Accepted Manuscript

Synthesis and Antiproliferative Activity of Imidazo[1,2-a]pyrimidine Mannich bases

Raghunath Aeluri, Manjula Alla, Sowjanya Polepalli, Nishant Jain

PII: S0223-5234(15)30064-7
DOI: 10.1016/j.ejmech.2015.05.037
Reference: EJMECH 7916

To appear in: European Journal of Medicinal Chemistry

Received Date: 8 October 2014

Revised Date: 22 May 2015
Accepted Date: 23 May 2015

Please cite this article as: R. Aeluri, M. Alla, S. Polepalli, N. Jain, Synthesis and Antiproliferative Activity
of Imidazo[1,2-a]pyrimidine Mannich bases, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.05.037.

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 ACCEPTED MANUSCRIPT
Graphical Abstract:

Design, Synthesis and Antiproliferative Activity of Imidazo[1,2-

a]pyrimidine Mannich bases
Raghunath Aeluria, Manjula Allaa, *, Sowjanya Polepallib, Nishant Jainb
a
Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
500607, India. Tel.: + 91-40-27191441; Fax: + 91-40-27193382. E-mail: manjula@iict.res.in,

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manjula_alla@yahoo.com.
b
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607,

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India. E-mail: nishant@iict.res.in.

A series of 28 novel imidazo[1,2-a]pyrimidine Mannich bases have been designed,

synthesized and their antiproliferative activity evaluated. Among them 5e, 6b, 7k were found

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to be potent with GI50 values similar to doxorubicin.

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N N N N N N
CH3
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N N N N
H3C
N N
N N
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N N
H3C 6b 7k
5e
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GI50 ~ 0.05 µM GI50 ~ 0.01 µM GI50 ~ 0.01 µM

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C EP
AC
 ACCEPTED MANUSCRIPT
Synthesis and Antiproliferative Activity of Imidazo[1,2-a]pyrimidine
Mannich bases
Raghunath Aeluria, Manjula Allaa, *, Sowjanya Polepallib, Nishant Jainb
a
Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
500607, India. Tel.: +91-40-27191441; Fax: +91-40-27160387. E-mail: manjula@iict.res.in,
manjula_alla@yahoo.com.
b
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607,

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India. E-mail: nishant@iict.res.in.

Abstract. A series of imidazo[1,2-a]pyrimidine Mannich bases were designed, synthesized in

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two phases. Mannich bases were obtained by one pot three component condensation of
imidazo[1,2-a]pyrimidine with secondary amine or piperazine and excess of formaldehyde
solution in methanol. The synthesized Mannich bases were screened for in vitro growth

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inhibition against a panel of 3 different human cancer cell lines. Most of the synthesized
compounds exhibited antiproliferative activity with GI50 values ranging from 0.01-79.4 µM.
Compounds 5e, 6b and 7k were found to be effective inhibitors of growth of all cell lines,

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with GI50 values similar to that of standard drug. The structure and activity relationship has
been disclosed.
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Key words: Mannich reaction, Imidazo[1,2-a]pyrimidine, Piperazine, Antiproliferative
activity, Growth inhibition.
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1. Introduction.

The major challenge in modern drug discovery has been the design and development of
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new antiproliferative drugs with improved efficacy and minimal side effects, especially due
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to a rapid rise in multidrug resistant tumours. A thorough review of recent literature reveals
that small heterocyclic frameworks, such as nitrogen heterocycles, offer an advantage in
design of antiproliferative agents as they mimic many biomolecules. Among them imidazo
fused heterocycles such as imidazopyrimidines are important scaffolds in medicinal
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chemistry, as they exhibit a broad spectrum of biological activity such as anticancer [1], anti-
tubercular [2], antiviral [3], antimicrobial [4], antifungal [5], anti-inflammatory [6],
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Parasiticidal activity [7], calcium channel blockers [8], benzodiazepine receptor agonists [9],
and potent P38 MAP kinase inhibitors [10]. Furthermore, fused pyrimidine derivatives have
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an established anticancer activity. Several pyrimidine analogues have been approved or in use
as drugs for the treatment of cancers. For example, all four new antiproliferative drugs i.e.,
Gefitinib (Iressa), Erlotinib (Tarceva), Lapatinib (Tykerb) and Vandetanib (Caprelsa) (Fig. 1)
approved by the US FDA for the treatment of cancer possess a benzofused pyrimidine core
moiety. Moreover, fused heterocycles like pyrazolopyrimidines [11], pyridopyrimidines [12],
pyrrolopyrimidnes [13], imidazopyrazines [14], imidazopyrimidines [15], imidazopyridines
[16] etc., have also been reported as antitumor agents. An earlier successful experience with
imidazo fused rings as potential antiproliferative agents led us to explore the potential of
imidazo[1,2-a]pyrimidine derivatives for this purpose.

<Figure 1>

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2. Results and Discussions.

2.1. Chemistry

As mentioned earlier, know-how on working with imidazopyrazines uncovered that

substituents at C-2 and C-3 of imidazo ring as well as substituents next to nitrogen atom of
six membered ring have an influence on the anticancer activity [17]. Therefore, a new library

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of imidazo[1,2-a]pyrimidine molecules having C-2, C-3 & C-7 (next to nitrogen of six
membered ring) substituents has been designed (Fig. 2). The C-3 position would be the most
suitable position for electrophilic attack. Therefore it was proposed to introduce an

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aminoalkyl substitution at C-3. Mannich reaction, one of the most familiar C-C bond forming
reactions in organic synthesis, provides a suitable method to introduce amino alkyl

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substituent on the molecule [18]. Recently, Istanbullu et al reported a series of uracil C-
Mannich bases as potent cytotoxic agents [19]. It has been widely accepted that Mannich
derivatives have significantly improved activity profile compared to their parent analogues.
The aminocarbonyl Mannich products are useful in the construction of β-peptides and β-

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lactams. Several bioactive molecules, such as taxol [20], an antitumour agent possess
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aminocarbonyl appendages. In addition Mannich derivatives have better bioavailability and
are amenable to develop water based formulations. Further it was envisaged that variation of
substituents at C-2 and C-7 would add to the diversity of library.
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<Figure 2>

At the outset the construction of crucial intermediate i.e., fused imidazo[1,2-

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a]pyrimidine has been achieved using a simple protocol of condensation of the acyl bromide
with various 2-aminopyrimidines. As is evident, the reaction needs (substituted) 2-
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aminopyrimidine and acyl bromide as raw materials for introduction of substituents at C-2
and C-7 positions of imidazo[1,2-a]pyrimidines. The pool of acyl bromides commercially
available has been found to be limited for this purpose. Therefore, various acyl bromides
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required for construction of diverse imidazo[1,2-a]pyrimidines were prepared by bromination

of corresponding acetophenones with copper(II)bromide in ethyl acetate [21] (scheme 1).
Bromination by CuBr2 was found to be a versatile as well as clean process, providing the
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corresponding acyl bromides in pure form. Coupling of acyl bromides thus obtained with
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different 2-aminopyrimidines has been accomplished in acetone at ambient temperatures

[22]. Various imidazo[1,2-a]pyrimidines 4a-o have been synthesized without the necessity of
either a base or catalyst (scheme 1). Finally, the targeted aminoalkyl imidazo[1,2-
a]pyrimidine analogues 5a-m have been successfully obtained via the Mannich reaction. A
one pot three component reaction of imidazo[1,2-a]pyrimidines 4a-o with various secondary
amines (such as piperidine, pyrrolidine, morpholine, N-methyl aniline etc.) in excess
formaldehyde in methanol under reflux conditions (scheme 2) gave target compounds 5a-m.
The isolated yields (table 1) of the products varied from good to excellent (74-92%).

<Scheme 1>

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<Scheme 2>

<Table 1>

2.2. In vitro antiproliferative activity:

2.2.1. Preliminary activity

To begin with, in vitro antiproliferative activity of the synthesized compounds 5a-m

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was tested against a panel of 4 human cancer cell lines viz., human epithelial lung carcinoma
(A-549) cervical (HeLa), pancreatic (Panc-1) and breast adenocarcinoma (MDA-MB-231)
using Doxorubicin as standard drug. All the compounds showed significant inhibition of

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cancer cell line growth in the concentration range 0.02–7.38 µM as illustrated in table 2.

<Table 2>

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All the tested compounds inhibited growth of at least one cancer cell line. Nine of the
thirteen tested compounds inhibited growth of two or more cell lines. This justifies the design
of imidazo[1,2-a]pyrimidine Mannich bases as a new class of antiproliferative agents.

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Among the amino alkyl groups on C-3 carbon, pyrrolidine and piperidine substitutions gave
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the best inhibition. Compound 5e inhibited the growth of all four cancer cell lines with GI50
values comparable to standard drug Doxorubicin. Compound 5m was also effective in growth
inhibition of all four cancer cell lines but to a lesser extent than compound 5e. In both the
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cases (5e and 5m) the imidazopyrimidine framework had no substitution on six membered
ring, while the C-2 carbon had an aryl substituent (phenyl in 5e and thiophenyl in 5m) and
pyrrolidin-1-yl-methyl as the amino alkyl at C-3 carbon, respectively. On the other hand
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compound 5i which inhibits growth of 3 cell lines had piperidine-1-yl-methyl at C-3 carbon
with a methyl substituent on C-7 carbon and tert-butyl substituent at C-2 position,
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respectively. No decipherable effect of other ring substitutions on growth inhibition could be

made at this stage. However, it is apparent that Mannich bases of imidazo[1,2-a]pyrimidines
incorporating cycloalkyl amines (pyrrolidin-1-yl-methyl, piperidine-1-yl-methyl) were
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definitely best candidates for further exploitation.

Encouraged by the positive antiproliferative activity results of above series of

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compounds, a focused redesign of these Mannich bases was undertaken. From the activity
results it was clear that pyrrolidin-1-yl-methyl and piperidine-1-yl-methyl substituted
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analogues gave the best growth inhibition of cancer cell lines. Therefore, it was decided to
focus on similar Mannich bases. A cycloalkyl diamine like piperazine was chosen.
Piperazines and substituted piperazines are important pharmacophores that have been found
in many marketed drugs such as the Merck HIV protease inhibitor Crixivan [23]. Piperazinyl
linked ciprofloxacin dimers have been shown as potent antibacterial agents [24] against
resistant strains. In the last decade a number of piperazine derivatives have been reported for
their cytotoxic activity [25]. Moreover clinical/preclinical drug development studies of
piperazine compounds by US National Cancer Institute (NCI) demonstrated that they have
capability to suppress experimental tumors. Recently, Akkoc et al also reported that indole
based 1,4-disubstituted piperazines act as cytotoxic agents [26].

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In this context, earlier synthetic protocol has been extended to construct imidazo[1,2-
a]pyrimidine Mannich bases incorporating N-substituted piperazine. The series of compounds
would have only variation in C-2 and C-7 substitutions, making it easier to analyse the
substituent effects. Mannich reaction of imidazopyrimidine, formaldehyde and N-substituted
piperazines resulted in amino alkylation at C-3 carbon (6a-c). However, Mannich reaction
with N-unsubstituted piperazine resulted in the formation of symmetrical imidazo[1,2-
a]pyrimidine dimers connected by piperazine linker as a by product in 1:1 ratio. Further
optimization of the reaction conditions led to exclusive construction of symmetrical 1,4-

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bis((imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine analogues 7a-l. They were obtained by
treatment of two equivalents of imidazo[1,2-a]pyrimidines with one equivalent of piperazine
and excess of formaldehyde (37%) solution in methanol under reflux conditions (scheme 3).

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Structures of all the synthesized compounds [6a-c and 7a-l, table 3] have been confirmed by
their spectral data.

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<Scheme 3>

<Table 3>

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2.2.2. Antiproliferative activity of redesigned Mannich bases:
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Preliminary SRB assay results indicated that A-549 cell line was poorly inhibited by the
imidazo[1,2-a]pyrimidine Mannich bases when compared to other cell lines. Thus,
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antiproliferative assay of the redesigned series of compounds was not extended to this cell
line. As a result, antiproliferative activity of second set of Mannich bases 6a-c and 7a-l was
evaluated against a panel of 3 human cancer cell lines i.e., HeLa, Panc-1 and MDA-MB-231
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only. Growth inhibitory activities (GI50 values) thus determined have been summarized in
table 4.
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Much to our satisfaction the redesigned series of compounds also showed inhibition of
cancer cell line growth. Nine of the 15 tested compounds inhibited the growth of Panc-1 cell
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line. Five compounds each out of fifteen synthesized compounds inhibited HeLa cell line or
MDA-MB-231 cell line growth. The compound 3-((4-methylpiperazin-1-yl)methyl)-2-
phenylimidazo[1,2-a]pyrimidine (6b) inhibited the growth of all three cell lines. The other
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best compound in the series was 7k, a symmetrical dimer which had a 2-methyl phenyl
substitution on C-2 carbon and no substituent on C-7 carbon. Compounds 6b and 7k
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exhibited a GI50 of 0.01 µM against MDA-MB-231 while 7l inhibited Panc-1 with a GI50 of
0.06 µM. In all three cases GI50 values have been found to be comparable to that of standard
drug. As mentioned earlier, significant number of compounds inhibited the Panc-1 cell line (9
out of 15) indicating that this series of compounds may be specific growth inhibitors of Panc-
1 cell line. It was interesting to note that both the simple piperazine Mannich bases as well as
the symmetrical dimers were equally effective in growth inhibition. The C-7 substituents of
the imidazo[1,2-a]pyrimidine Mannich bases such as H, methyl or methoxy didn’t show any
specific variation on growth inhibition of cancer cell lines and all of them were equally
effective. However, compounds with bulkier substituents such as naphthyl, tert-butyl and
hetero aryl (2-thiophenyl) on C-2 carbon were found to be poor inhibitors of cell line growth

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in the dimers. SRB assays of both the series of Mannich bases gave a clear indication that
phenyl substitution on C-2 carbon and cycloalkyl amino group on C-3 carbon are essential for
effective growth inhibition of cancer cell lines.

<Table 4>

3. Conclusions.

In summary, a series of imidazo[1,2-a]pyrimidine Mannich bases have been designed

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and synthesized, in two phases, following simple reaction protocols. All the synthesized
compounds were screened for their growth inhibitory activity against a panel of 3 different
human cancer cell lines viz., HeLa, Panc-1 and MDA-MB-231. Most of the tested Mannich

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bases displayed promising growth inhibitory activity against cancer cell lines. Among all the
synthesized compounds, 5e, 6b and 7k showed maximum growth inhibitory activity against

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all the cell lines at low concentrations. Their GI50 values were comparable to those of
standard drug. Further studies involving redesign of the active moieties and target
identification are in progress.

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Acknowledgements:
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We thank the Director, CSIR-IICT and Head, CPC division for facilities. ARN thanks
the Council of Scientific and Industrial Research (CSIR), New Delhi, India for research
fellowship. SP and NJ thank the 12th FYP-SMiLE (CSC-0111) for funding.
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References.
D

1. a) B. Cosimelli, S. Laneri, C. Ostacolo, A. Sacchi, E. Severi, E. Porcu, E. Rampazzo,

E. Moro, G. Basso, G. Viola, Eur. J. Med. Chem. 83 (2014) 45-56. b) A. Linton, P.
TE

Kang, M. Ornelas, S. Kephart, Q. Hu, M. Pairish, Y. Jiang, C. Guo, J. Med. Chem. 54

(2011) 7705-7712.
2. N. Margiotta, R. Ostuni, R. Ranaldo, N. Denora, V. Laquintana, G. Trapani, G. Liso,
EP

G. Natile, J. Med. Chem. 50 (2007) 1019-1027.

3. A. Gueiffier, M. Lhassani, A. Elhakmaoui, R. Snoeck, G. Andrei, O. Chavignon, J. C.
Teulade, A. Kerbal, E. M. Essassi, J. C. Debouzy, M. Witvrouw, Y. Blache, J.
C

Balzarini, E. D. Clercq, J. P. Chapat, J. Med. Chem. 39 (1996) 2856-2859.

4. Y. Rival, G. Grassy, G. Michel, Chem. Pharm. Bull. 40 (1992) 1170-1176.
AC

5. Y. Rival, G. Grassy, A. Taudou, R. Ecalle, Eur. J. Med. Chem. 26 (1991) 13-18.

6. A. Vidal, M. L. Ferrándiz, A. Ubeda, A. Acero-Alarcón, J. Sepulveda-Arques, M. J.
Alcaraz, Inflamm. Res. 50 (2001) 317-320.
7. J. Jiricek, F. Liang, C. J. N. Mathison, P. K. Mishra, V. Molteni, A. S. Nagle, F.
Supek, L. J. Tan, A. Vidal, V. Yeh, PCT Int. Appl. (2014) WO2014151784 A1.
8. P. J. Sanfilippo, M. Urbanski, J. B. Press, B. Dubinsky, J. B. Moore, J. Med. Chem.
31 (1988) 2221-2227.
9. W. R. Tully, C. R. Gardner, R. J. Gillespie, R. Westwood, J. Med. Chem. 34 (1991)
2060-2067.

5
 ACCEPTED MANUSCRIPT
10. a) K. Nacro, A. J. Duraiswamy, L. R. Chennamaneni, PCT Int. Appl. (2013)
WO2013147711 A1. b) K. C. Rupert, J. H. Henry, J. H. Dodd, S. A. Wadsworth, D.
E. Cavender, G. C. Olini, B. Fahmy, J. J. Siekierka, Bioorg. Med. Chem. Lett. 13
(2003) 347-350.
11. a) M. M. Kandeel, L. W. Mohamed, M. K. Abd El Hamid, A. T. Negmeldin, Sci.
Pharm. 80 (2012) 531-545. b) H. Y. He, J. N. Zhao, R. Jia, Y. L. Zhao, S. Y. Yang, L.
T. Yu, L. Yang, Molecules 16 (2011) 10685-10694.
12. M. A. Azam, B. R. P. Kumar, S. Shalini, B Suresh, T. K. Reddy, C. D. Reddy, Indian

PT
J. Pharm. Sci. 70 (2008) 672-677.
13. a) V. Spano, A. Montalbano, A. Carbone, B. Parrino, P. Diana, G. Cirrincione, I.
Castagliuolo, P. Brun, O. G. Issinger, S. Tisi, I. Primac, D. Vedaldi, A. Salvador, P.

RI
Barraja, Eur. J. Med. Chem. 74 (2014) 340-357. b) P. Barraja, L. Caracausi, P. Diana,
A. Montalbano, A. Carbone, A. Salvador, P. Brun, I. Castagliuolo, S. Tisi, F.

SC
DallAcqua, D. Vedaldi, G. Cirrincione, ChemMedChem 6 (2011) 1238-1248.
14. K. Zurbonsen, A. Michel, P. A. Bonnet, M. N. Mathieu, C. Chevillard, Gen. Pharmac.
32 (1999) 135-141.

U
15. A. Kamal, J. S. Reddy, M. J. Ramaiah, D. Dastagiri, E. V. Bharathi, M. V. Prem
Sagar, S. N. C. V. L. Pushpavalli, P. Ray, M. Pal-Bhadra, Med. Chem. Commun., 1
AN
(2010) 355-360.
16. S. Muniyan, Y. W. Chou, M. A. Ingersoll, A. Devine, M. Morris, V. A. O. Marah, S.
A. Khan, W. G. Chaney, X. R. Bu, M. F. Lin, Cancer Lett. 353 (2014) 59-67.
M

17. M. Shailaja, A. Manjula, S. Venkateshwarlu, B. Vittal Rao, A. Anthony, Eur. J. Med.

Chem. 45 (2010) 5208-5216.
18. a) M. Tramontini, L. Angiolini, Chemistry and Uses, CRC, BocaRaton, FL, 1994. b)
D

M. Tramontini, L. Angiolini, Tetrahedron 46 (1990) 1791-1837. c) M. Tramontini, L.

Angiolini, N. Ghedini, Polymer 29 (1988) 771-788. d) M. Tramontini, Synthesis 12
TE

(1973) 703-775.
19. H. Istanbullu, I. Zupko, V. Alptuzun, E. Erciyas, Turk J Chem 36 (2012) 583-592.
20. K. C. Nicolaou, W. M. Dai, R. K. Guy, Angew. Chem. Int. Ed. 33 (1994) 15-44.
EP

21. a) V. S. Miguel, C. G. Bochet, A. D. Campo, J. Am. Chem. Soc. 133 (2011) 5380-
5388. b) Q. Jiang, W. Sheng, C. Guo, Green Chem. 15 (2013) 2175-2179.
22. S. Holder, A. Zulch, T. Bar, T. Maier, M. Zim, T. Beckers, V. Gekeler, H. Joshi, Y.
C

Munot, U. Bhise, S. Chavan, S. Shivatare, S. Patel, V. Gore, WO 2009/021990 A1.

AC

23. a) J. P. Vacca, B. D. Dorsey, W. A. Schleif, R. B. Levine, S. L. McDaniel, P. L.

Darke, J. Zugay, J. C. Quintero, O. M. Blahy, E. Roth, V. V. Sardana, A. J.
Schlabach, P. I. Graham, J. H. Condra, L. Gotlib, M. K. Holloway, J. Lin, I. W. Chen,
K. Vastag, D. Ostovic, P. S. Anderson, E. A. Emini, J. R. Huff, Proc. Natl. Acad. Sci.
U.S.A. 91 (1994) 4096-4100. b) B. D. Dorsey, R. B. Levin, S. L. McDaniel, J. P.
Vacca, J. P. Guare, P. L. Darke, J. A. Zugay, E. A. Emini, W. A. Schleif, J. C.
Quintero, J. H. Lin, I. W. Chen, M. K. Holloway, P. M. D. Fitzgerald, M. G. Axel, D.
Ostovic, P. S. Anderson, J. R. Huff, J. Med. Chem. 37 (1994) 3443-3451. c) D. Askin,
K. K. Eng, K. Rossen, R. M. Purick, K. M. Wells, R. P. Volante, P. J. Reider,
Tetrahedron Lett. 35 (1994) 673-676. d) K. Rossen, S. A. Weissman, J. Sager, R. A.

6
 ACCEPTED MANUSCRIPT
Reamer, D. Askin, R. P. Volante, P. J. Reider, Tetrahedron Lett. 36 (1995) 6419-
6422.
24. R. J. Kerns, M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, V. U.
Diwadkar, Bioorg. Med. Chem. Lett. 13 (2003) 2109-2112.
25. a) C. S. A. Kumar, S. N. Swamy, N. R. Thimmegowda, S. B. B. Prasad, G. W. Yip,
K. S. Rangappa, Med. Chem. Res. 16 (2007) 179-187. b) B. Chetan, M. Bunha, M.
Jagrat, B. N. Sinha, P. Saiko G. Graser, T. Szekeres, G. Raman, P. Rajendran, D.
Moorthy, A. Basu, V. Jayaprakash, Bioorg. Med. Chem. Lett. 20 (2010) 3906-3910.

PT
26. M. K. Akkoc, M. Y. Yuksel, I. Durmaz, R. C Atalay, Turk J Chem 36 (2012) 515-
525.

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Figure Captions:

Figure 1. Cancer drugs containing Pyrimidine skeleton.

Figure 2. Imidazo[1,2-a]pyrimidine skeleton.

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Tables:

Table 1: Synthesized Mannich bases of imidazo[1,2-a]pyrimidines.

R1

N
N N

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R
N
R 2 R3

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Entry Product R R1 R2R3NH
1. 5a C6H5 H morpholine

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2. 5b C6H5 CH3 piperidine
3. 5c 4-OHC6H4 H piperidine
4. 5d 4-OHC6H4 CH3 pyrrolidine

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5. 5e C6H5 H pyrrolidine
6. 5f C6H5 H piperidine
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7. 5g tert-butyl H piperidine
8. 5h C6H5 H N-methylaniline
9. 5i tert-butyl CH3 piperidine
10. 5j C6H5 CH3 morpholine
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11. 5k 4-ClC6H5 CH3 morpholine

12. 5l C6H5 CH3 pyrrolidine
13. 5m 2-thiophenyl H pyrrolidine
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Table 2: Growth inhibition (GI50 in µM) valuesa of compounds 5a-m on different human
cancer cell lines.
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GI50 ± SD values against cancer cell lines (µM)

Entry
A-549 HeLa Panc-1 MDA-MB-231
C

5a 0.21 ± 0.01 1.67 ± 0.02 0.77 ± 0.02 1.41 ± 0.02

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5b 4.04 ± 0.02 1.71 ± 0.03 0.02 ± 0.02 0.03 ± 0.01

5c 6.89 ± 0.02 4.03 ± 0.03 0.2 ± 0.02 7.23 ± 0.03
5d 2.48 ± 0.02 4.76 ± 0.02 7.28 ± 0.03 1.2 ± 0.03
5e 0.05 ± 0.02 0.07 ± 0.01 0.06 ± 0.02 0.07 ± 0.01
5f 0.26 ± 0.02 2.28 ± 0.03 1.31 ± 0.01 0.03 ± 0.03
5g 0.33 ± 0.01 0.03 ± 0.02 4.51 ± 0.03 1.49 ± 0.02
5h 5.19 ± 0.01 3.12 ± 0.03 5.76 ± 0.02 0.32 ± 0.01
5i 7.39 ± 0.02 0.45 ± 0.02 0.96 ± 0.03 0.35 ± 0.01

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5j 1.09 ± 0.02 0.85 ± 0.01 1.83 ± 0.02 1.43 ± 0.03

5k 2.57 ± 0.03 0.16 ± 0.01 1.17 ± 0.03 0.32 ± 0.02
5l 4.15 ± 0.02 0.18 ± 0.01 0.15 ± 0.01 4.83 ± 0.02
5m 0.18 ± 0.03 0.24 ± 0.01 0.68 ± 0.02 0.2 ± 0.02
Doxorubicin <0.01 ± 0.002 0.031 ± 0.003 <0.01 ± 0.001 0.016 ± 0.002
a
Cell lines were treated with different concentrations of Mannich bases for 48 hrs as described in experimental protocols.
Cell viability was measured employing SRB assay. GI50 values indicated are a mean of three independent experiments.

PT
Table 3: Synthesized Mannich bases of imidazo[1,2-a]pyrimidines incorporating Piperazine:

RI
U SC
Entry Product R R1 R2
AN
6a H
1 C6H5 H
(R3=H)
M

6b H
2 C6H5 H
(R3=methyl)
6c H
3 C6H5 H
D

(R3=tosyl)
4 C6H5 H H
7a
TE

H
5 7b 4-CH3-C6H4 H

6 4-Cl-C6H4 H H
7c
EP

7 tert-butyl H H
7d

8 C6H5 CH3 H
7e
C

9 tert-butyl CH3 H
7f
AC

10 2-naphthyl H H
7g

11 2,4-C6H3Cl2 H H
7h
12 7i C6H5 OCH3 OCH3
13 2-thiophenyl H H
7j

14 2-CH3-C6H4 H H
7k

15 4-Br-C6H4 H H
7l

10
 ACCEPTED MANUSCRIPT

Table 4: Growth inhibition (GI50 in µM) valuesa of compounds 6a-c & 7a-l on different
human cancer cell lines.

GI50 ± SD values against cancer cell lines (µM)

Entry
HeLa Panc-1 MDA-MB-231

PT
6a 0.72±0.03 9.4±0.8 26.7±0.9

6b 0.93±0.03 0.35±0.01 <0.01

RI
6c 4.5±0.2 0.11±0.02 12.9±0.1

7a 3.24±0.05 0.14±0.05 0.27±0.02

SC
7b 2.8±0.01 0.38±0.04 2.3±0.03

7c 25.6±0.4 0.24±0.01 4.7±0.2

7d 2.4±0.3
U 3.9±0.1 1.1±0.08
AN
7e 12.7±0.9 0.6±0.07 15.9±0.4
M

7f 14.5±0.08 1.4±0.06 25.7±0.09

7g 12.6±0.7 79.4±0.3 31.6±0.2

D

7h 2.72±0.21 27.7±0.1 0.21±0.06

TE

7i 9.32±0.04 0.51±0.09 0.12±0.05

7j 0.93±0.01 3.7±0.2 7.1±0.06

EP

7k 0.48±0.04 0.12±0.03 <0.01

7l 0.89±0.02 0.06±0.004 1.0±0.03

C

Doxorubicin 0.092±0.005 0.04±0.002 <0.01

AC

a
Cell lines were treated with different concentrations of Mannich bases for 48 hrs as described in
experimental protocols. Cell viability was measured employing SRB assay. GI50 values indicated are a mean
of three independent experiments.

11
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Figures:

Figure 1:

F
O N
H 3CO
O HN Cl
H 3CO N

PT
N O O
N
HN C CH
H 3CO N

RI
Gefitinib Erlotinib

SC
N
N
O N
O NH
N

U
H 3CO
O NH
O NH
S
AN
O Cl
F
Br
M

F
Lapatinib Vandetanib
D

Fig. 1 Cancer drugs containing Pyrimidine skeleton

TE

Figure 2:
C EP
AC

12
 ACCEPTED MANUSCRIPT
Schemes:

Scheme 1:

PT
RI
U SC
AN
M

Scheme 2:
D

R1
R1
TE

N
N
MeOH
R2R3NH N N
N N HCHO
reflux
4-6 hrs
EP

R
R N R
3
R2
5a R = phenyl, R1 = H, R2R3N = morpholinyl 5a-m
5b R = phenyl, R1 = methyl, R2R3N = piperidinyl
C

5c R = 4-OH-phenyl, R1 = H, R2R3N = piperidinyl

AC

5d R = 4-OH-phenyl, R1 = methyl, R2R3N = pyrrolidinyl

5e R = phenyl, R1 = H, R2R3N = pyrrolidinyl
5f R = phenyl, R1 = H, R2R3N = piperidinyl
5g R = tert-butyl, R1 = H, R2R3N = piperidinyl
5h R = phenyl, R1 = H, R2 = methyl, R3 = phenyl
5i R = tert-butyl, R1 = methyl, R2R3N = piperidinyl
5j R = phenyl, R1 = methyl R2R3N = morpholinyl
5k R = 4-Cl-phenyl, R1 = methyl, R2R3N = morpholinyl
5l R = phenyl, R1 = methyl, R2R3N = pyrrolidinyl
5m R = 2-thiophenyl, R1 = H, R2R3N = pyrrolidinyl.

Scheme 2: Synthesis of Mannich bases of imidazo[1,2-a]pyrimidine

13
 ACCEPTED MANUSCRIPT
Scheme 3:

PT
RI
U SC
AN
M
D
TE
C EP
AC

14
 ACCEPTED MANUSCRIPT
Highlights:

A series of 28 novel imidazo[1,2-a]pyrimidine Mannich bases have been synthesized.

Library includes simple Mannich bases as well as 1,4-symmetrical dimers.

Antiproliferative activity of 28 Mannich bases was evaluated against 3 cell lines.

5e, 6b, 7k exhibited excellent growth inhibitory activity comparable to doxorubicin.

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

Design, Synthesis and Antiproliferative Activity of Imidazo[1,2-a]pyrimidine

Mannich bases
Raghunath Aeluria, Manjula Allaa, *, Sowjanya Polepallib, Nishant Jainb
a
Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
500607, India. Tel.: + 91-40-27191441; Fax: + 91-40-27160387. E-mail: manjula@iict.res.in,

PT
manjula_alla@yahoo.com.
b
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607, India.
E-mail: nishant@iict.res.in.

RI
4. Experimental section.
4.1. Chemistry

SC
All the chemicals used for the synthesis are of the synthetic grade procured from
Spectrochem, Aldrich, SD-fine chemicals. Completion of the reaction was monitored by thin
layer chromatography (TLC) using E-Merck 0.25 mm silica gel plates using ethyl acetate/hexane

U
(5:5) as eluting medium. Visualization was accomplished by using UV light (256 nm) and iodine
chamber. Melting points were determined in open capillaries and are uncorrected. 1H-NMR (300
AN
MHz and 500 MHz) and 13C-NMR (75 MHz) spectra were recorded on Avance 300 MHz,
Avance 500 MHz and JCAMP DX-50 in CDCl3 and DMSO-d6 unless otherwise mentioned.
Chemical shifts are reported in δ (ppm) downfield from internal standard Tetramethylsilane
M

(TMS) and coupling constant (J) values are in Hz. IR spectra were recorded on a Thermo Nicolet
NEXUS 670 spectrometer in KBr with absorption in cm-1. Electronic ionization Mass spectra
were recorded on Thermo Finnigan ESI ion trap mass spectrometer and HRMS done on high
D

resolution QSTAR XL Hybrid/MS system. HPLC data of compounds was recorded on Waters
2998 instrument and Waters 2998 Photodiode Array Detector using Kromasil 100 C18, 5u
TE

column (Dimensions: 150 X 4.6 mm) at 210-400 nm wavelength and water/methanol as the
mobile phase (composition mentioned in the data).
EP

4.2. General procedure for synthesis of α-haloketones (2): To the solution of

alkyl/aryl/heteroaryl ketone 1 (1 mmol) in ethyl acetate (10 mL), copper(II)bromide (1.2 mmol)
was added. The reaction mixture was allowed to stir at room temperature for about 24 hrs. After
C

completion of the reaction (as monitored by TLC), reaction mixture was filtered off. The filtrate
was concentrated under reduced pressure to get the desired product in 95-99% yield.
AC

4.3. General procedure for synthesis of imidazo[1,2-a]pyrimidines 4a-o: Various 2-

aminopyrimidines 3 (1 mmol) dissolved in acetone (15 mL) and α-haloketones 2 (1 mmol) was
added to the above solution with constant stirring at ambient temperatures. After completion of
the reaction (as monitored by TLC), the solid product was filtered off and washed with acetone
to get the pure imidazo[1,2-a]pyrimidine without any purification. The isolated yields are 85-
98%.
 ACCEPTED MANUSCRIPT

4.3.1. 2-Phenylimidazo[1,2-a]pyrimidine(4a): White solid, Yield: 98%; M. P. 248-250 0C; 1H

NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 7.44-7.60(m, 3H), 7.68(dd, J = 4.34, 6.61 Hz, 1H),
7.99(d, J = 6.42 Hz, 2H), 8.99(dd, J = 1.70, 4.34 Hz, 1H), 9.12(s, 1H), 9.83(d, J = 6.61 Hz, 1H);
ESI-MS: m/z 196 [M+H]+.

4.3.2. 2-p-Tolylimidazo[1,2-a]pyrimidine(4b): White solid, Yield: 97%; M. P. 294-296 0C; 1H

PT
NMR (DMSO-d6, 300 MHz) δ ppm 2.45(s, 3H), 7.35(d, J = 8.12 Hz, 2H), 7.66(dd, J = 4.34,
6.61 Hz, 1H), 7.86(d, J = 8.30 Hz, 2H), 8.94-9.02(m, 2H), 9.74(d, J = 6.61 Hz, 1H); ESI-MS:
m/z 210 [M+H]+.

RI
4.3.3. 2-(4-Chlorophenyl)imidazo[1,2-a]pyrimidine(4c): White solid, Yield: 96%; M. P. 267-269
0
C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 7.62(d, J = 7.72 Hz, 3H), 8.03(d, J = 8.83 Hz, 2H),

SC
8.82(s, 1H), 8.99(d, J = 4.41 Hz, 1H), 9.39(dd, J = 4.41, 6.62 Hz, 1H); ESI-MS: m/z 230
[M+H]+.

4.3.4. 2-(2,4-Dichlorophenyl)imidazo[1,2-a]pyrimidine (4d): brown solid, Yield: 88%; M. P.

U
208-210 0C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 7.25(dd, J = 4.27, 6.71 Hz, 1H), 7.45(dd, J =
AN
1.98, 8.24 Hz, 1H), 7.62( dd, J = 1.98, 8.24 Hz, 2H), 8.67(s, 1H), 8.87(dd, J = 1.98, 4.27 Hz,
1H), 9.84(dd, J = 2.13, 6. 71 Hz, 1H); ESI-MS: m/z 264 [M+H]+.

4.3.5. 2-(Naphthalen-2-yl)imidazo[1,2-a]pyrimidine(4e): White solid, Yield: 91%; M. P. 276-

M

278 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 7.57-7.71(m, 3H), 7.89-8.08(m, 4H),
8.57(s, 1H), 8.96-9.02(m, 2H), 9.61(dd, J = 1.70, 6.79 Hz, 1H); ESI-MS: m/z 246 [M+H]+.
D

4.3.6. 2-(Thiophen-2-yl)imidazo[1,2-a]pyrimidine(4f): Dirty white solid, Yield: 92%; M. P. 240-

242 0C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 7.23(t, J = 4.57 Hz, 1H), 7.61(q, J = 4.57 Hz,
TE

1H), 7.67-7.71(m, 1H), 7.85(d, J = 3.66 Hz, 1H), 8.63(s, 1H), 8.96(d, J = 3.20 Hz, 1H), 9.41-
9.46(m, 1H); ESI-MS: m/z 202 [M+H]+.
EP

4.3.7. 2-o-Tolylimidazo[1,2-a]pyrimidine(4g): Light red solid, Yield: 86%; M. P. 265-267 0C; 1H

NMR (DMSO-d6, 300 MHz) δ ppm 2.56(s, 3H), 7.33-7.53(m, 3H), 7.58-7.74(m, 2H), 8.89(s,
1H), 8.98-9.06(m, 1H), 9.96(dd, J = 1.70, 6.79 Hz, 1H); ESI-MS: m/z 210 [M+H]+.
C

4.3.8. 2-(4-Bromophenyl)imidazo[1,2-a]pyrimidine(4h): White solid, Yield: 89%; M. P. 287-289

AC

0
C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 7.27(t, J = 7.91 Hz, 2H), 7.62-7.67(m, 1H), 8.03-
8.10(m, 2H), 8.94-9.01(m, 2H), 9.64(d, J = 5.93 Hz, 1H); ESI-MS: m/z 274 [M+H]+.

4.3.9. 2-(tert-Butyl)imidazo[1,2-a]pyrimidine(4i): White solid, Yield: 87%; M. P. 296-298 0C; 1H

NMR (CDCl3, 500 MHz) δ ppm 1.42(s, 9H), 6.79 (dd, J = 4.12, 6.71 Hz, 1H), 7.30(s, 1H),
8.36(dd, J = 2.13, 6.71 Hz, 1H), 8.46(dd, J = 2.13, 4.12 Hz, 1H); ESI-MS: m/z 176 [M+H]+.

4.3.10. 4-(Imidazo[1,2-a]pyrimidin-2-yl)phenol(4j): White solid, Yield: 90%; M. P. 290-292 0C;

1
H NMR (DMSO-d6, 500 MHz) δ ppm 6.97(d, J = 8.24 Hz, 2H), 7.57-7.66(m, 1H), 7.74-7.81(m,
 ACCEPTED MANUSCRIPT

2H), 8.63(s, 1H), 8.93-8.97(m, 1H), 9.42-9.48(m, 1H), 9.83-10.17(brs, 1H); ESI-MS: m/z 212
[M+H]+.

4.3.11. 7-Methyl-2-phenylimidazo[1,2-a]pyrimidine(4k): White solid, Yield: 95%; M. P. 270-

272 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 2.82(s, 3H), 7.50-7.62(m, 4H), 7.89-
7.97(m, 2H), 8.88(s, 1H), 9.56(d, J = 6.79 Hz, 1H); ESI-MS: m/z 210 [M+H]+.

PT
4.3.12. 2-(4-Chlorophenyl)-7-methylimidazo[1,2-a]pyrimidine(4l): White solid, Yield: 90%; M.
P. 246-248 0C; 1H NMR (DMSO-d6, 300 MHz) δ ppm 2.70(s, 3H), 7.48(d, J = 6.79 Hz, 1H),
7.67(d, J = 8.49 Hz, 2H), 8.00(d, J = 8.49 Hz, 2H), 8.65(s, 1H), 8.13(d, J = 6.79 Hz, 1H); ESI-

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MS: m/z 244 [M+H]+.

4.3.13. 4-(7-Methylimidazo[1,2-a]pyrimidin-2-yl)phenol(4m): White solid, Yield: 88%; M. P.

SC
290-292 0C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 2.74(s, 3H), 6.93(d, J = 8.39 Hz, 2H), 7.31-
7.41(m, 1H), 7.71-7.79(m, 2H), 8.41(s, 1H), 9.15-9.20(m, 1H) 9.83(brs, 1H); ESI-MS: m/z 226
[M+H]+.

U
4.3.14. 2-(tert-Butyl)-7-methylimidazo[1,2-a]pyrimidine(4n): White solid, Yield: 85%; M. P. 75-
AN
77 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 1.46(s, 9H), 2.78(s, 3H), 7.43(d, J = 6.98
Hz, 1H), 8.09(d, J = 2.26 Hz, 1H), 9.39(dd, J = 4. 15, 6.79 Hz, 1H); ESI-MS: m/z 190 [M+H]+.
M

4.3.15. 5,7-Dimethoxy-2-phenylimidazo[1,2-a]pyrimidine(4o): White solid, Yield: 89%; M. P.

281-283 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 3.41(s, 3H), 3.88(s, 3H), 5.23(s,
1H), 7.33-7.50(m, 3H), 7.82(d, J = 7.17 Hz, 2H), 7.91(s, 1H); ESI-MS: m/z 256 [M+H]+.
D
TE

4.4. General procedure for synthesis of imidazo[1,2-a]pyrimidine Mannich bases 5a-m: To the
solution of secondary amine (1 mmol) in methanol excess formaldehyde solution (37%) (10
mmol) was added. After five minutes imidazo[1,2-a]pyrimidine (1 mmol) was added with
EP

constant stirring. Then the reaction mixture was subjected to reflux. After completion of the
reaction (as monitored by TLC), methanol was evaporated under reduced pressure, extracted
with ethyl acetate. The combined organic layers were concentrated and the crude product was
C

purified by column chromatography using solvent system hexane and ethyl acetate (4:6) to give
the desired product. The yields are 74-94%.
AC

4.4.1. 4-((2-Phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)morpholine (5a): Colourless solid,

Yield: 92%; M. P. 134-136 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.49(t, J = 4.53 Hz, 4H),
3.68(t, J = 4.53 Hz, 4H), 4.03(s, 2H), 6.90(dd, J = 4.53, 6.79 Hz, 1H), 7.42(d, J = 6.79 Hz, 1H),
7.48(t, J = 7.55 Hz, 2H), 7.85(d, J = 6.79 Hz, 2H), 8.59(dd, J = 2.26, 4.53 Hz, 1H), 8.79(dd, J =
1.51, 6.79 Hz, 1H). 13C NMR (CDCl3, 75 MHz) δ ppm 51.87, 53.06, 66.76, 108.10, 114.33,
128.13, 128.41, 128.84, 133.03, 133.50, 146.42, 148.04, 149.70; IR (KBr) cm-1 3513, 3395,
 ACCEPTED MANUSCRIPT

2962, 2849, 1612, 1503, 1231, 1117, 706; ESI-MS: m/z 295 [M+H]+; ESI-HRMS (m/z): [M+H]+
calcd for C17H19ON4, 295.1553; obsd 295.1549.

4.4.2. 7-Methyl-2-phenyl-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5b): Off-white

solid, Yield: 84%; M. P. 117-119 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 1.38-1.60(m, 6H),
2.29-2.47(brs, 4H), 2.64(s, 3H), 3.93(s, 2H), 6.72(d, J = 6.86 Hz, 1H), 7.33-7.56(m, 3H), 7.87(d,

PT
J = 8.08 Hz, 2H), 8.68(d, J = 7.01 Hz, 1H). 13C NMR (CDCl3, 75 MHz) δ ppm 24.24, 24.85,
25.96, 52.45, 54.12, 108.69, 114.97, 127.63, 128.22, 128.77, 132.67, 134.22, 145.01, 148.06,
159.45; IR (KBr) cm-1 3420, 2934, 1622, 1510, 1346, 1205, 1105, 769, 703; ESI-MS: m/z 307

RI
[M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C19H23N4, 307.1917; obsd 307.1914.

4.4.3. 4-(3-(Piperidin-1-ylmethyl)imidazo[1,2-a]pyrimidin-2-yl)phenol (5c): Light yellow solid,

SC
Yield: 85%; M. P. 172-174 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 1.36-1.61(m, 6H), 2.33-
2.47(m, 4H), 3.93(s, 2H), 6.80-7.02(m, 3H), 7.54-7.67(m, 2H), 8.53(dd, J = 2.26, 4.53 Hz, 1H),
8.83(dd, J = 2.26, 6.79 Hz, 1H); 13C NMR (CDCl3+DMSO-d6, 75 MHz) δ ppm 23.64, 25.34,

U
51.44, 53.40, 107.59, 114.97, 115.08, 129.33, 129.43, 133.11, 137.97, 148.75, 157.16, 157.36;
IR (KBr) cm-1 3416, 2929, 1615, 1505, 1380, 1238, 1101, 840, 766; ESI-MS: m/z 309 [M+H]+;
AN
ESI-HRMS (m/z): [M+H]+ calcd for C18H21ON4, 309.1709; obsd 309.1708.

4.4.4. 4-(7-Methyl-3-(pyrrolidin-1-ylmethyl)imidazo[1,2-a]pyrimidin-2-yl)phenol (5d): Brown

solid, Yield: 78%; M. P. 162-164 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm 1.73(brs,
M

4H), 2.42-2.64(m, 8H), 4.07(s, 2H), 6.81-6.91(m, 3H), 7.65(d, J = 8.30 Hz, 2H), 8.72(d, J = 7.55
Hz, 1H); 13C NMR (CDCl3+DMSO-d6, 75 MHz) δ ppm 23.00, 24.19, 47.56, 52.93, 108.36,
D

114.77, 114.98, 124.87, 129.36, 132.41, 143.29, 146.86, 156.98, 158.36; IR (KBr) cm-1 3445,
2958, 2771, 1613, 1502, 1388, 1285, 1213, 833; ESI-MS: m/z 309 [M+H]+; ESI-HRMS (m/z):
TE

[M+H]+ calcd for C18H21ON4, 309.1709; obsd 309.1702.

4.4.5. 2-Phenyl-3-(pyrrolidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5e): White solid, Yield:

86%; M. P. 123-125 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 1.67-1.82(m, 4H), 2.38-2.57(m,
EP

4H), 4.14(s, 2H), 6.86 (dd, J = 4.53, 6.79 Hz, 1H), 7.38(m, 1H), 7.43-7.51(m, 2H), 7.86(d, J =
6.79 Hz, 2H), 8.56(dd, J = 2.26, 4.53 Hz, 1H), 8.81-8.89(dd, J = 1.51, 6.79 Hz, 1H); 13C NMR
(CDCl3, 75 MHz) δ ppm 23.44, 48.66, 53.61, 107.90, 116.42, 127.83, 128.23, 128.93, 133.18,
C

133.84, 144.96, 147.75, 149.38; IR (KBr) cm-1 3423, 2960, 2780, 1607, 1502, 1387, 1217, 1116,
AC

767, 695; ESI-MS: m/z 279 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C17H19N4, 279.1604;
obsd 279.1598.

4.4.6. 2-Phenyl-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5f): Brown solid, Yield:

90%; M. P. 116-118 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 1.38-1.61(m, 6H), 2.31-2.53(m,
4H), 3.97(s, 2H), 6.86(dd, J = 4.15, 6.79 Hz, 1H), 7.38(t, J = 7.17 Hz, 1H), 7.47(t, J = 6.98 Hz,
2H), 7.86(d, J = 6.98 Hz, 2H), 8.56(dd, J = 2.07, 4.15 Hz, 1H), 8.86(dd, J = 2.07, 6.98 Hz, 1H);
13
C NMR (CDCl3, 75 MHz) δ ppm 23.64, 25.36, 51.65, 53.55, 107.46, 115.08, 127.36, 127.79,
128.32, 133.00, 133.38, 145.11, 147.40, 148.98. IR (KBr) cm-1 3428, 2925, 1607, 1502, 1340,
 ACCEPTED MANUSCRIPT

1217, 1102, 765, 694; ESI-MS: m/z 293 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for
C18H21N34, 293.1760; obsd 293.1750.

4.4.7. 2-(tert-Butyl)-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5g): Brown solid, Yield:

83%; M. P. 98-100 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 1.14-1.76(m, 15H), 2.39(s, 4H),
3.92(s, 2H), 6.78(dd, J = 3.77, 6.79 Hz, 1H), 8.47(dd, J = 2.26, 4.53 Hz, 1H), 8.78(dd, J = 2.26,

PT
6.79 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm 24.17, 25.89, 31.06, 33.58, 52.85, 53.99,
107.34, 113.63, 133.37, 146.71, 148.28, 154.68; IR (KBr) cm-1 3415, 2926, 1606, 1501, 1332,
1235, 1097, 1038, 985, 803, 763; ESI-MS: m/z 273 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for

RI
C16H25N4, 273.2073; obsd 273.2067.

4.4.8. N-Methyl-N-((2-phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)aniline (5h): Light yellow

SC
viscous liquid, Yield: 74%; 1H NMR (CDCl3, 300 MHz) δ ppm 3.40(s, 3H), 4.87(s, 2H), 6.59-
7.01(m, 4H), 7.32-7.56(m, 5H), 7.78-7.89(m, 2H), 8.46-8.59(m, 2H); 13C NMR (CDCl3, 125
MHz) δ ppm 40.51, 55.77, 93.47, 108.41, 113.11, 114.74, 117.27, 128.21, 128.35, 128.62,

U
128.89, 131.29, 144.95, 147.63, 149.27, 149.66; IR (KBr) cm-1 3425, 2926, 1612, 1503, 1339,
1185, 1085, 767, 704; ESI-MS: m/z 315 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C20H19N4,
AN
315.1604; obsd 315.1598.

4.4.9. 2-(tert-Butyl)-7-methyl-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5i): Light

yellow solid, Yield: 87%; M. P. 136-138 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 1.36-1.62(m,
M

15H), 2.26-2.52(brs, 4H), 2.57(s, 3H), 3.87(s, 2H), 6.64(d, J = 7.01 Hz, 1H), 8.59(d, J = 7.01 Hz,
1H); 13C NMR (CDCl3, 75 MHz) δ ppm 24.27, 24.44, 25.96, 31.09, 33.55, 52.88, 53.95, 107.92,
D

112.97, 132.65, 146.75, 153.93, 157.98; IR (KBr) cm-1 3419, 2935, 1620, 1505, 1445, 1338,
1212, 1103, 986, 792, 765; ESI-MS: m/z 287 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for
TE

C17H27N4, 287.2230; obsd 287.2217.

4.4.10. 4-((7-Methyl-2-phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)morpholine (5j): White solid,

Yield: 88%; M. P. 82-84 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.35-2.63(m, 7H), 3.62(brs,
EP

4H), 3.95(s, 2H), 6.76(t, J = 7.55 Hz, 1H), 7.24-7.50(m, 3H), 7.79(t, J = 8.30, 9.06 Hz, 2H),
8.59-8.67(m, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm 24.67, 51.65, 52.86, 66.61, 108.83,
113.71, 127.68, 128.15, 128.52, 132.19, 133.70, 145.22, 147.93, 159.62; IR (KBr) cm-1 3038,
C

2929, 1620, 1504, 1339, 1111, 1001, 863; ESI-MS: m/z 309 [M+H]+; ESI-HRMS (m/z): [M+H]+
AC

calcd for C18H21ON4, 309.1709; obsd 309.1703.

4.4.11. 4-((2-(4-Chlorophenyl)-7-methylimidazo[1,2-a]pyrimidin-3-yl)methyl)morpholine (5k):

White solid, Yield: 92%; M. P. 205-207 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 2.42-2.51(m,
4H), 2.65(s, 3H), 3.63-3.70(m, 4H), 3.96(s, 2H), 6.77(d, J = 7.01 Hz, 1H), 7.43(d, J = 8.54 Hz,
2H), 7.81(d, J = 8.54 Hz, 2H), 8.59(d, J = 7.01 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm
24.72, 51.66, 52.91, 66.61, 109.12, 113.98, 128.43, 129.75, 129.78, 132.19, 133.67, 144.08,
147.93, 160.09; IR (KBr) cm-1 3424, 2928, 2845, 1620, 1339, 1208, 1111, 1001, 863, 793; ESI-
 ACCEPTED MANUSCRIPT

MS: m/z 343 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C18H20ON4Cl, 343.1320; obsd
343.1312.

4.4.12. 7-Methyl-2-phenyl-3-(pyrrolidin-1-ylmethyl)imidazo[1,2-a]pyrimidine (5l): Brown solid,

Yield: 86%; M. P. 131-133 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 1.67-1.82(brs, 4H), 2.45-
2.58(brs, 4H), 2.63(s, 3H), 4.13(s, 2H), 6.75(d, J = 6.98 Hz, 1H), 7.33-7.50(m, 3H), 7.80(d, J =

PT
6.98 Hz, 2H), 8.72(d, J = 6.98 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm 23.39, 24.69, 48.46,
53.50, 63.82, 108.94, 115.78, 127.71, 128.23, 128.76, 129.57, 132.62, 133.82, 144.06, 159.80;
IR (KBr) cm-1 3388, 2927, 2780, 1621, 1513, 1387, 1209, 1112, 768, 697; ESI-MS: m/z 293

RI
[M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C18H21N4, 293.1760; obsd 293.1756.

4.4.13. 3-(Pyrrolidin-1-ylmethyl)-2-(thiophen-2-yl)imidazo[1,2-a]pyrimidine (5m): Light yellow

SC
solid, Yield: 74%; M. P. 114-116 0C; 1H NMR (CDCl3+DMSO-d6, 500 MHz) δ ppm 1.79(s, 4H),
2.59(s, 4H), 4.24(s, 2H), 6.92-7.00(m, 1H), 7.12-7.18(m, 1H), 7.40-7.45(m, 1H), 7.59-7.64(m,
1H), 8.50-8.59(m, 1H), 8.80-8.88 (m, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm 23.18, 48.08,

U
53.38, 63.56, 108.10, 108.21, 125.99, 126.22, 127.30, 132.93, 136.05, 139.02, 149.67; IR (KBr)
cm-1 3403, 2958, 1613, 1505, 1318, 1196, 1045, 795, 708; ESI-MS: m/z 285 [M+H]+; ESI-
AN
HRMS (m/z): [M+H]+ calcd for C15H17N4S, 285.1168; obsd 285.1165.

4.5. Procedure for synthesis of 2-phenyl-3-(piperazin-1-ylmethyl)imidazo[1,2-a]pyrimidine (6a):

M

To the solution of piperazine (1 mmol) in methanol, formaldehyde solution (37%) (3 mmol) was
added. 2-phenylimidazo[1,2-a]pyrimidine (1 mmol) was added to the above reaction mixture
with constant stirring. Then the reaction mixture was stirred under reflux conditions. After
D

completion of the reaction, as monitored by TLC, methanol was evaporated under reduced
pressure. The crude product was purified by column chromatography using ethyl
TE

acetate/methanol (9:1) as eluent to get the desired product in 75% yield. Light red solid, M. P.
183-185 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 2.49(brs, 1H), 2.93(s, 8H), 4.03(s, 2H), 6.95(dd,
J = 4.15, 6.61 Hz, 1H), 7.40(d, J = 7.17 Hz, 1H), 7.42-7.50(m, 2H), 7.81(d, J = 6.98 Hz, 2H),
EP

8.57(dd, J = 1.88, 3.96 Hz, 1H), 8.81(dd, J = 1.51, 6.79 Hz, 1H); 13C NMR (CDCl3+DMSO-d6,
75 MHz) δ ppm 9.07, 46.37, 52.73, 108.73, 115.61, 128.26, 128.76, 128.84, 128.93, 134.22,
145.48, 148.01, 150.23; IR (KBr) cm-1 3423, 2937, 2806, 2677, 1610, 1498, 1399, 1226, 1170,
C

1034, 766, 701; ESI-MS: m/z 294 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C17H20N5,
294.1713; obsd 294.1707.
AC

4.6. Procedure for synthesis of 3-((4-methylpiperazin-1-yl)methyl)-2-phenylimidazo[1,2-

a]pyrimidine (6b): To the solution of 4-methylpiperazine (1 mmol) in methanol, excess
formaldehyde solution (37%) (6 mmol) was added. 2-Phenylimidazo[1,2-a]pyrimidine (1 mmol)
was added to the above reaction mixture with constant stirring. Then the reaction mixture was
stirred under reflux conditions. After completion of the reaction, as monitored by TLC, methanol
was evaporated under reduced pressure. The crude product was purified by column
chromatography using ethyl acetate/methanol (9:1) as eluent to get the desired product in 79%
 ACCEPTED MANUSCRIPT

yield. White solid, M. P. 124-126 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.28(s, 3H), 2.32-
2.70(m, 8H), 4.02(s, 2H), 6.88(dd, J = 4.15, 6.79 Hz, 1H), 7.40(d, J = 7.17 Hz, 1H), 7.47(t, J =
7.55 Hz, 2H), 7.87(d, J = 6.98 Hz, 2H), 7.57(dd, J = 1.88, 3.96 Hz, 1H), 8.79(dd, J = 1.88, 6.79
Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ ppm 45.73, 51.50, 52.54, 54.87, 107.95, 114.82, 127.99,
128.32, 128.83, 133.07, 133.62, 146.15, 148.00, 149.52; IR (KBr) cm-1 3447, 2927, 2792, 1608,
1502, 1449, 1342, 1151, 1008, 767, 693; ESI-MS: m/z 308 [M+H]+; ESI-HRMS (m/z): [M+H]+

PT
calcd for C18H22N5, 308.1869; obsd 308.1862.

4.7. Procedure for synthesis of 2-phenyl-3-((4-tosylpiperazin-1-yl)methyl)imidazo[1,2-

RI
a]pyrimidine (6c): To the solution of 4-tosylpiperazine (1 mmol) in methanol, formaldehyde
solution (37%) (5 mmol) was added. 2-Phenylimidazo[1,2-a]pyrimidine (1 mmol) was added to
the above reaction mixture with constant stirring. Then the reaction mixture was stirred under

SC
reflux conditions. After completion of the reaction, as monitored by TLC, methanol was
evaporated under reduced pressure. The crude product was purified by column chromatography
using ethyl acetate/methanol (9:1) as eluent to get the desired product in 71% yield. White solid,

U
M. P. 185-187 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.43(d, J = 7.55 Hz, 7H), 3.08(s, 4H),
4.95(s, 2H), 6.93(dd, J = 3.77, 6.79 Hz, 1H), 7.28-7.38(m, 5H), 7.60(d, J = 7.55 Hz, 2H), 7.73(d,
AN
J = 7.55 Hz, 2H), 8.54(dd, J = 1.51, 6.79 Hz, 1H), 8.59(dd, J = 1.51, 4.53 Hz, 1H); 13C NMR
(CDCl3+DMSO-d6, 75 MHz) δ ppm 22.05, 46.79, 50.70, 52.15, 79.25, 109.58, 121.01, 128.46,
129.04, 129.43, 129.51, 130.74, 130.80, 132.92, 134.51, 134.86, 144.72, 151.30; IR (KBr) cm-1
M

3446, 2925, 2855, 1347, 1165, 1128, 733, 546; ESI-MS: m/z 448 [M+H]+; ESI-HRMS (m/z):
[M+H]+ calcd for C24H26O2N5S, 448.1801; obsd 448.1784.
D

4.8. General procedure for synthesis of 1,4-bis((imidazo[1,2-a]pyrimidin-3-

yl)methyl)piperazines 7a-l: To the solution of piperazine (1 mmol) in methanol, excess
TE

formaldehyde solution (37%) (10 mmol) was added. Imidazo[1,2-a]pyrimidine (2 mmol) was
added to the above reaction mixture with constant stirring. Then the reaction mixture was stirred
under reflux conditions. After completion of the reaction, as monitored by TLC, methanol was
EP

evaporated under reduced pressure. The crude product was purified by column chromatography
using ethyl acetate/methanol (9:1) as eluent to get the desired product in 70-93% yield. Poor
solubility of some imidazopyrimidine dimers in NMR solvents resulted in incomplete spectral
C

data.
AC

4.8.1. 1,4-bis((2-Phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7a): White solid, Yield:

92%; M. P. 265-268 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.46(brs, 8H), 4.01(s, 4H), 6.89(dd,
J = 4.15, 6.79 Hz, 2H), 7.40(d, J = 7.17 Hz, 2H), 7.46(t, J = 6.98, 7.36 Hz, 4H), 7.80(d, J = 6.79
Hz, 4H), 8.58(dd, J = 2.07, 4.15 Hz, 2H), 8.74(dd, J = 2.07, 6.79 Hz, 2H); IR (KBr) cm-1 3450,
2950, 2810, 1612, 1499, 1344, 1226, 1008, 771, 703; ESI-MS: m/z 501 [M+H]+; ESI-HRMS
(m/z): [M+H]+ calcd for C30H29N8, 501.2509; obsd 501.2491.
 ACCEPTED MANUSCRIPT

4.8.2. 1,4-bis((2-(4-Tolyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7b): White solid,

Yield: 93%; M. P. 272-275 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.41(brs, 10H), 3.49(s, 4H),
4.00(s, 4H), 6.86(dd, J = 4.15, 6.79 Hz, 2H), 7.27(d, J = 7.93 Hz, 4H), 7.70(d, J = 8.12 Hz, 4H),
8.56 (dd, J = 1.88, 3.96 Hz, 2H), 8.73(dd, J = 1.88, 6.79 Hz, 2H); IR (KBr) cm-1 3388, 3023,
2805, 1614, 1499, 1342, 1214, 1117, 1010, 828, 764; ESI-MS: m/z 529 [M+H]+; ESI-HRMS
(m/z): [M+H]+ calcd for C32H33N8, 529.2822; obsd 529.2809.

PT
4.8.3. 1,4-bis((2-(4-Chlorophenyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7c): White
solid, Yield: 86%; M. P. 207-209 0C; 1H NMR (DMSO-d6, 300 MHz) δ ppm 2.11-2.44(m, 4H),

RI
2.71-3.15(m, 4H), 4.02(brs, 2H), 5.00(brs, 2H), 7.08-7.32(m, 2H), 7.54(d, J = 8.30 Hz, 4H),
7.78-7.99(m, 4H), 8.58-8.73(m, 2H), 8.90-9.29(m, 2H); IR (KBr) cm-1 2983, 1610, 1502, 1335,
1215, 1128, 1005, 832, 766; ESI-MS: m/z 569 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for

SC
C30H27N8Cl2, 569.1730; obsd 569.1721.

4.8.4. 1,4-bis((2-(tert-Butyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7d): White solid,

U
Yield: 70%; M. P. 287-290 0C; 1H NMR (CDCl3+DMSO-d6, 500 MHz) δ ppm 1.44(s, 18H),
AN
2.22-2.51(brs, 8H), 3.97(s, 4H), 6.88-6.96(m, 2H), 8.41-8.47(m, 2H), 8.75-8.82(m, 2H); 13C
NMR (CDCl3+DMSO-d6, 75 MHz) δ ppm 32.10, 34.60, 52.23, 53.40, 108.56, 114.16, 134.58,
147.32, 149.42, 155.22; IR (KBr) cm-1 3425, 2948, 2802, 1611, 1499, 1336, 1236, 1146, 1005,
767; ESI-MS: m/z 461 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C26H37N8, 461.3135; obsd
M

461.3134.
D

4.8.5. 1,4-bis((7-Methyl-2-phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7e): White

solid, Yield: 71%; M. P. 290-292 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 1.80(s, 6H), 2.47(brs,
TE

8H), 4.01(s, 4H), 6.89(dd, J = 4.53, 6.79 Hz, 2H), 7.39(d, J = 6.79 Hz, 2H), 7.42-7.50(m, 4H),
7.80(d, J = 7.55 Hz, 4H), 8.58(dd, J = 1.51, 3.77 Hz, 2H); IR (KBr) cm-1 3442, 2950, 2808, 1610,
1499, 1342, 1225, 1121, 1008, 771, 703; ESI-MS: m/z 529 [M+H]+; ESI-HRMS (m/z): [M+H]+
EP

calcd for C32H33N8, 529.2822; obsd 529.2809.

4.8.6. 1,4-bis((2-(tert-Butyl)-7-methylimidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7f):

C

White solid, Yield: 73%; M. P. 218-221 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm
2.46(brs, 4H), 3.05(brs, 4H), 3.14(s, 28H), 7.37(d, J = 8.12 Hz, 2H), 7.59(d, J = 8.30 Hz, 2H);
AC

13
C NMR (CDCl3+DMSO-d6, 75 MHz) δ ppm 24.44, 31.19, 33.66, 67.33, 78.60, 107.90, 108.62,
121.74, 132.58, 157.91, 161.82; IR (KBr) cm-1 2954, 1622, 1507, 1448, 1341, 1216, 1146, 1004,
801; ESI-MS: m/z 489 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C28H41N8, 489.3400; obsd
489.3400.

4.8.7. 1,4-bis((2-(Naphthalen-2-yl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7g): Yellow

solid, Yield: 82%; M. P. 260-262 0C; 1H NMR (DMSO-d6, 300 MHz) δ ppm 3.34(s, 8H), 4.01(s,
4H), 6.95-7.04(m, 2H), 7.39-7.50(m, 5H), 7.68-7.88(m, 9H), 8.54-8.62(m, 2H), 8.79-8.88(m,
 ACCEPTED MANUSCRIPT

2H); IR (KBr) cm-1 3421, 3078, 1613, 1507, 1336, 1268, 1140, 1004, 758; ESI-MS: m/z 601
[M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C38H33N8, 601.2822; obsd 601.2826.

4.8.8. 1,4-bis((2-(2,4-Dichlorophenyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7h):

White solid, Yield: 72%; M. P. 99-101 0C; 1H NMR (DMSO-d6, 500 MHz) δ ppm 1.82-2.11(m,
8H), 3.58-3.77(m, 4H), 6.82-6.93(m, 2H), 7.31-7.59(m, 6H), 7.93-7.98(m, 1H), 8.26-8.32(m,

PT
1H), 8.52-8.65(m, 2H); IR (KBr) cm-1 3074, 3021, 1610, 1527, 1461, 1344, 1199, 1099, 782,
711; ESI-MS: m/z 637 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C30H25N8Cl4, 637.0950;
obsd 637.0951.

RI
4.8.9. 1,4-bis((5,7-Dimethoxy-2-phenylimidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7i):
White solid, Yield: 70%; M. P. 273-275 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 2.68(s, 4H),

SC
3.08(s, 4H), 3.67(s, 6H), 3.78(s, 6H), 4.41(s, 4H), 5.47(s, 2H), 6.93-7.14(m, 6H), 7.39-7.56(m,
4H); IR (KBr) cm-1 3402, 2947, 1665, 1526, 1381, 1204, 995, 770, 699; ESI-MS: m/z 621
[M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C34H37N8O4, 621.2409; obsd 621.2415.

U
AN
4.8.10. 1,4-bis((2-(Thiophen-2-yl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7j): White
solid, Yield: 73%; M. P. 201-203 0C; 1H NMR (CDCl3, 500 MHz) δ ppm 3.40(s, 6H), 5.00(s,
4H), 5.57(s, 2H), 6.90(dd, J = 4.12, 6.71 Hz, 2H), 7.13-7.17(m, 2H), 7.41-7.46(m, 2H), 7.55-
7.66(m, 4H), 8.49(dd, J = 1.83, 6.71 Hz, 2H), 8.57(dd, J = 1.83, 3.96 Hz, 2H); 13C NMR
M

(DMSO-d6, 75 MHz) δ ppm 48.59, 49.04, 108.87, 113.88, 120.58, 126.13, 127.52, 128.27,
136.10, 146.27, 151.53, 151.77; IR (KBr) cm-1 3345, 2840, 1616, 1516, 1431, 1318, 1126, 763,
D

702; ESI-MS: m/z 513 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C26H25N8S2, 513.1638;
obsd 513.1635.
TE

4.8.11. 1,4-bis((2-(2-Tolyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7k): Yellow solid,

Yield: 83%; M. P. 110-112 0C; 1H NMR (CDCl3, 300 MHz) δ ppm 1.95-2.35(m, 6H), 2.35-
EP

2.74(m, 4H), 3.25-4.02(m, 8H), 6.76-6.92(m, 2H), 7.16-7.48(m, 8H), 7.87(dd, J = 2.26, 6.79 Hz,
1H), 8.51-8.64(m, 2H), 8.77-8.90(m, 1H); 13C NMR (DMSO-d6, 75 MHz) δ ppm 19.01, 52. 44,
54.66, 108.85, 120.64, 126.07, 126.80, 129.31, 130.35, 131.67, 132.21, 137.01, 138.34, 146.80,
C

149.49; IR (KBr) cm-1 3392, 3066, 2926, 1616, 1497, 1327, 1133, 1005, 765; ESI-MS: m/z 529
[M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for C32H33N8, 529.2822; obsd 529.2811.
AC

4.8.12. 1,4-bis((2-(4-Bromophenyl)imidazo[1,2-a]pyrimidin-3-yl)methyl)piperazine (7l): Light

orange solid, Yield: 85%; M. P. 115-117 0C; 1H NMR (CDCl3+DMSO-d6, 300 MHz) δ ppm
2.47(brs, 8H), 3.99(s, 4H), 6.88-7.02(m, 2H), 7.54-7.83(m, 8H), 8.52-8.64(m, 2H), 8.73-8.90(m,
2H); 13C NMR (DMSO-d6, 75 MHz) δ ppm 50.87, 52.05, 108.51, 116.03, 121.28, 130.31,
131.47, 133.15, 133.93, 143.20, 147.26, 150.37; IR (KBr) cm-1 3422, 2926, 1615, 1498, 1343,
1141, 1004, 828, 765; ESI-MS: m/z 657 [M+H]+; ESI-HRMS (m/z): [M+H]+ calcd for
C30H27N8Br2, 657.0719; obsd 657.0740.
 ACCEPTED MANUSCRIPT

4.9. Evaluation of the antiproliferative activity against A-549, HeLa, Panc-1and MDA-MB-231
cell lines.
4.9.1. Materials and Methods, Cell Cultures, Maintenance and Antiproliferative Evaluation:
The cell lines, A-549, HeLa, Panc-1 and MDA-MB-231(lung carcinoma, cervical, pancreatic,
and neuroblastoma) which were used in this study were procured from American Type Culture

PT
Collection (ATCC), United States. The synthesized test compounds were evaluated for their in
vitro antiproliferative activity in these four different human cancer cell lines. A protocol of 48
hrs continuous drug exposure was used, and a SRB cell proliferation assay was used to estimate

RI
cell viability or growth. All the cell lines were grown in Dulbecco's modified Eagle's medium
(containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 0C). Cells were trypsinized
when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-well plates in 100 µL

SC
aliquots at plating densities depending on the doubling time of individual cell lines. The
microtiter plates were incubated at 37 0C, 5% CO2, 95% air, and 100% relative humidity for 24
hrs prior to addition of experimental drugs and were incubated for 48 hrs with different doses

U
(0.01, 0.1, 1, 10, 100 µM) of prepared derivatives where DMSO treated cells and DMF treated
AN
cells were used as negative controls for simple Mannich bases and symmetrical dimers
respectively. After 48 hrs incubation at 37 0C, cell monolayers were fixed by the addition of 10%
(wt/vol) cold trichloroacetic acid and incubated at 4 0C for 1hr and were then stained with
0.057% SRB dissolved in 1% acetic acid for 30 min at room temperature. Unbound SRB was
M

washed with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution
for OD determination at 510 nm using a micro plate reader (Enspire, Perkin Elmer, and USA).
D

Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth
in the presence of drug at the five concentration levels (Ti)], the percentage growth was
TE

calculated at each of the drug concentrations levels. Percentage of growth inhibition was
calculated as:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti ≥ Tz

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[(Ti-Tz)/Tz] x 100 for concentrations for which Ti < Tz.

C

The dose response parameter, growth inhibition of 50% (GI50) was calculated from [(Ti-Tz)/(C-
Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein
AC

increase (as measured by SRB staining) in control cells during the drug incubation. Values were
calculated for this parameter if the level of activity is reached. However, if the effect is not
reached or is exceeded, the value for that parameter was expressed as greater or less than the
maximum or minimum concentrations tested.
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Spectras:

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H NMR (CDCl3, 300 MHz) spectrum of compound 5a
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C NMR (CDCl3, 75 MHz) spectrum of compound 5a
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H NMR (CDCl3, 500 MHz) spectrum of compound 5b

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C NMR (CDCl3, 75 MHz) spectrum of compound 5b
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H NMR (CDCl3, 300 MHz) spectrum of compound 5c
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C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 5c
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H NMR (CDCl3+DMSO-d6, 300 MHz) spectrum of compound 5d
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C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 5d
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H NMR (CDCl3, 300 MHz) spectrum of compound 5e
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C NMR (CDCl3, 75 MHz) spectrum of compound 5e
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H NMR (CDCl3, 500 MHz) spectrum of 5f
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C NMR (CDCl3, 75 MHz) of compound 5f
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H NMR (CDCl3, 500 MHz) spectrum of compound 5g
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C NMR (CDCl3, 75 MHz) spectrum of compound 5g
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H NMR (CDCl3, 300 MHz) spectrum of compound 5h
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C NMR (CDCl3, 125 MHz) spectrum of compound 5h
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H NMR (CDCl3, 500 MHz) spectrum of compound 5i
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C NMR (CDCl3, 75 MHz) spectrum of compound 5i
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H NMR (CDCl3, 300 MHz) spectrum of compound 5j
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C NMR (CDCl3, 75 MHz) spectrum of compound 5j
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H NMR (CDCl3, 500 MHz) spectrum of compound 5k
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C NMR (CDCl3, 75 MHz) spectrum of compound 5k
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H NMR (CDCl3, 300 MHz) spectrum of compound 5l
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C NMR (CDCl3, 75 MHz) spectrum of compound 5l
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H NMR (CDCl3+DMSO-d6,, 500 MHz) spectrum of compound 5m
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C NMR (CDCl3, 75 MHz) spectrum of compound 5m
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H NMR of (CDCl3, 500 MHz) spectrum of compound 6a
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13
C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 6a
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H NMR (CDCl3, 300 MHz) spectrum of compound 6b
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C NMR (CDCl3, 75 MHz) spectrum of compound 6b
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H NMR (CDCl3, 300 MHz) spectrum of compound 6c
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C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 6c
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H NMR (CDCl3, 300 MHz) spectrum of compound 7a
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H NMR (CDCl3, 300 MHz) spectrum of compound 7b
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H NMR (DMSO-d6, 300 MHz) spectrum of compound 7c
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H NMR (CDCl3+DMSO-d6, 500 MHz) spectrum of compound 7d
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C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 7d
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H NMR (CDCl3, 300 MHz) spectrum of compound 7e
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H NMR (CDCl3+DMSO-d6, 300 MHz) spectrum of compound 7f
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13
C NMR (CDCl3+DMSO-d6, 75 MHz) spectrum of compound 7f
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H NMR (DMSO-d6, 300 MHz) spectrum of compound 7g
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H NMR (DMSO-d6, 500 MHz) spectrum of compound 7h
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H NMR (CDCl3, 300 MHz) spectrum of compound 7i
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H NMR (CDCl3, 500 MHz) spectrum of compound 7j
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C NMR (DMSO-d6, 75 MHz) spectrum of compound 7j
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H NMR (CDCl3, 300 MHz) spectrum of compound 7k
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C NMR (DMSO-d6, 75 MHz) spectrum of compound 7k

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1H NMR (CDCl3+DMSO-d6, 300 MHz) spectrum of compound 7l

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C NMR (DMSO-d6, 75 MHz) spectrum of compound 7l
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HPLC data
Conditions:
Column: Kromasil 100 C18, 5u

Dimensions: 150 X 4.6 mm

Detection: 210-400 nm

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Instrument: Waters 2998

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HPLC of compound 7a

Mobile phase: A: 0.1 M CH3COOH and 0.1M CH3COONa (PH 5) in water

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B: Methanol (20 : 80)

Flow Rate: 0.6 mL/min

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HPLC of compound 7b

Mobile phase: A: 0.1 M CH3COOH and CH3COONa (PH 5) in water

B: Methanol (5 : 95)

Flow Rate: 0.6 mL/min

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HPLC of compound 7c

Mobile phase: A: water

B: Methanol (60 : 40)

Flow Rate: 1 mL/min

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HPLC of compound 7e

Mobile phase: A: 0.1 M CH3COOH and CH3COONa (PH 5) in water

B: Methanol (5 : 95)

Flow Rate: 0.6 mL/min

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HPLC of compound 7g

Mobile phase: A: 0.1 M CH3COOH and CH3COONa (PH 5) in water

B: Methanol (5 : 95)

Flow Rate: 0.6 mL/min

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HPLC of compound 7h

Mobile phase: A: water

B: Methanol (30 : 70)

Flow Rate: 0.6 mL/min

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HPLC of compound 7i

Mobile phase: A: Water

B: Methanol (10 : 90)

Flow Rate: 0.6 mL/min

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