Structure activity relationship studies of imidazo[1,2-a]pyrazine derivatives against cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2010.08.035

Designed novel imidazo[1,2-a]pyrazine based inhibitors, synthesized by condensing α-aminopyrazines with α-halocarbonyl compounds followed by electrophilic substitutions. Cytotoxic effects on four cancer cell lines evaluated. Based on preliminary data, imidazo[1,2-a]pyrazine template redesigned to improve activity.

Full text of DOI:10.1016/j.ejmech.2010.08.035

European Journal of Medicinal Chemistry 45 (2010) 5208e5216
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure activity relationship studies of imidazo[1,2-a]pyrazine derivatives
against cancer cell lines
,
b
a,
Shailaja Myadaraboina a, Manjula Alla a , Venkateshwarlu Saddanapu , Vittal Rao Bommena
,
**
*
,
Anthony Addlagatta b
***
a Organic Chemistry Division-II, Indian Institute of Chemical Technology, Tarnaka-uppal road, Tarnaka, Hyderabad 500607, India
b Center for Chemical Biology, Indian Institute of Chemical Technology, Tarnaka-uppal road, Tarnaka, Hyderabad 500607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Designed novel imidazo[1,2-a]pyrazine based inhibitors, synthesized by condensing a-aminopyrazines
with a-halocarbonyl compounds followed by electrophilic substitutions. Cytotoxic effects on four cancer
cell lines evaluated. Based on preliminary data, imidazo[1,2-a]pyrazine template redesigned to improve
activity.
Received 21 April 2010
Received in revised form
11 August 2010
Accepted 11 August 2010
Available online 18 August 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Inhibitor design
Synthesis
Imidazo[1,2-a]pyrazine
Anticancer activity
Structure activity relationship
1. Introduction
activity [14], antiproliferative activity [15], controlling allergic reac-
tions [16], smooth muscle relaxant properties [17] and phosphodi-
Discovery of new drugs for treatment of cancer has been gaining
a great deal of interest mainly due to a universal resistance to
conventional single drug chemotherapeutic agents. Multidrug
resistance [1] characterized by resistance not only to drugs that are
similar structurally and functionally but also cross-resistance to
unrelated drugs like doxorubicin, vincristine, vinblastine, colchi-
cine and actinomycin has been documented. Thus, search for novel
anticancer agents with diverse chemical structure is need of the
hour. Herein, we report the synthesis and evaluation of a series of
imidazo[1,2-a]pyrazines as potent anticancer agents.
Imidazo[1,2-a]pyrazines have been gaining attention in drug
discovery realm especially as structural analogues of purines [2e4]
(Fig. 1). Derivatives of imidazo[1,2-a]pyrazines exhibit various phar-
macological activities such as antibacterial [5], anti-inflammatory
[6e8], uterine relaxing activity [9], antibronchospastic [10], antiulcer
[11], cardiac stimulating [12], antidepressant [13], hypoglycemic
esterase inhibitory activity [18a]. They have also been shown to
inhibit the receptor tyrosine kinase EphB4 recently [19].
2. Rationale for designing
The imidazo[1,2-a]pyrazine is an interesting skeleton to design
inhibitors with variable substitutions because of its planar shape.
While C3, C5 and C6 form one edge of the plane, and the N7, C8 and
N1 form the other. The C2 is positioned at the middle where these
two edges meet. Note that N1 and N7 are hydrogen bond acceptors.
Larger substitutions on C8 may be deleterious on the activity due
probably to affecting the hydrogen bonding capacity of N1 and N7.
We decided not to modify edge 2. On the edge1, C3 (R3) and C6 (R6)
are selected for modification. In addition we wanted to explore the
role of the substitutions on C2 (R2) in conjunction with the varia-
tion on the face1 on the cytotoxic effects. As a starting set, we have
designed and synthesized imidazo[1,2-a]pyrazine derivatives with
various modifications (Table 1). The substitution on C6 is limited to
only methyl group as it is close to N7, which may have effect on the
hydrogen bonding capacity of this molecule. Bulky substitutions
were placed on C2 expecting that they may explore the spatial
volume of the target receptor on either side. Substitutions on C3
* Corresponding author. Tel.: þ91 040 27193150; fax: þ91 040 27193382.
** Corresponding author.
*** Corresponding author.
E-mailaddresses:manjula@iict.res.in(M. Alla),vittalrao@iict.res.in(V.R. Bommena),
anthony@iict.res.in (A. Addlagatta).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.035

S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
5209
sluggish and low yielding. Amongst the two pyrazines used,
2-amino-5-methylpyrazine (1b) was more reactive and corre-
sponding products were obtained in higher yields. All products were
completely characterized before proceeding further.
N
R6
N
N
8
6
5
N
3
N
N
A
series of electrophilic substitutions were performed on
2-substitutedimidazo[1,2-a]pyrazines(3)toobtain2,3-disubstituted
imidazo[1,2-a]pyrazines (4). Since the ring is highly deactivated
towards electrophiles, the reactions gave moderate yields of corre-
sponding products. To begin with bromination was attempted and
NBS/Ethanol was found to be ideal method [13,23] when compared
to direct bromination with molecular bromine (Scheme 1).
The nitrile substitution was achieved by nucleophilc attack on
the bromo group of 3-bromo-2-substituted imidazo[1,2-a]pyr-
azines (4) with CuCN in DMF [24] leading to 3-cyano-2-aryl imi-
dazo[1,2-a]pyrazines in moderate yields(5aee) (Scheme 1).
Hydroxymethylation was carried out on 2-substituted imidazo
[1,2-a]pyrazines (3) employing formaldehyde, acetic acid and sodium
acetate [25] (Scheme 2). The corresponding 3-hydroxymethyl-2-
substituted imidazo[1,2-a]pyrazines (6aeb) were obtained in
moderate yields.
2
R3
R2
Fig. 1. Schematic diagram of imidazo[1,2-a]pyrazine skeleton. In the left panel, the
IUPAC numbering is provided and in the right panel the substitution position is
identified.
also include large variations since it is far from hydrogen bond
forming atoms (Table 2, entries 1e11).
3. Synthetic chemistry
A series of substituted imidazo[1,2-a]pyrazines have been
synthesized with substitutions at 2,3,6-ring positions being varied
generating mono-,di-,trisubstituted imidazo[1,2-a]pyrazines pos-
sessing functional groups like halo, hydroxymethyl, amine, alkyl,
aryl, heteroaryl etc.
Amination of the 2-substituted imidazo[1,2-a]pyrazines (3)
occurred at 3-position as shown in Scheme 3 and was found to
proceed with better yields compared to hydroxymethylation
[26,27]. All newly synthesized compounds were completely char-
acterized by their spectral data (compounds listed in Table 1).
A generally established method for synthesis of 3-substituted
imidazo[1,2-a]pyrazine derivatives is by condensation of
a-halo-
genocarbonyl compounds with aminopyrazines [20e22]. Accord-
ingly, a series of 3-substituted imidazo[1,2-a]pyrazines were made
by condensing 2-aminopyrazine (1a) and 2-amino-5-methylpyr-
azine (1b) with 3-bromoacetophenones or halo substituted
carbonyls (2aei) (Scheme 1). In all reactions, the products i.e, 2-
substituted imidazo[1,2-a]pyrazines (3aei) were obtained in
moderate to good yields. Of the acyl bromides, 4-flourophenacyl
bromide was very effective and the corresponding 2-(4-fluo-
rophenyl)-6-methyl imidazo[1,2-a]pyrazine was obtained in high
yield (70%). The condensation with heterocyclic acyl bromides was
4. Pharmacological screening
All the compounds were characterized by the methods
described in Section 5. Cytotoxic effects of these compounds were
tested against four different cancer cell lines and their activities are
summarized in the Table 2. Interesting results were observed
(entries 1e11). Compound 4c exhibited the most cytotoxic effects
on all the four cells lines with best against MCF-7 and SK-N-SH as
13.0 and 13.8
is 5c with activities close to 100
m
M, respectively. The next best molecule in this series
Table 1
mM. All the other compounds
Yields and physical properties of Imidazo[1,2-a]pyrazine derivatives synthesized.
displayed much lower efficacies. Compounds 4c and 5b are iden-
tical except for the substitution on C3, a bromo in the former and
the cyano in the later. Altogether, the data point out that bromo
substitution is better than the cyano on C3, when the other
substituents are identical. Note that p-fluorophenyl is the preferred
substituent among all the others on C2. The role of methyl group on
C6 is not clear, since except for 7c all the other molecules have it.
To further explore the role of substituents on C2 and C6 while it is
either bromo or cyano on the C3, we have synthesized (Table 2,
entries 12e23) and evaluated several more derivatives. Surprisingly,
removing the p-fluoro substitution on the C3-phenyl group in the
compound 4a, improvedtheactivityby morethanten-foldagainstall
cell lines except in MCF-7, where it is only two-fold improvement.
Several derivatives differ by the C6-methyl group. However, no
systematic variation hasbeen seenin theactivities based onpresence
or absence of C6-methyl group (Table 2). A fluoro to chloro substi-
tution between 4g and 4h, the efficiency of cytotoxicity improves by
about eight folds in two cell lines while it remains almost the same in
other two (Table 2). Of the four new C3-cyano derivatives only one
i.e., 5d displayed appreciable efficacy against HEPG-2 cell lines.
To summarize, we have designed, synthesized and evaluated
imidazo[1,2-a]pyrazine derivatives systematically by varying
substitutions on C2, C3 and C6 and established a structure activity
relationship. Although, the role of C6 methyl is still not clear,
substitution of bromo on C3 and a phenyl on the C2 seems to be
important for their activity against cancer cell lines. Further studies
are required to identify the target protein, which will enable the
design of more specific and efficient molecules.
Compound R6
R2
R3
Yield (%) M.p. (ꢀC)
3a
3b
3c
3d
3e
3f
CH3 Ph
CH3 4-C6H4CH3
CH3 4-C6H4F
H
H
H
H
H
H
50
67
70
65
30
45
123e125
173e175
138e140
66e68
108e110
90e92
CH3 t-Butyl
CH3 3-Coumarinyl
CH3 1-((2-Nitrophenoxy)
methyl)phenyl
3g
3h
3i
H
H
H
C6H5
4-C6H4CH3
4-C6H4F
H
H
H
40
52
55
87
86
74
70
68
57
65
89
73
51
60
46
100e102
104e106
92e94
105e107
72e75
172e174
Liquid
70e72
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH3 Ph
Br
Br
Br
Br
Br
Br
Br
Br
Br
CN
CN
CH3 4-C6H4CH3
CH3 4-C6H4F
CH3 3-Coumarinyl
CH3 t-Butyl
H
H
H
H
Ph
78e82
4-C6H4F
4-C6H4Cl
t-Butyl
154e156
103e105
85e87
162e164
187e190
68e70
5a
5b
5c
CH3 Ph
CH3 4-C6H4F
CH3 1-((2-Nitrophenoxy) CN
methyl)phenyl
5d
5e
6a
6b
H
H
4-C6H4F
4-C6H4Cl
CN
CN
CH2OH
50
48
45
48
158e160
146e148
134e136
150e152
CH3 3-Coumarinyl
CH3 1-((2-Nitrophenoxy) CH2OH
methyl)phenyl
7a
7b
7c
CH3 Ph
CH3 4-C6H4F
H
Piperidinyl
66
Morpholinyl 72
Morpholinyl 56
148e150
140e145
138e140
4-C6H4CH3

5210
S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
Table 2
Inhibition values (IC50) of Imidazo[1,2-a]pyrazines on human tumor cell lines.
Entry
Compound
IC50 values against cancer cell lines (mM)
MDAMB-231
SK-N-SH
HepG-2
129.8
MCF-7
N
N
N
1
>1000
154.6
900.0
O
3f NO2
N
N
N
O
2
N/A
>1000
102.0
>1000
>1000
O
3e
N
N
N
3
4
N/A
95.7
13.8
992.0
615.0
3d
N
N
N
F
191.8
12.9
Br
4c
N
O
N
N
5
>1000
101.6
900.0
730
O
Br
4e
N
N
N
F
6
7
116.0
107.6
103.8
106.7
99.5
77.6
CN
N
5c
N
N
798.0
98.3
232.8
121.6
N
7a
N
N
N
F
8
217.5
162.3
871.0
7b
N
O
N
N
N
9
112.9
126.0
>1000
N
7c
O

S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
5211
Table 2 (continued)
Entry
Compound
IC50 values against cancer cell lines (mM)
MDAMB-231
SK-N-SH
HepG-2
853.0
MCF-7
N
N
N
O
6a
10
124.5
145.9
232.6
O
HO
N
N
N
11
N/Aa
91.6
>1000
>1000
O
OH
6b
NO2
N
N
N
12
13
>1000
>1000
9.6
>1000
>1000
>1000
>1000
3a
N
N
N
10.8
3b
N
N
F
N
14
15
104.0
159.2
106.6
96.32
3c
N
N
N
>1000
>1000
>1000
960.0
140.8
3g
N
N
N
F
N
N
16
17
135.9
117.9
737.0
103.9
3i
N
3l
>1000
>1000
>1000
N
N
N
18
19
20
15.2
168.9
171.2
1.0
9.5
61.1
85.1
6.2
Br
4a
N
N
N
24.9
Br
4b
N
N
N
123.2
123.0
107.8
Br
4d
(continued on next page)

5212
Table 2 (continued)
S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
Entry
Compound
IC50 values against cancer cell lines (mM)
MDAMB-231
SK-N-SH
HepG-2
108.7
MCF-7
N
N
N
21
142.4
91.1
555.0
Br
4f
N
N
F
N
22
23
101.3
80.6
87.6
81.1
Br
4g
N
N
N
111.3
2.3
113. 9
1.1
190.0
282.4
2.4
Br
4j
24
Doxorubicin
0.954
a
N/A indicates that the activity could not be measured.
5. Experimental section
Silica gel 60 F254 plates. Phenacyl bromide and 4-methylphenacyl
bromide purchased from Spectrochem chemicals, India. 3-Nitro-2-
benzyloxyphenacyl bromide [29] and 5-methyl-2-amino pyrazine
were prepared in the laboratory [30]. Rest of the acyl bromides and
2-aminopyrazine was purchased from SigmaeAldrich.
5.1. Chemistry
Melting points were determined in open capillary tubes with
a Cintex Industrial Corporation melting point apparatus and are
uncorrected. IR spectra were obtained on a Perkin-Elmer FTIR
spectrophotometer neat or as KBr pellets. 1H NMR spectra were
determined on Avance 300 MHz instrument. Chemical shifts are
5.1.1. General procedure for substituted 2-phenylimidazo[1,2-a]
pyrazines (3aei)
To a stirred solution of 2-aminopyrazine (1 mmol) in 30 mL of
ethanol, phenacyl bromide (1 mmol) and NaHCO3 (3 mmol) were
added and heated to reflux. After completion of the reaction
(monitored by TLC), the reaction mixture was cooled to room
temperature. The solid was filtered from reaction mixture and
given in
d values referenced to the solvent. Mass spectra were
recorded on Waters quadruple mass spectrometry. HRMS was
recorded on high resolution QSTAR XL Hybrid/MS system. Analyt-
ical thin layer chromatography (TLC) was performed on Merck
R6
N
N
N
R6
N
N
R6
b
a
Br
N
NH2
N
N
O
R2
R3
R2
3 (a-i)
R2
4 (a-i)
1a: R6= H
1b: R6= CH3
2 (a-i)
2a, 3a: R2 = Ph, R6= CH3
4a: R3= Br, R2 = Ph, R6= CH3
2b, 3b: R2 = 4-C6H4CH3, R6= CH3
2c, 3c: R2 = 4-C6H4F, R6= CH3
2d, 3d: R2 = t-butyl, R6= CH3
2e, 3e: R2 =3-coumarinyl, R6= CH3
2f, 3f: R2 = 1-((2-nitrophenoxy)methyl)
phenyl, R6= CH3
4b: R3= Br, R2 =4-C6H4CH3, R6= CH3
4c: R3= Br, R2 =4-C6H4F, R6= CH3
4d: R3= Br,R2 =3-coumarinyl,R6=CH3
4e: R3= Br, R2 =t-butyl, R6= CH3
4f: R3= Br, R2 =Ph, R6= H
4g: R3= Br, R2 =4-C6H4F, R6= H
4h :R3= Br, R2 =4-C6H4Cl, R6= H
4i: R3= Br, R2 =t-butyl, R6= H
2g, 3g: R2 = Ph, R6= H
2h, 3h: R2 = 4-C6H4CH3, R6= H
2i, 3i: R2 = 4-C6H4F, R6= H
R6
N
N
5a: R3=CN, R2 = Ph, R6= CH3
c
5b: R3= CN, R2 =4-C6H4F, R6= CH3
5c: R3= CN,R2 =1-((2- nitrophenoxy)methyl)phenyl,
R6= CH3
N
R3
5 (a-e)
5d: R3= CN, R2 =4- C6H4Cl, R6= H
5e: R3= CN, R2 =4-C6H4F, R6= H
R2
Scheme 1. Synthesis of 2-/2, 3-di substituted imidazo[1,2-a]pyrazine (3ae3i, 4ae4i, 5ae5e). Reagents and conditions: (a) acyl bromide, NaHCO3, EtOH, reflux; (b) N-bromo-
succinimide, EtOH, RT; and (c) CuCN, DMF, reflux.

S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
5213
N
N
a
N
N
R2
R2
N
N
R6
R6
OH
3e:R2 =3-coumarinyl, R6= CH3
3f: R2 =1-((2-nitrophenoxy)methyl)phenyl,
6a: R =3-coumarinyl, R = CH
2
3
6
6b: R =1-((2-nitrophenoxy)methyl)phenyl,
2
R6= CH3
R = CH
3
6
Scheme 2. Synthesis of 3-hydroxymethyl-2-substituted imidazo[1,2-a]pyrazine. Reagents and conditions: (a) formaldehyde, acetic acid, acetic anhydride, 60 ꢀC.
ethanol was removed on rotovapor. The residue was extracted with
ethyl acetate and washed with water, dried over anhydrous sodium
sulphate, filtered and concentrated. Further purification by column
chromatography using neutral alumina (hexane/ethyl acetate, 70/
30) gave the desired product.
128.85, 137.72, 141.13; Mass (ESI-MS): 190 [M þ 1]þ; HRMS:
Observed value C11H15N3 [M þ 1]: 190.1352; Calculated Value:
190.1344.
5.1.1.5. 3-(6-Methylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one
(3e). brown solid, Yield: 30%. M.p.108e110 ꢀC; IR (KBr, cmꢁ1): 3335,
2924,1725, 1657,1025, 874, 447; 1H NMR (CDCl3 þ DMSO, 300 MHz,
5.1.1.1. 6-Methyl-2-phenylimidazo[1,2-a]pyrazine (3a). Yellow solid,
Yield: 50%; M.p.123e125 ꢀC; IR (KBr, cmꢁ1): 3414, 3313, 2918,
d); 8.97 (s, 1H, Ar), 8.81 (s, 1H, Ar), 8.59 (s, 1H, Ar), 7.86 (s, 1H, Ar),
1417,821, 744; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d
): 9.13 (s,1H, Ar),
7.82 (d, 1H, Ar, J ¼ 7.62 Hz), 7.32 (t, 1H, Ar, J ¼ 8.34 Hz), 7.19 (d, 2H,
Ar, J ¼ 7.62 Hz), 2.39 (s, 3H, CH3); 13C NMR (CDCl3 þ DMSO,
75 MHz): 22.34, 108.23, 114.84, 120.87, 122.67, 124.77, 125.86,
126.45, 127.88, 128.47, 128.99, 140.73, 144.12, 146.23, 152.56, 158.96;
Mass (ESI-MS): 278 [M þ 1]þ; HRMS: Observed value C16H11N3O2
[M þ Na]: 300.0752; Calculated Value: 300.0748.
8.21 (s, 1H, Ar), 8.17(s, 1H, Ar), 7.54e7.50 (m, 5H, Ar), 2.16 (s, 3H,
CH3); 13C NMR (CDCl3 þ DMSO, 75 MHz): 20.62, 108.47, 113.30,
127.45, 128.10, 128.44, 129.42, 131.56, 138.86, 141.73, 152.63; Mass
(EI-MS): 209 [M]þ; HRMS: Observed value C13H11N3 [M þ 1]:
210.1024; Calculated Value: 210.1031.
5.1.1.2. 6-Methyl-2-p-tolylimidazo[1,2-a]pyrazine (3b). Light yellow
solid, Yield 52%; M.p.173e175 ꢀC; IR (KBr, cmꢁ1): 3418, 2921, 1417,
5.1.1.6. 2-(4-(Benzyloxy)-3-nitrophenyl)-6-methylimidazo[1,2-a]pyr-
azine (3f). Brown solid, yield 45%; M.p. 90e92 ꢀC; IR (KBr, cmꢁ1):
3440, 2925, 1511, 1340, 1250, 983, 705; 1H NMR (CDCl3 þ DMSO,
1108, 825, 741; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d ppm): 8.95 (s,
1H, Ar), 7.80- 7.74 (m, 4H, Ar), 7 .21 (d, 2H, Ar, J ¼ 8.210 Hz), 2.49 (s,
3H, CH3), 2.40 (s, 3H, CH3); 13C NMR (CDCl3 þ DMSO,
75 MHz):19.58, 20.15, 107.85, 114.86, 124.94, 128.35, 132.15, 136.94,
137.29, 140.59, 143.87, 152.56; Mass(ESI-MS): 223 [M]þ; HRMS:
Observed value C14H13N3 [M þ 1]: 224.1184: Calculated Value:
224.1187.
300 MHz, d); 8.47 (s, 1H, Ar), 8.42 (s, 1H, Ar), 8.13 (s, 1H, Ar), 8.09 (s,
1H, Ar), 7.47e7.45 (m, 7H, Ar), 3.60 (s, 2H, OCH2), 2.44 (s, 3H, CH3);
13CNMR (CDCl3 þ DMSO, 75 MHz): 26.64, 61.03, 109.50, 114.24,
115.08, 122.82, 126.40, 126.66, 128.00, 128.26, 129.31, 131.29, 132.45,
135.01, 141.09, 142.60, 154.88; Mass (ESI-MS): 361 [M]þ.
5.1.1.7. 2-Phenylimidazo[1,2-a]pyrazine (3g). Yellow solid, yield:
40%; M.p.100e102 ꢀC; IR (KBr, cmꢁ1): 3337, 3014,1658, 730, 432; 1H
5.1.1.3. (4-Fluorophenyl)-6-methylimidazo[1,2-a]pyrazine (3c). Yellow
solid, yield: 58%; M.p.138e140 ꢀC; IR (KBr, cmꢁ1): 3338, 3129, 2925,
NMR (CDCl3 þ DMSO, 300 MHz,
d): 8.98 (s, 1H, Ar), 8.30 (d, 1H, Ar,
1487, 1229, 841, 743; 1H NMR: (CDCl3 þ DMSO, 300 MHz,
d
): 9.07
J ¼ 3.77 Hz), 8.20 (s, 1H, Ar), 7.96 (d, 2H, J ¼ 7.932 Hz), 7.81 (d, 1H,
J ¼ 4.532 Hz), 7.43 (t, 3H, Ar, J ¼ 7.458 Hz); Mass (ESI-MS):196
[M þ 1]þ.
(s,1H, Ar), 8.05 (s,1H, Ar), 8.1 (d, 2H, Ar, J ¼ 7.932 Hz), 7.31 (d, 2H, Ar,
J ¼ 7.932 Hz), 7.25 (s, 1H, Ar), 2.61 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 20.25, 105.72, 113.60, 113.65, 115.62,
126.90, 127.75, 135.47, 141.26, 152.37, 158.90; Mass (ESI-MS): 228
[M þ 1]þ; HRMS: observed value C13H10N3F: 228.0948; calculated
Value: 228.0937.
5.1.1.8. 2-p-Tolylimidazo[1,2-a]pyrazine (3h). Yellow solid, yield
53%; M.p.104e106 ꢀC; IR (KBr, cmꢁ1): 3336, 3144, 1658, 1531, 811,
510; 1H NMR(CDCl3 þ DMSO, 300 MHz,
d): 8.95 (s, 1H, Ar), 8.45 (d,
2H, Ar, J ¼ 8.650 Hz), 8.13 (s, 1H, Ar), 7.40 (d, 2H, Ar, J ¼ 8.210 Hz),
7.21 (d, 2H, Ar, J ¼ 8.210 Hz), 2.30 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 20.91, 108.83, 118.52, 125.76, 127.13,
128.96, 129.08, 132.45, 136.89, 141.32, 155.00; Mass (ESI-MS): 210
[M þ 1]þ; HRMS: observed value C13H11N32[M þ 1]: 210.1040;
calculated value: 210.1031.
5.1.1.4. 2-tert-Butyl-6-methylimidazo[1,2-a]pyrazine (3d). Brown
solid, Yield: 65%. M.p. 65e68 ꢀC; IR (KBr, cmꢁ1): 3435, 2925, 1744,
816; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d): 8.87 (s, 1H, Ar), 7.74 (s,
1H, Ar), 7.29 (s, 1H, Ar), 2.45 (s, 3H, CH3), 1.37 (s, 9H, t-bu); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 20.51, 29.97, 30.47, 108.37, 115.35, 125.39,
N
N
R6
R6
N
N
a
N
N
R3
R2
R2
7a: R3= piperdinyl R2 = Ph, R6= CH3
7b: R3=morpholinyl R2 =4-C6H4F, R6= CH3
7c: R3= morpholinyl R2 =4-C6H4CH3, R6= H
3a: R2 = Ph, R6= CH3
3c: R2 =4-C6H4F, R6= CH3
3h:R2 =4-C6H4CH3, R6= H
Scheme 3. Synthesis of 3-aminomethyl-2-substituted imidazo[1,2-a]pyrazine. Reagents and conditions: (a) secondary amine, formaldehyde, acetic acid, methanol, reflux.

5214
S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
5.1.1.9. 2-(4-Fluorophenyl)imidazo[1,2-a]pyrazine (3i). Yellow solid,
yield: 55%; M.p.92e94 ꢀC; IR(KBr,cmꢁ1): 3360, 2925, 2854, 1144,
HRMS: observed value C11H14BrN3: 268.0444; calculated value:
268.0449.
840, 741, 516; 1HNMR (CDCl3 þ DMSO, 300 MHz,
d): 9.07 (s,1H, Ar),
8.05 (s, 1H, Ar), 8.10 (d, 2H, Ar, J ¼ 8.120 Hz), 7.31 (d, 2H, Ar,
J ¼ 8.120 Hz), 7.25 (d, 2H, Ar, J ¼ 7.932 Hz); Mass (ESI-MS): 214
[M þ 1]þ; HRMS: observed value C12H8FN3 [M þ 1]: 214.0783;
calculated value 214.0780.
5.1.2.6. 3-Bromo-2-phenylimidazo[1,2-a]pyrazine (4f). Light yellow
solid, yield: 57%; M.p. 93e95 ꢀC; IR (KBr, cmꢁ1): 3312, 3189, 2925,
1623, 1450, 873, 689, 495; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d):
8.99 (s, 1H, Ar), 8.17 (d, 1H, Ar, J ¼ 4.532 Hz), 8.11 (d, 2H, Ar,
J ¼ 6.987 Hz), 8.02 (d, 1H, Ar, J ¼ 4.532 Hz), 7.79e7.82 (m, 3H, Ar);
5.1.2. General procedure for substituted 3-bromo-2-phenylimidazo
[1,2-a]pyrazine (4aei)
13C NMR (CDCl3 þ DMSO, 75 MHz): 93.45, 116.34, 127.31, 127.89,
þ
128.35, 129.69, 131.61, 142.41, 143.10; Mass (ESI-MS): 272 [M]
;
To a solution of imidazopyrazine (1 mmol) in ethanol, N-bro-
mosuccinimide (1.5 mmol) was added and stirred at room
temperature. Following the completion of the reaction (monitored
by TLC), ethanol was removed from the reaction mixture on roto-
vapour. Water was added to the residue and extracted with ethyl
acetate. Combined organic layers were washed with water, dried
over anhydrous sodium sulphate, filtered and concentrated. Further
purification by column chromatography using silica gel (hexane/
ethyl acetate 90/10) gave a desired product.
HRMS: observed value C12H8BrN3 [M þ 1]: 273.9973; calculated
value: 273.9979.
5.1.2.7. 3-Bromo-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine
(4g). Light yellow solid, yield: 65%; M.p. 98e100 ꢀC; IR (KBr, cmꢁ1):
3199, 2924, 2853, 1464, 1206, 833,784, 551: 1H NMR
(CDCl3 þ DMSO, 300 MHz,
d): 8.82 (s, 1H, Ar), 7.94 (d, 2H, Ar,
J ¼ 9.253 Hz), 7.75 (d, 2H, Ar, J ¼ 8.120 Hz), 7.14(d, 2H, Ar,
J ¼ 7.932 Hz); 13C NMR (CDCl3 þ DMSO, 75 MHz): 92.57, 144.18,
143.34, 131.73, 131.76, 130.45, 130.05, 129.90, 115.61, 116.67; Mass
(ESI-MS): 291 [M]þ, 293 [M þ 2]þ; HRMS: Observed value
C12H7BrFN3; 291.9875; Calculated value: 291.9885.
5.1.2.1. 3-Bromo-6-methyl-2-phenylimidazo[1,2-a]pyrazine
(4a). Brown solid; yield: 87%; M.p.105e107 ꢀC; IR (KBr, cmꢁ1):
2962, 2926, 2857, 1729, 1156, 1014; 1H NMR (CDCl3 þ DMSO,
300 MHz,
d
): 8.96 (s, 1H, Ar), 8.0 (d, 2H, Ar, J ¼ 8.309 Hz), 7.89 (s, 1H,
5.1.2.8. 3-Bromo-2-(4-chlorophenyl)imidazo[1,2-a]pyrazine
Ar), 7.30e7.27 (m, 3H, Ar), 2.61 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 20.60, 93.15, 113.22, 127.47, 128.10,
128.20, 128.49, 131.49, 138.84, 139.33, 141.75; Mass (ESI-MS): 288
[M] þ; HRMS: observed value C13H10BrN3: 288.0143; calculated
value: 288.0136.
(4h). Red colour solid, yield: 89%; M.p. 100e102 ꢀC; IR (KBr, cmꢁ1):
3097, 2981, 1726, 1387, 1175, 756, 665; 1H NMR (CDCl3 þ DMSO,
300 MHz,
d
): 8.81 (s, 1H, Ar), 7.94 (d, 2H, Ar, J ¼ 9.253 Hz), 7.74 (d,
2H, Ar, J ¼ 8.120 Hz),7.14 (d, 2H, Ar, J ¼ 8.120 Hz); 13CNMR
(CDCl3 þ DMSO, 75 MHz): 96.45, 108.15, 116.86, 128.35, 128.49,
128.56, 128.63,128.98, 130.21, 130.58; Mass (ESI-MS): 308 [M]þ;
HRMS: observed value C12H7BrClN3: 307.9585; calculated value:
307.9590.
5.1.2.2. 3-Bromo-6-methyl-2-p-tolylimidazo[1,2-a]pyrazine
(4b). Brown solid, yield: 86%; M.p. 72e75 ꢀC; IR (KBr, cmꢁ1): 2922,
2852, 1609, 1450, 1151, 821, 716, 508; 1H NMR (CDCl3 þ DMSO,
300 MHz,
d
): 8.84 (s, 1H, Ar), 7.94 (d, 2H, Ar, J ¼ 7.806 Hz), 7.91 (s,
5.1.2.9. 2-tert-Butyl-3-bromoimidazo[1,2-a]pyrazine
(4i). Light
1H, Ar), 7.22 (d, 2H, Ar, J ¼ 8.782 Hz), 2.59 (s, 3H, CH3), 2.43 (s, 3H,
CH3); 13C NMR (CDCl3 þ DMSO, 75 MHz): 20.79, 28.31, 115.84,
123.89, 126.567, 128.05, 129.047, 129.061, 131.134, 141.482, 142.348,
153.80; Mass (ESI-MS): 302 [M þ 1] þ; HRMS: observed value
C14H12BrN3 [M þ 1]: 302.0286; calculated value: 302.0294.
yellow solid, yield: 73%; M.p. 85e87 ꢀC; IR (KBr, cmꢁ1): 2962, 2926,
1725, 1262, 1013, 800; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d): 8.94
(s, 1H, Ar), 8.02 (d, 1H, Ar, J ¼ 5.05 Hz), 7.94 (d, 1H, Ar, J ¼ 4.532 Hz),
1.52 (s, 9H, t-bu); 13C NMR (CDCl3 þ DMSO, 75 MHz): 28.89, 30.67,
93.54, 115.67,124.78, 129.63, 131.78, 142.35; Mass (ESI-MS): 254
[M]þ; HRMS: observed value C10H12BrN3 [M þ 1]: 254.0296;
calculated value: 254.0294.
5.1.2.3. 3-Bromo-2(4-fluorophenyl)-6-methylimidazo[1,2-a]pyrazine
(4c). Light yellow solid, yield: 74%; M.p.173e175 ꢀC; IR (KBr, cmꢁ1):
3030, 2923, 1710, 1153, 937, 768, 691; 1H NMR (CDCl3 þ DMSO,
5.1.3. General procedure for substituted 3-cyano-2-phenylimidazo
[1,2-a]pyrazine (5aee)
300 MHz, d): 8.82 (s, 1H, Ar), 7.94 (s, 1H, Ar), 7.75 (d, 2H, Ar,
J ¼ 7.932 Hz), 7.14 (d, 2H, Ar, J ¼ 7.932 Hz), 2.42 (s, 3H, CH3); 13C
NMR (CDCl3 þ DMSO, 75 MHz): 29.63,115.66,115.99,116.81,129.88,
129.98,130.42,131.72,143.33,144.20; Mass (ESI-MS): 306 [M]þ, 308
[M þ 2]þ; HRMS: observed value C13H9BrFN3: 306.0048; calculated
value: 306.0042.
To a stirred mixture 3-bromoimidazopyrazine (1 mmol), cuprous
cyanide (1.2 mmol) and DMF (5 mL) were added and refluxed till the
reaction was completed (monitored by TLC). After completion of the
reaction resulting dark brown solution, while still hot, was poured in
to a beaker containing 25% ammonia solution. Toluene was added to
the above solution and stirred well until all the lumps have dis-
integrated. To the cold solution ether was added and filtered
through sintered glass funnel. The ether-toluene layer was sepa-
rated and washed successfully with dilute ammonia solution until
the organic layer was colorless. The ether-toluene layer was further
washed with diluted HCl followed by water and brine solution,
finally dried with anhydrous sodium sulphate, filtered and
concentrated. Pure product was obtained by column chromatog-
raphy using silica gel (hexane/ethyl acetate 85/15).
5.1.2.4. 3-(3-Bromo-6-methylimidazo[1,2-a]pyrazin-2-yl)-2H-chro-
men-2-one (4d). Liquid, yield: 70%; IR (KBr, cmꢁ1): 3321, 3182,
2924, 1710, 1616, 1462, 1373, 1093; 1H NMR (CDCl3, 300 MHz,
d);
8.97(s, 1H, Ar), 8.81(s, 1H, Ar), 8.59 (s, 1H, Ar), 7.82 (d, 2H, Ar,
J ¼ 7.62 Hz), 7.32 (t, 1H, Ar, J ¼ 8.34 Hz), 7.19 (d, 1H, Ar, J ¼ 7.62 Hz),
2.39 (s, 3H, CH3); 13C NMR (CDCl3 þ DMSO, 75 MHz): 27.23, 97.20,
114.96, 121.05, 121.57, 122.65, 123.43, 124.67, 126.42, 128.54, 134.43,
141.47, 144.89, 150.65, 152.6, 158.98; Mass (ESI-MS): 357 [M þ 1]þ.
5.1.2.5. 2-tert-Butyl-3-bromo-6-methylimidazo[1,2-a]pyrazine
(4e). Light yellow solid, yield: 80%; M.p. 70 ꢀC; IR (KBr, cmꢁ1): 3381,
2922,1709,1453,1160, 820, 719; 1H NMR (CDCl3 þ DMSO, 300 MHz,
5.1.3.1. 6-Methyl-2-phenylimidazo[1,2-a]pyrazine-3-carbonitrile
(5a). Brown solid, yield: 54%; M.p. 162e164 ꢀC; IR (KBr, cmꢁ1):
3434, 2923, 2215, 1501, 1241, 777, 699; 1H NMR (CDCl3 þ DMSO,
d
): 8.90 (s, 1H, Ar), 7.94 (s, 1H, Ar), 2.40 (s, 3H, CH3), 1.34 (s, 9H,
300 MHz, d): 9.18 (s, 1H, Ar), 8.37 (s, 1H, Ar), 8.10 (d, 2H, Ar,
t-bu); 13C NMR (CDCl3 þ DMSO, 75 MHz): 29.07, 33.06, 33.87, 92.57,
J ¼ 8.120 Hz), 7.50e7.40 (m, 3H, Ar), 2.62 (s, 3H, CH3); 13CNMR:
þ
115.67, 129.65,132,45, 142.35, 154.01; Mass (ESI-MS): 268 [M]
;
20.41, 111.05, 115.42, 123.98, 126.80, 128.63, 130.08, 139.57, 141.07,

S. Myadaraboina et al. / European Journal of Medicinal Chemistry 45 (2010) 5208e5216
5215
141.91, 152.81; Mass (ESI-MS): 235 [M]þ; HRMS: observed value
C14H11N4 [M þ 1]: 235.0977; calculated value: 235.0893.
Ar), 8.19 (s, 1H, Ar), 7.80 (d, 2H, Ar, J ¼ 6.583 Hz), 7.20 (d, 1H, Ar,
J ¼ 6.583 Hz), 7.32 (s, 1H, Ar), 7.09 (t, 1H, Ar, J ¼ 8.458 Hz), 5.25
(d, 2H, CH2, J ¼ 4.389 Hz), 4.59 (brs, 1H, OH), 2.29 (s, 3H, CH3); 13C
NMR (CDCl3 þ DMSO, 75 MHz): 24.18, 51.05, 115.68, 120.50, 122.43,
125.98, 126.48, 127.89,130.92, 134.57, 138.60, 138.68, 139.32, 141.29,
149.78, 153.84, 158.90; Mass (ESI-MS): 308 [M þ 1]þ.
5.1.3.2. 2-(4-Fluorophenyl)-6-methylimidazo[1,2-a]pyrazine-3-car-
bonitrile (5b). Yellow solid, yield: 60%; M.p.187e190 ꢀC; IR (KBr,
cmꢁ1): 3426, 2924, 2217, 1608, 1474, 1237, 837; 1H NMR
(CDCl3 þ DMSO, 300 MHz,
d): 9.09 (s, 1H, Ar), 8.37 (brs, 1H, Ar), 8.18
(t, 2H, Ar, J ¼ 8.687 Hz), 7.23 (t, 2H, Ar, J ¼ 8.682 Hz), 2.62 (s, 3H,
CH3); 13C NMR (CDCl3 þ DMSO, 75 MHz): 29.38, 115.33, 116.40,
116.14, 121.65, 129.34, 129.52, 142.64, 152.34, 165.77; Mass (ESI-
MS): 253 [M]þ; HRMS observed value: C14H9FN4: 253.0883;
calculated value: 253.0889.
5.1.5. General procedure for substituted 3-(amino methyl) imidazo
[1,2-a]pyrazine (7aec)
To a stirred solution of imidazopyrazine (1 mmol) and secondary
amine (1.2 mmol) in methanol (5 mL) and acetic acid (3 mL), form-
aldehyde (2 mL) was added and the resulting mixture was refluxed
for 8 h. The reaction was monitored by TLC. On disappearance of the
starting materials, the solvent was evaporated from the reaction
mixture under reduced pressure. The residue was dissolved in ethyl
acetate and excess acetic acid quenched with saturated NaHCO3
solution. Organic layer was washed with water, dried over anhydrous
Na2SO4 and filtered. The residue obtained on evaporation of the
solvent was purified by silica gel chromatography (light petroleum
ether/ethyl acetate 40/60) to give the desired product.
5.1.3.3. 2-(4-(Benzyloxy)-3-nitrophenyl)imidazo[1,2-a]pyrazine-3-
carbonitrile (5c). Liquid, yield: 46%; IR (KBr, cmꢁ1): 3364, 2926,
2855, 2217, 1739, 1617, 1290, 1173, 731; 1H NMR (CDCl3 þ DMSO,
300 MHz,
d): 8.47(s, 1H, Ar), 8.42 (s, 1H, Ar), 8.09 (s, 1H, Ar),
7.47e7.45 (m, 7H, Ar), 3.60 (s, 2H, OCH2), 2.44 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 26.64, 61.03, 109.50, 114.24, 115.08,
122.82, 123.10,126.40, 126.66, 128.00, 128.26, 129.31, 131.29, 132.45,
135.01, 141.09, 142.60, 154.88; Mass (ESI-MS): 386 [M þ 1]þ.
5.1.5.1. 6-Methyl-2-phenyl-3-((piperidin-1-yl)methyl)imidazo[1,2-a]
pyrazine (7a). Yellow solid, yield: 66%; M.p. 148e150 ꢀC; IR (KBr,
cmꢁ1): 3407, 3128, 2922, 1504, 1418, 1112, 815, 732, 695; 1H NMR
5.1.3.4. 2-(4-Chlorophenyl)-6-methylimidazo[1,2-a]pyrazine-3-car-
bonitrile (5d). Light brown solid, yield: 48%; M.p.146e148 ꢀC; IR
(KBr, cm-1): 3097, 2924, 2214, 1473, 1091, 835, 794; 1H NMR
(CDCl3 þ DMSO, 300 MHz,
d): 9.07 (s, 1H, Ar), 8.15 (s, 1H, Ar),
(CDCl3 þ DMSO, 300 MHz,
d
): 8.92 (s, 1H, Ar), 8.60 (s, 1H, Ar), 8.23
7.54e7.50 (m, 5H, Ar), 3.47 (s, 2H, CH2), 2.45 (t, 4H, eCH2,
J ¼ 7.658 Hz), 2.16 (s, 3H, CH3), 1.30 (m, 6H, eCH2); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 20.25, 52.83, 66.53, 81.86, 108.37, 114.45,
115.15, 125.76, 128.18, 128.44, 132.54, 137.93, 141.94, 152.46; Mass
(ESI-MS): 307 [M þ 1]þ; HRMS: Observed value C19H22N4 [M þ 1]:
307.1915; Calculated Value: 307.1922.
(d, 2H, Ar, J ¼ 7.63 Hz), 7.82 (s, 1H, Ar), 7.15 (d, 2H, Ar, J ¼ 7.63 Hz);
13C NMR: 119.36, 120.87, 122.68, 127.89, 128.01, 129.65, 133.43,
135.55, 142.22, 142. 87; Mass (ESI-MS): 255 [M þ 1]þ.
5.1.3.5. 2-(4-Fluorophenyl)imidazo[1,2-a]pyrazine-3-carbonitrile
(5e). Yellow solid, yield: 50%; M.p. 158e160 ꢀC; IR (KBr, cmꢁ1):
3055, 2924, 2216, 1606, 1478, 1236, 838; 1H NMR (CDCl3 þ DMSO,
5.1.5.2. 2-(4-Fluorophenyl)-6-methyl-3-(morpholinomethyl)imidazo
[1,2-a]pyrazine (7b). White solid, yield: 72%; M.p.140e145 ꢀC; IR
(KBr, cmꢁ1): 3416, 2922, 2856, 1492, 1113, 861; 1H NMR
300 MHz, d): 9.09 (s, 1H, Ar), 8.47 (s, 1H, Ar), 8.18 (d, 2H, Ar,
J ¼ 7.86 Hz), 7.92 (s, 1H, Ar), 7.23 (d, 2H, Ar, J ¼ 7.86 Hz); 13C NMR:
115.20, 125.50, 127.79, 128.73, 128.88, 132.97, 134.65, 136.43, 144.0,
141.82; Mass (ESI-MS): 238 [M]þ.
(CDCl3 þ DMSO, 300 MHz,
d): 8.85 (s, 1H, Ar), 8.10 (s, 1H, Ar), 7.77(d,
2H, Ar, J ¼ 8.458 Hz), 7.09 (d, 2H, Ar, J ¼ 8.458 Hz), 3.87 (s, 2H, CH2),
3.58 (t, 4H, mor, J ¼ 7.39 Hz), 2.49 (t, 4H, mor, J ¼ 7.39 Hz), 2.40 (s,
3H, CH3);13C NMR (CDCl3 þ DMSO, 75 MHz): 21.18, 29.73, 51.70,
53.30, 66.85, 114.86, 115.50, 130.57, 130.68, 138.68, 138.10, 139.32,
142.29, 151.56; Mass (ESI-MS): 327 [M þ 1] þ; HRMS C18H19FN4O
[M þ 1]: observed value: 327.1632; calculated Value: 327.1621.
5.1.4. General procedure for substituted 3- (imidazo[1,2-a]pyrazin-
3-yl)methanol (6aeb)
To a stirred solution of imidazopyrazine (1 mmol) in acetic acid
(5.22 mmol), sodium acetate (4.34 mmol) and formaldehyde
(8.8 mmol) were added at room temperature. The resulting solu-
tion was heated at 60 ꢀc for 24 h. Subsequently, on completion of
the reaction (monitored by TLC), reaction mixture was neutralized
with saturated NaHCO3 solution and extracted with ethyl acetate.
Organic layer was washed with water, dried over anhydrous Na2SO4
and filtered. On evaporation of the solvent the crude product
obtained was purified by silica gel chromatography (Hexane/ethyl
acetate 50/50) to the desired product.
5.1.5.3. 3,3-(Morpholinomethyl)-2-p-tolylimidazo[1,2-a]pyrazine
(7c). Yellow solid, yield: 56%; m.p. 138e140 ꢀC; IR (KBr, cmꢁ1):
3445, 2936, 1650, 1384, 1113, 638; 1H NMR (CDCl3 þ DMSO,
300 MHz,
d): 8.91 (s, 1H, Ar), 8.20 (d, 1H, Ar, J ¼ 7.432 Hz), 7.80 (d,
2H, Ar, J ¼ 7.176 Hz), 7.43 (d, 2H, Ar, J ¼ 7.432 Hz), 7.38 (d, 1H, Ar,
J ¼ 7.176 Hz), 3.97 (s, 2H, eCH2), 3.63 (t, 4H, eCH2, J ¼ 6.23 Hz), 2.46
(t, 4H, eCH2, J ¼ 6.23 Hz), 2.55 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 23.45, 45.78, 52.83, 66.53, 122.10, 126.45,
128.47, 129.76, 132.27, 134.52, 137.80, 142.98, 144.54, 153.87; Mass
(ESI-MS): 309 [M þ 1]þ; HRMS: observed value C18H20N4O [M þ 1]:
309.1712; calculated value: 309.17150.
5.1.4.1. (6-Methyl-2-(3-nitro-4-phenethylphenyl)imidazo[1,2-a]pyr-
azin-3-yl)methanol (6a). Brown solid, yield: 48%; M.p.: 150e152 ꢀC;
IR (KBr, cmꢁ1): 3412, 3019, 2919, 1622, 1509, 1261, 740; 1H NMR
(CDCl3 þ DMSO, 300 MHz,
d): 8.43 (s,1H, Ar), 8.39 (s,1H, Ar), 8.09 (s,
1H, Ar), 7.47e7.45 (m, 7H, Ar), 5.02 (d, 2H, CH2, J ¼ 4.267 Hz), 4.40 (br
S, 1H, OH), 3.60 (s, 2H, OCH2), 2.44 (s, 3H, CH3); 13C NMR
(CDCl3 þ DMSO, 75 MHz): 26.64, 52.32, 61.03,109.50,114.24,115.08,
122.82, 126.40, 126.66, 128.00, 128.26, 129.31, 131.29, 132.45, 135.01,
5.2. Cytotoxicity against four different cancer cell lines
Cellular viability in the presence of test compounds was deter-
mined by MTT-microcultured tetrazolium assay following the
reportedprotocol[28]. Twohumanbreastcancercelllines, MDA-MB-
231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-
positive), a human neuroblastoma cell line, SK-N-SH, and a human
hepatocellular liver carcinoma cell line, Hep G2, are employed in the
current study. All the four types of cancer cell lines are seeded to flat
141.09, 142.60, 151.20, 154.88; Mass (ESI-MS): 390 [M] þ
.
5.1.4.2. 3-(3-(Hydroxymethyl)-6-methylimidazo[1,2-a]pyrazin-2-yl)-
2H-chromen-2-one (6b). Dark brown solid, yield: 45%;
M.p.136e138 ꢀC; IR (KBr, cmꢁ1): 3322, 2925, 2857,1674, 1592,
1341,1027, 755; 1H NMR (CDCl3 þ DMSO, 300 MHz,
d): 8.29 (s, 1H,
bottom 96 (10,000 cells/100 ml) well plate and cultured in the

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