Purine and Purine Isostere Derivatives of Ferrocene: An Evaluation of ADME, Antitumor and Electrochemical Properties.

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 potent cytotoxic effect on colorectal adenocarcinoma (SW620) was exerted by the 6-chloro-7-deazapurine 11c (IC50 = 9.07 µM), 6-chloropurine 13a (IC50 = 14.38 µM) and 15b (IC50 = 15.50 µM) ferrocenylalkyl derivatives. The N-9 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.

1. Introduction

Nucleoside analogues were among the first chemotherapeutic agents that were introduced for the medical treatment of cancer [1,2]. Cytotoxic nucleobase-derived compounds have gained a lot of importance in recent years for combating cancer of different types, usually in combination with other agents [3,4,5]. They were shown to interact with various biological targets, such as cellular kinases, ribonucleotide reductase, and cellular polymerases in order to produce cytotoxic effects [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 [8,9]. The antitumor activity of ferrocene-containing compounds 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 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 in tamoxifen with ferrocene, exhibits a strong effect against both hormone-dependent (ERα+) and hormone-independent (ERα−) breast cancer cell types in contrast to tamoxifen, which is only active against ERα+ cells [13,14]. Furthermore, the antimalarial drug ferroquine was recently found to enhance anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy [15]. It negatively regulates hypoxia-inducible factor-1α (HIF-1α) and showed to be effective under starved and hypoxic conditions frequently observed in advanced solid cancers. The anticancer potential of organometallic nucleoside analogues was explored [16,17,18] and showed that nucleoside analogues of ferrocene exhibited potent antineoplastic activity [19,20,21,22]. 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, 12a−12c, 13a−13c, 14a−14c, 15a−15c, 16a, 23a−23c, 24a−24c, 25a−25c) and a ferrocene 4-substituted 1,2,3-triazole moiety (34a−34c and 35a−35c) was carried out as shown in Scheme 1, Scheme 2 and Scheme 3.

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, 24 h; (ii) corresponding azide, Cu(OAc)2, methanol, room temperature, 24 h.

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, 24 h; (ii) corresponding azide, Cu(OAc)2, methanol, room temperature, 24 h.

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, H2O, MW: 100 °C, 400 W; (ii) heterocyclic base, NaH, 1,2-dibromoethane, anhydrous DMF, room temperature, 24 h; (iii) N-alkylated heterocyclic base, NaN3, acetone, 60 °C, 12 h; (iv) corresponding terminal azide, Cu(OAc)2, ethynylferrocene, methanol, room temperature, 12 h.

The key intermediates, the N-9- (5−8) [28,29,30,31] 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 obtained by N-alkylation of the corresponding heterocyclic base as reported in the literature [32,33]. Target regioselective 1,4-disubstituted 1,2,3-triazoles (11a−11c, 12a−12c, 13a−13c, 14a−14c, 15a−15c, 16a, 23a−23c, 24a−24c, 25a−25c) (Scheme 1 and Scheme 2) were prepared by copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition of alkynyl derivatives of corresponding purine and purine isosteres with 1-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 1 and 3 with the corresponding amines in the presence of KOH under microwave irradiation gave 6-pyrrolidinyl and 6-piperidinyl-substituted intermediates 26−29. N-alkylation of the corresponding heterocyclic base 1, 3, 26−29 with 1,2-dibromoethane in the presence of NaH provided the 2-bromoethyl heterocyclic derivatives 30a−30c [35], 31a−31c. Precursors 30a−30c and 31a−31c were converted in situ, using NaN3, to the 2-azidoethyl heterocyclic derivatives 32a−32c [35], 33a−33c. 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).

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, 23a−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.

Table 1

In vitro growth inhibitory effects of compounds 11a−11c, 12a−12c, 13a−13c, 14a−14c, 15a−15c, 16a, 23a−23c, 24a−24c, 25a−25c, 34a−34c and 35a−35c on selected tumor cell lines and their measured kinetic solubility range and Chrom logD.

Compd	Heterocycle	R	IC50 (µM)					Kinetic Solubility (µM)	Chrom logD
			SW620	CFPAC-1	HepG2	HeLa	HFF
11a			31.58 ± 0.84	32.91 ± 2.46	41.96 ± 5.93	38.25 ± 2.30	8.57 ± 1.95	<1	5.62
11b			31.96 ± 0.34	31.26 ± 4.51	34.52 ± 1.36	31.38 ± 1.04	8.88 ± 3.46	3–10	5.94
11c			9.07 ± 1.21	35.03 ± 5.44	31.94 ± 4.32	27.74 ± 3.23	5.89 ± 1.88	3–10	5.62
12a			>100	>100	>100	>100	56.63 ± 15.60	30–100	2.85
12b			74.95 ± 8.93	70.15 ± 6.50	68.60 ± 9.15	76.40 ± 3.41	18.44 ± 2.64	>100	3.09
12c			>100	>100	>100	>100	54.50 ± 4.96	3–10	2.64
13a			14.38 ± 3.02	22.83 ± 4.31	19.89 ± 5.12	25.20 ± 4.31	3.98 ± 0.17	>100	4.27
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
13c			51.78 ± 1.90	54.67 ± 5.13	54.66 ± 3.21	46.78 ± 6.72	6.77 ± 2.78	>100	4.19
14a			60.23 ± 24.36	49.47 ± 5.03	60.13 ± 5.73	71.43 ± 16.06	>100	30–100	3.62
14b			98.74 ± 19.71	67.36 ± 9.12	77.10 ± 5.92	>100	11.53 ± 5.09	30–100	3.90
14c			81.33 ± 1.23	90.50 ± 1.83	>100	>100	24.55 ± 4.45	<1	3.50
15a			27.21 ± 5.30	34.39 ± 0.43	36.01 ± 1.81	28.62 ± 8.28	13.80 ± 4.17	>100	3.82
15b			15.50 ± 3.24	28.59 ± 2.68	37.11 ± 1.84	28.97 ± 0.09	0.88 ± 1.06	>100	4.15
15c			45.47 ± 4.52	72.25 ± 13.70	81.71 ± 17.05	69.25 ± 25.47	29.59 ± 4.28	>100	3.68
16a			>100	>100	>100	>100	5.30 ± 5.36	<1	3.37
23a			96.98 ± 13.55	64.57 ± 12.18	59.90 ± 10.94	>100	23.83 ± 3.32	10–30	4.56
23b			>100	68.03 ± 13.67	83.60 ± 8.61	89.63 ± 13.65	31.80 ± 9.81	30–100	4.84
23c			>100	>100	>100	>100	>100	30–100	4.45
24a			61.99 ± 8.79	78.05 ± 6.61	53.55 ± 5.05	68.72 ± 2.91	16.72 ± 8.61	3–10	>6.57
24b			>100	87.63 ± 1.79	>100	89.64 ± 7.64	>100	3–10	>6.57
24c			>100	84.54 ± 15.76	90.87 ± 11.51	88.12 ± 1.18	>100	3–10	>6.57
25a			>100	>100	>100	78.52 ± 9.36	>100	1–3	>6.57
25b			>100	>100	>100	>100	>100	1–3	>6.57
25c			>100	>100	71.79 ± 3.44	79.44 ± 1.82	4.75 ± 1.70	1–3	>6.57
34a			>100	>100	>100	>100	>100	10–30	4.92
34b			>100	>100	>100	>100	>100	10–30	4.30
34c			57.65 ± 6.55	45.78 ± 9.11	57.61 ± 13.80	71.12 ± 15.89	20.16 ± 1.54	10–30	5.49
35a			78.18 ± 5.69	65.28 ± 6.23	48.09 ± 7.85	63.00 ± 6.06	6.00 ± 2.99	10–30	3.70
35b			>100	>100	>100	>100	>100	30–100	3.58
35c			>100	>100	>100	>100	6.83 ± 2.17	10–30	5.02

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 15b with ferrocenyleth-yl moiety exhibited enhancement of the activity in comparison to its N-9 isomer 13b. 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, 24b, 24c and 25c containing ferrocenylethyl and ferrocenyl directly linked to the N-1 of 1,2,3-triazole showed reduced activity.

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 35a). 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 [36,37], was determined for novel purine and purine isosteres with a ferrocene motif as 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 15a−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, 6-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.

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.

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.

Compd	Heterocycle	R	Papp (AB) (×10−6 cm/s)	Papp (BA) (×10−6 cm/s)	Efflux Ratio (BA/AB)	Papp (AB) (×10−6 cm/s)	Papp (BA) (×10−6 cm/s)	Efflux Ratio (BA/AB)
			without P-gp inhibitor			with P-gp inhibitor
11a			8.4	18.0	2.2	29.4	21.1	0.7
11b			11.8	17.4	1.5	19.7	20.5	1.1
11c			5.3	14.9	2.9	15.5	16.5	1.1
13a			8.3	23.8	2.9	29.8	25.3	0.9
15a			4.8	33.5	7.0	16.9	13.7	0.8
15b			3.2	33.2	10.7	11.7	12.9	1.1

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 permeability in the range from 8.3 to 8.48 × 10−6 cm/s.

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.

Compd	Heterocycle	R	Mouse			Human
			t1/2 (min)	In Vivo CLh (ml/min/kg)	In Vivo CLh (% LBF)a	t1/2 (min)	In Vivo CLh (ml/min/kg)	In Vivo CLh (% LBF)
11a			4.4	25.2	98	7.8	23.2	90
11b			0	25.7	100	2.3	24.9	97
11c			1.3	25.6	99	4.1	24.3	95
13a			5.5	25.1	98	38.6	16.8	65
15a			8.8	24.8	97	51.9	15.0	58
15b			2.9	25.4	99	19.0	20.4	79

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 11a−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).

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 [43,44,45,46,47], 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 ≤ pH ≤ 10. The pH of the solution affects the voltammetric response of this compound, i.e., the oxidation of evaluated compounds 11c, 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.

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). (A) Esw = 50 mV, Es = 2 mV, f = 100 Hz. Dotted line represents SW voltammogram of the supporting electrolyte; (B) v = 100 mV s−1, Es = 2 mV. Arrows indicate the scan direction.

Figure 2 shows square-wave voltammograms for the oxidation of 11c, 13a and 15b on glassy carbon electrode, in 0.5 mol/L NaClO4 at pH 9. The forward (if) and backward (ib) components of the net response (Δi = if − ib) are shown as well (inset in Figure 2). Two peaks at ~0.4 V (P1) and ~1.1 V (P2) 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 can be ascribed to the following charge transfer reaction [FeII(C5H5)2] [FeIII(C5H5)2]+ + 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.

Figure 2

Square-wave voltammograms for the oxidation of 11c, 13a and 15b in 0.5 mol/L NaClO4 at pH 9. Conditions: c(13a) = 0.1 mmol/L, c(11c) = c(15b) = 0.05 mmol/L, Esw = 50 mV, Es = 2 mV, f = 100 Hz. Inset: The net SWV response (Δi) and its forward (if) and backward (ib) current components, for the oxidation of 13a. Arrow indicates the scan direction.

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 (if and ib) of the peak P2 are oxidative currents (see inset in Figure 2), which indicate that the electro-oxidation of 1,2,3-triazole in 11c, 13a and 15b at pH 9 is totally irreversible electrode reaction. The same results 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].

Figure 3

Square-wave voltammograms for the oxidation of ethynyl ferrocene (i), 6-chloropurine (ii) and 13a in 0.5 mol/L NaClO4 at pH 9. Conditions: c(i) = c(ii) = c(13a) = 0.1 mmol/L, Esw = 50 mV, Es = 2 mV, f = 100 Hz. Arrow indicates the scan direction.

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 CaH2 and stored over 3Å molecular sieves [50]. Thin layer chromatography (TLC) was performed on 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, Austria). 1H and 13C-NMR spectra were recorded on a Bruker 300 and 600 MHz spectrometers (Bruker, Billerica, MA, USA). All data were recorded in dimethyl sulfoxide (DMSO)-d6 at 298 K. NMR chemical shifts were referenced to the residual solvent signal of DMSO at δ 2.50 ppm for 1H and δ 39.50 ppm for 13C. Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances, and H–H coupling constants. 1H and 13C-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 (5) [29], 6-amino-9-(prop-2-yn-1-yl)-9H-purine (6) [30], 6-chloro-9-(prop-2-yn-1-yl)-9H-purine (7) [31], 2-amino-6-chloro-9-(prop-2-yn-1-yl)-9H-purine (8) [32], 6-chloro-9-(prop-2-yn-1-yl)-9H-purine (9) [31], 2-amino-6-chloro-7-(prop-2-yn-1-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], 6-(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.

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)2 (0.05 eq.) were added. The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography (CH2Cl2:CH3OH = 60:1).

4-Chloro-7-[1-(1-ferrocenymethyl-1,2,3-triazol-4-yl)methyl]-7H-pyrrolo[2,3-d]pyrimidine (11a) Compound 11a was prepared using the above-mentioned procedure using compound 5 (100 mg, 0.52 mmol) and 1-methylazidoferrocene (150 mg, 0.62 mmol) to obtain 11a as orange oil (60.3 mg, 33 %). 1H-NMR (300 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, CH2), 5.25 (2H, s, CH2), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.14 2H, (m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO-d6) δ 151.12 (C4), 150.87 (C2), 150.82 (C7a), 143.04 (C4′), 131.77 (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 (CH2), 40.05 (CH2). Anal. calcd. for C20H17ClFeN6: C, 55.52; H, 3.96; N, 19.42. Found: C, 55.46; H, 3.95; N, 19.44.

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 %). 1H-NMR (300 MHz, DMSO-d6) δ 8.65 (1H, s, H2), 8.09 (1H, s, H5′), 7.80 (1H, d, J = 3.6 Hz, H6), 6.67(1H, d, J = 3.6 Hz, H5), 5.65 (1H, q, J = 7.0 Hz, CH), 5.56 (2H, s, CH2), 4.33–4.28 (1H, m, CH-Fc), 4.17–4.15 (2H, m, CH-Fc), 4.08 (5H, H,s, Cp-Fc), 1.78 (3H, d, J = 7.0 Hz, CH3). 13C-NMR (151 MHz, DMSO-d6) δ 150.60 (C4), 150.36 (C2), 150.30 (C7a), 142.26 (C4′), 131.25 (C6), 121.58 (C5′), 116.70 (C4a), 98.82 (C5), 88.53 (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), 39.54 (CH2), 20.83 (CH3). Anal. calcd. for C21H19ClFeN6: C, 56.46; H, 4.29; N, 18.81. Found: C, 56.35; 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-azidoferrocene (142 mg, 0.62 mmol) to obtain 11c as orange oil (39.4 mg, 18 %). 1H-NMR (600 MHz, DMSO-d6) 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, H5), 5.64 (2H, s, CH2), 5.00 (2H, t, J = 1.9 Hz, CH-Fc), 4.35–4.31 (2H, m, CH-Fc), 4.18 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO-d6) δ 150.85 (C4), 150.39 (C2), 150.19 (C7a), 142.95 (C4′), 130.83 (C6), 123.33 (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 (CH2). Anal. calcd. for C19H15ClFeN6: C, 54.51; H, 3.61; N, 20.07. Found: C, 55.40; H, 3.90; N, 20.03.

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 6 (100 mg, 0.58 mmol) and 1-methylazidoferrocene (168 mg, 0.70 mmol) to obtain 12a as orange powder (132 mg, 76%, m.p. = 107 °C). 1H-NMR (300 MHz, DMSO-d6) δ 8.18 (1H, s, H8), 8.12 (1H, s, H2), 8.07 (1H, s, H5′), 7.21 (2H, s, NH2), 5.41 (2H, s, CH2), 5.26 (2H, s, CH2), 4.30 (2H, dd, J = 3.4, 1.6 Hz, CH-Fc), 4.174.14 (2H, m, CH-Fc), 4.13 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO-d6) δ 155.91 (C6), 152.50 (C2), 149.21 (C4), 142.54 (C4′), 140.59 (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 (CH2), 37.96 (CH2). Anal. calcd. for C19H18FeN8: C, 55.09; H, 4.38; N, 27.05. Found: C, 54.98; H, 4.39; N, 27.00.

6-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 6 (100 mg, 0.58 mmol) and 1-azidoethylferrocene (177 mg, 0.70 mmol) to obtain 12b as orange powder (53.7 mg, 31 %, m.p. = 223 °C). 1H-NMR (600 MHz, DMSO) δ 8.57 (1H, s, H8), 8.26 (1H, s, H5′), 8.15 (1H, s, H2), 7.27 (2H, s, NH2), 5.49 (2H, s, CH2), 5.01 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 156.46 (C6), 153,17 (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 (CH2). Anal. calcd. for C20H20FeN8: C, 56.09; H, 4.71; N, 26.16. Found: C, 56.17; H, 4.69; N, 26.11.

6-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 6 (100 mg, 0.58 mmol) and 1-azidoferrocene (160 mg, 0.70 mmol) to obtain 12c as orange powder (13.5 mg, 5.8 %, m.p. > 250 °C). 1H-NMR (600 MHz, DMSO) δ 8.57 (1H, s, H8), 8.26 (1H, s, H5′), 8.15 (1H, s, H2), 7.27 (2H, s, NH2), 5.49 (2H, s, CH2), 5.01 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 156.46 (C6), 153,17 (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 (CH2). Anal. calcd. for C18H16FeN8: C, 54.02; H, 4.03; N, 28.00. Found: 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 7 (55 mg, 0.28 mmol) and 1-methylazidoferrocene (81 mg, 0.34 mmol) to obtain 13a as orange powder (53.2 mg, 43 %, m.p. = 156 °C). 1H-NMR (300 MHz, DMSO) δ 8.79 (1H, s, H8), 8.77 (1H, s, H2), 8.15 (1H, s, H5′), 5.60 (2H, s, CH2), 5.26 (2H, s, CH2), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.15 (2H, m, CH-Fc), 4.13 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 152.15 (C6), 152.13 (C2), 149.53 (C4), 147.92 (C8), 142.18 (C4′), 131.22 (C5), 123.69 (C5′), 82.75 (Cq-Fc), 69.10 (CH-Fc), 69.05 (Cp-Fc), 68.78 (CH-Fc), 49.43 (CH2), 39.42 (CH2). Anal. calcd. for C19H16ClFeN7: C, 52.62; H, 3.72; N, 22.61. Found: C, 52.46; H, 3.73; N, 22.66.

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-azidoethylferrocene (157 mg, 0.62 mmol) to obtain 13b as orange oil (55.5 mg, 23 %). 1H-NMR (600 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, CH2), 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 (3H, d, J = 7.0 Hz, CH3). 13C-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), 66.70 (CH-Fc), 56.19 (CH), 39.42 (CH2), 21.33 (CH3). Anal. calcd. for C20H18ClFeN7: C, 53.65; H, 4.05; 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, 0. 62 mmol) to obtain 13c as orange oil (43.2 mg, 19 %). 1H-NMR (600 MHz, DMSO) δ 8.85 (1H, s, H8), 8.81 (1H, s, H2), 8.59 (1H, s, H5′), 5.68 (2H, s, CH2), 4.99 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.19 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 151.77 (C6), 151.65 (C2), 149.05 (C4), 142.18 (C4′), 130.77 (C5), 123.45 (C5′), 93.17 (Cp-Fc), 69.91 (Cp-Fc), 66.61 (CH-Fc), 61.84 (CH-Fc). Anal. calcd. for C18H14ClFeN7: 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). 1H-NMR (300 MHz, DMSO) δ 8.17 (1H, s, H8), 8.05 (1H, s, H5′), 6.93 (2H, s, NH2), 5.33 (2H, s, CH2), 5.27 (2H, s, CH2), 4.31 (2H, t, J = 1.8 Hz, CH-Fc), 4.18–4.15 (2H, m, CH-Fc), 4.14 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 160.32 (C2), 154.33 (C6), 149.85 (C4), 143.46 (C8) 142.74 (C4′), 123.64 (C5), 123.40 (C5′), 82.75 (Cq-Fc), 69.12 (CH-Fc), 69.06 (Cp-Fc), 68.80 (CH-Fc), 49.45 (CH2), 38.71 (CH2). Anal. calcd. for C19H17ClFeN8: C, 50.86; H, 3.82; N, 24.97. Found: C, 50.91; H, 3.80; N, 24.92.

2-Amino-6-chloro-9-[1-(1-ferrocenyl-1-methylmethyl-1,2,3-triazol-4-yl)methyl]-9H-purine (14b) Compound 14b was prepared using the above-mentioned procedure using compound 8 (100 mg, 0.48 mmol) and 1-azidoethylferrocene (147 mg, 0.58 mmol) to obtain 14b as orange powder (55 mg, 24 %, m.p. = 214 °C). 1H-NMR (300 MHz, DMSO) δ 8.17 (1H, s, H8), 8.07 (1H, s, H5′), 6.93 (2H, s, NH2), 5.66 (1H, q, J = 7.0 Hz, CH), 5.33 (2H, s, CH2), 4.32 (2H, d, J = 2.1 Hz, CH-Fc), 4.23–4.13 (3H, m, CH-Fc), 4.10 (5H, s, Cp-Fc), 1.80 (3H, d, J = 7.0 Hz, CH3). 13C-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), 67.66 (CH-Fc), 67.23 (CH-Fc), 66.17 (CH-Fc), 55.70 (CH), 38.24 (CH2), 20.77 (CH3). Anal. calcd. for C20H19ClFeN8: C, 51.91; H, 4.14; N, 24.22. Found: C, 51.75; H, 4.13; N, 24.24.

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 8 (100 mg, 0.48 mmol) and 1-azidoferrocene (131 mg, 0.58 mmol) to obtain 14c as orange powder (44 mg, 21 %, m.p. > 250 °C). 1H-NMR (300 MHz, DMSO) δ 8.50 (1H, s, H8), 8.24 (1H, s, H5′), 6.96 (2H, s, NH2), 5.41 (2H, s, CH2), 4.99 (2H, t, J = 1.9 Hz, CH-Fc), 4.384.30 (2H, m, CH-Fc), 4.19 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 159.83 (C2), 153.93 (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), 61.86 (CH-Fc), 38.24 (CH2). Anal. calcd. for C18H15ClFeN8: C, 49.74; H, 3.48; N, 25.78. Found: C, 49.78; 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 9 (50 mg, 0.26 mmol) and 1-methylazidoferrocene (75 mg, 0.31 mmol) to obtain 15a as orange powder (56.5 mg, 50 %, m.p. = 96 °C). 1H-NMR (300 MHz, DMSO) δ 8.94 (1H, s, H8), 8.80 (1H, s, H2), 8.16 (1H, s, H5′), 5.80 (2H, s, CH2), 5.26 (2H, s, CH2), 4.30 (2H, t, J = 1.8 Hz, CH-Fc), 4.204.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 161.57 (C4), 151.74 (C2), 151.17 (C8), 142.54 (C4′), 142.29 (C6), 122.82 (C5′), 121.98 (C5), 82.36 (Cq-Fc), 68.53 (Cp-Fc), 68.51 (CH-Fc), 68.26 (CH-Fc), 48.93 (CH2), 41.89 (CH2). Anal. calcd. for C19H16ClFeN7: C, 52.62; H, 3.72; N, 22.61. Found: C, 52.56; H, 3.71; N, 22.57.

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 9 (90 mg, 0.47 mmol) and 1-azidoethylferrocene (145 mg, 0.56 mmol) to obtain 15b as orange powder (56.5 mg, 27 %, m.p. = 68 °C). 1H-NMR (300 MHz, DMSO) δ 8.94 (1H, s, H8), 8.80 (1H, s, H2), 8.19 (1H, s, H5′), 5.80 (2H, s, CH2), 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), 1.78 (3H, d, J = 7.0 Hz, CH3). 13C-NMR (151 MHz, DMSO) δ 161.57 (C4), 151.72 (C2), 142.34 (C4′), 142.30 (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 (CH-Fc), 66.23 (CH-Fc), 55.70 (CH), 41.97 (CH2), 20.94 (CH3). Anal. calcd. for C20H18ClFeN7: C, 53.65; H, 4.05; N, 21.90. Found: C, 53.68; H, 4.03; N, 21.86.

6-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 9 (90 mg, 0.47 mmol) and 1-azidoferrocene (129 mg, 0.56 mmol) to obtain 15c as orange powder (44.7 mg, 22 %, m.p. = 76 °C). 1H-NMR (600 MHz, DMSO) δ 9.00 (1H, s, H8), 8.83 (1H, s, H2), 8.58 (1H, s, H5′), 5.87 (2H, s, CH2), 4.99 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.17 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 161.71 (C4), 151.77 (C2), 151.24 (C8), 143.23 (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), 42.00 (CH2). Anal. calcd. for C18H14ClFeN7: C, 51.52; H, 3.36; N, 23.36. Found: C, 51.55; H, 3.34; N, 23.39.

2-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. >250 °C). 1H-NMR (300 MHz, DMSO) δ 8.49 (1H, s, H8), 8.09 (1H, s, H5′), 6.64 (2H, s, NH2), 5.60 (2H, s, CH2), 5.26 (2H, s, CH2), 4.29 (2H, t, J = 1.8 Hz, CH-Fc), 4.174.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 164.27 (C4), 159.98 (C2), 142.82 (C6), 142.33 (C4′), 122.74 (C5′), 82.48 (Cq-Fc), 68.54 (CH-Fc), 68.46 (CH-Fc), 68.22 (CH-Fc), 48.89 (CH2), 41.56 (CH2). Anal. calcd. for C19H17ClFeN8: C, 50.86; H, 3.82; N, 24.97. Found: C, 50.80; H, 3.83; N, 24.91.

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)2 (0.05 eq.) were added. The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography (CH2Cl2:CH3OH = 60:1).

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 1-methylazidoferrocene (130.2 mg, 0.54 mmol) to obtain 23a as orange powder (113 mg, 63 %, m.p. = 163 °C). 1H-NMR (300 MHz, DMSO) δ 8.32 (1H, s, H2), 8.14 (1H, s, H5′), 7.65 (2H, dd, J = 7.1, 1.9 Hz, H4/7), 7.287.16 (2H,m, H5/6), 5.56 (2H, s, CH2), 5.28 (2H, s, CH2), 4.354.28 (2H, m, CH-Fc), 4.194.17 (2H, m, CH-Fc), 4.15 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 143.87 (C2), 143.40 (C3a), 142.54 (C4′), 133.45 (C7a), 123.09 (C5′), 122.28 (C6), 121.54 (C4), 119.37 (C5), 110.66 (C7), 82.24 (Cq-Fc), 68.56 (Cp-Fc), 68.29 (CH-Fc), 48.88 (CH2). Anal. calcd. for C21H18FeN5: C, 63.65; H, 4.58; N, 17.67. 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-azidoethylferrocene (163.2 mg, 0.64 mmol) to obtain 23b as brown powder (37.48 mg, 17 %, m.p. = 140 °C). 1H-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, 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, CH2), 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, CH3). 13C-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), 66.64 (CH-Fc), 56.12 (CH), 39.86 (CH2), 21.37 (CH3). Anal. calcd. for C22H20FeN5: C, 64.40; H, 4.91; N, 17.07. 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 mg, 0.64 mmol) to obtain 23c as orange powder (25 mg, 12 %, m.p. > 250 °C). 1H-NMR (600 MHz, DMSO) 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 = 7.2 Hz, H5), 5.62 (2H, s, CH2), 4.99 (2H, s, CH-Fc), 4.33 (2H, s, CH-Fc), 4.17 (5H, s, Cp-Fc). 13C-NMR (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 C20H17FeN5: C, 62.68; H, 4.47; N, 18.27. Found: C, 62.74; H, 4.46; N, 18.29.

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 (130.2 mg, 0.54 mmol) to obtain 24a as orange powder (25 mg, 14 %, m.p. = 135 °C). 1H-NMR (600 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, CH2), 5.24 (2H, s, CH2), 4.28 (2H, t, J = 1.8 Hz, CH-Fc), 4.15 (2H, t, J = 1.8 Hz, CH-Fc), 4.13 (5H, s, Cp-Fc). 13C-NMR (101 MHz, DMSO) δ 144.07 (C4′), 135.98 (C7a), 129.04 (C2), 128.68 (C3a), 123.18 (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 (CH2), 41.25 (CH2). Anal. calcd. for C22H20FeN4: C, 66.68; H, 5.09; N, 14.14. Found: C, 66.48; 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-azidoethylferrocene (147.9 mg, 0.58 mmol) to obtain 24b as brown oil (40 mg, 20 %). 1H-NMR (300 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, CH2), 4.314.27 (1H, m, CH-Fc), 4.174.12 (3H, m, CH-Fc), 4.08 (5H, s, Cp-Fc), 1.77 (3H, d, J = 7.0 Hz, CH3). 13C-NMR (75 MHz, DMSO) δ 143.75 (C4′), 135.98 (C7a), 128.99 (C2), 128.65 (C3a), 121.79 (C5′), 121.52 (C6), 120.84 (C4), 119.55 (C5), 110.53 (C7), 101.39 (C3), 89.00 (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), 41.25 (CH2), 21.37 (CH3). Anal. calcd. for C23H22FeN4: C, 67.33; H, 5.40; N, 13.66. Found: C, 67.19; H, 5.41; 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, 1.02 mmol) to obtain 24c as brown crystals (32 mg, 9.8 %, m.p. = 120 °C). 1H-NMR (300 MHz, DMSO) 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), 7.14 (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, CH2), 4.97 (2H, t, J = 1.9 Hz, CH-Fc), 4.354.27 (2H, m, CH-Fc), 4.17 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 144.37 (C4′), 136.01 (C7a), 129.14 (C2), 128.77 (C3a), 123.71 (C5′), 121.58 (C6), 120.91 (C4), 119.63 (C5), 110.57 (C7), 101.50 (C3), 93.86 (Cq-Fc), 70.35 (Cp-Fc), 67.06 (CH-Fc), 62.40 (CH-Fc), 41.23 (CH2). Anal. calcd. for C21H18FeN4: C, 65.99; H, 4.75; N, 14.66. Found: C, 66.06; H, 4.76; N, 14.63.

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). 1H-NMR (300 MHz, DMSO) 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, CH2), 5.24 (2H, s, CH2), 4.304.24 (2H, m, CH-Fc), 4.164.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 143.77 (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 (CH2), 41.35 (CH2). Anal. calcd. for C22H19FeIN4: C, 50.60; H, 3.67; N, 10.73. Found: C, 50.65; H, 3.68; N, 10.76.

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-azidoethylferrocene (242.3 mg, 0.95 mmol) to obtain 25b as orange powder (125 mg, 29 %, m.p. = 118 °C). 1H-NMR (300 MHz, DMSO) 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, CH2), 5.24 (2H, s, CH2), 4.304.24 (2H, m, CH-Fc), 4.164.14 (2H, m, CH-Fc), 4.12 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 143.77 (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 (CH2), 41.35 (CH2). Anal. calcd. for C23H21FeIN4: C, 51.52; H, 3.95; N, 10.45. Found: C, 51.57; H, 3.96; N, 10.49.

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, 1.48 mmol) to obtain 25c as brown crystal (53 mg, 10 %, m.p. = 169 °C). 1H-NMR (600 MHz, DMSO) δ 8.52 (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, CH2), 4.97 (2H, s, CH-Fc), 4.32 (2H, s, CH-Fc), 4.17 (5H, s, Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 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), 40.81 (CH2). Anal. calcd. for C21H17FeIN4: C, 49.64; H, 3.73; N, 11.03. Found: C, 49.54; H, 3.72; N, 10.99.

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, 95 %, m.p. > 250 °C). 1H-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, CH2-pyrrolidine), 1.95 (4H, bs, CH2-pyrrolidine). 13C-NMR (151 MHz, DMSO) δ 154.77 (C4), 151.18 (C2), 150.89 (7a), 120.31 (C6), 102.43 (4a), 100.79 (C5), 47.38 (CH2-pyrrolidine). Anal. calcd. for C10H12N4: C, 63.81; H, 6.43; N, 29.77. Found: C, 63.87; H, 6.44; N, 29.65.

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, 84 %, m.p. = 189 °C). 1H-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, CH2-piperidine), 1.73–1.62 (2H, m, CH2-piperidine), 1.62–1.50 (4H, m, CH2-piperidine). 13C-NMR (151 MHz, DMSO) δ 156.27 (C4), 151.90 (C7a), 150.65 (C2), 120.99 (C6), 101.99 (C4a), 100.90 (C5), 46.23 (CH2-piperidine), 25.45 (CH2-piperidine), 24.25 (CH2-piperidine). Anal. calcd. for C11H14N4: C, 65.32; H, 6.98; N, 27.70. Found: C, 65.12; H, 6.99; N, 27.76.

3.5. General Procedure for N-alkylation of Compounds 30b, 30c, 31a–31c

To a solution of the corresponding heterocyclic base 1, 3, 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 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 powder (106 mg; 16 %, m.p.= 109 °C). 1H-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, CH2), 3.85 (2H, t, J = 6.3 Hz, CH2), 3.70 (4H, bs, CH2-pyrrolidine), 1.96 (4H, bs, CH2-pyrrolidine). 13C-NMR (101 MHz, DMSO) δ 155.17 (C6), 151.61 (C2), 150.27 (C4), 124.29 (C8), 103.18 (C5), 101.08 (C7), 47.96 (CH2-pyrrolidine), 45.93 (CH2), 32.35 (CH2). Anal. calcd. for C12H15BrN4: C, 48.83; H, 5.12; N, 18.98. Found: C, 48.73; H, 5.11; N, 18.94.

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 powder (87 mg; 14 %, m.p. = 70 °C). 1H-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, CH2), 3.84 (6H, m, CH2, CH2-piperidine), 1.65 (2H, m, CH2-piperidine), 1.59–1.54 (4H, m, CH2-piperidine). 13C-NMR (151 MHz, DMSO) δ 156.20 (C6), 150.73 (C4), 150.67 (C2), 124.32 (C8), 102.21 (C5), 100.65 (C7), 46.21 (CH2-piperidine), 45.50 (CH2), 31.74 (CH), 25.46 (CH2-piperidine), 24.20 (CH2-piperidine). Anal. calcd. for C13H17BrN4: C, 50.50; H, 5.54; N, 18.12. Found: C, 50.60; H, 5.52; N, 18.06.

9-(2-Bromoethyl)-6-chloro-9H-purine (31a) Compound 31a was prepared using the above-mentioned procedure using compound 3 (500 mg; 3.23 mmol) to obtain 31a as white powder (312 mg; 36 %; m.p.= 111°C). 1H-NMR (400 MHz, DMSO) δ 8.82 (1H, s, H2), 8.77 (1H, s, H8), 4.75 (2H, t, J = 6.1 Hz, CH2), 4.01 (2H, t, J = 6.1 Hz, CH2). 13C-NMR (101 MHz, DMSO) δ 152.42 (C6), 152.14 (C2), 149.59 (C4), 147.99 (C8), 131.26 (C5), 45.82 (CH2), 31.64 (CH2). Anal. calcd. for C7H6BrClN4: C, 32.15; H, 2.31; N, 21.42. Found: C, 32.05; H, 2.32; N, 21.47.

9-(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 (208 mg; 27 %; m.p.= 163 °C). 1H-NMR (400 MHz, DMSO) δ 8.22 (1H, s, H2), 8.17 (1H, s, H8), 4.58 (2H, t, J = 6.1 Hz, CH2), 4.07 (2H, bs, CH2-pyrrolidine), 3.95 (2H, t, J = 6.1 Hz, CH2), 3.64 (2H, s, CH2-pyrrolidine), 1.95 (4H, s, CH2-pyrrolidine). 13C-NMR (101 MHz, DMSO) δ 152.95 (C4), 152.70 (C2), 150.37 (C6), 140.71 (C8), 119.81 (C5), 45.07 (CH2), 32.03 (CH2). Anal. calcd. for C11H14BrN5: C, 44.61; 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 (200 mg; 27 %; m.p. = 149 °C. 1H-NMR (400 MHz, DMSO) δ 8.23 (1H, s, H2), 8.20 (1H, s, H8), 4.58 (2H, t, J = 6.1 Hz, CH2), 4.20 (4H, bs, CH2-piperidine), 3.94 (2H, t, J = 6.1 Hz, CH2), 1.73–1.63 (2H, m, CH2-piperidine), 1.63–1.55 (4H, m, CH2-piperidine). 13C-NMR (101 MHz, DMSO) δ 153.58 (C4), 152.39 (C2), 150.95 (C6), 140.18 (C8), 119.34 (C5), 46.08 (CH2-piperidine), 45.13 (CH2), 31.94 (CH2), 26.15 (CH2-piperidine), 24.74 (CH2-piperidine). Anal. calcd. for C16H16BrN5: C, 46.46; H, 5.20; N, 22.58. 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 room temperature overnight. Solvent was evaporated and the residue was purified by column chromatography (CH2Cl2:CH3OH = 60:1).

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 (193.2 mg, 0.92 mmol) to obtain 34a as orange powder (20.7 mg, 6 %, m.p. = 196 °C). 1H-NMR (600 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, CH2), 4.81 (2H, dd, J = 6.7, 4.6 Hz, CH2), 4.59 (2H, t, J = 1.8 Hz, CH-Fc), 4.27–4.21 (2H, m, CH-Fc), 3.91 (5H, s,Cp-Fc). 13C-NMR (151 MHz, DMSO) δ 150.70 (C4), 150.59 (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 (Cp-Fc), 68.18 (CH-Fc), 66.13 (CH-Fc), 48.80 (CH2), 44.54 (CH2). Anal. calcd. for C20H17ClFeN6: C, 55.52; H, 3.96; N, 19.42. Found: C, 55.56; H, 3.95; N, 19.37.

4-(Pyrrolidin-1-yl)-7-[2-(4-ferrocenyl-1,2,3-triazol-1-yl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (34b) Compound 34b 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 °C). 1H-NMR (300 MHz, DMSO) δ 8.12 (1H, s, H2), 7.89 (1H, s, H5′), 6.96 (1H, d, J = 3.6 Hz, H6), 6.53 (1H, d, J = 3.6 Hz, H5), 4.80 (2H, t, J = 5.5 Hz, CH2), 4.73–4.64 (2H, m, CH2), 4.63–4.59 (2H, m, CH-Fc), 4.29–4.24 (2H, m, CH-Fc), 3.94 (5H, s, Cp-Fc), 3.66 (4H, s, CH2-pyrrolidine), 1.91 (4H, s, CH2-pyrrolidine). 13C-NMR (75 MHz, DMSO) δ 155.19 (C4), 151.71 (C2), 150.31 (C7a), 145.56 (C4′), 123.87 (C6), 121.03 (C5′), 103.11 (C4a), 101.23 (C5), 76.26 (Cq-Fc), 69.64 (Cp-Fc), 68.63 (CH-Fc), 66.63 (CH-Fc), 49.46 (CH2), 47.90 (CH2-pyrrolidine), 44.27 (CH2). Anal. calcd. for C24H25FeN7: C, 61.68; H, 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 34c 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). 1H-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, CH2), 4.67 (2H, t, J = 5.7 Hz, CH2), 4.61 (2H, d, J = 1.5 Hz, CH-Fc), 4.28–4.24 (2H, m, CH-Fc), 3.93 (5H, s, Cp-Fc), 3.82–3.79 (4H, m, CH2-pipridin), 1.64–1.60 (2H, m, CH2-pipridin), 1.52 (4H, d, J = 3.7 Hz, CH2-pipridin). 13C-NMR (75 MHz, DMSO) δ 156.63 (C4), 151.27 (C7a), 151.20 (C2), 145.60 (C4′), 124.33 (C6), 121.05 (C5′), 102.59 (C4a), 101.40 (C5), 76.22 (Cq-Fc), 69.65 (Cp-Fc), 68.63 (CH-Fc), 66.63 (CH-Fc), 49.41 (CH2), 46.63 (CH2-piperidine), 44.30 (CH2), 25.87 (CH2-piperidine), 24.65 (CH2-piperidine). Anal. calcd. for C25H27FeN7: C, 62.38; H, 5.65; N, 20.37. Found: C, 62.42; H, 5.64; N, 20.31.

6-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, 0.92 mmol) to obtain 35a as orange powder (123 mg, 37 %, m.p. = 240 °C).1H-NMR (300 MHz, DMSO) δ 8.76 (1H, s, H2), 8.50 (1H, s, H5′), 8.02 (1H, s, H8), 4.94–4.90 (2H, m, CH2), 4.84 (2H, d, J = 6.1 Hz, CH2), 4.60 (2H, d, J = 1.6 Hz, CH-Fc), 4.28–4.25 (2H, m, CH-Fc), 3.94 (5H, s, Cp-Fc). 13C-NMR (75 MHz, DMSO) δ 152.43 (C6), 152.06 (C2), 149.52 (C4), 147.73 (C8), 145.87 (C4′), 131.16 (C5), 121.23 (C5′), 76.02 (Cq-Fc), 69.64 (Cp-Fc), 68.70 (CH-Fc), 66.67 (CH-Fc), 48.88 (CH2), 44.37 (CH2). Anal. calcd. for C19H16ClFeN7: C, 52.62; H, 3.72; N, 22.61. Found: C, 52.56; H, 3.71; N, 22.66.

6-(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 mg, 0.82 mmol) to obtain 35b as orange powder (124 mg, 39 %, m.p. = 230 °C). 1H-NMR (300 MHz, 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, CH2), 4.70 (2H, t, J = 5.6 Hz, CH2), 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, s, CH2-pyrrolidine), 1.89 (4H, s, CH2-pyrrolidine). 13C-NMR (75 MHz, DMSO) δ 152.90 (C6), 152.72 (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 (CH-Fc), 66.63 (CH-Fc), 48.94 (CH2-pyrrolidine), 43.45 (CH2-pyrrolidine). Anal. calcd. for C23H24FeN8: C, 58.98; H, 5.17; N, 23.93. Found: C, 58.92; H, 5.16; N, 23.96.

6-(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 mg, 0.77 mmol) to obtain 35c as orange powder (47 mg, 15 %, m.p. = 233 °C). 1H-NMR (300 MHz, 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, CH2), 4.70 (2H, t, J = 5.7 Hz, CH2), 4.66–4.55 (2H, m, CH-Fc), 4.30–4.22 (2H, m, CH-Fc), 4.14 (4H, bs, CH2-piperidin), 3.94 (5H, s, Cp-Fc), 1.62 (2H, s, CH2-piperidine), 1.52 (4H, s, CH2-piperidine). 13C-NMR (75 MHz, DMSO) δ 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), 68.66 (CH-Fc), 66.64 (CH-Fc), 48.93 (CH2), 43.48 (CH2), 26.04 (CH2-piperidine), 24.68 (CH2-piperidine). Anal. calcd. for C24H26FeN8: C, 59.76; H, 5.43; N, 23.23. Found: C, 59.70; H, 5.42; N, 23.28.

3.8. Kinetic Solubility Assay

The compounds’ DMSO stock solutions in concentration of 10 mM were serially diluted by factor 3.3×, 3×, 3.3× and 3× in DMSO. Prepared solutions were spiked into phosphate-buffered saline (PBS, 100 mM) to obtain final compounds’ concentrations of 100, 30, 10, 3 and 1 µM (3 µL of prepared dilutions were spiked to 297 µL 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 × 3 mm i.d., 5 µm particle size (Phenomenex, Torrance, CA, USA) was used for analysis. Sample injection volume was 2 µL and the flow rate was 1 mL min−1. All compounds were analysed in duplicates. Fast-gradient elution was as follows: from 0 to 100% of acetonitrile (0−3 min), 100% of acetonitrile (3.0−3.5 min), from 100 to 0% of acetonitrile (3.5−3.7 min), and re-equilibration with 100% of aqueous mobile phase (3.7−5.0 min). All measurements were performed on an HPLC-DAD 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]

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% CO2) in MDCK cell culture medium containing 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 seeded 4 days prior to the experiment in concentration of 0.3 × 106 cells mL−1 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 CO2 incubator in D-PBS containing 1% DMSO (1% v/v) with or without Elacridar (2 µM). Compounds, in final concentration of 10 µM, were prepared in transport medium containing Lucifer yellow (100 µM), with DMSO contentment of 1% (v/v) and in the presence and without Elacridar. Lucifer yellow calibration curve was prepared by serial dilution (12 points, with 100 µM as the highest point). Monolayer integrity was examined by fluorescent 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 (C0) was sampled 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 µL) were injected onto a reversed phase HPLC column Luna Omega Polar C18, 30 × 2.1 mm i.d., 1.6 µm 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 (90/10/0.1, v/v/v). Flow rate was kept at 0.7 mL min−1 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 compound concentration. Apparent permeability coefficient, Papp (nm s−1) values were calculated by using the following Equation (1):P where: dQ/dT = permeability rate; C0 = initial concentration in donor compartment; A = surface area of the cell monolayer (0.7 cm2).

The efflux ratio in the presence or absence of the P-gp inhibitor was calculated from Papp values, using following Equation (2):Efflux ratio = P

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 µM, were incubated in phosphate buffer (50 mM, pH 7.4) for 60 min at 37 °C together with liver microsomes and NADPH generating system (nicotinamide adenine dinucleotide phosphate (NADP, 0.5 mM), glucose-6-phosphate (G6P, 5 mM), glucose-6-phosphate dehydrogenase (1.5 U mL−1) 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): where 52.5 mg protein/g liver is used as a constant.

Predicted in vivo hepatic clearance, in vivo CLh was calculated as follows: where: LW/BW = liver weight/body weight [g kg−1]; 25.7 (human), 87.5 (mouse); Q = LBF = liver blood flow [mL min−1 kg−1]; 21/1.26 (human), 131/7.86 (mouse).

Predicted in vivo hepatic clearance can be expressed as %LBF and calculated as follows:

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 11c, 13a, 15b (c = 2.0 × 10−3 mol/L) and 6-chloropurine (c = 1.2 × 10−2 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 × 10−2 mol/L) was prepared from dry pure substance in ethanol (Kemika). For the supporting electrolyte, analytical grade NaClO4 (Kemika) was used. Water was deionized by the Millipore Milli-Q system to the resistivity ≥ 18 MΩcm. Voltammetric 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”. 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 NaClO4), adjusted to the desired pH value, 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.

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, 16a, 23a−23c, 24a−24c, 25a−25c) were prepared using Cu-promoted azide-alkyne cycloaddition (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. 6-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 µM, respectively. 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 11a−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 13a 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.