Synthesis of 2,6-diphenylpyrazine derivatives and their DNA binding and cytotoxic properties

Article abstract of DOI:10.1016/j.ejmech.2005.07.003

A series of 2,6-diphenylpyrazine derivatives was synthesized from 2,6-dichloropyrazine and 4-methoxyphenylboronic acid using palladium(0) as catalyst in a Suzuki methodology. After deprotection of the hydroxyl, alkylation reactions with different halides

Full text of DOI:10.1016/j.ejmech.2005.07.003

European Journal of Medicinal Chemistry 40 (2005) 1206–1213
www.elsevier.com/locate/ejmech
Original article
Synthesis of 2,6-diphenylpyrazine derivatives
and their DNA binding and cytotoxic properties
Nathalie Dias b, Ulrich Jacquemard a, Brigitte Baldeyrou b, Amélie Lansiaux b,
Jean-François Goossens c, Christian Bailly b,1, Sylvain Routier a,*, Jean-Yves Mérour a
a Institut de Chimie Organique et Analytique, UMR CNRS 6005, Université d’Orléans, B.P. 6759, 45067 Orléans cedex 2, France
b Inserm U-524, Centre Oscar Lambret, IRCL, Place de Verdun, 59045 Lille, France
c Laboratoire de Chimie Analytique, Faculté de Pharmacie, Université de Lille 2, 59006 Lille, France
Received 6 April 2005; received in revised form 22 June 2005; accepted 1 July 2005
Available online 08 September 2005
Abstract
A series of 2,6-diphenylpyrazine derivatives was synthesized from 2,6-dichloropyrazine and 4-methoxyphenylboronic acid using palla-
dium(0) as catalyst in a Suzuki methodology. After deprotection of the hydroxyl, alkylation reactions with different halides afforded com-
pounds 5–8 bearing hydrophilic chains. DNA binding and cytotoxic properties were investigated. Compound 11 bearing imidazoline terminal
groups was found to be a potent AT-specific DNA minor groove binder but there was no relationship between DNA interaction and cytotox-
icity. However, in all cases the incorporation of the pyrazine ring was found to promote the cytotoxicity of the molecules compared to the
corresponding pyridine analogues, previously synthesized.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Pyrazine derivatives; Cytotoxicity; DNA binding; Anticancer agents
1. Introduction
pound furamidine which is a highly promising anti-infectious
agent and a potent AT-selective DNA binder [9]. This latter
bis-amidine series has been considerably expanded over the
past 10 years to provide a repertoire of potent DNA recogni-
tion molecules all possessing an extended unfused aromatic
system. This approach has yield a considerable variety of
therapeutically active compounds useful to combat various
types of infectious diseases, such asAfrican trypanosomiasis
and Pneumocystis carinii pneumonia for examples [9]. All
these molecules represent a rich source of information from
which we can design novel categories of DNA recognition
compounds. With this idea in mind, we have recently reported
the synthesis of a series of bis-phenylpyridine derivatives sub-
stituted with different side chains, neutral or cationic [10]. A
few cytotoxic molecules were identified but their antiprolif-
erative activity did not correlate with DNA binding. In an
extension of this work, we report here the synthesis of a fol-
low up series of molecules for which the pyridine central ring
has been replaced with a pyrazine ring. Eight molecules
(Scheme 1) have been prepared and the DNA binding and
cytotoxic properties were investigated for six of them which
could be directly compared to their pyridinic analogs.
DNA remains an attractive target for the design of antitu-
mor and antiparasitic agents [1,2]. For more than 20 years,
medicinal chemists have elaborated small molecules capable
of reading the genetic information embedded in the DNA base
pairs. Successful strategies have been built in four broad,
structurally distinct series of molecules that bind to the minor
groove of DNA: (i) hairpin polyamide essentially composed
of pyrrole- and imidazole-carboxamide units that remark-
ably sense the minor groove surface of DNA to distinguish
almost all types ofA-T and G-C base pair combinations [3,4],
(ii) bis and ter-benzimidazoles showing tight binding to DNA
and in some cases inhibition of topoisomerase I coupled with
potent antitumor activities [5,6], (iii) pyrrolobenzodiazepine
and cyclopropylpyrroloindole DNA damaging agents [7,8]
and (iv) bis-amidines derived from the diphenylfuran com-
* Corresponding author.
E-mail address: sylvain.routier@univ-orleans.fr (S. Routier).
1 Present address: Centre de Recherche en Oncologie Expérimentale, Ins-
titut de Recherche Pierre Fabre, 3, rue des satellites, 31400 Toulouse, France.
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.07.003

N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
1207
Scheme 1. a) 4-Methoxyphenylboronic acid 2 (2.5 eq.), Pd(PPh3)4 , toluene, EtOH, NaHCO3 satd., 14 h., quant.; b) BBr3 (4.2 eq.), CH2Cl2, 0 °C to r.t., 3 h.,
98%; c) 2-dimethyl aminoethyl chloride hydrochloride (2.2 eq.), Cs2CO3 (4.4 eq.), DMF, r.t. to 100 °C, 2 h., 65%; d) same procedure as c) with
3-dimethylaminopropyl chloride hydrochloride (2.2 eq.), 67%; e) 1-bromo-2-benzyloxyethane (2.2 eq.), Cs2CO3 (2.2 eq.), DMF, r.t. to 100 °C, 1 h., 63%; f)
ClCH2CN (3.0 eq.), K2CO3 (2.2 eq.), acetone, 24 h, 70%; g) from 7, BBr3 (6.2 eq.), CH2Cl2, 0 °C to r.t., 2 h., 97%; from 8 : h) BH3.THF (10.0 eq.), THF, rflx,
15 h, 50% i) ethylenediamine, P2S5 (cat.), 120 °C, 4 h, 80%.
2. Chemistry
detectable trace of the slight excess of 1-benzyloxy-2-
bromoethane used. The last alkylation reaction was per-
formed with chloroacetonitrile [15]. The optimization of this
reaction leads us to use K2CO3 as a base in refluxing acetone
for 24 h to afford compound 8 in 70% yield. In a general
manner, one can imagine that the moderate yield of all the
aforementioned phenol alkylation results from an unwanted
reaction of the nitrogen pyrazine atoms providing highly water
soluble compounds which were lost during the washes or
make the purification more difficult by flash chromatogra-
phy.
We have performed some reactions to generate the useful
hydrophilic functions masked on compounds 7 and 8 (Scheme
1). Debenzylation was performed on 7 using BBr3 at room
temperature for 2 h to afford the bis alcohol 9 in 98% yield.
Nitrile functions on 8 were first used to obtain primary amines
and then to generate dihydroimidazole rings. In each case,
we adapted the experimental conditions to obtain only sym-
metrical compounds. First reduction of the nitrile 8 was real-
ized in the presence of a large excess of 1 M borane solution
in refluxing THF for 15 h. Despite our numerous efforts, the
compound 10 was obtained only in a 50% yield. Other reduc-
ing agents such as LiAlH4 gave lower yields. Heating 8 in
freshly distillated ethylenediamine with a catalytic amount
of P2S5 led to the imidazoline compound 11 in 80% yield
after only 4 h [16].
In a recent article we reported the synthesis of various 2,5-
and 2,6-diphenylpyridinyl derivatives [10]. A similar syn-
thetic sequence was d eveloped to prepare the new 2,6-
disubstituted pyrazine compounds (Scheme 1). Starting from
2,6-dichloropyrazine 1, a Suzuki biphasic cross coupling pro-
cedure using the boronic acid 2 in excess (2.5 eq) afforded
the bis derivative 3 in a quantitative yield [11–13]. The
replacement of 2,6-dichloropyrazine 1 by the 2,6-dibromo-
pyridine gave equivalent results with similar yields and times
of reaction to obtain the bis-substituted compound 3. The dem-
ethylation of 3 with BBr3 at room temperature led to the phe-
nolic compound 4 in an excellent 98% yield. A synthesis of
compounds 3 and 4, using Grignard derivatives in presence
of dichloropyrazine and Ni(dpp)2Cl2 in THF at room tem-
perature, has been previously reported in the literature [14].
In this case, the yield (only given for compound 4) was lim-
ited to 55%.
The alkylation of 4 was performed using different halides
and cesium carbonate as a base in DMF at 100 °C. The
2-chloroethyldimethylamine hydrochloride salt is first neu-
tralized for 30 min at room temperature using cesium carbon-
ate in DMF prior to add this solution to compound 4. Com-
pound 5 was obtained after 2 h in 65% yield. It is important
to follow this two-steps procedure because we noted that the
yield of this reaction dropped dramatically when all compo-
nents were added altogether in one time. Modifying the nature
of the base or the replacement of DMF by THF gave much
lower yields. Similar conditions used for the 3-dimethyl-
aminopropyl chloride hydrochloride salt afforded compound
6 in 57% yield. The other alkylation reactions did not require
any pretreatment of the halogenated compound. Thus the
1-benzyloxy-2-bromoethane was reacted with 4 in the pres-
ence of cesium carbonate to give 7. This reaction was com-
pleted after 2 h but the yield of 7 was relatively modest (63%).
No starting material was recovered and there was also no
3. Results
3.1. DNA interaction
Fig. 1 illustrates the absorption spectra of compounds 5
and 11 in an aqueous buffer in the presence of increasing
amounts of calf thymus DNA. In both cases, the binding of
the drugs to DNA results in considerable spectral changes,
characterized by a large bathochromic shift (16 nm) and a

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N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
Fig. 1
.
(Top) Absorption and (bottom) CD DNA titration of compounds 5 and 11 in BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA).
Aliquots of a concentrated calf thymus DNA solution were added to 1 ml of a drug solution (20 µM for the absorbance and 50 µM for the CD measurements).
The drug/DNA ratios increased from 0 to 20 (arrows).
marked hypochromism (about 30%). The red-shift of the drug
absorption band was very pronounced with 5, 6 and 11,
whereas there was no shift with 4 and 9 (Table 1). These two
latter compounds bear OH terminal groups on both sides of
the diphenylpyrazine core and are uncharged. The extent of
binding is increased when the OH terminal groups of 9 are
replaced with NH2 groups in 10 and the substitution of these
amines with methyl substituents, as in 5, further promotes
DNA interaction, as expected. The length of the alkyl chain
with two or three CH2 units makes no difference in terms of
spectral changes and the melting temperature measurements
also indicate that these two compounds 5 and 6 exhibit simi-
homopolymer poly(dAT)2. Fig. 2 illustrates the concentration-
dependence for the thermal stabilization of poly(dAT)2 by
the different compounds. The magnitude of the melting tem-
perature shifts measured with poly(dAT)2 suggests that the
affinity of the compounds for DNA rank in the order: 11 > 5,
6 > 10 >>> 9, 4. The imidazoline compound shows stronger
binding to DNA than the other compounds in the series. The
DTm value reaches 30 °C at a 11/poly(dAT)2 ratio of 0.5.
This corresponds to a reasonably high affinity for DNA. For
this compound, an apparent binding constant of Kapp = 4.3
(
0.5) 105 M−1 was measured using an ethidium displace-
ment fluorescence assay. This value is six times higher than
that calculated for 10 (Kapp = 7.3 ( 0.8) 104 M−1) for
example.
The important spectral changes (large red-shift and hypo-
chromism) observed with 11 or 5 (Fig. 1) in the presence of
DNA can be attributed to the binding of the drugs in a lower
lar affinities for DNA. The DTm values (Tmdrug–DNA complex
–
TmDNA alone) reach about 16 °C calf thymus DNA (42% GC
bp) at a drug/DNA ratio of 0.5 whereas a DTm of about 7 °C
was recorded with the free amino compound 10 (Table 1).
There was no increase of the Tm in the presence of the
uncharged OH compounds 4 and 9, whatever the DNA used,
be it with calf thymus DNA or the synthetic alternating
Table 1
DNA binding properties
kmax
Bound
(nm)
343
Hypochromism DTma
%
(°C)
Free
(nm)
343
338
340
342
338
334
Dk
(nm)
0
4
5
6
9
10
11
9.6
0
354
16
16
0
33.7
33.5
0.7
15.2
17.1
0
356
342
Fig. 2. ,
Melting temperature variation DTm (Tmdrug–DNA complex – TmDNA alone
350
12
7.7
6.8
20.5
in °C) of poly(dAT)2 after incubation with the diphenylpyrazines at increasing
drug/DNA-phosphate. Tm measurements were performed in BPE buffer pH
7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), in 1 cm quartz cuvet-
tes at 260 nm with a heating rate of 1 °C min–1. The Tm values were obtai-
ned from first-derivative plots.
350
16
28.2
a Tm measurements were performed in BPE buffer pH 7.1 (6 mM
Na2HPO4, 2 mM Na2H2PO4, 1 mM EDTA) using 10 µM drug and 20 µM
calf thymus DNA (nucleotide concentration) with a heating rate of 1 °C min–1
.

N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
1209
polarity environment relative to the bulk aqueous solvent. But
different binding modes can account for the spectral pertur-
bation and it is never possible to distinguish the mode of inter-
action of a drug with DNA from absorption measurements
alone. Intercalation and groove binding can lead to similar
and important spectral variations. Therefore, to approach the
drug binding mechanism, we used circular dichroism (CD)
which is one of the spectroscopic methods suitable to deter-
mine drug binding mode. As shown in Fig. 1, the addition
calf thymus DNA to a solution of 11 induces a marked increase
of the CD signal centered at 350 nm. A high positive induced
CD is typically observed with DNA minor groove binders. In
contrast, intercalating agents generally give no induced CD
or weakly negative signals in the drug absorption band [17].
A positive CD signal was only recorded with 11; there was
very little induced CD with 5 at 345 nm (Fig. 1) and the same
weak signals with 6 (data not shown). The imidazoline com-
pound 11 which exhibits the highest affinity for DNA is also
the only potent minor groove binder in the series, probably
because of the terminal imidazole groups which may prevent
the rapid dissociation of the drug–DNA complexes. Consid-
ering this, it is therefore easy to explain the footprinting gel
presented in Fig. 3 which shows most clearly that 11 is the
only molecule in the series which blocks the cleavage of DNA
by the endonuclease DNase I at-specific sites. Clear foot-
prints develop at several sites along the sequence of the 265-bp
DNA restriction fragment used in these experiments in the
presence of 11 at 5, 10 or 20 µM, whereas there was abso-
lutely no effect with the other molecules, even at the highest
concentration tested (20 µM). For the dimethylamino com-
pounds 5 and 6, either their affinity for DNA is not suffi-
ciently high, or most likely they form insufficiently stable
drug–DNA minor groove complexes. The densitometric
analysis of the gel presented in Fig. 4 reveals that the dihy-
droimidazole molecule preferentially recognizesAT-rich DNA
sequences. The footprints coincides with the position of three
strictlyAT sites: 5′-ATTA, 5′-ATTAA and 5′-AAAA (Fig. 4).
Fig. 3
.
DNase I footprinting of the diphenylpyrazines on the 265-bp EcoRI-
PvuII DNA fragment from pBS. The DNA was 3′-end labeled with
[a-32P]dATP in the presence of AMV reverse transcriptase. The products of
nuclease digestion were resolved on an 8% polyacrylamide gel containing
8 M urea. Each drug was tested at 5, 10 or 20 µM. Control tracks (Ct) contai-
ned no drug. The tracks labeled “G” represent dimethylsulfate-piperidine
markers specific for guanines.
3.2. Cytotoxicity
The antiproliferative activity of the molecules was inves-
tigated using human leukemia CEM cells after treatment for
72 h with the test drugs. IC50 values are collated in Table 2.
As in the previous series of diphenylpyridine compounds [10],
we regrettably found no direct correlation between DNA inter-
action and cytotoxicity for the pyrazine molecules. The potent
DNA binder 11 shows no higher cytotoxicity than the two
uncharged molecules 4 and 9 that practically do not bind to
DNA. The most cytotoxic compounds are the dimethylami-
noalkyl molecules 5 and 6, with IC50 values in the ~5 µM
range. An interesting point, however, is to compare the cyto-
toxicity of the pyrazine molecules with that of the corre-
spond compounds A–F in the pyridine series (Table 2). In all
cases, the pyrazines proved to be more cytotoxic that the
pyridines. The two alcohol compounds A and D which were
totally inactive in the pyridine series now become relatively
cytotoxic in the pyrazine series. The same is true for the imi-
dazoline compound for which the IC50 value is also signifi-
cantly reduced as a consequence of the pyridine → pyrazine
substitution. The additional heterocyclic nitrogen thus plays
a role to promote the cytotoxic action of the molecules. This
effect cannot be attributed to DNA recognition (the pyrazine
and pyridine molecules show roughly comparable affinities
for DNA) but it may be a consequence of a facilitated uptake
in cells and/or interaction with targets other than nucleic acids.
Some compounds in the series were found to inhibit two pro-
tein kinases (Dr. L. Meijer, CNRS, Roscoff, France, personal
communication) and we cannot exclude the possibility that
kinase inhibition contribute to the cytotoxic action in some
cases.

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N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
Fig. 4
.
Differential cleavage plots comparing the susceptibility of the 265-mer DNA fragment to DNase I cutting in the presence of 10 µM (×) 11, (C) 9 and (n)
10. Negative values correspond to a ligand-protected site and positive values represent enhanced cleavage. Vertical scales are in units of ln(fa) – ln(fc), where fa
is the fractional cleavage at any bond in the presence of the drug and fc is the fractional cleavage of the same bond in the control, given closely similar extents
of overall digestion. Each line drawn represents a three-bond running average of individual data points, calculated by averaging the value of ln(fa) – ln(fc) at any
bond with those of its two nearest neighbors. Only the region of the restriction fragment analyzed by densitometry is shown.
reported in cm−1. MS spectra (Ion Spray) were performed on
Table 2
Cytotoxicity [IC50
a
]
a Perkin Elmer Sciex PI 300. Monitoring of the reactions was
performed using silica gel TLC plates (silica Merck 60 F254).
Spots were visualized by UV light at 254 and 356 nm. Col-
umns chromatography were performed using silica gel 60
(0.063–0.200 mm, Merck).
Pyridine (X = CH)
Pyrazine (X = N)
14 1.9
4.1.1. 2,6-Bis-(4-methoxy-phenyl)-pyrazine (3) [12,13]
A solution of 2,6-dichloropyrazine 1 (500 mg, 3.36 mmol)
and 4-methoxyphenylboronic acid (1.28 g, 8.39 mmol) in a
mixture of toluene (33 ml), ethanol (20 ml) and aqueous satu-
rated NaHCO3 solution (14 ml) was degassed by argon bub-
bling for 20 min. Pd(PPh3)4 (10% mol) was added and the
mixture was immediately transferred in an pre-heated oil bath
and refluxed for 14 h. After hydrolysis (50 ml), the aqueous
layers were extracted with ethyl acetate (2 × 50 ml), washed
with brine (50 ml) then dried over MgSO4, and filtered. The
solvents were removed under reduced pressure and the crude
material was purified by flash chromatography (ether
petroleum/ethyl acetate 7:3) to afford compound 3 as a white
solid (900 mg, quant.). Rf (ether petroleum/ethyl acetate 7:3):
0.38; m.p. 137 °C [18]; IR (KBr, cm−1) m 1605, 1513, 1426,
1305, 1249, 1165, 1017, 829; 1H-NMR (CDCl3, 250 MHz):
d 3.85 (s, 6H), 7.01 (d, 4H, J = 11.4 Hz), 8.07 (d, 4H,
J = 11.4 Hz), 8.81 (s, 2H); 13C-NMR (CDCl3, 62.5 MHz): d
55.4 (2 × CH3), 114.4 (4 × CH), 128.4 (4 × CH), 129.2 (2 ×
Cq), 138.5 (2 × CH), 151.0 (2 × Cq), 161.2 (2 × Cq); MS
(IS): 293 (M + 1)+;Anal. calcd for C18H16N2O2: C, 73.96; H,
5.52; N, 9.58. Found: C, 73.68; H, 5.67; N, 9.69.
R = OH
A
> 30
4
5
6
9
R = O–(CH2)2–N(CH3)2
R = O–(CH2)3–N(CH3)2
R = O–(CH2)2–OH
B
C
D
E
F
16.3 1.4
5.4 0.5
> 70
5.7 1.6
4.8 1.8
16.3 0.3
R = O–(CH2)2–NH2
25.6 2.3
40.9 1.2
10
11
12.7 2.5
15.5 3.1
R = O–CH2-imidazole
a Drug concentration (µM) that inhibits CEM leukemia cell growth by 50%
after incubation in liquid medium for 72 h.
In conclusion, we have synthesized novel diphenylpyra-
zine molecules and investigated their DNA binding and cyto-
toxic properties. Three major points can be raised from this
study: (1) One compound, bearing imidazoline terminal
groups, acts as a potent AT-specific DNA minor groove rec-
ognition element. (2) There is no relationship between DNA
interaction and cytotoxicity but the replacement of the pyri-
dine by a pyrazine ring promotes the cytotoxicity of the mol-
ecules. (3) A versatile synthetic method has been elaborated
to build the bis-phenol-pyrazine unfused aromatic core which
can easily substituted to afford a repertoire of symmetrical
molecules functionalized with various side chains.
4. Experimental section
4.1.2. 2,6-Bis-(4-hydroxy-phenyl)-pyrazine (4) [12,13]
To a solution of compound 3 (1 g, 3.73 mmol) in CH2Cl2
(20 ml) at 0 °C, BBr3 (15.67 ml, 1 M in CH2Cl2, 15.67 mmol)
was added dropwise. After 3 h at room temperature, the reac-
tion mixture was poured into ice (100 g), the aqueous layers
were neutralized with NaHCO3 then successively extracted
with ethyl acetate (2 × 50 ml), dried over MgSO4, and fil-
tered. The solvents were removed under reduced pressure and
the crude material was purified by flash chromatography col-
umns (dichloromethane/methanol 96:4) to afford compound
4 as a yellow solid (900 mg, 98%). Rf (dichloromethane/
methanol 96:4): 0.25; m.p. 155 °C Dec.; IR (KBr, cm−1) m
4.1. Chemistry
1H-NMR and 13C-NMR spectra were recorded on a Bruker
DPX 250 instrument using CDCl3 or DMSO-d6. The chemi-
cal shifts are reported in ppm (d scale) and all coupling con-
stants (J) values are in hertz (Hz). The splitting patterns are
designated as follows: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and dd (doublet doublet). Melting
points are uncorrected. IR absorption spectra were obtained
on a Perkin Elmer PARAGON 1000PC and values were

N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
1211
3418, 2648, 1609, 1590, 1489, 1268, 1207, 1176, 833;
4.1.5. 2,6-Bis-[4-(2-benzyloxy-ethoxy)-phenyl]-pyrazine
(7)
A solution of 4 (500 mg, 1.89 mmol) and Cs2CO3 (1.36 g,
1H-NMR (DMSO-d6, 250 MHz) : d 6.93 (d, 4H, J = 8.5 Hz);
8.10 (d, 4H, J = 8.5 Hz); 8.98 (s, 2H); 13C-NMR (DMSO-d6,
62.5 MHz): d 115.8 (4 × CH), 126.8 (2 × Cq), 128.3 (4 ×
CH), 137.6 (2 × CH), 150.4 (2 × Cq), 159.3 (2 × Cq); MS
(IS): 265 (M + 1)+;Anal. calcd for C16H12N2O2: C, 72.72; H,
4.58; N, 10.60. Found: C, 73.05; H, 4.41; N, 10.73.
4.17 mmol) in DMF (30 ml) was stirred under argon at room
temperature, for 30 min. 1-Bromo-2-benzyloxyethane (719 µl,
4.54 mmol) was added and the mixture was heated up to
100 °C. After 2 h, water (30 ml) was added and the aqueous
layers were extracted with ethyl acetate (3 × 50 ml). The com-
bined organic layers were washed with brine (3 × 150 ml),
dried over MgSO4, and filtered. The solvents were removed
under reduced pressure and the crude material was purified
by flash chromatography (ethyl acetate/ether petroleum 1:1)
to afford compound 7 as a white solid (630 mg, 63%). Rf
(ethyl acetate/ether petroleum 1:1): 0.39; m.p. 87 °C; IR (KBr,
cm−1) m 2882, 1606, 1514, 1431, 1244, 1100, 831; 1H-NMR
(CDCl3, 250 MHz): d 3.85 (dd, 4H, J = 3.2, 5.0 Hz), 4.21 (t,
4H, J = 4.6 Hz), 4.64 (s, 4H), 7.05 (d, 4H, J = 8.7 Hz), 7.28–
7.39 (m, 10H), 8.08 (dd, 4H, J = 1.8, 7.7 Hz), 8.82 (s, 2H);
13C-NMR (CDCl3, 62.5 MHz): d 67.6 (2 × CH2), 68.5 (2 ×
CH2), 73.5 (2 × CH2), 115.1 (4 × CH), 127.8 (5 × CH), 128.4
(4 × CH), 128.5 (5 × CH), 129.4 (2 × Cq), 138.0 (2 × Cq),
138.5 (2 × CH), 151.1 (2 × Cq), 160.4 (2 × Cq); MS (IS) :
533 (M + 1)+;Anal. calcd for C34H32N2O4: C, 76.67; H, 6.06;
N, 5.26. Found: C, 76.34; H, 6.21; N, 5.38.
4.1.3. 2,6-Bis-[4-(2-Dimethylamino-ethoxy)-phenyl]-pyra-
zine (5)
To a solution of compound 4 (200 mg, 0.76 mmol) was
added in one portion Cs2CO3 (543 mg, 1.67 mmol) in dry
DMF (10 ml) under argon at room temperature. In parallel, a
solution of 2-chloroethyldimethylamine hydrochloride
(240 mg, 1.67 mmol) in DMF (10 ml) was stirred in presence
of Cs2CO3 (741 mg, 2.27 mmol). After 30 min, this solution
was added to the pyrazine solution. The mixture was heated
up to 100 °C for 2 h. After 2 h, water (50 ml) was added and
the aqueous layers were extracted successively with ethyl
acetate (2 × 25 ml) and dichloromethane (2 × 25 ml). The
combined organic layers were dried over MgSO4, and fil-
tered. The solvents were removed under reduced pressure and
the crude material was purified by flash chromatography
(dichloromethane/methanol/triethylamine 80:20:0.01) to
afford compound 5 as a white solid (200 mg, 65%). Rf
(dichloromethane/methanol/triethylamine 80:20:0.01): 0.43;
m.p. 97 °C; IR (KBr, cm−1) m 2771, 1608, 1513, 1437, 1247,
4.1.6. 2,6-Bis-(4-cyanomethoxy-phenyl)-pyrazine (8)
To a solution of compound 4 (1.20 g, 4.54 mmol) in acetone
(50 ml), K2CO3 (1.38 g, 10.00 mmol) and chloroacetonitrile
(864 µl, 13.65 mmol) were added. After 24 h at reflux, the
reaction mixture was cooled to room temperature, water was
added (100 ml), and the mixture was extracted with EtOAc
(3 × 50 ml). The organic layers were then dried over MgSO4,
and filtered. The solvents were removed under reduced pres-
sure and the residue was crystallized in a mixture of ethyl
acetate/petroleum ether 90:10, to afford compound 8 as a
white solid (1.09 mg, 70%). Rf (petroleum ether/ethyl acetate
1:1): 0.13; m.p. 148 °C; IR (KBr, cm−1) m 2980, 2250, 1607,
1
1170, 1023, 833; H-NMR (CDCl3, 250 MHz): d 2.37 (s,
12H), 2.78 (t, 4H, J = 5.6 Hz), 4.15 (t, 4H, J = 5.7 Hz), 7.06
(dd, 4H, J = 2.0, 6.9 Hz), 8.09 (dd, 4H, J = 1.8, 6.8 Hz), 8.84
(s, 2H); 13C-NMR (CDCl3, 62.5 MHz): d 46.0 (4 × CH3),
58.3 (2 × CH2), 66.2 (2 × CH2), 115.1 (4 × CH), 128.4 (4 ×
CH), 129.3 (2 × Cq), 138.5 (2 × CH), 151.1 (2 × Cq), 160.5
(2 × Cq); MS (IS): 407 (M + 1)+;Anal. calcd for C24H30N4O2:
C, 70.91; H, 7.44; N, 13.78. Found: C, 71.23; H, 7.30; N,
13.95.
4.1.4. 2,6-Bis-[4-(3-Dimethylamino-propoxy)-phenyl]-
pyrazine (6)
1
1512, 1437, 1287, 1225, 1172, 831; H-NMR (CDCl3,
Same procedure as described for 5: Compound 4 (200 mg,
0.76 mmol), Cs2CO3 (543 mg, 1.67 mmol), DMF (10 ml),
room temperature, 3-chloropropyldimethylamine hydrochlo-
ride (240 mg, 1.67 mmol), Cs2CO3 (741 mg, 2.27 mmol),
2 h, flash chromatography (dichloromethane/methanol/
triethylamine 80:20:0.01). Compound 6 was obtained as a
white solid (188 mg, 57%). Rf (dichloromethane/methanol/
triethylamine 80:20:0.01): 0.53; m.p. 76 °C; IR (KBr, cm−1)
m 2942, 2768, 1607, 1515, 1432, 1248, 1176, 1055, 828;
1H-NMR (CDCl3, 250 MHz) : d 1.99 (q, 4H, J = 6.5 Hz),
2.27 (s, 12H), 2.48 (t, 4H, J = 6.9 Hz), 4.09 (t, 4H, J = 6.6 Hz),
7.03 (d, 4H, J = 8.7 Hz), 8.08 (d, 4H, J = 8.9 Hz), 8.82 (s,
2H); 13C-NMR (CDCl3, 62.5 MHz): d 27.6 (2 × CH2), 45.6
(4 × CH3), 56.4 (2 × CH2), 66.4 (2 × CH2), 114.9 (4 × CH),
128.3 (4 × CH), 129.1 (2 × Cq), 138.5 (2 × CH), 151.1 (2 ×
Cq), 160.6 (2 × Cq); MS (IS): 435 (M + 1)+; Anal. calcd for
C26H34N4O2: C, 71.86; H, 7.89; N, 12.89. Found: C, 71.54;
H, 7.70; N, 13.07.
250 MHz): d 4.86 (s, 4H), 7.14 (d, 4H, J = 8.7 Hz), 8.16 (d,
4H, J = 8.7 Hz), 8.91 (s, 2H); 13C-NMR (CDCl3, 62.5 MHz) :
d 53.7 (2 × CH2), 115.0 (2 × Cq), 115.6 (4 × CH), 128.9 (4 ×
CH), 131.7 (2 × Cq), 139.4 (2 × CH), 150.2 (2 × Cq), 158.15
(2 × Cq); MS (IS): 343 (M + 1)+;Anal. calcd for C20H14N4O2:
C, 70.17; H, 4.12; N, 16.36. Found: C, 69.80; H, 4.26; N,
16.50.
4.1.7. 2,6-Bis-[4-(2-hydroxy-ethoxy)-phenyl]-pyrazine (9)
To a solution of compound 7 (531 mg, 1 mmol) in CH2Cl2
(20 ml) at 0 °C, BBr3 (6.18 ml, 1 M in CH2Cl2, 6.18 mmol)
was added dropwise. After 3 h at room temperature, the reac-
tion mixture was poured into ice (100 g), extracted with
EtOAc (2 × 30 ml) then dried over MgSO4, and filtered. The
solvents were removed under reduced pressure and the crude
material was purified by flash chromatography (dichloro-
methane/methanol 90:10) to afford compound 9 as a yellow
solid (350 mg, 98%). Rf (dichloromethane/methanol 90:10):

1212
N. Dias et al. / European Journal of Medicinal Chemistry 40 (2005) 1206–1213
0.54; m.p. 191 °C Dec.; IR (KBr, cm−1) m 3380, 2926, 1607,
1514, 1433, 1253, 1177, 1052, 827; H-NMR (DMSO-d6,
RTE111 cryostat. The Tm measurements were performed in
BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA). The temperature inside the cuvette (10 mm path-
length) was increased over the range 20–100 °C with a heat-
ing rate of 1 °C min–1. The “melting” temperature Tm was
taken as the mid-point of the hyperchromic transition.
1
250 MHz): d 3.75 (t, 4H, J = 5.2 Hz), 4.08 (t, 4H, J = 4.7 Hz),
7.12 (d, 4H, J = 8.7 Hz), 8.21 (d, 4H, J = 9.3 Hz), 9.07 (s,
2H); 13C-NMR (DMSO-d6, 62.5 MHz): d 59.5 (2 × CH2),
69.7 (2 × CH2), 114.9 (4 × CH), 128.3 (4 × CH), 129.2 (2 ×
Cq), 138.3 (2 × CH), 150.1 (2 × Cq), 160.3 (2 × Cq); MS
(IS): 353 (M + 1)+;Anal. calcd for C20H20N2O4: C, 68.17; H,
5.72; N, 7.95. Found: C, 67.86; H, 5.89; N, 8.08.
4.2.2. CD spectroscopy
CD spectra were recorded on a J-810 Jasco dichrograph.
Solutions of drugs, DNA and their complexes (1 ml in BPE
buffer) were scanned in 1 cm quartz cuvettes. Measurements
were made by progressive addition of concentrated calf thy-
mus DNA to a drug solution at 50 µM to obtain the desired
drug/DNA ratio. Five scans were accumulated and automati-
cally averaged.
4.1.8. 2,6-Bis-[4-(2-amino-ethoxy)-phenyl]-pyrazine (10)
To a solution of compound 8 (200 mg, 0.58 mmol) in THF
(15 ml) at room temperature, BH3·THF (5.85 ml, 1 M in THF,
5.85 mmol) was added dropwise. After 15 h at reflux, the
reaction mixture was cooled to 0 °C, and then methanol was
added dropwise to neutralize the excess of BH3·THF. The
solvents were removed under reduced pressure and the crude
material was purified by flash chromatography (dichloro-
methane/methanol/triethylamine 1:1:0.01) to afford
compound 10 as a hygroscopic solid (102 mg, 50%). Rf
(dichloromethane/methanol/triethylamine 1:1:0.01): 0.15;
m.p. > 250 °C IR (KBr, cm−1) m 3418, 2934, 2866, 2360, 1606,
4.2.3. Fluorescence measurements
An experimental procedure previously described was fol-
lowed [19,20].
4.2.4. DNase I footprinting
The experimental procedure has been recently detailed
[21].
1
1516, 1244, 1168, 828; H-NMR (DMSO-d6, 250 MHz): d
2.88 (t, 4H, J = 5.1 Hz), 4.02 (t, 4H, J = 5.2 Hz), 7.12 (d, 4H,
J = 8.7 Hz), 8.21 (d, 4H, J = 8.5 Hz), 9.07 (s, 2H); 13C-NMR
(DMSO-d6, 62.5 MHz) : d 59.3 (2 × CH2), 69.3 (2 × CH2),
113.9 (4 × CH), 127.3 (4 × CH), 127.0 (2 × Cq), 137.1 (2 ×
CH), 149.1 (2 × Cq), 160.2 (2 × Cq); MS (IS): 351 (M + 1)+;
Anal. calcd for C20H22N4O2: C, 68.55; H, 6.33; N, 15.99.
Found: C, 68.82; H, 6.17; N, 15.84.
4.2.5. Cell cultures and survival assay
CEM human leukemia cells were grown at 37 °C in a
humidified atmosphere containing 5% CO2 in RPMI
1640 medium, supplemented with 10% fetal bovine serum,
glutamine (2 mM), penicillin (100 UI ml–1) and streptomy-
cin (100 µg ml–1). The cytotoxicity of the studied molecules
was assessed using a cell proliferation assay developed by
Promega (CellTiter 96® AQueous one solution cell prolifera-
tion assay). Briefly, 2 × 104 exponentially growing cells were
seeded in 96-well microculture plates with various drug con-
centrations in a volume of 100 µl. After 72 h incubation at
37 °C, 20 µl of the tetrazolium dye solution were added to
each well and the samples were incubated for a further 2 h at
37 °C. Plates were analyzed on a Labsystems Multiskan MS
(type 352) reader at 492 nm.
4.1.9. 2,6-Bis-[4-(4,5-dihydro-1H-imidazol-2-yl-methoxy)-
phenyl]-pyrazine (11)
A stirred mixture of compound 8 (200 mg, 0.58 mmol),
ethylenediamine (10 ml) (freshly distilled on KOH) and P2S5
(5 mg, 0.02 mmol) was heated at 120 °C in an oil bath for 4 h.
The reaction mixture was then cooled, and the solvent was
removed under reduced pressure. The crude material was puri-
fied by flash chromatography (dichloromethane/methanol/
triethylamine 1:1:0.01) to afford compound 11 as a yellow
solid (200 mg, 80%). Rf (dichloromethane/methanol/
triethylamine 1:1:0.01): 0.15; m.p. 159 °C; IR (KBr, cm−1) m
3266, 2936, 2866, 1622, 1516, 1244, 1176, 1062, 828;
1H-NMR (CD3OD, 250 MHz): d 3.67 (s, 8H), 4.78 (s, 4H),
7.14 (d, 4H, J = 8.9 Hz), 8.16 (d, 4H, J = 8.9 Hz), 8.89 (s,
2H); 13C-NMR (CD3OD, 62.5 MHz): d 49.2 (2 × CH2), 64.1
(4 × CH2), 115.1 (4 × CH), 128.2 (4 × CH), 128.9 (2 × Cq),
138.8 (2 × CH), 149.9 (2 × Cq), 159.6 (2 × Cq), 163.2 (2 ×
Cq); MS (IS): 429 (M + 1)+; Anal. calcd for C24H24N6O2: C,
67.27; H, 5.65; N, 19.61. Found: C, 67.53; H, 5.48; N, 19.50.
Acknowledgments
This work was supported by grants (toA.L. and S.R.) from
the Ligue Nationale Française contre le Cancer (comité du
Nord, comité du Centre), (to S.R.) the Fondation pour la
Recherche Médicale, the Cancéropôle grand Ouest and (to
U.J.) from the Conseil Régional du Centre and CNRS. N.D.
is the recipient of a fellowship from the Ligue Nationale Con-
tre le Cancer. We thanks Dr. L. Meijer and O. Lozach for the
CDK evaluation (CNRS, Roscoff, France).
4.2. Biological studies
4.2.1. Absorption spectroscopy and melting temperature
studies
References
Absorption spectra and melting curves were measured
using an Uvikon 943 spectrophotometer coupled to a Neslab
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