Next Article in Journal
Optimisation by Design of Experiment of Benzimidazol-2-One Synthesis under Flow Conditions
Next Article in Special Issue
Impact of the Position of the Chemically Modified 5-Furyl-2′-Deoxyuridine Nucleoside on the Thrombin DNA Aptamer–Protein Complex: Structural Insights into Aptamer Response from MD Simulations
Previous Article in Journal
Antioxidant and Lipoxygenase Inhibitory Activities of Essential Oils from Endemic Plants of Côte d’Ivoire: Zanthoxylum mezoneurispinosum Ake Assi and Zanthoxylum psammophilum Ake Assi
Previous Article in Special Issue
(F)uridylylated Peptides Linked to VPg1 of Foot-and- Mouth Disease Virus (FMDV): Design, Synthesis and X-Ray Crystallography of the Complexes with FMDV RNA-Dependent RNA Polymerase
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 24
Issue 13
10.3390/molecules24132446
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

New HSV-1 Anti-Viral 1′-Homocarbocyclic Nucleoside Analogs with an Optically Active Substituted Bicyclo[2.2.1]Heptane Fragment as a Glycoside Moiety

by
Constantin I. Tănase
1,*,
Constantin Drăghici
2,
Anamaria Hanganu
2,
Lucia Pintilie
1,
Maria Maganu
2,
Alexandrina Volobueva
3,
Ekaterina Sinegubova
3,
Vladimir V. Zarubaev
3,
Johan Neyts
4,
Dirk Jochmans
4 and
Alexander V. Slita
4
1
National Institute for Chemical-Pharmaceutical Research and Development, Department of Bioactive Substances and Pharmaceutical Technologies, 112 Vitan Av., 031299 Bucharest-3, Romania
2
Organic Chemistry Center “C.D.Nenitescu”, Spectroscopy Laboratory, 202 B Splaiul Independentei, 060023 Bucharest, Romania
3
Department of Virology, Pasteur Institute of Epidemiology and Microbiology, 197101 St. Petersburg, Russia
4
KU Leuven Department of Micobiology, Immunology and Transplantation, Rega Institute, Laboratory of Virology and Chemotherapy, Herestraat 49, BE-3000 Leuven, Belgium
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(13), 2446; https://doi.org/10.3390/molecules24132446
Submission received: 6 June 2019 / Revised: 28 June 2019 / Accepted: 30 June 2019 / Published: 3 July 2019
(This article belongs to the Special Issue Discovery, Design, Synthesis, and Application of Nucleoside/Nucleotides)
Browse Figures
Review Reports Versions Notes

Abstract

:
New 1′-homocarbanucleoside analogs with an optically active substituted bicyclo[2.2.1]heptane skeleton as sugar moiety were synthesized. The pyrimidine analogs with uracil, 5-fluorouracil, thymine and cytosine and key intermediate with 6-chloropurine (5) as nucleobases were synthesized by a selective Mitsunobu reaction on the primary hydroxymethyl group in the presence of 5-endo-hydroxyl group. Adenine and 6-substituted adenine homonucleosides were obtained by the substitution of the 6-chlorine atom of the key intermediate 5 with ammonia and selected amines, and 6-methoxy- and 6-ethoxy substituted purine homonucleosides by reaction with the corresponding alkoxides. No derivatives appeared active against entero, yellow fever, chikungunya, and adeno type 1viruses. Two compounds (6j and 6d) had lower IC50 (15 ± 2 and 21 ± 4 µM) and compound 6f had an identical value of IC50 (28 ± 4 µM) to that of acyclovir, suggesting that the bicyclo[2.2.1]heptane skeleton could be further studied to find a candidate for sugar moiety of the nucleosides.

Graphical Abstract

1. Introduction

Nucleosides are a chemical class of active substances used as efficient antitumor [1,2,3] or antiviral drugs [4,5,6,7]. The resistance which appears after prolonged use and their toxicity are two principal secondary effects that request the development of newer, more safe candidates. An impressive number of modifications of the sugar moiety or replacements with carbocyclic fragment, replacement of the base or both have been done and some of the corresponding molecules had antiviral or anticancer activity and became useful drugs [2,3,6,7].
Another direction pursued in search for new active nucleosides was to introduce a methylene group between the heterocyclic base and the sugar moiety. The new compounds are, in fact, acyclic nucleosides, but in the literature, their most common name is “1′-homonucleosides”. The insertion of the methylene group alters the chemical and biological characteristics of the molecules, as follows: (1) The anomeric carbon atom C1′ in a tetrahydrofuran moiety has no more reactivity and the compounds are resistant to the enzyme hydrolysis of C1′-CH2Base bond, (2) the methylene group allows free rotation between the base and sugar moiety, increasing the flexibility of the molecule in the enzyme pocket(s), (3) electronic and steric interactions between the base and sugar moiety are decreased, (4) the lipophilicity of the compounds is slightly increased, a factor sometimes important for the transport of the molecule across the cell wall, (5) the separation between HO-C5′ of the (un)modified sugar and the N1 or N9 nitrogen atoms of the heterocyclic bases is slightly increased and many times this is the cause of the biological activity or of the lack of activity of the analogs [8].
In the 1′-homonucleoside, the biological activity mainly decreased compared with that of the parent nucleoside [9,10]. The substitution of oxygen with nitrogen [11] or sulfur [12] did not lead to active compounds. However, some changes in the sugar moiety have led to carbocyclic compounds with potent antiviral or anticancer activity.
The introduction of a cyclopropyl fragment led to the 1′-homocarbanucleosides I [13] with “extremely potent antiherpetic activity” [14] and II, with anti-VZV activity, IC50 = 0.027 µg/mL [14] (Figure 1). It is interesting that the introduction of a double bond between the cyclopropyl ring and the methylene group, linked to the N-9 nitrogen, in a Z-conformation of the hydroxymethyl and nucleobase, gave potent active compounds III and IV against HCMV (IC50 = 1–2.1 μM and 0.04–2.1 μM) and EpsteinBarr virus (IC50 = 0.2 μM and 0.3 μM) (Figure 1) [15,16].
1′-Homocarbonucleosides with a cyclobutene ring had no significant activity against HIV-1 and HSV-1 [17]. In the series of 2′- or 3′-hydroxymetylcyclopentane-1′-homocarbanucleosides, a few compounds presented a slight antiviral or anticancer activity [18,19], while 1′,3′-disubstituted cyclopentene analogs [20] or 2′,3′-cis diols [21], were found to be inactive. Other analogs synthesized had low or no antiviral activity [22,23,24], with the exception of the adenine analog [24]. However, a few chemical structures were fruitfully used to obtain active 1′-homocarbanucleosides (Figure 2), like for example:
-
V, with a 2,2,3-trimethylcyclopentanol, active against HIV-1 and HIV-2 at an EC50 = 4–14 µg/mL [25,26]. Some of these compounds exhibited considerable cytostatic activity, on Molt4/C8 (IC50 = 13.2 and 3.8 µg/mL with nucleobase 6-Cl-2-amino-purine and 2,6 diamino-purine) and L1210/0 (IC50 = 16.2 and 13.9 µg/mL with nucleobase 6-Cl-2-amino-purine and 2-chloro-purine) cancer lines [25,26]. It is interesting that compound VI with the opposite conformation of the cyclopentane ring is inactive (Figure 2) [27].
-
VII, with a 3-hydroxymethyl-indane scaffold, most active being on L1210/0, Molt4/C8 (IC50 = 1.4–5.7 µg/mL) and CEM (IC50 = 0.63–3.3 µg/mL) cancer cell lines [28,29].
-
VIII and IX, with a cyclopenta[c]pyrazole moiety [30] or cyclopenta[d]pyrazole moiety and hydroxymethyl protected as TBDMS [31,32]. Compounds VIII, with adenine or 6-Cl-2-aminopurine as the base, are more active against VZV/TK- strain (EC50 = 1.5 and 2.1 µmol) than acyclovir, used as reference (EC50 = 27 µmol) [30]. The same compounds are as active as ganciclovir (EC50 = 0.25 and 0.40 µmol for both compounds) against cytomegalovirus AD169 and DAVIS 07/1 strains (EC50 = 0.50 and 0.50 µmol for adenine analog and EC50 = 0.44 and 0.39 µmol for 6-Cl-2-amino-purine analog). Compounds IX, with 6-Cl-Pu, 6-Cl-2-amino-Pu, Uracil, and with the opposite position of N-Me, present significant anticancer activity on L1210/0, Molt4/C8 and CEM cell lines (IC50 = 3.2–11 µg/mL) [8,31,32].
In our previous papers [33,34,35,36,37,38] we used an optically active bicyclo[2.2.1]heptane scaffold to obtain a library of L-type carbocyclic nucleosides X and tested them for antiviral and anticancer activity (Figure 3). Compounds Xa and Xb were “the most prospective for their antiviral activity against influenza virus due to their low toxicity and high activity” [35]. Compound Xc was very active against coxsackievirus B4, with EC50 = 0.6 µg/mL and selective index of 141, compound Xb being also promising. The compounds had low anticancer activity.
The promising results with the specified substituted constrained bicyclo[2.2.1]heptane scaffold, gave us the idea to use it for obtaining 1′-homocarbanucleosides, linking the nucleobase to the hydroxymethyl group and keeping the 5-endo-OH group free, in search to obtain potentially antiviral compounds. The works in this direction are presented below.

2. Results and Discussion

2.1. Molecular Design

The docking studies were performed using CLC Drug Discovery Workbench Software (professional software). The score and hydrogen bonds formed with the amino acids from the group interaction atoms were used to predict the binding modes, the binding affinities and the orientation of the docked ligands in the active site of the protein receptor. The protein-ligand complex was realized based on the X-ray structure of herpes simplex type-1 thymidine kinase (TK) in complex with acyclovir (AC2), who was downloaded from the Protein Data Bank (PDB ID: 2KI5, for a 1.9Å resolution) [39]. The TK enzyme was studied for binding capabilities in the development of new, more effective HSV-1 inhibitors in many papers (for example [40]). The binding site and binding pockets, used to guide the molecular docking of the ligands, were established, and the search was carried out inside the binding site volume (the green sphere, Figure S1a).
Firstly, the binding site and docking pose of the co-crystallized AC2 interacting with amino acids residues are shown in Figure S1a–c. Then hydrogen bonds between amino acids residues of thymidine kinase and co-crystallized AC2 and docking validation (for the co-crystallized) were done (see Figure S2). The new 1′-homocarbacyclonucleosides were finally docked, and the results of the calculated properties are presented in Table 1 (flexible bonds, Lipinski violations, the number of hydrogen bond donors, the number of hydrogen bond acceptors and log P). These parameters can predict if a molecule possesses properties that might turn it into an active drug, according to the Lipinski’s rule of five. The number of violations of the Lipinski rules allows to evaluate drug-likeness for a molecule. According to the data presented in Table 1, all of the compounds comply with the Lipinski rules (Lipinski violation is 0) [41]. In Table 1, the results of the docking study (docking score, RMSD < 2Å) are also presented.
The docking score (PLANTPLP score) is a function described in Korb et al. [42]. For a strong binding, the score has a negative value, for weak or non-existing binding the score has a less negative or even positive value.
Additionally, group interaction, hydrogen bonds of ligands with amino acid residues were determined and hydrogen bond length was calculated. These are presented in the Table S1.
The data presented in the Table 1 and Table S1 and Figure 4 show that compounds 4a–4d, 5, 6a–6b, 6e, 6i–6k, 7a–7b have a docking score greater than that of acyclovir (−49.29, RMSD 0.71).
Compounds 6i and 6k presented the best scores, −70.21, respectively −70.07. The docking pose of the two ligands interacting with the amino acids residues is presented in Figure S3. In fact, all new ligands (1′-homocarbanucleosides) were found to have the same orientation as AC2 (Figure S4a), as it can be observed in Table S1 for linking to a group of interaction with the amino acids.
The orientation and the docking pose of AC2 with the pyrimidine ligands 4a–4d, 6a–6k N6-substituted adenine ligands and N6-alkoxy-purine 7a–7b ligands are presented in Figures S4b, and S5a,b, and show a good overlay.
The docking score was correlated with the experimental HSV-1 antiviral results (IC50), and the best matching is for compound 6j (score −62.08, RMSD 1.57, IC50 = 15 ± 2 µM) (Figure 5). The second compound (6d) with lower IC50 (21 ± 4 µM) than acyclovir (28 ± 4 µM) had a little lower value for the docking score (−39.94, RMSD 0.13) than that for acyclovir (−49.29, RMSD 0.71).
The compounds with the best scores: 6k and 6i (−70.07, RMSD 0.09, respectively, −70.21, RMSD 0.07), presented greater values for IC50 (48 ± 6 and 155 ± 13 µM) which are not in agreement with the experimental anti-HSV-1 activity. Therefore, the molecular design should be taken with caution, because the results are prediction values, not experimental ones.

2.2. Chemistry

The synthesis of these new 1′homocarbanucleosides started from the diol 2, obtained as a major isomer by sodium borohydride reduction of the optically active keto-alcohol intermediate 1 by our previous procedure [36]. The bulk of 5-endo-OH isomer 2 was obtained in pure form by crystallization, in yield greater than 77%. By low pressure chromatography (LPC) purification of the mother liquors, the total yield was increased to 91% (Scheme 1).
We chose to use a Mitsunobu reaction to alkylate the pyrimidine bases with the primary hydroxyl group of the diol 2, as the most direct chemical option to obtain the 1′-homocarba-pyrimidine nucleosides 4 (Scheme 1). For this goal, we relied on the fact that in the Mitsunobu reaction, the primary hydroxyl group has greater reactivity than the secondary hydroxyl group, linked endo to C5 carbon atom. Certainly, the protection of the secondary hydroxyl group would lead to greater yields of the final products 4, but this would make the sequence of reactions longer, and at that time the yield was not considered so important. Compounds 4a, 4b, and 4c were obtained in 20.3, 34.5 and respectively 36.6% yields. Compound 4d was obtained in 23.5% yield by the Mitsunobu reaction of diol 2 with N4-Cytosine benzoate, followed by the deprotection of the benzoate group on crude alkylation reaction product by transesterification (MeONa/MeOH). The isolation of the pure compound 4d was realized by LPC. The reduced yield for the pyrimidine analogs is attributed to the formation of O2,O4- and N1,O4-bis-akylated secondary compounds, as we observed previously [43] for the alkylation of the diol 2 protected at the primary hydroxyl as benzoate. In this case, alkylated compounds to both hydroxyls of the diol 2 could also be formed. All secondary compounds had higher mobility on TLC, in the domain of triphenylphosphine oxide as Rf, and were not isolated pure for characterization.
The second part of the paper is focused on obtaining 1′-homocarbanucleoside analogs with a purine base. In this case, we also chose the most direct synthesis, which implies two steps. In the first step, we obtained the key optically active intermediate 5 in 67.6% yield by a slight modification of the Mitsunobu reaction of 6-chloropurine [44] with our diol 2 (Scheme 2). In the second step, the 6-chlorine atom of the key intermediate 5 was substituted with ammonia to obtain the adenine analog 6a, and with selected amines, used previously in our papers, where 6-chloropurine was linked exo to the C5 carbon atom [33,34], as in X (Figure 3, R = Cl), to 1′-homocarba-N6-substituted adenine nucleosides 6a–6k, in good yields (from 77.9% for 6k to 95.1% for 6d). Only the compound 6g was obtained in a moderate yield of 49.2%. The chlorine atom was also substituted by a methoxy (7a) or ethoxy group (7b), by the reaction of the key intermediate 5 with sodium methoxide in methanol or sodium ethoxide in ethanol in good yield (70.9%, respectively 83.6%).
The pure compounds were fully characterized and have been screened for antiviral activity.

2.3. 1H, 13C-NMR, MS, Elemental Analysis and Optical Rotation

All new compounds were purified by pressure chromatography and analyzed by optical rotation, IR, 1H-, 13C-NMR, and 2D-NMR spectra, presented at the experimental part. The analytical data were in full agreement with the proposed structures. In 1H-NMR spectra, the assignment was performed on the basis of chemical shifts, signal intensity and multiplicity of H-H coupling constants. 13C-NMR and complementary 2D-NMR and decoupling spectra gave the correct signal for each proton and carbon atom in the molecules. The 1H-, 13C-, and 2D-NMR spectra of the key intermediate 5 and of the new 1′homocarbanucleosides 4, 6 and 7 are presented in the Supplementary Material.

2.4. Antiviral Activity of the Compounds

The new 1′-homocarbanucleosides analogs were tested on different viruses:
1)
adeno-, herpes- and influenza viruses, at Pasteur Institute of Epidemiology and Microbiology, Department of Virology, St. Petersburg, Russia, and
2)
enteroviruses type: enterovirus 71 (EV71), yellow fever and Chikungunya viruses, at Rega Institute, Laboratory of Virology and Chemotherapy, Leuven-Belgium
The results of anti-viral testing of novel 1′-homocarbanucleosides analogs against adeno-, influenza, and herpesvirus type 1are summarized in Table 2.
As can be seen from the data presented, no derivatives appeared active against adenovirus. Only one compound demonstrated moderate inhibiting activity against influenza virus while 5 out of 18 compounds were active against the herpes virus. Importantly, there was no correlation between activity against influenza virus and herpes virus. Indeed, the cytosine compound 4d, the only derivative active against influenza virus, was ineffective against HSV-1. On another hand, the compounds active against herpes (5-chloro-purine 5, and 6 alkyl-substituted 6c, 6d, 6f, 6j) did not show the same activity against influenza virus. These differences may reflect the specificity in their targets. With high probability, the targets for nucleoside analogs should be the enzymes participating in the nucleic acids metabolism, in particular, viral polymerases. Herpes virus and influenza virus have a different organization of their genomes. The former has double-stranded DNA genome while the latter—single strand RNA genome of negative polarity. Due to differences in genomes of influenza virus and herpes virus, one can suggest that different inhibitors, although belonging to one chemical class, are needed to interfere with the activities of two different enzymes.
It is known that the best-known anti-herpes compound acyclovir is phosphorylated in infected cells by viral thymidine kinase following by incorporation of its triphosphate form into the DNA resulting in chain termination and death of infected virus cells [45]. Probably, the compounds we studied have a similar mechanism of activity, and for this reason, are ineffective against influenza virus who does not encode its own kinase. Further experiments are needed for deciphering the mechanism of activity and identifying the target(s) of lead compounds, including but not limited to time-of-addition experiments, virus yield reduction assay as well as tests for specific viral enzymes and selection and analysis of resistant mutants.
By analyzing the structures of active compounds, one can infer that even minor differences in the substituents R result in a dramatic change in the anti-viral properties. Indeed, the similar compounds 6f and 6g differing in one nitrogen bridge atom demonstrate almost a 30-fold difference in the virus-inhibiting activity against HSV-1. The introduction of an amino group between 4-methylpyperazine and N6 nitrogen atom resulted in the complete loss of virus inhibition in 6g compared to 6f. Change of the hexane group to unsaturated phenyl group (6d to 6k) resulted in the decrease of virus inhibition (IC50 from 21 to 48 µM), while the addition of the metoxy group (6j) restored the activity (IC50 15). Even the change of cyclohexyl for cyclo-pentyl group (6d to 6c) decreased the virus inhibition (IC50 from 21 to 47 µM). Chorine- and amino- substituted derivatives 5 and 6a did not differ significantly in their properties. Taking into account only the IC50 of the new 1′-homocarbacyclonucleosides in comparison with that of acyclovir (28 ± 4 µM), compounds 6j and 6d have a lower IC50 (15 ± 2 and 21 ± 4 µM), compound 6f—an identic value (28 ± 4 µM) and another three compounds had the values of IC50 about twice higher. Five of them have also a SI greater than 10 (See Table 3). Importantly, compounds 6f, 6d, and 6j showed values of IC50 against herpes virus similar to that of acyclovir, but their cytotoxicity was higher. This suggests that further optimization of the structure directing to decrease the toxicity could provide good pharmacologic characteristics of the resulting compounds.
No compounds were active against yellow fever and Chikungunya viruses. Compounds 6c, 6d, 6j, and 6k presented low activity against EV 71 virus (IC50 20–80 µM) (Table 3).
Taken together, our findings suggest that the bicyclo[2.2.1]heptane skeleton could be a candidate for sugar moiety of the nucleosides with potential virus-inhibiting properties.

3. Materials and Methods

3.1. Experimental-Chemistry

Melting points (uncorrected) were determined in open capillary on an OptiMelt melting point apparatus (MPA 100, Stanford Research System, Inc., Sunnyvale, CA, USA). The progress of the reaction was monitored by TLC on silica gel 60 or 60F254 plates (Merck, Darmstadt, Germany) eluted with the solvent systems: I dichloromethane-methanol, 9:1, II dichloromethane-methanol, 95:5, III dichloromethane-methanol, 4:1. Spots developed in UV and with 15% H2SO4 in MeOH (heating at 110 °C, 10 min). IR spectra were recorded on FT-IR-100 Perkin Elmer spectrometer (Perkin Elmer, Shelton, CT, USA), in solid phase by ATR and frequencies were expressed in cm−1, with the following abbreviations: w = weak, m = medium, s = strong, v = very, br = broad. 1H-NMR and 13C-NMR spectra are recorded on Bruker Fourier 300 MHz spectrometer (300 MHz for 1H and 75 MHz for 13C, Karlsruhe, Germany), or Bruker Avance III 500 MHz spectrometer (500 MHz for 1H and 125 MHz for 13C), spectrometer chemical shifts are given in ppm relative to TMS as internal standard. Complementary spectra: 2D-NMR and decoupling were done for the correct assignment of NMR signals. The numbering of the atoms in the compounds is presented in schemes. Diol 2 was obtained by sodium borohydride reduction of the keto group of compound 1, as previously presented [36], having mp 116–117.2 °C, [α]D = 11.3° (1% MeOH).

3.1.1. Synthesis of 1-(((1S,2S,4S,7R)-2-Chloro-5-hydroxybicyclo[2.2.1]heptan-7-yl)methyl)-5-fluoropyrimidine-2,4(1H,3H)-dione, 4a

Triphenylphosphine (Ph3P) (5.246 g, 20 mmol) and 5-fluorouracil (2.62 g, 20 mmol) were suspended in 120 mL anh tetrahydrofuran and stirred at rt for 20 min under anh. argon atmosphere, then cooled to 0 °C with an ice-water bath. DIAD (4.16 mL, 20 mmol) was added dropwise. After one hour, a solution of diol 2 (1.766 g, 10 mmol) in tetrahydrofuran (80 mL) was added during 50 min and stirred overnight monitoring the end of the reaction by TLC (I, Rf 2 = 0.43, Rf = 0.55). A secondary compound with Rf = 0.63 was also formed in the reaction and was not isolated pure. Solvent was distilled under reduced pressure, the residue was purified by low pressure chromatography (LPC) (solvent system, I), resulting a pure fraction of 587 mg (20.3%) 4a, mp 212.3–213.7 °C (EtOH), [α]D = 39.4° (1% EtOH), IR: 3377s, 3170w, 3045w, 2962w, 2878w, 2819w, 1762w, 1703s, 1663vs, 1475w, 1434w, 1375m, 1339m, 1237s, 1113m, 1077m, 1006m, 900w, 817m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 11.77 (d, 1H, NH, 4.8), 8.10 (d, 1H, H-6, JH-F = 2.8 Hz), 4.73 (brs, 1H, OH) deuterable, 4.21 (dd, 1H, H-8, 11.0, 14.0), 4.08 (brdd+TFA, 1H, H-2, 3.0, 8.1), 3.92 (brd+TFA, 1H, H-5, 9.6), 3.62 (dd, 1H, H-8, 3.6, 14.0), 2.66 (dd, 1H, H-3, 8.0, 13.7), 2.26 (d, 1H, H-1, 4.5), 2.13 (s, 1H, H-4), 2.11 (brd, 1H, H-3, 13.4), 2.01–1.94 (m, 2H, H-6, H-7), 0.79 (dd+TFA, 1H, H-6, 2.2, 13.4), 13C-NMR (DMSO-d6, δ ppm): 157.48 (d, C-4′, J = 24.8 Hz), 149.72 (C-2′), 139.67 (d, C-5, J = 227.3 Hz), 130.13 (d, C-6, J = 33 Hz), 68.11 (C-5), 61.48 (C-2), 48.76 (C-1) from COSY and HMBC, 47.99 (C-7), 44.37 (C-8), 45.01 (C-4), 38.78 (C-6), 32.14 (C-3).

3.1.2. Synthesis of 1-(((1S,2S,4S,5R,7R)-2-Chloro-5-hydroxybicyclo[2.2.1]heptan-7-yl)methyl)pyrimidine-2,4(1H,3H)-dione, 4b

Starting from 10 mmol diol 2 and uracil (2.24 g, 20 mmol) as in the example 3.1, 935 mg (34.5%) 4b, were obtained as a foam, mp 202.2–204.5 °C (EtOH), [α]D = 35.0° (1% EtOH), 4b, IR: 3353m, 3194w, 3103w, 2968w, 2881w, 1700s, 1657vs, 1461m, 1337m, 1241m, 1073m, 1007w, 814m, 612m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 11.25 (1H, NH), 7.68 (d, 1H, H-6′, 7.8), 5.55 (d, 1H, H-5′, 7.8), 4.73 (d, 1H, OH, 3.7), 4.20 (dd, 1H, H-8, 10.4, 13.8), 4.09 (brd, 1H, H-2, 7.9), 3.92 (brd, 1H, H-5, 5.8), 3.70 (dd, 1H, H-8, 4.2, 13.8), 2.70 (dd, 1H, H-3, 7.9, 14.0), 2.26 (d, 1H, H-1, 4.5), 2.11 (s, 1H, H-4), 2.09 (brd, 1H, H-3, 14.0), 2.02–1.93 (m, 2H, H-6, H-7), 0.79 (dd, 1H, H-6, 2.3, 13.3), 13C-NMR (DMSO-d6, δ ppm): 163.75 (C-4′), 151.06 (C-2′), 145.70 (C-6′), 100.96 (C-5′), 68.06 (C-5), 61.44 (C-2), 48.59 (C-1), 48.14 (C-7), 46.09 (C-8), 45.05 (C-4), 38.82 (C-6), 32.18 (C-3).

3.1.3. Synthesis of 1-(((1S,2S,4S,7R)-2-Chloro-5-hydroxybicyclo[2.2.1]heptan-7-yl)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione, 4c

Starting from 10 mmol diol 2 and tymine (2.522 g, 20 mmol) as in the example 3.1, 1.043 g (36.6%) 4c, were obtained as a foam, Rf = 0.50 (I). A sample was crystallized from EtOH, mp 211.3–212.0 °C (dec.), [α]D = 43.2 ° (1% EtOH), IR: 3390s, 3188m, 3064m, 2891m, 2815w, 1679vs, 1660m, 1335m, 1249w, 1125w, 1004m, 833m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 11.23 (1H, NH), 7.56 (s, 1H, H-6′), 4.72 (d, 1H, OH, 3.4), 4.20 (dd, 1H, H-8, 10.8, 13.9), 4.09 (brd, 1H, H-2, 4.5, 8.0), 3.91 (brd, 1H, H-5, 6.1), 3.64 (dd, 1H, H-8, 3.3, 13.9), 2.67 (dd, 1H, H-3, 8.0, 14.7), 2.26 (d, 1H, H-1, 4.5), 2.12–2.10 (m, 2H, H-4, H-3), 2.02–1.92 (m, 2H, H-6, H-7), 1.75 (s, 3H, CH3), 0.79 (dd, 1H, H-6, 2.2, 13.3), 13C-NMR (DMSO-d6, δ ppm): 164.33 (C-4′), 151.00 (C-2′), 141.46 (C-6′), 108.60 (C-5), 68.10 (C-5), 61.44 (C-2), 48.67 (C-1), 48.21 (C-7), 45.84 (C-8), 45.03 (C-4), 38.81 (C-6), 32.20 (C-3), 11.98 (CH3).

3.1.4. Synthesis of 4-Amino-1-(((1S,2S,4S,7R)-2-chloro-5-hydroxybicyclo[2.2.1]heptan-7-yl)methyl)pyrimidin-2(1H)-one, 4d

Starting from 5 mmol diol 2, with Ph3P (10 mmol, 2.623 g), N4-Cytosine benzoate (10 mmol, 2.152 g), DIAD (10 mmol, 2.1 mL) in THF (90 mL), as in the example 3.1, followed by the cleavage of the benzoate group on the crude product in 1 M MeONa (100 mL) and methanol (100 mL) overnight and LPC (I), 317 mg (23.5%) 4d were obtained as a foam, Rf 4d = 0.30, [α]D = 33.2° (1% EtOH), IR: 3532m, 3335s, 3209m, 3100m, 2959m, 2874w, 1629vs, 1605vs, 1523m, 1480vs, 1436s, 1381s, 1270s, 1196m, 1074m, 994m, 784m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 7.61 (d, 1H, H-6′, 7.1), 7.01 (s, 2H, NH), 5.64 (d, 1H, H-5′, 7.1), 4.72 (d, 1H, OH, 3.2), 4.15 (dd, 1H, H-8, 10.0, 13.5), 4.09 (brdd, 1H, H-2, 3.1, 8.0), 3.88 (brd, 1H, H-5, 6.5), 3.69 (dd, 1H, H-8, 4.2, 13.5), 2.66 (dd, 1H, H-3, 8.0, 13.5), 2.23 (d, 1H, H-1, 4.7), 2.11 (brd, 1H, H-3, 13.5), 2.09 (s, 1H, H-4), 2.00–1.93 (m, 2H, H-6, H-7), 0.78 (dd, 1H, H-6, 1.9, 13.1), 13C-NMR (DMSO-d6, δ ppm): 165.89 (C-4′), 155.85 (C-2′), 146.04 (C-6′), 93.22 (C-5′), 68.13 (C-5), 61.54 (C-2), 48.49, 48.38 (C-1, C-7), 47.18 (C-8), 45.00 (C-4), 38.82 (C-6) in DMSO, see HETCOR, 32.30 (C-3).

3.1.5. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-chloro-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 5

Compound 5 was synthesized by the slightly modified procedure used in example 3.1.1: Diol 2 (3.53 g, 20 mmol) was dissolved in tetrahydrofuran (160 mL), and the solution was added during one hour to the complex formed by the addition of DIAD (8.3 mL, 40 mmol) to a solution of 6-chloropurine (10.49 g, 40 mmol) and Ph3P (6.18 g, 40 mmol) in tetrahydrofuran (280 mL) at −6 °C over 40 min, followed by a one-hour waiting time for the complete formation of the betaine complex. The reaction mixture was stirred over weekend, monitoring the end of the reaction by TLC (I, Rf 6-ClPu = 0.39, Rf 2 = 0.43, Rf 5 = 0.55), the solvent was removed under reduced pressure and the residue was purified by LPC (solvent system I). The compound was crystallized from CH2Cl2-hexane, resulting 4.24 g (67.7%) of crystallized 5, mp 198.0–199.0 °C (>200.5 °C dec.), [α]D = 7.35° (1% EtOH), IR: 3394m, 3312m, 3076w, 2956w, 2899w, 1596s, 1563s, 1444m, 1402m, 1335vs, 1262m, 1212m, 1168m, 1079m, 1045m, 935s, 640m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 8.79 (s, 1H, H-8′), 8.77 (s, 1H, H-2′), 4.74 (d, 1H, OH, 3.1), 4.73 (dd, 1H, H-8, 11.0, 14.2), 4.49 (dd, 1H, H-8, 5.0, 14.2), 4.16 (brdd, 1H, H-2, 3.1, 7.6), 3.86 (m, 1H, H-5), 2.75 (dd, 1H, H-3, 7.6, 14.2), 2.36 (d, 1H, H-1, 4.5), 2.30–2.22 (m, 2H, H-3, H-7), 2.05–1.94 (m, 2H, H-6, H-4), 0.82 (dd, 1H, H-6, 2.0, 13.3), 13C-NMR (DMSO-d6, δ ppm): 151.98 (C-6′), 151.52 (C-2′), 148.99 (C-4′), 147.44 (C-8′), 130.73 (C-5′), 67.89 (C-5), 61.25 (C-2), 48.48 (2C, C-1, C-7), 45.32 (C-8), 42.64 (C-4), 38.84 (C-6), 32.16 (C-3).

General Procedure for Synthesis of Carbocyclic 1′-Homonucleosides 6a–7k

The key intermediate 5 (0.4 or 0.8 mmol) was stirred with the specified amount of amine, without or in the presence of ethanol (3.5 or 7 mL) as solvent, without or in the presence of triethylamine, for the time frame mentioned in each example. The solvent (ethanol) and the volatile amines were removed under reduced pressure, the residue was taken in CH2Cl2, washed with water, organic phase dried (Na2SO4) and concentrated to dryness (the aqueous phases were extracted with CH2Cl2, and unified with the crude product). The crude product was purified by LPC and the pure compound was obtained as foam or waxy. Some of the compounds were crystallized in the solvent(s) specified in each case.

3.1.6. Synthesis of (1S,4S,5S,7R)-7-((6-Amino-9H-purin-9-yl)methyl)-5-chlorobicyclo[2.2.1]heptan-2-ol, 6a

Compound 5 (0.8 mmol), 25% NH4OH (40 mL), methanol (20 mL) were heated at 80–90 °C in a pressure vessel for 24 h; TLC (I, Rf = 0.29), 197 mg (83.8%) of pure 6a were obtained, mp 223.0–223.5 °C (EtOH), [α]D = 1.3° (0.5% EtOH), IR: 3307s, 3163s, 2991m, 2961w, 2916w, 2880w, 1730w, 1651vs, 1583vs, 1514m, 1452w, 1328m, 1302m, 1241m, 1200w, 1172w, 1076m, 1002m, 894m, 647m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 8.19 (s, 1H, H-8′), 8.14 (s, 1H, H-2′), 7.22 (s, 2H, NH2), 4.72 (d, 1H, OH, 3.5), 4.59 (dd, 1H, H-8, 10.1, 14.1), 4.31 (dd, 1H, H-8, 5.1, 14.1), 4.15 (dd, 1H, H-2, 3.6, 7.8), 3.87 (m, 1H, H-5), 2.73 (dd, 1H, H-3, 8.1, 14.1), 2.33 (d, 1H, H-1, 4.5), 2.26–2.18 (m, 2H, H-3, H-7), 2.01 (m, 1H, H-4), 1.98 (ddd, 1H, H-6, 5.0, 9.9, 13.5), 0.81 (dd, 1H, H-6, 2.2, 13.5), 13C-NMR (DMSO-d6, δ ppm): 156.42 (C-6′), 152.92 (C-2′), 149.96 (C-4′), 141.17 (C-8′), 119.03 (C-1′), 68.40 (C-5), 61.76 (C-2), 49.38 (C-7), 48.80 C-1), 45.74 (C-4), 42.14 (C-8), 39.17 in DMSO (C-6), 32.18 (C-3).

3.1.7. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(cyclopropylamino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6b

Compound 5 (0.8 mmol), 2 mL cyclopropylamine, 72 h, rt, Rf = 0.39). LPC (II) gave 252 mg (94.4%) of pure 6b, as a foam, [α]D = −15.0° (1% EtOH), IR: 3256brm, 3089w, 3035m, 2962s, 1613vs, 1476m, 1450m, 1348m, 1295m, 1260s, 1082s, 1009vs, 793s, 1H-NMR (DMSO-d6, δ ppm, J Hz): 8.25 (s, 1H, H-2′), 8.19 (s, 1H, H-8′), 7.88 (brs, NH), 4.72 (d, 1H, OH, 3.5), 4.60 (dd, 1H, H-8, 10.1, 14.0), 4.32 (dd, 1H, H-8, 5.1, 14.0), 4.14 (brdd, 1H, H-2, 3.3, 8.0), 3.85 (m, 1H, H-5), 3.03 (m, 1H, H-1′′), 2.73 (dd, 1H, H-3, 8.0, 14.1), 2.32 (d, 1H, H-1, 4.4), 2.26–2.20 (m, 2H, H-3, H-7), 2.02–1.93 (m, 2H, H-6, H-4), 0.81 (brd, 1H, H-6, 2.0, 14.0), 0.73–0.60 (m, 4H, CH2-Cyclopropyl), 13C-NMR (DMSO-d6, δ ppm): 155.53 (C-6′), 152.39 (C-2′), 148.95 (C-4′), 140.58 (C-8′), 118.95 (C-5′), 67.96 (C-5), 61.33 (C-2), 48.96 (C-7), 48.36 C-1), 45.31 (C-4), 41.70 (C-8), 38.66 in DMSO (C-6), 32.20 (C-3), 23.91 (C-1″), 6.42 (C-2″, C-3″).

3.1.8. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(cyclopentylamino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6c

Compound 5 (0.8 mmol), cyclopentylamine (2.2 mL), stirring at rt for 72 h; TLC (I, Rf = 0.60). LPC (II) gave 234.5 mg (81.0%) of pure compound 6c as a foam, [α]D = −18.3° (1% EtOH), IR: 3319brm, 2955m, 2868w, 1613vs, 1534m,1477w, 1333m, 1296m, 1083w, 1002w, 854w, 647w, 1H-NMR (CDCl3, δ ppm, J Hz): 8.37 (s, 1H, H-2′), 7.94 (s, 1H, H-8c′), 5.96 (brs, 1H, NH), 4.61 (dd, 1H, H-8, 8.2, 14.3), 4.57 (m, 1H, H-1″) from Hetcor, 4.54 (dd, 1H, H-8, 7.8, 14.3), 4.14–4.11 (m, 2H, H-2, H-5), 2.97 (dd, 1H, H-3, 8.0, 14.4), 2.34 (d, 1H, H-1, 4.5), 2.33–2.28 (m, 2H, H-4, H-7), 2.21–2.02 (m, 4H, H-6, H-4, 2H-2″), 1.80–1.65 (m, 4H, H-3″), 1.56 (m, 2H, H-2″), 0.96 (brd, 1H, H-6, 14.0), 13C-NMR (CDCl3, δ ppm): 155.54 (C-6′), 152.19 (C-2′), 148.74 (C-4′), 139.65 (C-8′), 119.53 (C-5′), 69.24 (C-5), 60.55 (C-2), 52.40 (C-1″), 49.07 (C-7), 48.03 C-1), 45.73 (C-4), 42.59 (C-8), 39.36 (C-6), 33.40 (C-2″), 32.73 (C-3), 23.66 (C-3″).

3.1.9. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(cyclohexylamino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6d

Compound 5 (0.8 mmol), cyclohexylamine (4.8 mL), stirring for six days at rt, TLC (I, Rf = 0.55). LPC gave 286 mg (95.1%) of pure compound 6d as a foam, [α]D = 2.9° (1% EtOH), IR: 3320brm, 2926s, 2852m, 1612vs, 1474m, 1446m, 1331m, 1295m, 1253m, 1083m, 1001w, 895w, 646w, 1H-NMR (CDCl3, δ ppm, J Hz): 8.35 (s, 1H, H-2′), 7.84 (s, 1H, H-8′), 5.89 (brs, 1H, NH), 4.61 (dd, 1H, H-8, 8.2, 14.4), 4.54 (dd, 1H, H-8, 8.0, 14.4), 4.14–4.11 (m, 2H, H-2, H-5), 2.97 (dd, 1H, H-3, 8.0, 14.5), 2.41–2.28 (m, 3H, H-1, H-4, H-7), 2.17 (brd, 1H, H-3, 14.5), 2.10–2.05 (m, 3H, H-6, 2H-2″), 1.80–1.75 (m, 2H, H-4″), 1.48–1.24 (m, 6H, 2H-2″, 4H-3″), 0.95 (brd, 1H, H-6, 13.2), 13C-NMR (CDCl3, δ ppm): 154.10 (C-6′), 153.19 (C-2′), 148.83 (C-4′), 139.66 (C-8′), 119.41 (C-5′), 69.17 (C-5), 60.58 (C-2), 50.43 (C-1″), 49.05 (C-7), 48.00 C-1), 45.73 (C-4), 42.57 (C-8), 39.38 (C-6), 33.30 (C-2″), 32.74 (C-3), 26.88 (C-3″), 24.79 (C-4″).

3.1.10. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(((R)-1-hydroxy-3-phenylpropan-2-yl)amino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6e

Compound 5 (0.4 mmol), phenylalaninol (165 mg, 1 mmol), ethanol (4 mL), Et3N (0.3 mL) stirred for 72 h at 60 °C, TLC (I, Rf = 0.29). LPC (I) gave 161 mg (94.1%) of pure compound 6e as a waxy, [α]D = −89.9° (1% EtOH), IR: 3297brs, 2956brm, 1810w, 1695w, 1610vs, 1477m, 1447m, 1330m, 1296m, 1260m, 1079m, 1002m, 896w, 734m, 644w, 1H-NMR (CDCl3, δ ppm, J Hz): 8.32 (s, 1H, H-2′), 7.88 (s, 1H, H-8′), 7.30–7.21 (m, 5H, H-Ar), 6.49 (brs, 1H, NH), 4.60 (dd, 1H, H-8, 7.8, 14.3), 4.57 (m, 1H, H-2″), 4.53 (dd, 1H, H-8, 8.0, 14.3), 4.16–4.10 (m, 2H, H-5, H-2), 3.88 (dd, 1H, H-1″, 2.0, 10.9), 3.74 (dd, 1H, H-1″, 5.4, 10.9), 3.04 (d, 2H, H-3″, 7.1), 2.95 (dd, 1H, H-3, 8.2, 14.7), 2.39 (d, 1H, H-1, 4.3), 2.31–2.27 (m, 2H, H-4, H-7), 2.19 (brdt, 1H, H-3, 14.7), 2.06 (ddd, 1H, H-6, 5.0, 10.3, 13.5), 0.94 (brd, 1H, H-6, 2.5, 13.5), 1H-NMR (CDCl3 + TFA, δ ppm, J Hz): 9.65 (d, 1H, NH, 9.3), 8.68 (s, 1H, H-2′), 8.33 (s, 1H, H-8′), 7.24–7.16 (m, 5H, H-Ar), 4.85 (dd, 1H, H-8, 7.0, 14.3), 4.78 (dd, 1H, H-8, 8.2, 14.3), 4.61 (m, 1H, H-2″), 4.34 (m, 1H, H-2 or 5), 4.23–4.13 (m, 2H, H-1″), 3.93 (t, 1H, H-5 or 2, 11.4), 3.11 (dd, 1H, H-3″, 5.3, 14.2), 3.02 (dd, 1H, H-3″, 9.0, 14.2), 2.88 (dd, 1H, H-3, 8.0, 15.2), 2.51 (s, 1H, H-1), 2.44 (d, 1H, H-4, 4.2), 2.30 (m, 1H, H-7), 2.26 (brdt, 1H, H-3, 15.2), 2.14 (ddd, 1H, H-6, 4.9, 10.4, 14.4), 1.06 (brd, 1H, H-6, 2.4, 14.4), 13C-NMR (CDCl3, δ ppm): 154.58 (C-6′), 152.72 (C-2′), 149.03 (C-4′), 140.05 (C-8′), 137.89 (Cq-Ar), 129.29 (C-m Ar), 128.56 (C-o Ar), 126.57 (C-p Ar), 119.46 (C-1′), 69.35 (C-5), 64.49 (C-1″), 60.49 (C-2), 54.57 (C-2″), 49.03 (C-7), 47.93 C-1), 45.72 (C-4), 42.60 (C-8), 39.44 (C-6), 37.60 (C-3″), 32.74 (C-3).

3.1.11. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6f

Compound 5 (0.8 mmol), N-methyl-piperazine (2 mL), ethanol (8 mL), Et3N (0.3 mL), stirred for 72 h at rt, TLC (I, Rf = 0.35). After work-up, the crude compound was crystallized from CH2Cl2-hexane, and 258.5 mg (74.7%) of pure compound 6f resulted as needles, mp 172.9–174.0 °C (dec.) [LPC (I) of mother liquors gave another 82 mg of pure 6f as a foam, total yield: 90.3%], [α]D = 18.4° (1% EtOH), IR: 3317s, 3002m, 2970m, 2933m, 2864m, 2796m, 2471w, 2057w, 1917w, 1657w, 1584vs, 1565vs, 1442s, 1365m, 1294s, 1248s, 1140m, 1078m, 996s, 851w, 646m, 1H-NMR (CDCl3, δ ppm, J Hz): 8.31 (s, 1H, H-2′), 7.89 (s, 1H, H-8′), 4.59 (dd, 1H, H-8, 7.8, 14.3), 4.52 (dd, 1H, H-8, 8.4, 14.3), 4.33 (brs, 4H, H-1″ +TFA), 4.14–4.08 (m, 2H, H-5, H-2), 3.19 (t, H-2″ in free N-Me-Piperazine, 4.5), 2.92 (dd, 1H, H-3, 8.1, 14.5), 2.69 (br.t, H-2″ in free N-Me-Piperazine, 4.5), 2.57 (t, 4H, H-2″, 4.5), 2.37–2.27 (m, 6H, H-1, H-4, H-7, CH3), 2.15 (brdt, 1H, H-3, 4.7, 14.5), 2.03 (ddd, 1H, H-6, 5.0, 10.0, 13.4), 0.92 (brd, 1H, H-6, 1.8, 13.4), 13C-NMR (CDCl3, δ ppm): 153.78 (C-6′), 152.22 (C-2′), 150.82 (C-4′), 138.99 (C-8′), 119.98 (C-1′), 69.27 (C-5), 60.56 (C-2), 54.98 (C-2″), 51.82 (CH2, C-2″ in free N-Me-Piperazine), 48.90 (C-7), 47.87 C-1), 46.00 (C-4), 45.74 (CH3-N), 44.78 (C-1″), 43.51 (CH2, C-1″ in free N-Me-Piperazine), 42.45 (C-8), 39.57 (C-6), 32.74 (C-3), the molecule crystalized with ~0.5 molecule of N-me-piperazine. After washing with water, the signals of free N-methyl piperazine disappeared.

3.1.12. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-((4-methylpiperazin-1-yl)amino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6g

Compound 5 (0.8 mmol), 4-methy-1-amino-piperazine (0.22 mL, 1.83 mmol), ethanol (7 mL), Et3N (0.6 mL) stirred for 72 h at rt, TLC (I, Rf = 0.08). LPC (III + 0.5 mL Et3N/100 mL system) gave 193 mg (49.2%) of pure compound 6g as a foam, [α]D = −14.3° (1% EtOH), IR: 3374brs, 3211s, 3073m, 2961s, 2859m, 1711w, 1607vs, 1592vs, 1516m, 1444m, 1410s, 1334m, 1299s, 1263s, 1237m, 1087m, 1008m, 992m, 796w, 649m, 1H-NMR (DMSO-d6, δ ppm, J Hz): 8.71 (s, 1H, NH), 8.22, 8.20 (s, 1H, H-2′, H-8″), 4.59 (dd, 1H, H-8, 10.0, 14.0), 4.32 (dd, 1H, H-8, 5.1, 14.0), 4.15 (dd, 1H, H-2, 3.7, 8.0), 3.84 (m, 1H, H-5), 2.84 (m, 4H, H-1′′), 2.74 (dd, 1H, H-3, 8.0, 14.0), 2.47–2.40 (m, 5H, H-4, 4H-2″), 2.35 (d, 1H, H-1, 4.6), 2.28–2.15 (m, 5H, H-3, H-7, CH3), 2.10 (m, 2H, H-4, H-6), 0.83 (dd, 1H, H-6, 2.2, 13.7), 13C-NMR (DMSO-d6, δ ppm): 172.66 (C-6′), 153.59 (C-4′), 152.40 (C-2′), 140.89 (C-8′), 117.66 (C-5′), 67.94 (C-5), 61.32 (C-2), 54.44 (C-1″), 48.94 (C-7), 48.36 C-1), 45.58 (CH3N), 45.55 (C-2″), 45.31 (C-4), 41.67 (C-8), 38.75 in DMSO (C-6), 32.18 (C-3).

3.1.13. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-morpholino-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6h

Compound 5 (0.8 mmol), morpholine (4 mL) stirred for 72 h at rt, TLC (I, Rf = 0.53). By crystallization of the crude product, 283.5 mg (66.1%) of pure compound 6h, crystallized with two molecules of morpholine/mol of 6h, were obtained as needless, mp 162.3–163.2 °C [LPC (I) of the mother liquors gave 75 mg (25.8%) of pure 6h, total yield 91.9%], [α]D = −15.8° (1% THF), IR: 1H-NMR (CDCl3, δ ppm, J Hz): 9.9 (brs, 1H, NH), 8.34 (s, 1H, H-2′), 7.90 (s, 1H, H-8′), 4.62 (dd, 1H, H-8, 7.6, 14.0), 4.54 (dd, 1H, H-8, 8.3, 14.0), 4.30 (brs, 4H, H-1″), 4.17–4.09 (m, 2H, H-5, H-2), 3.99 (t, 4H, H-1″ 4.6,), 3.82 (t, 4H, H-2″, in free morpholine, 4.4), 3.23 (t, 8H, H-1″, 4.6), 2.93 (dd, 1H, H-3, 8.1, 14.4), 2.38 (d, 1H, H-1, 5.0), 2.34 (d, 1H, H-4, 5.9), 2.30 (t, 1H, H-7, 8.3), 2.17 (brdt, 1H, H-3, 4.1, 14.4), 2.06 (ddd, 1H, H-6, 5.0, 10.1, 13.6), 0.92 (brdd, 1H, H-6, 2.4, 13.6), 13C-NMR (CDCl3, δ ppm): 153.90 (C-6′), 152.24 (C-2′), 150.82 (C-4′), 138.99 (C-8′), 119.98 (C-5′), 69.44 (C-5), 67.01 (C-2″), 63.73 (C-2″ in morpholine), 60.51 (C-2), 48.91 (C-7), 47.88 C-1), 45.71 (C-4), 45.56 (C-1″ in morpholine), 43.24 (C-1″), 42.46 (C-8), 39.55 (C-6), 32.72 (C-3). The molecule crystalized with ~2 molecule of morpholine. After washing NaOH in D2O, the proton signals of free morpholine moved to 3.65 and 2.87 ppm, both as triplet with J = 4.7 Hz.

3.1.14. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-((4-hydroxyphenethyl)amino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6i

Compound 5 (0.4 mmol), tyramine hydrochloride (0.8 mmol, 140 mg), ethanol (3.5 mL), Et3N (0.3 mL) were stirred for six days at rt, TLC (I, Rf = 0.49). LPC (I) gave 151.5 mg (91.5%) of pure compound 6i as a foam, [α]D = 6.4° (1% EtOH), IR: 3268br, 2934m, 2805w, 2678w, 2593w, 1890w, 1616vs, 1542w, 1512m, 1482m, 1446m, 1338m, 1298m, 1228s, 1170w, 1082m, 1050w, 900w, 648w, 1H-NMR (DMSO-d6 + trases CDCl3, δ ppm, J Hz): 9.18 (s, 1H, OH), 8.23 (s, 1H, H-2′), 8.17 (s, 1H, H-8′), 7.72 (brs, 1H, NH), 7.05 (d, 2H, H-o, 7.8), 6.98 (d, 2H, H-m, 7.8), 4.71 (brs, 1H, 5-OH), 4.59 (dd, 1H, H-8, 10.0, 14.0), 4.32 (dd, 1H, H-8, 4.7, 14.0), 4.14 (m, 1H, H-2), 3.84 (m, 1H, H-5), 3.63 (brs, 2H, CH2-NH), 2.79 (t, 2H, CH2-Ph, 7.6), 2.73 (dd, 1H, H-3, 7.8, 13.9), 2.33 (d, 1H, H-1, 4.4), 2.26–2.21 (m, 2H, H-3, H-7), 2.03 (brs, 1H, H-4), 1.98 (m, 1H, H-6), 0.81 (d, 1H, H-6, 13.2), 13C-NMR (DMSO-d6, δ ppm): 155.60 (C-p Ar), 154.42 (C-6′), 152.48 (C-2′), 148.70 (C-4′), 140.47 (C-8′), 129.52 (2C-C-o), 118.94 (C-5′), 115.11 (2C, C-m), 67.95 (C-5), 61.30 (C-2), 48.95 (C-7), 48.35 C-1), 45.30 (C-4), 41.70 (C-8), 39.47 (C-6), 32.19 (C-3).

3.1.15. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-((4-hydroxyphenethyl)amino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6j

Compound 5 (0.4 mol), 4-methoxyphenethylamine (121 mg, 0.8 mmol), ethanol (3,5 mL), Et3N (0.3 mL) were stirred for six days at rt, TLC (I, Rf = 0.68). LPC (I) gave 134.4 mg (78.5%) of pure compound 6j as a foam, [α]D = −4.0° (1% EtOH), IR: 3400m, 3293m, 2997m, 2939m, 2872m, 2834m, 1969w, 1704w, 1614vs, 1576s, 1510m, 1463m, 1440m, 1333s, 1294s, 1236s, 1079m, 1003w, 896w, 644w, NMR (CDCl3, δ ppm, J Hz): 8.39 (s, 1H, H-2′), 7.86 (s, 1H, H-8′), 7.72 (brs, 1H, NH), 7.17 (d, 2H, H-o, 8.2), 6.85 (d, 2H, H-m, 8.2), 5.90 (brs, 1H, 5-OH), 4.62 (dd, 1H, H-8, 8.0, 14.3), 4.55 (dd, 1H, H-8, 8.3, 14.0), 4.17–4.10 (m, 2H, H-2, H-5), 3.89 (brs, 2H, CH2-NH), 3.79 (s, 3H, CH3O), 2.96 (m, 1H, H-3), 2.93 (t, 2H, CH2-Ph, 7.0), 2.40 (d, 1H, H-1, 4.4), 2.18 (brdt, 1H, H-3, 14.6), 2.36–2.30 (m, 2H, H-4, H-7), 2.07 (ddd, 1H, H-6, 5.1, 9.9, 13.7), 0.91 (brd, 1H, H-6, 13.7), 13C-NMR (CDCl3, δ ppm): 158.24 (C-p Ar), 154.79 (C-6′), 153.06 (C-2′), 149.16 (C-4′), 140.01 (C-8′), 130.80 (C-1Ar), 130.80 (2C-C-o), 129.75 (C-o), 119.70 (C-5′), 114.03 (2C, C-m), 69.40 (C-5), 60.54 (C-2), 55.26 CH3O), 49.05 (C-7), 47.95 C-1), 45.73 (C-4), 42.55 (C-8), 42.10 (CH2NH), 39.51 (C-6), 35.00 (CH2-Ph), 32.71 (C-3).

3.1.16. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-(phenethylamino)-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 6k

Compound 5 (0.4 mmol), 2-phenylethylamine (1 mL), were stirred for six days at rt, TLC (I, Rf = 0.54). LPC (I) gave 124 mg (77.9%) of pure compound 6k as a foam, [α]D = −25.8° (1% EtOH), IR: 3268brm, 3029m, 2952m, 1616vs, 1538w, 1483m, 1448m, 1332m, 1296m, 1226m, 1082m, 1001m, 900w, 647w, 1H-NMR (CDCl3, δ ppm, J Hz): 8.39 (s, 1H, H-8′), 7.85 (s, 1H, H-2′), 7.33–7.22 (m, 5H, H-Ar), 6.01 (s, 1H, NH), 4.62 (dd, 1H, H-8, 8.1, 14.2), 4.54 (dd, 1H, H-8, 8.1, 14.2), 4.17–4.08 (m, 2H, H-5, H-2), 3.97–3.86 (m, 2H, H-1′), 2.99 (m, 1H, H-3), 2.95 (t, 2H, H-2″, 8.3, 16.0), 2.40 (d, 1H, H-1, 4.5), 2.35–2.30 (m, 2H, H-4, H-7), 2.30 (t, 1H, H-7, 8.3), 2.18 (brdt, 1H, H-3, 4.1, 17.6), 2.06 (ddd, 1H, H-6, 5.0, 10.1, 13.8), 0.93 (brd, 1H, H-6, 2.2, 13.8), 13C-NMR (CDCl3, δ ppm): 154.75 (C-6′), 153.05 (C-2′), 148.91 (C-4′), 139.92 (Cq-Ar), 138.82 (C-8′), 128.80 (2C-C-m), 128.60 (2C, C-o), 119.66 (C-5′), 69.31 (C-5), 60.54 (C-2), 49.03 (C-7), 47.95 C-1), 45.72 (C-4), 42.56 (C-8), 41.80 (C-1″), 39.47 (C-6), 35.88 (C-2″), 32.72 (C-3).

General Procedure for Obtaining the 6-Alkoxy-purine Analogs 7a and 7b

Sodium (25 mg) was reacted with methanol or ethanol (3.5 mL), compound 5 (0.4 mmol) was added and stirred for four days monitoring the end of the reaction by TLC (I). The base was neutralized (1 M HCl), volatiles were removed under reduced pressure and the residue was purified by LPC (solvent system I).

3.1.17. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-methoxy-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 7a

Compound 5 (0.4 mmol), methanol (3.5 mL), Na (25 mg) were stirred for four days at rt, TLC (I, Rf = 0.50). By LPC, 87.6 mg (70.9%) of crystallized 7a, mp 196.2–197.2 °C (CH2Cl2-Hexane), [α]D = 25.2° (1% EtOH), IR: 3328m, 3072w, 3006w, 2964w, 2940w, 2874w, 2142w, 1812w, 1596vs, 1478m, 1417w, 1374m, 1315s, 1231s, 1083s, 1003m, 960m, 892w, 647w, 1H-NMR (DMSO-d6, δ ppm, J Hz): 8.54 (s, 1H, H-2′), 8.44 (s, 1H, H-8′), 4.72 (d, 1H, OH, 2.8), 4.70 (dd, 1H, H-8, 10.3, 14.2), 4.42 (dd, 1H, H-8, 5.2, 14.2), 4.15 (brdd, 1H, H-2, 3.1, 8.0), 4.09 (s, 3H, CH3), 3.85 (m, 1H, H-5), 2.74 (dd, 1H, H-3, 8.0, 14.2), 2.34 (d, 1H, H-1, 4.6), 2.25 (d, 1H, H-3, 14.2), 2.23 (m, 1H, H-7), 2.04–1.94 (m, 2H, H-6, H-4), 0.82 (brdd, 1H, H-6, 2.2, 13.3), 13C-NMR (DMSO-d6, δ ppm): 160.27 (C-6′), 152.06 (C-2′), 151.50 (C-4′), 136.00 (C-8′), 120.45 (C-5′), 67.97 (C-5), 62.25 (C-2), 53.90 (OCH3), 48.74 (C-7), 48.41 C-1), 45.30 (C-4), 42.16 (C-8), 38.90 (C-6) in DMSO, 32.17 (C-3).

3.1.18. Synthesis of (1S,4S,5S,7R)-5-Chloro-7-((6-ethoxy-9H-purin-9-yl)methyl)bicyclo[2.2.1]heptan-2-ol, 7b

Compound 5 (0.4 mmol), ethanol (3.5 mL), Na (25 mg) were stirred for four days at rt, TLC (I, Rf = 0.59). By LPC, 108 mg (83.6%) 7b were obtained as a foam, [α]D = 11.6° (1% EtOH), IR: 3317brm, 2962m, 2728w, 1907w, 1598vs, 1508w, 1456s, 1412m, 1317s, 1223s, 1145w, 1053s, 1002m, 900w, 648m, 1H-NMR (CDCl3, δ ppm, J Hz): 8.51 (s, 1H, H-2′), 8.05 (s, 1H, H-8′), 4.73–4.59 (m, 3H, H-8, 2H-1″), 4.58 (dd, 1H, H-8, 7.9, 14.8), 4.18–4.11 (m, 1H, H-2, H-5), 3.85 (m, 1H, H-5), 2.96 (dd, 1H, H-3, 8.0, 14.6), 2.41 (d, 1H, H-1, 4.7), 2.25 (d, 1H, H-3, 14.2), 2.35–2.30 (m, 2H, H-4, H-7), 2.21 (brt, 1H, H-3, 4.2, 14.9), 2.07 (ddd, 1H, H-6, 5.0, 10.0, 13.8), 0.95 (brdd, 1H, H-6, 2.2, 13.8), 13C-NMR (CDCl3, δ ppm): 160.88 (C-6′), 152.04 (C-4′), 151.96 (C-2′), 142.32 (C-8′), 121.44 (C-5′), 69.43 (C-5), 63.16 (C-1″), 60.44 (C-2), 49.08 (C-7), 48.03 C-1), 45.76 (C-4), 42.82 (C-8), 36.51 (C-6), 32.67 (C-3), 14.52 (C-2″).

3.2. Anti-Viral Testing of the Compounds Viruses and Cells

Influenza virus (Flu A, strain A/Puerto Rico/8/34 (H1N1)), human adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1) were obtained from the collection of viruses of the Pasteur Institute (St. Petersburg, Russia). Prior to the experiment, the influenza virus was propagated in the allantoic cavity of 10–12 days old chicken embryos for 48 h at 36 °C. AdV5 and HSV-1 were grown in Vero cells for three days at 36 °C and 5% CO2. Infectious titers of FluA was determined in MDCK cells (ATCC # CCL-34) cells, of AdV5 and HSV-1—in Vero cells (ATCC #CCL-81). Cells were seeded into 96-wells plates in Eagle′s minimal essential medium (MEM).

3.2.1. Cytotoxicity Assay.

The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds (Mossman, 1983). Briefly, a series of three-fold dilutions of each compound (1000–4 µg/mL) in MEM were prepared. Cells were incubated for 48 h (MDCK) or 72 h (Vero) at 36 °C in 5% CO2 in the presence of the dissolved substances. The cells were then washed twice with saline, and a solution of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, OH, USA) (0.5 µg/mL) in saline was added to the wells. After 1 h of incubation, the wells were washed, and the formazan residue was dissolved in DMSO (0.1 mL per well). The optical density of cells was then measured on a Victor21440 multifunctional reader (Perkin Elmer, Turku, Finland) at a wavelength of 535 nm and plotted against the concentration of the compounds. Each compound concentration was tested in three parallels. The 50% cytotoxic dose (CC50) of each compound (i.e., the compound concentration that causes the death of 50% of cells in a culture, or decreases the optical density twice as compared to the control wells) was calculated from the data obtained.

3.2.2. Virus Titration.

The compounds in appropriate concentrations were dissolved in MEM with 1 µg/mL trypsin for Flu A and without additives for AdV5 and HSV-1, and incubated with cells for 1 h at 36 °C. The cell culture was then infected with either virus (moi 0.01). The plates were incubated for 48 h (Flu A) or 72 h (AdV5 and HSV-1) at 36 °C in the presence of 5% CO2. The activity of the viruses was evaluated by the MTT test, as described above, as its ability to decrease cell viability. Each concentration of the compounds was tested in triplicate. The antiviral activity of the compounds was estimated by the protection of cells from virus-induced death as compared with the control. The 50% inhibiting concentration (IC50) of the drug, that is, the concentration at which 50% of cells were protected, compared to control wells, and the selectivity index (the ratio of CC50 to IC50) were calculated from the data obtained.
Antiviral assays with EV71, YFV, and Chikungunya where performed as described earlier [46,47,48].

4. Conclusions

New 1′homocarbanucleoside analogs with pyrimidines, 6-chloropurine, adenine and 6-substituted adenine, 6-methoxy and ethoxy-purine as nucleobase were synthesized from an optically active 5-endo-hydroxy-8-hydroxymetil- bicyclo[2.2.1]heptane skeleton as sugar moiety. The 1′-homonucleosides with uracil, 5-fluorouracil, thymine, cytosine, and 6-chloropurine as nucleobases were synthesized by a selective Mitsunobu reaction on the primary hydroxymethyl group in the presence of 5-endo-hydroxyl group of the norbornane starting compound. Adenine and 6-substituted adenine homonucleosides were obtained by substitution of the 6-chlorine atom of the key intermediate 5 with ammonia and selected amines, and 6-methoxy- and 6-ethoxy substituted purine analogs by reaction with the corresponding alkoxides. The compounds were inactive against entero-, yellow fever, Chikungunya and adenoviruses. Two compounds (6j and 6d) have lower IC50 (15 ± 2 and 21 ± 4 µM) and one compound has IC50 similar to that of acyclovir (28 ± 4 µM).
These suggest that the bicyclo[2.2.1]heptane fragment can be effectively used instead of ribose or deoxyribose moiety to give nucleoside analogs with virus-inhibiting activity. The exact mechanism(s) of their anti-viral activity should be further studied in order to optimize the structure to create derivatives with low toxicity and high selectivity against specific viruses and with broad range of activity.

5. Patents

A patent pending dealing with the synthesis of the compounds was also filled.

Supplementary Materials

The following are available online at https://www.mdpi.com/1420-3049/24/13/2446/s1, copies of 1H- and 13C-NMR spectra for the key intermediate 5 and 1′-homocarbanucleoside analogs. Figures S1–S5, Table S1.

Author Contributions

C.I.T. and L.P. conceived and designed the experiments, performed the experiments, and analyzed the data; C.I.T., V.V.Z. and C.D. wrote the paper; A.H. and M.M. did the physico-chemical experiments of the synthesized compounds, A.V., E.S., A.V.S., D.J. for anti-viral screening, V.Z. and J.N. supervised anti-viral screening.

Funding

We thank the Romanian Ministry for Research and Innovations for contract PN 19-41 01 03/ 34N/2019 and for funds to cover the cost of publishing the article in open access.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Galmarini, C.M.; Popowycz, F.; Joseph, B. Cytotoxic nucleoside analogues: Different strategies to improve their clinical efficacy. Curr. Med. Chem. 2008, 15, 1072–1082. [Google Scholar] [CrossRef] [PubMed]
  2. Blagosklonny, M.V. Analysis of FDA Approved Anticancer Drugs Reveals the Future of Cancer Therapy. Cell Cycle 2004, 3, 1035–1042. [Google Scholar] [CrossRef] [PubMed]
  3. Shelton, J.; Lu, X.; Hollenbaugh, J.A.; Cho, J.H.; Amblard, F.; Schinazi, R.F. Metabolism, Biochemical Actions, and Chemical Synthesis of Anticancer Nucleosides, Nucleotides, and Base Analogs. Chem. Rev. 2016, 116, 14379–14455. [Google Scholar] [CrossRef] [PubMed]
  4. De Clercq, E. Highlights in the discovery of antiviral drugs: A personal retrospective. J. Med. Chem. 2010, 53, 1438–1450. [Google Scholar] [CrossRef] [PubMed]
  5. Deval, J. Antimicrobial strategies: Inhibition of viral polymerases by 3′-hydroxyl nucleosides. Drugs 2009, 69, 151–166. [Google Scholar] [CrossRef]
  6. De Clercq, E. Antiviral drugs in current clinical use. J. Clin. Virol. 2004, 30, 115–133. [Google Scholar] [CrossRef] [PubMed]
  7. Seley-Radtke, K.L.; Yates, M.K. The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold. Antiviral Res. 2018, 154, 66–86. [Google Scholar] [CrossRef]
  8. Wroblewski, A.E.; Glowacka, I.E.; Piotrowska, D.G. 1′-homonucleosides and their structural analogues: A Review. Eur. J. Med. Chem. 2016, 118, 121–142. [Google Scholar] [CrossRef]
  9. Lecerclé, D.; Clouet, A.; Al-Dabbagh, B.; Crouvoisier, M.; Bouhss, A.; Gravier-Pelletier, C.; Le Merrer, Y. Bacterial transferase MraY inhibitors: Synthesis and biological evaluation. Bioorg. Med. Chem. 2010, 18, 4560–4569. [Google Scholar] [CrossRef]
  10. Lee, H.W.; Choi, W.J.; Jacobson, K.A.; Jeong, L.S. Synthesis and Binding Affinity of Homologated Adenosine Analogues as A3 Adenosine Receptor Ligands. Bull. Korean Chem. Soc. 2011, 32, 961–964. [Google Scholar] [CrossRef]
  11. Rinaldo-Matthis, A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Tyler, P.C.; Evans, G.B.; Furneaux, R.H.; Almo, S.C.; Wang, C.C.; et al. Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues. Biochemistry 2007, 46, 659–668. [Google Scholar] [CrossRef] [PubMed]
  12. Lee, H.W.; Kim, H.O.; Choi, W.J.; Choi, S.; Lee, J.H.; Park, S.; Loo, L.; Jacobson, K.A.; Jeong, L.S. Design, synthesis, and binding of homologated truncated 4′-thioadenosine derivatives at the human A3 adenosine receptors. Bioorg. Med. Chem. 2010, 18, 7015–7021. [Google Scholar] [CrossRef] [PubMed]
  13. Sekiyama, T.; Hatsuya, S.; Tanaka, Y.; Uchiyama, M.; Ono, N.; Iwayama, S.; Oikawa, M.; Suzuki, K.; Okunishi, H.; Tsuji, T. Synthesis and Antiviral Activity of Novel Acyclic Nucleosides: Discovery of a Cyclopropyl Nucleoside with Potent Inhibitory Activity against Herpesviruses. J. Med. Chem. 1998, 41, 1284–1298. [Google Scholar] [CrossRef] [PubMed]
  14. Onishi, T.; Mukai, C.; Nakagawa, R.; Sekiyama, T.; Aoki, M.; Suzuki, K.; Nakazawa, H.; Ono, N.; Ohmura, Y.; Iwayama, S.; et al. Synthesis and Antiviral Activity of Novel Anti-VZV 5-Substituted Uracil Nucleosides with a Cyclopropane Sugar Moiety. J. Med. Chem. 2000, 43, 278–282. [Google Scholar] [CrossRef] [PubMed]
  15. Qiu, Y.L.; Ksebati, M.B.; Ptak, R.G.; Fan, B.Y.; Breitenbach, J.M.; Lin, J.S.; Cheng, Y.C.; Kern, E.R.; Drach, J.C.; Zemlicka, J. (Z)- and (E)-2-((hydroxymethyl)cyclo-propylidene)methyladenine and -guanine. New nucleoside analogues with a broad- spectrum antiviral activity. J. Med. Chem. 1998, 41, 10–23. [Google Scholar] [CrossRef] [PubMed]
  16. Baldanti, F.; Sarasini, A.; Drach, J.C.; Zemlicka, J.; Gerna, G. Z-isomers of 2-hy-droxymethylcyclopropylidenemethyl adenine (synadenol) and guanine (synguanol) are active against ganciclovir- and foscarnet-resistant human cytomegalovirus UL97 mutants. Antivir. Res. 2002, 56, 273–278. [Google Scholar] [CrossRef]
  17. Danappe, S.; Pal, A.; Alexandre, C.; Aubertin, A.-M.; Bourgougnon, N.; Huet, F. Synthesis of new nucleoside analogues comprising a methylenecyclobutane unit. Tetrahedron 2005, 61, 5782–5787. [Google Scholar] [CrossRef]
  18. Santana, L.; Teijeira, M.; Uriarte, E.; Teran, C.; Andrei, G.; Snoeck, R.; Balzarini, J.; De Clercq, E. Synthesis and Biological Evaluation of 1,2-Disubstituted Carbonucleosides of 6-Substituted Purine and 8-Azapurine. Nucl. Nucl. 1999, 18, 733–734. [Google Scholar] [CrossRef] [PubMed]
  19. Balo, M.C.; Fernandez, F.; Lens, E.; Lopez, C.; De Clercq, E.; Andrei, G.; Snoeck, R.; Balzarini, J. Synthesis and Antiviral Activities of Some Novel Carbocyclic Nucleosides. Nucl. Nucl. 1996, 15, 1335–1346. [Google Scholar] [CrossRef]
  20. Balo, M.C.; Blanco, J.M.; Fernandez, F.; Lens, E.; Lopez, C. Synthesis of novel carbocyclic nucleosides with a cyclopentenyl ring: Homocarbovir and analogues. Tetrahedron 1998, 54, 2833–2842. [Google Scholar] [CrossRef]
  21. Rajapan, V.P.; Yin, X.; Schneller, S.W. A flexible synthesis of carbanucleosides and 5′-nor-1′-homo carbanucleosides from a common precursor. Tetrahedron 2002, 58, 9889–9895. [Google Scholar] [CrossRef]
  22. Kim, A.; Hong, J.H. Synthesis and Anti-HCMV Activity of Novel 5′-Norcarboacyclic Nucleosides. Arch. Pharm. Chem. Life Sci. 2005, 338, 522–527. [Google Scholar] [CrossRef] [PubMed]
  23. Bianco, A.; Celona, D.; Di Rita, S.; Guiso, M.; Melchioni, C.; Umani, F. Chiral Synthons from the Iridoid Glucoside Antirrhinoside−Synthesis of a Carbocyclic Homonucleoside Analogue. Eur. J. Org. Chem. 2001, 21, 4061–4066. [Google Scholar] [CrossRef]
  24. Franzyk, H.; Rasmussen, J.H.; Mazzei, R.A.; Jensen, S.R. Synthesis of Carbocyclic Homo-N-Nucleosides from Iridoids. Eur. J. Org. Chem. 1998, 12, 2931–2935. [Google Scholar] [CrossRef]
  25. Blanco, J.M.; Caamano, O.; Fernandez, F.; Gomez, M.; Nieto, M.I.; Balzarini, J.; Padalko, E.; De Clerq, E. Synthesis and Antiviral and Cytostatic Activities of Carbocyclic Nucleosides Incorporating a Modified Cyclopentane Ring. I: Guanosine Analogues. Nucl. Nucl. 1997, 16, 159–171. [Google Scholar] [CrossRef]
  26. Nieto, M.J.; Blanco, J.M.; Caamano, O.; Fernandez, F.; Garcia-Mera, X.; Balzarini, J.; Padalko, E.; Neyts, J.; De Clerq, E. Synthesis, Antiviral and Cytostatic Activities of Carbocyclic Nucleosides Incorporating a Modified Cyclopentane Ring. Part 2:1 Adenosine and Uridine Analogues. Nucl. Nucl. 1998, 17, 1255–1266. [Google Scholar] [CrossRef] [PubMed]
  27. Nieto, M.J.; Caamano, O.; Fernandez, F.; Gomez, M.; Balzarini, J.; De Clerq, E. Synthesis, Antiviral and Cytostatic Activities, of Carbocyclic Nucleosides Incorporating a Modified Cyclopentane Ring. IV. Adenosine and Uridine Analogues. Nucl. Nucl. Nucleic Acids 2002, 21, 243–255. [Google Scholar] [CrossRef] [PubMed]
  28. Fernandez, F.; Garcia-Mera, X.; Morales, M.; Rodriguez-Borges, De Clerq, E. Synthesis and Cytostatic Activities of New 6-Substituted Purinylcarbonucleosides Derived from Indan. Synthesis 2002, 21, 1084–1090. [Google Scholar] [CrossRef]
  29. Yao, S.-W.; Lopez, V.H.C.; Fernandez, F.; Garcia-Mera, X.; Morales, M.; Rodriguez-Borges, J.E.; Cordeiro, M.N. Synthesis and QSAR study of the anticancer activity of some novel indane carbocyclic nucleosides. Bioorg. Med. Chem. 2003, 11, 4999–5006. [Google Scholar] [CrossRef]
  30. Garcia, M.D.; Caamano, O.; Fernandez, F.; Lopez, M.C.; De Clerck, E. Synthesis of Purinyl homo-Carbonucleoside Derivatives of 2-Benzylcyclopenta[c]pyrazol. Synthesis 2005, 925–932. [Google Scholar]
  31. Garcia, M.D.; Caamano, O.; Fernandez, F.; Abeijon, P.; Blanco, J.M. Synthesis of New 1′(N)-Homocarbanucleosides Based on 1-Methylcyclopenta[c]pyrazole Scaffold. Synthesis 2006, 73–80. [Google Scholar]
  32. Garcia, M.D.; Caamano, O.; Fernandez, F.; Garcia-Mera, X.; Perez-Castro, I. Synthesis of Purinyl and Pyrimidinyl 1′(N)-Homocarbanucleosides Based on a 1-Methylcyclopenta[c]pyrazole Scaffold; Part 2. Synthesis 2006, 3967–3972. [Google Scholar]
  33. Tănase, C.; Drăghici, C.; Căproiu, M.T.; Shova, S.; Mathe, C.; Cocu, F.G.; Enache, C.; Maganu, M. New carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, X-ray crystallography and anticancer activity. Bioorg. Med. Chem. 2014, 22, 513–522. [Google Scholar] [CrossRef] [PubMed]
  34. Tănase, C.; Drăghici, C.; Cojocaru, A.; Galochkina, A.V.; Orshanskaya, J.R.; Zarubaev, V.V.; Shova, S.; Enache, C.; Maganu, M. New carbocyclic N6-substituted adenine and pyrimidine nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and X-ray crystallography. Bioorg. Med. Chem. 2015, 23, 6346–6354. [Google Scholar] [CrossRef] [PubMed]
  35. Tănase, C.; Cocu, F.G.; Neagu, M.; Manda, G. New Bicyclo[2.2.1]heptane Nucleoside Analogues as Antitumor Agents. Rev. Chim. (Bucharest) 2009, 60, 147–151. [Google Scholar]
  36. Tănase, C.; Cojocaru, A.; Drăghici, C. New 1′-homocarbonucleosides analogs with a constrained bicyclo[2.2.1]heptane fragment. Patent RO 141479 A2, 13 May 2015. [Google Scholar]
  37. Tănase, C.; Drăghici, C.; Neguț, C.; Pintilie, L. New constrained Amines in a Bicyclo[2.2.1]heptane Skeleton. Rev. Chim. (Bucharest) 2018, 69, 2448–2453. [Google Scholar]
  38. Tănase, C.; Drăghici, C.; Neguț, C.; Pintilie, L. Bicyclo[2.2.1]heptane Amines Protected at the Hydroxyl Group. Patent RO 133248 A2, 30 April 2019. [Google Scholar]
  39. Bennett, M.S.; Wien, F.; Champness, J.N.; Batuwangala, T.; Rutherford, T.; Summers, W.C.; Sun, H.; Wright, G.; Sanderson, M.R. Structure to 1.9 Å resolution of a complex with herpex simplex virus type-1 thymidine kinase of a novel, non-substrate inhibitor: X-ray crystallographic comparison with binding of acyclovir. FEBS Lett. 1999, 443, 121–125. [Google Scholar] [CrossRef]
  40. Al-Salahi, R.; Alswaidan, I.; Ghabbour, H.A.; Ezzeldin, E.; Elaasser, M.; Marzouk, M. Docking and antiherpetic activity of 2-aminobenzo[de]-isoquinoline-1,3-diones. Molecule 2015, 20, 5099–5111. [Google Scholar] [CrossRef] [PubMed]
  41. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2001, 46, 3–26. [Google Scholar] [CrossRef]
  42. Korb, O.; Stützle, T.; Exner, T.E. Empirical scoring functions for advanced protein-ligand docking with plants. J. Chem. Inf. Model 2009, 49, 84–96. [Google Scholar] [CrossRef]
  43. Tănase, C.; Căproiu, M.T.; Drăghici, C.; Enache, C.; Cocu, F.G.; Manda, G. Secondary Compounds and Intermediates in Deprotection of the Benzoate Groups of O2,O4- and N1,O4-Nucleoside Analogues With Optically Active Bicyclo[2.2.1]Heptane Sugar Moieties. Rev. Chim. (Bucharest) 2011, 62, 380–385. [Google Scholar]
  44. Hamon, N.; Kaci, M.; Uttaro, J.-P.; Perigaud, C.; Mathe, C. Synthesis of 3′-halo-5′-norcarbocyclic nucleoside phosphonates as potential anti-HIV agents. Eur. J. Med. Chem. 2018, 150, 642–654. [Google Scholar] [CrossRef] [PubMed]
  45. Shiraki, K. Antiviral Drugs Against Alphaherpesvirus. Adv. Exp. Med. Biol. 2018, 1045, 103–122. [Google Scholar] [PubMed]
  46. Lacroix, C.; Querol-Audi, J.; Roche, M.; Franco, D.; Froen, M.; Guerra, P.; Terme, T.; Vanelle, P.; Verdaguer, N.; Neyts, J.; et al. A novel benzonitrile analogue inhibits rhinovirus replication. J. Antimicrob. Chemother. 2014, 69, 2723–2732. [Google Scholar] [CrossRef] [PubMed]
  47. Saudi, M.; Zmurko, J.; Kaptein, S.; Rozenski, J.; Neyts, J.; Van Aerchot, A. Syntesis and evaluation of imidazole-4,5- and pyrazine-2,3-dicarboxamides targeting dengue and yellow fever virus. Eur. J. Med. Chem. 2014, 87, 529–539. [Google Scholar] [CrossRef] [PubMed]
  48. Delang, L.; Li, C.; Tas, A.; Querat, G.; Albulescu, I.C.; De Burghgraeve, T.; Segura Guerrero, N.A.; Gigante, A.; Piorkowski, G.; Decroly, E.; et al. The viral capping enzyme nsP1: A novel target for the inhibition of chikungunya virus infection. Sci. Rep. 2016, 6, 31819. [Google Scholar] [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 4, 5, 6 and 7 (with exception of 4d and 6g) are available in amount of 5-to 10 mg from the authors, for a year.
Molecules 24 02446 g001 550
Figure 1. 1′-Homonucleosides with a cyclopropane ring in the sugar moiety.
Figure 1. 1′-Homonucleosides with a cyclopropane ring in the sugar moiety.
Molecules 24 02446 g001
Molecules 24 02446 g002 550
Figure 2. 1′-Homocarbanucleosides with modified sugar with 2,2,3-trimethylcyclopentane, indane, cyclopenta[c]pyrazole, cyclopenta[d]pyrazole fragments.
Figure 2. 1′-Homocarbanucleosides with modified sugar with 2,2,3-trimethylcyclopentane, indane, cyclopenta[c]pyrazole, cyclopenta[d]pyrazole fragments.
Molecules 24 02446 g002
Molecules 24 02446 g003 550
Figure 3. l-Carbanucleosides with an optically active bicyclo[2.2.1]heptane scaffold with potential anti-influenza and coxsackie B4 viruses.
Figure 3. l-Carbanucleosides with an optically active bicyclo[2.2.1]heptane scaffold with potential anti-influenza and coxsackie B4 viruses.
Molecules 24 02446 g003
Molecules 24 02446 g004 550
Figure 4. Docking score of the compounds.
Figure 4. Docking score of the compounds.
Molecules 24 02446 g004
Molecules 24 02446 g005 550
Figure 5. Docking pose of the 6j ligand interacting with amino acids residues.
Figure 5. Docking pose of the 6j ligand interacting with amino acids residues.
Molecules 24 02446 g005
Molecules 24 02446 sch001 550
Scheme 1. Synthesis of pyrimidine N′-homocarbanucleoside analogs 4a–4c: 1) 1, NaBH4, MeOH, −16 °C [36], 2) 5FU, U or T, Ph3P, THF 0 °C, DIAD added dropwise, 1 h stirring, 2 in THF added in 50 min, stirring overnight; for 4d, N4-benzoyl cytosine crude product hydrolyzed with MeONa/MeOH, rt, overnight.
Scheme 1. Synthesis of pyrimidine N′-homocarbanucleoside analogs 4a–4c: 1) 1, NaBH4, MeOH, −16 °C [36], 2) 5FU, U or T, Ph3P, THF 0 °C, DIAD added dropwise, 1 h stirring, 2 in THF added in 50 min, stirring overnight; for 4d, N4-benzoyl cytosine crude product hydrolyzed with MeONa/MeOH, rt, overnight.
Molecules 24 02446 sch001
Molecules 24 02446 sch002 550
Scheme 2. Synthesis of 6-purine substituted 1′-homocarbanucleoside analogs: 5, 6a–6k, 7a–7b: 1) 6-Cl-Pu, Ph3P in THF, DIAD, −6 °C, 1 h, 2 in THF added in 1h, then stirring overnight, 2) for 6a: 5, 25% NH4OH, MeOH, 80–90 °C (pressure vessel) 24h, for 6b–6d, 6h, 6k: 5, amine, rt, 72 h, six days, for compounds 6e–6g, 6i–6g: 5, amine, EtOH, EtN3, 72h (6f, 6g) to six days (6i, 6g), rt, (60 °C for 6e).
Scheme 2. Synthesis of 6-purine substituted 1′-homocarbanucleoside analogs: 5, 6a–6k, 7a–7b: 1) 6-Cl-Pu, Ph3P in THF, DIAD, −6 °C, 1 h, 2 in THF added in 1h, then stirring overnight, 2) for 6a: 5, 25% NH4OH, MeOH, 80–90 °C (pressure vessel) 24h, for 6b–6d, 6h, 6k: 5, amine, rt, 72 h, six days, for compounds 6e–6g, 6i–6g: 5, amine, EtOH, EtN3, 72h (6f, 6g) to six days (6i, 6g), rt, (60 °C for 6e).
Molecules 24 02446 sch002
Table
Table 1. Docking score and calculated properties of ligands.
Table 1. Docking score and calculated properties of ligands.
CompoundAtomsWeight [Daltons]ScoreRMSD (Å)Flexible BondsLipinski ViolationsHydrogen DonorsHydrogen AcceptorsLog P
Acyclovir26224.20−49.290.7140380.50
4a33288.70−63.160.0120252.68
4b33270.71−59.490.00820252.58
4c36284.74−62.370.0220252.94
4d34269.73−65.530.0120452.76
534313.18−58.790.0320362.02
6a36293.75−59.370.0220360.71
6b43333.82−51.590.0640261.93
6c49361.87−38.450.0240262.64
6d52375.90−39.940.1340263.19
6e56427.93−55.540.2970372.71
6f51376.88−26.860.0230171.33
6g53391.90−19.260.0240281.42
6h47363.84−32.120.4730171.14
6i53413.90−70.210.0960372.97
6j56427.93−62.081.5770273.30
6k52397.90−70.070.0760263.33
7a38308.76−57.270.0230161.36
7b41322.79−53.110.5540161.73
Table
Table 2. Cytotoxic and virus-inhibiting properties of 1′-homocarbanucleosides analogs against influenza virus, human herpesvirus type 1 and human adenovirus type 5 in cell culture. Values of selectivity indices 10 and higher are marked by color.
Table 2. Cytotoxic and virus-inhibiting properties of 1′-homocarbanucleosides analogs against influenza virus, human herpesvirus type 1 and human adenovirus type 5 in cell culture. Values of selectivity indices 10 and higher are marked by color.
CmpdMwAnti-Viral Activity
Influenza VirusHerpes Simplex Virus Type 1Human Adenovirus Type 5
CC50, µM aIC50, µM bSI cCC50, µMIC50, µMSICC50, µMIC50, µMSI
4a288.71>1039173 ± 216>1039>10391>1039>10391
4b270.72>1108369 ± 2631108705 ± 812>1108>11081
4c284.74>1054>10541>1054>10541>1054>10541
4d269.73>1112102 ± 1211>1112727 ± 772>1112>11121
6a293.76>1021328 ± 293975 ± 64102 ± 1110977 ± 103>3403
6b333.82752 ± 44419 ± 512>773599 ± 641>899>8991
6c361.87227 ± 19>833605 ± 4247 ± 613824 ± 91>2763
6d375.9042 ± 3>272521 ± 4821 ± 425697 ± 75123 ± 146
6e427.93188 ± 22>267>608380 ± 462>701>7011
6f376.89>796241 ± 303>71928 ± 426>796105 ± 118
6g391.90>766>7661>766540 ± 321>766>7661
6h500.00>600196 ± 153>600341 ± 402>600>6001
6i413.91124 ± 9>722447 ± 31155 ± 133217 ± 17>723
6j427.93467 ± 28>2342205 ± 1915 ± 214241 ± 22>703
6k397.91254 ± 2174 ± 83185 ± 1648 ± 64245 ± 18>753
5313.19>958254 ± 144>83069 ± 912>958>9581
7a308.77>972233 ± 334>972>9721>972>9721
7b322.78>929>9291>929524 ± 632>929>9291
AcyclovirN/T>113128 ± 440N/T
Oseltamivir>1000.3 ± 0.1>333N/TN/T
a CC50, cytotoxic concentration, the concentration resulting in 50% death of cells. b IC50, effective concentration, the concentration resulting in 50% inhibition of virus replication. c SI, selectivity index, ratio CC50/IC50.
Table
Table 3. Cytotoxic and virus-inhibiting properties of 1′-homocarbanucleosides analogs against Yellow Fever virus, Chikungunya virus and Enterovirus 71 in cell culture.
Table 3. Cytotoxic and virus-inhibiting properties of 1′-homocarbanucleosides analogs against Yellow Fever virus, Chikungunya virus and Enterovirus 71 in cell culture.
CmpdMwAnti-Viral Activity
Yellow Fever VirusChikungunya VirusEnterovirus 71
CC50, µM aIC50, µM bSI cCC50, µMIC50, µMSICC50, µMIC50, µMSI
4a288.71>100>1001>100>1001>100>1001
4b270.72>100>1001>100>1001>100>1001
4c284.74>100>1001>100>1001>100>1001
4d269.73>100>1001>100>1001>100>1001
6a293.76>100>1001>100>1001>100711
6b333.82>100>1001>100>1001>100>1001
6c361.87>100>1001>100>1001>10028 ± 164
6d375.90>100>1001>100>1001>10028 ± 134
6e427.93>100>1001>100>1001>100>1001
6f376.89>100>1001>100>1001>100>1001
6g391.90>100>1001>100>1001>100>1001
6h500.00>100>1001>100>1001>100>1001
6i413.91>100>1001>100>1001>100791
6j427.93>100>1001>100>1001>10042 ± 3.22
6k397.91>100>1001>100>1001>10025 ± 5.64
5313.19>100>1001>100>1001>100>1001
7a308.77>100>1001>100>1001>100>1001
7b322.78>100>1001>100>1001>100>1001
a CC50, cytotoxic concentration, the concentration resulting in 50% death of cells. b IC50, effective concentration, the concentration resulting in 50% inhibition of virus replication. c SI, selectivity index, ratio CC50/IC50.

Share and Cite

MDPI and ACS Style

Tănase, C.I.; Drăghici, C.; Hanganu, A.; Pintilie, L.; Maganu, M.; Volobueva, A.; Sinegubova, E.; Zarubaev, V.V.; Neyts, J.; Jochmans, D.; et al. New HSV-1 Anti-Viral 1′-Homocarbocyclic Nucleoside Analogs with an Optically Active Substituted Bicyclo[2.2.1]Heptane Fragment as a Glycoside Moiety. Molecules 2019, 24, 2446. https://doi.org/10.3390/molecules24132446

AMA Style

Tănase CI, Drăghici C, Hanganu A, Pintilie L, Maganu M, Volobueva A, Sinegubova E, Zarubaev VV, Neyts J, Jochmans D, et al. New HSV-1 Anti-Viral 1′-Homocarbocyclic Nucleoside Analogs with an Optically Active Substituted Bicyclo[2.2.1]Heptane Fragment as a Glycoside Moiety. Molecules. 2019; 24(13):2446. https://doi.org/10.3390/molecules24132446

Chicago/Turabian Style

Tănase, Constantin I., Constantin Drăghici, Anamaria Hanganu, Lucia Pintilie, Maria Maganu, Alexandrina Volobueva, Ekaterina Sinegubova, Vladimir V. Zarubaev, Johan Neyts, Dirk Jochmans, and et al. 2019. "New HSV-1 Anti-Viral 1′-Homocarbocyclic Nucleoside Analogs with an Optically Active Substituted Bicyclo[2.2.1]Heptane Fragment as a Glycoside Moiety" Molecules 24, no. 13: 2446. https://doi.org/10.3390/molecules24132446

Article Metrics

Back to TopTop