Purine and purine isostere derivatives of ferrocene: An evaluation of ADME, antitumor and electrochemical properties

Article abstract of DOI:10.3390/molecules25071570

Novel purine and purine isosteres containing a ferrocene motif and 4,1-disubstituted (11a?11c, 12a?12c, 13a?13c, 14a?14c, 15a?15c, 16a, 23a?23c, 24a?24c, 25a?25c) and 1,4-disubstituted (34a?34c and 35a?35c) 1,2,3-triazole rings were synthesized. The most

Full text of DOI:10.3390/molecules25071570

molecules
Article
Purine and Purine Isostere Derivatives of Ferrocene:
An Evaluation of ADME, Antitumor and
Electrochemical Properties
1
1
1
2
3
Valentina Rep , Martina Piškor , Helena Šimek , Petra Mišeti c´ , Petra Grb cˇ i c´ ,
Jasna Padovan 2 , Vesna Gabelica Markovi c´ , Dijana Jadreško , Krešimir Paveli c´ ,
Sandra Kraljevi c´ Paveli c´ 3 and Silvana Rai c´ -Mali c´
4
5
6
1,
*
1
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
Zagreb 10000, Croatia; vrep@fkit.hr (V.R.); mpiskor@fkit.hr (M.P.); hsimek@fkit.hr (H.Š.)
Fidelta d.o.o., Zagreb 10000, Croatia; Petra.Misetic@fidelta.eu (P.M.); Jasna.Padovan@fidelta.eu (J.P.)
Department of Biotechnology, Center for High-Throughput Technologies, University of Rijeka, Rijeka 51000,
Croatia; petra.grbcic@biotech.uniri.hr (P.G.); sandrakp@biotech.uniri.hr (S.K.P.)
International Relations Office, Faculty of Chemical Engineering and Technology, University of Zagreb,
Zagreb 10000, Croatia; vesnagm@fkit.hr
Division for Marine and Environmental Research, Ruđer Boškovi c´ Institute, Zagreb 10000, Croatia;
djadresko@irb.hr
Faculty of Medicine, Juraj Dobrila University of Pula, Pula 52100, Croatia; pavelic@unipu.hr
Correspondence: sraic@fkit.hr; Tel.: +385-1-4597213
2
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5
6
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Paula B. Andrade
Received: 13 March 2020; Accepted: 26 March 2020; Published: 29 March 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Novel purine and purine isosteres containing a ferrocene motif and 4,1-disubstituted
(
(
11a−11c
34a−34c and 35a−35c) 1,2,3-triazole rings were synthesized. The most potent cytotoxic effect on
M),
M) ferrocenylalkyl derivatives. The N-9
,
12a−12c
,
13a−13c
,
14a−14c
,
15a−15c
,
16a
,
23a−23c
,
24a−24c
,
25a−25c) and 1,4-disubstituted
colorectal adenocarcinoma (SW620) was exerted by the 6-chloro-7-deazapurine 11c (IC50 = 9.07
-chloropurine 13a (IC50 = 14.38 M) and 15b (IC50 = 15.50
µ
6
µ
µ
isomer of 6-chloropurine 13a containing ferrocenylmethylene unit showed a favourable in vitro
physicochemical and ADME properties including high solubility, moderate permeability and good
metabolic stability in human liver microsomes.
Keywords: purine; ferrocene; cytostatic activity; permeability; microsomal stability
1
. Introduction
Nucleoside analogues were among the first chemotherapeutic agents that were introduced for
the medical treatment of cancer [
of importance in recent years for combating cancer of different types, usually in combination with
other agents [ ]. They were shown to interact with various biological targets, such as cellular
kinases, ribonucleotide reductase, and cellular polymerases in order to produce cytotoxic effects [ ].
1,2]. Cytotoxic nucleobase-derived compounds have gained a lot
3–5
6,7
The interest in metal complexes of ferrocene-based ligands is due to their favourable physicochemical
properties and reversible redox properties that enable ferrocene derivatives to be used as excellent
candidates for drug development [
is shown to be related to cytotoxic ferrocenium cation and generation of reactive oxygen species
ROS), especially hydroxyl radicals that can be produced under oxidizing conditions through a
Fenton-type reaction, whose presence may lead to the damage of the cells [10 11]. To enhance
8,9]. The antitumor activity of ferrocene-containing compounds
(
,
the cytotoxic activity, the ferrocenyl moiety has been successfully incorporated into molecules that
have biological activity [12]. In this regard, ferrocifen, obtained by replacing the phenyl group
Molecules 2020, 25, 1570; doi:10.3390/molecules25071570
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1570
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in tamoxifen with ferrocene, exhibits a strong effect against both hormone-dependent (ERα+) and
−
hormone-independent (ER
α
) breast cancer cell types in contrast to tamoxifen, which is only active
+
against ER
anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant
to existing anticancer therapy [15]. It negatively regulates hypoxia-inducible factor-1 (HIF-1
and showed to be effective under starved and hypoxic conditions frequently observed in advanced
α cells [13,14]. Furthermore, the antimalarial drug ferroquine was recently found to enhance
α
α)
solid cancers. The anticancer potential of organometallic nucleoside analogues was explored [16
–18
]
and showed that nucleoside analogues of ferrocene exhibited potent antineoplastic activity [19
–
22]. Combined application of ferrocenylalkyl nucleobase with anticancer drug cyclophosphamide
demonstrated therapeutic synergism of antitumor activity against Lewis lung carcinoma (LLC) [23].
Besides, ferrocene derivatives of thieno[2,3-d]pyrimidine, as purine isosteres, exhibited potent anticancer
effects in several breast cancer and AML (acute myeloid leukemia) cell lines, despite a loss of
mitogen-activated protein kinase-interacting kinase (MNK) potency [24]. On the other hand, ferrocenes
incorporating cinchona or carbohydrate moiety through 1,2,3-triazole linker showed significant
cytostatic activity [25,26]. Some ferrocene-cinchona hybrids increased reactive oxygen species (ROS)
production and induced mitochondrial damage in human multidrug resistant (MDR) cancer cells [27].
Taking into consideration the biological relevance of nucleoside analogues of ferrocene, here we
have described the synthesis of novel ferrocene-tagged purine and purine isosteres with the aim to
evaluate their antiproliferative activity. Preliminary ADME properties and electrochemical oxidation
potential of compounds that exhibited best growth-inhibitory activity were also assessed.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of novel purine and purine isosteres (pyrrolo[2,3-d]pyrimidine, benzimidazole
and indole) containing a ferrocene N-1-substituted 1,2,3-triazole moiety (11a−11c
16a
34a−34c and 35a−35c) was carried out as shown in Schemes 1–3.
The key intermediates, the N-9- (5−8) [28 31] and N-7-propargylated purine and 7-deazapurine
9−10) [30 31] (Scheme 1) and N-1-propargylated benzimidazole and indole 20−22 (Scheme 2) were
33].
15a−15c
,
12a−12c
,
13a−13c
,
1
(
4a−14c
,
15a−15c
,
,
23a−23c
,
24a−24c
,
25a−25c) and a ferrocene 4-substituted 1,2,3-triazole moiety
–
(
,
obtained by N-alkylation of the corresponding heterocyclic base as reported in the literature [32
,
Target regioselective 1,4-disubstituted 1,2,3-triazoles (11a−11c
,
12a−12c
,
13a−13c
,
14a−14c
,
,
1
1
1
6a
,
23a−23c
,
24a−24c 25a−25c) (Schemes 1 and 2) were prepared by copper(I)-catalysed Huisgen
,
,3-dipolar cycloaddition of alkynyl derivatives of corresponding purine and purine isosteres with
-methylazidoferrocene, 1-azidoethylferrocene and 1-azidoferrocene, which were prepared from
ferrocene methanol, 1-hydroxyethylferrocene and 1-bromoferrocene, respectively [34].
The structural diversity of target compounds was further expanded to the synthesis of heterocyclic
bases linked through 1-ethyl-1,2,3-triazole to ferrocene (Scheme 3). Reaction of 6-chloro-substituted
heterocyclic bases
irradiation gave 6-pyrrolidinyl and 6-piperidinyl-substituted intermediates 26−29. N-alkylation of the
corresponding heterocyclic base
26−29 with 1,2-dibromoethane in the presence of NaH provided
1 and 3 with the corresponding amines in the presence of KOH under microwave
1, 3,
the 2-bromoethyl heterocyclic derivatives 30a−30c
[
35], 31a−31c. Precursors 30a−30c and 31a−31c
were converted in situ, using NaN , to the 2-azidoethyl heterocyclic derivatives 32a−32c
[
35], 33a−33c
.
3
Target purine and 7-deazapurine and ferrocene conjugates 34a−34c 35a−35c were obtained by a
,
Cu(I)-catalysed click reaction of ethynylferrocene and corresponding N-(2-azidoethyl) 6-substituted
purine and 7-deazapurine derivatives (Scheme 3).

Molecules 2020, 25, 1570
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Scheme 1. Synthesis of novel purine and 7-deazapurine derivatives linked to C-4 of 1,2,3-triazolyl
◦
ferrocene. Reagents and conditions: (i) propargyl bromide, NaH, anhydrous DMF, Ar atmosphere, 60 C,
4 h; (ii) corresponding azide, Cu(OAc) , methanol, room temperature, 24 h.
2
2
Scheme 2. Synthesis of novel benzimidazole and indole derivatives linked to C-4 of 1,2,3-triazolyl
◦
ferrocene. Reagents and conditions: (i) propargyl bromide, NaH, anhydrous DMF, Ar atmosphere, 60 C,
4 h; (ii) corresponding azide, Cu(OAc) , methanol, room temperature, 24 h.
2
2

Molecules 2020, 25, 1570
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Scheme 3. Synthesis of novel purine and 7-deazapurine derivatives linked to N-1 of 1,2,3-triazolyl
◦
ferrocene. Reagents and conditions: (i) heterocyclic base, corresponding amine, KOH, H O, MW: 100 C,
2
4
00 W; (ii) heterocyclic base, NaH, 1,2-dibromoethane, anhydrous DMF, room temperature, 24 h; (iii)
◦
N-alkylated heterocyclic base, NaN , acetone, 60 C, 12 h; (iv) corresponding terminal azide, Cu(OAc) ,
3
2
ethynylferrocene, methanol, room temperature, 12 h.
2
.2. Biological Profiling
2
.2.1. Evaluation of Antiproliferative Activity and Physicochemical Properties
Antiproliferative activity of compounds 11a−11c
,
12a−12c
,
13a−13c
,
14a−14c
,
15a−15c
, 16a,
2
3a−23c 24a−24c 25a−25c 34a−34c and 35a−35c was evaluated in vitro on four human tumor cell
,
,
,
lines (American Type Culture Collection, Manassas, VA, USA): metastatic colorectal adenocarcinoma
SW620), ductal pancreatic adenocarcinoma (CFPAC-1), hepatocellular carcinoma (HepG2) and cervical
(
carcinoma (HeLa) as well as normal skin fibroblasts (HFF). The results are presented in Table 1.
Among all tested compounds, 6-chloro-7-deazapurines 11a−11c, 6-chloropurines 13a−13c and
ferrocenylalkyl derivatives 15a−15c exhibited the best inhibitory effects, particularly on colorectal
adenocarcinoma (SW620) cells. However, compounds that exhibited cytostatic activity were also
cytotoxic in the non-tumor HFF cell line. 6-Chloro-7-deazapurine 11c with ferrocene linked directly
to the N-1 position of 1,2,3-trazole exhibited the highest activity on SW620 cells (IC50 = 9.07 µM).
Replacement of the 6-chloro with the 6-amino group in 12a−12c reduced their inhibitory activity in all
cell lines. While 6-chloropurine 13a with the ferrocenylmethylene unit showed better potency compared
to its purine isostere 11a, 6-chloropurine derivatives 13b and 13c had lower activity than 7-deazapurine
analogues 11b and 11c. Similarly, among 2-amino-6-chloropurine and ferrocene conjugates 14a−14c
compound 14a with the ferrocenylmethylene unit had moderate growth-inhibitory activity, while 14b
with ferrocenylethyl and 14c with ferrocenyl directly linked to 1,2,3-triazole showed reduced inhibitory
effect. Interestingly, among N-7 regioisomers of purine-based ferrocene derivatives, only compound
,
1
1
5b with ferrocenyleth-yl moiety exhibited enhancement of the activity in comparison to its N-9 isomer
3b. Among the benzimidazole, indole and 5-iodoindole derivatives, only benzimidazole 23a and
indole 24a, both with ferrocenylmethylene unit, had moderate cytostatic activity. Their analogues 23b
4b 24c and 25c containing ferrocenylethyl and ferrocenyl directly linked to the N-1 of 1,2,3-triazole
showed reduced activity.
,
2
,

Molecules 2020, 25, 1570
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,
Table 1. In vitro growth inhibitory effects of compounds 11a−11c
6a 24a−24c 25a−25c 34a−34c and 35a−35c on selected tumor cell lines and their measured
23a−23c
kinetic solubility range and Chrom logD.
,
12a−12c
,
13a−13c
,
14a−14c
,
15a−15c
1
,
,
,
,
IC50 (µM)
Kinetic Solubility
Chrom
logD
Compd Heterocycle
R
SW620
CFPAC-1
HepG2
HeLa
HFF
(µM)
1
1a
31.58 ± 0.84
32.91 ± 2.46
41.96 ± 5.93
34.52 ± 1.36
38.25 ± 2.30
8.57 ± 1.95
<1
5.62
5.94
11b
31.96 ± 0.34
31.26 ± 4.51
31.38 ± 1.04
8.88 ± 3.46
3–10
1
1c
9.07 ± 1.21
>100
35.03 ± 5.44
>100
31.94 ± 4.32
>100
27.74 ± 3.23
>100
5.89 ± 1.88
3–10
5.62
2.85
12a
56.63 ± 15.60
30–100
12b
74.95 ± 8.93
70.15 ± 6.50
68.60 ± 9.15
76.40 ± 3.41
18.44 ± 2.64
>100
3.09
1
2c
>100
>100
>100
>100
54.50 ± 4.96
3.98 ± 0.17
3–10
2.64
4.27
13a
14.38 ± 3.02
22.83 ± 4.31
19.89 ± 5.12
25.20 ± 4.31
>100
13b
36.56 ± 3.42
43.83 ± 10.94
38.77 ± 10.11
40.34 ± 4.52
6.79 ± 1.06
30–100
4.64
1
3c
51.78 ± 1.90
60.23 ± 24.36
54.67 ± 5.13
49.47 ± 5.03
54.66 ± 3.21
60.13 ± 5.73
46.78 ± 6.72
71.43 ± 16.06
6.77 ± 2.78
>100
>100
4.19
3.62
14a
30–100
14b
98.74 ± 19.71
67.36 ± 9.12
77.10 ± 5.92
>100
11.53 ± 5.09
30–100
3.90
1
4c
81.33 ± 1.23
27.21 ± 5.30
90.50 ± 1.83
34.39 ± 0.43
>100
>100
24.55 ± 4.45
13.80 ± 4.17
<1
3.50
3.82
15a
36.01 ± 1.81
28.62 ± 8.28
>100
1
5b
15.50 ± 3.24
45.47 ± 4.52
28.59 ± 2.68
72.25 ± 13.70
37.11 ± 1.84
81.71 ± 17.05
28.97 ± 0.09
69.25 ± 25.47
0.88 ± 1.06
>100
>100
4.15
3.68
1
5c
29.59 ± 4.28
1
6a
3a
>100
>100
>100
>100
5.30 ± 5.36
<1
3.37
2
96.98 ± 13.55
>100
64.57 ± 12.18
68.03 ± 13.67
59.90 ± 10.94
83.60 ± 8.61
>100
23.83 ± 3.32
31.80 ± 9.81
10–30
4.56
4.84
23b
89.63 ± 13.65
30–100
2
3c
>100
>100
>100
>100
>100
30–100
3–10
4.45
24a
61.99 ± 8.79
78.05 ± 6.61
53.55 ± 5.05
68.72 ± 2.91
16.72 ± 8.61
>6.57
24b
>100
87.63 ± 1.79
>100
89.64 ± 7.64
>100
3–10
>6.57
2
4c
>100
>100
84.54 ± 15.76
>100
90.87 ± 11.51
>100
88.12 ± 1.18
78.52 ± 9.36
>100
>100
3–10
1–3
>6.57
>6.57
25a
2
5b
>100
>100
>100
>100
>100
>100
>100
1–3
1–3
>6.57
>6.57
2
5c
71.79 ± 3.44
79.44 ± 1.82
4.75 ± 1.70
3
4a
4b
4c
5a
5b
5c
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10–30
10–30
4.92
4.30
3
3
57.65 ± 6.55
78.18 ± 5.69
>100
45.78 ± 9.11
65.28 ± 6.23
>100
57.61 ± 13.80
48.09 ± 7.85
>100
71.12 ± 15.89
63.00 ± 6.06
>100
20.16 ± 1.54
6.00 ± 2.99
>100
10–30
10–30
30–100
5.49
3.70
3.58
3
3
3
>100
>100
>100
>100
6.83 ± 2.17
10–30
5.02

Molecules 2020, 25, 1570
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6-Chloro-7-deazapurine 34a with ferrocene connected to the C-4 position of 1,2,3-triazole showed
a lack of activity, while its 6-chloropurine structural analogue 35a displayed only a marginal effect.
A comparison of inhibitory activity between 1,4- and 4,1-disubstituted 1,2,3-triazoles showed that a
4,1-disubstituted triazole (as in 11c and 13c) is favored over a 1,4-disubstituted triazole (as in 34a and
3
5a). Transformation of 6-chloro to 6-cyclic amine in 34a 34c 35a and 35c did not lead to significant
,
,
changes in the antiproliferative activity. 6-Pyrrolidinyl (34b and 35b) and 6-piperidinyl-substituted
(35c) 7-deazapurines and purines were deprived of any activity.
The distribution coefficient Chrom logD, a parameter used to assess the lipophilicity of
compounds [36
shown in Table 1. Correlation between lipophilicity and antiproliferative activity was observed and
showed that compounds 11a−11c 13a 15a and 15b with the highest cytostatic effect had Chrom logD
values in the 3.82–5.94 range. A 6-chloro-7-deazapurine moiety increased the lipophilicity of 11a−11c
5.62–5.94) compared to a 6-chloropurine ring of 13a−13c (4.19–4.64). Furthermore, N-7 regioisomers
,37], was determined for novel purine and purine isosteres with a ferrocene motif as
,
,
(
1
5a−15c and 16a showed lower Chrom logD values than their N-9 regioisomers 13a−13c and 14a
.
Indole 24a−24c, 5-iodoindole 25a−25c and ferrocene conjugates that exhibited only marginal inhibition
had the highest lipophilicity with Chrom logD values above 6.57. The highest hydrophilicity was
determined for adenine-based ferrocene derivatives 12a−12c (Chrom logD = 2.64–3.09) that showed
low or did not exhibit any growth-inhibitory effects.
The turbidimetric solubility assay, which allows a rapid determination of the kinetic solubility of
newly synthesized compounds using small amounts of their DMSO solutions, was used to assess the
kinetic solubility [38]. Results showed that nitrogen heterocycles had the highest impact on solubility
(
Table 1). Generally, both N-9 and N-7 isomers of 6-chloropurine had high solubility. Conversely,
-chloro-7-deazapurine 11a−11c, indole 24a−24c and 5-indole 25a−25c derivatives were less soluble.
Among the most active compounds, 6-chloropurine derivatives 13a 15a and 15b had high solubility,
whereas the solubility of 6-chloro-7-deazapurine derivatives 11a−11c was significantly decreased.
6
,
2
.2.2. Evaluation of Permeability and Metabolic Stability
Compounds 11a−11c
,
13a 15a and 15b with highest cytostatic activities were further screened
,
for permeability and P-glycoprotein (P-gp) substrate assessment, as well as metabolic stability in
liver microsomes (human and mouse). These properties have been shown to significantly affect
the bioavailability of drugs and to be important in the early drug discovery phases and the lead
optimization process [39].
The Madin-Darby canine kidney cells with overexpressed human multidrug-resistant gene
(MDCKII-hMDR1) have been frequently used to study bidirectional transport of compounds and
assess P-gp substrate potential [40]. Apparent permeability (Papp) values were determined from the
amount permeated through the MDCKII-hMDR1 cell monolayer in both the apical-basolateral (AB)
and basolateral-apical (BA) direction. Papp values and the efflux ratios with and without P-gp inhibition
with Elacridar are listed in Table 2.
Compound 11b is characterized by a high permeability, with an efflux ratio of 1.5 suggesting no
influence of P-pg on permeability. All the remaining compounds exhibited a moderate permeability
(PappA/B), with efflux ratios above 2, classifying them as P-gp substrates, which was further confirmed
when they were incubated with a P-gp inhibitor, elacridar. As previously reported in the literature [41],
a comparison of efflux ratio generated in the presence and absence of a P-gp inhibitor can be used for
the identification of P-gp substrates. A compound is typically considered to be a P-gp substrate when
the efflux ratio in the absence of inhibitor is greater than 2 and/or is at least 50% drop is observed in the
presence of inhibitor [42]. Among compounds with moderate permeability the 6-chloro-7-deazapurine
derivative 11c with a directly connected ferrocene moiety and N-7 regioisomers of 6-chloropurine 15a
and 15b showed moderate AB permeability, while compounds 11a and 13a had moderate to high AB
−
6
permeability in the range from 8.3 to 8.48 × 10 cm/s.

Molecules 2020, 25, 1570
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Table 2. The apparent permeability (Papp) of 11a−11c
, 13a, 15a, 15b in MDCKII-hMDR1 cell assay
from apical-to-basolateral (AB) and basolateral-to-apical (BA) side without and with inhibition of
P-glycoprotein (P-gp) efflux pump with Elacridar.
Papp (AB)
Papp (BA)
Efflux Ratio
(BA/AB)
Papp (AB)
Papp (BA)
Efflux Ratio
(BA/AB)
Compd Heterocycle
R
(×10−6 cm/s)
(×10−6 cm/s)
(×10−6 cm/s)
(×10−6 cm/s)
without P-gp inhibitor
with P-gp inhibitor
1
1a
8.4
18.0
2.2
1.5
29.4
19.7
21.1
0.7
1.1
11b
11.8
17.4
14.9
20.5
16.5
1
1c
5.3
8.3
2.9
2.9
15.5
29.8
1.1
0.9
1
1
3a
5a
23.8
25.3
4.8
3.2
33.5
33.2
7.0
16.9
11.7
13.7
12.9
0.8
1.1
15b
10.7
Incubation with mouse liver microsomes suggested that that all tested compounds are metabolically
unstable with a predicted in vivo clearance above 97% of liver blood flow as summarized in Table 3.
Table 3. Metabolic stability parameters of 11a−11c
,
13a
,
15a
,
15b in mouse and human liver microsomes.
Mouse
Human
Compd
Heterocycle
R
In Vivo CLh
ml/min/kg)
In Vivo CL
(% LBF)a
In Vivo CLh
(ml/min/kg)
In Vivo CLh
(% LBF)
h
t1/2 (min)
t1/2 (min)
(
1
1a
4.4
25.2
98
100
99
7.8
23.2
24.9
24.3
90
97
95
11b
0
25.7
25.6
2.3
4.1
1
1c
1.3
1
1
3a
5a
5.5
25.1
98
38.6
16.8
65
8.8
2.9
24.8
25.4
97
99
51.9
19.0
15.0
20.4
58
79
15b
a
Liver Blood Flow.
No substantial differences in clearance values for these compounds were observed in mouse
microsomes. On the other hand, incubation with human liver microsomes showed the significant
impact of type of nitrogen heterocycle on stability. Namely, 6-chloro-7-deazapurine derivatives
1
1a−11c exhibiting high clearance values (>90% LBF) can be considered as highly labile compounds.
On the contrary, 6-chloropurine derivative 13a, as structural analogue of 6-chloro-7-deazapurine 11a
,
with clearance value of 65% LBF showed to be moderately stable. Additionally, N-7 regioisomer 15a
had somewhat increased microsomal stability (58% LBF) than its N-9 analogue 13a (65% LBF).

Molecules 2020, 25, 1570
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2
.3. Electrochemical Properties of Selected Compounds by Voltammetric Assays
Compounds 11c 13a and 15b with strong antiproliferative activity and good physicochemical
,
properties were further evaluated for their electrochemical properties under a wide range of solution
conditions. Although the electrochemical behaviour of ferrocene, triazole and their derivatives has
been studied in both protic and aprotic solvents [43–47], we have been interested to examine the redox
mechanism of the above-mentioned compounds, which may be influenced by the type of nitrogen
heterocycle, ferrocene, and alkyl spacer between ferrocene and triazole unit.
The electrochemical properties of 6-chloro-7-deazapurine and 6-chloropurine derivatives of
ferrocene 11c, 13a and 15b on a glassy carbon electrode were studied using cyclic (CV) and square-wave
voltammetry (SWV) in aqueous electrolyte solutions over a wide pH range. The pH was varied from 2
to 10 in order to determine the effect of pH on the voltammograms (net peak currents and potentials).
Figure 1 shows square-wave and cyclic voltammograms for the oxidation of 0.1 mM of 13a in aqueous
buffer electrolytes 3
compound, i.e., the oxidation of evaluated compounds 11c
≤
pH
≤
10. The pH of the solution affects the voltammetric response of this
13a and 15b is strongly dependent on it.
,
Furthermore, as can be seen, the best results were obtained at pH 9, so this value was selected for the
following experiments.
1
00
B
A
60
80
60
40
20
0
d
d
40
20
0
P2
P2
a
c
c
b
b
P1
P1
a
0
.0
0.5
E / V
1.0
1.5
-0.5
0.0
0.5
1.0
1.5
E / V
Figure 1.
(
A
) Square-wave and (
B) cyclic voltammograms of 0.1 mmol/L 13a in 0.5 mol/L NaClO4
at pH 3 (a), 7 (b), 9 (c) and 10 (d). (
SW voltammogram of the supporting electrolyte; (
scan direction.
A
) Esw = 50 mV, Es = 2 mV, f = 100 Hz. Dotted line represents
−
1
B) v = 100 mV s , Es = 2 mV. Arrows indicate the
Figure 2 shows square-wave voltammograms for the oxidation of 11c
, 13a and 15b on glassy
carbon electrode, in 0.5 mol/L NaClO at pH 9. The forward (i ) and backward (i ) components of the
4
f
b
net response (
can be observed, indicating that these molecules have two redox active centres. The peak P1 occurs
in all supporting electrolytes (i.e., at 2 pH 10), while the peak P2 is only observed at pH 9 (see
Figure 1). The potential of peak P1 is pH-independent. Comparing to the literature data, peak P1
∆
i = if
−
ib) are shown as well (inset in Figure 2). Two peaks at ~0.4 V (P1) and ~1.1 V (P2)
≤
≤
≥
II
III
+
−
can be ascribed to the following charge transfer reaction [Fe (C H ) ]
ꢀ
[Fe (C H ) ] + e [44
,
48].
The forward and backward components of peak P1 are oxidative and reductive currents, respectively
see inset in Figure 2), which indicates that the oxidation of ferrocene is reversible electrode reaction.
5
5 2
5
5 2
(

Molecules 2020, 25, 1570
9 of 24
8
0
0
0
13a
P2
1
1
1
40
20
00
4
P1

i
if
ib
-
0.5
0.0
0.5
1.0
1.5
80
E / V
P2
6
4
2
0
0
0
0
1
5b
1
3a
P1
1
1c
-20
0
.0
0.5
1.0
1.5
E / V
Figure 2. Square-wave voltammograms for the oxidation of 11c
pH 9. Conditions: c(13a) = 0.1 mmol/L, c(11c) = c(15b) = 0.05 mmol/L, Esw = 50 mV, Es = 2 mV, f =
00 Hz. Inset: The net SWV response ( i) and its forward (i ) and backward (i ) current components,
for the oxidation of 13a. Arrow indicates the scan direction.
, 13a and 15b in 0.5 mol/L NaClO at
4
1
∆
f
b
Furthermore, in order to clarify the origin of peak P2, ethynyl ferrocene (i) and 6-chloropurine
ii) (bearing no triazole moiety) were analysed as well (under otherwise identical conditions, Figure 3).
(
No anodic peak P2 was detected on SWV responses of these compounds within the investigated potential
window. Comparing with the SWV response of 13a (black curve in Figure 3), it can be concluded
that the peak P2 corresponds to the electrode reaction of triazole ring. Both components (i and i ) of
f
b
the peak P2 are oxidative currents (see inset in Figure 2), which indicate that the electro-oxidation of
,2,3-triazole in 11c 13a and 15b at pH 9 is totally irreversible electrode reaction. The same results
1
,
were obtained by cyclic voltammetry (under otherwise identical conditions). These observations are in
agreement with existing literature on the electrochemical oxidation of triazole-acridine conjugates [49].
1
00
80
60
40
20
0
1
3a
i
ii
-0.5
0.0
0.5
1.0
1.5
E / V
Figure 3. Square-wave voltammograms for the oxidation of ethynyl ferrocene (i), 6-chloropurine
ii) and 13a in 0.5 mol/L NaClO at pH 9. Conditions: c(i) = c(ii) = c(13a) = 0.1 mmol/L, Esw = 50 mV,
(
4
Es = 2 mV, f = 100 Hz. Arrow indicates the scan direction.

Molecules 2020, 25, 1570
10 of 24
In addition, it can be seen from Figure 2 that all investigated compounds provided similar
electrochemical responses. More precisely, the electro-oxidation of the 1,2,3-triazole moiety (i.e., peak P2)
in 11c, 13a and 15b takes place at almost the same potential. Taking into account the structural difference
between 13a and 15b in type of the alkyl bridge between the two redox-active centres triazole and
ferrocene, it is reasonable to conclude that its effect on the oxidation of the triazole in studied compounds
is negligible. However, in the case of 11c, where the triazole ring is directly attached to the ferrocene,
the electrochemical oxidation of ferrocene (i.e., peak P1) occurs at more positive potentials. This result
indicates that iron(II) in 11c is more difficult to oxidize due to the stronger electron-withdrawing effect
of the 1,2,3-triazoles ring directly attached to the ferrocene nucleus, or due to its steric hindrance effect
on the Fe(II) ion in 11c.
3
. Materials and Methods
3
.1. General Information
All chemicals and solvents were purchased from Aldrich (St. Louis, MO, USA), Fluorochem
(
Hadfield, UK) and Acros (Geel, Belgium). Anhydrous dimethyl formamide (DMF) was prepared using
CaH and stored over 3Å molecular sieves [50]. Thin layer chromatography (TLC) was performed on
2
pre-coated silica gel 60F-254 plates (Merck, Kenilworth, NJ, USA), while glass column slurry-packed
under gravity with 0.063–0.2 mm silica gel (Fluka, Seelze, Germany) was employed for column
chromatography. Melting points were determined using a Kofler micro hot-stage (Reichert, Vienna,
1
13
Austria). H and C-NMR spectra were recorded on a Bruker 300 and 600 MHz spectrometers (Bruker,
Billerica, MA, USA). All data were recorded in dimethyl sulfoxide (DMSO)-d at 298 K. NMR chemical
6
1
shifts were referenced to the residual solvent signal of DMSO at
δ
2.50 ppm for H and
δ
39.50 ppm
13
for C. Individual resonances were assigned on the basis of their chemical shifts, signal intensities,
1
13
multiplicity of resonances, and H–H coupling constants. H and C-NMR spectra of compounds are
available in Supplementary Materials. Microwave-assisted syntheses were carried out in a microwave
◦
oven (Milestone Start S, Sorisole, BG, Italy) at 100 C and pressure of 1 bar.
4
-Chloro-7-(prop-2-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine (
H-purine ( ) [30], 6-chloro-9-(prop-2-yn-1-yl)-9H-purine ( ) [31], 2-amino-6-chloro-9-(prop-2-yn-1-yl)-
H-purine ( ) [32], 6-chloro-9-(prop-2-yn-1-yl)-9H-purine ( ) [31], 2-amino-6-chloro-7-(prop-2-yn-
-yl)-7H-purine (10) [32], 1-(prop-2-yn-1-yl)-1H-benzimidazole (20) [34], 1-(prop-2-yn-1-yl)-1H-
indole (21) [51], 5-iodo-1-(prop-2-yn-1-yl)-1H-indole (22) [34], 6-(pyrrolidin-1-yl)-9H-purine (28) [52],
-(piperidin-1-yl)-9H-purine (29) [53], and 9-(2-bromoethyl)-6-chloro-7H-pyrrolo[2,3-d]pyrimidine
30a) [36] were synthesized in accordance with procedures given in the literature.
5) [29], 6-amino-9-(prop-2-yn-1-yl)-
9
9
1
6
8
7
9
6
(
3
.2. General Procedure for the Synthesis of Purine and Purinomimetics with Ferrocene at N-1 of 1,2,3-Triazole
The corresponding N-propargylated heterocyclic base 5−10 (1 eq.) was dissolved in methanol,
and the corresponding terminal azide (1.2 eq.) and Cu(OAc) (0.05 eq.) were added. The reaction
2
mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure
and the residue was purified by column chromatography (CH Cl :CH OH = 60:1).
2
2
3
4
-Chloro-7-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-7H-pyrrolo[2,3-d]pyrimidine (11a) Compound
1
1a was prepared using the above-mentioned procedure using compound
5
(100 mg, 0.52 mmol) and
1
1
-methylazidoferrocene (150 mg, 0.62 mmol) to obtain 11a as orange oil (60.3 mg, 33 %). H-NMR (300
0
MHz, DMSO-d6)
δ
8.65 (1H, s, H2), 8.06 (1H, s, H5 ), 7.80 (1H, d, J = 3.6 Hz, H6), 6.67 (1H, d, J = 3.6 Hz,
H5), 5.56 (2H, s, CH ), 5.25 (2H, s, CH ), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.14 2H, (m, CH-Fc), 4.12
2
2
5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO-d )
δ 151.12 (C4), 150.87 (C2), 150.82 (C7a), 143.04 (C4 ),
0
(
1
6
0
31.77 (C6), 123.51 (C5 ), 117.22 (C4a), 99.30 (C5), 82.83 (Cq-Fc), 69.08 (CH-Fc), 69.04 (Cp-Fc), 68.76
(
CH-Fc), 49.35 (CH ), 40.05 (CH ). Anal. calcd. for C H ClFeN : C, 55.52; H, 3.96; N, 19.42. Found:
2 2 20 17 6
C, 55.46; H, 3.95; N, 19.44.

Molecules 2020, 25, 1570
11 of 24
4-Chloro-7-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-7H-pyrrolo[2,3-d] pyrimidine (11b)
Compound 11b was prepared using the above-mentioned procedure using compound
5 (100 mg, 0.52
mmol) and 1-azidoethylferrocene (159 mg, 0.62 mmol) to obtain 11b as orange oil (82.6 mg, 35 %).
1
0
δ 8.65 (1H, s, H2), 8.09 (1H, s, H5 ), 7.80 (1H, d, J = 3.6 Hz, H6), 6.67(1H,
H-NMR (300 MHz, DMSO-d6)
d, J = 3.6 Hz, H5), 5.65 (1H, q, J = 7.0 Hz, CH), 5.56 (2H, s, CH ), 4.33–4.28 (1H, m, CH-Fc), 4.17–4.15
2
13
(
2H, m, CH-Fc), 4.08 (5H, H,s, Cp-Fc), 1.78 (3H, d, J = 7.0 Hz, CH ). C-NMR (151 MHz, DMSO-d ) δ
3 6
0
0
1
8
3
50.60 (C4), 150.36 (C2), 150.30 (C7a), 142.26 (C4 ), 131.25 (C6), 121.58 (C5 ), 116.70 (C4a), 98.82 (C5),
8.53 (Cq-Fc), 68.61 (Cp-Fc), 68.09 (CH-Fc), 67.62 (CH-Fc), 67.15 (CH-Fc), 66.21 (CH-Fc), 55.60 (CH),
9.54 (CH ), 20.83 (CH ). Anal. calcd. for C H ClFeN : C, 56.46; H, 4.29; N, 18.81. Found: C, 56.35;
2
3
21 19
6
H, 4.28; N, 18.87.
4-Chloro-7-[1-(1-ferrocenyl-1,2,3-triazol-1-yl)methyl]-7H-pyrrolo[2,3-d]pyrimidine (11c) Compound 11c
was prepared using the above-mentioned procedure using compound
5
(100 mg, 0.52 mmol) and
1
1
-azidoferrocene (142 mg, 0.62 mmol) to obtain 11c as orange oil (39.4 mg, 18 %). H-NMR (600 MHz,
0
δ 8.68 (1H, s, H2), 8.55 (1H, s, H5 ), 7.81 (1H, d, J = 3.6 Hz, H6), 6.70 (1H, d, J = 3.6 Hz,
DMSO-d6)
H5), 5.64 (2H, s, CH ), 5.00 (2H, t, J = 1.9 Hz, CH-Fc), 4.35–4.31 (2H, m, CH-Fc), 4.18 (5H, s, Cp-Fc).
2
1
3
0
C-NMR (151 MHz, DMSO-d )
C5 ), 116.83 (C4a), 99.33 (C5), 93.32 (Cq-Fc), 69.87 (Cp-Fc), 66.57 (CH-Fc), 61.86 (CH-Fc), 34.42 (CH ).
δ
150.85 (C4), 150.39 (C2), 150.19 (C7a), 142.95 (C4 ), 130.83 (C6), 123.33
6
0
(
2
Anal. calcd. for C H ClFeN : C, 54.51; H, 3.61; N, 20.07. Found: C, 55.40; H, 3.90; N, 20.03.
19
15
6
6
-Amino-9-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (12a) Compound 12a was prepared
using the above-mentioned procedure using compound (100 mg, 0.58 mmol) and
-methylazidoferrocene (168 mg, 0.70 mmol) to obtain 12a as orange powder (132 mg, 76%, m.p. = 107
6
1
◦
1
0
C). H-NMR (300 MHz, DMSO-d ) δ 8.18 (1H, s, H8), 8.12 (1H, s, H2), 8.07 (1H, s, H5 ), 7.21 (2H, s,
6
NH ), 5.41 (2H, s, CH ), 5.26 (2H, s, CH ), 4.30 (2H, dd, J = 3.4, 1.6 Hz, CH-Fc), 4.174.14 (2H, m, CH-Fc),
2
2
2
13
0
4
1
.13 (5H, s, Cp-Fc). C-NMR (151 MHz, DMSO-d )
40.59 (C8), 123.05 (C5 ), 118.51 (C5), 82.35 (Cq-Fc), 68.57 (CH-Fc), 68.56 (Cp-Fc), 68.26 (CH-Fc), 48.85
δ
155.91 (C6), 152.50 (C2), 149.21 (C4), 142.54 (C4 ),
6
0
(
CH ), 37.96 (CH ). Anal. calcd. for C H FeN : C, 55.09; H, 4.38; N, 27.05. Found: C, 54.98; H, 4.39;
2 2 19 18 8
N, 27.00.
-Amino-9-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (12b) Compound 12b was
prepared using the above-mentioned procedure using compound (100 mg, 0.58 mmol) and
-azidoethylferrocene (177 mg, 0.70 mmol) to obtain 12b as orange powder (53.7 mg, 31 %, m.p.
6
6
1
=
◦
1
0
223 C). H-NMR (600 MHz, DMSO) δ 8.57 (1H, s, H8), 8.26 (1H, s, H5 ), 8.15 (1H, s, H2), 7.27 (2H, s,
13
NH ), 5.49 (2H, s, CH ), 5.01 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s, Cp-Fc). C-NMR (75
MHz, DMSO)
2
2
0 0
δ 156.46 (C6), 153,17 (C8), 153.01 (C2), 149.87 (C4), 143.39 (C4 ), 123.94 (C5 ), 119.13 (C5),
9
3.79 (Cq-Fc), 70.38 (Cp-Fc), 67.08 (CH-Fc), 62.38 (CH-Fc), 38.46 (CH ). Anal. calcd. for C H FeN :
2 20 20 8
C, 56.09; H, 4.71; N, 26.16. Found: C, 56.17; H, 4.69; N, 26.11.
-Amino-9-[1-(1-ferrocenyl-1,2,3-triazol-1-yl)methyl]-9H-purine (12c) Compound 12c was prepared using
the above-mentioned procedure using compound (100 mg, 0.58 mmol) and 1-azidoferrocene (160
6
6
◦
1
mg, 0.70 mmol) to obtain 12c as orange powder (13.5 mg, 5.8 %, m.p. > 250 C). H-NMR (600 MHz,
8.57 (1H, s, H8), 8.26 (1H, s, H5 ), 8.15 (1H, s, H2), 7.27 (2H, s, NH ), 5.49 (2H, s, CH ), 5.01
2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s, Cp-Fc). C-NMR (75 MHz, DMSO)
C8), 153.01 (C2), 149.87 (C4), 143.39 (C4 ), 123.94 (C5 ), 119.13 (C5), 93.79 (Cq-Fc), 70.38 (Cp-Fc), 67.08
CH-Fc), 62.38 (CH-Fc), 38.46 (CH ). Anal. calcd. for C H FeN : C, 54.02; H, 4.03; N, 28.00. Found:
0
DMSO)
(
(
(
δ
2
2
1
3
δ
156.46 (C6), 153,17
0
0
2
18 16
8
C, 53.86; H, 4.04; N, 27.97.
6
-Chloro-9-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (13a) Compound 13a was prepared
using the above-mentioned procedure using compound (55 mg, 0.28 mmol) and
-methylazidoferrocene (81 mg, 0.34 mmol) to obtain 13a as orange powder (53.2 mg, 43 %, m.p. =
7
1
1
◦
1
0
56 C). H-NMR (300 MHz, DMSO) δ 8.79 (1H, s, H8), 8.77 (1H, s, H2), 8.15 (1H, s, H5 ), 5.60 (2H, s,
CH ), 5.26 (2H, s, CH ), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.15 (2H, m, CH-Fc), 4.13 (5H, s, Cp-Fc).
2
2

Molecules 2020, 25, 1570
12 of 24
1
3
0
C-NMR (75 MHz, DMSO)
δ
152.15 (C6), 152.13 (C2), 149.53 (C4), 147.92 (C8), 142.18 (C4 ), 131.22 (C5),
0
1
23.69 (C5 ), 82.75 (Cq-Fc), 69.10 (CH-Fc), 69.05 (Cp-Fc), 68.78 (CH-Fc), 49.43 (CH ), 39.42 (CH ). Anal.
2
2
calcd. for C H ClFeN : C, 52.62; H, 3.72; N, 22.61. Found: C, 52.46; H, 3.73; N, 22.66.
19
16
7
6
-Chloro-9-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (13b) Compound 13b was
prepared using the above-mentioned procedure using compound
7
(100mg, 0.52 mmol) and
1
1
-azidoethylferrocene (157 mg, 0.62 mmol) to obtain 13b as orange oil (55.5 mg, 23 %). H-NMR (600
0
MHz, DMSO)
δ
8.79 (1H, s, H8), 8.78 (1H, s, H2), 8.16 (1H, s, H5 ), 5.66 (1H, q, J = 7.0 Hz, CH), 5.60 (2H,
s, CH ), 4.31 (1H, s, CH-Fc), 4.17 (2H, d, J = 1.7 Hz, CH-Fc), 4.15 (1H, s, CH-Fc), 4.09 (5H, s, Cp-Fc), 1.78
2
13
(
(
6
3H, d, J = 7.0 Hz, CH ). C-NMR (75 MHz, DMSO)
δ
152.17 (C6), 152.12 (C2), 149.52 (C4), 141.95
C4 ), 131.21 (C5), 122.23 (C5 ), 88.99 (Cq-Fc), 69.13 (Cp-Fc), 68.61 (CH-Fc), 68.15 (CH-Fc), 67.67 (CH-Fc),
6.70 (CH-Fc), 56.19 (CH), 39.42 (CH ), 21.33 (CH ). Anal. calcd. for C H ClFeN : C, 53.65; H, 4.05;
3
0
0
2
3
20 18
7
N, 21.90. Found: C, 53.54; H, 4.04; N, 21.86.
6
-Chloro-9-[1-(1-ferrocenyl-1,2,3-triazol-1-yl)methyl]-9H-purine (13c) Compound 13c was prepared using
the above-mentioned procedure using compound
7
(100 mg, 0.52 mmol) and 1-azidoferrocene (138 mg,
1
0
8
. 62 mmol) to obtain 13c as orange oil (43.2 mg, 19 %). H-NMR (600 MHz, DMSO)
δ
8.85 (1H, s, H8),
0
.81 (1H, s, H2), 8.59 (1H, s, H5 ), 5.68 (2H, s, CH ), 4.99 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s,
2
13
0
Cp-Fc). C-NMR (151 MHz, DMSO)
δ
151.77 (C6), 151.65 (C2), 149.05 (C4), 142.18 (C4 ), 130.77 (C5),
0
1
23.45 (C5 ), 93.17 (Cp-Fc), 69.91 (Cp-Fc), 66.61 (CH-Fc), 61.84 (CH-Fc). Anal. calcd. for C H ClFeN :
18
14
7
C, 51.52; H, 3.36; N, 23.36. Found: C, 51.57; H, 3.38; N, 23.33.
2
-Amino-6-chloro-9-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (14a) Compound 14a was
prepared using the above-mentioned procedure using compound
8 (100 mg, 0.48 mmol) and
1
-methylazidoferrocene (139 mg, 0.58 mmol) to obtain 14a as orange powder (123 mg, 57 %; m.p. = 222
C). H-NMR (300 MHz, DMSO)
◦
1
0
δ 8.17 (1H, s, H8), 8.05 (1H, s, H5 ), 6.93 (2H, s, NH ), 5.33 (2H, s,
2
CH ), 5.27 (2H, s, CH ), 4.31 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.15 (2H, m, CH-Fc), 4.14 (5H, s, Cp-Fc).
2
2
1
3
0
δ 160.32 (C2), 154.33 (C6), 149.85 (C4), 143.46 (C8) 142.74 (C4 ), 123.64 (C5),
C-NMR (75 MHz, DMSO)
23.40 (C5 ), 82.75 (Cq-Fc), 69.12 (CH-Fc), 69.06 (Cp-Fc), 68.80 (CH-Fc), 49.45 (CH ), 38.71 (CH ). Anal.
0
1
2
2
calcd. for C H ClFeN : C, 50.86; H, 3.82; N, 24.97. Found: C, 50.91; H, 3.80; N, 24.92.
19
17
8
2
1
1
-Amino-6-chloro-9-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (14b) Compound
4b was prepared using the above-mentioned procedure using compound (100 mg, 0.48 mmol) and
-azidoethylferrocene (147 mg, 0.58 mmol) to obtain 14b as orange powder (55 mg, 24 %, m.p. = 214
8
◦
1
0
δ 8.17 (1H, s, H8), 8.07 (1H, s, H5 ), 6.93 (2H, s, NH ), 5.66 (1H, q, J =
2
C). H-NMR (300 MHz, DMSO)
.0 Hz, CH), 5.33 (2H, s, CH ), 4.32 (2H, d, J = 2.1 Hz, CH-Fc), 4.23–4.13 (3H, m, CH-Fc), 4.10 (5H, s,
7
2
13
Cp-Fc), 1.80 (3H, d, J = 7.0 Hz, CH ). C-NMR (151 MHz, DMSO)
δ
159.82 (C2), 153.84 (C6), 149.34
C4), 142.96 (C8), 141.95 (C4 ), 123.15 (C5), 121.47 (C5 ), 88.49 (Cq-Fc), 68.64 (Cp-Fc), 68.12 (CH-Fc),
7.66 (CH-Fc), 67.23 (CH-Fc), 66.17 (CH-Fc), 55.70 (CH), 38.24 (CH ), 20.77 (CH ). Anal. calcd. for
3
0
0
(
6
2
3
C H ClFeN : C, 51.91; H, 4.14; N, 24.22. Found: C, 51.75; H, 4.13; N, 24.24.
20
19
8
2
-Amino-6-chloro-9-[1-(1-ferrocenyl-1,2,3-triazol-1-yl)methyl]-9H-purine (14c) Compound 14c was prepared
using the above-mentioned procedure using compound (100 mg, 0.48 mmol) and 1-azidoferrocene
8
◦
1
(
131 mg, 0.58 mmol) to obtain 14c as orange powder (44 mg, 21 %, m.p. > 250 C). H-NMR (300 MHz,
0
δ 8.50 (1H, s, H8), 8.24 (1H, s, H5 ), 6.96 (2H, s, NH ), 5.41 (2H, s, CH ), 4.99 (2H, t, J = 1.9 Hz,
2 2
DMSO)
13
CH-Fc), 4.384.30 (2H, m, CH-Fc), 4.19 (5H, s, Cp-Fc). C-NMR (151 MHz, DMSO)
C6), 149.38 (C4), 142.63 (C4 ), 123.28 (C5 ), 123.23 (C5), 93.34 (Cq-Fc), 69.89 (Cp-Fc), 66.58 (CH-Fc),
δ
159.83 (C2), 153.93
0
0
(
6
1.86 (CH-Fc), 38.24 (CH ). Anal. calcd. for C H ClFeN : C, 49.74; H, 3.48; N, 25.78. Found: C, 49.78;
2 18 15 8
H, 3.47; N, 25,74.
6
-Chloro-7-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-7H-purine (15a) Compound 15a was prepared
using the above-mentioned procedure using compound (50 mg, 0.26 mmol) and
-methylazidoferrocene (75 mg, 0.31 mmol) to obtain 15a as orange powder (56.5 mg, 50 %, m.p. = 96
9
1
◦
1
0
δ 8.94 (1H, s, H8), 8.80 (1H, s, H2), 8.16 (1H, s, H5 ), 5.80 (2H, s, CH ),
2
C). H-NMR (300 MHz, DMSO)

Molecules 2020, 25, 1570
13 of 24
1
5
(
(
.26 (2H, s, CH ), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.204.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 3C-NMR
2
0 0
δ 161.57 (C4), 151.74 (C2), 151.17 (C8), 142.54 (C4 ), 142.29 (C6), 122.82 (C5 ), 121.98
151 MHz, DMSO)
C5), 82.36 (Cq-Fc), 68.53 (Cp-Fc), 68.51 (CH-Fc), 68.26 (CH-Fc), 48.93 (CH ), 41.89 (CH ). Anal. calcd.
2
2
for C H ClFeN : C, 52.62; H, 3.72; N, 22.61. Found: C, 52.56; H, 3.71; N, 22.57.
19
16
7
6
-Chloro-7-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-7H-purine (15b) Compound 15b was
prepared using the above-mentioned procedure using compound (90 mg, 0.47 mmol) and
-azidoethylferrocene (145 mg, 0.56 mmol) to obtain 15b as orange powder (56.5 mg, 27 %, m.p.
9
1
=
◦
1
0
68 C). H-NMR (300 MHz, DMSO) δ 8.94 (1H, s, H8), 8.80 (1H, s, H2), 8.19 (1H, s, H5 ), 5.80 (2H, s,
CH ), 5.66 (1H, q, J = 7.0 Hz, CH), 4.334.27 (1H, m, CH-Fc), 4.16 (3H, m, CH-Fc), 4.07 (5H, s, Cp-Fc),
2
13
0
1
1
.78 (3H, d, J = 7.0 Hz, CH3). C-NMR (151 MHz, DMSO)
42.30 (C6), 122.02 (C5), 121.37 (C5 ), 88.62 (Cq-Fc), 68.59 (Cp-Fc), 68.08 (CH-Fc), 67.61 (CH-Fc), 67.01
δ
161.57 (C4), 151.72 (C2), 142.34 (C4 ),
0
(
CH-Fc), 66.23 (CH-Fc), 55.70 (CH), 41.97 (CH ), 20.94 (CH ). Anal. calcd. for C H ClFeN : C, 53.65;
2 3 20 18 7
H, 4.05; N, 21.90. Found: C, 53.68; H, 4.03; N, 21.86.
-Chloro-7-[1-(1-ferrocenyl-1,2,3-triazol-1-yl)methyl]-7H-purine (15c) Compound 15c was prepared using
the above-mentioned procedure using compound (90 mg, 0.47 mmol) and 1-azidoferrocene (129 mg,
6
9
◦
1
0
δ
.56 mmol) to obtain 15c as orange powder (44.7 mg, 22 %, m.p. = 76 C). H-NMR (600 MHz, DMSO)
0
9.00 (1H, s, H8), 8.83 (1H, s, H2), 8.58 (1H, s, H5 ), 5.87 (2H, s, CH ), 4.99 (2H, s, CH-Fc), 4.33 (2H, s,
2
1
3
CH-Fc), 4.17 (5H, s, Cp-Fc). C-NMR (151 MHz, DMSO)
C6), 142.29 (C4 ), 122.75 (C5 ), 122.13 (C5), 93.24 (Cq-Fc), 69.89 (Cp-Fc), 66.61 (CH-Fc), 61.74 (CH-Fc),
δ
161.71 (C4), 151.77 (C2), 151.24 (C8), 143.23
0
0
(
4
2
2
2.00 (CH ). Anal. calcd. for C H ClFeN : C, 51.52; H, 3.36; N, 23.36. Found: C, 51.55; H, 3.34; N,
2 18 14 7
3.39.
-Amino-6-chloro-7-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-7H-purine (16a) Compound 16a was
prepared using the above-mentioned procedure using compound 10 (70 mg, 0.34 mmol) and
1
>
-methylazidoferrocene (98 mg, 0.41 mmol) to obtain 16a as orange powder (76 mg, 50 %, m.p.
◦ 0
250 C). H-NMR (300 MHz, DMSO) δ 8.49 (1H, s, H8), 8.09 (1H, s, H5 ), 6.64 (2H, s, NH ), 5.60
2
1
(
2H, s, CH ), 5.26 (2H, s, CH ), 4.29 (2H, t, J = 1.8 Hz, CH-Fc), 4.174.14 (2H, m, CH-Fc), 4.12 (5H, s,
2 2
Cp-Fc). 13C-NMR (151 MHz, DMSO)
δ
0 0
164.27 (C4), 159.98 (C2), 142.82 (C6), 142.33 (C4 ), 122.74 (C5 ),
8
2.48 (Cq-Fc), 68.54 (CH-Fc), 68.46 (CH-Fc), 68.22 (CH-Fc), 48.89 (CH ), 41.56 (CH ). Anal. calcd. for
2
2
C H ClFeN : C, 50.86; H, 3.82; N, 24.97. Found: C, 50.80; H, 3.83; N, 24.91.
19
17
8
3
.3. General Procedure for the Synthesis of Purinomimetics with Ferrocene at N-1 of 1,2,3-Triazole
The corresponding N-propargylated heterocyclic base 20−22 (1 eq.) was dissolved in methanol,
and the corresponding terminal azide (1.2 eq.) and Cu(OAc) (0.05 eq.) were added. The reaction
2
mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure
and the residue was purified by column chromatography (CH Cl :CH OH = 60:1).
2
2
3
1
-[1-(1-Ferrocenylmethyl-1,2,3-triazol-4-yl)methyl]-1H-benzimidazole (23a) Compound 23a was prepared
using the above-mentioned procedure using compound 20 (70 mg, 0.45 mmol) and
-methylazidoferrocene (130.2 mg, 0.54 mmol) to obtain 23a as orange powder (113 mg, 63 %,
1
◦
1
0
m.p. = 163 C). H-NMR (300 MHz, DMSO)
δ
8.32 (1H, s, H2), 8.14 (1H, s, H5 ), 7.65 (2H, dd, J =
7
4
1
.1, 1.9 Hz, H4/7), 7.287.16 (2H,m, H5/6), 5.56 (2H, s, CH ), 5.28 (2H, s, CH ), 4.354.28 (2H, m, CH-Fc),
.194.17 (2H, m, CH-Fc), 4.15 (5H, s, Cp-Fc). C-NMR (151 MHz, DMSO) δ 143.87 (C2), 143.40 (C3a),
0 0
42.54 (C4 ), 133.45 (C7a), 123.09 (C5 ), 122.28 (C6), 121.54 (C4), 119.37 (C5), 110.66 (C7), 82.24 (Cq-Fc),
2
2
13
6
8.56 (Cp-Fc), 68.29 (CH-Fc), 48.88 (CH ). Anal. calcd. for C H FeN : C, 63.65; H, 4.58; N, 17.67.
2 21 18 5
Found: C, 63.71; H, 4.59; N, 17.69.
1-[1-(1-Ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-1H-benzimidazole (23b) Compound 23b was
prepared using the above-mentioned procedure using compound 20 (82 mg, 0.53 mmol) and
1
1
-azidoethylferrocene (163.2 mg, 0.64 mmol) to obtain 23b as brown powder (37.48 mg, 17 %, m.p. =
40 C). H-NMR (600 MHz, DMSO) δ 8.33 (1H, s, H2), 8.15 (1H, s, H5 ), 7.64 (2H, dd, J = 7.4, 4.7 Hz,
◦
1
0

Molecules 2020, 25, 1570
14 of 24
H4/), 7.23 (1H, t, J = 7.4 Hz, H6), 7.19 (1H, t, J = 7.5 Hz, H5), 5.66 (1H, q, J = 7.0 Hz, CH), 5.53 (2H, s,
CH ), 4.31 (1H, s, CH-Fc), 4.214.13 (3H, m, CH-Fc), 4.09 (5H, s, Cp-Fc), 1.78 (3H, d, J = 7.0 Hz, CH ).
2
3
13
0
0
C-NMR (75 MHz, DMSO)
δ 143.94 (C3a), 142.73 (C4 ), 133.99 (C7a), 122.77 (C6), 122.19 (C5 ), 122.03
(C4), 119.88 (C5), 111.15 (C7), 88.96 (Cq-Fc), 69.13 (Cp-Fc), 68.61 (CH-Fc), 68.15 (CH-Fc), 67.65 (CH-Fc),
6
6.64 (CH-Fc), 56.12 (CH), 39.86 (CH ), 21.37 (CH ). Anal. calcd. for C H FeN : C, 64.40; H, 4.91; N,
2
3
22 20
5
1
7.07. Found: C, 64.14; H, 4.92; N, 17.10.
1-[1-(1-Ferrocenyl-1,2,3-triazol-4-yl)methyl]-1H-benzimidazole (23c) Compound 23c was prepared using
the above-mentioned procedure using compound 20 (83 mg, 0.53 mmol) and 1-azidoferrocene (144.7
◦
1
mg, 0.64 mmol) to obtain 23c as orange powder (25 mg, 12 %, m.p. > 250 C). H-NMR (600 MHz,
0
DMSO)
7
δ
8.64 (1H, s, H5 ), 8.39 (1H, s, H2), 7.66 (2H, s, H4/7), 7.24 (1H, t, J = 7.4 Hz, H6), 7.20 (1H, t, J =
.2 Hz, H5), 5.62 (2H, s, CH ), 4.99 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.17 (5H, s, Cp-Fc). C-NMR
2
13
0
(75 MHz, DMSO)
δ
144.01 (C3a), 143.39 (C4 ), 134.00 (C7a), 124.01 (C5’), 122.84 (C6), 122.12 (C4),
119.96 (C5), 111.15 (C7), 93.78 (Cq-Fc), 70.37 (Cp-Fc), 67.11 (CH-Fc), 62.41 (CH-Fc). Anal. calcd. for
C H FeN : C, 62.68; H, 4.47; N, 18.27. Found: C, 62.74; H, 4.46; N, 18.29.
20
17
5
1-[1-(1-Ferrocenylmethyl-1,2,3-triazol-4-yl)methyl]-1H-indole (24a) Compound 24a was prepared using
the above-mentioned procedure using compound 21 (70 mg, 0.45 mmol) and 1-methylazidoferrocene
◦
1
(
130.2 mg, 0.54 mmol) to obtain 24a as orange powder (25 mg, 14 %, m.p. = 135 C). H-NMR (600
0
MHz, DMSO)
δ
7.99 (1H, s, H5 ), 7.56 (1H, d, J = 7.9 Hz, H7), 7.51 (1H, d, J = 7.9 Hz, H4), 7.42 (1H, d, J
=
3.1 Hz, H2), 7.11 (1H, t, J = 7.2 Hz, H6), 7.00 (1H, t, J = 7.0 Hz,H5), 6.42 (1H, d, J = 2.7 Hz, H3), 5.42
(
2H, s, CH ), 5.24 (2H, s, CH ), 4.28 (2H, t, J = 1.8 Hz, CH-Fc), 4.15 (2H, t, J = 1.8 Hz, CH-Fc), 4.13 (5H,
2
1
2
3
0
s, Cp-Fc). C-NMR (101 MHz, DMSO)
(
δ
144.07 (C4 ), 135.98 (C7a), 129.04 (C2), 128.68 (C3a), 123.18
0
C5 ), 121.53 (C6), 120.85 (C4), 119.56 (C5), 110.55 (C7), 101.40 (C3), 82.83 (Cq-Fc), 69.07 (Cp-Fc), 68.77
(
CH-Fc), 49.32 (CH ), 41.25 (CH ). Anal. calcd. for C H FeN : C, 66.68; H, 5.09; N, 14.14. Found: C,
2
2
22 20
4
6
6.48; H, 5.08; N, 14.18.
1
-[1-(1-Ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-1H-indole (24b) Compound 24b was prepared
using the above-mentioned procedure using compound 21 (75 mg, 0.48 mmol) and
1
1
-azidoethylferrocene (147.9 mg, 0.58 mmol) to obtain 24b as brown oil (40 mg, 20 %). H-NMR (300
0
MHz, DMSO)
δ
8.02 (1H, s, H5 ), 7.57 (1H, d, J = 7.6 Hz, H7), 7.51 (1H, d, J = 7.8 Hz, H4), 7.41 (1H, d, J
=
3.1 Hz, H2), 7.11 (1H, t, J = 7.0 Hz, H6), 7.00 (1H, t, J = 7.0 Hz, H5), 6.42 (1H, dd, J = 3.1, 0.6 Hz, H3),
5
.64 (1H, q, J = 7.0 Hz, CH), 5.41 (2H, s, CH ), 4.314.27 (1H, m, CH-Fc), 4.174.12 (3H, m, CH-Fc), 4.08
2
13
0
(
5H, s, Cp-Fc), 1.77 (3H, d, J = 7.0 Hz, CH ). C-NMR (75 MHz, DMSO)
δ
143.75 (C4 ), 135.98 (C7a),
3
0
128.99 (C2), 128.65 (C3a), 121.79 (C5 ), 121.52 (C6), 120.84 (C4), 119.55 (C5), 110.53 (C7), 101.39 (C3),
8
4
5
9.00 (Cq-Fc), 69.14 (Cp-Fc), 68.59 (CH-Fc), 68.13 (CH-Fc), 67.66 (CH-Fc), 66.62 (CH-Fc), 56.02 (CH),
1.25 (CH ), 21.37 (CH ). Anal. calcd. for C H FeN : C, 67.33; H, 5.40; N, 13.66. Found: C, 67.19; H,
2
3
23 22
4
.41; N, 13.63.
1-[1-(1-Ferrocenyl-1,2,3-triazol-4-yl)methyl]-1H-indole (24c) Compound 24c was prepared using the
above-mentioned procedure using compound 21 (132 mg, 0. 85 mmol) and 1-azidoferrocene (230.6 mg,
◦
1
1
.02 mmol) to obtain 24c as brown crystals (32 mg, 9.8 %, m.p. = 120 C). H-NMR (300 MHz, DMSO)
0
δ
7
4
δ
8.53 (1H, s, H5 ), 7.61 (1H, d, J = 8.2 Hz, H7), 7.55 (1H, d, J = 7.8 Hz, H4), 7.48 (1H, d, J = 3.2 Hz, H2),
.14 (1H, t, J = 7.6 Hz, H6), 7.02 (1H, t, J = 7.0 Hz, H5), 6.47 (1H, d, J = 3.1 Hz, H3), 5.50 (2H, s, CH ),
.97 (2H, t, J = 1.9 Hz, CH-Fc), 4.354.27 (2H, m, CH-Fc), 4.17 (5H, s, Cp-Fc). C-NMR (75 MHz, DMSO)
144.37 (C4 ), 136.01 (C7a), 129.14 (C2), 128.77 (C3a), 123.71 (C5 ), 121.58 (C6), 120.91 (C4), 119.63 (C5),
10.57 (C7), 101.50 (C3), 93.86 (Cq-Fc), 70.35 (Cp-Fc), 67.06 (CH-Fc), 62.40 (CH-Fc), 41.23 (CH ). Anal.
2
13
0
0
1
2
calcd. for C H FeN : C, 65.99; H, 4.75; N, 14.66. Found: C, 66.06; H, 4.76; N, 14.63.
21
18
4
5-Iodo-1-[1-(1-ferrocenylmethyl-1,2,3-triazol-4-yl)methyl]-1H-indole (25a) Compound 25a was prepared
using the above-mentioned procedure using compound 22 (70 mg, 0.25 mmol) and
1
=
-methylazidoferrocene (72.3 mg, 0.30 mmol) to obtain 25a as brown crystals (79 mg, 15 %, m.p.
109 C). H-NMR (300 MHz, DMSO) δ 7.97 (1H, s, H5 ), 7.89 (1H, s, H4), 7.44 (2H, m, H2/7), 7.37
◦
1
0

Molecules 2020, 25, 1570
15 of 24
(
(
(
(
1H, dd, J = 8.6, 1.5 Hz, H6), 6.39 (1H, d, J = 3.0 Hz, H3), 5.42 (2H, s, CH ), 5.24 (2H, s, CH ), 4.304.24
2 2
13
2H, m, CH-Fc), 4.164.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). C-NMR (75 MHz, DMSO)
C4 ), 135.10 (C7a), 131.44 (C3a), 130.17 (C2), 129.43 (C6), 129.25 (C4), 123.19 (C5 ), 113.17 (C7), 100.84
C3), 83.49 (C5), 82.79 (Cq-Fc), 69.06 (Cp-Fc), 68.77 (CH-Fc), 49.32 (CH ), 41.35 (CH ). Anal. calcd. for
δ
143.77
0
0
2
2
C H FeIN : C, 50.60; H, 3.67; N, 10.73. Found: C, 50.65; H, 3.68; N, 10.76.
22
19
4
5-Iodo-1-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-1H-indole (25b) Compound 25b was
prepared using the above-mentioned procedure using compound 22 (223 mg, 0.79 mmol) and
1
1
-azidoethylferrocene (242.3 mg, 0.95 mmol) to obtain 25b as orange powder (125 mg, 29 %, m.p. =
18 C). H-NMR (300 MHz, DMSO)
◦
1
0
δ 7.97 (1H, s, H5 ), 7.89 (1H, s, H4), 7.44 (2H, m, H2/7), 7.37 (1H,
dd, J = 8.6, 1.5 Hz, H6), 6.39 (1H, d, J = 3.0 Hz, H3), 5.42 (2H, s, CH ), 5.24 (2H, s, CH ), 4.304.24 (2H,
2
2
m, CH-Fc), 4.164.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO)
1
δ
143.77 (C4 ),
0
0
35.10 (C7a), 131.44 (C3a), 130.17 (C2), 129.43 (C6), 129.25 (C4), 123.19 (C5 ), 113.17 (C7), 100.84 (C3),
8
3.49 (C5), 82.79 (Cq-Fc), 69.06 (Cp-Fc), 68.77 (CH-Fc), 49.32 (CH ), 41.35 (CH ). Anal. calcd. for
2
2
C H FeIN : C, 51.52; H, 3.95; N, 10.45. Found: C, 51.57; H, 3.96; N, 10.49.
23
21
4
5
-Iodo-1-[1-(1-ferrocenyl-1,2,3-triazol-4-yl)methyl]-1H-indole (25c) Compound 25c was prepared using the
above-mentioned procedure using compound 22 (345 mg, 1.23 mmol) and 1-azidoferrocene (334.5 mg,
◦
1
1
8
.48 mmol) to obtain 25c as brown crystal (53 mg, 10 %, m.p. = 169 C). H-NMR (600 MHz, DMSO)
δ
0
.52 (1H, s, H5 ), 7.92 (1H, s, H4), 7.517.46 (2H, m, H2/7), 7.39 (1H, d, J = 8.6 Hz, H6), 6.44 (1H, d, J = 3.0
Hz, H3), 5.49 (2H, s, CH ), 4.97 (2H, s, CH-Fc), 4.32 (2H, s, CH-Fc), 4.17 (5H, s, Cp-Fc). C-NMR (151
143.60 (C4 ), 134.63 (C7a), 131.01 (C3a), 129.77 (C2), 128.97 (C6), 128.81 (C4), 123.22
C5 ), 112.67 (C7), 100.43 (C3), 93.32 (Cq-Fc), 83.09 (C5), 69.86 (Cp-Fc), 66.58 (CH-Fc), 61.91 (CH-Fc),
13
2
0
MHz, DMSO)
(
δ
0
4
0.81 (CH ). Anal. calcd. for C H FeIN : C, 49.64; H, 3.73; N, 11.03. Found: C, 49.54; H, 3.72; N,
2 21 17 4
1
0.99.
3
.4. General Procedure for N-alkylation of Compounds 26 and 27
To a solution of potassium hydroxide (2 eq.) in water, corresponding heterocyclic base 1, 3 (1
eq.) and corresponding amine (4 eq.) were added. Reaction mixture was stirred for 10 min under
◦
microwave irradiation at 100 C and 400 W. Formed precipitate was filtered off and dried.
4-(Pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine (26) Compound 26 was prepared using the above-
mentioned procedure using compound 1 (500 mg; 3.26 mmol) to obtain 26 as white powder (613 mg,
◦
1
9
5 %, m.p. > 250 C). H-NMR (300 MHz, DMSO)
δ 11.56 (1H, bs, NH), 8.08 (1H, s, H2), 7.09 (1H, d, J =
3
.5 Hz, H6), 6.56 (1H, d, J = 3.5 Hz, H5), 3.71 (4H, bs, CH -pyrrolidine), 1.95 (4H, bs, CH -pyrrolidine).
2
2
1
3
C-NMR (151 MHz, DMSO)
δ 154.77 (C4), 151.18 (C2), 150.89 (7a), 120.31 (C6), 102.43 (4a), 100.79 (C5),
4
6
7.38 (CH -pyrrolidine). Anal. calcd. for C H N : C, 63.81; H, 6.43; N, 29.77. Found: C, 63.87; H,
.44; N, 29.65.
2
10 12
4
4-(Piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine (27) Compound 27 was prepared using the above-
mentioned procedure using compound 1 (500 mg; 3.26 mmol) to obtain 27 as white powder (659 mg,
◦
1
8
4 %, m.p. = 189 C). H-NMR (300 MHz, DMSO)
δ 11.64 (1H, bs, NH), 8.12 (1H, s, H2), 7.15 (1H,
d, J = 3.5 Hz, H6), 6.55 (1H, d, J = 3.5 Hz, H5), 3.96–3.76 (4H, m, CH -piperidine), 1.73–1.62 (2H, m,
2
13
CH -piperidine), 1.62–1.50 (4H, m, CH -piperidine). C-NMR (151 MHz, DMSO)
δ
156.27 (C4), 151.90
2
2
(
C7a), 150.65 (C2), 120.99 (C6), 101.99 (C4a), 100.90 (C5), 46.23 (CH -piperidine), 25.45 (CH -piperidine),
2
2
2
4.25 (CH -piperidine). Anal. calcd. for C H N : C, 65.32; H, 6.98; N, 27.70. Found: C, 65.12; H, 6.99;
2 11 14 4
N, 27.76.
3
.5. General Procedure for N-alkylation of Compounds 30b, 30c, 31a–31c
To a solution of the corresponding heterocyclic base 26 29 (1 eq.) in anhydrous DMF, NaH (1.2
eq.) was added and stirred for 30 min under argon atmosphere. 1,2-Dibromoethane (1 eq.) was added
1,
3,
–

Molecules 2020, 25, 1570
16 of 24
and the reaction mixture was stirred at room temperature for 24 h. Solvent was evaporated and the
residue was purified by column chromatography (hexane:ethyl acetate = 8:1).
9-(2-Bromoethyl)-6-(pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine (30b) Compound 30b was prepared
using the above-mentioned procedure using compound 26 (400 mg; 2.13 mmol) to obtain 30b as yellow
◦
1
powder (106 mg; 16 %, m.p.= 109 C). H-NMR (600 MHz, DMSO) δ 8.11 (1H, s, H2), 7.24 (1H, d, J =
3
.5 Hz, H8), 6.61 (1H, d, J = 3.5 Hz, H7), 4.52 (2H, t, J = 6.4 Hz, CH ), 3.85 (2H, t, J = 6.3 Hz, CH ), 3.70
2
2
13
(
4H, bs, CH -pyrrolidine), 1.96 (4H, bs, CH -pyrrolidine). C-NMR (101 MHz, DMSO)
δ
155.17 (C6),
2
2
151.61 (C2), 150.27 (C4), 124.29 (C8), 103.18 (C5), 101.08 (C7), 47.96 (CH -pyrrolidine), 45.93 (CH ),
2 2
3
2.35 (CH ). Anal. calcd. for C H BrN : C, 48.83; H, 5.12; N, 18.98. Found: C, 48.73; H, 5.11; N, 18.94.
2
12 15
4
9
-(2-Bromoethyl)-6-(piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine (30c) Compound 30c was prepared using
the above-mentioned procedure using compound 27 (400 mg; 1.97 mmol) to obtain 30c as yellow
◦
1
powder (87 mg; 14 %, m.p. = 70 C). H-NMR (600 MHz, DMSO)
δ 8.14 (1H, s, H2), 7.29 (1H, d, J = 3.6
Hz, H8), 6.59 (1H, d, J = 3.7 Hz, H7), 4.52 (2H, t, J = 6.4 Hz, CH ), 3.84 (6H, m, CH , CH -piperidine),
2
2
2
13
1
.65 (2H, m, CH -piperidine), 1.59–1.54 (4H, m, CH -piperidine). C-NMR (151 MHz, DMSO)
δ
156.20
2
2
(
C6), 150.73 (C4), 150.67 (C2), 124.32 (C8), 102.21 (C5), 100.65 (C7), 46.21 (CH -piperidine), 45.50 (CH ),
2
2
31.74 (CH ), 25.46 (CH -piperidine), 24.20 (CH -piperidine). Anal. calcd. for C H BrN : C, 50.50; H,
2 2 2 13 17 4
5
.54; N, 18.12. Found: C, 50.60; H, 5.52; N, 18.06.
-(2-Bromoethyl)-6-chloro-9H-purine (31a) Compound 31a was prepared using the above-mentioned
(500 mg; 3.23 mmol) to obtain 31a as white powder (312 mg; 36 %; m.p.=
11 C). H-NMR (400 MHz, DMSO) 8.82 (1H, s, H2), 8.77 (1H, s, H8), 4.75 (2H, t, J = 6.1 Hz, CH2),
.01 (2H, t, J = 6.1 Hz, CH2). 13C-NMR (101 MHz, DMSO)
152.42 (C6), 152.14 (C2), 149.59 (C4), 147.99
C8), 131.26 (C5), 45.82 (CH ), 31.64 (CH ). Anal. calcd. for C H BrClN : C, 32.15; H, 2.31; N, 21.42.
9
procedure using compound
1
4
(
3
◦
1
δ
δ
2
2
7
6
4
Found: C, 32.05; H, 2.32; N, 21.47.
-(2-Bromoethyl)-6-(pyrrolidin-1-yl)-9H-purine (31b) Compound 31b was prepared using the
above-mentioned procedure using compound 28 (500 mg; 2.65 mmol) to obtain 31b as white powder
9
◦
1
(
(
208 mg; 27 %; m.p.= 163 C). H-NMR (400 MHz, DMSO) δ 8.22 (1H, s, H2), 8.17 (1H, s, H8), 4.58
2H, t, J = 6.1 Hz, CH ), 4.07 (2H, bs, CH -pyrrolidine), 3.95 (2H, t, J = 6.1 Hz, CH ), 3.64 (2H, s,
CH -pyrrolidine), 1.95 (4H, s, CH -pyrrolidine). C-NMR (101 MHz, DMSO)
2
2
2
13
δ
152.95 (C4), 152.70
2
2
(
C2), 150.37 (C6), 140.71 (C8), 119.81 (C5), 45.07 (CH ), 32.03 (CH ). Anal. calcd. for C H BrN : C,
2
2
11 14
5
4
4.61; H, 4.76; N, 23.65. Found: C, 44.65; H, 4.75; N, 23.60.
9-(2-Bromoethyl)-6-(piperidin-1-yl)-9H-purine (31c) Compound 31c was prepared using the
above-mentioned procedure using compound 29 (500 mg; 2.46 mmol) to obtain 31c as white powder
◦
1
(
(
200 mg; 27 %; m.p. = 149 C. H-NMR (400 MHz, DMSO)
δ 8.23 (1H, s, H2), 8.20 (1H, s, H8), 4.58
2H, t, J = 6.1 Hz, CH ), 4.20 (4H, bs, CH -piperidine), 3.94 (2H, t, J = 6.1 Hz, CH ), 1.73–1.63 (2H,
m, CH -piperidine), 1.63–1.55 (4H, m, CH -piperidine). C-NMR (101 MHz, DMSO)
2
2
2
13
δ
153.58 (C4),
2
2
1
2
2
52.39 (C2), 150.95 (C6), 140.18 (C8), 119.34 (C5), 46.08 (CH -piperidine), 45.13 (CH ), 31.94 (CH ),
2
2
2
6.15 (CH -piperidine), 24.74 (CH -piperidine). Anal. calcd. for C H BrN : C, 46.46; H, 5.20; N,
2
2
16 16
5
2.58. Found: C, 46.36; H, 5.19; N, 22.62.
3
.6. General Procedure for the Synthesis of Azidoethyl Derivatives of Purinomimetics
The corresponding N-alkylated heterocyclic base 30a 30c 31a 31c (1 eq.) was dissolved in acetone.
–
,
–
To suspension was added dropwise solution of sodium azide (4 eq.) in water. The reaction mixture
◦
was stirred at 60 C overnight. Solvent was evaporated and crude product was used as is in next step.
3
.7. General Procedure for the Synthesis of Purinomimetics and Purines with Ferrocene at C-4 of 1,2,3-Triazole
The corresponding terminal azide 32a 32c 33a 33c (1 eq.) was dissolved in methanol, Cu(OAc)2
0.05 eq), and the ethynylferrocene (1.2 eq.) were added. The reaction mixture was stirred at
–
,
–
(

Molecules 2020, 25, 1570
17 of 24
room temperature overnight. Solvent was evaporated and the residue was purified by column
chromatography (CH Cl :CH OH = 60:1).
2
2
3
4
-Chloro-7-(2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine (34a). Compound 34a was
prepared using the above-mentioned procedure using compound 32a (0.76 mmol) and ethynylferrocene
◦
1
(
193.2 mg, 0.92 mmol) to obtain 34a as orange powder (20.7 mg, 6 %, m.p. = 196 C). H-NMR (600
0
MHz, DMSO)
δ
8.61 (1H, s, H2), 7.93 (1H, s, H5 ), 7.62 (1H, d, J = 3.6 Hz, H6), 6.63 (1H, d, J = 3.6 Hz,
H5), 4.88 (2H, dd, J = 6.7, 4.5 Hz, CH ), 4.81 (2H, dd, J = 6.7, 4.6 Hz, CH ), 4.59 (2H, t, J = 1.8 Hz,
2
2
13
CH-Fc), 4.27–4.21 (2H, m, CH-Fc), 3.91 (5H, s,Cp-Fc). C-NMR (151 MHz, DMSO)
C7a), 150.27 (C2), 145.16 (C4 ), 131.09 (C6), 120.58 (C5 ), 116.69 (C4a), 98.76 (C5), 75.60 (Cq-Fc), 69.12
δ
150.70 (C4), 150.59
0
0
(
(
Cp-Fc), 68.18 (CH-Fc), 66.13 (CH-Fc), 48.80 (CH ), 44.54 (CH ). Anal. calcd. for C H ClFeN : C,
2
2
20 17
6
5
5.52; H, 3.96; N, 19.42. Found: C, 55.56; H, 3.95; N, 19.37.
4
3
-(Pyrrolidin-1-yl)-7-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (34b) Compound
4b was prepared using the above-mentioned procedure using compound 32b (0.68 mmol) and
ethynylferrocene (172.2 mg, 0.82 mmol) to obtain 34b as orange powder (122 mg, 38 %, m.p. = 200
◦
1
0
δ 8.12 (1H, s, H2), 7.89 (1H, s, H5 ), 6.96 (1H, d, J = 3.6 Hz, H6),
C). H-NMR (300 MHz, DMSO)
.53 (1H, d, J = 3.6 Hz, H5), 4.80 (2H, t, J = 5.5 Hz, CH ), 4.73–4.64 (2H, m, CH ), 4.63–4.59 (2H,
6
2
2
m, CH-Fc), 4.29–4.24 (2H, m, CH-Fc), 3.94 (5H, s, Cp-Fc), 3.66 (4H, s, CH -pyrrolidine), 1.91 (4H, s,
2
13
0
CH -pyrrolidine). C-NMR (75 MHz, DMSO)
1
δ
155.19 (C4), 151.71 (C2), 150.31 (C7a), 145.56 (C4 ),
23.87 (C6), 121.03 (C5 ), 103.11 (C4a), 101.23 (C5), 76.26 (Cq-Fc), 69.64 (Cp-Fc), 68.63 (CH-Fc), 66.63
2
0
(
CH-Fc), 49.46 (CH ), 47.90 (CH -pyrrolidine), 44.27 (CH ). Anal. calcd. for C H FeN : C, 61.68; H,
2 2 2 24 25 7
5
.39; N, 20.98. Found: C, 61.63; H, 5.38; N, 21.04.
4
-(Piperidin-1-yl)-7-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (34c) Compound
3
4c was prepared using the above-mentioned procedure using compound 32c (0.64 mmol) and
◦
ethynylferrocene (162.8 mg, 0.77 mmol) to obtain 34c as orange powder (55 mg, 17 %, m.p. = 171 C).
1
0
H-NMR (600 MHz, DMSO) δ 8.16 (1H, s, H2), 7.89 (1H, s, H5 ), 7.03 (1H, d, J = 3.6 Hz, H6), 6.54 (1H,
d, J = 3.6 Hz, H5), 4.81 (2H, t, J = 5.8 Hz, CH ), 4.67 (2H, t, J = 5.7 Hz, CH ), 4.61 (2H, d, J = 1.5 Hz,
2
2
CH-Fc), 4.28–4.24 (2H, m, CH-Fc), 3.93 (5H, s, Cp-Fc), 3.82–3.79 (4H, m, CH -pipridin), 1.64–1.60 (2H,
2
1
3
m, CH -pipridin), 1.52 (4H, d, J = 3.7 Hz, CH -pipridin). C-NMR (75 MHz, DMSO)
δ
156.63 (C4),
51.27 (C7a), 151.20 (C2), 145.60 (C4 ), 124.33 (C6), 121.05 (C5 ), 102.59 (C4a), 101.40 (C5), 76.22 (Cq-Fc),
9.65 (Cp-Fc), 68.63 (CH-Fc), 66.63 (CH-Fc), 49.41 (CH ), 46.63 (CH -piperidine), 44.30 (CH ), 25.87
2
2
0
0
1
6
2
2
2
(
CH -piperidine), 24.65 (CH -piperidine). Anal. calcd. for C H FeN : C, 62.38; H, 5.65; N, 20.37.
2 2 25 27 7
Found: C, 62.42; H, 5.64; N, 20.31.
-Chloro-9-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-9H-purine (35a) Compound 35a was prepared using
the above-mentioned procedure using compound 33a (0.76 mmol) and ethynylferrocene (193.2 mg,
6
◦
1
0
δ
.92 mmol) to obtain 35a as orange powder (123 mg, 37 %, m.p. = 240 C). H-NMR (300 MHz, DMSO)
0
8.76 (1H, s, H2), 8.50 (1H, s, H5 ), 8.02 (1H, s, H8), 4.94–4.90 (2H, m, CH ), 4.84 (2H, d, J = 6.1 Hz,
2
13
CH ), 4.60 (2H, d, J = 1.6 Hz, CH-Fc), 4.28–4.25 (2H, m, CH-Fc), 3.94 (5H, s, Cp-Fc). C-NMR (75
2
0 0
δ 152.43 (C6), 152.06 (C2), 149.52 (C4), 147.73 (C8), 145.87 (C4 ), 131.16 (C5), 121.23 (C5 ),
MHz, DMSO)
6.02 (Cq-Fc), 69.64 (Cp-Fc), 68.70 (CH-Fc), 66.67 (CH-Fc), 48.88 (CH ), 44.37 (CH ). Anal. calcd. for
7
2
2
C19H16ClFeN7: C, 52.62; H, 3.72; N, 22.61. Found: C, 52.56; H, 3.71; N, 22.66.
-(Pyrrolidin-1-yl)-9-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-9H-purine (35b) Compound 35b was prepared
using the above-mentioned procedure using compound 33b (0.68 mmol) and ethynylferrocene (172.2
6
◦
1
mg, 0.82 mmol) to obtain 35b as orange powder (124 mg, 39 %, m.p. = 230 C). H-NMR (300 MHz,
0
DMSO)
δ
8.21 (1H, s, H2), 7.96 (1H, s, H5 ), 7.84 (1H, s, H8), 4.86 (2H, t, J = 5.6 Hz, CH ), 4.70 (2H, t, J =
2
5
.6 Hz, CH ), 4.62 (2H, t, J = 1.8 Hz, CH-Fc), 4.31–4.21 (2H, m, CF-Fc), 3.95 (7H, s, Cp-Fc), 3.59 (2H,
2
13
s, CH -pyrrolidine), 1.89 (4H, s, CH -pyrrolidine). C-NMR (75 MHz, DMSO)
δ
152.90 (C6), 152.72
2
2
(C2), 150.36 (C4), 145.73 (C4’), 140.32 (C8), 121.12 (C5’), 119.71 (C5), 76.13 (Cq-Fc), 69.64 (Cp-Fc), 68.65

Molecules 2020, 25, 1570
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(
CH-Fc), 66.63 (CH-Fc), 48.94 (CH -pyrrolidine), 43.45 (CH -pyrrolidine). Anal. calcd. for C H FeN :
2 2 23 24 8
C, 58.98; H, 5.17; N, 23.93. Found: C, 58.92; H, 5.16; N, 23.96.
-(Piperidin-1-yl)-9-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-9H-purine (35c) Compound 35c was prepared
using the above-mentioned procedure using compound 33c (0.32 mmol) and ethynylferrocene (162.8
6
◦
1
mg, 0.77 mmol) to obtain 35c as orange powder (47 mg, 15 %, m.p. = 233 C). H-NMR (300 MHz,
0
DMSO)
δ
8.22 (1H, s, H2), 7.97 (1H, s, H5 ), 7.88 (1H, s, H8), 4.87 (2H, t, J = 5.5 Hz, CH ), 4.70 (2H, t, J =
2
5
.7 Hz, CH ), 4.66–4.55 (2H, m, CH-Fc), 4.30–4.22 (2H, m, CH-Fc), 4.14 (4H, bs, CH -piperidin), 3.94
2 2
13
(
5H, s, Cp-Fc), 1.62 (2H, s, CH -piperidine), 1.52 (4H, s, CH -piperidine). C-NMR (75 MHz, DMSO) δ
2 2
0
0
153.52 (C6), 152.39 (C2), 150.95 (C4), 145.75 (C4 ), 121.14 (C5 ), 119.22 (C5), 76.10 (Cq-Fc), 69.65 (Cp-Fc),
6
8.66 (CH-Fc), 66.64 (CH-Fc), 48.93 (CH ), 43.48 (CH ), 26.04 (CH -piperidine), 24.68 (CH -piperidine).
2
2
2
2
Anal. calcd. for C H FeN : C, 59.76; H, 5.43; N, 23.23. Found: C, 59.70; H, 5.42; N, 23.28.
24
26
8
3
.8. Kinetic Solubility Assay
The compounds’ DMSO stock solutions in concentration of 10 mM were serially diluted by factor
, 3 , 3.3 and 3 in DMSO. Prepared solutions were spiked into phosphate-buffered saline (PBS,
M (3 L of prepared
3.3
×
×
×
×
100 mM) to obtain final compounds’ concentrations of 100, 30, 10, 3 and 1
µ
µ
dilutions were spiked to 297
µ
L of PBS, with final DMSO content of 1%, v/v). All compounds were
◦
tested in two replicas per concentration. Prepared solutions were incubated at 37 C for 2 h with
gentle shaking. Blank solutions were also prepared by spiking DMSO in PBS buffer (1% of DMSO,
v/v). After the incubation, absorbance of suspensions was measured by Microplate reader Infinite
F500 (Tecan, Männedorf, Switzerland) at 620 nm and compared to a blank control to obtain the tested
solutions’ absorbance, which is proportionally increased with concentration of insoluble particles.
Standard compounds α-naphtoflavone (low solubility) and sulfaphenazole (high solubility) were used
as controls. Compound/control samples are compared to the blank solution. Precipitation occurred
if significant increase of sample absorbance is observed, i.e., when its absorbance is 3-fold standard
deviation of average blank absorbance. Results were expressed as an estimated solubility range (lower
and upper bound).
3
.9. Chrom logD Determination
The distribution coefficient, Chrom logD, was determined from compounds’ gradient retention
times by employing a high-performance liquid chromatography method with fast acetonitrile gradient.
Compounds were prepared for analysis in final concentration of 1.25 mM by dilution of 10 mM stock
solutions in DMSO with acetonitrile. Aqueous mobile phase was 50 mM ammonium acetate adjusted
with ammonia solution to pH 7.4 and the organic mobile phase was acetonitrile. A reversed phase
HPLC column Luna C18, 50
for analysis. Sample injection volume was 2
analysed in duplicates. Fast-gradient elution was as follows: from 0 to 100% of acetonitrile (0
×
3 mm i.d., 5 µm particle size (Phenomenex, Torrance, CA, USA) was used
−1
µ
L and the flow rate was 1 mL min . All compounds were
3 min),
3.7 min), and re-equilibration with
5.0 min). All measurements were performed on an HPLC-DAD
−
1
1
00% of acetonitrile (3.0
00% of aqueous mobile phase (3.7
−
3.5 min), from 100 to 0% of acetonitrile (3.5
−
−
instrument Agilent 1100 Series (Agilent Technologies, Santa Clara, CA, USA) coupled with a mass
spectrometer Micromass Quattro API (Waters, MA, USA). MassLynx software, version 4.1 (Waters,
Manchester, UK) was used for data acquisition and processing. Before compound analysis, a calibration
step was performed. A set of standard compounds with literature CHI values [36] were plotted against
gradient retention times to obtain a calibration equation, which was used to determine CHI value from
the compounds’ retention times. Chrom logD values were calculated from the CHI value using the
equation Chrom logD = 0.0857 × CHI − 2. [38]

Molecules 2020, 25, 1570
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3
.10. ADME Properties
3
.10.1. Permeability and P-glicoprotein Substrate Assessment
MDCKII-hMDR1 cell monolayer was used for determination of permeability and P-gp substrate
assessment. MDCKII-hMDR1 cells (Solvo Biotechnology, Szeged, Hungary) were grown in the
◦
controlled atmosphere (37 C, 95% humidity, 5% CO ) in MDCK cell culture medium containing
2
Dulbecco’s Modified Eagle Medium (DMEM) + 10% Fetal bovine serum (FBS, inactivated) + 1%
glutamax-100 + 1% antibiotic/antimycotic + 1% of Non-essential amino acids Minimum Essential
Medium (MEM NEAA). Cultures were split every 3–4 days using 0.05% Trypsin-EDTA. Cells were
6
−1
seeded 4 days prior to the experiment in concentration of 0.3
×
10 cells mL and cultured in CO2
incubator. On the experiment day, cell monolayers were washed with Dulbecco’s phosphate buffer
saline (D-PBS) and equilibrated for 45 min in CO incubator in D-PBS containing 1% DMSO (1% v/v)
2
with or without Elacridar (2
transport medium containing Lucifer yellow (100
the presence and without Elacridar. Lucifer yellow calibration curve was prepared by serial dilution
12 points, with 100 M as the highest point). Monolayer integrity was examined by fluorescent
µM). Compounds, in final concentration of 10
µM, were prepared in
µM), with DMSO contentment of 1% (v/v) and in
(
µ
measurement on a Microplate reader Infinite F500 (Tecan) by using an excitation of 485 nm and
emission of 530 nm. Experiment was started by applying the solutions containing test compounds to
apical and basolateral side of the cell monolayer. Starting compound concentration (C ) was sampled
0
at the start of the experiment and apical and basolateral compartments were sampled after incubation
◦
at 37 C with gentle shaking for 60 min. Compound were tested in duplicate. Amprenavir was used
as a control with low permeability and as a P-gp substrate without inhibitor presence, turning into a
highly permeable compound with the inhibitor present. Diclofenac was used as a control with high
permeability and no interaction with P-gp.
Samples were analyzed on an ABSciex API 4000 Triple Quadrupole Mass Spectrometer (Sciex,
Division of MDS Inc., Toronto, ON, Canada) coupled to a UHPLC Nexera X2 (Shimadzu, Kyoto, Japan).
Samples (1
i.d., 1.6
µ
L) were injected onto a reversed phase HPLC column Luna Omega Polar C18, 30
×
2.1 mm
◦
µ
m particle size (Phenomenex) which was kept at 50 C. Aqueous solution was 0.1% formic
acid in deionized water and the organic mobile phase was water/acetonitrile/formic acid mixture
−1
(
90/10/0.1, v/v/v). Flow rate was kept at 0.7 mL min for all measurements. Fast-gradient elution
was as follows: 2% of organic mobile phase through 0.15 min, from 2 to 95% of organic mobile phase
(
0.15
−
0.7 min), 95% of organic mobile phase (0.7 1.1 min), from 95 to 2% of organic mobile phase
−
(1.1
−
1.11 min), and re-equilibration with 98% of aqueous mobile phase (1.11
−1.5 min). Positive ion
◦
mode with turbo spray, an ion source temperature of 550 C and a dwell time of 75 ms were utilized for
mass spectrometric detection. Quantitation was performed using multiple reaction monitoring (MRM)
at the specific transition corresponding to the compound of interest. Warfarin was used as an internal
standard. The ratios between compound and internal standard peak areas were used instead of real
−1
compound concentration. Apparent permeability coefficient, Papp (nm s ) values were calculated by
using the following Equation (1):
−
1
Papp [nm s ] = (dQ/dT) × (1/C ) × (1/A)
(1)
0
where: dQ/dT = permeability rate; C = initial concentration in donor compartment; A = surface area
0
2
of the cell monolayer (0.7 cm ).
The efflux ratio in the presence or absence of the P-gp inhibitor was calculated from Papp values,
using following Equation (2):
Efflux ratio = Papp (BA)/Papp(AB)
(2)

Molecules 2020, 25, 1570
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3
.10.2. Metabolic Stability in Liver Microsomes
Metabolic stability of compounds was assessed in human and mouse liver microsomes (Corning,
Tewksbury, MA, USA). Compounds, in final concentration of 1
buffer (50 mM, pH 7.4) for 60 min at 37 C together with liver microsomes and NADPH generating
µ
M, were incubated in phosphate
◦
system (nicotinamide adenine dinucleotide phosphate (NADP, 0.5 mM), glucose-6-phosphate (G6P,
−1
5
mM), glucose-6-phosphate dehydrogenase (1.5 U mL ) and magnesium chloride (0.5 mM)). Also,
compounds were incubated without the presence of NADPH cofactor, as a buffer stability control.
Metabolic activity of liver microsomes was verified by including testosterone and propranolol as
positive controls, as well as caffeine as a negative control. Sampling was performed at six time points
(0, 10, 20, 30, 45 and 60 min), followed by reaction termination by addition of acetonitrile/methanol
mixture (2:1, v/v) containing dicofenac as internal standard. Samples were analyzed as previously
described for samples from permeability assay. The in vitro half-life (t1/2) was calculated from the
slope of the linear regression by plotting ln % remaining of parent compound against incubation time.
In vitro intrinsic clearance, CLint was calculated from half-life using following Equation (3):
ln 2
mL per incubation
t1/2 mg protein per incubation
CLint [µL min 1 mg ] =
−
−1
×
(3)
where 52.5 mg protein/g liver is used as a constant.
Predicted in vivo hepatic clearance, in vivo CL was calculated as follows:
h
CLint × (mg protein/g liver) × Q × (LW/BW)
Q + (CLint × (mg protein/g liver) × (LW/BW))
−
1
−1
in vivo CL [mL min kg ] =
(4)
h
−
1
where: LW/BW = liver weight/body weight [g kg ]; 25.7 (human), 87.5 (mouse); Q = LBF = liver blood
−1
−1
flow [mL min kg ]; 21/1.26 (human), 131/7.86 (mouse).
Predicted in vivo hepatic clearance can be expressed as %LBF and calculated as follows:
in vivo CLh
in vivo CL [%LBF] =
× 100
(5)
h
LBF
3
.11. Voltammetric Measurements
All chemicals used in the experiments were of the best grade commercially available
Sigma-Aldrich) and were used without further purification. Stock standard solutions of compounds
(
−
3
−2
1
1c
,
13a
,
15b (c = 2.0
×
10 mol/L) and 6-chloropurine (c = 1.2
×
10 mol/L) were prepared from dry
pure substances in dimethyl sulfoxide (DMSO, p.a.), purchased from Kemika (Zagreb, Croatia). Stock
standard solution of ethynyl ferrocene (c = 1.1
in ethanol (Kemika). For the supporting electrolyte, analytical grade NaClO (Kemika) was used.
−
2
×
10 mol/L) was prepared from dry pure substance
4
Water was deionized by the Millipore Milli-Q system to the resistivity
measurements were carried out using the computer-controlled electrochemical system „PGSTAT 101“
Eco-Chemie, Utrecht, The Netherlands), controlled by the electrochemical software “NOVA 1.5”.
≥
18 MΩcm. Voltammetric
(
A three-electrode system (BioLogic, Claix, France) with glassy carbon electrode (GCE) of 3 mm in
diameter as a working electrode, Ag/AgCl (3 mol/L NaCl) as a reference electrode and a platinum
wire as a counter electrode were used. All potentials were expressed versus Ag/AgCl (3 mol/L NaCl)
reference electrode. The supporting electrolyte (0.5 mol/L NaClO ), adjusted to the desired pH value,
4
was placed in the electrochemical cell and the required aliquot of the standard analyte solution was
added. The solution in the electrochemical cell was degassed with high purity nitrogen for 10 min
before measurement, and the nitrogen blanket was maintained thereafter. Before each run, the glassy
carbon working electrode was polished with diamond suspension in spray (grain size 6 µm) and
rinsed with ethanol and deionized water. All experiments were performed at room temperature.
The presented results are reported as the mean value of three independent measurements.

Molecules 2020, 25, 1570
21 of 24
4
. Conclusions
Novel purine, pyrrolo[2,3-d]pyrimidine, benzimidazole and indole derivatives with ferrocenylalkyl
or ferrocenyl directly linked to N-1 of 1,2,3-triazole (11a−11c 12a−12c 13a−13c 14a−14c 15a−15c
6a 24a−24c 25a−25c) were prepared using Cu-promoted azide-alkyne cycloaddition
23a−23c
CuAAC) reaction of alkylated nitrogen heterocycles and azidoferrocene precursors. Purine and
pyrrolo[2,3-d]pyrimidine with ferrocenyl attached to C-4 of 1,2,3-triazole (34a−34c and 35a−35c
were obtained by CuAAC reaction of N-azidoethyl heterocycle derivatives and ethynyl-ferrocene.
-Chloro-7-deazapurine 11a−11c and 6-chloropurine 13a 15a and 15b ferrocenylalkyl derivatives
exhibited pronounced cytostatic activities. Compounds 11c 13a and 15b found to be the most
potent on colorectal adenocarcinoma (SW620) cells with IC50 of 9.07, 14.38 and 15.50 M, respectively.
,
,
,
,
,
1
,
,
,
(
)
6
,
,
µ
However, preliminary assessment of permeability and metabolic stability in liver microsomes (mouse
and human) showed that these compounds have a moderate to high permeability and are P-gp
substrates. The 6-chloropurine moiety and N-7 regiosomerism are favored for metabolic stability
although the overall instability was moderate to high, suggesting further optimization is required.
Overall, N-9 and N-7 isomers of 6-chloropurine 13a and 15a containing ferrocenylmethylene unit
are characterized with high solubility, good permeability and moderate stability in human liver
microsomes. Although 15b showed good physicochemical properties, this compound, together with
1
1a−11c, is metabolically unstable. Voltammetric analysis showed that the oxidation of purine and
pyrrolo[2,3-d]pyrimidine derivatives of ferrocene is influenced by the spacer between these moieties,
showing that ferrocenyl group directly attached to N-1 of triazole in 11c requires higher potential than
1
3a and 15b with ferrocene linked to triazole through an alkyl bridge, which may also contribute to
their higher antioxidant properties.
Taken together, we may conclude that compound 13a is highlighted for further structural
optimization to obtain more effective and selective ferrocene-tagged purine and/or purine isostere
derivatives against SW620 cell lines.
Supplementary Materials: The following are available online, Figures S1–S38: scanned H and 13C-NMR spectra
1
of compounds.
Author Contributions: Conceptualization, S.R.-M., K.P., and S.K.P.; syntheses and structure characterization, M.P.,
H.Š. and V.R.; antiproliferative evaluation, P.G., S.K.P. and K.P.; ADME profiling, P.M. and J.P.; electrochemical
evaluation, D.J.; writing—original draft preparation, S.R.-M., V.R. and D.J.; writing—review and editing, V.R.,
M.P., P.M., P.G., J.P., V.G.M., D.J., K.P., S.K.P. and S.R.-M.; supervision, J.P., V.G.M., K.P., S.K.P., and S.R.-M.; project
administration, S.R.-M.; funding acquisition, S.R.-M. and K.P. All authors have read and agreed to the published
version of the manuscript.
Funding: Financial support from the Croatian Science Foundation under the project HRZZ-IP-2018-01-4682 is
gratefully acknowledged.
Acknowledgments: We would like to also thank Croatian Government and the European Union (European
Regional Development Fund—the Competitiveness and Cohesion Operational Program—KK.01.1.1.01) for funding
this research through project Bioprospecting of the Adriatic Sea (KK.01.1.1.01.0002) granted to The Scientific Centre
of Excellence for Marine Bioprospecting—BioProCro. We also acknowledge the project “Research Infrastructure
for Campus-based Laboratories at University of Rijeka”, co-financed by European Regional Development Fund
(ERDF) and the University of Rijeka research grant UNIRI-biomed-18-133 (1277).
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds 11a−11c
,
12a−12c
,
13a−13c
,
14a−14c
,
15a−15c
,
16a
,
23a−23c
,
2
4a−24c, 25a−25c, 34a−34c and 35a−35c are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(