimics the N-1 nitro-
gen of the adenine ring of ATP.
Introduction
Mitogen-activated protein (MAP) kinases are important
signaling molecules. They participate in diverse cellular
events and are potential targets for intervention in in-
flammation, arthritis and other joint diseases, septic
shock and myocardial injury [1]. Activation of p38 MAP
kinase results in the production of IL-1 and TNF-α, sug-
gesting that inhibition of this kinase may provide a useful
therapeutic target for intervention in various diseases
mediated by these cytokines [2, 3].
In continuing our interest in the investigation of bi- and
tricyclic hetero compounds, the influences of the tricyclic
system pyridinylimidazole on the inhibition of p38 MAP
kinase were studied. Only few 2-phenylimidazo[2,1-
a]phthalazines are reported in the literature [12, 13], and
were designed as CNS agents.To the best of our knowl-
edge, no 2,3-diaryl compounds have been described. In
the light of our experience, imidazo[2,1-a]phthalazine
compounds have been prepared by our general method
previously described in the imidazopyridine series [14].
The rapid and accurate calculation of binding free ener-
gy of putative protein-ligand complexes is difficult to eval-
uate but important to consider in the structure-based
drug design. The LUDI program (MSI/Byosim, San
Diego, USA) has a simple scoring function to predict
binding constants for protein-ligand complexes of known
three-dimensional structure [4–7]. The LUDI program
positions molecular fragments or small molecules into
protein binding sites in such a way that hydrogen bonds
are formed with the protein, and lipophilic groups are
placed into hydrophobic pockets. Ionic interactions and
the number of rotatable bonds in the ligand are also
taken into account. It was shown that a very significant
correlation between the sum of atom pair potentials and
total binding free energy exists, and the sum is therefore
a good measure for estimating binding affinities [8]. It
was demonstrated for a great number of structures that
the LUDI predictive function is quite a good model for
nanomolar to 100 nM activities [9].
Results
The 2-arylimidazophthalazines 4a–c were prepared in
good yield from aminophthalazine by reaction with phen-
acyl bromide or p-fluorophenacyl bromide in refluxing
ethanol (Scheme 1). Iodination of 4a–c with N-iodosuc-
cinimide in acetonitrile at room temperature, according
to the procedure described in the imidazo[1,2-a]pyridine
series [15], gave the 3-iodo derivatives in good yield.
Treatment of iodoimidazophthalazine 5a–c by Pd(0)-
catalyzed cross coupling with aryl- or heteroarylboronic
acid under Suzuki condition [16, 17] yielded diarylimid-
azophthalazine 6a–h. Benzene, 4-fluorobenzene and
3-pyridyl boronic acids were used as well as pyridine-3-
boronic acid 1,3-propanediol cyclic ester. For this last-
mentioned compound, under the same reaction condi-
tions, yields were equivalent. The imidazophthalazine
structures are listed in Table 1.
Different pyridinylimidazole compounds, such as
SB203580, were found to be selective inhibitors of p38
MAP kinase [10, 11].These compounds have effects on
eicosanoid production, consistently inhibiting IL-1 and
TNF synthesis in human monocytes at concentrations in
the low µM range [2]. X-ray crystallographic studies
Discussion
Four distinct members of the p38 MAPK family have
been identified to date, standardized in the nomencla-
ture as p38α, β, χ and δ. These kinases are 60 to 70%
identical to each other [19, 20].Thus, the X-ray crystallo-
Correspondence: Sylvie Mavel, Laboratoire de Chimie
Thérapeutique, Faculté de Pharmacie, 31 Av. Monge,
37200 Tours, France. Phone: (+)33(0)2 47 36 72 42, Fax:
(+)33(0)2 47 36 72 39, e-mail: mavel@univ-tours.fr.
© WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
0365-6233/01/0007 $ 17.50+.50/0

8
Mavel et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Scheme 1. (a) Reaction condition:(i): KOH, phenol, 100°C, 2 h; (ii) for 3b: CH3CO2NH4, 160°C, 1 h; (iii) for 3a: NH4OH,
Parr apparatus, 120°C, 4 h;(iv):phenacyl bromide or fluorophenacyl bromide, EtOH, reflux, 4 h;(v):N-iodosuccinimide,
acetonitrile, room temperature, 45 min; (vi): Pd(PPh3)4, aryl- or heteroarylboronic acid, DME, NaOH, H2O, reflux, 8 h.
Table 1. Physical data for compounds 4, 5, 6.
R1
R2
R3
X
Yield
[%]
Mp
[°C]
Formula
Analysis
Calcd./found [%]
C, H, N
4a
4b
4c
Cl
Cl
H
F
–
–
–
–
–
–
81
75
68
156–158
236–238
210–212a
C16H10ClN3
C16H9ClFN3
C16H12N4
68.70, 3.60, 15.02
68.98, 3.61, 15.01
64.55, 3.05, 14.11
64.65, 3.06, 14.09
NH2
H
73.83, 4.65, 21.52
73.99, 4.65, 21.60
5a
5b
5c
Cl
Cl
H
F
–
–
–
–
–
–
67
68
60
190–192
206–208
194–196
C16H9ClIN3
C16H8ClFIN3
C16H11IN4
47.38, 2.24, 10.36
47.58, 2.25, 10.32
45.37, 1.90, 9.92
45.57, 1.90, 9.90
NH2
H
49.76, 2.87, 14.51
49.95, 2.88, 14.49

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Synthesis of Imidazo[1,2-a]phthalazines 9
Table 1. (continued)
R1
R2
R3
X
Yield
[%]
Mp
[°C]
Formula
Analysis
Calcd./found [%]
C, H, N
6a
6b
6c
6d
6e
6f
Cl
Cl
H
H
H
F
H
F
CH
CH
N
63
51
46
61
43
49
48
53
49
218–220
216–218
220–224
168–170
220–222
222–224
250–252
234–236
156–158
C22H14ClN3,
H2O
70.68, 4.31, 11.24
70.75, 4.30, 11.25
C22H13ClFN3
70.69, 3.51, 11.24
70.69, 3.53, 11.21
Cl
H
H
F
C21H13ClN4,
H2O
67.29, 4.03, 14.95
67.15, 4.03, 14.89
Cl
CH
CH
N
C22H13ClFN3
C22H12ClF2N3
C21H12ClFN4
C22H16N4
70.69, 3.51, 11.24
70.49, 3.52, 11.23
Cl
F
67.44, 3.09, 10.72
67.14, 3.11, 10.71
Cl
F
H
H
F
67.30, 3.23, 14.95
66.98, 3.24, 14.93
6g
6h
6i
NH2
NH2
NH2
H
H
H
CH
CH
N
78.55, 4.79, 16.66
78.26, 4.79, 16.70
C22H15FN4
74.56, 4.27, 15.81
74.21, 4.28, 15.81
H
C22H15N5,
H2O
70.97, 4.82, 19.71
71.18, 4.81, 19.70
a
in the literature 215–216°C [12].
graphic structures of native unactivated p38α (unphos-
phorylated form), or the pyrimidinylimidazole p38 com-
plex were used for the molecular modeling studies [20,
21]. It was demonstrated that the arylpyridinylimidazole
structures bind in the same site on the kinase, as does
ATP [22, 23]. Biochemical studies combined with en-
zyme mutagenesis support this model [24].The structur-
al relevance of the inactivated form of p38 to the phos-
phorylated active form is not known, although members
of the pyridinylimidazole class of inhibitors, which com-
pete with ATP, have similar affinity for the active and inac-
tive form of the enzyme [25, 26].The p38-binding site is
formed by Val38, Val52, Lys53, Glu71, Leu75, Ile84,
Leu104, Thr106, and Asp168 [20, 22, 23]. Mutagenesis
showed that a single residue difference between p38
MAPK and other MAPK is sufficient to confer selectivity
among pyridinylimidazole compounds [19, 23, 26]. The
p38 kinase inhibitors contain a common set of structural
features.A pharmacophore derived from a series of sev-
eral tri- and tetra-substituted imidazole inhibitors of p38
has been described by Gallagher et al. [27]. In general,
the inhibitors contain a 4-aryl-5-pyridinylimidazole struc-
ture [20, 21]. The 4-pyridyl substituent has been shown
to be essential for activity.
Further substitution of the imidazole at N-1 adjacent to
the pyridinyl group or at the C-2 is allowed, while substi-
tution of the imidazole nitrogen adjacent to the aryl group
is not [27]. The structure-activity relationships (SAR)
have been described with pyrrole [26] and pyrazolinone
triaryl substituents, suggesting a similar binding mode.
Optimization of binding occurs with pyrrole substituents.
Substituted pyrimidines as replacement for the 4-pyridi-
nyl group have demonstrated improved activity [28]. In
attempt to develop a ligand-binding model for inhibition
of p38 enzyme, the LUDI scoring program was used to
identify which compounds could fit into this enzyme’s ac-
tive site. We investigated, by the LUDI program, binding
interactions of the different 6a–e imidazophthalazine in
the ATP pocket of p38 and calculated the empirical bind-
ing affinities at physiological pH. To have a comparative
level, we studied some diaryl imidazole Ia–e (Table 4) in-
hibitors of p38 MAP kinase described by Liverton et al.
[29].The binding affinities Ki are only a predictive value,
with an error range of about 1–4 log Ki units. In our

10 Mavel et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Table 2. 1H-NMR data for compounds 4, 5, 6: δ (ppm).
H(3) H(7) H(8) H(9) H(10)
Others
4aa
4ba
4cb
8.08 8.15 7.68 7.88
8.27 8.54 7.92 8.27
8.28 8.28 7.72 7.90
8.63
8.76
8.38
7.42 (3 Harom), 8.00 (2 Harom
7.33 (2 Harom), 8.10 (2 Harom
6.90 (NH2), 7.25 (1 Harom), 7.43 (2 Harom), 7.90 (2 Harom
)
)
)
)
5aa
5ba
5cb
–
–
–
8.29 7.92 7.99
8.18 7.82 7.99
8.31 7.74 7.92
8.70
8.66
8.41
7.52 (3 Harom), 8.20 (2 Harom
7.23 (2 Harom), 8.26 (2 Harom
7.21 (NH2), 7.35 (1 Harom), 7.50 (2 Harom), 8.12 (2 Harom
)
)
6aa
6ba
6ca
–
–
–
8.24 7.78 7.97
8.26 7.98 7.80
8.28 7.76 7.92
8.75
8.76
8.80
7.39 (3 Harom), 7.54 (3 Harom), 7.65 (2 Harom), 7.78 (2 Harom
7.23 (2 Harom), 7.36 (3 Harom), 7.63 (2 Harom), 7.73 (2 Harom
7.57 (4 Harom), 7.92 (1 Harom), 8.13 (1 Harom), 8.28 (1 Harom),
)
)
8.88 (1 Harom), 9.05 (1 Harom
7.63 (7 Harom), 7.07 (2 Harom
)
)
6da
6ea
6fa
–
–
–
8.23 7.77 7.96
8.23 7.74 7.68
8.28 7.82 7.38
8.73
8.70
8.73
7.07 (2 Harom), 7.23 (2 Harom), 7.65 (4 Harom
7.10 (2 Harom), 7.48 (1 Harom), 7.67 (2 Harom), 8.00 (1 Harom),
8.73 (1 Harom), 8.87 (1 Harom
6.87 (NH2), 7.30 (3 Harom), 7.52 (7 Harom
6.93 (2 Harom), 7.40 (NH2, 5 Harom), 7.74 (2 Harom
7.15 (1 Harom), 7.36 (NH2, 4 Harom), 7.72 (3 Harom), 8.48 (1 Harom),
8.66 (1 Harom
)
)
6gb
6ha
6ia
–
–
–
8.26 7.75 7.92
8.38 7.77 7.96
8.37 7.73 7.95
8.46
8.72
8.70
)
)
)
a
b
solvent: CDCl3, solvent: DMSO-d6.
studies, for the structures Ia–e, with a supposed Ki Х
IC50/2, a difference of 1.5 log Ki units was found. This is
the reason why the IC50 values are given in nM when the
predictive Ki are in µM, but it permits arrangement of the
values in order of size and enables comparison with the
different structures.The predictive activity for 6c falls into
the range of experimental values reported in the imida-
zole series (between 50 and 150 nM).For 6f, g, i and 6b,
IC50 might be between 0.5 and 1 µM. “No score” means
that the LUDI program failed to place the structure into
the ATP pocket (corresponding to poor activity as for Ie
with a IC50 > 10,000 nM).The imidazophthalazine skele-
ton seemed to be an excellent structure for the inhibition
of p38 MAPK, and a 3-pyridin-3-yl substituent led to a
predictive Ki < 20 µM.The importance of a fluorine atom
on the aryl ring at C(2) could not be demonstrated (no
score for 6d and 6e).Substitution at C(6) on the phthala-
zine system by a chlorine atom or an amino group does
not contribute significantly to activity.
The binding interactions defined by the docked struc-
tures (Figure 1) are in good agreement with these de-
scribed by X-ray crystallographic studies of the pyridi-
nylimidazole inhibitor [20–23] or in the quinazoline class
[30]:
Fig. 1. ATP pocket of p38 with residues which bind, in
predictive studies, with Ia (in black) and 6c (in gray).
Lys53 and Met109 gave hydrogen bonds, Tyr35, Val38,
Leu75, Ile84, and Asp168 formed the aryl binding pock-
et.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Synthesis of Imidazo[1,2-a]phthalazines 11
Table 3. 13C-NMR for compounds 4, 5, 6: δ (ppm), J (Hz).
C(2) C(3) C(6) C(6a) C(7) C(8) C(9) C(10) C(10a) C(10b)
Others
4aa 143.0 112.8 146.9 125.9 126.7 129.1 133.5 122.8 121.6 136.2 125.7, 127.9, 128.7, 133.2
4bb 142.2 112.6 147.2 126.0 126.9 129.3 133.8 121.8 121.8 136.4 115.7 (J = 21), 127.4 (J = 8), 129.5
(J = 3), 162.7 (J = 249)
4cb 140.4 113.5 153.1 117.9 125.3 128.8 133.0 122.4 126.2 134.9 125.3, 128.8, 129.0, 135.1
5aa 147.8 98.2 140.1 125.8 127.0 129.6 133.9 122.4 121.9 136.6 128.5, 127.5, 128.3, 132.6
5ba 139.3 97.8 146.5 125.8 127.1 129.8 134.0 122.4 122.0 137.7 115.5 (J = 21), 128.1 (J = 3),
129.3 (J = 8), 162.5 (J = 248)
5cb 141.7 95.3 153.2 118.0 125.2 129.1 133.0 121.9 125.7 134.6 127.5, 127.6, 128.6, 137.2
6aa 140.2 125.1 146.8 121.8 126.9 129.1 133.5 122.9 125.8 135.7* 127.6, 128.2, 128.4, 128.6, 130.6,
133.5*
6ba 140.4 124.6 146.9 121.9 126.8 129.3 133.7 123.0 126.3 135.8 115.8 (J = 21), 124.6 (J = 3),
127.7, 128.2, 128.5, 133.8 (J = 8),
133.9, 162.8 (J = 249)
6ca 140.4 125.4 152.1 122.2 127.7 129.2 133.1 122.6 125.9 137.4 123.5, 127.7, 128.2, 128.5, 131.1,
133.0, 137.4, 150.5, 150.8
6da 139.4 125.4 146.9 121.9 126.9 129.3 133.7 122.9 126.3 135.7, 115.4 (J = 21), 128.3, 128.7, 128.7,
129.9 (J = 8), 130.2 (J = 3), 130.6,
162.5 (J = 247)
6ea 139.4 124.4 147.0 121.9 126.9 129.3 133.7 122.9 126.2 135.8, 115.4 (J = 21), 115.9 (J = 21),
124.3 (J = 3), 128.3, 130.0 (J = 8),
130.1 (J = 3), 132.4 (J = 8),
162.5 (J = 248), 162.9 (J = 248)
6fa 140.5 124.9 147.3 122.1 127.0 129.6 133.9 123.0 126.1 136.4 115.7 (J = 21), 123.5, 129.7,
130.0 (J = 8), 137.6, 130.1, 149.4,
151.0, 162.7 (J = 234)
6gb 137.4 124.9 152.6 117.8 125.0 128.7 132.8 122.3 126.0 130.2 127.0, 127.3, 128.5, 128.9, 131.1,
134.3, 135.1
6ha 139.7 124.3 153.2 117.7 124.7 128.7 133.3 122.8 127.2 134.7* 115.0 (J = 21), 121.5, 124.7 (J = 3),
127.4, 128.4 (J = 7), 129.4, 135.4*,
162.2 (J = 248)
6ia 140.6 122.0 152.9 117.8 124.6 128.9 133.3 122.8 125.2 134.2* 121.4, 122.6, 127.0, 127.7, 128.8,
135.9*, 136.9, 148.3, 150.4
a
b
solvent: CDCl3, : solvent: DMSO-d6.
– A hydrogen bond between the 3-pyridyl nitrogen (for
6c, 6f, 6i) and the amide N-H of Met109 (a hydrogen
bond between the 4-pyridyl nitrogen for SB203580 and
Met109) with a N-NH (Met109) distance of 2.2 Å. It is
analogous to the H bond which is seen for the N-1 ade-
nine of ATP in all available kinase crystal structures.
– H-bond-like interaction between Lys53 and the nitro-
gen N(1) for 6c, 6f, 6g, 6i.
(H-bond-like interaction between Lys53 and the un-
alkylated imidazole N-H with SB203580)
– Potential H-bond-like interaction with Asp168 (with
6g)

12 Mavel et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Table 4. p38 inhibition data for literature compounds [30] and predictive binding of complex p38-ligand.
a Literature inhibitory concentration in nM required to inhibit 50% of p38 MAPK. b Predictive Ki (score = –100 log Ki).
c
“-”means “no score”. ND: not determined yet.
– Hydrophobic contacts between potent inhibitor and
Val38 (with 6g), Tyr35 (with 6b), Leu75 (with 6b),
Ile84 (with 6b).The distances between the potent in-
hibitor and the enzyme are all within 4 Å.
Compounds 2, 3a and 3b were prepared following reported
methods [31–33].The structure of all the new compounds was
determined using 1H (Table 2) and 13C-NMR (Table 3) spectros-
copy and when necessary by 13C-1H correlation (XHCOR) and
long-range 13C-1H correlation (LRHETCOR).
In conclusion, we have prepared in an efficient way new
2,3-diarylimidazo[2,1-a]phthalazine derivatives which,
in their conception, are of great potential biological inter-
est as inhibitors of p38 MAP kinase.Biological evaluation
will be performed in order to confirm the model obtained
by molecular modeling studies.
6-Chloro-2-arylimidazo[2,1-a]phthalazine 4a,b and 6-amino-
2-arylimidazo[2,1-a]phthalazine 4c
A solution of aminophthalazine 3a–c (10 mmol) and 2′-bro-
moacetophenone (or 2′-bromo-4-fluoroacetophenone, or 2′-
bromopyridyn-3-ylethanone) (9 mmol) was heated under reflux
in ethanol (20 mL) for 4 h. After cooling, the solution was con-
centrated, the precipitate was filtered and washed with diethyl
ether.The crude product was separated by flash chromatogra-
phy on silica gel, eluted with ethyl acetate/hexane (5/5 v/v) to
give 4a–c.
Experimental Section
6-Chloro-3-iodo-2-arylimidazo[2,1-a]phthalazine 5a,b and 6-
amino-3-iodo-2-arylimidazo[2,1-a]phthalazine 5c
All melting points were determined on a Kofler hot-plate melting
point apparatus and were uncorrected. 1H and 13C-NMR spec-
tra were recorded on a Bruker DPX-200 spectrometer (SAVIT,
Tours, France). Elemental analysis were performed by Micro-
analytical Center, ENSCM, Montpellier, France.
N-Iodosuccinimide (2.2 mmol) was added to a solution of imid-
azophthalazine 4a–c (2 mmol) in 5 mL acetonitrile.The result-
ing mixture was stirred at room temperature for 45 min.The pre-

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 7–14
Synthesis of Imidazo[1,2-a]phthalazines 13
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column and eluted with ethyl acetate/hexane (10/90 v/v) to give
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6-Chloro-2,3-diarylimidazo[2,1-a]phthalazine 6a–f and 6-ami-
no-2,3-diarylimidazo[2,1-a]phthalazine 6g–i
To a mixture of 3-iodoimidazophtalazine 5a–c (0.5 mmol) in
4 mL dimethoxyethane (DME), under nitrogen, Pd(PPh3)4
(0.025 mmol), aryl boronic acid or pyridine-3-boronic acid 1,3-
propanediol cyclic ester (0.55 mmol), and 2 mL of 4 N aqueous
sodium hydroxide solution were added. The reaction mixture
was heated under reflux for 8 h. After cooling, the mixture was
poured into a brine solution (100 mL) and then extracted with
dichloromethane. The organic layers were dried over an-
hydrous magnesium sulfate and evaporated to dryness under
vacuo. The crude product was separated by chromatography
on a flash silica gel column and eluted with dichloromethane/
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