Title: Ferrocene bridged nucleosides: synthesis, electrochemistry, antitrypanosomal and antiviral activity studies
Subject: Journal of Organometallic Chemistry, 1050 (2026) 124081. doi:10.1016/j.jorganchem.2026.124081
Author: Rafa› Karpowicz
Keywords: ferrocene,CuAAC reaction,nucleosides,antitrypanosomal,HCoV-OC43 coronavirus,bioorganometallics

R. Karpowicz et al.	J o u rn al o f O rga n o m e ta llic C h em is try 1 050 (2026) 124081

Journal of Organometallic Chemistry 1050 (2026) 124081

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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Ferrocene bridged nucleosides: synthesis, electrochemistry, antitrypanosomal and antiviral activity studies
Rafał Karpowicz ^a, Dietmar Steverding ^b, Judith Füllborn-Ott ^c, Ingo Ott^c, Sansthita Dhital ^d, Tim Albrecht ^d, Konrad Kowalski ^a,*
^a University of Ło´d´z, Faculty of Chemistry, Department of Organic Chemistry, Tamka 12, 91-403 Ł´od´z, Poland
^b Bob Champion Research & Education Building, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7UQ, UK
^c Technische Universita¨t Braunschweig, Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstr. 55, 38106 Braunschweig, Germany

ABSTRACT

^d School of Chemistry, University of Birmingham, Edgbaston Campus, Birmingham B15 2TT, UK

Keywords:
ferrocene
CuAAC reaction
nucleosides
antitrypanosomal
HCoV-OC43 coronavirus
bioorganometallics

ARTICLE INFO
Five ferrocenyl–containing dinucleosides were obtained using the copper(I)–catalyzed azide–alkyne cycloaddition reaction of a 1,1’–dipropargylated ferrocenyl synthon with nucleosides or nucleobase azides. Cyclic voltammetry measurements revealed that the newly synthesized compounds can undergo reversible, one–electron oxidation–reduction at potential E^0 of ca. +0.5 V vs. decamethyl ferrocene, and thus behave similar to ferrocene. Antitrypanosomal evaluation using Trypanosoma brucei bloodstream forms showed overall low activity with the exception for the diadenosine compound 1 for which a GI50 of 42 ± 5 µM was determined. Likewise, 1 showed also weak activity against the HCoV–OC43 coronavirus with an IC_50 of 211 ± 68 µM. This activity was only slightly better than that of the diadenine (3) and the thymidine (4) dinucleosides. Despite the lack of strong antimicrobial activity, ferrocene–bridged dinucleosides add new knowledge to a growing field of organometallic nucleic acid components.

* Corresponding author.
E-mail address: kondor15@wp.pl (K. Kowalski).
https://doi.org/10.1016/j.jorganchem.2026.124081
Received 1 December 2025; Received in revised form 20 February 2026; Accepted 21 February 2026
Available online 22 February 2026
0022-328X/Crown Copyright © 2026 Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

1.		Introduction
Bioorganometallic [1–4] compounds have been shown to exhibit anticancer [5–10], antibacterial [4], antiparasitic [11] and antiviral [12, 13] activity. In addition, bioorganometallic compounds can be useful tools for bioimaging [14] and radiotherapy [15]. Of particular interest are bioorganometallic conjugates of biopolymers, such as peptides, proteins [16] and nucleic acids [17].
Ferrocene (FcH = Fe(η^5–C5H5)2) [18,19] is an aromatic sandwich complex, which has been the most widely studied organometallic compound for biological applications [20,21] because of several reasons and physicochemical traits. For instance, FcH and many ferrocenyl (Fc) derivatives are characterized by resistance against dioxygen, moisture and elevated temperatures, which enables handling them safely without the danger of quick decomposition. Also, FcH and many of its simple derivatives are available from commercial sources, making the synthesis of complex derivatives less time–consuming. Another advantage of FcH is that it can be functionalized using a variety of organic reactions [18, 19].
It has been shown that linking the Fc group with biologically relevant compounds and macromolecules generates conjugates with useful properties. Firstly, its redox activity (due to reversible Fe^2+ oxidation) allows for electrochemical studies and detection [22]. Secondly, its ability to produce reactive oxygen species (ROS) inside cancer cells enables for its elimination via the induction of apoptosis and ferroptosis [20,21]. Yet another relates to lipophilicity increase and the alternation of the overall 3D conformation of ferrocenyl conjugates vs. its organic congeners.
Due to their biological importance, nucleic acids and their components have long been recognized as attractive ligands for Fc conjugation [17,21–23]. Such conjugates have been shown to display anticancer [21], antimicrobial [24], and antiviral [25] activities, or to be useful tools for electrochemical studies [22]. In addition, nucleic acids and its components can be modified by ferrocenylation in many different ways. The privileged modification sites are at the C5– and the N3–position of pyrimidines [22,26,27], whereas ferrocenylation of purines [26,28], the (deoxy)ribose backbone [29], or the phosphodiester bridge are less common [30]. Another interesting example of a ferrocene–modified
oligonucleotide is an oligomer, in which the entire deoxyribose backbone had been replaced with suitably modified ferrocenyl units creating an organometallic structural mimic of DNA [31].
The copper(I)–catalysed Huisgen azide–alkyne 1,3–dipolar (3+2)– cycloaddition reaction (CuAAC) together with a copper–free cycloaddition reactions (all collectively named as click reactions or click chemistry) [32] plays an important role in nucleic acid chemistry [33]. We also have previously adopted the CuAAC and an inverse–electron–demand Diels–Alder (4+2)–(IEDDA) cycloaddition reaction as synthetic approaches to modify nucleic acid components [21,34–37]. This resulted in the preparation of dinucleosides A [35], B [36] and C [37] (Fig. 1), all of which share the same CH2–N–(CH2–1H–1,2,3-tri- azolyl)_2 linker that substitutes the canonical phosphodiester bridge. In addition, due to the presence of the [Re_2(µ–Cl)_2(CO)_6] and the pyrenyl entities, compounds A and C show luminescence properties allowing to track their distribution in human cancer cells by confocal microscopy imaging [35,37]. On the other hand, ferrocenyl dinucleoside B is redox active [36]. Another example of click nucleosides reported by our group is the biferrocenyl thymidine conjugate D (Fig. 1) [21,34]. It showed anticancer activity in cisplatin (cisPt) resistant lung cancer cells, which was mechanistically due to ROS generation and ferroptosis induction [21,34]. It was also shown that the oxidised D^þ belongs to class II mixed-valence species [34].
In this communication we report on new 1,1’–ferrocenyl– (1,2,3–triazolyl)_2–bridged dinucleosides 1–3, 5 and 6 as well as the synthetic intermediate 4 (Scheme 1 and 2). The electrochemistry of the newly synthesised compounds was studied by cyclic voltammetry and their biological activity was investigated with Trypanosoma brucei bloodstream forms and HCoV–OC43 coronavirus (surrogate of SARS–CoV–2). Selection of these two pathogens was justify by our previous findings which showed that some adenine–ferrocenyl complexes exhibit anti–trypanosomal activity in the mid–micromolar range [24] and that thymine-ferrocene conjugates are weak inhibitors of the coronavirus S protein and ACE2 receptor interaction [25].
2.		Results and discussion
2.1.		Synthesis of 1–6
Compounds 1-6 were obtained according to Schemes 1 and 2 using the CuAAC reaction of 1,1’–dimethyloxypropargyl ferrocene (E) [38] with	5′–azido–2′,3′–O–isopropylidene adenosine F [39], 3′–azido–3′–deoxythymidine G (purchased from commercial source), and 9–(2–azidoethyl)–adenine H [40]. In order to obtain the symmetrically substituted products 1–3, a two–fold molar excess of F, G, or H over ferrocene E was used (Scheme 1). The first step in the synthesis of 5 and 6 involved preparation of the common alkyne intermediate 4. In a second step, compound 4 reacted with stoichiometric amounts of F or H under CuAAC reaction conditions to afford 5 and 6, respectively (Scheme 2). Compounds 1-6 were obtained in 52, 65, 44, 27, 19 and 26 % yield, respectively. Nucleoside analogs 1-6 are stable in the solid state and as such can be stored for months in the fridge. The structures and purity of all compounds were confirmed by spectroscopic analysis (^1H, ^13C NMR, HRMS, FTIR). The ^1H, ^13C NMR, and HRMS spectra are shown in Figs. S1–S51 (Supplementary Materials).
2.2.		Electrochemistry of E, and 1–6

Fig. 1. Structures of CH_2–N–(CH_2–1H–1,2,3–triazolyl)_2–bridged dinucleoside analogs A–C and biferrocenyl compound D.

The ferrocenyl entity is an excellent redox-active unit which can be utilized as a building block for the construction of single–molecule electronics [41,42], molecular ring–like structures [43], and hal- oferrocenes [44]. Presence of the ferrocenyl linker in compounds 1-6 makes them well suited for electrochemical studies. The electrochemical characterization of compounds E and 1–6 was performed in a three–electrode configuration under anaerobic conditions in 0.1 M tetrabutyl ammonium perchlorate TBAP/DMF (Autolab, working electrode: glassy carbon (area: 0.07 cm^2); Pt wire as counter- and quasi reference electrodes, respectively; purging with Ar > 15 min.). The working electrode was polished with 0.25 µm alumina slurry prior to each experiment and rinsed with copious amounts of H_2O, acetone and subsequently dried in an oven at 60^◦C. Decamethyl ferrocene (DmFc) was added to the electrolyte at the end of an experiment, as an internal redox standard (E^0(DmFc) vs. E^0(Fc) = -0.532 V in our experiments). Cyclic

Scheme 1. Synthesis of compounds 1–3. Reagents and conditions: CuSO_4 _* 5H_2O, Na ascorbate, DMF–H_2O (9:1 v/v), 80^◦C, 24 h.

Scheme 2. Synthesis of compounds 4–6. Reagents and conditions a: CuSO_4 _* 5H_2O, Na ascorbate, DMF–H_2O (9:1 v/v), ambient temperature, 24 h; b: CuSO_4 _* 5H_2O, Na ascorbate, DMF–H2O (9:1 v/v), 80^◦C, 24.

voltammetry (CV) are shown in Fig. S52–58. All E^0 and peak potential values, E_p,a and E_p,c, were corrected for uncompensated resistance effects, iRu. To this end, Ru was estimated in the background electrolyte using frequency-dependent impedance spectroscopy (f = 0.1 Hz – 1 MHz) at fixed potential (with no apparent redox activity, E ≈ 0 V vs. Pt), via extrapolation of the Nyquist plot, -Z_im vs. Z_re, towards the real axis (Ru ≈ 910 Ω). The concentrations of ferrocene derivatives and of DmFc were comparable and ranged from 140 μM for 2 to 1.5 mM for 3. Table 1 contains E^0, and peak separation values Ep,a - Ep,c, recorded at a scan rate v = 0.25 V/s. The latter are close to 0.06 V and thus broadly in line with expectations for a reversible, one-electron transfer process. The CV of 1–6 have shown that redox potential of ferrocenyl linkers is almost identical to that of redox potential of the precursor compound E. It illustrates negligible influence of the “nucleic acid” components on the electronic density of the ferrocene unit in studied compounds.
Table 1
Electrochemistry data for compounds E, 1–6.
Compound | E^0 (V) vs. DmFc/DmFc^+ | Ep,a – Ep,c (V)
E | 0.53 | 0.07
1 | 0.49 | 0.06
2 | 0.51 | 0.07
3 | 0.49 | 0.09
4 | 0.52 | 0.08
5 | 0.51 | 0.09
6 | 0.52 | 0.06

2.3.		Antitrypanosomal activity of E and 1–6
In the course of earlier studies, we discovered that some adenine–ferrocenyl conjugates exhibit anti–trypanosomal activity in the mid–micromolar range [24]. Justify by that, the trypanocidal activity of the ferrocene bridged nucleosides 1–6 and the parent compound E was evaluated with Trypanosoma brucei bloodstream forms using the resa- zurin viability assay [24]. Except for 1, all other nucleoside analogues were less active compared to the parent compound E (Table 2). Thus, only symmetrical substitution of E with 5′–azido–2′,3′–O–isopropylidene adenosine yielded a derivative with increased trypanocidal activity with a 50 % growth inhibition (GI_50) value in the mid-molar range (1.7–times more trypanocidal). To establish the selectivity of the ferrocene bridged nucleosides, their cytotoxicity was determined with human myeloid leukaemia HL–60 cells using the same viability assay. Except for 1, all other compounds were nontoxic to the human cells (Table 2). With a value of 1.95, the selectivity of 1 was found to be only moderate (Table 2). The commercial drugs suramin and ethidium bromide used for the treatment of human and animal African trypanosomiasis, respectively, are much more potent with GI50 values in the sub–molar range and selectivity indices of 20 and >2600 (Table 2). Except for 1, all other modifications reduce the activity of the parent compound E. However, E itself was already not very active. Only 1 displayed cytotoxic activity and 1 was also the most trypanocidal.
2.4.		Antiviral activity of E and 1–6
The antiviral activity of E and the bioorganometallics 1–6 was determined by an in–cell ELISA method in rhabdomyosarcoma (RD) cells infected with the human coronavirus HCoV–OC43, which represents a surrogate for the SARS–CoV–2 coronavirus (Table 3, Figure 2) [45]. The antiviral activity is given as IC_50 value and the cytotoxicity of the agents against the RD host cells was expressed as CC50 value. While E, 2, 5 and 6 were inactive up to the highest evaluated dosage of 500 µM, compounds 1, 3, and 4 triggered weak antiviral effects with IC50 values in the range of 200–250 µM. With CC_50 values above 500 µM for all test compounds, the observed antiviral effects were not accompanied by relevant cytotoxic effects against the host cells. On the other hand, the broad-spectrum antiviral drug remdesivir was more than 200–times more effective in inhibiting viral replication in RD cells than compounds 1, 3, and 4 (Table 3, Figure 2).
3.		Conclusion
In this work we report on a new family of ferrocene–containing dinucleoside analogs. They were obtained via CuAAC reaction and all share the ferrocenyl–ether–triazole linker which replaced natural phosphodiester bond. The presence of the ferrocenyl entity confers the dinucleosides with redox activity. They undergo a reversible one–electron oxidation–reduction processes at ca. 50 mV vs. DmFc/DmFc^+. as shown by CV. Biological studies of the dinucleosides revealed overall weak trypanocidal activity and HCoV–OC43 antivirus activity. Of all studied compounds only dinucleoside 1 bearing two adenosine entities
Table 2
Trypanocidal and cytotoxic activity of E and 1–6.

Compound

T. brucei
GI50 (μM)

HL-60
GI50 (μM)

Selectivity^b

E | 71±37 | >100 (7%)^a | >1.4
1 | 42±5 | 82±2 | 2
2 | >100 (17%)^a | >100 (0%)^a | 1
3 | >100 (19)^a | >100 (0%)^a | 1
4 | 100±42 | >100 (0%)^a | >1
5 | >100 (20%)^a | >100 (0%)^a | 1
6 | >100 (33%)^a | >100 (0%)^a | 1
Suramin | 0.038±0.003 | >100 (0%)^a | >2631
Ethidium bromide | 0.16±0.06 | 3.3±0.2 | 20.6
^a , % inhibition at 100 μM
^b , selectivity = ratio of GI50(T. brucei)/GI50(HL-60)

Table 3
Antiviral activity (IC_50; µM) and cytotoxic effect (CC_50; µM) of compounds E and 1–6 in RD cells infected with human coronavirus HCoV–OC43 at an MOI of 0.1; results are presented as mean values ± standard deviation of IC_50 or CC_50 values from 3 to 4 independent experiments.

Compound

Antiviral activity
IC50 [µM]

Cytotoxicity
CC50 [µM]

E | >500 | >500
1 | 211 ± 68 | >500
2 | >500 | >500
3 | 250 ± 12 | >500
4 | 254 ± 36 | >500
5 | >500 | >500
6 | >500 | >500
Remdesivir | 0.09 ± 0.02 | >10

showed moderate activity against T. brucei bloodstream forms with a GI_50 = 42 ± 5 µM. Remarkably the same compound 1 was also the most active derivative against HCoV–OC43 coronavirus, but its antiviral activity was likewise weak (IC50 = 211 ± 68 µM) and similar to that of its diadenine (3) and monothymidine (4) congeners. Irrespectively of the weak antiparasite and antiviral activities, the studied nucleosides represent new scaffold in the field of organometallic nucleic acid components. This scaffold is worth further examination by means of chemical modification and conjugation with natural nucleic acid oligonucleotides.
4.		Experimental
4.1.		General remarks
All preparations were carried out using standard Schlenk techniques. Chromatographic separations were carried out on silica gel 60 (Merck, 230–400 mesh ASTM). Toluene was distilled and purged with argon prior to use. Other solvents were of reagent grade and used without prior purification. Ferrocene, propargyl bromide, adenine, 2′,3′–O–isopropylidene adenosine, 3′–azido–3′–deoxythymidine (G) and Na ascorbate were purchased from commercial suppliers and used without further purification. 1,1’–Dimethyloxypropargyl ferrocene (E) [38], 5′–azido–2′,3′–O–isopropylidene adenosine (F) [39] and 9– (2-azidoethyl)–adenine (H) [40] were synthesized according to literature.
^1H NMR (600 MHz) and ^13C{1H} NMR (150 MHz) spectra were recorded on a Bruker Avance III 600 spectrometer operating at 298 K in Fourier transform mode. Chemical shifts are reported in δ units (ppm) using the residual signal of DMSO (^1H δ 2.50 ppm, ^13C δ 39.5) as reference. High–resolution mass spectrometry (HRMS) measurements were performed using a Synapt G2-Si mass spectrometer (Waters) equipped with an ESI source and quadrupole–Time–of–Flight mass analyzer. The mass spectrometer was operated in positive or negative ion detection modes. The optimized source parameters were: capillary voltage 2.7 kV, cone voltage 30 V, source temperature 110^◦C, desolvation gas (nitrogen) flow rate 600 L/h with a temperature of 350^◦ C, and nebulizer gas pressure 6.5 bar. To ensure accurate mass measurements, data were collected in centroid mode, and mass was corrected during acquisition using leucine encephalin solution as an external reference, which generated reference ions at m/z 554.2615 ([M-H]^-) in negative ESI mode and at m/z 556.2771 Da ([M+H]^+) in positive ESI mode. The results of the measurements were processed using the MassLynx 4.1 software (Waters). IR spectra (KBr pellets) were measured on an FTIR Nexus Nicolet apparatus. Microanalyses were carried out by the Analytical Services of the Polish Academy of Sciences (Ło´d´z). DCM = dichloromethane, MeOH = methanol, Adenine = ade, cyclopentadienyl = Cp, thymine = thym, triazole = triaz, isoprop = isopropylidene.

Fig. 2. Antiviral activity and cytotoxicity of E and 1–6 in HCoV–OC43 infected RD cells (MOI = 0.1, n = 3–4); the IC_50 and CC_50 values given as inserts were obtained from the depicted functions.

4.2.		Synthesis of compound 1–3
The same synthetic procedure was applied for the synthesis of compounds 1–3.
Alkyne E (50 mg, 0.15 mmol, 1.0 eq.), sodium ascorbate (61 mg, 0.31 mmol, 2.0 eq.) and CuSO_4 × 5H_2O (23 mg, 0.09 mmol, 0.6 eq.), were added to a stirred solution of azide F (103 mg, 0.31 mmol, 2.0 eq. for 1), G (82 mg, 0.31 mmol, 2.0 eq. for 2) or H (75 mg, 0.31 mmol, 2.0 eq. for 3) in 10 mL of DMF–H_2O mixture (9/1 v/v). The resulting reaction mixture was stirred at 80^◦C for 24 h. Then H2O (20 mL) was added and the mixture was extracted with DCM (3×30 mL). The organic layer was separated, dried over anhydrous Na2SO4, transferred to round bottom flask and all volatiles were evaporated under reduced pressure. After evaporation, the remaining oil was subjected to column chromatography on SiO2 (eluting mixture: DCM/MeOH 25:2–25:3 v/v for 1, DCM/ MeOH 25:2 v/v for 2, DCM/MeOH 25:2–25:5 v/v for 3) to afford analytically pure compound 1 (yellow solid), 2 (orange solid) and 3 (yellow solid) in 52% (79 mg), 65% (84 mg) and 44% (50 mg) yield, respectively. Rf = 0.24 (1), 0.04 (2), 0.05 (3) (DCM/MeOH, 25:2 v/v).
1:		^1H NMR (600 MHz, DMSO–d6): δ = 8.28 (s, 2H, 1,2,3–triaz), 8.19 (s, 2H, ade), 7.90 (s, 2H, ade), 7.35 (bs, 4H, NH2 ade), 6.21 (d, JH,H = 2.0 Hz, 2H, H1′ ribose), 5.45 (dd, JH,H = 6.1, 2.1 Hz, 2H, H2′ ribose), 5.13 (dd, JH,H = 6.2, 3.0 Hz, 2H, H3′ ribose), 4.75 (dd, JH,H = 14.1, 5.3 Hz, 2H, H5′ or H5’’ ribose), 4.66 (dd, JH,H = 14.1, 7.9 Hz, 2H, H5’ or H5′’ ribose), 4.55–4.52 (m, 2H, H4′ ribose), 4.40 (dd, JHH = 12.1, 3.5 Hz, 4H, CH2), 4.20 (s, 4H, CH2), 4.14 (d, JH,H =1.6 Hz, 4H, Cp), 4.08 (pt, JH,H = 1.6 Hz, 4H, Cp), 1.52 (s, 6H, isoprop), 1.31 (s, 6H, isoprop) ppm. ^13C NMR (150 MHz, DMSO–d6): δ = 156.1, 152.7, 148.5, 144.1, 140.0, 124.1, 119.1, 113.6, 89.0, 84.4, 83.4, 83.2, 81.4, 69.6, 68.5, 67.3, 62.2, 50.9, 26.8, 25.1 ppm. MS (TOF ESI+): m/z = 987.3441 [M+H^+] (calcd. for C44H51N16O8Fe: 987.3425). FTIR (KBr ν [cm^-1]): 3152, 2987, 2905, 1736, 1661, 1598, 1480, 1375, 1329, 1235, 1214, 1077. Anal. calcd for C44H50N16O8Fe: C, 53.55; H, 5.11, N, 22.71%. Found: C, 53.25; H, 5.39, N, 22.38%.
2:		^1H NMR (600 MHz, DMSO–d6): δ = 11.32 (s, 2H, NH thym), 8.25 (s, 2H, 1,2,3–triaz), 7.80 (s, 2H, thym), 6.40 (pt, JH,H = 6.8 Hz, 2H, H1’ deoxyribose), 5.35 (dt, JH,H = 8.5, 5.3 Hz, 2H, H3’ deoxyribose), 5.25 (t, JH,H =5.1 Hz, 2H, OH), 4.50 (s, 4H, CH2), 4.29 (s, 4H, CH2), 4.21 (pt, JH, H = 1.5 Hz, 4H, Cp), 4.20 (app q, JH,H = 3.6 Hz, 2H, H4’ deoxyribose), 4.14 (pt, JH,H = 1.5 Hz, 4H, Cp), 3.71–3.67 (m, 2H, H5’ or H5’’ deoxyribose), 3.62–3.59 (m, 2H, H5’ or H5’’ deoxyribose), 2.75–2.70 (m, 2H, H2’ or H2” deoxyribose), 2.66–2.61 (m, 2H, H2’ or H2’’ deoxyribose), 1.80 (s, 6H, CH_3 thym) ppm. ^13C NMR (150 MHz, DMSO–d6): δ = 163.6, 150.3, 144.3, 136.1, 123.3, 109.5, 84.4, 83.8, 83.5, 69.7, 68.6, 67.5, 62.3, 60.6, 59.0, 37.0, 12.1 ppm. MS (TOF ESI+): m/z = 857.2672 [M+H^+] (calcd. for C38H45N10O10Fe: 857.2670). FTIR (KBr ν [cm^-1]): 2930, 2866, 1693, 1470, 1273, 1095. Anal. calcd for C38H44N10O10Fe: C, 53.28; H, 5.18, N, 16.35%. Found: C, 52.98; H, 5.42, N, 16.09%.
3:		^1H NMR (600 MHz, DMSO–d6): δ = 8.11 (s, 2H, ade), 7.97 (s, 2H, ade), 7.78 (s, 2H, 1,2,3–triaz), 7,17 (bs, 4H, NH2), 4.85 (t, JH,H = 5.9 Hz, 4H, CH2), 4.63 (t, JH,H =5.9 Hz, 4H, CH2), 4.41 (s, 4H, CH2), 4.18 (s, 4H, CH2), 4.18 (pt, JH,H = 1.6 Hz, 4H, Cp), 4.12 (pt, JH,H = 1.6 Hz, 4H, Cp) ppm. ^13C NMR (150 MHz, DMSO‑d6): δ = 155.8, 152.4, 149.4, 144.1, 140.4, 123.9, 118.5, 83.4, 69.6, 68.6, 67.2, 62.1, 48.5, 42.9 ppm. (TOF ESI+): m/z = 731.2466 [M+H^+] (calcd. for C32H35N16O2Fe: 731.2478). FTIR (KBr ν [cm^-1]): 3321, 3138, 2954, 2860, 1660, 1602, 1489, 1421, 1330, 1253, 1077, 797. Anal. calcd for C32H34N16O2Fe: C, 52.61; H, 4.69, N, 30.68%. Found: C, 52.59; H, 4.71, N, 30.39%.
4.3.		Synthesis of compound 4
Compound G (236 mg, 0.88 mmol, 1.0 eq.), sodium ascorbate (175 mg, 0.88 mmol, 1.0 eq.) and CuSO_4 × 5H_2O (66 mg, 0.26 mmol, 0.3 eq.), were added to a stirred solution of compound E (284 mg, 0.88 mmol, 1.0 eq.) in 10 mL of DMF–H_2O mixture (9/1 v/v). The resulted reaction mixture was stirred at ambient temperature for 24 h. Then H_2O (20 mL) was added and mixture was extracted with dichloromethane (3×30 mL). The organic layer was separated, dried over anhydrous Na2SO4, transferred to round bottom flask and all volatiles were evaporated under reduced pressure. After evaporation, the remaining oil was subjected to column chromatography on SiO_2 (DCM/MeOH 25:2 v/v) to afford analytically pure compound 4 as an orange oil in 27% (140 mg) yield. Rf = 0.29 (4) (DCM/MeOH, 25:2 v/v).
4:		^1H NMR (600 MHz, DMSO–d6): δ = 11.32 (s, 1H, NH thym), 8.25 (s, 1H, 1,2,3–triaz), 7.80 (s, 1H, thym), 6.40 (pt, JH,H = 6.6 Hz, 1H, H1’ deoxyribose), 5.35 (dt, J_H,H = 8.5, 5.5 Hz, 1H, H3’ deoxyribose), 5.25 (t, JH,H =5.5 Hz, 1H, OH), 4.50 (s, 2H, CH2), 4.30 (s, 2H, CH2), 4.26 (s, 2H, CH2), 4.23 (pt, JH,H = 1.6 Hz, 2H, Cp), 4.19 (pt, JH,H = 1.6 Hz, 3H, Cp and H4’ deoxyribose), 4.15–4.14 (m, 4H, Cp), 4.07 (d, JH,H =2.3 Hz, 2H, CH2), 3.71–3.67 (m, 1H, H5’ or H5’’ deoxyribose), 3.62–3.59 (m, 1H, H5’ or H5’’ deoxyribose), 3.41 (t, JH,H =2.4 Hz, 1H, CH), 2.75–2.70 (m, 1H, H2’ or H2’’ deoxyribose), 2.66–2.61 (m, 1H, H2’ or H2’’ deoxyribose), 1.80 (s, 3H, CH3 thym) ppm. ^13C NMR (150 MHz, DMSO–d6): δ = 163.6, 150.3, 144.3, 136.1, 123.3, 109.5, 84.4, 83.8, 83.5, 82.9, 80.3, 76.9, 69.7, 69.6, 68.7, 68.6, 67.5, 66.8, 62.3, 60.6, 59.0, 56.1, 37.0, 12.1 ppm. MS (TOF ESI+): m/z = 612.1521 [M+Na^+] (calcd. for C28H31N5O6FeNa: 612.1521). FTIR (KBr ν [cm^-1]): 3254, 3075, 2929, 2863, 2111, 1693, 1469, 1372, 1272, 1237, 1069. Anal. calcd for C28H31N5O6Fe: C, 57.06; H, 5.30, N, 11.88%. Found: C, 56.77; H, 5.51, N, 11.83%.
4.4.		Synthesis of compounds 5 and 6
Compound 4 (85 mg/192 mg, 0.14 mmol/0.32 mmol, 1.0 eq. for 5/ 6), sodium ascorbate (29 mg/65 mg, 0.14 mmol/0.32 mmol, 1.0 eq.) and CuSO4 × 5H2O (11 mg/24 mg, 0.04 mmol/0.09 mmol, 0.3 eq.) were added to a stirred solution of azide F (48 mg, 0.14 mmol, 1.0 eq. for 5) or H (79 mg, 0.32 mmol, 1.0 eq. for 2) in 10 mL of DMF–H2O mixture (9/1 v/v). The resulting reaction mixture was stirred at 80^◦C for 24 h. Then H_2O (20 mL) was added and mixture was extracted with DCM (3×30 mL). The organic layer was separated, dried over anhydrous Na2SO4, transferred to round bottom flask and all volatiles were evaporated under reduced pressure. After evaporation, the remaining oil was subjected to column chromatography on SiO_2 (eluting mixture: DCM/ MeOH 25:2–50:5 v/v for 5, DCM/MeOH 25:2–100:15 v/v for 6) to afford analytically pure compound 5 (yellow solid) or 6 (yellow solid) in 19% (25 mg) and 26% (66 mg) yield, respectively. Rf = 0.08 (5), 0.03 (6) (DCM/MeOH, 25:2 v/v).
5:		^1H NMR (600 MHz, DMSO–d6): δ = δ = 11.32 (s, 1H, NH thym), 8.28 (s, 1H, 1,2,3–triaz), 8.24 (s, 1H, 1,2,3–triaz), 8.19 (s, 1H, ade), 7.91 (s, 1H, ade), 7.80 (s, 1H, thym), 7.35 (bs, 2H, NH2 ade), 6.40 (pt, JH,H = 6.6 Hz, 1H, H1’ deoxyribose), 6.22 (d, J_H,H = 2.2 Hz, 1H, H1′ ribose), 5.45 (dd, JH,H = 6.2, 2.2 Hz, 1H, H2′ribose), 5.35 (dt, JH,H = 8.6, 5.5 Hz, 1H, H3’ deoxyribose), 5.25 (t, JH,H = 5.2 Hz, 1H, OH), 5.13 (dd, JH,H = 6.3, 3.2 Hz, 1H, H3′ ribose), 4.76 (dd, JH,H = 14.1, 8.3 Hz, 1H, H5′ or H5’’ ribose), 4.66 (dd, J_H,H = 14.1, 8.1 Hz, 1H, H5′ and H5’’ ribose), 4.55-4.53 (m, 1H, H4′ ribose), 4.48 (s, 2H, CH2), 4.42 (dd, JHH = 12.1, 3.4 Hz, 2H, CH2), 4.27 (s, 2H, CH2), 4.22 (s, 2H, CH2), 4.19 (pt, JH,H = 1.6 Hz, 3H, Cp and H4’ deoxyribose), 4.17-4.16 (m, 2H, Cp), 4.11 (pt, J_H, H = 1.6 Hz, 4H, Cp), 3.70-3.67 (m, 1H, H5’ or H5’’ deoxyribose), 3.623.59 (m, 1H, H5’ or H5’’ deoxyribose), 2.74–2.70 (m, 1H, H2’ or H2’’ deoxyribose), 2.65–2.61 (m, 1H, H2’ or H2’’ deoxyribose), 1.80 (s, 3H, CH3 thym), 1.52 (s, 3H, isoprop), 1.31 (s, 3H, isoprop) ppm. ^13C NMR (150 MHz, DMSO–d6): δ = 163.6, 156.1, 152.7, 150.3, 148.5, 144.4, 144.1, 140.0, 136.1, 124.2, 123.3, 119.2, 113.6, 109.5, 89.0, 84.4, 83.8, 83.4, 83.4, 83.2, 81.5, 69.7, 69.6, 68.6, 68.5, 67.6, 67.3, 62.3, 62.2, 60.6, 59.0, 50.9, 37.1, 26.8, 25.1, 12.1 ppm. MS (TOF ESI+): m/z = 922.3046 [M+H^+] (calcd. for C41H48N13O9Fe: 922.3047). FTIR (KBr ν [cm^-1]): 3200, 2987, 2937, 2864, 1693, 1473, 1376, 1274, 1075, 870, 838, 801. Anal. calcd for C41H47N13O9Fe: C, 53.42; H, 5.14, N, 19.75%. Found: C, 53.14; H, 5.31, N, 19.42%.
6:		^1H NMR (600 MHz, DMSO–d6): δ = 11.33 (s, 1H, NH thym), 8.25 (s, 1H, 1,2,3–triaz), 8.11 (s, 1H, ade), 7.97 (s, 1H, ade), 7.80 (s, 1H, thym), 7.78 (s, 1H, 1,2,3–triaz), 7,16 (bs, 2H, NH2), 6.40 (pt, JH,H =6.6 Hz, 1H, H1’ deoxyribose), 5.35 (dt, J_H,H = 8.3, 5.2 Hz, 1H, H3’ deoxyribose), 5.24 (t, JH,H = 5.2 Hz, 1H, OH), 4.85 (t, JH,H = 5.8 Hz, 2H, CH2), 4.63 (t, JH,H = 5.6 Hz, 2H, CH2), 4.49 (s, 2H, CH2), 4.41 (s, 2H, CH2), 4.28 (s, 2H, CH2), 4.21 (t, JH,H =1.7 Hz, 2H, CH2), 4.19-4.18 (m, 5H, Cp and H4’ deoxyribose), 4.13 (m, 4H, Cp) 3.70–3.67 (m, 1H, H5’ or 5” deoxyribose), 3.62–3.58 (m, 1H, H5’ or 5’’ deoxyribose), 2.74–2.70 (m, 1H, H2’ or H2’’ deoxyribose), 2.65–2.61 (m, 1H, H2’ or H2’’ deoxyribose), 1.80 (s, 6H, CH3 thym) ppm. ^13C NMR (150 MHz, DMSO–d6): δ = 163.6, 155.8, 152.4, 150.3, 149.4, 144.4, 144.1, 140.4, 136.1, 123.9, 123.3, 118.5, 109.5, 84.4, 83.8, 83.5, 83.4, 69.7, 69.6, 68.6, 68.5, 67.6, 67.1, 62.3, 62.1, 60.6, 59.0, 48.5, 42.9, 37.1, 12.1 ppm. (TOF ESI+): m/z = 794.2569 [M+H^+] (calcd. for C35H40N13O6Fe: 794.2574). FTIR (KBr ν [cm^-1]): 3326, 3192, 2956, 2863, 1695, 1600, 1473, 1367, 1329, 1273, 1051, 817. Anal. calcd for C35H39N13O6Fe: C, 52.97; H, 4.95, N, 22.94%. Found: C, 52.95; H, 5.02, N, 22.89%.
4.5.		Antiviral activity against HCoV-OC43
The antiviral activity of the test compounds against HCoV–OC43 was determined according to a recently reported protocol [45]. In short: A virus dilution at an MOI (multiplicity of infection) of 0.1 was simultaneously added with the test compounds to an almost confluent layer of RD cells. After 24h, incubation was stopped and the viral load was quantified by an ELISA using an N–protein primary antibody (N–protein HCoV–OC43, Sino Biological) and an HRP–labeled secondary antibody (Sino biological). Following the determination of N–positive cells, the plates were washed twice with phosphate buffered saline and a crystal violet counter screen was performed to determine cytotoxicity.
4.6.		Trypanocidal and cytotoxic assays
Bloodstream forms of the T. brucei clone 427–221a [46] and human myeloid leukemia HL–60 cells [47] were incubated with various concentration of test compounds in 200 μl of Baltz medium [48] supplemented 16.7% bovine serum in 96-well plates in the presence of 0.9% DMSO. Controls were incubated in medium containing only 0.9% DMSO. The seeding densities were 1 × 10^4/ml for trypanosomes and 5 × 10^4/ml for HL–60 cells. After 24 h incubation at 37^◦C in a humidified atmosphere containing 5% of CO_2, 20 µL of a 0.5 mM resazurin solution prepared in PBS was added to the cultures. After a further 48 h incubation, the absorbance of the cultures was read at 570 nm (test wavelength) and 630 nm (reference wavelength) using a microplate reader. GI_50 values, i.e., the concentration of a test compound that reduces the growth rate of cells by 50% compared to the growth rate of the control cells, were calculated using the method described by Huber and Koella [49].
CRediT authorship contribution statement
Rafał Karpowicz: Investigation, Data curation. Dietmar Steverd- ing: Writing – original draft, Investigation, Data curation. Judith Füllborn-Ott: Investigation, Data curation. Ingo Ott: Writing – original draft, Investigation, Data curation. Sansthita Dhital: Writing – original draft, Investigation, Data curation. Tim Albrecht: Writing – original draft, Investigation, Data curation. Konrad Kowalski: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
K.K. thanks the University of Ł´od´z for support.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jorganchem.2026.124081.
Data availability
Data will be made available on request.
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