Adenine Derivatives: Promising Candidates for Breast Cancer Treatment

Article abstract of DOI:10.1002/ejoc.201800629

The cascade reaction of N-aryl-5-{[(dimethylamino)methylene]amino}-4-cyanoimidazoles with primary aromatic amines, under reflux in acetic acid, proved to be a general and efficient method for the regioselective synthesis of 6-arylaminopurines. A selection of these compounds was tested in breast cancer cell models MCF-7 and Hs578t and five compounds proved to be highly active and non-toxic to the non-neoplastic cell line MCF-10A. Furthermore, the compounds demonstrated apoptosis-inducing capacity in the cancer cells.

Full text of DOI:10.1002/ejoc.201800629

A Journal of
Accepted Article
Title: Adenine derivatives: promising candidates for breast cancer
treatment
Authors: Pedro Figueiredo, Marta Costa, Olivia Pontes, Fátima
Baltazar, and Maria Fernanda de Jesus Rego Paiva Proença
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800629
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800629
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10.1002/ejoc.201800629
European Journal of Organic Chemistry
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Adenine derivatives: promising candidates for breast cancer
treatment
Pedro Figueiredo,[a] Marta Costa,* [b,c] Olívia Pontes,[b,c] Fátima Baltazar[b,c] and Fernanda Proença*[a]
Abstract:
The
cascade
reaction
of
N-aryl-5-
Diverse methodologies for the synthesis of purine libraries
continue to be developed, essentially centered on modifications
of the substituents in halogenated purines or other active purine-
based intermediates.[2] Adenine derivatives were also prepared
from amino-substituted pyrimidines via N-acylation and
intramolecular cyclization,[2b] from acyclic precursors[2c] or from
appropriately substituted imidazole derivatives.[14] 5-Amino-4-
cyanoimidazoles can be considered convenient adenine
precursors as they are stable and easily available starting
materials. The N-1 substituent can be an alkyl,[15] aryl,[16] amino[17]
or alkoxyl[18] group, widening the scope of these precursors.
(((dimethylamino)methylene)amino)-4-cyanoimidazoles with primary
aromatic amines, under reflux in acetic acid, proved to be a general
and efficient method for the regioselective synthesis of 6-
arylaminopurines. A selection of these compounds was tested in
breast cancer cell models MCF-7 and Hs578t and three compounds
proved to be highly active and non-toxic to the non-neoplastic cell line
MCF-10A. Further, the compounds demonstrated apoptosis-inducing
capacity in the cancer cells.
Introduction
Results and Discussion
Synthesis
The purine scaffold is present in a wide range of molecules, either
isolated from natural sources[1] or from synthetic origin.[2] The
literature reveals a diverse number of applications for purine-
based compounds, making them impressive targets in the
preparation of new therapeutic agents.[3] Molecules containing the
purine core were reported as interferon inducers[4] and tested in
the therapy of chronic hepatitis C.[5] They may be used as
cytostatic antitumor agents due to their potent microtubule-
disassembling properties.[6] They were also identified as Hsp90
inhibitors, inducing the proteasomes degradation of diverse
Hsp90 client proteins associated to cancer.[7] Adenine derivatives,
in particular, were identified as inhibitors of cysteine protease
cathepsin k,[8] representing promising therapeutic agents for the
treatment of osteoporosis[9] and as potent and selective
phosphodiesterase inhibitors[10] demonstrating efficacy in various
gastrointestinal and neurologic diseases.
In this work, we explored the reactivity of N-aryl-5-amino-4-
cyanoimidazoles 4, prepared from the corresponding amidine 3
by intramolecular cyclization in the presence of aqueous base
(Scheme 1).
Adenosine receptors are involved in several pathophysiological
processes that include inflammation and cancer[11] and diverse
antagonists were already identified in the literature.[12] Several N-
substituted adenine derivatives were reported as protein kinase
inhibitors.[13] The biological activity of all these molecules highly
depends on the nature and position of the substituents on the
heterocyclic ring. The capacity to generate libraries of
compounds, manipulating their substitution pattern, continues to
be a relevant topic for research, diversifying the possible
derivatives to be prepared.
Scheme 1. Synthetic sequence for the preparation of 5-amino-4-
cyanoimidazoles 4.
Amidine 3 is generated from the reaction of imidate 2 with primary
amines, following an experimental procedure previously
developed by the group.[15-18] A diversity of primary aryl amines 6
was selected, contributing to widen the library of compounds that
can be prepared by this synthetic approach.
The imidate function was built on the amino group of o-
aminonitrile 4 by reaction with triethylorthoformate, following a
reported procedure (Scheme 2).[16] Compound 5, isolated in 85%
yield, was used to select the optimized conditions for the
synthesis of adenine derivatives 8 (Table 1).
[a]
[b]
[c]
Chemistry Department
University of Minho
Campus de Gualtar, Braga, Portugal
E-mail: fproenca@quimica.uminho.pt; cq.uminho.pt
Life and Health Sciences Research Institute (ICVS)
University of Minho
Campus de Gualtar, Braga, Portugal
E-mail: martasilviacosta@gmail.com
ICVS/3B’s – PT Government Associate Laboratory
Braga/Guimarães, Portugal
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201800629
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sulfuric acid. The activated aniline 6a led to 78% of the
corresponding product 8a after heating in a 0.1M ethanolic
Table 2. Synthesis of 1H-imidazol-5-yl)-N,N-dimethylformimidamides 7.
Entry
1
R
7
Yield (%) a)
82
7a
7b
7c
7d
88
79
50
2
3
4
7e
7f
78
78
58
5
6
7
Scheme 2. Alternative synthetic pathways used to generate adenine derivatives
9.
7g
7h
7i
65
66
62
8
9
Table 1. Synthesis of 1,6-dihydropurines 8 and evolution to purines 9.
7j
10
7k
36
11
[a] Isolated yield.
solution of the reagent 5 for 1.5 h. The deactivated aniline 6c
resulted in only 44% isolated yield of 8c after 4 h, while aniline 6b
generated 53% of 8b, under the same experimental conditions.
One of the draw-backs of this method is the acid-catalyzed
cleavage of the imidate function in imidazole 5, a competitive
reaction that could never be prevented even at room temperature,
as the precursor imidazole 4 was always isolated from the mother
liquor. The halogenated aromatic amines (3-bromo-, 4-bromo-, 3-
chloro- and 4-chloro-anilines) failed to generate the
corresponding adenine even when the reaction was performed at
room temperature, with acid catalysis, leading to the quantitative
recovery of imidazole 4. The Dimroth rearrangement of 8a proved
to be effective in DMF and TFA (1.5 molar equiv.) as adenine
derivative 9a was isolated in 87% yield after 2 h under reflux.
Entry
1
R
Product
Yield (%) a)
78
8a
8b
8c
9a
53
44
87
2
3
4
[a] Isolated yield.
The experiments were performed with aromatic amines 6a, 6b
and 6c, testing the effect of strongly electron-withdrawing and
electron-donating groups of the aromatic ring, on the reaction
outcome. The reaction required heating at 80 ºC and catalysis of
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Table 3. Synthesis of N,9-diaryl-9H-purin-6-amines 9 by a one-pot
procedure.
9r
78
79
17
18
9s
9t
9u
9v
93
78
92
19
20
21
Entry
1
R1
R2
9
Yield (%) a)
29
9b
9c
9d
50
46
2
3
9w
9x
83
95
80
78
75
22
23
24
25
26
9e
47
4
9y
9f
9g
9h
80
86
84
5
6
7
9z
9aa
9ab
9ac
94
74
27
28
9i
9j
79
82
92
72
8
9
9ad
87
29
9k
9l
10
11
9ae
9af
73
56
39
30
31
32
9ag
9m
9n
62
92
12
13
9ah
45
33
9o
9p
9q
75
97
70
14
15
16
[a] Isolated yield.
Nevertheless, this method was abandoned as it could not be used
as a general approach to prepare a library of purines in good yield.
Inspired by the method reported by Tsou et al.[19] for the synthesis
of quinazolines from the reaction of the formamidine of 5-
nitroanthranilonitrile with aromatic amines,
a selection of
formylated imidazoles (7a-k) was prepared from the reaction of 4
with dimethylformamide diethyl acetal (Table 2), performed under
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10.1002/ejoc.201800629
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reflux in the absence of solvent. The product was isolated after
approximately 1 h, in 36-88% yield.
Table 4. Anticancer potency and selectivity of the selected compounds in
MCF-7, Hs578t and MCF-10A cell lines.
Compound 7 evolved to the corresponding purine 9 when
combined with a substituted aniline 6 in acetic acid, under reflux
conditions. The product was isolated in 29-95% yield after 1-3 h
(Table 3).
The phenolic purine derivatives 9ac, 9ad, 9ae and 9af were
acetylated, aiming at the preparation of a pro-drug that would
release acetic acid upon cleavage of the acetyl group in the
vicinity of the tumor cell. According to the literature,
microelectrode measurements indicate that the extracellular pH
of the tumor cells is acidic[20] and these conditions would
selectively favor the ester hydrolysis. This controlled acidification
process of the tumor cellular medium could complement the drug
effect by changing the intracellular pH.
IC50 (µM) a)
SI
MCF7
vs
MCF-
10A
MCF-7
(luminal
A)
Hs578t
(basal-
like)
MCF-10A
(non-
neoplastic)
Compd
9a
14.38
3.03
10.29
8.60
---
25.76
---
---
41.39
---
9h
9j
9k
13
19.12
˃60
˃6
9o
9r
9s
9t
15.21
8.48
31.49
11.90
32.00
---
25.91
---
---
---
---
˃50
---
---
---
˃5
A selection of substituted adenines was tested on MCF-7 breast
cancer cells (luminal A) for cell viability at 10 and 30 M
concentrations. The anticancer potency of the most active
molecules was also tested on basal-like cells (Hs578t) and non-
neoplastic (MCF-10A) cell lines.
9x
9ac
10a
Doxorubicin
4.96
3.52
1.37
12.43
12.73
---
˃60
˃60
1.54
˃11
˃16
---
Bioassays
[a] Each compound was tested at least in triplicate and the data are presented
as mean values. SI selectivity index.
Effect on cell viability
The anticancer activity of a series of compounds was analyzed in
a breast cancer cell model, using the MCF-7 (estrogen receptor
positive breast cancer cells) cell line. The Sulforhodamine B
assay was used to evaluate cell viability. An initial screening was
performed for the compounds identified in Table 4, using
doxorubicin as a model drug.[21] The compounds were added to
cells at two different concentrations (10 and 30 M) and the effect
was evaluated after 72h of incubation (Table 1, Supporting
Information). Compounds presenting higher inhibitory effects on
cell growth were further studied in the MCF-7 cell line for IC50
determination (Table 4). The 95% confidence intervals for the IC50
values were included in the supporting information (Table 2).
Compounds presenting IC50 lower than 10 M were also
evaluated in the breast cancer cell line Hs578t (triple negative).
The non-neoplastic cell line MCF-10A was used to compare the
selectivity of the compounds towards tumor cells MCF-7.
Compounds 9h, 9k, 9r, 9ac and 10a presented lower IC50 values
for MCF-7 cell lines than for Hs578t, suggesting a higher potency
of these compounds for the luminal subtype of breast cancer
model. SI value was also calculated in order to assess cytotoxicity
of the compounds toward non-tumoral cells. A SI value higher
than 1 shows a higher affinity to tumoral cells, compared to
healthy non-malignant cells. The tested compounds do not
present significant cytotoxicity for MCF-10A non-neoplastic cell
line at the tested concentrations, presenting high SI values and
clearly demonstrating selectivity for the breast cancer cell lines.
Effect on apoptosis protein expression: Western
blot
An initial study of the mechanism of action of compounds 9h, 9k
and 9r was performed by immunoblot and MCF-7 cells were used
since the compounds showed higher activity for this cell line.
Malignant cells show the capacity to avoid apoptosis and the use
of compounds capable to induce this type of cell death is
important.[22] An apoptosis kit was used to determine if the
compounds could exert their toxicity through specific proteins
involved in programmed cell death: Caspase 3, Caspase 9 and
Poly (ADP-ribose) polymerase (Parp). Cells were treated with the
IC50 and half of the IC50 concentration (Figure 1).
The tested compounds induced cleavage of Caspase 3 and 9,
however the expression does not appear to be significantly
different from the control (Figure 1A). On the contrary, cleaved
Parp was expressed by the cells treated with the compounds at
the IC50 and half of the IC50 concentration but not by the control
(Figure 1B and C). The mechanism of action of the compounds
appears to involve cleavage of Parp, a final step of caspase
activation and considered to be a hallmark of apoptosis,[23] as
demonstrated by Western-blot. The tested compounds proved to
have apoptosis-inducing capacity and although further studies are
required to characterize their mechanism of action, they certainly
present potential as anticancer agents.
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10.1002/ejoc.201800629
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active on the luminal A MCF-7 cell model, with IC50 values of 3.03,
8.60, 8.48, 4.96 and 3.52 M respectively. Doxorubicin, a
commonly used chemotherapeutic agent for breast cancer
treatment but with serious side effects, was used as a model
compound. The doxorubicin IC50 value determined for the MCF-7
cell line was 1.37 M, in the same range as the values obtained
for our adenine derivatives, however with lower cytotoxicity for the
MCF-10A non-neoplastic cells. The SI values calculated for these
compounds are all higher than 1, demonstrating selectivity for the
breast cancer cells, compared to the non- neoplastic cells.
This new synthetic methodology can be expanded to the
preparation of a diversified library of adenine derivatives with an
interesting potential as anticancer drug candidates.
Experimental Section
Chemistry
General information: All the chemicals and solvents were used
as received from commercial suppliers, without further
purification. The (Z)-ethyl N-((Z)-2-amino-1,2-dicyanovinyl)-
formimidate
2,
the
(Z)-N'-(1-amino-2,2-dicyanovinyl)-N-
arylformimidamide derivatives 3 and the 5-amino-1-aryl-1H-
imidazole-4-carbonitrile derivatives 4 used in this work were
prepared by a previously described procedure.[16] The reactions
were performed in an IKAMAG RCT basic, at different
temperatures with magnetic stirring between 300-500 RPM. The
1H NMR spectra were recorded in a Bruker Advance II+ 400
spectrometer at 400 MHz, at 20ºC and in some cases at 80ºC.
The 13C NMR spectra were obtained in the Bruker spectrometer
1
at 100 MHz and the H-15N HMBC correlation spectra were
obtained at 40.6 MHz. The chemical shifts () were registered in
ppm relative to the residual solvent signal (DMSO-d6). The
elemental analysis was obtained in a LECO instrument and the
melting points were determined in a Stuart SMP3 digital
apparatus.
Synthesis of ethyl N-4-cyano-1-p-tolyl-1H-imidazole-5-yl-
formimidate 5: Acetic anhydride (141 μL, 3 eq.) was added to a
suspension of 5-amino-1-(p-tolyl)-1H-imidazole-4-carbonitrile) 4f
(0.10 g, 0.5 mmol) in triethyl orthoformate (664 μL, 8 eq.). The
reaction mixture was refluxed for 12 minutes. When TLC showed
the absence of starting material, cold n-hexane was added
leading to a brownish solid precipitate, which was filtered and
washed with cold n-hexane. The product was identified as ethyl
N-4-cyano-1-p-tolyl-1H-imidazol-5-yl-formimidate 5 (0.10 g, 0.43
mmol, 85%); M.p. 96-98 ºC; 1H NMR (400 MHz, DMSO-d6)  8.41
(s, 1H, H6) 7.98 (s, 1H, H2) 7.37 (dd, 2H, H2’ e H6’, J 2.4 and 8.4
Hz), 7.33 (dd, 2H, H3’ e H5’, J 2.4 and 8.4 Hz), 4.21 (q, 2H, CH2, J
7.2 Hz), 2.31 (s, 3H, CH3), 1.23 (t, 3H, CH3, J 7.2 Hz); 13C NMR
(100 MHz, DMSO-d6) 161.46 (C6), 145.43 (C5) 138.23
(C4’),136.56 (C2), 131.61 (C1’), 129.79 (C3’ e C5’), 124.83 (C2’ e C6’)
116.01 (CN), 99.05 (C4), 62.74 (CH2), 20.59 and 13.77 (CH3);
C14H14N4O(254) calcd: C 66.11, H 5.56, N 22.04; found: C 66.14,
H 5.67, N 22.12.
Figure 1. Effect of compounds 9h, 9k and 9r on MCF-7 cells on the expression
of A: Total and cleaved Caspases 3 and 9, using 15% polyacrylamide gel; B:
total Parp and cleaved Parp, using 12% polyacrylamide gel; 72 hours treatment
by Western blot analysis. Similar blots were obtained in 3 independent
experiments. Actin was used as loading control. C: Quantification of cleaved-
Parp, normalized to actin, results are presented as mean±SD of at least three
independent experiments, *p<0.05 compared to control (DMSO, 0.3%).
Conclusions
In summary, we developed a simple and efficient method to
synthesize adenine derivatives
9
from 5-amino-4-cyano
imidazoles directly prepared from commercial low-cost
4
reagents. Reaction with DMFDEA incorporated an amidine
function in the 5-position and these compounds were used in a
cascade reaction with primary aromatic amines, under reflux in
acetic acid, leading to the desired adenine products. A selection
of compounds was tested on the breast cancer cell lines MCF-7
and Hs578t using the Sulforhodamine B assay to evaluate cell
viability. Compounds 9h, 9k, 9r, 9ac and 10a proved to be highly
General procedure for the synthesis of N’-(4-cyano-1-aryl-
1H-imidazol-5-yl)-N,N-dimethylformimidamides 7
Method A: DMFDEA (8 eq.) was added to 5-amino-1-aryl-1H-
imidazole-4-carbonitrile 4, under a nitrogen atmosphere and the
suspension was refluxed in a closed vessel for 1 hour (with
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10.1002/ejoc.201800629
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heating, all solid went into solution). After cooling the mixture to
room temperature, cold diethyl ether was added leading to a
precipitate that was filtered, washed with cold diethyl ether and
identified as the corresponding N'-(4-cyano-1-aryl-1H-imidazol-5-
yl)-N,N-dimethylformimidamide 7.
mmol), DMFDEA (3.7 mL, off-white suspension). Product 7d was
isolated as a light brown solid (0.32 g, 1.34 mmol, 50%); M.p. 145-
146 ºC; 1H NMR (400 MHz, DMSO-d6) 8.09 (s, 1H, H7), 7.80 (s,
1H, H2), 7.47-7.53 (m, 4H, H2’, H3’, H5’ and H6’), 7.39-7.43 (m, 1H,
R (H4’)), 3.05 (s, 3H, CH3, 10), 2.86 (d, 3H, CH3, 9, J 0.8Hz); 13C
NMR (100 MHz, DMSO-d6) 156.17 (C7), 150.36 (C5), 134.82
(C1’), 134.75 (C2), 129.12 (C3’ and C5’), 127.85 (C4), 124.80 (C2’
and C6’), 117.69 (CN), 95.91 (C4), 39.92 (C10), 34.00 (C9)15N
NMR (40 MHz, DMSO-d6) 188.94 (N6), 178.42 (N1), 95.21 (N8);
C13H13N5 (239) calcd: C 65.25, H 5.48, N 29.27; found: C 65.12,
H 5.24, N 29.04.
Method B: DMFDEA (8 eq.) was added to 5-amino-1-aryl-1H-
imidazole-4-carbonitrile 4 under a nitrogen atmosphere and the
suspension was heated in a closed vessel at 60 ºC (with heating,
all solid went into solution). After cooling the mixture to room
temperature, cold diethyl ether was added and the precipitate was
filtered, washed with cold diethyl ether and identified as the
corresponding
dimethylformimidamide derivative 7.
N'-(4-cyano-1-aryl-1H-imidazol-5-yl)-N,N-
N'-(4-Cyano-1-(3-bromophenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7e: Prepared by the general method
A. 5-Amino-1-(3-bromophenyl)-1H-imidazole-4-carbonitrile 4b
(0.50 g, 1.89 mmol), DMFDEA (2.5 mL, light brown suspension).
Product 7e was isolated as a light brown solid (0.47 g, 1.48 mmol,
78%); M.p. 151-152 ºC; 1H NMR (400 MHz, DMSO-d6) 8.11 (s,
1H, H7), 7.87 (s, 1H, H2), 7.84 (t, 1H, H2’, J 2.0Hz), 7.57 (dm, 1H,
H6’), 7.62 (dm, 1H, H4’), 7.46 (t, 1H, H5’, J 8.0Hz), 3.06 (s, 3H, CH3,
10), 2.89 (d, 3H, CH3, 9, J 0.4Hz); 13C NMR (100 MHz, DMSO-d6)
156.18 (C7), 150.17 (C5), 136.12 (C1’), 134.57 (C2), 130.95 (C5’),
130.57 (C4’), 127.40 (C2’), 123.75 (C6’), 121.49 (C3’), 117.55 (CN),
95.81 (C4), 40.01 (C10), 34.01 (C9)15N NMR (40 MHz, DMSO-d6)
189.97 (N6), 177.12 (N1), 98.07 (N8); C13H12BrN5 (317) calcd: C
49.07, H 3.80, N 22.01; found: C 48.95, H 4.07, N 22.35.
N'-(4-Cyano-1-(p-tolyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7a: Prepared by the general method
A. 5-Amino-1-(p-tolyl)-1H-imidazole-4-carbonitrile 4f (0.50 g, 2.53
mmol), DMFDEA (3.5 mL, dark blue suspension). Product 7a was
isolated as a light brown solid (0.52 g, 2.05 mmol, 82%); M.p. 132-
134 ºC; 1H NMR (400 MHz, DMSO-d6) 8.08 (s, 1H, H7), 7.75 (s,
1H, H2), 7.38 (d, 2H, H2’ and H6’, J 4.0Hz), 7.29 (d, 2H, H3’ and H5’,
J 4.0Hz), 3.04 (s, 3H, CH3, 10), 2.86 (d, 3H, CH3, 9, J 0.2Hz), 2.33(s,
3H, R (Me)); 13C NMR (100 MHz, DMSO-d6) 156.11 (C7), 150.37
(C5), 137.39 (C1’), 134.73 (C2), 132.34 (C4’), 129.53 (C3’ and C5’),
124.63 (C2’ and C6’), 117.73 (CN), 95.83 (C4), 39.71 (C10), 33.98
(C9), 20.57 (Me)15N NMR (40 MHz, DMSO-d6) 190.70 (N6),
179.62 (N1), 96.91 (N8); C14H15N5 (253) calcd: C 66.38, H 5.97, N
27.65; found: C 66.03, H 6.11, N 27.89.
N'-(4-Cyano-1-(4-bromophenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7f: Prepared by the general method
A. 5-Amino-1-(4-bromophenyl)-1H-imidazole-4-carbonitrile 4c
(0.30 g, 1.14 mmol), DMFDEA (1.5 mL, grey suspension). Product
7f was isolated as a light brown solid (0.28 g, 0.88 mmol, 78%);
M.p. 150-151 ºC; 1H NMR (400 MHz, DMSO-d6) 8.10 (s, 1H, H7),
7.82 (s, 1H, H2), 7.70 (d, 2H, H2’ and H6’, J 8.8Hz), 7.50 (d, 2H, H3’
and H5’, J 8.8Hz), 3.06 (s, 3H, CH3, 10), 2.88 (s, 3H, CH3, 9); 13C
NMR (100 MHz, DMSO-d6) 156.19 (C7), 150.20 (C5), 134.51 (C2
and C1’), 132.00 (C2’ and C6’), 126.82 (C3’ and C5’), 120.71 (C4’),
95.85 (C4), 117.57 (CN), 39.98 (C10), 34.05 (C9)15N NMR (40
MHz, DMSO-d6) 258.12 (N3), 189.76 (N6), 178.08 (N1), 97.91
(N8); C13H12BrN5 (317) calcd: C 49.07, H 3.80, N 22.01; found: C
49.25, H 4.02, N 22.36.
N'-(4-Cyano-1-(4-methoxyphenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7b: Prepared by the general method
A. 5-Amino-1-(4-methoxyphenyl)-1H-imidazole-4-carbonitrile 4g
(0.50 g, 2.34 mmol), DMFDEA (3.2 mL, off-white suspension).
Product 7b was isolated as a light brown solid (0.56 g, 2.07 mmol,
88%); M.p. 116-117 ºC; 1H NMR (400 MHz, DMSO-d6) 8.07 (s,
1H, H7), 7.71 (s, 1H, H2), 7.40 (d, 2H, H2’ and H6’, J 8.8Hz), 7.03
(s, 2H, H3’ and H5’, J 9.2Hz), 3.78 (s, 3H, R (OMe)), 3.04 (s, 3H,
CH3, 10), 2.85 (s, 3H, CH3, 9); 13C NMR (100 MHz, DMSO-d6)
158.65 (C4’), 156.09 (C7), 150.45 (C5), 134.85 (C2), 127.69 (C1’),
126.26 (C2’ and C6’), 117.76 (CN), 114.21 (C3’ and C5’), 95.66 (C4),
55.44 (R (OMe), 39.91 (C10), 33.96 (C9)15N NMR (40 MHz,
DMSO-d6) 191.23 (N6), 178.96 (N1), 96.35 (N8); C14H15N5O
(269) calcd: C 62.44, H 5.61, N 26.01; found C 62.58, H 5.74, N
26.20.
N'-(4-Cyano-1-(3-fluorophenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7g: Prepared by the general method
A. 5-Amino-1-(3-fluorophenyl)-1H-imidazole-4-carbonitrile 4d
(0.50 g, 2.46 mmol), DMFDEA (3.3 mL, brown suspension).
Product 7g was isolated as a light brown solid (0.37 g, 1.43 mmol,
58%); M.p. 124-126 ºC; 1H NMR (400 MHz, DMSO-d6) 8.09 (s,
1H, H7), 7.79 (s, 1H, H2), 7.53-7.58 (m, 2H, H2’ and H6’), 7.34 (tt,
2H, H3’ and H5’, J 3.2 and 4.4Hz), 3.05 (s, 3H, CH3, 10), 2.86 (d, 3H,
CH3, 9, J 0.4Hz); 13C NMR (100 MHz, DMSO-d6) 160.54 (C4’, J
243.0Hz), 156.19 (C7), 150.37 (C5), 134.77 (C2), 131.18 (C1’, J
3.0Hz), 127.16 and 127.07 (C2’ and C6’, J 9.0Hz), 115.81
and116.04 (C3’ and C5’, J 23.0Hz), 117.65 (CN), 95.71 (C4), 39.97
(C10) 34.01 (C9)15N NMR (40 MHz, DMSO-d6) 189.98 (N6),
178.34 (N1), 97.49 (N8); C13H12FN5 (257) calcd: C 60.69, H 4.70,
N 27.22; found: C 60.78, H 4.81, N 27.36.
N'-(4-Cyano-1-(4-cyanophenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7c: Prepared by the general method
A. 5-Amino-1-(4-cyanophenyl)-1H-imidazole-4-carbonitrile 4l
(0.50 g, 2.39 mmol), DMFDEA (3.2 mL, dark suspension). Product
7c was isolated as a brownish solid (0.50 g, 1.88 mmol, 79%);
M.p. 196-197 ºC; 1H NMR (400 MHz, DMSO-d6) 8.15 (s, 1H, H7),
8.01 (d, 2H, H3’ and H5’, J 8.8Hz), 7.95 (s, 1H, H2), 7.83 (d, 2H, H2’
and H6’, J 9.2Hz), 3.04 (s, 3H, CH3, 10), 2.92 (d, 3H, CH3, 9, J
0.4Hz); 13C NMR (100 MHz, DMSO-d6) 156.30 (C7), 150.24 (C5),
138.57 (C1’), 134.46 (C2), 133.34 (C3’ and C5’), 125.26 (C2’ and
C6’), 118.24 (R (CN), 117.43 (CN), 110.24 (C4’), 96.12 (C4), 40.07
(C10), 34.15 (C9)15N NMR (40 MHz, DMSO-d6) 188.96 (N6),
178.38 (N1), 99.22 (N8); C14H12N6 (264) calcd: C 63.62, H 4.58, N
31.80; found: C 63.45, H 4.72 N 31.96.
N'-(4-Cyano-1-(4-fluorophenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7h: Prepared by the general method
A. 5-Amino-1-(4-fluorophenyl)-1H-imidazole-4-carbonitrile 4e
(0.50 g, 2.46 mmol), DMFDEA (3.3 mL, brown suspension).
Product 7h was isolated as a light orange solid (0.41 g, 1.58
mmol, 65%); M.p. 122-123 ºC; 1H NMR (400 MHz, DMSO-d6)
N'-(4-Cyano-1-phenyl-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7d: Prepared by the general method
A. 5-Amino-1-phenyl-1H-imidazole-4-carbonitrile 4a (0.50 g, 2.71
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10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
8.09 (s, 1H, H7), 7.79 (s, 1H, H2), 7.53-7.58 (m, 2H, H2’ and H6’),
7.34 (tt, 2H, H3’ and H5’, J 4.4 and 3.2 Hz), 3.05 (s, 3H, CH3, 10),
2.86 (s, 3H, CH3, 9, J 0.4 Hz); 13C NMR (100 MHz, DMSO-d6)
161.35 (C4’, J 243.0 Hz), 156.19 (C7), 150.37 (C5), 134.77 (C2),
131.15 (C1’, J 3.0 Hz), 127.11 (C2’ and C6’, J 9.0 Hz), 117.65 (CN),
95.71 (C4), 115.92 (C3’ and C5’, J 23.0 Hz), 39.97 (C10), 34.01
(C9)15N NMR (40 MHz, DMSO-d6) 189.98 (N6), 178.34 (N1),
97.49 (N8); C13H12FN5 (257) calcd: C 60.69, H 4.70, N 27.22;
found: C 60.50, H 4.61, N 27.06.
at 80 °C for 1.5-4 hours. The beige solid precipitate was filtered,
washed with cold ethanol and identified as 1-phenyl-9-(p-tolyl)-
1H-purin-6(9H)-imine derivative 8.
1-(4-Methoxyphenyl)-9-(p-tolyl)-1H-purin-6(9H)-imine 8a:
Ethyl N-4-cyano-1-p-tolyl-1H-imidazol-5-yl-formimidate 5 (0.09 g,
0.38 mmol), ethanol (1 mL), p-anisidine 1.3g (0.06 g), sulfuric acid
(4 μL), 1.5 hours. Product 8a was isolated as a light brown solid
(0.10 g, 0.3 mmol; 78%); 1H NMR (400 MHz, DMSO-d6) 8.32 (s,
1H, H8), 7.94 (s, 1H, H2), 7.63 (d, 2H, H2’ and H6’, J 8.4 Hz), 7.38
(d, 2H, H3’ and H5’, J 8.4 Hz), 7.37 (d, 2H, H2’’ and H6’’, J 8.8 Hz),
7.06 (d, 2H, H3’’ and H5’’, J 8.8 Hz), 7.06 (s, 1H), 3.80 (s, 3H, R
(OMe)), 2.37 (s, 3H, Me); 13C NMR (100 MHz, DMSO-d6) 20.59
(Me), 55.45 (R (OMe)), 114.33 (C3’’ and C5’’), 123.37 (C2’ and C6’),
129.25 (C2’’ and C6’’), 129.89 (C3’ and C5’), 138.37 (C8), 140.97
(C4), 119.27 (C5), 154.77 (C6), 148.17 (C2); C19H17N5O (311)
calcd: C 68.87, H 5.17, N 21.13; found: C 69.01, H 5.22, N 21.03.
N'-(4-Cyano-1-(4-ethoxyphenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7i: Prepared by the general method
A. 5-Amino-1-(4-hydroxyphenyl)-1H-imidazole-4-carbonitrile 4i
(0.3 g, 1.5 mmol), DMFDEA (2 mL, beige suspension). Product 7i
was isolated as a light orange solid (0.28 g, 283 g/mol, 66%); M.p.
141-143 ºC; 1H NMR (400 MHz, DMSO-d6) 8.07 (s, 1H, H7), 7.71
(s, 1H, H2), 7.39 (d, 2H, H2’ and H6’, J 9.2Hz), 7.01 (s, 2H, H3’ and
H5’, J 9.2Hz), 4.05 (q, 2H J 6.8Hz, R (OEt)), 3.04 (s, 3H, CH3, 10),
2.85 (d, 3H, CH3, 9, J 0.4Hz), 1.32 (t, 3H, J 6.8Hz, R (OEt)); 13C
NMR (100 MHz, DMSO-d6) 157.92 (C4’), 156.07 (C7), 150.42
(C5), 134.83 (C2), 127.55 (C1’), 126.24 (C2’ and C6’), 117.75 (CN),
114.62 (C3’ and C5’), 95.65 (C4), 39.92 (C10), 33.94 (C9), 14.56 and
63.38 (OEt)15N NMR (40 MHz, DMSO-d6) 256.67 (N3), 191.10
(N6), 179.03 (N1), 96.39 (N8); C15H17N5O (283) calcd: C 63.59, H
6.05, N 24.72; found: C 63.42, H 6.29, N 24.56.
1-Phenyl-9-(p-tolyl)-1H-purin-6(9H)-imine 8b: Ethyl N-4-
cyano-1-p-tolyl-1H-imidazol-5-yl-formimidate
5 (0.05 g, 0.21
mmol), ethanol (0.5 mL), aniline 1.3a (23 μL), sulfuric acid (3 μL),
4 hours. Product 8b was isolated as a light brown solid (0.03 g,
0.11 mmol; 53%); 1H NMR (400 MHz, DMSO-d6) 10.10 (s, 1H,
NH), 8.94 (s, 1H, H8), 8.66 (s, 1H, H2), 7.70 (d, 2H, H2’ and H6’, J
8.4 Hz), 7.46 (d, 2H, H3’ and H5’, J 8.4 Hz), 7.33 (d, 2H, H2’’ and
H6’’, J 7.6 Hz), 7.25 (t, 2H, H3’’ and H5’’, J 7.6 Hz), 6.95-6.92 (m,
1H, H4’’), 2.41 (s, 3H, Me); 13C NMR (100 MHz, DMSO-d6)
151.20 (C6), 147.54 (C2), 146.44 (C4), 143.62 (C8), 138.75 (C4’),
135.02 (C1’’), 130.96 (C1’), 130.68 (C3’’ and C5’’), 130.22 (C3’ and
C5’), 127.71 (C2’’ and C6’’), 123.85 (C2’ and C6’), 121.88 (C4’’),
119.29 (C5), 20.65 (Me); C19H14N6 (326) calcd: C 69.92, H 4.32,
N 25.75; found: C 69.99, H 4.13, N 25.51.
N'-(4-Cyano-1-(4-hydroxyphenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7j: Prepared by the general method
B. 5-Amino-1-(4-hydroxyphenyl)-1H-imidazole-4-carbonitrile 4i
(0.19 g, 0.99 mmol), DMFDEA (1.3 mL, off-white suspension), 1.5
hours. Product 7j was isolated as a beige solid (0.16 g, 0.62
mmol, 62%); M.p. 120-121 ºC; 1H NMR (400 MHz, DMSO-d6)
9.75 (S, 1H, R (OH)), 8.05 (s, 1H, H7), 7.66 (s, 1H, H2), 7.26 (d,
2H, H2’ and H6’, J 8.8 Hz), 6.83 (d, 2H, H3’ and H5’, J 8.8 Hz), 3.03
(d, 3H, CH3, 9, J 0.4Hz), 2.85 (s, 3H, CH3, 10); 13C NMR (100 MHz,
DMSO-d6) 157.05 (C4’), 156.02 (C7), 150.45 (C5), 134.87 (C2),
126.33 (C2’ and C6’), 126.21 (C1’), 117.80 (CN), 115.42 (C3’ and
C5’), 95.59 (C4), 39.92 (C10), 33.93 (C9)15N NMR (40 MHz,
DMSO-d6) 256.46 (N3), 191.29 (N6), 179.57 (N1), 96.10 (N8);
C13H13N5O (255) calcd: C 61.17, H 5.13, N 27.43; found: C 61.04,
H 5.26, N 27.28.
4-(6-Imino-9-(p-tolyl)-6,9-dihydro-1H-purin-1-
yl)benzonitrile 8c: Ethyl N-4-cyano-1-p-tolyl-1H-imidazol-5-yl-
formimidate 5 (0.05 g, 0.21 mmol), ethanol (0.5 mL), 4-
aminobenzonitrile 1.3l (0.03 g), sulfuric acid (3 μL), 4 hours.
Product 8c was isolated as a light grey solid (0.03 g, 0.09 mmol;
44%); 1H NMR (400 MHz, DMSO-d6)  NH not seen, 8.96 (s, 1H,
H8), 8.69 (s, 1H, H2), 8.23 (d, 2H, H3’’ and H5’’, J 8.8 Hz), 7.95 (d,
2H, H2’’ and H6’’, J 8.8 Hz), 7.69 (d, 2H, H2’ and H6’, J 8.4 Hz), 7.47
(d, 2H, H3’ and H5’, J 8.4 Hz), 2.35 (s, 3H, Me); 13C NMR (100 MHz,
DMSO-d6) 151.17 (C6), 147.01 (C2), 146.35 (C4), 143.73 (C8),
138.86 (C4’), 138.32 (C1’’), 134.91 (C3’’ and C5’’), 132.59 (C1’),
130.26 (C3’ and C5’), 129.44 (C2’’ and C6’’), 123.85 (C2’ and C6’),
119.36 (C5), 117.96 (R (CN)), 114.17 (C4’’), 20.67 (Me); C18H15N5
(301) calcd: C 71.74, H 5.02, N 23.24; found: C 72.05, H 4.91, N
23.33.
N'-(4-Cyano-1-(3-hydroxyphenyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7k: Prepared by the general method
B. 5-Amino-1-(3-hydroxyphenyl)-1H-imidazole-4-carbonitrile 4h
(0.05 g, 0.25 mmol), DMFDEA (342 μL, off-white suspension), 40
minutes. Product 7k was isolated as a beige solid (0.02 g, 0.08
mmol, 36%); M.p. 155-157 ºC; 1H NMR (400 MHz, DMSO-d6)
9.84 (R (OH)), 8.07 (s, 1H, H7), 7.75 (s, 1H, H2), 7.26 (t, 1H, H6’,
J 8.0 Hz), 6.88-6.93 (m, 2H, H2’ and H5’), 6.80 (dm, 1H, H4’), 3.05
(s, 3H, CH3, 10), 2.89 (s, 3H, CH3, 9); 13C NMR (100 MHz, DMSO-
d6)  157.84 (C3’), 156.09 (C7), 150.34 (C5), 135.77 (C1’),
134.69(C2), 129.87 (C5’), 117.69 (CN), 115.10 (C4’), 114.79 (C6’),
111.80 (C2’), 96.00 (C4), 39.91 (C10), 34.03 (C9)15N NMR (40
MHz, DMSO-d6) 256.32 (N3), 190.02 (N6), 179.24 (N1), 98.35
(N8); C13H13N5O (255) calcd: C 61.17, H 5.13, N 27.43; found: C
61.23, H 5.19, N 27.59.
Synthesis of N-(4-methoxyphenyl)-9-(p-tolyl)-9H-purin-6-
amine 9a by Dimroth rearrangement of 8a: TFA (1.5 eq., 16
μL) was added to a solution of 8a (0.006 g, 0.14 mmol) in DMF
(1mL). The mixture was refluxed for 2 hours. Addition of water led
to a light brown solid precipitate that was filtered, washed with
cold water and identified as N-(4-methoxyphenyl)-9-(p-tolyl)-9H-
purin-6-amine 9a (0.004g, 0.12 mmol, 87 %): M.p. 157-159 ºC; 1H
NMR (400 MHz, DMSO-d6) 9.81 (s, 1H, NH), 8.66 (s, 1H, H8),
8.37 (s, 1H, H2), 7.80 (d, 2H, H2’’ and H6’’, J 9.2Hz), 7.78 (d, 2H,
H2’ and H6’, J 8.0Hz), 6.91 (d, 2H, H3’’ and H5’’, J 8.8Hz), 7.39 (d,
2H H3’ and H5’, J 8.0Hz), 3.74 (s, 3H, R2 (OMe)), 2.38 (s, 3H, R1
(Me)); 13C NMR (100 MHz, DMSO-d6) 155.16 (C4’’), 152.61 (C2),
152.45 (C6), 149.05 (C4), 140.25 (C8), 137.09 (C4’), 132.48 (C1’
and C1’’), 129.88 (C3’ and C5’), 122.95 (C2’ and C6’), 122.77 (C2’’
and C6’’), 119.99 (C5), 113.60 (C3’’ and C5’’), 55.19 (R2 (OMe)),
General procedure for the synthesis of 1-aryl-9-(p-tolyl)-
1H-purin-6(9H)-imines 8
Aromatic amine 6 (1.2 eq.) and sulfuric acid (0.2 eq.) were added
to
a solution of ethyl N-4-cyano-1-p-tolyl-1H-imidazol-5-yl-
formimidate 5 in ethanol. The mixture was stirred in a water bath
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10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
20.59 (R1 (Me)); 15N NMR (40 MHz, DMSO-d6) 233.47(N1),
230.48 (N3), 171.64 (N9), 103.32 (NH); C19H17N5O (321) calcd: C
68.87, H 5.17, N 21.13; found C 68.99, H 5.23, N 21.44.
acetic acid (1 mL, blue suspension), 3-bromoaniline (1.1 eq., 48
µL), 2 hours. Product 9d was isolated as a white solid (0.07 g,
46%); M.p. 187-189 ºC; 1H NMR (400 MHz, DMSO-d6) 10.14 (s,
1H, NH), 8.73 (s, 1H, H8), 8.46 (s, 1H, H2), 7.98 (d, 2H, H2’’ and
H6’’, J 8.8Hz), 7.78 (d, 2H, H2’ and H6’, J 8.8Hz), 7.50 (d, 2H, H3’’
and H5’’, J 8.8Hz), 7.44 (d, 2H, H3’ and H5’, J 8.8Hz), 2.38 (s, 3H,
R1 (Me)); 13C NMR (100 MHz, DMSO-d6) 152.41 (C2), 151.99
(C6), 149.44 (C4), 141.03 (C1’’ and C8), 137.29 (C4’), 132.79 (C3’’),
132.33 (C1’), 130.04 (C5’’), 129.93 (C3’ and C5’), 123.07 (C2’ and
C6’), 122.12 (C4’’), 120.44 (C5), 119.84 (C2’’), 118.93 (C6’’), 20.61
(R1 (Me)); 15N NMR (40 MHz, DMSO-d6) 234.67 (N1), 231.99
(N3), 171.89 (N9), 105.53 (NH) ; C18H14BrN5 (380) calcd: C 56.86,
H 3.71, N 18.42; found C 57.02, H 3.88, N 18.03.
General procedure for the synthesis of N,9-diaryl-9H-
purin-6-amines 9
Method A: The aromatic amine 6 (1.0-1.2 eq.) was added to a
solution
of
the
N'-(4-cyano-1-aryl-1H-imidazol-5-yl)-N,N-
dimethylformimidamide derivative 7 in acetic acid and the mixture
was refluxed in a closed vessel. The reaction was controlled by
TLC using a mixture of dichloromethane:ethyl acetate:acetic acid,
16:8:1 as eluent.
Method B: The aromatic amine 6 (0.6 eq.) was added to a
N-(3-Chlorophenyl)-9-(p-tolyl)-9H-purin-6-amine
9e:
solution
of
the
N'-(4-cyano-1-aryl-1H-imidazol-5-yl)-N,N-
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), 3-chloroaniline (1.0 eq., 42
µL), 2 hours. Product 9e was isolated as a beige solid (0.10 g,
75%); M.p. 161-163 ºC; 1H NMR (400 MHz, DMSO-d6) 10.19 (s,
1H, NH), 8.51 (s, 1H, H2), 8.74 (s, 1H, H8) 8.24 (t, 1H, H2’’, J 2.0Hz),
7.92 (dm, 1H, H4’’ or H6’’), 7.79 (d, 2H, H2’ and H6’, J 8.8Hz), 7.38
(d, 2H, H3’ and H5’, J 8.8Hz), 7.35 (t,1H, H5’’, J 8.0Hz), 7.08 (dm,
1H, H4’’ or H6’’), 2.38 (s, 3H, R1 (Me)); 13C NMR (100 MHz, DMSO-
d6) 157.37 (C3’’), 152.47 (C2),152.32 (C6), 149.19 (C4), 140.58
(C1’’), 140.51 (C8), 137.15 (C4’), 132.43 (C1’), 129.89 (C3’ and C5’),
128.95 (C5’’), 123.00 (C2’ and C6’), 120.29 (C5), 111.76 (C2’’),
109.88 (C4’’), 107.99 (C6’’), 20.60 (R1 (Me)); 15N NMR (40 MHz,
DMSO-d6) 235.82 (N1), 231.72 (N3), 171.94 (N9); C18H14ClN5
(335) calcd: C 64.38, H 4.20, N 20.86; found C 64.00, H 4.32, N
21.01.
dimethylformimidamide derivative 7 in acetic acid and the mixture
was refluxed in a closed vessel. The reaction was controlled by
TLC and after complete consumption of the aromatic amine,
another 0.6 eq. were added and the mixture was further refluxed.
After cooling to room temperature, under stirring, water was
added until the product was precipitated from the reaction mixture.
The solid was filtered, washed with water and a small amount of
cold ethanol and identified as the N,9-diaryl-9H-purin-6-amine
derivative 9.
4-((9-(p-Tolyl)-9H-purin-6-yl)amino)benzonitrile
9b:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), 4-aminobenzonitrile (1.2 eq.,
56 mg), 1 hour. Product 9b was isolated as a light grey solid (0.36
g, 29%); M.p. 254-255 ºC; 1H NMR (400 MHz, DMSO-d6) 10.51
(s, 1H, NH), 8.79 (s, 1H, H8), 8.55 (s, 1H, H2), 8.26 (d, 2H, H2’ and
H6’, J 8.8Hz), 7.77 (d, 4H, H2’’, H3’’, H5’’ and H6’’, J 7.6Hz), 7.40 (d,
2H, H3’ and H5’, J 8.4 Hz), 2.38 (s, 3H, R1 (Me)); 13C NMR (100
MHz, DMSO-d6) 152.22 (C2), 151.63 (C6), 149.74 (C4), 144.21
(C1’ or C1’’), 141.50 (C8), 137.35 (C1’ or C1’’), 132.85 (C2’’ and C6’’
or C3’’ and C5’’), 132.22 (C4’’), 129.91 (C3’ and C5’), 123.09 (C2’’ and
C6’’ or C3’’ and C5’’), 120.81 (C5), 120.09 (C2’ and C6’), 119.39 (R2
(CN)), 103.66 (C4’’), 20.60 (R1 (Me)); 15N NMR (40 MHz, DMSO-
d6) 237.20 (N1), 235.26 (N3), 172.13 (N9), 108.24 (NH); C19H14N6
(328) calcd: C 69.92, H 4.32, N 25.75; found C 69.82, H 4.12, N
25.70.
N-(4-Chlorophenyl)-9-(p-tolyl)-9H-purin-6-amine
9f:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), 4-chloroaniline (1.2 eq., 60
mg), 2.5 hours. Product 9f was isolated as a beige solid (0.10 g,
75%); M.p. 217-219 ºC; 1H NMR (400 MHz, DMSO-d6) 10.13 (s,
1H, NH), 8.72 (s, 1H, H8), 8.46 (s, 1H, H2), 8.03 (d, 2H, H2’’ and
H6’’, J 8.8Hz), 7.78 (d, 2H, H2’ and H6’, J 8.4Hz), 7.38 (d, 2H, H3’
and H5’, J 8.4Hz), 7.31 (d, 2H, H3’’ and H5’’, J 8.8Hz), 2.38 (s, 3H,
R1 (Me)); 13C NMR (100 MHz, DMSO-d6) 153.35 (C4’’), 152.67
(C2), 152.57 (C6), 148.96 (C4), 140.08 (C8), 137.06 (C4’), 132.52
(C1’), 130.87 (C1’’), 129.88 (C3’ and C5’), 123.15 (C2’’ and C6’’),
122.94 (C2’ and C6’), 119.91 (C5), 114.85 (C3’’ and C5’’), 20.60 (R1
(Me)); 15N NMR (40 MHz, DMSO-d6) 235.82 (N1), 229.41 (N3),
171.83 (N9), 103.53 (NH) ; C18H14ClN5 (335) calcd: C 64.38, H
4.20, N 20.86; found C 64.44, H 4.15, N 20.99.
N-Phenyl-9-(p-tolyl)-9H-purin-6-amine 9c: Prepared by the
general method A. N'-(4-cyano-1-(p-tolyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7a (0.035 g, 0.14 mmol), acetic acid (400
µL, blue suspension), aniline (1.1 eq., 15 µL), 1 hour. Product 9c
was isolated as a light orange solid (0.03 g, 50%); M.p. 162-164
ºC; 1H NMR (400 MHz, DMSO-d6) 9.96 (s, 1H, NH), 8.70 (s, 1H,
H8), 8.44 (s, 1H, H2), 7.96 (d, 2H, H2’ and H6’, J 7.6Hz), 7.79 (d,
2H, H3’ and H5’, J 8.0Hz), 7.39 (d, 2H, H2’’ and H6’’, J 8.0Hz), 7.33
(t, 2H, H3’’ and H5’’, J 8.0Hz), 7.04 (t, 1H, R2 (H), J 6.8Hz), 2.38 (s,
3H, R1 (Me)); 13C NMR (100 MHz, DMSO-d6) 152.49 (C2),
152.31 (C6), 149.25 (C4), 140.58 (C8), 139.57 (C1’’), 137.16 (C1’),
132.42 (C4’), 129.89 (C2’’ and C6’’), 128.37 (C3’’ and C5’’), 123.00
(C3’ and C5’), 122.67 (C4’’), 120.86 (C2’ and C6’), 120.26 (C5),
20.59 (R1 (Me)); 15N NMR (40 MHz, DMSO-d6) 234.67 (N1),
231.99 (N3), 171.89 (N9), 105.53 (NH) ; C18H15N5 (302) calcd: C
71.74, H 5.02, N 23.24; found C 71.85, H 5.22, N 22.95.
N-(4-Bromophenyl)-9-(p-tolyl)-9H-purin-6-amine
9g:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), 4-bromoaniline (1.1 eq., 75
mg), 2 hours. Product 9g was isolated as a light yellow solid (0.13
g, 86%); M.p. 162-163 ºC; 1H NMR (400 MHz, DMSO-d6) 10.17
(s, 1H, NH), 8.74 (s, 1H, H8), 8.50 (s, 1H, H2), 8.38 (t, 1H, H6’’, J
2.0Hz) 7.96 (dm, 1H, H2’’), 7.78 (d, 2H, H2’ and H6’, J 8.4Hz), 7.39
(d, 2H, H3’ and H5’, J 8.4Hz), 7.29 (t, 1H, H5’’, J 8.0Hz), 7.21 (dm,
1H, H4’’), 2.38 (s, 3H, R1 (Me)); 13C NMR (100 MHz, DMSO-d6)
152.40 (C2), 152.03 (C6), 149.44 (C4), 140.85 (C8), 138.24 (C1’’),
137.23 (C4’), 132.36 (C1’), 129.90 (C3’ and C5’), 128.53 (C3’’ and
C5’’), 126.19 (C4’’), 123.03 (C2’ and C6’), 122.14 (C2’’ and C6’’),
120.44 (C5), 20.60 (R1 (Me)); 15N NMR (40 MHz, DMSO-d6)
235.24 (N1), 232.90 (N3), 171.92 (N9), 104.50 (NH) ; C18H14BrN5
N-(3-Bromophenyl)-9-(p-tolyl)-9H-purin-6-amine
9d:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
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10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
(380) calcd: C 56.86, H 3.71, N 18.42; found C 56.98, H 3.65, N
18.26.
H6’’, J 2.4Hz), 7.39 (d, 2H, H3’ and H5’, J 8.0 Hz), 7.25 (dd, 1H, H2’’,
J 2.4Hz and 8.4Hz), 6.87 (d, 1H, H5’’, J 8.8Hz), 3.74 (s, 3H, R2
(OMe)), 2.38 (s, 3H, R1 (Me)); 13C NMR (100 MHz, DMSO-d6)
152.58 (C2), 152.42 (C6), 149.00 (C4), 146.17 (C3’’), 143.76 (C4’’),
140.19 (C8), 137.09 (C4’), 132.99 (C1’’), 132.50 (C1’), 129.88 (C3’
and C5’), 122.95 (C2’ and C6’), 120.04 (C5), 112.36 (C5’’), 112.16
(C2’’), 109.84 (C6’’), 56.00 (R2 (OMe)), 20.59 (R1 (Me)); 15N NMR
(40 MHz, DMSO-d6) 234.00 (N1), 230.00 (N3), 171.97 (N9),
103.94 (NH) ; C19H17N5O2 (347) calcd: C 65.69, H 4.93, N 20.16;
found C 66.00, H 4.85, N 20.02.
N,9-Bis-p-tolyl-9H-purin-6-amine 9h: Prepared by the
general method A. N'-(4-cyano-1-(p-tolyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7a (0.10 g, 0.40 mmol), acetic acid (1 mL,
blue suspension), p-toluidine (1.1 eq., 46 mg), 1 hour. Product 9h
was isolated as a light beige solid (0.11 g, 84%); M.p. 188-189 ºC;
1H NMR (400 MHz, DMSO-d6) 8.86 (s, 1H), 8.68 (s, 1H, H8),
8.41 (s, 1H, H2), 7.82 (d, 2H, H2’’ and H6’’, J 8.4Hz), 7.77 (d, 2H,
H2’ and H6’, J 8.4Hz), 7.39 (d, 2H, H3’ and H5’, J 8.0Hz), 7.13 (d,
2H, H3’’ and H5’’, J 8.0Hz), 2.28 (s, 3H, R1 (Me)), 2.27 (s, 3H, R2
(Me)); 13C NMR (100 MHz, DMSO-d6) 152.54 (C2), 152.36 (C6),
149.14 (C4), 140.40 (C8), 137.11 (C1’), 136.98 (C1’’), 132.45 (C4’),
131.64 (C4’’), 129.88 (C3’ and C5’), 128.78 (C3’’ and C5’’), 122.96
(C2’ and C6’), 120.97 (C2’’ and C6’’), 120.15 (C5), 20.59 (R1 (Me)),
20.54 (R2 (Me)); 15N NMR (40 MHz, DMSO-d6) 234.74 (N1),
231.08 (N3), 171. 86 (N9), 104.53 (NH) ; C19H17N5 (315) calcd: C
72.36, H 5.43, N 22.21; found C 72.06, H 5.55, N 22.00.
3-((9-(p-Tolyl)-9H-purin-6-yl)amino)phenol 9l: Prepared by
the general method A. N'-(4-cyano-1-(p-tolyl)-1H-imidazol-5-yl)-
N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol), acetic acid
(1 mL, blue suspension), 3-aminophenol (1.1 eq., 44 mg), 2 hours.
Product 9l was isolated as a light grey solid (0.11 g, 72%); M.p.
1
253-254 ºC; H NMR (400 MHz, DMSO-d6) 9.81 (s, 1H, NH),
9.33 (bs, 1H, R2 (OH)), 8.69 (s, 1H, H8), 8.43 (s, 1H, H2), 7.78 (d,
2H, H2’ and H6’, J 8.0Hz), 7.54 (t, 1H, H6’’, J 2.4Hz), 7.39 (d, 2H,
H3’ and H5’, J 8.4Hz), 7.34 (dm, 1H, H2’’), 7.09 (t, 1H, H5’’, J 8.0Hz),
6.46 (dm, 1H, H4’’), 2.38 (s, 3H, R1 (Me)); 13C NMR (100 MHz,
DMSO-d6) 152.39 (C2), 152.00 (C6), 149.30 (C4), 140.86 (C8),
139.10 (C1’’), 137.22 (C4’), 132.35 (C1’), 131.43 (C3’ and C5’),
131.15 (C3’’ and C5’’), 123.02 (C2’ and C6’), 122.54 (C2’’ and C6’’),
120.38 (C5), 114.19 (C4’’), 20.59 (R1 (Me)); 15N NMR (40 MHz,
DMSO-d6) 235.24 (N1), 239.90 (N3), 171.92 (N9), 104.53 (NH) ;
C18H15N5O (317) calcd: C 68.13, H 4.76, N 22.07; found C 67.89,
H 4.55, N 22.16.
N-(3-Methoxyphenyl)-9-(p-tolyl)-9H-purin-6-amine
9i:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), m-anisidine (1.1 eq., 50 µL),
1 hour. Product 9i was isolated as a white solid (0.11 g, 79%);
M.p. 124-126 ºC; 1H NMR (400 MHz, DMSO-d6) 9.93 (s, 1H,
NH), 8.71 (s, 1H, H8), 8.46 (s, 1H, H2), 7.78 (d, 2H, H2’ and H6’, J
8.4 Hz), 7.69 (t, 1H, H6’’, J 2.0Hz), 7.60 (dm, 1H, H2’’), 7.39 (d, 2H,
H3’ and H5’, J 8.0Hz), 7.23 (t, 1H, H5’’, J 8.0Hz), 6.63 (dm, 1H, H4’’),
3.75 (s, 3H, R2 (OMe)), 2.38 (s, 3H, R1 (Me)); 13C NMR (100 MHz,
DMSO-d6) 159.36 (C3’’), 152.45 (C6), 152.26 (C2), 149.24 (C4),
140.80 (C1’’), 140.64 (C8), 137.17 (C4’), 132.40 (C1’), 129.89 (C3’
and C5’), 129.11 (C5’’), 123.00 (C2’ and C6’), 120.34 (C5), 113.04
(C2’’), 107.84 (C4’’), 106.71 (C6’’), 54.96 (R2 (OMe)), 20.59 (R1
(Me)); 15N NMR (40 MHz, DMSO-d6) 235.84 (N1), 232.28 (N3),
171.96 (N9), 105.73 (NH) ; C19H17N5O (321) calcd: C 68.87, H
5.17, N 21.13; found C 69.02, H 5.45, N 21.22.
4-((9-(p-Tolyl)-9H-purin-6-yl)amino)phenol 9m: Prepared by
the general method A. N'-(4-cyano-1-(p-tolyl)-1H-imidazol-5-yl)-
N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol), acetic acid
(1 mL, blue suspension), 4-aminophenol (1.1 eq., 44 mg), 2 hours.
Product 9m was isolated as a light orange solid (0.09 g, 62%);
1
M.p. 275-276 ºC; H NMR (400 MHz, DMSO-d6) 9.67 (s, 1H,
NH), 9.01 (bs, 1H, R2 (OH)), 8.63 (s, 1H, H8), 8.33 (s, 1H, H2),
7.77 (d, 2H, H2’ and H6’, J 8.4Hz), 7.63 (d, 2H, H2’’ and H6’’, J
8.8Hz), 7.39 (s, 2H, H3’ and H5’, J 8.8Hz), 6.73 (d, 2H, H3’’ and H5’’,
J 8.8Hz), 2.38 (s, 3H, R1 (Me)); 152.38 (C2), 151.94 (C6), 149.42
(C4), 141.39 (C1’’), 140.99 (C8), 137.24 (C4’), 132.32 (C1’), 130.32
(C5’’), 129.90 (C3’ and C5’), 124.99 (C4’’), 123.00 (C2’ and C6’),
122.65 (C6’’), 121.28 (C3’’ and C5), 119.30 (C2’’), 20.59 (R1 (Me));
15N NMR (40 MHz, DMSO-d6) 235.80 (N1), 234.01 (N3), 171.97
(N9), 104.42 (NH) ; C18H15N5O (319) calcd: C 68.13, H 4.76, N
22.07; found C 68.25, H 4.87, N 22.41.
N-(m-Tolyl)-9-(p-tolyl)-9H-purin-6-amine 9j: Prepared by the
general method A. N'-(4-cyano-1-(p-tolyl)-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7a (0.10 g, 0.40 mmol), acetic acid (1 mL,
blue suspension), m-anisidine (1.1 eq., 53 µL), 1 hour. Product 9j
was isolated as a light beige solid (0.10 g, 82%); M.p. 148-149 ºC;
1H NMR (400 MHz, DMSO-d6) 9.85 (s, 1H, NH), 8.69 (s, 1H, H8),
8.44 (s, 1H, H2), 7.80 (bs, 1H, H2’’), 7.78 (d, 2H, H2’ and H6’, J
8.4Hz), 7.74 (dm, 1H, H6’’), 7.39 (d, 2H, H3’ and H5’, J 8.4Hz), 7.21
(t, 1H, H5’’, J 8.0Hz), 6.87 (dt, 1H, H4’’, J 0.8Hz and 7.6Hz), 2.38
(s, 3H, R1 (Me)), 2.30 (s, 3H, R2 (Me)); 13C NMR (100 MHz,
DMSO-d6) 152.53 (C2), 152.36 (C6), 149.21 (C4), 140.52 (C8)
139.48 (C1’’), 137.48 (C3’’), 137.14 (C4’), 132.43 (C1’), 129.89 (C3’
and C5’), 128.22 (C5’’), 123.46 (C4’’), 122.98 (C2’ and C6’), 121.37
(C2’’), 120.25 (C5), 118.14 (C6’’), 21.32 (R2 (Me)), 20.60 (R1 (Me));
15N NMR (40 MHz, DMSO-d6) 235.25 (N1), 231.67 (N3), 171.93
(N9) ; C19H17N5 (315) calcd: C 72.36, H 5.43, N 22.21; found C
72.44, H 5.71, N 22.35.
N,9-Bis(4-methoxyphenyl)-9H-purin-6-amine 9n: Prepared
by the general method B. N'-(4-cyano-1-(4-methoxyphenyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7b (0.10 g, 0.37 mmol),
acetic acid (1 mL, beige suspension), p-anisidine (0.6 eq., 26 mg),
2 hours. Product 9n was isolated as a light brown solid (0.12 g,
92%); M.p. 240-242 ºC; 1H NMR (400 MHz, DMSO-d6) 9.79 (s,
1H, NH), 8.60 (s, 1H, H8), 8.35 (s, 1H, H2), 7.79 (d, 2H, H2’’ and
H6’’, J 9.2Hz), 7.77 (d, 2H, H2’ and H6’, J 9.2Hz), 7.13 (d, 2H, H3’
and H5’, J 9.2Hz), 6.91 (d, 2H, H3’’ and H5’’, J 9.2Hz), 3.82 (s, 3H,
R1 (OMe)), 3.74 (s, 3H, R2 (OMe)); 13C NMR (100 MHz, DMSO-
d6) 158.57 (C4’), 155.17 (C4’’), 152.57 (C2), 152.45 (C6), 149.16
(C4), 140.45 (C8), 132.52 (C1’’), 127.83 (C1’), 124.85 (C2’ and C6’),
122.77 (C2’’ and C6’’), 119.85 (C5), 114.63 (C3’ and C5’), 113.62 (C3’’
and C5’’), 55.52 (R1 (OMe)), 55.20 (R2 (OMe)); 15N NMR (40 MHz,
DMSO-d6) 233.55 (N1), 229.61 ( N3), 170.84 (N9), 103.01 (NH) ;
C19H17N5O2 (347) calcd: C 65.69, H 4.93, N 20.16; found C 65.62,
H 4.87, N 20.37.
2-Methoxy-5-((9-(p-tolyl)-9H-purin-6-yl)amino)phenol 9k:
Prepared by the general method A. N'-(4-cyano-1-(p-tolyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7a (0.10 g, 0.40 mmol),
acetic acid (1 mL, blue suspension), 5-amino-2-methoxyphenol
(1.1 eq., 60 mg), 1 hour. Product 9k was isolated as a light grey
solid (0.13 g, 92%); M.p. 198-199 ºC; 1H NMR (400 MHz, DMSO-
d6) 9.68 (s, 1H, NH), 8.94 (bs, 1H, R2 (OH)), 8.65 (s, 1H, H8),
8.37 (s, 1H, H2), 7.77 (d, 2H, H2’ and H6’, J 8.4Hz), 7.47 (d, 1H,
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10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
9-(4-Methoxyphenyl)-N-(m-tolyl)-9H-purin-6-amine
9o:
(H), J 1.2Hz and 7.2Hz), 7.09 (d, 2H, H2’ and H6’, J 2.0Hz), 6.92
(d, 2H, H3’’ and H5’’, J 8.8Hz), 3.74 (s, 3H, R2 (OMe)); 13C NMR
(100 MHz, DMSO-d6) 155.22 (C4’’), 152.70 (C2), 152.51 (C6),
149.08 (C4), 140.27 (C8), 134.96 (C1’), 133.63 (C3’’ and C5’’),
132.46 (C1’’), 129.54 (C3’ and C5’), 127.63 (R1 (H)), 123.12 (C2’ and
C6’), 122.84 (C2’’ and C6’’), 120.06 (C5), 55.21 (R2 (OMe)); 15N
NMR (40 MHz, DMSO-d6) 234.06 (N1), 229.96 (N3), 171.98 (N9),
103.34 (NH) ; C18H15N6O (317) calcd: C 68.13, H 4.76, N 22.07;
found C 68.03, H 4.79, N 21.82.
Prepared by the general method B. N'-(4-cyano-1-(4-
methoxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide
7b (0.10 g, 0.37 mmol), acetic acid (1 mL, beige suspension), m-
toluidine (0.6 eq., 23 µL), 3 hours. Product 9o was isolated as a
1
beige solid (0.09 g, 75%); M.p. 143-144 ºC; H NMR (400 MHz,
DMSO-d6) 9.83 (s, 1H, NH), 8.63 (s, 1H, H8), 8.42 (s, 1H, H2),
7.79 (bs, 1H, H2’’), 7.76 (d, 2H, H2’ and H6’, J 12.4Hz), 7.74 (dd,
1H, H6’’, J 1.2Hz and 8.0Hz), 7.20 (t, 1H, H5’’, J 7.6Hz), 7.13 (d,
2H, H3’ and H5’, J 9.2Hz), 6.86 (dd, 1H, H4’’, J 0.4Hz and 6.8Hz),
3.82 (s, 3H, R1 (OMe)), 2.30 (s, 3H, R2 (Me)); 13C NMR (100 MHz,
DMSO-d6) 158.64 (C4’), 152.53 (C2), 152.36 (C6), 149.33 (C4),
140.75 (C8), 139.50 (C1’’), 137.56 (C3’’), 128.29 (C1’), 127.78 (C5’’),
124.92 (C2’ and C6’), 123.50 (C4’’), 121.38 (C2’’), 120.11 (C5),
118.15 (C6’’), 114.66 (C3’ and C5’), 55.55 (R1 (OMe)), 21.35 (R2
(Me)); 15N NMR (40 MHz, DMSO-d6) 235.21 (N1), 231.13 (N3),
171.32 (N9) ; C19H17N5O (331) calcd: C 68.87, H 5.17, N 21.13;
found C 68.99, H 5.41, N 21.00.
9-Phenyl-N-(m-tolyl)-9H-purin-6-amine 9s: Prepared by the
general method B. N'-(4-cyano-1-phenyl-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7d (0.10 g, 0.42 mmol), acetic acid (1 mL,
beige suspension), m-toluidine (0.6 eq., 27 µL), 2 hours. Product
9s was isolated as a light brown solid (0.10 g, 79%); M.p. 129-130
ºC; 1H NMR (400 MHz, DMSO-d6) 9.87 (s, 1H, NH), 8.75 (s, 1H,
H8), 8.45 (s, 1H, H2), 7.92 (d, 2H, H2’ and H6’, J 7.6 Hz), 7.80 (bs,
1H, H2’’), 7.74 (bd, 1H, H6’’, J 7.6 Hz), 7.60 (t, 2H, H3’ and H5’, J
7.60 Hz), 7.46 (t, 1H, H5’’, J 7.2 Hz), 7.21 (t, 1H, H4’, J 7.6 Hz),
6.87 (bd, 1H, H4’’, J 7.2 Hz), 2.31 (s, 3H, R2 (Me)); 13C NMR (100
MHz, DMSO-d6) 152.60 (C2), 152.39 (C6), 149.22 (C4), 140.53
(C8), 139.46 (C1’’), 137.49 (C3’’), 134.89 (C1’), 129.52 (C3’ and C5’),
128.23 (C5’’), 127.64 (C4’), 123.49 (C4’’), 123.13 (C2’ and C6’),
121.42 (C2’’), 120.31 (C5), 118.17 (C6’’), 21.32 (R2 (Me)); 15N NMR
(40 MHz, DMSO-d6) 240.97 (N7), 235.49 (N1), 231.63 (N3),
172.24 (N9), 105.75 (NH); C18H15N6 (301) calcd: C 71.74, H 5.02,
N 23.24; found C 71.79, H 4.87, N 23.02.
4-(6-((4-Methoxyphenyl)amino)-9H-purin-9-yl)benzonitrile
9p: Prepared by the general method B. N'-(4-cyano-1-(4-
cyanophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7c
(0.10 g, 0.38 mmol), acetic acid (1 mL, dark suspension), p-
anisidine (0.6 eq., 28 mg), 2 hours. Product: 9p was isolated as a
1
light brown solid (0.13 g, 97%); M.p. 236-237 ºC; H NMR (400
MHz, DMSO-d6) 9.89 (s, 1H, NH), 8.87 (s, 1H, H8), 8.41 (s, 1H,
H2), 8.27 (d, 2H, H3’ and H5’, J 9.2 Hz), 8.08 (d, 2H, H2’ and H6’, J
9.2 Hz), 7.77 (d, 2H, H2’’ and H6’’, J 9.2 Hz), 6.91 (d, 2H, H3’’ and
H5’’, J 9.2 Hz), 3.71 (s, 3H, R2 (OMe)); 13C NMR (100 MHz, DMSO-
d6) 155.31 (C4’’), 152.95 (C2), 152.55 (C6), 149.00 (C4), 139.88
(C8), 138.85 (C1’), 132.27 (C1’’), 132.26 (C2’ and C6’), 122.94 (C3’
and C5’), 122.84 (C2’’ and C6’’), 120.24 (C5), 118.35 (R1 (CN)),
113.63 (C3’’ and C5’’), 109.64 (C4’), 55.21 (R2 (OMe)); 15N NMR (40
MHz, DMSO-d6) 243.26 (N7), 234.68 (N1), 229.63 (N3), 170.62
(N9), 104.12 (NH) ; C19H14N6O (342) calcd: C 66.66, H 4.12, N
24.55; found C 66.29, H 3.97, N 24.36.
9-(3-Bromophenyl)-N-(4-methoxyphenyl)-9H-purin-6-
amine 9t: Prepared by the general method B. N'-(1-(3-
bromophenyl)-4-cyano-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7e (0.10 g, 0.31 mmol), acetic acid (1 mL,
brown suspension), p-anisidine (0.6 eq., 23 mg), 2 hours. Product
9t was isolated as a light brown solid (0.11 g, 93%); M.p. 166-167
ºC; 1H NMR (400 MHz, DMSO-d6) 9.86 (s, 1H, NH), 8.78 (s, 1H,
H8), 8.40 (s, 1H, H2), 8.26 (t, 1H, H2’, J 2.0 Hz), 8.00 (dm, 1H, H4’),
7.78 (d, 2H, H2’’ and H6’’, J 8.8 Hz), 7.65 (dm, 1H, H6’), 7.55 (t, 1H,
H5’, J 8.4 Hz), 6.92 (d, 2H, H3’’ and H5’’, J 8.8 Hz), 3.74 (s, 3H, R2
(OMe)); 13C NMR (100 MHz, DMSO-d6) 155.27 (C4’’), 152.86
(C2), 152.52 (C6), 148.99 (C4), 140.10 (C8), 136.40 (C1’), 132.36
(C1’’), 131.44 (C5’), 130.26 (C6’), 125.36 (C2’), 122.89 (C2’’ and C6’’),
122.05 (C3’), 121.74 (C4’), 120.06 (C5), 113.65 (C3’’ and C5’’), 55.22
(R2 (OMe)); 15N NMR (40 MHz, DMSO-d6) 234.48 (N1), 229.35
(N3), 170.13 (N9), 103.52 (NH) ; C18H14BrN5O (396) calcd: C 54.56,
H 3.56, N 17.67; found C 54.88, H 3.84, N 17.98.
4-(6-(m-Tolylamino)-9H-purin-9-yl)benzonitrile
9q:
Prepared by the general method B. N'-(4-cyano-1-(4-
cyanophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7c
(0.10 g, 0.38 mmol), acetic acid (1 mL, dark suspension), m-
toluidine (0.6 eq., 25 µL), 2 hours. Produc 9q was isolated as a
1
light brown solid (0.09 g, 70%); M.p. 235-236 ºC; H NMR (400
MHz, DMSO-d6) 9.94 (s, 1H, NH), 8.91 (s, 1H, H8), 8.48 (s, 1H,
H2), 8.28 (d, 2H, H3’ and H5’, J 8.8 Hz), 8.09 (d, 2H, H2’ and H6’, J
8.8 Hz), 7.78 (bs, 1H, H2’’), 7.72 (bd, 1H, H6’’, J 7.6 Hz), 7.21 (t,
1H, H5’’, J 7.6 Hz), 6.88 (bd, 1H, H4’’, J 7.6 Hz), 2.30 (s, 3H, R2
(Me)); 13C NMR (100 MHz, DMSO-d6) 152.85 (C2), 152.46 (C6),
149.15 (C4), 140.14 (C8), 139.28 (C1’’), 138.79 (C1’), 137.52 (C3’’),
133.80 (C2’ and C6’), 128.24 (C5’’), 123.66 (C4’’), 122.87 (C3’ and
C5’), 121.52 (C1’’), 120.49 (C5), 118.34 (R1 (CN)), 118.28 (C6’’),
109.68 (C4’), 21.31 (R2 (Me)); 15N NMR (40 MHz, DMSO-d6)
243.34 (N7), 236.63 (N1), 231.21 (N3), 170.26 (N9), 106.73 (NH);
C19H14N6 (326) calcd: C 69.92, H 4.32, N 25.75; found C 69.59, H
4.48, N 25.85.
9-(3-Bromophenyl)-N-(m-tolyl)-9H-purin-6-amine
9u:
Prepared by the general method B. N'-(1-(3-bromophenyl)-4-
cyano-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7e (0.10 g,
0.31 mmol), acetic acid (1 mL, brown suspension), m-toluidine
(0.6 eq., 20 µL), 2 hours. Product 9u was isolated as a beige solid
1
(0.09 g, 78%); M.p. 169-170 ºC; H NMR (400 MHz, DMSO-d6)
9.90 (s, 1H, NH), 8.81 (s, 1H, H8), 8.47 (s, 1H, H2), 8.26 (t, 1H,
H2’, J 2.0 Hz) 8.00 (dm, 1H, H4’), 7.78 (bs, 1H, H2’’), 7.72 (bd, 1H,
H6’’, J 8.0 Hz), 7.65 (dm, 1H, H6’), 7.56 (t, 1H, H5’, J 8.0 Hz), 7.21
(t, 1H, H5’’, J 8.0 Hz), 6.87 (bs, 1H, H4’’, J 8.0 Hz), 2.30 (s, 3H, R2
(Me)); 13C NMR (100 MHz, DMSO-d6) 152.79 (C2), 152.42 (C6),
149.15 (C4), 140.37 (C8), 139.38 (C1’’), 137.56 (C3’’), 136.34 (C1’),
N-(4-Methoxyphenyl)-9-phenyl-9H-purin-6-amine
Prepared by the general method B. N'-(4-cyano-1-phenyl-1H-
9r:
imidazol-5-yl)-N,N-dimethylformimidamide 7d (0.10 g, 0.42 mmol), 131.44 (C5’), 130.32 (C6’), 128.29 (C5’’), 125.41 (C2’), 123.63 (C4’’),
acetic acid (1 mL, beige suspension), p-anisidine (0.6 eq., 31 mg),
2 hours. Product 9r was isolated as a light brown solid (0.10 g,
78%); M.p. 168-169 ºC; 1H NMR (400 MHz, DMSO-d6) 9.83 (s,
1H, NH), 8.71 (s, 1H, H8), 8.38 (s, 1H, H2), 7.78 (d, 2H, H2’’ and
H6’’, J 9.2Hz), 7.59 (t, 2H, H3’ and H5’, J 7.6Hz), 7.45 (tt, 1H, R1
122.05 (C3’), 121.79 (C4’),121.47 (C2’’), 120.31 (C5), 118.24 (C6’’),
21.34 (R2 (Me)); 15N NMR (40 MHz, DMSO-d6) 235.86 (N1),
230.35 (N3), 170.15 (N9) ; C18H14BrN5 (380) calcd: C 56.86, H 3.71,
N 18.42; found C 56.49, H 3.50, N 18.66.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
9-(3-Fluorophenyl)-N-(4-methoxyphenyl)-9H-purin-6-
amine 9v: Prepared by the general method B. N'-(4-cyano-1-(3-
fluorophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7g
(0.10 g, 0.39 mmol), acetic acid (1 mL, brown suspension), p-
anisidine (0.6 eq., 29 mg), 2 hours. Product 9v was isolated as a
light pink solid (0.12 g, 92%); M.p. 150-152 ºC; 1H NMR (400 MHz,
DMSO-d6) 9.86 (s, 1H, NH), 8.78 (s, 1H, H8), 8.40 (s, 1H, H2),
7.93 (dt, 1H, H2’, J 10.4 and 4.4 Hz), 7.87 (dm, 1H, H5’), 7.78 (d,
2H, H2’’ and H6’’, J 8.8 Hz), 7.64 (q, 1H, H5’, J 8.4 and 6.4 Hz), 7.30
(dm, 1H, H4’), 6.92 (d, 2H, H3’’ and H5’’, J 8.8 Hz), 3.74 (s, 3H, R2
(OMe)); 13C NMR (100 MHz, DMSO-d6) 162.21 (C3’, J 243.0 Hz),
155.26 (C4’’), 152.83 (C2), 152.52 (C6), 149.00 (C4), 140.08 (C8),
136.46 (C1’, J 10.0 Hz), 132.35 (C1’’), 131.30 (C5’, J 9.0 Hz),
122.88 (C2’’ and C6’’), 120.11 (C5), 118.60 (C6’, J 3.0 Hz), 114.19
(C2’, J 20.0 Hz), 113.62 (C3’’ and C5’’), 110.04 (C4’, J 26.0 Hz),
55.20 (R2 (OMe)); 15N NMR (40 MHz, DMSO-d6) 234.62 (N1),
229.37 (N3), 170.17 (N9), 103.71 (NH) ; C18H14FN5O (335) calcd:
C 64.47, H 4.21, N 20.88; found C 64.10, H 4.33, N 21.06.
7.97-7.93 (m, 2H, H2’ and H6’), 7.79 (bs, 1H, H2’’), 7.73 (d, 1H, H6’’,
J 7.6 Hz), 7.45 (t, 2H, H3’ and H5’, J 8.8 Hz), 7.21 (t, 1H, H5’’, J 7.6
Hz), 6.87 (d, 1H, H4’’, J 7.6 Hz), 2.30 (s, 3H, R2 (Me)); 13C NMR
(100 MHz, DMSO-d6) 161.02 (C4’, J 243.0 Hz), 152.63 (C2),
152.38 (C6), 149.23 (C4), 140.58 (C8), 139.43 (C1’’), 137.50 (C3’’),
131.25 (C1’, J 2.7 Hz), 128.23 (C5’’), 125.39 (C2’ and C6’, J 8.6 Hz),
123.50 (C4’’), 121.41 (C2’’), 120.14 (C5), 118.18 (C6’’), 116.21 (C3’
and C5’, J 22.8 Hz), 21.32 (R2 (Me)); 15N NMR (40 MHz, DMSO-
d6) 235.53 (N1), 230.54 (N3), 170.15 (N9), 105.70 (NH) ;
C18H14FN5 (319) calcd: C 67.70, H 4.42, N 21.93; found C 68.01,
H 4.39, N 21.84.
9-(4-Bromophenyl)-N-(4-methoxyphenyl)-9H-purin-6-
amine 9z: Prepared by the general method B. N'-(1-(4-
bromophenyl)-4-cyano-1H-imidazol-5-yl)-N,N-
dimethylformimidamide 7f (0.10 g, 0.31 mmol), acetic acid (1 mL,
dark red suspension), p-anisidine (0.6 eq., 23 mg), 3 hours.
Product 9z was isolated as a light brown solid (0.10 g, 78%); M.p.
1
210-211 ºC; H NMR (400 MHz, DMSO-d6) 9.85 (s, 1H, NH),
9-(3-Fluorophenyl)-N-(m-tolyl)-9H-purin-6-amine
9w:
8.74 (s, 1H, H8), 8.39 (s, 1H, H2), 7.93 (d, 2H, H3’ and H5’, J 9.2
Hz), 7.79 (d, 2H, H2’ and H6’, J 9.2 Hz), 7.78 (d, 2H, H2’’ and H6’’, J
9.2 Hz), 6.91 (d, 2H, H3’’ and H5’’, J 9.2 Hz), 3.74 (s, 3H, R2 (OMe));
13C NMR (100 MHz, DMSO-d6) 155.23 (C4’’), 152.77 (C2),
152.49 (C6), 148.94 (C4), 140.01 (C8), 134.31 (C1’), 132.40 (C2’
and C6’), 129.00 (C1’’), 124.83 (C3’ and C5’), 122.84 (C2’’ and C6’’),
120.15 (C5), 120.05 (C4’), 113.61 (C3’’ and C5’’), 55.20 (R2 (OMe));
15N NMR (40 MHz, DMSO-d6) 241.75 (N7), 23.53 (N1), 230.84
(N3), 170.17 (N9), 105.78 (NH) ; C18H14BrN5O (396) calcd: C 54.56,
H 3.56, N 17.67; found C 54.78, H 3.74, N 17.53.
Prepared by the general method B. N'-(4-cyano-1-(3-
fluorophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7g
(0.10 g, 0.39 mmol), acetic acid (1 mL, brown suspension), m-
toluidine (0.6 eq., 25 µL), 2 hours. Product 9w was isolated as a
1
beige solid (0.10 g, 83%); M.p. 148-149 ºC; H NMR (400 MHz,
DMSO-d6) 9.91 (s, 1H, NH), 8.82 (s, 1H, H8), 8.47 (s, 1H, H2),
7.94 (ddd, 1H, H4’, J 10.4 Hz), 7.88 (dm, 1H, H6’), 7.79 (bs, 1H,
H2’’), 7.73 (bd, 1H, H6’’, J 8.0 Hz), 7.65 (td, 1H, H5’, J 8.4 and 6.8
Hz), 7.31 (tdd, 1H, H2’, J 8.8, 3.2 and 0.8 Hz), 7.21 (t, 1H, H5’’, J
8.0 Hz), 6.88 (dm, 1H, H4’’), 2.31 (s, 3H, R2 (Me)); 13C NMR (100
MHz, DMSO-d6) 162.20 (C3’, J 243.0 Hz), 152.74 (C2), 152.41
(C6), 149.15 (C4), 140.34 (C8), 139.36 (C1’’), 136.38 (C1’, J 11.2
Hz), 131.30 (C5’, J 9.0 Hz), 130.50 (C3’’), 128.23 (C5’’), 123.58 (C4’’),
121.46 (C1’’), 120.35 (C5), 118.65 (C6’, J 3.0 Hz), 118.23 (C6’’),
114.23 (C2’, J 20.7 Hz), 110.08 (C4’, J 26.0 Hz), 21.31 (R2 (Me));
15N NMR (40 MHz, DMSO-d6) 241.78 (N7), 236.24 (N1), 230.75
(N3), 170.62 (N9), 105.73 (NH) ; C18H14FN5 (319) calcd: C 67.70,
H 4.42, N 21.93; found C 67.85, H 4.36, N 22.07.
9-(4-Bromophenyl)-N-(m-tolyl)-9H-purin-6-amine
9aa:
Prepared by the general method B. N'-(1-(4-bromophenyl)-4-
cyano-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7f (0.1 g,
0.31 mmol), acetic acid (1 mL, dark red suspension), m-toluidine
(0.6 eq., 20 µL), 3 hours. Product 9aa was isolated as a light grey
solid (0.09 g, 75%); M.p. 189-190 ºC; 1H NMR (400 MHz, DMSO-
d6) 9.90 (s, 1H, NH), 8.77 (s, 1H, H8), 8.45 (s, 1H, H2), 7.94 (d,
2H, H3’ and H5’, J 8.8 Hz), 7.80 (d, 2H, H2’ and H6’, J 8.8 Hz), 7.79
(bs, 1H, H2’’), 7.72 (bd, 1H, H6’’, J 8.0 Hz), 7.21 (t, 1H, H5’’, J 8.0
Hz), 6.87 (bd, 1H, H4’’, J 8.0 Hz), 1.89 (s, 3H, R2 (Me)); 13C NMR
(100 MHz, DMSO-d6) 152.68 (C2), 152.40 (C6), 149.09 (C4),
140.28 (C8), 139.39 (C1’’), 137.50 (C3’’), 134.26 (C1’), 132.40 (C2’
and C6’), 128.23 (C5’’), 124.86 (C3’ and C5’), 123.55 (C4’’), 121.43
9-(4-Fluorophenyl)-N-(4-methoxyphenyl)-9H-purin-6-
amine 9x: Prepared by the general method B. N'-(4-cyano-1-(4-
fluorophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7h
(0.10 g, 0.39 mmol), acetic acid (1 mL, brown suspension), p-
anisidine (0.6 eq., 29 mg), 2 hours. Product 9x was isolated as a
(C2’’), 120.31 (C5), 120.20 (C4’), 118.20 (C6’’), 21.32 (R2 (Me)); 15
N
1
light brown solid (0.13 g, 95%); M.p. 178-179 ºC; H NMR (400
NMR (40 MHz, DMSO-d6) 241.12 (N7), 235.53 (N1), 230.84 (N3),
170.17 (N9), 105.78 (NH) ; C18H14BrN5 (380) calcd: C 56.86, H
3.71, N 18.42; found C 57.11, H 3.88, N 18.52.
MHz, DMSO-d6) 9.83 (s, 1H, NH), 8.68 (s, 1H, H8), 8.37 (s, 1H,
H2), 7.95-7.92 (m, 2H, H2’ and H6’), 7.78 (d, 2H, H2’’ and H6’’, J 9.2
Hz), 7.44 (t, 2H, H3’ and H5’, J 8.8 Hz), 6.92 (d, 2H, H3’’ and H5’’, J
9.2 Hz), 3.74 (s, 3H, R2 (OMe)); 13C NMR (100 MHz, DMSO-d6)
155.21 (C4’’), 152.72 (C2), 152.49 (C6), 149.08 (C4), 140.31 (C8),
132.43 (C1’’), 131.34 (C4’), 131.31 (C1’), 125.34 (C2’ and C6’ J 9.0
Hz), 122.83 (C2’’ and C6’’), 119.90 (C5), 116.21 (C3’ and C5’, J 23.0
Hz), 113.61 (C3’’ and C5’’), 55.20 (R2 (OMe)); 15N NMR (40 MHz,
DMSO-d6) 233.96 (N1), 229.37 (N3), 170.16 (N9), 103.35 (NH) ;
C18H14FN5O (335) calcd: C 64.67, H 4.21, N 20.88; found C 64.57,
H 4.11, N 21.00.
9-(4-Ethoxyphenyl)-N-(4-methoxyphenyl)-9H-purin-6-
amine 9ab: Prepared by the general method B. N'-(4-cyano-1-
(4-ethoxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide
7i (0.2 g, 0.71 mmol), acetic acid (2 mL, dark brown suspension),
p-anisidine (0.6 eq., 52 mg), 3 hours. Product 9ab was isolated
as an off-white solid (0.24g, 94%); M.p. 219-220 ºC; 1H NMR (400
MHz, DMSO-d6) 9.79 (s, 1H, NH), 8.60 (s, 1H, H8), 8.35 (s, 1H,
H2), 7.89 (d, 2H, H2’ and H6’, J 9.2 Hz), 7.75 (d, 2H, H2’’ and H6’’, J
8.8 Hz), 7.12 (d, 2H, H3’ and H5’, J 9.2 Hz), 6.91 (d, 2H, H3’’ and
H5’’, J 8.8 Hz), 4.09 (q, 2H, R2 (OEt), J 6.8 and 7.2 Hz), 3.74 (s,
3H, R2 (OMe)), 1.35 (t, 3H, R2 (OEt), J 6.8 Hz); 13C NMR (100
MHz, DMSO-d6) 157.81 (C4’), 155.14 (C4’’), 152.55 (C2), 152.43
(C6), 149.13 (C4), 140.42 (C8), 132.52 (C1’’), 127.69 (C1’), 124.81
(C2’’ and C6’’), 122.76 (C2’ and C6’), 119.83 (C5), 115.06 (C3’ and
C5’), 113.60 (C3’’ and C5’’), 63.47 (R2 (OEt)), 55.20 (R2 (OMe)),
14.59 (R2 (OEt)); 15N NMR (40 MHz, DMSO-d6) 240.21 (N7),
9-(4-Fluorophenyl)-N-(m-tolyl)-9H-purin-6-amine
9y:
Prepared by the general method B. N'-(4-cyano-1-(4-
fluorophenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7h
(0.10 g, 0.39 mmol), acetic acid (1 mL, brown suspension), m-
toluidine (0.6 eq., 25 µL), 2 hours. Product 9y was isolated as a
1
beige solid (0.10 g, 80%); M.p. 178-179 ºC; H NMR (400 MHz,
DMSO-d6) 9.88 (s, 1H, NH), 8.71 (s, 1H, H8), 8.44 (s, 1H, H2),
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
233.22 (N1), 230.07 (N3), 170.86 (N9), 103.53 (NH) ; C20H19N5O2
(361) calcd: C 66.47, H 5.30, N 19.38; found C 66.19, H 5.22, N
19.51.
aminophenol (0.6 eq., 38 mg), 2 hours. Product 9af was isolated
as a beige solid (0.11 g, 56%); M.p. 290-291 ºC; H NMR (400
1
MHz, DMSO-d6) 9.63 (s, 1H, NH), 8.51 (s, 1H, H8), 8.31 (s, 1H,
H2), 7.62 (d, 2H, H2’’ and H6’’, J 8.8 Hz), 7.60 (d, 2H, H2’ and H6’, J
8.8 Hz), 6.93 (d, 2H, H3’ and H5’, J 8.8 Hz), 6.73 (d, 2H, H3’’ and
H5’’, J 8.8 Hz); 13C NMR (100 MHz, DMSO-d6) 152.53 (C2 and
C6), 149.08 (C4), 140.35 (C8), 125.04 (C2’ and C6’), 123.13 (C2’’
and C6’’), 119.69 (C5), 115.85 (C3’ and C5’), 114.82 (C3’’ and C5’’);
15N NMR (40 MHz, DMSO-d6) 239.80 (N7), 233.30 (N1), 229.10
(N3), 172.00 (N9), 102.80 (NH) ; C17H13N5O2 (319) calcd: C 63.94,
H 4.10, N 21.93; found C 63.87, H 4.26, N 21.81.
4-(6-((4-Methoxyphenyl)amino)-9H-purin-9-yl)phenol 9ac:
Prepared by the general method B. N'-(4-cyano-1-(4-
hydroxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7j
(0.06 g, 0.24 mmol), acetic acid (600 µL, light brown suspension),
p-anisidine (0.6 eq., 17 mg), 2 hours. Product 9ac was isolated as
a light brown solid (0.06 g, 73%); M.p. 292-293 ºC; 1H NMR (400
MHz, DMSO-d6) 9.80 (s, 1H, NH), 9.77 (s, 1H, R1 (OH)), 8.54 (s,
1H, H8), 8.34 (s, 1H, H2), 7.79 (s, 2H, H2’’ and H6’’), 7.61 (s, 2H, H2’
and H5’, J 8.8 Hz), 6.95 (d, 2H, H3’ and H5’, J 8.8 Hz), 6.91 (d, 2H,
H3’’ and H5’’, J 9.2 Hz), 3.74 (s, 3H, R2 (OMe)); 13C NMR (100 MHz,
DMSO-d6) 156.96 (C4’), 155.10 (C4’’), 152.47 (C2), 152.39 (C6),
149.15 (C4), 140.49 (C8), 132.54 (C1’’), 129.71 (C2’ and C6’),
126.32 (C1’), 125.02 (C2’’ and C6’’), 119.77 (C5), 115.80 (C3’ and
C5’), 113.58 (C3’’ and C5’’), 55.10 (R2 (OMe)); 15N NMR (40 MHz,
DMSO-d6) 239.80 (N7), 233.70 (N1), 230.40 (N3), 171.90 (N9),
103.20 (NH) ; C18H15N5O2 (333) calcd: C 64.86, H 4.54, N 21.01;
found C 65.07, H 4.56, N 20.68.
3-(6-((4-Methoxyphenyl)amino)-9H-purin-9-yl)phenol 9ag:
Prepared by the general method B. N'-(4-cyano-1-(3-
hydroxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7k
(0.06 g, 0.24 mmol), acetic acid (1 mL, grey suspension), p-
anisidine (0.6 eq., 18 mg), 2 hours. Product 9ag was isolated as
a light brown solid (0.03 g, 39%); M.p. 235-236 ºC; 1H NMR (400
MHz, DMSO-d6) 9.81 (s, 1H, NH), 9.91 (s, 1H, R1 (OH)), 8.66 (s,
1H, H8), 8.32 (s, 1H, H2), 7.79 (d, 2H, H2’’ and H6’’, J 9.2 Hz), 7.38-
7.34 (m, 2H, H2’ and H5’), 7.29 (dm, 1H, H6’), 6.92 (d, 2H, H3’’ and
H5’’, J 9.2 Hz), 6.85 (dm, 1H, H4’), 3.74 (s, 3H, R2 (OMe)); 13C NMR
(100 MHz, DMSO-d6) 158.17 (C3’), 155.19 (C4’’), 152.61 (C2),
152.48 (C6), 148.99 (C4), 140.25 (C8), 135.92 (C1’), 132.45 (C1’’),
130.31 (C5’), 122.82 (C2’’ and C6’’), 120.08 (C5), 114.57 (C4’),
113.61 (C3’’ and C5’’), 113.32 (C6’), 110.13 (C2’), 55.19 (R2 (OMe));
15N NMR (40 MHz, DMSO-d6) 240.04 (N7), 233.91 (N1), 230.41
(N3), 172.21 (N9), 103.37 (NH) ; C18H15N5O2 (333) calcd: C 64.86,
H 4.54, N 21.01; found C 65.05, H 4.83, N 20.66.
9-(4-Hydroxyphenyl)-N-(m-tolyl)-9H-purin-6-amine 9ad:
Prepared by the general method B. N'-(4-cyano-1-(4-
hydroxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7j
(0.06 g, 0.24 mmol), acetic acid (600 µL, beige suspension), m-
toluidine (0.6 eq., 15 µL), 2 hours. Product 9ad was isolated as a
1
beige solid (0.07 g, 87%); M.p. 253-254 ºC; H NMR (400 MHz,
3-(6-(m-Tolylamino)-9H-purin-9-yl)phenol 9ah: Prepared by
the general method B. N'-(4-cyano-1-(3-hydroxyphenyl)-1H-
imidazol-5-yl)-N,N-dimethylformimidamide 7k (0.06 g, 0.24 mmol),
acetic acid (1 mL, grey suspension), m-toluidine (0.6 eq., 16 µL),
2 hours. Product 9ah was isolated as a light brown solid (0.04 g,
45%); M.p. 235-236 ºC; 1H NMR (400 MHz, DMSO-d6) 9.92 (s,
1H, R1 (OH)), 9.85 (s, 1H, NH), 8.70 (s, 1H, H8), 8.45 (s, 1H, H2),
7.80 (bs, 1H, H2’’), 7.73 (d, 1H, H6’’, J 8.0 Hz), 7.39-7.37 (m, 2H,
H2’ and H5’), 7.21 (t, 1H, H5’’, J 8.0 Hz), 7.29 (dm, 1H, H6’), 6.88-
6.84 (m, 2H, H3’ and H4’’), 2.31 (s, 3H, R2 (Me)); 13C NMR (100
MHz, DMSO-d6) 158.18 (C3’),152.54 (C2), 152.38 (C6), 149.15
(C4), 140.51 (C8), 139.46 (C1’’), 137.50 (C3’’), 135.86 (C1’), 130.32
(C5’), 128.23 (C5’’), 123.49 (C4’’), 121.41 (C2’’), 120.35 (C5), 118.17
DMSO-d6) 9.81 (s, 2H, NH and R1 (OH)), 8.57 (s, 1H, H8), 8.41
(s, 1H, H2), 7.80 (bs, 1H, H2’’), 7.74 (d, 1H, H6’’, J 8.0 Hz), 7.62 (d,
2H, H2’ and H6’, J 8.8 Hz), 7.20 (t, 1H, H5’’, J 8.0 Hz), 6.94 (d, 2H,
H3’ and H5’, J 8.8 Hz), 6.86 (d, 1H, H4’’ J 8.0 Hz), 2.30 (s, 3H, R2
(Me)); 13C NMR (100 MHz, DMSO-d6) 157.01 (C4’), 152.41 (C2),
152.31 (C6), 149.33 (C4), 140.78 (C8), 139.54 (C1’’), 137.48 (C3’’),
128.33 (C5’’), 126.27 (C1’), 125.07 (C2’ and C6’), 123.41 (C4’’),
121.32 (C2’’), 120.04 (C5), 118.10 (C6’’), 115.82 (C3’ and C5’), 21.33
(R2 (Me)); 15N NMR (40 MHz, DMSO-d6) 239.40 (N7), 235.00
(N1), 231.70 (N3), 171.50 (N9), 105.00 (NH) ; C18H15N5O (317)
calcd: C 68.13, H 4.76, N 22.07; found C 68.01, H 4.88, N 21.87.
3-((9-(4-Hydroxyphenyl)-9H-purin-6-yl)amino)phenol 9ae:
Prepared by the general method B. N'-(4-cyano-1-(3-
hydroxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7j
(0.15 g, 0.59 mmol), acetic acid (1.5 mL, light green suspension),
3-hydroxyaniline (0.6 eq., 38 mg), 2 hours. Product 9ae was
isolated as a beige solid (0.14 g, 73%); M.p. 315-317 ºC; 1H NMR
(400 MHz, DMSO-d6) 9.77 (s, 1H, NH), 9.38 (s, 1H, R2 (OH)),
9.33 (s, 1H, R1 (OH)), 8.57 (s, 1H, H8), 8.40 (s, 1H, H2), 7.61 (d,
2H, H2’ and H6’, J 8.8 Hz), 7.53 (t, 1H, H2’’, J 2.0 Hz), 7.33 (dm, 1H,
H4’’), 7.08 (t, 1H, H5’’, J 8.0 Hz), 6.94 (d, 2H, H3’ and H5’, J 8.8 Hz),
6.45 (dm, 1H, H6’’); 13C NMR (100 MHz, DMSO-d6) 157.39 (C4’’),
157.04 (C4’), 152.38 (C2), 152.29 (C6), 149.34 (C4), 140.81 (C8),
140.66 (C1’’), 129.00 (C5’’), 126.28 (C1’), 125.13 (C2’ and C6’),
120.08 (C5), 115.85 (C3’ and C5’), 111.75 (C6’’), 109.87 (C4’’),
107.97 (C2’’); 15N NMR (40 MHz, DMSO-d6) 239.45 (N7), 235.41
(N1), 231.61 (N3), 171.58 (N9), 105.71 (NH) ; C17H13N5O2 (319)
calcd: C 63.94, H 4.10, N 21.93; found C 64.13, H 4.01, N 22.06.
(C6’’), 114.61 (C4’), 113.35 (C6’), 110.15 (C2’), 21.32 (R2 (Me)); 15
N
NMR (40 MHz, DMSO-d6) 240.85 (N7), 235.52 (N1), 231.55 (N3),
172.19 (N9), 105.38 (NH) ; C18H15N5O (317) calcd: C 68.14, H
4.76, N 22.07; found C 67.98, H 4.69, N 21.97.
General procedure for acetylation of the phenolic
hydroxyl groups: Acetic anhydride and triethylamine were
added to a brown suspension of 4-(6-(arylamino)-9H-purin-9-
yl)phenol 9(ac-af) in acetonitrile. The mixture was stirred at room
temperature for 2-3 days. After partial removal of the solvent in
the rotary evaporator, addition of cold diethyl ether led to a solid
that was filtered, washed with cold diethyl ether and identified as
the 4-(6-(arylamino)-9H-purin-9-yl)phenyl acetate derivative 10(a-
d).
4-(6-((4-Methoxyphenyl)amino)-9H-purin-9-yl)phenyl
acetate
10a:
4-(6-((4-methoxyphenyl)amino)-9H-purin-9-
yl)phenol 9ac (0.04 g, 0.12 mmol), acetic anhydride (1.1 eq., 12
µL), triethylamine (1 eq., 17 µL), acetonitrile (0.5 mL, brown
suspension), 2 days. Product 10a was isolated as an off-white
solid (0.04 g, 76%); M.p. 195-197 ºC; 1H NMR (400 MHz, DMSO-
4-((9-(4-Hydroxyphenyl)-9H-purin-6-yl)amino)phenol 9af:
Prepared by the general method B. N'-(4-cyano-1-(4-
hydroxyphenyl)-1H-imidazol-5-yl)-N,N-dimethylformimidamide 7j
(0.15 g, 0.59 mmol), acetic acid (1.5 mL, brown suspension), 4-
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
d6) 9.84 (s, 1H, NH), 8.71 (s, 1H, H8), 8.54 (s, 1H, H2), 7.94 (d,
2H, H2’ and H6’, J 9.2 Hz), 7.79 (d, 2H, H2’’ and H6’’, J 9.2 Hz), 7.36
(d, 2H, H3’ and H5’, J 9.2 Hz), 6.92 (d, 2H, H3’’ and H5’’, J 9.2 Hz),
132.35 (C1’), 124.46 (C2’ and C6’), 123.01 (C3’ and C5’), 121.89 (C2’’
and C6’’), 121.67 (C3’’ and C5’’), 120.16 (C5), 20.86 (Me), 20.84
(Me); 15N NMR (40 MHz, DMSO-d6) 240.94 (N7), 234.69 (N1),
3.74 (s, 3H, R2 (OMe)), 2.30 (s, 3H, R1 (OAc)); 13C NMR (100 MHz, 231.53 (N3), 170.82 (N9), 104.36 (NH) ; C21H17N5O4 (403) calcd:
DMSO-d6) 155.21 (C4’’), 152.71 (C2), 152.49 (C6), 149.47 (C4’),
149.08 (C4), 140.32 (C1’), 140.29 (C8), 132.42 (C1’’), 124.33 (C2’
and C6’), 122.96 (C2’’ and C6’’), 122.84 (C3’ and C5’), 119.95 (C5),
114.61 (C4’), 113.60 (C3’’ and C5’’); 15N NMR (40 MHz, DMSO-d6)
241.00 (N7), 233.33 (N1), 230.00 (N3), 170.66 (N9), 103.47 (NH) ;
C20H17N5O3 (375) calcd: C 63.99, H 4.56, N 18.66; found C 64.14,
H 4.77, N 18.75.
C 62.53, H 4.25, N 17.36; found C 62.33, H 4.36, N 17.26.
Biology
Cell lines and culture conditions: Two human breast cancer
cell lines Hs578T (basal-like subtype) and MCF-7 (luminal A
subtype) and a normal breast cell line MCF-10A, were obtained
from ATCC (American Type Culture Collection). The two cancer
cell lines were cultured in Dulbecco’s modified Eagle medium,
4.5g/l glucose (DMEM, Gibco) supplemented with 10% heating
activated Fetal Bovine Serum (FBS, Gibco) and 1% antibiotic
solution (Penicillin-Streptomycin, Gibco). The non-neoplastic cell
line was cultured in Dulbecco's Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F12, Gibco) supplemented with 5% heating
activated Fetal Bovine Serum (FBS, Gibco), 1% antibiotic solution
4-(6-(m-Tolylamino)-9H-purin-9-yl)phenyl acetate 10b: 9-
(4-hydroxyphenyl)-N-(m-tolyl)-9H-purin-6-amine 9ad (0.04 g,
0.13 mmol), acetic anhydride (1.1 eq., 13 µL), triethylamine (1 eq.,
18 µL), acetonitrile (0.5 mL, brown suspension), 2 days. Product
10b was isolated as an off-white solid (0.03 g, 71%); M.p. 194-
196 ºC; 1H NMR (400 MHz, DMSO-d6) 9.88 (s, 1H, NH), 8.74 (s,
1H, H8), 8.45 (s, 1H, H2), 7.95 (d, 2H, H3’ and H5’, J 8.8 Hz), 7.80
(bs, 1H, H2’’), 7.73 (d, 2H, H6’’, J 8.0 Hz), 7.37 (d, 2H, H2’ and H6’,
J 8.8 Hz), 7.21 (t, 1H, H5’’, J 8.0 Hz), 6.87 (s, 2H, H4’’, J 8.0 Hz),
2.31 (s, 6H, R1 (OAc) and R2 (Me)); 13C NMR (100 MHz, DMSO-
d6) 169.21 (R1 (OAc)), 152.63 (C2), 152.39 (C6), 149.51 (C4’),
149.24 (C4), 140.58 (C8), 139.44 (C1’’), 137.51 (C3’’), 132.36 (C1’),
128.24 (C5’’), 124.37 (C2’ and C6’), 123.53 (C4’’), 122.97 (C3’ and
C5’), 121.43 (C2’’), 120.11 (C5), 118.20 (C6’’), 21.32 (R2 (Me)),
20.84 (R1 (OAc)); 15N NMR (40 MHz, DMSO-d6) 240.93 (N7),
235.59 (N1), 230.90 (N3), 170.80 (N9), 105.35 (NH) ; C20H17N5O2
(359) calcd: C 66.84, H 4.77, N 19.89; found C 66.51, H 4.84, N
20.00.
(Penicillin-Streptomycin,
Gibco),
1%
steroid
hormone
(Hydrocortisone, Sigma-Aldrich), 0.1% peptide hormone (Insulin,
Sigma-Aldrich) and 0.01% protein complex (Cholera Toxin,
Gibco). Cells were grown in a humidified incubator at 37C and
5% CO2. For all assays, DMSO (Dimethyl Sulfoxide, Sigma-
Aldrich) controls were used.
Cell viability assays - Sulforhodamine B assay: To
calculate the IC50 value, cells were plated in 96-multiwell culture
plates at 3000 cells per 100 µL density per well (MCF-7, Hs578T
and MCF-10A) and allowed to adhere for 18 to 20 hours
(overnight) in a complete DMEM (MCF-7 and Hs578T) or
DMEM/F12 (MCF-10A) medium. Cells were then treated with 7
different concentrations (from 0.1 to 40 µM or 5 to 60 µM) of
compounds in fresh medium and incubated for 72h. Controls were
performed with 0.3% of DMSO (compound vehicle). The
compound cytotoxic effect was evaluated by the Sulforhodamine
B assay (SRB, TOX-6, Sigma-Aldrich). After cell incubation with
the different compound concentrations, culture medium was
removed and cells were fixed with cold 10% Trichloroacetic Acid
(TCA) for 1h at 4°C. Plates were rinsed tree times with distilled
water to eliminate TCA excess and then were dried overnight at
room temperature. Cells were stained with 0.4% Sulforhodamine
B solution during 30 minutes and, after the dying period, plates
were again rinsed 3 times, with 1% acetic acid until the
unincorporated dye was totally removed. After drying overnight,
the fixed Sulforhodamine B was solubilized using a 10M Tris
Base Solution, stirring in the plate shaker for 20 to 30 minutes at
room temperature until a homogenous colour was obtained.
Spectrophotometric measurement of absorbance was read at
490nm, using 690nm as background absorbance (Tecan Infinite
M200). The results were expressed as percentage relative to the
control. The concentration of each compound that decreased the
number of viable cells to 50% (IC50) relative to control was
calculated using GraphPad Prism 6 software. IC50 values were
obtained from three independent experiments (at least), each one
in triplicate, applying a sigmoidal dose-response (variable slope)
non-linear regression, after logarithmic transformation.
3-((9-(4-Acetoxyphenyl)-9H-purin-6-yl)amino)phenyl
acetate
10c:
3-((9-(4-hydroxyphenyl)-9H-purin-6-
yl)amino)phenol 9ae (0.07 g, 0.22 mmol), acetic anhydride (3 eq.,
62 µL), triethylamine (2 eq., 61 µL), acetonitrile (0.7 mL, brown
suspension), 3 days. Product 10c was isolated as an off-white
solid (0.08 g, 79%); M.p. 220-221 ºC; 1H NMR (400 MHz, DMSO-
d6) 10.16 (s, 1H, NH), 8.77 (s, 1H, H8), 8.49 (s, 1H, H2), 7.95 (d,
2H, H2’ and H6’, J 9.2 Hz), 7.92 (t, 1H, H5’’, J 2.0 Hz), 7.80 (dm, 1H,
H6’’), 7.37 (d, 2H, H3’ and H5’, J 9.2 Hz), 7.33 (s, 1H, H2’’), 6.81 (dm,
1H, H4’’), 2.30 (s, 3H, R1 (OAc) or R2 (OAc)), 2.28 (s, 3H, R1 (OAc)
or R2 (OAc)); 13C NMR (100 MHz, DMSO-d6) 169.27 (R1 (OAc)
or R2 (OAc)), 169.25 (R1 (OAc) or R2 (OAc)), 152.57 (C2), 152.15
(C6), 150.51 (C3’’), 149.61 (C4’), 149.41 (C4), 140.97 (C8), 140.77
(C1’’), 132.30 (C1’), 129.14 (C2’’), 124.50 (C2’ and C6’), 123.03 (C3’
and C5’), 120.34 (C5), 117.89 (C6’’), 115.98 (C4’’), 113.82 (C5’’),
20.92 (R1 (OAc) or R2 (OAc)), 20.87 (R1 (OAc) or R2 (OAc)); 15N
NMR (40 MHz, DMSO-d6) 240.67 (N7), 235.55 (N1), 232.38 (N3),
174.94 (N9), 105.22 (NH) ; C21H17N5O4 (403) calcd: C 62.53, H
4.25, N 17.36; found C 62.64, H 4.02, N 17.48.
4-((9-(4-Acetoxyphenyl)-9H-purin-6-yl)amino)phenyl
acetate
10d:
4-((9-(4-hydroxyphenyl)-9H-purin-6-
yl)amino)phenol 9af (0.07 g, 0.22 mmol), acetic anhydride (3 eq.,
62 µL), triethylamine (2 eq., 61 µL), acetonitrile (0.7 mL, brown
suspension), 3 days. Product 10d was isolated as an off-white
solid (0.07 g, 76%); M.p. 224-225 ºC; 1H NMR (400 MHz, DMSO-
d6)  10.08 (s, 1H, NH), 8.75 (s, 1H, H8), 8.44 (s, 1H, H2), 7.95 (d,
2H, H2’ and H6’ or H2’’ and H6’’, J 9.2 Hz), 7.94 (d, 2H, H2’ and H6’
or H2’’ and H6’’, J 9.2 Hz), 7.37 (d, 2H, H3’ and H5’, J 9.2 Hz), 7.09
(d, 2H, H3’’ and H5’’, J 9.2 Hz), 2.30 (s, 3H, R1 (OAc) or R2 (OAc)),
2.25 (s, 3H, R1 (OAc) or R2 (OAc));; 13C NMR (100 MHz, DMSO-
d6) 169.41 (OAc), 169.26 (OAc), 152.62 (C2), 152.29 (C6),
149.58 (C4’), 149.33 (C4), 145.71 (C4’’), 140.75 (C8), 137.09 (C1’’),
Selectivity index (SI):[37] SI value was calculated using the IC50
values of all compounds for MCF7 and MCF10A cell lines, using
the following mathematical formula: SI = (IC50normal cell line -
IC50tumor cell line)/ IC50tumor cell line. For SI values ˃1,
cytotoxicity for cancer cell line is higher than for normal cell line.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800629
European Journal of Organic Chemistry
FULL PAPER
Protein extraction and Western blot: MCF-7 cells were
grown in T25 flasks and, at 70-80% confluence, cells were treated
with compounds at the respective IC50, ½ IC50 or 0.5% DMSO
(controls) for 72 hours. After treatment, adherent and floating cells
were collected by scraping and centrifuged at 2000 rpm for 10
min, at 4 °C. The pallet was resuspended in PBS and again
centrifuged (1200 rpm; 5 min, 4 ºC). The supernatant was
discarded and the pellet was suspended in lysis buffer (50mM Tris
pH 7.6-8, 150mM NaCl, 5mM EDTA, 1mM Na3VO4, 10 M NaF,
1% NP-40, 1 % Triton-X100 and 1/7 protease inhibitor cocktail
(Roche Applied Sciences)) and incubated for 20 minutes on ice.
Lysates were centrifuged at 14000 rpm for 15 minutes at 4C and
the supernatants collected for protein concentration determination
using the DC Protein Assay Kit (BioRad). Fifty µg of total protein
of each sample were separated on 12 or 15% polyacrylamide gel
(100V for 90 minutes) and transferred to a nitrocellulose
membrane (100V for 60 minutes). Membranes were blocked with
5% milk in 1X TBS for 60 minutes before overnight incubation with
the specific primary antibodies at 4 C (Apoptosis Antibody
Sampler Kit - Cell Signaling (#9915): rabbit anti-PARP antibody
(#9542), 1:1000 5% milk; mouse anti-caspase-9 antibody
(#9508), 1:1000 5% milk; rabbit anti-caspase-3 antibody (#9665),
1:1000 5% milk and mouse anti--Actin antibody (Santa Cruz
Biotechnology, #E1314), 1:500 5% milk). After washing 5 minutes
(2 times) and more 15 minutes (1 time) with 0.1% Tween 20, blots
were incubated for 1 hour with the respective secondary
antibodies at room temperature (Apoptosis Antibody Sampler Kit
- Cell Signaling (#9915): anti-rabbit IgG-HRP (7074) and anti-
mouse IgG-HRP (7076) secondary antibodies, 1:2000 5% milk,
Cell Signaling). After washing 5 minutes (twice) and further 15
minutes (once) with TBS/0.1% Tween 20, immunoreactive bands
were detected with chemiluminescent SuperSignal West Femto
Kit (Pierce, Thermo Scientific) on ChemiDoc XRS+system
(BioRad). Quantification of immunoblot was performed by
Quantity One 4.6.9.
Costa (SFRH/BPD/79609/2011) and the PhD grant of Olívia
Pontes (SFRH/BD/128850/2017).
Keywords: Adenine derivatives • 5-amino-4-cyanoimidazoles •
cyclization • breast cancer • MCF-7 and Hs578t
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Acknowledgements
We acknowledge the financial support from University of Minho,
Fundação para a Ciência e a Tecnologia (FCT) and FEDER-
COMPETE through Centro de Química (UID/QUI/ 00686/2013
and UID/QUI/0686/2016). The NMR spectrometer Bruker Avance
III 400 is part of the National NMR Network (RNRMN) and was
purchased within the framework of the National Program for
Scientific Re-equipment, contract REDE/1517/RMN/2005 with
funds from POCI 2010 (FEDER) and FCT. This work was also
developed under the scope of the project NORTE-01-0145-
FEDER- 000013, supported by the Northern Portugal Regional
Operational Programme (NORTE 2020) under the Portugal
Partnership Agreement, through the European Regional
Development Fund (FEDER), and through the Competitiveness
Factors Operational Programme (COMPETE) and by National
funds, through FCT, under the scope of the project POCI-01-
0145-FEDER-007038. We also acknowledge the financial
support by FCT through the postdoctoral grant awarded to Marta
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Adenine derivatives
Pedro Figueiredo,[a] Marta Costa* [b,c]
Olívia Pontes,[b,c] Fátima Baltazar[b,c] and
Fernanda Proença* [a]
Page No. – Page No.
A general and efficient method was developed for the regioselective synthesis of 6-
arylaminopurines, from N-formylated 5-amino-4-cyanoimidazoles and substituted
anilines in acetic acid. Five of these compounds proved to be highly active in
cancer cell models MCF-7 and Hs578t and non-toxic to the non-neoplastic cell line
MCF-10A.
Adenine derivatives: promising
candidates for breast cancer
treatment
Keywords
Adenine derivatives
5-amino-4-cyanoimidazoles
cyclization
breast cancer
MCF-7 and Hs578t
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