Regioselective N-alkylation of some imidazole-containing heterocycles and their in vitro anticancer evaluation

Article abstract of DOI:10.1016/j.molstruc.2019.127673

Imidazole-containing heterocycles: Imidazopyridines, imidazopyrimidines and imidazopyra-zines can exist more tautomeric forms than benzimidazoles. Their regioselectivities were determined for N-alkylations with 4-fluorobenzyl bromide under basic conditions (K₂CO₃) in DMF. We observed that, regioisomers were mainly formed as a mixture in this reaction and N-benzylation occurs at a higher ratio on six membered heterocycles. Their structural assignments were made with the use of two-dimensional ¹H–¹H NOE (nuclear overhauser effect spectroscopy, NOESY). Complementary structural information was provided by 2D-HMBC spectra of the compounds. Synthesized compounds were tested for in vitro cytotoxic activities against Human colon cancer cell line (HCT-116) and leukemia cell lines (K562 and HL-60) by MTT test. Among them, imidazopyridine analogue 10, bearing bromine atom at the C-6 position of the pyridine moiety, gave the lowest IC₅₀ value with 6–7 μg/mL against all three cancer cell lines.

Full text of DOI:10.1016/j.molstruc.2019.127673

Journal of Molecular Structure 1205 (2020) 127673
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Regioselective N-alkylation of some imidazole-containing
heterocycles and their in vitro anticancer evaluation
Cigdem Karaaslan a, Fatima Doganc a, Mehmet Alp a, Asli Koc b, Arzu Zeynep Karabay b,
a, *
€
Hakan Goker
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan, Ankara, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Imidazole-containing heterocycles: Imidazopyridines, imidazopyrimidines and imidazopyra-zines can
exist more tautomeric forms than benzimidazoles. Their regioselectivities were determined for N-al-
kylations with 4-fluorobenzyl bromide under basic conditions (K2CO3) in DMF. We observed that,
regioisomers were mainly formed as a mixture in this reaction and N-benzylation occurs at a higher ratio
on six membered heterocycles. Their structural assignments were made with the use of two-dimensional
1He1H NOE (nuclear overhauser effect spectroscopy, NOESY). Complementary structural information was
provided by 2D-HMBC spectra of the compounds. Synthesized compounds were tested for in vitro
cytotoxic activities against Human colon cancer cell line (HCT-116) and leukemia cell lines (K562 and HL-
60) by MTT test. Among them, imidazopyridine analogue 10, bearing bromine atom at the C-6 position of
Received 1 November 2019
Received in revised form
27 December 2019
Accepted 29 December 2019
Available online 31 December 2019
Keywords:
Imidazo[4,5-b]pyridine
Imidazo[4,5-c]pyridine
imidazo[4,5-d]pyrimidine (purine)
Imidazo[4,5-b]pyrazine
Regioisomer
the pyridine moiety, gave the lowest IC50 value with 6e7
m
g/mL against all three cancer cell lines.
© 2019 Published by Elsevier B.V.
NOESY (nuclear overhauser effect
spectroscopy)
1. Introduction
phenyl substitution at the 2-position and several halogens substi-
tution on the benzene moiety. Examples are represented in Fig. 1
(1) [2]. One of the compound was similarly reported as Rheb in-
hibitors with moderate activity in patent number WO2018/191146
[3].
In 2008, Astrazeneca AB also patented a series of 3H-imidazo
[4,5-b]pyridine-7-carboxamides which were glycogen synthase
kinase 3 (GSK3) inhibitors. As it is known GSK3 inhibitors, inhibit
growth and survival of solid tumors in pancreatic, colon and
prostate cancers. A representative example is given in Fig. 1 (2)
showing best Ki value with 390 nM [4].
In patent number WO2009/111277, Array BioPharma Inc.
claimed a series of imidazo[4,5-b] pyridines as B-Raf inhibitors.
Certain hyperproliferative disorders are characterized by over
activation of Raf kinase function caused by mutation or over
expression of the protein. So discovery of this type of inhibitors is
useful in treatment of cancer disease. In this patent, 42 compounds
with several benzamides substitution at the 6-position were
Imidazopyridines, imidazopyrimidines (purine) and imidazo-
pyrazines are formed by fusing imidazole ring with pyridine, py-
rimidine and pyrazine moieties. Furthermore imidazopyridines
have various isomeric forms like imidazo[4,5-b]pyridines, imidazo
[4,5-c]pyridines, imidazo[1,5-a]pyridines and imidazo[1,2-a]pyri-
dines. In 2017, a review was reported about pharmacological pro-
files of imidazopyridines such as anti-tumoral, anti-microbial, anti-
inflammatory, anti-diabetic, anti-hypertensive and other pharma-
cological properties [1]. In US Patent number 2017/0210741,
Southern Research Institute claimed a series of benzimidazole and
imidazopyridine analogues that are capable of inhibiting Wnt
signaling. Although Wnt/b-catenin signaling plays a critical role in
embryonic development, it can lead to tumor formation and dis-
ease when it is aberrantly activated. Therefore Wnt signaling
pathways are therapeutic targets in cancer therapies. The patent
exemplified 38 compounds in the structure of benzimidazole and
imidazopyridine with (4-(piperazine/pyrrolidine/piperidin-1-yl)
exemplified and some of their IC50’s were found less than 1 mM.
Example is represented in Fig. 1 (3) [5].
Purine ring system is undoubtedly among the most ubiquitous
of all the heterocyclic compounds. The two most common well
* Corresponding author.
E-mail address: goker@ankara.edu.tr (H. Goker).
€
https://doi.org/10.1016/j.molstruc.2019.127673
0022-2860/© 2019 Published by Elsevier B.V.

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C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
Fig. 1. The structures of previously reported 2-phenyl imidazole containing heterocycles.
known purines are adenine and guanine which constitute the
structure of DNA and RNA. Other purines like xanthine, hypoxan-
thine and uric acid are also important naturally occurring metab-
olites, although they are far less abundant than adenine and
guanine. Purine analogues are commonly used to treat cancer by
interfering with DNA replication [6], such as Cladribine, Tioguanine,
Clofarabine, Mercaptopurine, Fludarabine and Nelarabine. Gilead
Sciences disclosed a library of imidazo-(pyrimidine/pyridine) de-
rivatives as inhibitors of TBK1 and/or IKKε. All the compounds were
characterized by the presence of extended substituents on C-4. A
representative example is given in Fig. 1 (4), which shows IC50
values of 1.299 and 2.637 nM on TBK1 and IKKε, respectively [7].
On the other hand, imidazopyrazines have also various isomeric
forms like imidazopyridines some of which were found highly
attractive as anticancer agents. In 2011, Mortensen et al [8] reported
the discovery of the imidazo[4,5-b]pyrazin-2-one series as selective
Mammalian Target of Rapamycin (mTOR) kinase inhibitors.
Recently, new 1H-imidazo[4,5-b]pyrazine derivatives were pub-
lished as potent and selective inhibitor of Mesenchymal-epithelial
transition factor (c-Met) kinase by Zhao et al. [9] and Cui et al.
[10]. Payne et al. [11,12] reported a congeneric series of 1H-imidazo
[4,5-b]pyrazines as dual inhibitors of Inducible/Neuronal Nitric
Oxide Synthase (iNOS/nNOS) simultaneously. These compounds
were primarily found effective to chronic pain state associated with
asthma, inflammatory bowel disease, arthritis and neuropathy.
Compound KD7332 Fig. 1 (5) was confirmed as one of the several
potent inhibitors.
Despite the importance of these structures, bicyclic heterocycles
are difficult to prepare in a regioselective manner, especially with
substitution at the N-positions. The electrons of these rings are
extensively delocalized, hence the alkylation of these unsubstituted
imidazole cores is remarkably unselective. The design of improved,
high efficient and regioselective approaches for these condensed
heterocycles’s preparation is of prime importance. These cores with
unsubstituted NH groups exhibit fast prototropic tautomerism
leading to equilibrium mixtures of asymmetrically substituted
compounds. The existence of this tautomerism has been proved by
several approaches including NMR spectroscopy. This migration is
not found when the N-attached hydrogen is replaced by an alkyl
group. In our recently published papers, we described the synthesis
of some regioisomers of benzimidazoles [13] and imidazopyridines
[14] and elucidated their structure by selective synthesis and/or
2D-NMR data such as nuclear overhauser effect spectroscopy
(NOESY), and heteronuclear multiple bond correlation (HMBC)
experiments. These bicyclic heterocycles were shown to have
inhibitory effect on various kinases as anti-cancer agents.
Prompted by the findings from the studies mentioned above, in
this research we set out to develop and synthesize new bicyclic
derivatives (imidazopyridines, imidazopyrimidines and imidazo-
pyrazines which of their structures are given in Fig. 1) and inves-
tigate their inhibitory activities against some cancer cell lines. The
other aim of this study is to investigate the regioisomer selectivity
of the synthesized compounds during the N-alkylation reaction.
2. Results and discussion
Targeted compounds were prepared using the methods outlined
in Scheme 1- 5. Cyclization of related (o-diamino)pyridine, py-
rimidine and pyrazine derivatives with sodyum metabisulfite
adduct of corresponding benzaldehydes, gave required imidazo-
pyridines 7e16, 24, 25, imidazopyrimidines 27, 28 and imidazo-
pyrazine 33. In these condensed systems, nitrogen bearing a
hydrogen atom (N1,3) resembles a pyrole N-atom; the other nitro-
gen atoms (N4,5,6,7) resemble a pyridine N-atom. Hydrogen atom
attached to nitrogen in the 1,3-position can easily tautomerise in
several positions as in compounds 16, 21, 25, 28 and 33 shown in
Schemes 2e5. Because of these tautomeric forms, both 1H and 13
C
NMR spectra of unsubstituted analogues cannot be clearly seen
unless hexamethylphosphoramide-d18 (HMPA-d18) [15] is used as a
deuterated solvent. Both appearance of some proton and carbon
signals as broad peaks and unobservable some hinge carbon signals
are normal. Elimination of NH proton and substitution of this ni-
trogen atom would prevent tautomerism and can lead to a sepa-
rable mixture of regioisomers [14]. The regioselective alkylation of
one within other nitrogen atoms is difficult in many cases and
giving rise to mixtures of the two regioisomers. We reported the N-
alkylation reaction of some imidazo [4,5-b]pyridines in our previ-
ous study [14]. It was declared that formation ratio of the
regioisomers could be changed with reaction conditions and sol-
vent effects. We aimed to explain some conflict resolutions on the
formation of regioisomers. In the literature there were two
different results about the regiosomer formation ratio under same
conditions. Zeinyeh et al. [16,17] stated that at RT using K2CO3 in
DMF, benzylation of imidazo [4,5-b] pyridine-4-oxide and 2-
methyl- imidazo[4,5-b]pyridine-4-oxide with benzyl bromide or
benzyl iodide gave the N-1/N-3 regioisomers in slightly different
ratios. But they never mentioned about the formation of N-4
regioisomer. In contrast, Ouzidan et al. [18e20] and Bourichi et al.
[21] reported obtaining N-4 regiosomers with X-Ray Data by using
the same reaction. Similarly, when we attempted [14] alkylation of

C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
3
Scheme 1. Synthesis of 2-substituted imidazo[4,5-b]pyridine analogues.
Scheme 2. N-Benzylation of 2-substitutedimidazo[4,5-b]pyridine (16,17) analogues.
some imidazo [4,5-b]pyridine with n-butyl bromide and 4-
fluorobenzyl bromide under same conditions, alkylations were
formed mainly in N-4 position. Hence, we are totally in agreement
with literature [18e21]. Meantime same researchers also reported
in other studies [22e24] that N-3 regioisomers were obtained. It is
concluded that both properties of the previously existing sub-
stituents and nature of the alkylating agent can affect formation of
regioisomers. This was proved in our previous study not only by
advanced NMR techniques but also by regioselective synthesis [14].
In this study we have prepared a series of some new 2-(piperazine,
piperidine and morpholin-substituted) phenylimidazo [4,5-b]pyr-
idines 7e19 to investigate their anti-cancer activities (Schemes 1
and 2). The chlorine atom of 2-chloro-3-nitropyridine was con-
verted to alkylamino group by aromatic nucleophilic substitution
reaction. Reduction of the nitro groups in 1 and 2 with H2/PdeC
gave pyridinediamines 3 and 4 (Scheme 1). By condensation of the
respective substituted pyridinediamines 3e6 with the corre-
sponding Na2S2O5 adducts of various arylaldehydes in DMF, we

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C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
Scheme 3. N-Benzylation of 2-substitutedimidazo[4,5-c]pyridine (22).
Scheme 4. N-alkylation of purines reported by Khanna et al. [29].
reached the targeted compounds 7e19, (Scheme 1 and 2). When
we attempted alkylation of 16 and 17 with 4-fluorobenzyl bromide
under basic conditions (K2CO3, DMF), alkylations were formed
mainly in N-4 position. 18b (50.8%) and 19b (48%) were obtained as
N-4 isomer similar to our previous study [14]. In same reaction
medium. 18a (7.5%) and 19a (6%) were isolated as N-3 regioisomer
in less amount (Scheme 2) by using column chromatography. 18c
(N-1) which is the third regioisomer of 18 and its’ analogue in 19
were not detected in LC-MS chromatograms of 18 and 19. This is
why we prepared 18c by selective regioisomer synthesis method,
between the reaction product 20 and sodium metabisulfite adduct
of 4-(morpholin-4-ylcarbonyl)benzaldehyde. It can be seen in LC-
MS chromatogram of compound 18 (Fig. 2), while Rt values of 18c
(N-1) (5.22 min) and 18a (N-3) (5.36 min) regioisomers are very
close to each other, Rt value of 18b (N-4) (4.55 min) regioisomer is
far from them (N-1 and N-3) and the peak area of N-4 is much more
than other isomer.
Hence, N-4 regioisomer could be easily separated from N-3
regioisomer as it is shown in our experiments. Characterisation of
the individual isomeric products was determined by observation of
2D-NOESY enhancements between the NeCH2 and H-5,7 protons.
In the NOESY spectra of compound;
ꢀ 18b (Fig. 3a) and 19b, strong correlations were observed be-
tween benzylic and H-5 protons.
ꢀ 18c, NOE cross peaks between NeCH2 and H-7 protons were
observed.
ꢀ 18a and 19a, while there was no NOE contours between benzylic
protons and pyridine protons, strong correlations were seen
between benzylic and H-20 and/or H-60 as expected.
Complementary structural information was provided by 2D-

C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
5
Scheme 5. N-Benzylation of 2-substitutedimidazo[4,5-c]pyrimidine (25).
Fig. 2. LC-MS chromatogram (ESIþ and SIR techniques for [MþH]. 417 m/e) for 18a, 18 b at the endpoint of synthesis and 18c.

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C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
Fig. 3. aPartial NOESY spectrum of compound 18 b b Partial NOESY spectrum of compound 23. c. Partial NOESY spectrum of compound 26b. d. Partial NOESY spectrum of
compound 30b.
HMBC spectra of 18(a-c). It was possible to determine the chemical
shift values ( ppm) of C-3a and C-7a, by their correlations with
In the same conditions, 4-fluorobenzylation of 22 gave only 5H
isomer 23 (Scheme 3) as previously reported [14,25,26]. Therefore
very strong NOE interactions were seen between NeCH2 and H-4,6
in the NOESY spectra of 23 (Fig. 3b). These results were also
confirmed by HMBC correlations.
d
pyridine hydrogens in HMBC spectra. Subsequently, it could be
available to determine other possible correlations (whether
NeCH2: C-3a or NeCH2: C-7a) in separation of regioisomers.
The results obtained from the HMBC spectra of 18 (aec) and 19a
(Fig. 4), 19b provided all expected correlations.
Purine is generally represented when a hydrogen atom is
attached to N-9 and it is called N(9)H tautomer. It is obvious that
structures may be considered in which the proton is attached to the
other nitrogens of the molecule, yielding the tautomers N(7)H; N(3)
H and N(l)H [27]. Alkylation of purines usually results with the
formation of N-9 and N-7 alkylpurines and frequently N-9
regiosomer is formed as major product. Bookser et al. [28] reported
that alkylation of some purines with CH3I and NaHMDS in THF or
DMSO resulted with the formation of N-7 and N-9 regiosomers.
Khanna et al. [29] reported that benzylation of purine with potas-
sium hydride formed mixture of possible N-7 and N-9 alkylated
products. In contrast, without potassium hydride, only N-1 alky-
lated product was obtained (Scheme 4). In US 2006/0052602,
Gilead sciences, Inc. claimed a series of imidazo [4,5-d]pyrimidine
analogues for their antiviral activities [30]. In this study, when 8-
(substitutedphenyl) purines were alkylated with several benzyl
Fig. 4. Partial gHMBC spectrum of compound 19a showing. cross peaks between
NeCH2/C-2, C-3a, C-100, C-200,6’.’.

C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
7
Table 1
chlorides in aqueous NaOH medium, alkylation was formed at N-1
In vitro cytotoxic activities of compounds 7e30b was assessed by MTT.
position of purines.
In similiar conditions (with dry K2CO3 in DMF, instead of
aqueous NaOH), we achieved the same result and compound 26b
(39%) was obtained as N-1 regioisomer (Scheme 5). In addition, N-3
regioisomer 26a (7%) was also isolated in our conditions. N-3
regioisomers of purines are scarcely reported, Mitsunobu reactions
could be given as an example [31,32]. In NOESY spectra of 26a and
26b (Fig. 3c), very strong NOE interactions was detected with
NeCH2: only H-2 and NeCH2: H-2,6, respectively. Another detail
supporting this finding was the absence of cross peaks between
NeCH2: H-20 or/and 6’. It clarified that these products could not be
N-7 or N-9 alkylated products. HMBC correlations also confirmed
the proposed structures of both 26a and 26b.
Finally, to our knowledge no study has been made about
alkylation of 1H-imidazo [4,5-b] pyrazines yet. So their alkylations
were also examined in this study. 27 was prepared by the nucleo-
philic substitution reaction in sealed tube [33] (Scheme 6).
Bromine atom was removed by catalytic hydrogenation in Parr
apparatus. Finally by the cyclization of 28 with sodyum meta-
bisulfite adduct of 4-(morpholin-4-ylcarbonyl] benzaldehyde gave
29. The same alkylation reaction was carried out to 29, and it was
observed that alkylation of 29 favorably formed N4-(4-F-benzyl)
30b (71%). Because of the symmetrical position of 29, second and
last choice, regioisomer 30a (11%) was formed. NOESY and HMBC
correlations confirmed the proposed structures of both 30a and
30b (Fig. 3d).
IC50 M)
(
m
Compound
K562
HL-60
HCT-116
7
8
9
10
11
12
13
14
15
16
17
18b
18c
19b
21
22
23
24
25
26b
30a
30b
Camptotechin
Imatinib
>100
8.28 0.99
>100
7.05 0.28
>100
33.96 1.44
>100
>100
>100
>100
>100
78.9 5.35
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.45 0.297
3.61 0.627
7.28 0.417
>100
41.53 1.37
>100
>100
>100
>100
>100
>100
>100
>100
64.50 4.79
>100
>100
>100
>100
>100
9.74 3.82
3.34 0.52
6.22 0.83
>100
73.23 0.53
>100
>100
>100
>100
15.49 2.35
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.118 0.007
e
>100
>100
0.441 0 .016
e
e
0.502 0.09
further dilutions were made with culture medium solution.
All described imidazole-containing heterocycles 7e30b were
None of the compounds exhibited cell growth inhibition as
much as reference compounds. However, some compounds
showed remarkable inhibitory activities. Compounds 8e10 having
halogen atom on the 6 position of the pyridine moiety were the
tested for in vitro cytotoxic activities against human leukemia cell
lines (K562 and HL-60) and human colon cancer cell line (HCT-116)
by MTT test in triplicate (Table 1). Camptotechin and imatinib
mesylate were used as reference cytotoxic drugs in the experi-
ments. The results were expressed as growth inhibitory concen-
tration (IC50) values. It represents the compound concentrations
required to produce 50% growth inhibition of cells growth after of
incubation compared with untreated controls. The synthesized
compounds and reference drugs were dissolved in DMSO and
most active derivatives with the IC50 values 3.3e9.7 mM. Compound
12, bearing a cyclohexyl group on N3 position, also showed mod-
erate activity against selected cancer cell lines. The other benzyl N3-
substituted analogues did not show significant inhibitory activities.
It can be concluded that tautomeric NH and halogens substitution
on pyridine moiety can be important to determine inhibitory
Scheme 6. N-alkylation 2-substitutedimidazo[4,5-b]pyrazine (29).

8
C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
activities.
evaporated. The residue was crystallised from ethylacetate/n-
hexane.
3. Conclusion
4.1.1.1. N2-cyclohexyl-3-nitro-2-amino pyridine (1). Prepared from
2-chloro-3-nitropyridine (0.47 g), cyclohexylamine (0.34 g) and
K2CO3 (0.41 g) as described in general method. Yield. 0.19 g, (30%),
It was found that Nebenzylation of imidazo[4,5-b]pyridine,
imidazo[4,5-c]pyridine, imidazo[4,5-d]pyrimidine (Purine) and
imidazo [4,5-b]pyrazine favorably was realized on the six
membered N-heterocycles in presence of anhydrous K2CO3 in DMF.
Benzylation of imidazole nitrogen (N1,3,7,9) atoms occur at a lower
ratio secondarily. Since polarities of imidazole N-alkylated products
are not much different from each other, they cannot be easily
separated by column chromatography. However, it is easy to
separate imidazole N-alkylated regioisomers from N-alkylated six
membered heterocycles. Since N-alkylated six membered hetero-
cycles adhere more strongly to silica gel, they move slower than
imidazole N-alkylated regioisomers having a weaker adhesion on
the column chromatography. For this reason, imidazole N-alkylated
regioisomers are obtained first. By continuing to this elution, N-
alkylated six membered heterocycles (polarity of mobil phase was
increased gradually using MeOH) are obtained. NOESY experiment
is the primary method for structural elucidation of these types of
yellow oily liquid. 1H-NMR
d
ppm (CDCl3): 1.22e1.49 (m,5H),
1.59e1.74 (m,1H), 1.75e1.79 (m,2H), 2.01e2.05 (m,2H), 4.21 (m,1H),
6.56 (m,1H), 8.20 (br.s,1H), 8.36e8.38 (m,2H). 13C-NMR
ppm
d
(CDCl3): 24.7, 25.6, 32.9, 49.4, 111.2, 127.6, 135.3, 152.0, 155.9. MS
(ESIþ) m/z: 222(M þ H, 100%), C11H15N3O2.
4.1.1.2. N2-(3,4-dimethoxybenzyl)-3-nitro-2-amino pyridine (2).
Prepared
from
2-chloro-3-nitropyridine
(0.47
g),
3,4-
dimethoxybenzylamine (0.45 mL) and K2CO3 (0.41 g), as
described in general method. Yield, 0.29 g, (34%), mp 85e88 ꢂC. 1H-
NMR
(dd,1H,J ¼ 8 & 4.8 Hz), 6.84 (d, 1H, J ¼ 8 Hz), 6.91e6.94 (m,2H),
8.42e8.45 (m,3H). 13C-NMR
ppm (CDCl3): 44.9, 55.8, 55.9, 111.1,
d
ppm (CDCl3): 3.87 (s,6H), 4.77 (d,2H,J ¼ 5.2 Hz), 6.67
d
111.3, 112.0, 120.0, 128.1, 130.6, 135.3, 148.5, 149.1, 152.3, 155.7. MS
(ESIþ) m/z: 290(M þ H, 100%), C14H15N3O4.
regioisomers. The cross peaks of NeCH2 (at around 5e6 d ppm) and
aromatic protons confirm their spatial proximity as an evidence for
which nitrogen atom is substituted. HMBC technique can be an
effective alternative method for assignment of regioisomers. The
complete structure elucidation of all synthesized compounds were
performed using 1D and 2D NMR experiments including COSY,
NOESY, gHSQC, and gHMBC techniques.
4.1.2. General synthesis of 3e4
Compounds 1e2 (1 mmol) in EtOH (25 mL) was reduced by
hydrogenation using 50 psi of H2 and 10% PdeC (15 mg) until
cessation of H2 uptake. The catalyst was filtered on a bed of Celite,
washed with EtOH and the filtrate was concentrated in vacuo. The
crude amines were used for the further steps without purification.
4. Material and method
4.1.2.1. N2-cyclohexylpyridine-2,3-diamine (3). Prepared from N2-
cyclohexyl-3-nitro-2-amino pyridine (0.22 g) as described in gen-
eral method. Yield, 0.17 g, (90%), dark purple colored powder. 1H-
4.1. Chemistry
NMR
d ppm (CD3OD): 1.14e1.29 (m,3H), 1.35e1.46 (m,2H),
Uncorrected melting points were measured using a Büchi B-540
capillary melting point apparatus. All NMR experiments were car-
ried out using VARIAN (Agilent) MERCURY 400 MHz (Varian, Palo
Alto, CA, USA) at a proton resonance frequency of 400 and 100 MHz
for carbon. The NMR spectrum optimisation was conducted using
Agilent VnmrJ version 3.2 revision A software and all parameters
were set in it. The samples (5e15 mg) were prepared in 0.7 mL of
DMSO‑d6, CDCl3 and CD3OD. TMS was used as an internal standart.
The liquid chromatography mass spectrometry (LC-MS) spectra
were taken on a Waters Micromass ZQ connected with Waters
Alliance HPLC (Waters Corporation, Milford, MA, USA), using the
1.62e1.65 (m,1H), 1.72e1.77 (m,2H), 1.99e2.02 (m,2H), 3.70e3.78
(m,1H), 6.43 (dd,1H,J ¼ 7.6 & 5.6 Hz), 6.87 (dd,1H,J ¼ 7.6 & 0.8 Hz),
7.38 (dd,1H,J ¼ 5.6 & 0.8 Hz). 13C-NMR
d ppm (CD3OD): 26.3, 26.9,
34.4, 51.0,113.5,121.5,131.8,134.7,148.9. MS (ESIþ) m/z: 192(M þ H,
100%), C11H17N3.
4.1.2.2. N2-(3,4-dimethoxybenzyl)pyridine-2,3-diamine
(4).
Prepared from N2-(3,4-dimethoxybenzyl)-3-nitro-2-amino pyri-
dine (0.28 g) as described in general method. Yield, 0.26 g, (92%),
dark brown colored powder. 1H-NMR
d
ppm (CD3OD): 3.77 (s,6H),
5.2 Hz), 6.60 (dd,1H,J 7.6 & Hz), 6.86
(d,1H,J ¼ 8 Hz), 6.92 (dd, 1H,J ¼ 8.4 & 1.6 Hz), 6.99e7.02 (m,2H),
7.35e7.37 (m,1H). 13C-NMR
ppm (CD3OD): 46.6, 56.50, 56.53,
4.57 (d,2H,J
¼
¼
6
ESI(þ) method with a C-18 column (XTerra®, 4.6 ꢁ 250 mm, 5
mm).
Analytical conditions of mass spectrometry were as follows:
capillary voltage, 3.11 kV; cone voltage, 29 V; source temperature,
100 ꢂC; and desolvation temperature, 300 ꢂC. Because of the
tautomeric effect of imidazole ring, some nmr data did not give
satisfactory results, in order to eliminate the tautomeric effect, the
related benzimidazoles were dissolved in CD3OD or DMSO‑d6, fol-
lowed by a tiny amount of dry NaH, and 2e3 drops of D2O were
added to the nmr tube and stirred well. As it is reported in the
experimental part, this time very satisfactory nmr results have been
observed since the tautomeric effect was totally disappeared. The
HCl salt of the synthesized compounds were prepared using etha-
nolic HCl. Compounds 5 and 6 [14], 20 [34] were prepared ac-
cording to the literature methods.
d
112.9, 113.1, 114.4, 121.3, 121.4, 129.3, 131.5, 133.7, 147.3, 150.0, 150.6.
MS (ESIþ) m/z: 260(M þ H, 100%), C14H17N3O2.
4.1.3. General synthesis of sodium metabisulphite adduct of
benzaldehydes
Related benzaldehydes (7.5 mmol) was dissolved in EtOH
(25 mL) and sodium metabisulfite (0.8 g) (in 5 mL of water) was
added in portions. The reaction mixture was stirred vigorously and
more EtOH was added. The mixture was kept in a refrigerator for a
while. The white precipitate, the obtained salts were gained by
filtration, dried and used for the further steps without purification
and characterisation.
4.1.1. General synthesis of 1e2
4.1.4. General synthesis of 7e16, 21, 24, 25, 27, 28, 33
The mixture of 2-chloro-3-nitropyridine (3 mmol), corre-
sponding anilines (3 mmol) and K2CO3 (3 mmol) were heated in
DMF at 120 ꢂC, for 3e4 h. The reaction mixture was allowed to cool
to room temperature then diethyl ether was added. The organic
layer was washed with water dried over MgSO4, filtered and
The mixture of sodium metabisulphite adduct of benzaldehydes
(1 mmol) and corresponding 2,3-diamines (1 mmol), in DMF (2 mL)
were heated at 120 ꢂC, for 3e4 h. The reaction mixture was cooled,
poured into water. The resulting precipitate was collected by
filtration and dried. If the product was not solid, it was purified with

C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
9
column chromatography.
0.5) as eluant, yield 0.19 g, (55%), mp > 300 ꢂC. 1H-NMR
d
ppm
(DMSO‑d6þNaH þ D2O): 3.18 (t,4H,J ¼ 4.8 Hz), 3.77 (t,4H,J ¼ 4.8 Hz),
6.98 (d,2H,J ¼ 8.8 Hz), 7.80 (d,1H,J ¼ 2 Hz), 7.94 (d,1H,J ¼ 2.4 Hz),
8.14 (d, 2H,J ¼ 8.8 Hz). 13C-NMR (DMSO‑d6þNaH þ D2O): 48.7, 66.6,
108.7, 115.1, 123.7, 127.4, 128.5, 138.8, 141.1, 151.5, 159.6, 165.1. MS
(ESIþ) m/z: 359(M þ H, 100%), 361(M þ Hþ2, 98%). Anal Calcd for
4.1.4.1. 2-[4-(4-Methylpiperazin-1-yl)phenyl]-3H-imidazo
[4,5-b]
pyridine HCl (7). Prepared from 2,3-diaminopyridine (0.1 g) and
Na2S2O5 adduct of 4-(4-methylpiperazin-1-yl) benzaldehyde
(0.308 g) as described in general method. Crude product was con-
verted to HCl salt, yield 0.14 g, (35%), mp > 300 ꢂC. 1H-NMR
d
ppm
(DMSO‑d6þNaH þ D2O): 2.23 (s,3H), 2.49 (t,4H,J ¼ 4.8 Hz), 3.20
(t,4H,J 4.8 Hz), 6.77 (dd,1H,J 7.6 4.8 Hz,H-6), 6.95
C
16H15BrN4O . 0.25H2O: C, 52.83; H, 4.29; N, 15.40. Found: C, 53.04;
H, 4.22; N, 15.79.
¼
¼
&
(d,2H,J ¼ 6.8 Hz), 7.64 (dd,1H,J ¼ 7.2 & 1.2 Hz), 7.89 (dd,1H,J ¼ 4.8 &
2 Hz), 8.15 (d,2H,J ¼ 6.8 Hz). 13C-NMR (DMSO‑d6þNaH þ D2O): 46.1,
48.2, 54.9, 113.5, 115.1, 121.8, 127.9, 128.2, 138.7, 139.5, 150.8, 160.8,
163.1. MS (ESIþ) m/z: 294(M þ H, 37%), 148 (100%). Anal Calcd for
4.1.4.6. 3-Cyclohexyl-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imi-
dazo [4,5-b]pyridine (12). Prepared from 3 (0.19 g) and Na2S2O5
adduct of 4-(4-methylpiperazin-1-yl)benzaldehyde (0.308 g) as
described in general method. Resulting precipitate was purified
with column chromatography using CH2Cl2: EtOH (100 : 0.5) as
C
17H19N5$2HCl . 2.25H2O: C, 50.18; H, 6.31; N, 17.21. Found: C, 50.13;
H, 6.60; N, 17.36.
eluant, yield 0.15 g, (40%), mp 151e154 ꢂC. 1H-NMR
d ppm (CD3OD):
1.26e1.40 (m,3H), 1.67e1.70 (m,1H), 1.82e1.90 (m,4H), 2.36 (s,3H),
2.62 (t,4H,J ¼ 4.4 Hz), 2.74e2.82 (m,2H), 3.35 (t,4H,J ¼ 4.4 Hz),
4.36e4.42 (m,1H), 7.11 (d,2H,J ¼ 8.8 Hz), 7.26 (dd,1H,J ¼ 7.6 &
5.2 Hz,H-6), 7.52 (d,2H,J ¼ 8.8 Hz), 7.96 (dd,1H,J ¼ 7.6 & 1.2 Hz), 8.32
(dd,1H,J ¼ 5.2 &1.6 Hz). 13C-NMR (CD3OD): 26.1, 27.1, 31.7, 46.1, 48.6,
55.8, 58.8, 116.2, 119.4, 120.9, 127.3, 131.5, 136.0, 144.2, 149.4, 153.9,
156.7. MS (ESIþ) m/z: 376(M þ H, 58%), 147(100%). Anal Calcd for
4.1.4.2. 6-Chloro-2-[4-(4-methylpiperazin-1-yl)phenyl]-3H-imidazo
[4,5-b]pyridine (8). Prepared from 5-chloro-2,3-diaminopyridine
(0.14 g) and Na2S2O5 adduct of 4-(4-methylpiperazin-1-yl)benzal-
dehyde (0.308 g) as described in general method. Resulting pre-
cipitate was purified with column chromatography using CH2Cl2:
MeOH (100 : 0.5) as eluant, yield 0.245 g, (75%), mp > 300 ꢂC. 1H-
NMR
d
ppm (DMSO‑d6þNaH
þ
D2O): 2.19 (s,3H), 2.44
C
23H29N5 . 1.5H2O . 0.25C2H5OH: C, 68.16; H, 8.15; N, 16.91. Found: C,
(t,4H,J ¼ 4,8 Hz), 3.16 (t,4H,J ¼ 4,8 Hz), 6.90 (d,2H,J ¼ 8.8 Hz), 7.58
(d,1H,J ¼ 2.4 Hz), 7.81 (d, 1H,J ¼ 2.4 Hz), 8.09 (d,2H,J ¼ 8.8 Hz). 13C-
NMR (DMSO‑d6þNaH þ D2O): 46.0, 48.1, 54.8, 115.0, 119.9, 120.5,
127.3, 128.3, 136.5, 140.3, 151.1, 159.8, 165.3. MS (ESIþ) m/z:
328(M þ H, 42%), 330.7(M þ Hþ2, 15%), 165(100%). Anal Calcd for
68.28; H, 7.83; N, 16.82.
4.1.4.7. 3-(3,4-Dimethoxybenzyl)-2-(4-(4-methylpiperazin-1-yl)
phenyl)-3H-imidazo [4,5-b]pyridine (13). Prepared from 4 (0.25 g)
and Na2S2O5 adduct of 4-(4-methylpiperazin-1-yl)benzaldehyde
(0.308 g) as described in general method. Resulting precipitate was
purified with column chromatography using CH2Cl2: MeOH (100 :
C
17H18ClN5 . 0.5H2O: C, 60.62; H, 5.68; N, 20.79. Found: C, 60.73; H,
5.78; N, 20.61.
0.5) as eluant, yield 0.23 g, (52%), mp 192e194 ꢂC. 1H-NMR
d ppm
4.1.4.3. 6-Chloro-2-[4-(piperidin-1-yl)phenyl]-3H-imidazo
[4,5-b]
(CDCl3): 2.47 (s,3H), 2.73(br.t,4H), 3.42 (t,4H,J ¼ 4.4 Hz), 3.74 (s,3H),
3.83 (s,3H), 5.54 (s,2H), 6.64 (dd,1H,J ¼ 8 & 2 Hz), 6.75e6.77 (m,2H),
6.96 (d,2H,J ¼ 7.2 Hz), 7.26 (dd, 1H,J ¼ 7.2 & 5.6 Hz,H-6), 7.65
(d,2H,J ¼ 6.8 Hz), 8.06 (dd,1H,J ¼ 7.6 & 1.2 Hz), 8.38 (dd,1H,J ¼ 4.8 &
1.2 Hz). 13C-NMR (CDCl3): 45.9, 46.5, 47.7, 54.7, 55.7, 55.8, 110.0,
111.3, 114.9, 118.5, 118.8, 120.0, 126.6, 129.5, 130.3, 135.3, 143.5,
148.4, 149.0, 149.1, 152.1, 155.2. MS (ESIþ) m/z: 444(M þ H, 100%).
Anal Calcd for C26H29N5O2: C, 70.41; H, 6.59; N, 15.79. Found: C,
70.54; H, 6.85; N, 15.78.
pyridine (9). Prepared from 5-chloro-2,3-diaminopyridine (0.14 g)
and Na2S2O5 adduct of 4-(piperidin-1-yl)benzaldehyde (0.29 g) as
described in general method. Resulting precipitate was purified
with column chromatography using CH2Cl2: MeOH (100 : 0.8) as
eluant, yield 0.27 g, (88%), mp, 280e282 ꢂC. 1H-NMR
d ppm
(CD3OD þ NaH þ D2O): 1.60e1.62 (m,2H), 1.71e1.74 (m,4H),
3.20e3.23 (m,4H), 7.08 (dd,2H,J ¼ 7.2 & 2 Hz), 7.76 (d,1H,J ¼ 2 Hz),
7.97 (d,1H,J ¼ 2 Hz), 8.13 (d,2H,J ¼ 6.4 & 2 Hz). 13C-NMR
(CD3OD þ NaH þ D2O): 25.2, 26.4, 51.9, 117.8, 122.8, 122.9, 127.7,
129.3, 138.6, 140.9, 153.8, 159.5, 167.0. MS (ESIþ) m/z: 313(M þ H,
4.1.4.8. 3-Benzyl-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo
[4,5-b]pyridine (14). Prepared from 5 (0.199 g) and Na2S2O5 adduct
of 4-(4-methylpiperazin-1-yl)benzaldehyde (0.308 g) as described
in general method. Resulting precipitate was purified with column
chromatography using CH2Cl2: MeOH (100 : 0.5) as eluant, yield
.
98%), 315(M þ Hþ2, 35%). Anal Calcd for C17H17ClN4 0.25H2O: C,
64.35; H, 5.55; N, 17.66. Found: C, 64.25; H, 5.54; N, 17.85.
4.1.4.4. 6-Bromo-2-[4-(4-methylpiperazin-1-yl)phenyl]-3H-imidazo
[4,5-b]pyridine (10). Prepared from 5-bromo-2,3-diaminopyridine
(0.14 g) and Na2S2O5 adduct of 4-(4-methylpiperazin-1-yl)benzal-
dehyde (0.308 g) as described in general method. Resulting pre-
cipitate was purified with column chromatography using CH2Cl2:
MeOH (100 : 0.5) as eluant, yield 0.245 g, (75%), mp > 300 ꢂC. 1H-
0.21 g, (55%), mp 159e161 ꢂC. 1H-NMR
2.66(br.t,4H), 3.37 (t,4H,J
2H,J ¼ 8.8 Hz), 7.12 (d,2H,J ¼ 7.2 Hz), 7.23e7.31 (m,4H), 7.60
(d,2H,J ¼ 8.4 Hz), 8.06 (dd,1H,J ¼ 8 & 1.6 Hz), 8.36 (dd,1H,J ¼ 5.2 &
1.6 Hz). 13C-NMR (CDCl3): 45.8, 46.8, 47.6, 54.6, 115.1, 118.6, 120.1,
126.3, 126.7, 127.5, 128.8, 130.3, 135.3, 137.1, 143.6, 149.1, 151.9, 155.1.
d
ppm (CDCl3): 2.41 (s,3H),
¼
4.8 Hz) 5.60 (s,2H), 6.92 (d,
NMR
d
ppm (DMSO‑d6þNaH
þ
D2O): 2.23 (s,3H), 2.48
.
(t,4H,J ¼ 4.8 Hz), 3.20 (t,4H,J ¼ 4.8 Hz), 6.94 (d,2H,J ¼ 8.4 Hz), 7.72
(d,1H,J ¼ 2 Hz), 7.90 (d, 1H,J ¼ 2.4 Hz), 8.13 (d,2H,J ¼ 8.8 Hz). 13C-
NMR (DMSO‑d6þNaH þ D2O): 45.9, 48.0, 54.7, 108.1, 114.8, 122.9,
127.4, 128.1, 138.2, 141.1, 150.9, 159.9, 165.0. MS (ESIþ) m/z:
MS (ESIþ) m/z: 384(M þ H, 100%). Anal Calcd for C24H25N5
0.25H2O: C, 74.29; H, 6.62; N, 18.05. Found: C, 74.40; H, 6.98; N,
17.74.
.
372(M þ H, %100), 374(M þ Hþ2, %95). Anal Calcd for C17H18BrN5
4.1.4.9. 1-Benzyl-2-[4-(4-methylpiperazin-1-yl)phenyl]-1H-imidazo
[4,5-b]pyridine (15). Prepared from 6 (0.199 g) and Na2S2O5 adduct
of 4-(4-methylpiperazin-1-yl)benzaldehyde (0.308 g) as described
in general method. Resulting precipitate was purified with column
chromatography using CH2Cl2: MeOH (100 : 0.5) as eluant, yield
0.5H2O: C, 53.55; H, 5.02; N, 18.36. Found: C, 54.08; H, 5.06; N,
18.59.
4.1.4.5. 6-Bromo-2-[4-(morpholin-4-yl)phenyl]-3H-imidazo [4,5-b]
pyridine (11). Prepared from 5-bromo-2,3-diaminopyridine (0.18 g)
and Na2S2O5 adduct of 4-(morpholino) benzaldehyde (0.29 g) as
described in general method. Resulting precipitate (0.28 g) was
purified with column chromatography using CH2Cl2: MeOH (100 :
0.23 g, (60%), mp 236e238 ꢂC. 1H-NMR
2.66(br.t,4H), 3.36 (t,4H,J
(d,2H,J 8.8 Hz), 7.08e7.12 (m,3H), 7.26e7.37 (m,3H), 7.43
(dd,1H,J ¼ 7.6 & 1.2 Hz), 7.67 (d,2H,J ¼ 8.8 Hz), 8.54 (dd,1H,J ¼ 4.8 &
d
ppm (CDCl3): 2.41 (s,3H),
¼
4.8 Hz), 5.48 (s,2H), 6.94
¼

10
C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
1.6 Hz). 13C-NMR (CDCl3): 45.8, 47.6, 48.6, 54.6, 115.1, 117.6, 117.9,
119.5, 125.9, 127.9, 128.6, 129.2, 130.6, 135.9, 145.1, 152.1, 156.2,
156.6. MS (ESIþ) m/z: 384(M þ H, 100%). Anal Calcd for C24H25N5: C,
75.17; H, 6.57; N, 18.26. Found: C, 74.77; H, 6.78; N, 18.10.
NMR (DMSO‑d6þD2O): 46.1, 48.1, 54.8, 115.3, 126.3, 129.1, 138.4,
141.1, 148.5, 151.9, 164.1, 166.3. MS (ESIþ) m/z: 295(M þ H, 100%).
Anal Calcd for C16H18N6$4HCl: C, 43.65; H, 5.03; N, 19.09. Found: C,
43.58; H, 4.68; N, 19.13.
4.1.4.10. [4-(4H-imidazo [4,5-b]pyridin-2-yl)phenyl](morpholin-4-yl)
methanone (16). Prepared from 2,3-diaminopyridine (0.1 g) and
Na2S2O5 adduct of 4-(morpholin-4-ylcar-bonyl)benzaldehyde
(0.323 g) as described in general method. Resulting precipitate was
boiled in MeOH, cooled and filtered, yield 0.21 g, (68%), mp,
4.1.4.15. Morpholin-4-yl[4-(9h-purin-8-yl)phenyl]methanone (25).
Prepared from 4,5-diaminopyrimidine (0.11 g) and Na2S2O5 adduct
of 4-(morpholin-4-yl)car-bonyl)benzaldehyde (0.323 g) as
described in general method. Resulting precipitate was boiled in
MeOH, cooled and filtered, yield 0.24 g, (77%), mp 246e249 ꢂC. 1H-
264e268 ꢂC. 1H-NMR
(dd,1H,J 4.8 Hz,H-6), 7.64 (d,2H,J
d
ppm (CD3OD): 3.49e3.78 (m,8H), 7.33
8.4 Hz), 8.04
NMR
8.31 (d,2H,J
d
ppm (DMSO‑d6 þD2O): 3.41e3.62(8H), 7.63 (d,2H,J ¼ 8 Hz),
¼
8.4 Hz), 8.91 (s,1H), 9.12 (s,1H). 13C-NMR
¼
8
&
¼
(d,1H,J ¼ 7.6 Hz), 8.25 (m,2H,J ¼ 8.4 Hz), 8.38 (dd,1H,J ¼ 5.2 &
1.2 Hz). 13C-NMR (CD3OD): 44.0, 50.9, 58.6, 61.0, 67.8, 70.8, 120.0,
128.5, 129.0, 132.1, 138.8, 145.6, 171.6. MS (ESIþ) m/z: 309(M þ H,
(DMSO‑d6þD2O): 65.9, 127.1, 127.7, 129.7, 132.9, 138.0, 145.1, 152.1,
153.8, 168.2. MS (ESIþ) m/z: 310(M þ H, 100%). Anal Calcd for
C16H15N5O2$2H2O: C, 55.64; H, 5.54; N, 20.27. Found: C, 55.35; H,
.
100%). Anal Calcd for C17H16N4O2 0.75H2O: C, 63.44; H, 5.48; N,
5.68; N, 20.20.
17.40. Found: C, 63.53; H, 5.19; N, 17.20.
4.1.4.16. (4-(1H-imidazo
methanone (29). 5-Bromopyrazine-2,3-diamine (27) was prepared
[4,5-b]pyrazin-2-yl)phenyl)(morpholino)
4.1.4.11. [4-(6-Chloro-4H-imidazo
[4,5-b]pyridin-2-yl)phenyl](-
morpholin-4-yl)methanone (17). Prepared from 5-chloro-2,3-
diaminopyridine (0.144 g) and Na2S2O5 adduct of 4-(morpholin-
4- ylcarbonyl) benzaldehyde (0.323 g) as described in general
method. Resulting precipitate was boiled in MeOH, cooled and
from 2-amino-3,5-dibromopyrazine and NH4OH (25%) in sealed
tube according to lit.33 1H-NMR
d ppm (CD3OD): 4.90 (s,4H,NH2),
7.24 (s,2H,H-5,6). 13C-NMR (CD3OD): 123.3, 129.8, 144.3, 145.9. MS
(ESIþ) m/z: 189(M þ H, 100%), 191(M þ Hþ2, 97%). 27 (0.303 g,
1.6 mmol) in EtOH (10 mL) was reduced by hydrogenation using 40
psi of H2 and 10% PdeC (15 mg) for 8 h. The catalyst was filtered on
a bed of Celite, the filtrate was concentrated in vacuo. Purple
colored powder 28 were used for the further steps without puri-
filtered, yield 0.215 g, (63%), mp > 300 ꢂC. 1H-NMR
d ppm (CD3OD):
3.29e3.77(8H), 7.77 (br.s,2H), 8.28 (br.s,2H), 8.41 (s,1H), 8.67 (s,1H).
13C-NMR (CD3OD): 67.9,124.9,126.6,128.8,129.7,129.9,130.3,141.7,
146.0, 146.7, 154.7, 170.6. MS (ESIþ) m/z: 343(M þ H, 100%),
345(M þ Hþ2, 31%). Anal Calcd for C17H15ClN4O2 . 0.5H2O: C, 58.04;
H, 4.58; N, 15.92. Found: C, 58.40; H, 4.41; N, 16.32.
fication. 1H-NMR
d ppm (CD3OD): 4.90 (s,4H,NH2), 7.24 (s,2H, H-
5,6). 13C-NMR (CD3OD): 123.7, 145.1. MS (ESIþ) m/z: 110(M þ H,
100%). 29 was prepared from 28 (0.11 g) and Na2S2O5 adduct of 4-
(morpholin-4-ylcarbonyl) benzaldehyde (0.323 g) as described in
general method. Resulting precipitate (0.155 g) was purified with
column chromatography using CH2Cl2: MeOH (95 : 5) as eluant,
4.1.4.12. 2-[4-(4-Methylpiperazine-1-yl)phenyl]-3H-imidazo [4,5-c]
pyridine (21). Prepared from 3,4-diaminopyridine (0.1 g) and
Na2S2O5 adduct of 4-(4-methylpiperazin-1-yl)benzaldehyde
(0.308 g) as described in general method. Resulting precipitate was
used without purification, yield 0.135 g, (46%), mp > 280 ꢂC. 1H-
yield 0.056 g, (18%), mp > 300 ꢂC. 1H-NMR
d
ppm (DMSO‑d6þD2O):
3.27e3.61 (m,8H), 7.62 (d,2H,J ¼ 8.4 Hz), 8.33 (d,2H,J ¼ 8.4 Hz), 8.40
(s,2H,H-5,6). 13C-NMR (DMSO‑d6þD2O): 66.0, 127.3, 127.8, 129.9,
138.2, 139.0, 154.7, 168.2. MS (ESIþ) m/z: 310(M þ H, 100%). Anal
Calcd for C16H15N5O2$H2O: C, 58.70; H, 5.23; N, 21.39. Found: C,
58.97; H, 5.01; N, 20.91.
NMR
d
ppm (DMSO‑d6þNaH
þ
D2O): 2.23 (s,3H), 2.49
(t,4H,J ¼ 4.8 Hz), 3.21 (t,4H,J ¼ 4.8 Hz), 6.95 (d,2H,J ¼ 8.8 Hz), 7.32
(dd,1H,J ¼ 5.6 & 1.2 Hz,H-6), 7.81 (d,1H,J ¼ 5.2 Hz,H-7), 8.15
(d,2H,J
¼
8.8 Hz), 8.57 (d,1H,
J
¼
1.2 Hz,H-4). 13C-NMR
(DMSO‑d6þNaH þ D2O): 45.9, 48.1, 54.8, 111.2, 115.1, 127.5, 128.4,
136.4, 137.6, 145.3, 150.9, 151.6, 163.5. MS (ESIþ) m/z: 294.5(M þ H,
100%). Anal Calcd for C17H19N5 . 1.5H2O: C, 63.72; H, 6.92; N, 21.85.
Found: C, 64.13; H, 7.03; N, 21.66.
4.1.4.17. General synthesis of 18a-b, 19a-b, 23, 26a-b, 30a-b.
K2CO3 (0.069 g, 0.5 mmol) was added to a suspension of the 16, 17,
22, 25 and 29 (0.36 mmol) in DMF (0.5 mL) and stirred. One hour
later, 4-fluorobenzyl bromide (0.095 g, 0.5 mmol) was added. After
overnight stirring at rt, water was added and precipitate was
filtered, if precipitate was not occurred, it was extracted with the
mixture CH2Cl2: MeOH (95 : 5).
(4-(3-(4-Fluorobenzyl)-3H-imidazo [4,5-b]pyridin-2-yl)phenyl)(-
morpholino)methanone (18a) & (4-(4-(4-Fluorobenzyl)-4H-imidazo
[4,5-b]pyridin-2-yl)phenyl)(morpholino)methanone (18b)
4.1.4.13. [4-(5H-imidazo [4,5-c]pyridin-2-yl)phenyl](morpholin-4-yl)
methanone (22). Prepared from 3,4-diaminopyridine (0.1 g) and
Na2S2O5 adduct of 4-(morpholin-4-ylcarbonyl) benzaldehyde
(0.323 g) as described in general method. Resulting precipitate was
purified with column chromatography using CH2Cl2: MeOH (100 :
5) as eluant yield 0.21 g, (65%), mp, 257e260 ꢂC. 1H-NMR
d ppm
(DMSO‑d6þNaH þ D2O): 3.48e3.67(8H), 7.46 (dd,1H,J ¼ 5.6 &
Prepared from 16 (0.11 g) as described in general method. Pu-
rification (Silicagel, CH2Cl2: MeOH 100 : 2) of crude product first
1.2 Hz,H-6), 7.48 (d,2H,J ¼ 8.4 Hz), 7.92 (d,1H,J ¼ 5.6 Hz,H-7), 8.34
(d,2H,J
¼
8
Hz), 8.69 (d,1H,J
¼
1.2 Hz,H-4). 13C-NMR
afforded 18a as oily semi-solid, yield 0.011 g (7.5%). 1H-NMR
d ppm
(DMSO‑d6þNaH þ D2O): 66.9, 112.5, 127.8, 128.1, 135.0, 137.5, 137.9,
138.9, 145.3, 151.8, 162.7, 170.8. MS (ESIþ) m/z: 309(M þ H, 100%)
Anal Calcd for C17H16N4O2 . 2H2O: C, 59.29; H, 5.85; N, 16.26. Found:
C, 59.13; H, 5.70; N, 16.11.
(CD3OD): 3.44, 3.65 and 3.77 (br.s,8H,morph.), 5.68 (s,2H,NeCH2),
6.93e6.97 (m,2H,H-300,500), 7.00e7.04 (m,2H,H-200,600), 7.43
(dd,1H,J ¼ 8 & 5.2 Hz,H-6), 7.58 (d, 2H,J ¼ 8.4 Hz,H-30,50), 7.78
(d,2H,J ¼ 8 Hz,H-20,60), 8.15 (dd,1H,J ¼ 8 & 1.2 Hz,H-7), 8.45 (dd,
1H,J ¼ 5.2 & 1.6 Hz,H-5). COSY: (H-5/H-6), (H-6/H-7), (H-20,6’/H-
30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/H-20,60), (NeCH2/H-
4.1.4.14. 8-[4-(4-Methylpiperazin-1-yl)phenyl]-9H-purine HCl (24).
Prepared from 4,5-diaminopyrimidine (0.11 g) and Na2S2O5 adduct
of 4-(4-methylpiperazin-1-yl)benzaldehyde (0.308 g) as described
in general method. Crude product was converted to HCl salt in
200,600), (morph./H-30,50). 13C-NMR & HSQC & HMBC
d ppm (CD3OD):
43.9 & 67.7(morph.), 47.0(NeCH2), 116.6(d,J ¼ 22 Hz,CH-300,500),
120.7(CH-6), 128.4(CH-7), 128.8(CH-30,50), 129.9(d,J ¼ 8.4 Hz,CH-
200,600),130.8(CH-20,60),132.2(C-10),134.0(d,J ¼ 3.2 Hz,C-100),135.7(C-
MeOH, yield 0.24 g, (55%), mp > 300 ꢂC. 1H-NMR
d
ppm (DMSO‑d6þ
D2O): 2.18 (s,3H), 2.45 (t,4H,J ¼ 4.8 Hz), 3.18 (t,4H,J ¼ 4.8 Hz), 6.95
(d,2H,J ¼ 8.8 Hz), 8.12 (d,2H, J ¼ 8.8 Hz), 8.39 (s,1H), 8.55 (s,1H). 13C-
7a),
138.8(C-40),
145.9(CH-5),
149.5(C-3a),
155.4(C-2),

C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
11
163.6(d,J ¼ 244 Hz,C-400), 171.4(CO). MS (ESIþ) m/z: 417(M þ H,
100%), C24H21FN4O2.
300,500), (H-4/H-6 secondary). NOESY: (NeCH2/H-4,6), (NeCH2/H-
200,600), (morph./H-30,50). 13C-NMR & HSQC & HMBC
d
ppm (CD3OD):
114.1(CH-7),
Continued elution (CH2Cl2: MeOH 100 : 4) provided 18b as a
43.9
117.2(d,J
&
¼
67.8(morph),
63.0(NeCH2),
white solid, yield 0.074 g, (50.8%), mp 78e80 ꢂC (bubling), HCl salt
21.8 Hz,CH-300,500), 128.7(CH-30,50), 129.5(CH-20,60),
mp 260e265 ꢂC. 1H-NMR
d ppm (CD3OD): 3.5, 3.62 and 3.76
131.6(d,J ¼ 8.3 Hz,CH-200,600), 132.9(d,J ¼ 3.2 Hz,C-100), 133.2 (CH-4),
133.6(CH-6),136.6(C-10),138.0(C-40),145.9(C-3a),156.6(C-7a),164.5
(d,J ¼ 245 Hz, C-400), 171.6 (C-2), 172.1(CO). MS (ESIþ) m/z:
417(M þ H, 100%). Anal. Calcd for C24H21FN4O2 . 3H2O: C, 61.27; H,
5.78; N, 11.90. Found: C, 61.35; H, 5.69; N, 12.07.
(br.s,8H,morph.), 5.47 (s,2H,NeCH2), 7.05e7.096 (m,2H,H-300,500),
7.26 (dd,1H,J ¼ 7.6 & 6.4 Hz,H-6), 7.56e7.59 (m,4H,H-200,600,30,50),
8.16 (d,1H,J ¼ 6.4 Hz,H-5), 8.23 (dd,1H,J ¼ 8 & 0.8 Hz,H-7), 8.46
(d,2H,J ¼ 8 Hz,H-20,60). COSY: (H-5/H-6), (H-6/H-7), (H-20,6’/H-
30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/H-5), (NeCH2/H-200,600),
(morph./H-30,50). 13C-NMR & HSQC & HMBC
67.8(morph.), 57.0(NeCH2), 115.5(CH-6), 116.8(d,J ¼ 21.8 Hz,CH-
d ppm (CD3OD): 43.9 &
4.1.4.19. (4-(3-(4-Fluorobenzyl)-3H-purin-8-yl)phenyl)(morpholino)
methanone 26a
& (4-(1-(4-fluorobenzyl)-1H-purin-8-yl)phenyl)(-
300,500),
128.6(CH-30,50),
129.45(CH-20,60),
129.7(CH-7),
morpholino)methanone 26b. Prepared from 25 (0.11 g) as described
in general method. Purification (Silicagel, CH2Cl2: MeOH 100 : 3.5)
first afforded compound 26a as a oily semi-solid, yield 0.011 g,
132.0(d,J ¼ 8.4 Hz,CH-200,600), 132.6(d,J ¼ 3.3 Hz,C-100), 133.1(CH-5),
136.9(C-10),
137.9(C-40),
145.7(C-7a),
154.6(C-3a),
164.4(d,J ¼ 245 Hz,C-400), 168.7(C-2), 172.1(CO). MS (ESIþ) m/z:
417(M þ H, 100%). Anal. Calcd for C24H21FN4O2$H2O: C, 66.34; H,
5.33; N, 12.89. Found: C, 66.72; H, 5.37; N, 13.21.
(7.3%), 1H-NMR
5.9 (s,2H,NeCH2),
d
ppm (CD3OD): 3.5, 3.66 & 3.78 (br.s,8H,morph),
7.09e7.14 (m,2H, 7.59
H-300,500),
(d,2H,J ¼ 8.4 Hz,H-30,50), 7.66e7.69 (m,2H,H-200,600) 8.5 (d,2H,
J ¼ 8.4 Hz,H-20,60), 8.94 (s,1H,H-6), 8.99 (s,1H,H-2). COSY: (H-20,6’/
H-30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/H-2), (NeCH2/H-200,600),
(4-(6-Chloro-3-(4-fluorobenzyl)-3H-imidazo [4,5-b]pyridin-2 yl)
phenyl)(morpholino) methanone (19a) and (4-(6-Chloro-4-(4-
fluorobenzyl)-4H-imidazo [4,5-b]pyridin-2-yl)phenyl) (morpholino)
methanone (19b)
Prepared from 17 (0.125 g) as described in general method.
Purification (Silicagel, CH2Cl2: MeOH 100 : 2) first afforded com-
pound 19a as a white solid, yield 0.010 g, (6%), mp 132e135 ꢂC. 1H-
(morph./H-30,50). 13C-NMR & HSQC & HMBC
d ppm (CD3OD): 43.9
and 67.8(morph.), 54.8(NeCH2), 116.9(d, J ¼ 21.8 Hz,CH-300,500),
128.7 (CH-30,50), 129.9(CH-20,60), 131.9(d,J
¼
3.2 Hz,C-100),
132.2(d,J ¼ 8.3 Hz,CH-200,600), 136.0 (C-10), 138.75(C-40), 143.15(C-2),
143.3(C-5), 144.8(C-6), 157.0(C-4), 164.3(d,J
¼
246 Hz, C-400),
NMR
d ppm (CDCl3): 3.42, 3.63 and 3.78 (br.s,8H,morph.), 5.53
171.2(C-8), 172.0(CO). MS (ESIþ) m/z: 418(M
23H20FN5O2.
Continued elution (CH2Cl2: MeOH 100 : 5) provided compound
26b as a white solid (0.059 g, 39%), mp 114e117 ꢂC. 1H-NMR
ppm
þ
H, 100%),
(s,2H,NeCH2), 6.95e6.99 (m,2H,H-300,500), 7.05e7.09 (m,2H,H-
200,600), 7.52 (d,2H,J ¼ 8.4 Hz,H-30,50), 7.7 (d,2H, J ¼ 8.4 Hz,H-20,60),
8.09 (dd,1H,J ¼ 2 Hz,H-7), 8.38 (d,1H,J ¼ 2.4 Hz,H-5). COSY: (H-5/H-
7, secondary), (H-20,6’/H-30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/
H-20,60), (NeCH2/H-200,600), (morph./H-30,50). 13C-NMR & HSQC &
C
d
(CD3OD): 3.48, 3.46 and 3.76 (br.s,8H, morph), 5.68 (s,2H,NeCH2),
7.14e7.19 (m,2H,H-300,500), 7.51e7.54 (m,2H,H-200,600), 7.57
(d,2H,J ¼ 8.4 Hz,H-30,50), 8.41 (d,2H,J ¼ 8.4 Hz,H-20,60), 8.95
(d,1H,J ¼ 2 Hz,H-6), 9.04 (d,1H,J ¼ 1.6 Hz,H-2) COSY: (H-20,6’/H-
30,50), (H-200,6’’/H-300,500) NOESY: (NeCH2/H-2,6), (NeCH2/H-200,600),
HMBC
d ppm (CDCl3): 42.6, 48.1 and 66.8(morph.), 46.4(NeCH2),
115.9(d,J ¼ 21.8 Hz,CH-300,500), 127.0(C-6), 127.1(CH-7), 127.6 (CH-
30,50), 128.35(d,J ¼ 8.4 Hz,CH-200,600), 129.5(CH-20,60), 130.9(C-10),
131.9(d,J ¼ 3.2 Hz, C-100), 135.4(C-7a), 137.5(C-40), 143.5(CH-5),
147.1(C-3a), 155.0(C-2), 162.3(d,J ¼ 246 Hz, C-400), 169.3(CO). MS
(ESIþ) m/z: 451(M þ H, 100%). Anal. Calcd for C24H20ClFN4O2$H2O:
C, 61.47; H, 4.72; N, 11.94. Found: C, 61.87; H, 4.69; N, 12.15.
Continued elution (CH2Cl2: MeOH 100 : 4) provided compound
(morph./H-30,50). 13C-NMR & HSQC & HMBC
d ppm (CD3OD): 43.9
and 67.7(morph.) 60.1(NeCH2), 117.3(d,J ¼ 22.5 Hz,CH-300,500),
128.7(CH-30,50), 129.7(CH-20,60), 131.8(d,J
¼
8.3 Hz,CH-200,600),
132.2(d,J
¼
3.2 Hz,C-100), 135.15(C-2), 135.8(C-10), 137.7(C-5),
138.7(C-40), 145.9(C-6), 164.6(d,J
¼
245 Hz,C-400), 166.7(C-4),
19b as a white solid (0.079 g, 48%), mp 118e122 ꢂC. 1H-NMR
(CD3OD): 3.5, 3.67 and 3.8 (br.s,8H,morph.), 5.77 (s,2H,NeCH2),
7.03e7.08(2H,H-300,500), (m,2H,H-200,600),
7.45e7.49 7.5
(d,2H,J 1.6 Hz,H-5), 8.07
Hz, H-30,50), 7.64 (d,1H,J
d ppm
171.8(CO), 173.8(C-8). MS (ESIþ) m/z: 418(M þ H, 100%). Anal. Calcd
.
for C23H20FN5O2 3.25H2O: C, 58.04; H, 5.61; N, 14.71. Found: C,
58.07; H, 5.62; N, 14.60.
¼
8
¼
(4-(1-(4-Fluorobenzyl)-1H-imidazo [4,5-b]pyrazin-2-yl)phenyl)(-
morpholino)methanone (30a) & 4-(4-(4-fluorobenzyl)-4H-imidazo
[4,5-b]pyrazin-2-yl)phenyl)(morpholino)methanone (30b)
Prepared from 29 (0.11 g) as described in general method. Pu-
rification (Silicagel, EtOAc: MeOH 100 : 4) first afforded compound
30a as a white solid, yield 0.012 g, (8%), mp 138e142 ꢂC. 1H-NMR
(d,1H,J ¼ 1.6 Hz,H-7), 8.50 (d,2H,J ¼ 8 Hz,H-20,60). COSY: (H-5/H-7
secondary), (H-20,6’/H-30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/H-
5), (NeCH2/H-200,600), (morph./H-30,50). 13C-NMR & HSQC & HMBC
d
ppm (CD3OD): 42.65, 48.15 & 66.8(morph.), 56.2(NeCH2),
116.3(d,J ¼ 22 Hz,CH-300,500), 120.5(C-6), 127.1 (CH-5), 127.4(CH-
30,50), 127.8(CH-7), 128.6(CH-20,60), 129.6(d,J ¼ 3.8 Hz,C-100), 130.8(d,
J ¼ 8.4 Hz, CH-200,600), 135.3(C-10), 136.8(C-40), 146.0(C-7a), 153.6(C-
3a), 163.2(d,J ¼ 244 Hz, C-400), 170.1(CO), 170.7(C-2). MS (ESIþ) m/z:
451(M þ H, 100%). Anal. Calcd for C24H20ClFN4O2$H2O: C, 61.47; H,
4.72; N, 11.94. Found: C, 61.23; H, 4.86; N, 12.04.
d
ppm (CD3OD): 3.44, 3.63 and 3.77 (m,8H,morph), 5.70
(s,2H,NeCH2), 6.95e6.99 (m,2H,H-300,500), 7.06e7.09 (m,2H,H-
200,600), 7.60 (d,2H,J ¼ 8.4 Hz,H-30,50), 7.85 (d,2H,J ¼ 8 Hz,H-20,60),
8.47 (d,1H,J ¼ 2.8 Hz,H-5), 8.57 (d,1H,J ¼ 2.8 Hz,H-6). COSY: (H-5/H-
6), (H-20,6’/H-30,50), (H-200,6’’/H-300,500). NOESY: (NeCH2/H-20,60),
(NeCH2/H-200,600), (morph./H-30,50). 13C-NMR & HSQC & HMBC
4.1.4.18. (4-(5-(4-Fluorobenzyl)-5H-imidazo [4,5-c]pyridin-2-yl)phe-
nyl)(morpholino)methanone (23). Prepared from 22 (0.11 g) as
described in general method. The resulting precipitate was crys-
tallised from EtOAceMeOH as a white solid, yield 0.067 g (45%), mp
109e112 ꢂC.
d
ppm (CD3OD): 43.9 and 67.8(morph), 47.4(NeCH2),
116.7(d,J ¼ 21.8 Hz,CH-300,500),128.8(CH-30,50), 130.1(d,J ¼ 8.3 Hz,CH-
200,600), 130.9 (CH-20,60), 131.7(C-10), 133.4(d,J
¼
3.2 Hz,C-100),
139.3(C-40), 140.6(CH-5), 141.8(CH-6), 143.2(C-3a), 149.4(C-7a),
158.5(C-2), 163.8(d,J ¼ 244 Hz,C-400), 171.3(CO). MS (ESIþ) m/z:
418(M þ H, 100%). Anal. Calcd for C23H20FN5O2 . 2H2O: C, 60.91; H,
5.33; N, 15.44. Found: C, 61.00; H, 5.57; N, 15.30.
1H-NMR
d ppm (CD3OD): 3.51, 3.66 and 3.78 (m,8H,morp), 5.68
(s,2H,NeCH2), 7.13e7.17 (m, 2H,H-300,500), 7.46e7.49 (m,2H,H-200,
600), 7.58 (d,2H,J ¼ 8.4 Hz,H-30,50), 7.79 (d,1H,J ¼ 7.2 Hz, H-7), 8.18
(dd,1H,J ¼ 6.8 & 1.6 Hz,H-6), 8.39 (d,2H,J ¼ 8.8 Hz,H-20,60), 8.93
(d,1H,J ¼ 1.2 Hz,H-4). COSY: (H-6/H-7), (H-20,6’/H-30,50), (H-200,6’’/H-
Continued elution (EtOAc: MeOH 100 : 5) provided compound
30b as a white solid (0.07 g, 46.6%) mp 85e88 ꢂC. 1H-NMR
(CD3OD): 3.48, 3.64 and 3.76 (m,8H,morph), 5.91 (s,2H,NeCH2),
d
ppm

12
C. Karaaslan et al. / Journal of Molecular Structure 1205 (2020) 127673
7.08e7.13 (m,2H,H-300,500), 7.59 (d,2H,J ¼ 8.4 Hz,H-30,50), 7.62e7.66
(m,2H, H-200,600), 8.09 (d,1H,J ¼ 3.6 Hz,H-5), 8.35 (d,1H,J ¼ 4 Hz,H-6),
8.54 (d,2H,J ¼ 8.4 Hz,H-20,60). COSY: (H-5/H-6), (H-20,6’/H-30,50), (H-
200,6’’/H-300,500). NOESY: (NeCH2/H-5), (NeCH2/H-200,600), (morph./H-
Untreated cells were used as a control group and the IC50 was
calculated using GraphPad Prism 7.0 software (GraphPad Software,
USA). NE indicates that no IC50 value was determined in the con-
centration range in which the cytotoxic activities were screened.
30,50). 13C-NMR & HSQC & HMBC
d ppm (CD3OD): 43.9 &
67.8(morph), 57.3(NeCH2), 117.0(d,J ¼ 22 Hz,CH-300,500), 124.4(CH-
4.3. Statistical analysis
5), 128.7(CH-30,50), 130.2(CH-20,60), 131.5(d,J
¼
3.2 Hz,C-100),
132.5(d,J ¼ 8.4 Hz,CH-200,600), 135.8(CH-6), 135.9 (C-10), 139.3(C-40),
145.6(C-3a), 161.7(C-7a), 164.6(d,J ¼ 246 Hz,C-400), 171.8(CO), 174.4
One-way ANOVA variance test of StatistiXL program (Broad-
wayeNedlands, Western Australia) was used to determine statis-
tical differences among groups. A p value of <0.05 was considered
(C-2). MS (ESIþ) m/z: 418(M
þ
H, 100%). Anal. Calcd for
as significant and results were expressed as mean
standard
C
23H20FN5O2$H2O: C, 63.43; H, 5.09; N, 16.08. Found: C, 63.10; H,
deviation.
5.44; N, 15.86.
CRediT authorship contribution statement
4.1.4.20. (4-(1-(4-Fluorobenzyl)-1H-imidazo
[4,5-b]pyridin-2-yl)
phenyl)(morpholino)methanone (18c). A mixture of 20 (0.044 g,
0.2 mmol) and Na2S2O5 adduct of 4-(morpholin-4-ylcarbonyl)
benzaldehyde (0.065 g, 0.2 mmol) in DMF (0.35 mL) was heated at
120 ꢂC for 2.5 h. Water was added, the resulting precipitate was
filtered, dried. Precipitate was purified with column chromatog-
raphy using CH2Cl2: MeOH (100 : 3) as eluant, mp 212e215 ꢂC, yield
Cigdem Karaaslan: Investigation, Data curation, Writing - re-
view & editing. Fatima Doganc: Investigation. Mehmet Alp:
Investigation. Asli Koc: Investigation. Arzu Zeynep Karabay:
€
Investigation. Hakan Goker: Conceptualization, Methodology, Su-
pervision, Writing - review & editing.
0.051 g (60%). 1H-NMR
d ppm (CD3OD): 3.45, 3.64 and 3.76
(br.s,8H,morph.), 5.62 (s,2H,NeCH2), 6.99e7.07 (m,4H,H-
300,500,200,600), 7.34 (dd,1H,J ¼ 8 & 5.2 Hz,H-6), 7.60 (d,2H,J ¼ 8 Hz,H-
30,50), 7.83 (d,2H,J ¼ 8 Hz,H-20,60), 7.91 (dd,1H,J ¼ 8.4 & 1.6 Hz,H-7),
8.48 (dd,1H,J ¼ 5.2 & 1.6 Hz,H-5). COSY: (H-5/H-6), (H-6/H-7), (H-
20,6’/H-30,50). NOESY: (NeCH2/H-7), (NeCH2/H-20,60), (NeCH2/H-
Acknowledgement
Central Laboratory of Faculty of Pharmacy, Ankara University
provided support for acquisition of the NMR, Mass Spectrometer
and Elemental Analyzer used in this work.
200,600), (morph./H-30,50). 13C-NMR & HSQC & HMBC
d ppm (CD3OD):
43.9 & 67.7(morph), 49.5(NeCH2), 116.9(d,J ¼ 21.8 Hz,CH-300,500),
120.15(CH-6), 121.5(CH-7), 128.8(CH-30,50), 129.5(d,J ¼ 8.3 Hz,CH-
200,600), 130.0(C-7a), 131.0(CH-20,60), 131.9(C-10), 133.2(d,J ¼ 3.3 Hz,C-
100), 138.8(C-40), 146.1(CH-5), 155.8(C-3a), 156.9(C-2), 163.8(d,
J ¼ 244 Hz,C-400), 171.3(CO). MS (ESIþ) m/z: 417(M þ H, 100%). Anal.
Calcd for C24H21FN4O2$H2O: C, 66.34; H, 5.33; N, 12.9. Found: C,
66.72; H, 5.37; N, 13.21.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127673.
References
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