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Article

Stereoselective Synthesis and Antimicrobial Studies of Allo-Gibberic Acid-Based 2,4-Diaminopyrimidine Chimeras

by
Dima Depp
1,
Noémi Regina Sebők
2,
András Szekeres
2 and
Zsolt Szakonyi
1,*
1
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös Utca 6, H-6720 Szeged, Hungary
2
Department of Microbiology, University of Szeged, Közép Fasor 52, H-6726 Szeged, Hungary
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2025, 18(2), 168; https://doi.org/10.3390/ph18020168
Submission received: 8 January 2025 / Revised: 23 January 2025 / Accepted: 24 January 2025 / Published: 26 January 2025
(This article belongs to the Topic New Compounds Discovery and Development in Medicine — Advances in Research on Potential Therapeutic Agents and Drug Candidates, 2nd Edition)
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Abstract

:
Background: Gibberellins (GAs) are a family of tetracyclic ent-kaurenoid diterpenes found widely in several commonly used plants. Besides agricultural applications, gibberellins play an important role in the synthesis of bioactive compounds, especially those with antiproliferative and antibacterial activity. Methods: A series of gibberellic acid-based 2,4-diaminopyrimidines was designed and synthesized from commercially available gibberellic acid. The antimicrobial activity of the prepared compounds was also explored in B. subtilis, S. aureus, E. coli, and P. aeruginosa bacteria, as well as in C. krusei and C. albicans fungi. Results: The treatment of gibberellic acid with hydrochloric acid under reflux conditions resulted in aromatization followed by rearrangement to form allo-gibberic acid. The key intermediate azido alcohol was prepared according to the literature methods. The second key intermediate azidotriol was synthesized by the stereoselective dihydroxylation of the allylic function by the osmium (VIII)-tetroxide/NMO system. Starting from azide intermediates, click reactions were also carried out with 4-monoamino- and 2,4-diaminopyrimidines functionalized with the N-propargyl group. The new chimeric compounds, coupled with gibberellins thus obtained, were characterized by 1D- and 2D-NMR techniques and HRMS measurements. While the 4-monoamino-substituted derivatives exhibited only weak antibacterial activity, they demonstrated significant antifungal effectiveness against C. krusei. In general, 5-chloro-substituted pyrimidine derivatives displayed more consistent biological activities compared to their 5-fluoro counterparts, with the exception of one derivative, which showed acceptable activity against both C. krusei and C. albicans. The two derivatives featuring 5-chloro and 2-((4-(trifluoromethyl)phenyl)amino substituents proved to be highly effective against P. aeruginosa, making them promising candidates for further research. Aiming to elucidate the molecular interactions between the active compounds and their potential targets, molecular docking studies were conducted using AutoDock Vina 1.1.2. involving the most active compounds against P. aeruginosa. Conclusions: The biological effects of 2-monoamino or 2,4-diamino substitution as well as the effect of chloro or fluoro substitution at position 5 of the pyrimidine ring combined with the allo-gibberic acid moiety were determined. Compounds with selective antibacterial activity against P. aeruginosa as well as selective antifungal activity against C. krusei and C. albicans fungi were identified.

Graphical Abstract

1. Introduction

Resistant microorganisms represent a significant global health challenge. According to a study published by the Global Research on Antimicrobial Resistance (GRAM) Project, more than 1.2 million people lost their lives in 2019 due to antibiotic-resistant bacterial infections [1]. Additionally, the Annual Epidemiological Report 2023 from the European Centre for Disease Prevention and Control highlights that, based on EARS-Net data from 2020, over 35,000 deaths occur annually in the EU/EEA as a direct result of antimicrobial-resistant infections. In 2023, the highest estimated incidence of invasive bacterial isolates reported by laboratories in the EU/EEA was attributed to E. coli (71.4 per 100,000 population), followed by S. aureus (36.9 per 100,000), K. pneumoniae (24.2 per 100,000), E. faecalis (14.1 per 100,000), E. faecium (10.7 per 100,000), P. aeruginosa (10.5 per 100,000), S. pneumoniae (7.2 per 100,000), and Acinetobacter spp. (4.6 per 100,000) [2]. Consequently, the search for new antimicrobials has become essential, as the number of non-susceptible organisms is gradually increasing, while the introduction of new antimicrobials to the market remains limited. In several cases, these were isolated from exotic marine organisms with complex structures [3,4]. Biologically validated products with a natural origin serve as an important resource for the development of new pharmaceuticals [5,6,7]. Their commercially available natural sources have historically provided a significant inspiration for the design and synthesis of bioactive compounds in medicinal chemistry. Gibberellins (GAs), a family of tetracyclic ent-kaurenoid diterpenes, are present widely in several commonly used plants, and some of them are used in agriculture as regulators to promote plant germination and growth. Gibberellic acid (GA3, I, Scheme 1) is nowadays produced commercially in large quantities by the fermentation of the fungus Gibberella fujikuroi and used in agriculture worldwide [8,9,10,11]. It reduces the time to flowering, and it also affects seed germination, leaf area expansion, stem elongation, and the maturation of plant sexual organs.
Besides agricultural applications, gibberellins play an important role in the synthesis of bioactive compounds, especially with antiproliferative activity [12,13,14]. In 2009, Koehler discovered that gibberellic acid and 9α-H allo-gibberic acid could modulate the NF-κB signaling pathway [15], a transcription pathway associated with various cancer diseases [16,17]. In recent years, we have reported the synthesis of allo-gibberic acid-based aminoalcohols, aminodiols, and aminotriols and studied their antiproliferative activity [18,19,20]. Similarly, the synthesis of 4-aryl-substituted triazole-coupled allo-gibberic acid derivatives with remarkable antiproliferative activities was reported by Wu et al. [21]. Since there are only a few articles addressing the antibacterial activity of allo-gibberic acid [22,23,24], this encouraged us to undertake investigation in this area.
2,4-Diaminopyrimidine derivatives possess biological and pharmaceutical importance as well as antimicrobial [25], antiprotozoal [26], and anticancer therapy uses [27,28,29]. Therefore, it seemed interesting to combine the allo-gibberic acid-based moiety with a mono- or diaminopyrimidine unit to obtain a promising chimeric structure with antimicrobial activity.

2. Results and Discussion

2.1. Synthesis of Allo-Gibberic Acid-Based Azides

allo-Gibberic acid 1 was obtained starting from commercially available gibberellic acid (I) through HCl-mediated hydrolysis, which involves lactone opening, decarboxylation, and the aromatization of ring A [14,19]. Afterwards, key intermediate azide 4 was prepared according to the literature, as shown in Scheme 1, in a four-step synthesis via esterification, followed by LiAlH4-mediated reduction, tosylation of the primary alcohol and, finally, tosyl–azide exchange [22]. Moreover, by the oxidation of the double bond using catalytic amounts of OsO4 and NMO, we were able to synthesize another key intermediate, triol-azide 5, under mild conditions [30].

2.2. Synthesis of 2,4-Diaminopyrimidine-Derived Alkynes

Nitrogen-containing heterocycles hold a unique and significant position as a valuable source of therapeutic agents in medicinal chemistry [25,28,31,32]. Among them, 2,4-diaminopyrimidines are particularly important in cancer chemotherapy and the development of new antitrypanosomal, antimalarial, and antibacterial drugs [26,33,34,35,36]. Compounds 8–13 were synthesized according to our recently published article starting from halogen-substituted pyrimidines and propargylamine in two steps [20]. Furthermore, we were able to synthesize new 5-fluoro- and 5-chloro-substituted 2,4-diaminopyrimidines with 4-(methoxycarbonyl)phenyl and 1-(4-morpholinophenyl) moieties, as shown in Scheme 2. The aim of these reactions was to couple the products with the diterpene skeleton. Compounds 14 and 15 have low solubility, and they precipitated in ethanol as the reaction mixture started to cool down.

2.3. Coupling the Diterpene Skeleton with Monoamino- and Diaminopyrimidines via Click Reaction

The CuAAC reaction (copper-catalyzed azide–alkyne cycloaddition) is a widely used click chemistry approach for the synthesis of 1,2,3-triazoles with high efficiency and regioselectivity. Its simplicity, versatility, and compatibility with various functional groups make it invaluable in medicinal chemistry [37,38]. In order to prepare monoamino- and diaminopyrimidines derived from allo-gibberic acid, both azides 4 and 5 were subjected to a CuAAC reaction with the previously prepared alkynes, as shown in Scheme 3. Reactions were carried out under mild conditions at 45 °C with catalytic amounts of Cu(OAc)2.H2O and sodium ascorbate. The solubility of the corresponding alkyne played a crucial role in determining the type and amount of solvent required for the reaction. As for monoaminopyrimidines and 2,4-diaminopyrimidines with 4-(trifluoromethyl)phenyl, 1-methyl-1H-pyrazol-4-yl, and 1-(4-morpholino)phenyl substituents, a mixture of tert-BuOH and water (2:1) was used. In contrast, for compounds 23, 24, 32, and 33, involving the use of 4-(methoxycarbonyl)phenyl alkynes, a mixture of i-PrOH and water (2:1) or THF and water (2:1) was employed due to the insolubility of these alkynes in the tert-BuOH/water mixture. Moreover, the choice of solvent mixture influenced the solubility of the products during the reactions. In fact, compound 32 precipitated in the reaction mixture, whereas the alkyne precipitated when preparing compound 23. Compounds 30 and 31 exhibited higher polarity than initially indicated by TLC analysis, necessitating the use of a more polar solvent system during column chromatography for effective separation.

2.4. Determination of the Relative Configuration of Azide 5

A new chiral center was generated by the dihydroxylation of the double bond, yielding azide 5. The relative configuration of the obtained new stereocenter at position 8 was established using 2D-NMR experiments (Figure 1).
Despite the complexity caused by overlapping peaks in the aliphatic range in 1H NMR, clear NOE signals were observed between the CH2 proton of the primary alcoholic function [3.83 ppm (dd)] and the Ha proton at position 9 and similarly with CH2 at positions 5 and 6. Moreover, clear NOE signals were observed between OH at position 8 and Hb proton at position 9 as well as with CH2 at position 11. The HO group at position 8 was determined by HMBC measurements.

2.5. In Vitro Studies of Antimicrobial Effects of 4-Amino- and 2,4-Diaminopyrimidine Derivatives and Their Structure–Activity Relationship (SAR)

Since several diterpenes with an allylic alcohol function as well as several 2,4-diaminopyrimidines exerted antimicrobial activities against various bacterial and fungal strains, the antimicrobial activities of the prepared mono- and trihydroxy-2,4-diaminopyrimidine analogues were tested against two yeasts as well as two Gram-positive and two Gram-negative bacteria (Table 1) [39]. Interestingly, key intermediate azide 4 with an allylic alcohol function showed more potential antimicrobial activity against both Gram-positive and Gram-negative bacteria and similarly against yeasts. It is also evident from the results presented in Table 1 that 4-monoamino-substituted derivatives (17, 18, 26, and 27) have only weak antibacterial activity. In contrast, these compounds possess remarkable antifungal activity against Candida krusei, especially compound 27, which exhibits better activity compared to the reference compound nystatine at a concentration of 5 μg/mL. Generally, 5-chloro-substituted pyrimidine derivatives have more reliable biological activities compared with 5-fluoro derivatives, with the only exception of 2-(pyrazol-4-yl)amino derivative 21, which possesses acceptable activity against both C. krusei and C. albicans. Derivative 29, the best diaminopyrimidine with 5-chloro and 2-((4-(trifluoromethyl)phenyl)amino substituents, proved to be excellent against both P. aeruginosa and C. krusei, and it would be an excellent lead molecule for further investigation. Similarly, compound 20 [monohydroxy, with 5-chloro and 2-((4-(trifluoromethyl)phenyl substitution] shows a selective antibacterial effect against P. aeruginosa, while compound 28 was effective and selective against fungus.

2.6. Molecular Docking

Aiming to elucidate the molecular interactions between the active compounds and their potential targets, molecular docking studies were conducted using AutoDock Vina [40]. Two compounds, 20 and 29, which demonstrated the highest activity against P. aeruginosa were selected for docking.
Compounds that strongly inhibit bacterial growth are likely to disrupt fundamental processes such as DNA replication. DNA gyrase is a well-recognized target for several antibiotics, including fluoroquinolones. While fluoroquinolones act by inhibiting the GyrA subunit of the enzyme, novobiocin uniquely targets the GyrB subunit. Targeting the GyrB subunit is particularly significant because it contains the ATP-binding site, which is essential for enzymatic activity. Additionally, the conserved nature of the ATP-binding site minimizes the likelihood of spontaneous resistance to ATP-competitive inhibitors [41]. Similarly, nitrogen-containing heterocycles have the ability to mimic the structure of ATP, enabling them to effectively compete for the ATP-binding site [42,43]. Furthermore, our previous research suggested that allo-gibberic acid derivatives containing nitrogen heterocycles exhibit affinity for the ATP-binding sites of certain kinase enzymes [20]. As such, selecting DNA gyrase B as a docking target aligns with the compound’s observed ability to completely inhibit P. aeruginosa growth. Moreover, the essential role of DNA gyrase B, its validated druggability, and the availability of high-resolution crystal structures make it an ideal target. These factors, combined with the compound’s potential ATP-binding site affinity, enable precise docking studies to be conducted.
The DNA gyrase B structure of P. aeruginosa involves two amino acids, Arg78 and Arg138, which are crucial for its enzymatic activity. Additionally, it contains a hydrophobic groove formed by Ile80, Pro81, and Ile96. Moreover, a solvent-exposed area opposite two conserved amino acids, Asn48 and Asp51, may further contribute to improving the binding affinity [44]. In Figure 2A, a 3D illustration shows the interactions between a known co-crystalized pyrido [2,3-b]indole-type inhibitor and P. aeruginosa DNA gyrase B. The co-crystalized ligand has reliable interactions with key residues, as shown in Figure 2B, such as Arg78, Ile80, Pro81, and Ile96, as well as Asn48. The redocking of the co-crystalized ligand with the protein for the validation of the docking protocol yielded similar poses and interactions with binding energies below –7.0 kcal/mol and RMSD below 3 Å (detailed information can be found in the Supplementary Materials).
The docking results indicate that compound 20 exhibits strong affinity with binding energies ranging from –8.9 to –8.2 kcal/mol. As suggested by the software and shown in Figure 3A, compound 20 may form two conventional hydrogen bonds: one between a trifluoromethyl fluorine atom and Asn48 and another between the amino group and Asp51. Moreover, van der Waals interactions with Arg78 can be observed. The diterpene portion of compound 20 can also interact with Ile96 within the hydrophobic pocket. Similarly, compound 29 demonstrated high affinity, with binding energies ranging from –9.0 to –8.1 kcal/mol. In addition to the interactions mentioned previously, compound 29 may be able to form cation–π and anion–π stacking interactions between the pyrimidine ring and Arg78 and Glu52, as visualized in Figure 3B, which might increase the affinity. All of these interactions may contribute to the binding affinity of compounds 20 and 29 with the protein, stabilizing the complex by enhancing the associations between the ligand and the receptor. On the other hand, it is worth mentioning that despite sharing the same structure with compounds 20 and 29, with the exception of a fluorine atom replacing chlorine, compounds 19 and 28 exhibited almost no antibacterial activity against P. aeruginosa compared to 20 and 29. This finding is consistent with previous results, which suggest that chlorine atom substitutions play a key role in modulating the antibacterial profile [45]. For better illustration, all 2D diagrams of the interactions of compounds with the proteins can be found in the Supplementary Materials.
To gain deeper insights into the structure–activity relationship, the docking of a non-active compound was performed to compare its binding interactions with those of active compounds. Compound 30 was selected for this analysis. The results revealed binding affinities ranging from –7.4 to –6.8 kcal/mol, which are noticeably lower than the scores observed for active compounds. Although the compound was able to form four hydrogen bonds with Tyr57, Ser59, Asn76, and His139, an unfavorable interaction occurred between the 1-methyl-1H-pyrazol-4-yl group and Arg138. Moreover, the results indicated that all poses with low binding energies (below –7.0 kcal/mol) were located outside the original ligand’s binding site. In contrast, all poses of the active compounds were confined to the same binding site. Figure 4 illustrates a comparison of the binding locations of the original ligand, compound 20, compound 29, and compound 30. Detailed 3D representations of all poses suggested by the program are provided in the Supplementary Materials.

3. Materials and Methods

3.1. General Methods

Commercially available reagents were used as obtained from suppliers (Novochem Co., Ltd., 1089 Budapest, Hungary, Orczy út 6.; Merck Ltd., Budapest, Hungary; and VWR International Ltd., Debrecen, Hungary), while solvents were dried according to standard procedures. Chromatographic separations and monitoring of reactions were carried out on Merck Kieselgel 60 (Merck Ltd., Budapest, Hungary). Optical rotations were measured in MeOH at 20 °C with a PerkinElmer 341 polarimeter (PerkinElmer Inc., Shelton, CT, USA). Melting points were determined with a Kofler apparatus (Nagema, Dresden, Germany). HRMS flow injection analysis was performed with a Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer coupled to a Waters Acquity I-Class UPLC™ (Waters, Manchester, UK). 1H-, 13C J-MOD-, and 19F-NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer (Bruker Biospin, Karlsruhe, Baden-Württemberg, Germany) [500 MHz (1H), 125 MHz (13C J-MOD), and 470 MHz (19F) δ = 0 (TMS)]. Chemical shifts are expressed in ppm (δ) relative to TMS as an internal reference. J values are given in Hz. All 1H-, 13C, J-MOD-, 19F-NMR, COSY, NOESY, 2D-HMBC, and 2D-HMQC spectra are available in the Supporting Information file.

3.2. Starting Materials

Natural gibberellin (GA3, I) was obtained from AK Scientific Inc., Union City, CA, USA. The preparation of allo-gibberic acid 1, intermediates 2–4, and azide 5 was accomplished according to the literature methods starting from GA3 with spectroscopic data identical to those reported therein. Compounds 8 and 9 were prepared according to procedures reported in the literature [20,46].

3.3. Synthesis of New Compounds

3.3.1. (7S,8S,9aR,10R)-10-(Azidomethyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (5)

To a solution of 4 (0.91 g, 3.08 mmol) in acetone (15 mL), an aqueous solution of NMO (1.8 mL, 50% aqueous solution) and a solution of OsO4 in tert-BuOH (1.08 mL, 2% solution) were added in one portion. The reaction mixture was stirred at room temperature for 24 h and then quenched by the addition of a saturated aqueous solution of Na2SO3 (25 mL) and extracted with EtOAc (3 × 50 mL). The organic layer was dried over Na2SO4 and evaporated. The crude product was purified by chromatography on silica gel by using EtOAc. Yield: 0.99 g, 98%; white crystals; m.p.: 161–163 °C; [α]D20 = –7.933 (c 0.3 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 1.23 (d, J = 14.8 Hz, 1H), 1.59–1.50 (m, 3H), 1.91 (dd, J = 6.2, 13.5 Hz, 1H), 2.01 (dt, J = 6.0, 13.1 Hz, 1H), 2.07 (t, J = 5.8 Hz, 1H), 2.32 (ddd, J = 6.3, 6.3, 13.1 Hz, 1H), 2.37 (s, 3H), 2.57 (s, 1H), 2.76–2.69 (m, 2H), 3.25 (t, J = 6.6 Hz, 1H), 3.28 (s, 1H), 3.59 (dd, J = 6.3, 11.3 Hz, 1H), 3.72 (dd, J = 7.3, 12.8 Hz, 1H), 3.83 (dd, J = 5.0, 11.4 Hz, 1H), 4.10 (dd, J = 6.0, 12.8 Hz, 1H), 6.89 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 20.6, 22.2, 33.5, 35.3, 45.8, 48.2, 50.9, 51.4, 53.7, 64.8, 78.0, 80.6, 120.1, 127.0, 129.73, 134.0, 140.6, 144.7. HRMS (ESI): m/z calcd. for C36H47N6O6 [2M + H]+: 659.3557. Found: 659.3552.

3.3.2. General Procedure for Preparing the N-Propargyl-Substituted Diaminopyrimidine Derivatives

In total, 1.0 mmol of 8 or 9 (obtained according to the literature method [20]) was dissolved in dry EtOH (8 mL), and the appropriate amine (1 eq.) was added to the solution. The reaction mixture was irradiated in a microwave reactor (200 W, 100 °C, 17 bar) for 2 h. After the completion of the reaction and cooling, the crude product was filtered off (14 and 15) or the mixture was evaporated, and the crude product was purified by column chromatography on silica gel (16).

Methyl 4-((5-Fluoro-4-(prop-2-yn-1-ylamino)pyrimidin-2-yl)amino)benzoate (14)

The reaction was accomplished starting from 8 (0.4 g, 1.0 mmol) and methyl 4-aminobenzoate (0.165 g, 1 mmol). The product precipitating in EtOH was obtained by filtration. Yield: 0.18 g, 28%; white crystals; m.p.: 212–214 °C; 1H NMR (500 MHz, DMSO-d6) δ (ppm): 3.14 (s, 1H), 3.80 (s, 3H), 4.17 (dd, J = 1.9, 5.6 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 8.01–7.95 (m, 2H), 9.62 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 29.8, 52.1, 73.2, 81.8, 117.6 (2C), 121.5, 130.5 (2C), 139.7 (d, J = 18.7 Hz), 141.8 (d, J = 246.5 Hz), 146.2, 152.4 (d, J = 12.5 Hz), 155.6 (d, J = 2.9 Hz), 166.6; 19F-NMR (470 MHz, DMSO-d6) δ (ppm): −167.8; HRMS (ESI): m/z calcd. for C15H14FN4O2 [M + H]+: 301.1101. Found: 301.1091.

Methyl 4-((5-Chloro-4-(prop-2-yn-1-ylamino)pyrimidin-2-yl)amino)benzoate (15)

The reaction was accomplished starting from 9 (0.202 g, 1.0 mmol) and methyl 4-aminobenzoate (0.165 g, 1 mmol). The product precipitating in EtOH was filtrated off as pure product. Yield: 0.257 g, 81%; white crystals; m.p.: 228–231 °C; 1H NMR (500 MHz, DMSO) δ (ppm): 3.12 (s, 1H), 3.81 (s, 3H), 4.17 (dd, J = 1.7, 5.6 Hz, 2H), 7.72 (t, J = 5.6 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 8.07 (s, 1H), 9.75 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 30.4, 52.1, 72.9, 81.9, 105.2, 118.1 (2C), 121.9, 130.4 (2C), 145.8, 153.9, 157.6, 157.9, 166.5; HRMS (ESI): m/z calcd. for C15H14ClN4O2 [M + H]+: 317.0805. Found: 317.0794.

5-Fluoro-N2-(4-Morpholinophenyl)-N4-(prop-2-yn-1-yl)pyrimidine-2,4-diamine (16)

The reaction was accomplished starting from 8 (0.40 g, 1.0 mmol) and 4-morpholino aniline (0.178 g, 1.0 mmol). The product was purified by column chromatography on silica gel with DCM/MeOH (39:1). Yield: 0.37 g, 52%; white crystals; m.p.: 161–164 °C; 1H NMR (500 MHz, CDCl3) δ (ppm): 2.28 (s, 1H), 3.11 (dd, J = 4.6, 4.6 Hz, 4H), 3.86 (dd, J = 4.6, 4.6 Hz, 4H), 4.26 (dd, J = 2.2, 5.4 Hz, 2H), 5.09 (s, 1H), 6.80 (s, 1H), 6.89 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 2.7 Hz, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 30.2, 50.2 (2C), 67.0 (2C), 71.6, 79.8, 116.6 (2C), 120.7 (2C), 133.0, 139.6 (d, J = 19.4 Hz), 141,3 (d, J = 244.5 Hz), 146.9, 151.7 (d, J = 12.0 Hz), 156.1 (d, J = 2.8 Hz); 19F-NMR (470 MHz, DMSO-d6) δ (ppm): −169.9; HRMS (ESI): m/z calcd. for C17H19FN5O [M + H]+: 328.1574. Found: 328.1563.

3.3.3. General Procedure for Preparation of 1,2,3-Triazols by Click Reaction

To a solution of azide 4 or 5 (0.304 mmol) in a mixture of tert-BuOH/H2O (2:1, 12 mL), THF/H2O (2:1, 12 mL), or i-PrOH/H2O (2:1, 12 mL), Cu(OAc)2·H2O (0.1 eq.), sodium ascorbate (0.1 eq.), and the appropriate acetylene derivative 17–34 (0.334 mmol, 1.1 eq.) were added. The mixture was stirred for 48 h at 45 °C. In the case of crystalline product separation, the crude product was obtained by filtration. In other cases, the organic solvent was evaporated, and the residue was dissolved in water (10 mL) and then extracted with DCM or CHCl3 (3 × 50 mL). The organic phase was dried over Na2SO4 and evaporated at low pressure, and the crude product was purified by column chromatography on silica gel.

(7S,9aS,10R)-10-((4-(((2-Chloro-5-fluoropyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (17)

The reaction was accomplished starting from 4 and alkyne 8 in tert-BuOH/H2O (2:1), and the product was purified by column chromatography on silica gel with DCM/MeOH (19:1). Yield: 0.061 g, 42%; white crystals; m.p.: 104–107 °C; [α]D20 = –87.1 (c 0.28 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.39 (dd, J = 1.9, 10.2 Hz, 1H), 1.40 (dd, J = 1.9, 10.2 Hz, 1H), 1.51 (dq, J = 5.4, 12.8 Hz, 1H), 1.65 (s, 1H), 1.82 (dt, J = 5.2, 12.2 Hz, 1H), 1.93 (dd, J = 1.6, 17.0 Hz, 1H), 2.19 (ddd, J = 5.9, 5.9, 12.5 Hz, 1H), 2.38 (s, 3H), 2.61 (d, J = 17.1 Hz, 1H), 2.68 (dd, J = 5.0, 12.4 Hz, 1H), 3.84 (dd, J = 4.9, 11.5 Hz, 1H), 4.82–4.70 (4H, m), 4.93 (s, 1H), 5.32 (dd, J = 5.1, 14.0 Hz, 1H), 6.49 (s, 1H), 6.95 (d, J = 7.4 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.77 (s, 1H), 7.89 (d, J = 2.6 Hz, 1H); 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.4, 21.9, 30.7, 35.9, 38.8, 47.8, 49.3, 49.6, 51.6, 53.2, 79.6, 104.1, 120.5, 123.1, 127.4, 129.9, 133.3, 139.0, 140.0 (d, J = 19.6 Hz), 144.2, 144.4, 145.1, 146.5, 153.3 (d, J = 12.7 Hz), 153.6; 19F-NMR (470 MHz, CDCl3) δ (ppm): −158.8; HRMS (ESI): m/z calcd. for C25H27ClFN6O [M + H]+: 481.1919. Found: 481.1905.

(7S,9aS,10R)-10-((4-(((2,5-Dichloropyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (18)

The reaction was accomplished starting from 4 and alkyne 9 in tert-BuOH/H2O (2:1). Purification with column chromatography on silica gel with DCM/MeOH gave compound 18. Yield: 0.050 g, 33%; white crystals; m.p.: 111–114 °C; [α]D20 = –72.87 (c 0.26 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.42 (dd, J = 2.0, 10.1 Hz, 1H), 1.43 (dd, J = 1.9, 10.3 Hz, 1H), 1.51 (dq, J = 5.3, 12.8 Hz, 1H), 1.64 (d, J = 12.2 Hz, 1H), 1.83 (dt, J = 5.2, 12.3 Hz, 1H), 1.93 (d, J = 17.1 Hz, 1H), 2.19 (ddd, J = 5.8, 5.8, 12.5 Hz, 1H), 2.38 (s, 3H), 2.58 (d, J = 17.2 Hz, 1H), 2.68 (dd, J = 4.9, 12.5 Hz, 1H), 3.86 (dd, J = 5.0, 11.3 Hz, 1H), 4.84–4.69 (4H, m), 4.93 (s, 1H), 5.31 (dd, J = 5.1, 14.1 Hz, 1H), 6.49 (t, J = 5.7 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.97 (d, J = 7.4 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.76 (s, 1H), 8.05 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.3, 21.9, 30.8, 36.4, 38.8, 47.9, 49.3, 49.5, 51.5, 53.3, 79.6, 104.1, 113.7, 120.5, 123.1, 127.3, 129.9, 133.3, 139.1, 144.1, 145.1, 153.7, 153.9, 158.1, 158.5; HRMS (ESI): m/z calcd. for C25H27Cl2N6O [M + H]+: 497.1623. Found: 497.1616.

(7S,9aS,10R)-10-((4-(((5-Fluoro-2-((4-(trifluoromethyl)phenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (19)

The reaction was accomplished starting from 4 and alkyne 10 in tert-BuOH/H2O (2:1). Purification was performed by column chromatography on silica gel with DCM/MeOH (39:1). Yield: 0.055 g, 30%; pink crystals; m.p.: 123–125 °C; [α]D20 = –74.4 (c 0.33 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.53 (dd, J = 2.0, 10.3 Hz, 1H), 1.43 (dd, J = 1.9, 10.2 Hz, 1H), 1.55–1.46 (1H, m), 1.73–1.59 (1H, m), 1.86–1.78 (1H, m), 1.91 (d, J = 17.3 Hz, 1H), 2.22–2.14 (1H, m), 2.32 (s, 3H), 2.56 (d, J = 17.1 Hz, 1H), 2.64 (dd, J = 4.9, 12.3 Hz, 1H), 3.77 (dd, J = 5.1, 11.0 Hz, 1H), 4.67 (dd, J = 11.2, 14.0 Hz, 1H), 4.87–4.75 (3H, m), 4.94 (s, 1H), 5.25 (dd, J = 5.1, 14.1 Hz, 1H), 5.84 (s, 1H), 6.93 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.21 (s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.55 (s, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.84 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.2, 21.9, 30.8, 36.2, 38.9, 47.9, 49.2, 49.4, 51.5, 53.3, 79.7, 104.0, 117.9 (2C), 120.4, 122.0, 123.5, 126.1 (q, J = 3.7 Hz, 2C), 127.3, 129.9, 133.2, 139.0, 139.1 (d, J = 20.3 Hz), 141.8 (d, J = 246.9 Hz), 143.2, 145.0, 145.1 (2C), 152.3 (d, J = 12 Hz), 153.7, 155.1 (d, J = 3.6 Hz); 19F-NMR (470 MHz, CDCl3) δ (ppm): −166.8, −61.6; HRMS (ESI): m/z calcd. for C32H32F4N7O [M + H]+: 606.2604. Found: 606.2589.

(7S,9aS,10R)-10-((4-(((5-Chloro-2-((4-(trifluoromethyl)phenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (20)

The reaction was accomplished starting from 4 and alkyne 11 in tert-BuOH/H2O (2:1). Purification was carried out with column chromatography on silica gel with DCM/MeOH (39:1). Yield: 0.112 g, 59%; white crystals; m.p.: 128–130 °C; [α]D20 = –110.67 (c 0.26 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.53 (dd, J = 2.1, 10.2 Hz, 1H), 1.44 (dd, J = 1.9, 10.1 Hz, 1H), 1.49 (dq, J = 5.4, 12.7 Hz, 1H), 1.69–1.65 (1H, m), 1.82 (dt, J = 5.2, 12.3 Hz, 1H), 1.91 (dd, J = 1.8, 17.3 Hz, 1H), 2.18 (ddd, J = 5.8, 5.8, 12.6 Hz, 1H), 2.32 (s, 3H), 2.54 (d, J = 17.2 Hz, 1H), 2.64 (dd, J = 4.9, 12.3 Hz, 1H), 3.77 (dd, J = 5.0, 11.1 Hz, 1H), 4.65 (dd, J = 11.0, 14.1 Hz, 1H), 4.76 (s, 1H), 4.82 (dq, J = 5.9, 18.6 Hz, 2H), 4.94 (s, 1H), 5.24 (dd, J = 5.2, 14.1 Hz, 1H), 6.01 (t, J = 5.7 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 6.94 (d, J = 7.3 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 7.22 (s, 1H), 7.53 (s, 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.98 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.2, 21.9, 30.8, 36.7, 38.8, 48.0, 49.2, 49.4, 51.5, 53.3, 79.7, 104.0, 106.2, 118.4 (2C), 120.4, 122.1 (2C), 123.9, 126.1 (q, J = 3.8 Hz, 2C), 127.3, 129.9, 133.3, 139.1, 142.8, 145.0, 145.2, 153.3, 153.8, 157.5, 157.7; 19F-NMR (470 MHz, CDCl3) δ (ppm): −61.7; HRMS (ESI): m/z calcd. for C32H32ClF3N7O [M + H]+: 622.2309. Found: 622.2293.

(7S,9aS,10R)-10-((4-(((5-Fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (21)

The reaction was accomplished starting from 4 (0.12 g, 0.406 mmol), alkyne 12, and tert-BuOH and water (2:1, 18 mL). Purification with silica gel with DCM/MeOH (19:1) gave compound 21. Yield: 0.099 g, 45%; white crystals; m.p.: 138–141 °C; [α]D20 = –78 (c 0.28 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.53 (dd, J = 1.6, 10.3 Hz, 1H), 1.44 (dd, J = 2.0, 10.3 Hz, 1H), 1.51 (dq, J = 5.3, 12.7 Hz, 1H), 1.65 (dd, J = 5.1, 11.7 Hz, 1H), 1.82 (dt, J = 5.2, 12.4 Hz, 1H), 1.91 (d, J = 17.2 Hz, 1H), 2.18 (ddd, J = 5.7, 5.7, 12.4 Hz, 1H), 2.36 (s, 3H), 2.58 (d, J = 17.2 Hz, 1H), 2.65 (dd, J = 4.9, 12.3 Hz, 1H), 3.81 (dd, J = 4.8, 11.4 Hz, 1H), 3.86 (s, 3H), 4.67 (dd, J = 11.6, 13.9 Hz, 1H), 4.84–4.71 (3H, m), 4.94 (s, 1H), 5.28 (dd, J = 5.0, 14.1 Hz, 1H), 5.73 (t, J = 5.8 Hz, 1H), 6.83 (s, 1H), 6.93 (d, J = 7.4 Hz, 2H), 6.96 (d, J = 7.4 Hz, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.50 (s, 1H), 7.55 (s, 1H), 7.70 (s, 1H), 7.79 (d, J = 3.1 Hz, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.3, 21.9, 30.8, 36.3, 38.9, 39.2, 48.1, 49.2, 49.4, 51.5, 53.3, 79.6, 104.0, 120.4, 121.1, 122.0, 123.4, 127.3, 129.9, 130.9, 133.3, 139.0, 139.1 (d, J = 19.5 Hz), 141.2 (d, J = 243 Hz), 145.1, 145.7, 152.5 (d, J = 12.7 Hz), 153.8, 155.7 (d, J = 2.5 Hz); 19F-NMR (470 MHz, CDCl3) δ (ppm): −169.8; HRMS (ESI): m/z calcd. for C29H33FN9O [M + H]+: 542.2792. Found: 542.2776.

(7S,9aS,10R)-10-((4-(((5-Chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (22)

The reaction was accomplished starting from 4 and alkyne 13 in tert-BuOH/H2O (2:1). Column chromatography on silica gel with DCM/MeOH (19:1) was used for purification. Yield: 0.080 g, 47%; white crystals; m.p.: 165–170 °C; [α]D20 = –71.6 (c 0.28 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.53 (d, J = 10.3 Hz, 1H), 1.45 (dd, J = 1.9, 10.2 Hz, 1H), 1.55–1.47 (1H, m), 1.65 (dd, J = 5.0, 12.0 Hz, 1H), 1.82 (dt, J = 5.3, 12.2 Hz, 1H), 1.91 (d, J = 17.2 Hz, 1H), 2.21–2.14 (1H, m), 2.36 (s, 3H), 2.57 (d, J = 17.2 Hz, 1H), 2.65 (dd, J = 4.9, 12.4 Hz, 1H), 3.81 (dd, J = 4.9, 11.3 Hz, 1H), 3.86 (s, 3H), 4.66 (dd, J = 11.7, 13.9 Hz, 1H), 4.85–4.72 (3H, m), 4.94 (s, 1H), 5.27 (dd, J = 5.0, 14.1 Hz, 1H), 5.92 (t, J = 5.8 Hz, 1H), 6.89 (s, 1H), 6.93 (d, J = 7.4 Hz, 1H), 6.96 (d, J = 7.3 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.51 (s, 1H), 7.52 (s, 1H), 7.68 (s, 1H), 7.91 (s, 1H); 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.3, 21.9, 29.7, 30.8, 36.8, 39.2, 48.1, 49.2, 49.4, 51.5, 53.3, 79.6, 103.9, 120.4, 121.4, 122.0, 122.9, 127.3, 129.9, 131.1, 133.3, 139.1, 145.1 (2C), 145.8, 153.4, 153.8, 157.8, 157.9; HRMS (ESI): m/z calcd. for C29H33ClN9O [M + H]+: 558.2497. Found: 558.2486.

Methyl 4-((5-Fluoro-4-(((1-(((7S,9aS,10R)-7-hydroxy-1-methyl-8-methylene-5,6,7,8,9,10-hexahydro-4bH-7,9a-methanobenzo[a]azulen-10-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)pyrimidin-2-yl)amino)benzoate (23)

The reaction was accomplished starting from 4 and alkyne 14 in tert-BuOH/H2O (2:1). Compound 28 was purified by column chromatography on silica gel with DCM/MeOH (19:1). Yield: 0.143 g, 79%; pale pink crystals; m.p.: 140–144 °C; [α]D20 = –89.3 (c 0.15 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.54 (d, J = 10.2 Hz, 1H), 1.43 (d, J = 10.2 Hz, 1H), 1.50 (dq, J = 5.3, 12.8 Hz, 1H), 1.65 (dd, J = 4.8, 11.9 Hz, 1H), 1.82 (dt, J = 5.2, 12.3 Hz, 1H), 1.90 (d, J = 17.0 Hz, 1H), 2.21–2.13 (m, 1H), 2.31 (s, 3H), 2.54 (d, J = 17.1 Hz, 1H), 2.63 (dd, J = 4.8, 12.4 Hz, 1H), 3.75–3.69 (m, 1H), 3.85 (s, 3H), 4.65 (dd, J = 11.2, 14.0 Hz, 1H), 4.87–4.73 (m, 3H), 4.95 (s, 1H), 5.22 (dd, J = 5.1, 14.1 Hz, 1H), 5.92 (t, J = 6.1 Hz, 1H), 6.92 (d, J = 7.5 Hz, 2H), 6.94 (d, J = 7.5 Hz, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.34 (s, 1H), 7.57 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.2, 21.9, 30.8, 36.2, 38.9, 48.0, 49.2, 49.4, 51.5, 51.8, 53.3, 79.6, 104.0, 117.5 (2C), 120.4, 122.3, 123.1, 127.3, 129.9, 130.8 (2C), 133.3, 139.1, 139.2 (d, J = 19.5 Hz), 141.8 (d, J = 247.5 Hz), 144.5, 145.1 (2C), 152.4 (d, J = 12.0 Hz), 153.7, 155.0 (d, J = 2.8 Hz), 166.8; 19F-NMR (470 MHz, CDCl3) δ (ppm): −166.7; HRMS (ESI): m/z calcd. for C33H35FN7O3 [M + H]+: 596.2785. Found: 596.2770.

Methyl 4-((5-Chloro-4-(((1-(((7S,9aS,10R)-7-hydroxy-1-methyl-8-methylene-4b,6,7,8,9,10-hexahydro-5H-7,9a-methanobenzo[a]azulen-10-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)pyrimidin-2-yl)amino)benzoate (24)

The reaction was accomplished starting from 4 and alkyne 15 in i-PrOH/H2O (2:1). Compound 24 was purified by column chromatography on silica gel with DCM/MeOH (19:1). Yield: 0.066 g, 36%; pale yellow crystals; m.p.: 135–138 °C; [α]D20 = –81.7 (c 0.17 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.54 (d, J = 10.3 Hz, 1H), 1.43 (d, J = 10.4 Hz, 1H), 1.50 (dq, J = 5.3, 12.8 Hz, 1H), 1.64 (dd, J = 5.2, 12.2 Hz, 1H), 1.82 (dt, J = 5.3, 12.3 Hz, 1H), 1.90 (d, J = 17.1 Hz, 1H), 2.21–2.14 (m, 1H), 2.31 (s, 3H), 2.52 (d, J = 17.2 Hz, 1H), 2.62 (dd, J = 5.0, 12.4 Hz, 1H), 3.72 (dd, J = 5.1, 10.9 Hz, 1H), 3.86 (s, 3H), 4.63 (dd, J = 11.1, 14.0 Hz, 1H), 4.90–4.74 (m, 3H), 4.94 (s, 1H), 5.21 (dd, J = 5.1, 14.1 Hz, 1H), 6.08 (t, J = 5.8 Hz, 1H), 6.92 (d, J = 7.4 Hz, 2H), 6.94 (d, J = 7.4 Hz, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.38 (s, 1H), 7.54 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.96 (s, 1H), 7.99 (d, J = 8.5 Hz, 2H); 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.2, 21.9, 30.8, 36.7, 38.9, 48.0, 49.2, 49.4, 51.5, 51.8, 53.3, 79.6, 104.0, 117.9 (2C), 120.4, 122.2, 123.4, 127.3, 129.9, 130.8 (2C), 133.3, 139.1 (2C), 144.0, 145.0 (2C), 153.1, 153.7, 157.3, 157.7, 166.7; HRMS (ESI): m/z calcd. for C33H35ClN7O3 [M + H]+: 612.2490. Found: 612.2478.

(7S,9aS,10R)-10-((4-(((5-Fluoro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-8-methylene-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulen-7-ol (25)

The reaction was accomplished starting from 4 and alkyne 16 in tert-BuOH/H2O (2:1). Purification by column chromatography on silica gel with DCM/MeOH (19:1) delivered compound 25. Yield: 0.172 g, 91%; pale yellow crystals; m.p.: 139–141 °C; [α]D20 = –87.41 (c 0.17 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.44 (d, J = 10.2 Hz, 1H), 1.36 (d, J = 10.2 Hz, 1H), 1.50 (dq, J = 5.3, 12.7 Hz, 1H), 1.68–1.59 (m, 1H), 1.81 (dt, J = 5.2, 12.2 Hz, 1H), 1.89 (d, J = 17.2 Hz, 1H), 2.21–2.14 (m, 1H), 2.33 (s, 3H), 2.57 (d, J = 17.2 Hz, 1H), 2.66 (dd, J = 4.7, 12.4 Hz, 1H), 3.08–2.97 (m, 4H), 3.64 (dd, J = 4.9, 11.1 Hz, 1H), 3.82–3.73 (m, 4H), 4.68–4.58 (m, 2H), 4.80–4.74 (m, 2H), 4.94 (s, 1H), 5.15 (dd, J = 4.9, 14.0 Hz, 1H), 5.79 (t, J = 6.0 Hz, 1H), 6.78 (s, 1H), 6.88 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 7.5 Hz, 2H), 6.95 (d, J = 7.4 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 7.40 (s, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.77 (s, 1H); 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.3, 21.9, 30.8, 36.0, 38.9, 47.9, 49.3 (2C), 50.0 (2C), 51.6, 53.2, 66.8 (2C), 79.6, 104.0, 116.4 (2C), 120.4, 121.8 (2C), 122.9, 127.3, 129.9, 133.0, 133.3, 139.2, 139.5 (d, J = 19.3 Hz), 141.4 (d, J = 244.5 Hz), 145.1, 145.3, 147.2, 152.2 (d, J = 12.3 Hz), 153.8, 156.3 (d, J = 2.7 Hz); 19F-NMR (470 MHz, CDCl3) δ (ppm): −169.3; HRMS (ESI): m/z calcd. for C35H40FN8O2 [M + H]+: 623.3258. Found: 623.3244.

(7S,8S,9aR,10R)-10-((4-(((2-Chloro-5-fluoropyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (26)

The reaction was accomplished starting from 5 and alkyne 8 in tert-BuOH/H2O (2:1). Compound 26 was purified by column chromatography on silica gel with DCM/MeOH (9:1). Yield: 0.040 g, 51%; white crystals; m.p.: 137–140 °C; [α]D20 = –6.28 (c 0.29 MeOH); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.20–1.12 (m, 2H), 1.53–1.42 (m, 3H), 1.70–1.62 (m, 2H), 2.15 (s, 3H), 2.20–2.17 (m, 1H), 2.63 (dd, J = 5.1, 11.8 Hz, 1H), 3.35 (d, J = 5.8 Hz, 2H), 3.69 (s, 1H), 3.86 (t, J = 6.9 Hz, 1H), 4.08 (s, 1H), 4.26 (t, J = 5.7 Hz, 1H), 4.69–4.54 (m, 3H), 5.16 (dd, J = 6.3, 14.3 Hz, 1H), 6.89 (d, J = 7.6 Hz, 2H), 6.91 (d, J = 7.5 Hz, 2H), 7.05 (t, J = 7.5 Hz, 1H), 8.12–8.10 (m, 2H), 8.65 (t, J = 5.7 Hz, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.6, 22.3, 33.4, 35.3, 36.1, 45.5, 48.9 (2C), 51.4, 53.6, 63.4, 78.4, 79.2, 120.6, 124.8, 127.1, 129.8, 133.6, 140.4, 140.9, 143.9, 144.7, 145.7, 153.7 (d, J = 13.00 Hz), 153.9 (d, J = 2.5 Hz). 19F-NMR (470 MHz, DMSO-d6): δ (ppm): −157.1; HRMS (ESI): m/z calcd. for C25H29ClFN6O3 [M + H]+: 515.1974. Found: 515.1960.

(7S,8S,9aR,10R)-10-((4-(((2,5-Dichloropyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (27)

The reaction was accomplished starting from 5 and alkyne 9 in tert-BuOH/H2O (2:1). The obtained crude product was purified by column chromatography on silica gel with toluene/EtOH (9:1). Yield: 0.055 g, 34%; white crystals; m.p.: 240–243 °C; [α]D20 = –3.4 (c 0.26 MeOH); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.20–1.13 (m, 2H), 1.55–1.45 (m, 3H), 1.71–1.63 (m, 2H), 2.14 (s, 3H), 2.20–2.17 (m, 1H), 2.63 (dd, J = 5.7, 11.7 Hz, 1H), 3.36 (d, J = 5.7 Hz, 2H), 3.66 (s, 1H), 3.86 (t, J = 6.7 Hz, 1H), 4.05 (s, 1H), 4.22 (t, J = 5.6 Hz, 1H), 4.57 (dd, J = 7.8, 14.4 Hz, 1H), 4.66 (ddd, J = 6.1, 15.5, 21.9 Hz, 2H), 5.15 (dd, J = 6.3, 14.3 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 8.08 (s, 1H), 8.19 (s, 1H), 8.38 (t, J = 5.9 Hz, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.6, 22.3, 33.4, 35.3, 36.7, 45.5, 48.9 (2C), 51.4, 53.6, 63.4, 78.4, 79.2, 113.5, 120.5, 124.7, 127.1, 129.8, 133.6, 140.9, 144.0, 145.7, 154.4, 157.8, 159.0. HRMS (ESI): m/z calcd. for C25H29Cl2N6O3 [M + H]+: 531.1678. Found: 531.1669.

(7S,8S,9aR,10R)-10-((4-(((5-Fluoro-2-((4-(trifluoromethyl)phenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (28)

The reaction was accomplished starting from 5 and alkyne 10 in tert-BuOH/H2O (2:1) mixture. The obtained crude product was purified by column chromatography on silica gel with toluene/EtOH (9:1). Yield: 0.10 g, 52%; white crystals; m.p.: 146–148 °C; [α]D20 = +3.2 (c 0.27 MeOH); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.19–1.12 (m, 2H), 1.53–1.43 (m, 2H), 1.55 (d, J = 10.2 Hz, 1H), 1.71–1.61 (m, 2H), 2.08 (s, 3H), 2.15 (q, J = 5.6 Hz, 1H), 2.60 (dd, J = 5.5, 11.9 Hz, 1H), 3.36 (d, J = 5.7 Hz, 2H), 3.72 (s, 1H), 3.84 (t, J = 6.8 Hz, 1H), 4.09 (s, 1H), 4.25 (t, J = 5.7 Hz, 1H), 4.55 (dd, J = 7.6, 14.3 Hz, 1H), 4.70 (ddt, J = 5.9, 14.2, 13.6 Hz, 2H), 5.11 (dd, J = 6.4, 14.4 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 7.3 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.97 (d, J = 3.5 Hz, 1H), 8.04 (t, J = 5.7 Hz, 1H), 8.15 (s, 1H), 9.50 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.5, 22.3, 33.5, 35.3, 36.2, 45.4, 48.8, 48.9, 51.4, 53.6, 63.4, 78.4, 79.2, 117.9 (2C), 120.5, 120.5, 120.7, 124.5, 126.0 (d, J = 3.6 Hz, 2C), 127.0, 129.7, 133.5, 139.2 (d, J = 18.8 Hz), 141.0, 141.8 (d, J = 246.1 Hz), 145.1, 145.3, 145.6, 152.4 (d, J = 12.5 Hz), 155.7 (d, 2.8 Hz). 19F-NMR (470 MHz, DMSO-d6) δ (ppm): −165.9, −59.7 HRMS (ESI): m/z calcd. for C32H34F4N7O3 [M + H]+: 640.2659. Found: 640.2642.

(7S,8S,9aR,10R)-10-((4-(((5-Chloro-2-((4-(trifluoromethyl)phenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (29)

The reaction was accomplished starting from 5 and alkyne 11 in tert-BuOH/H2O (2:1). The crude product was purified by column chromatography on silica gel with toluene/EtOH (9:1). Yield: 0.072 g, 36%; white crystals; m.p.: 185–189 °C; [α]D20 = +11.0 (c 0.27 MeOH); 1H NMR (500 MHz, DMSO) δ (ppm): 1.14 (d, J = 10.7 Hz, 1H), 1.17 (d, J = 13.5 Hz, 1H), 1.52–1.43 (m, 2H), 1.57 (d, J = 10.7 Hz, 1H), 1.73–1.64 (m, 2H), 2.06 (s, 3H), 2.19–2.11 (m, 1H), 2.60 (dd, J = 5.5, 11.8 Hz, 1H), 3.69 (s, 1H), 3.82 (t, J = 6.8 Hz, 1H), 4.09 (s, 1H), 4.25 (t, J = 5.6 Hz, 1H), 4.54 (dd, J = 7.5, 14.4 Hz, 1H), 4.72 (ddt, J = 5.9, 13.7, 12.9 Hz, 2H), 5.10 (dd, J = 6.3, 14.4 Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 7.4 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.76 (t, J = 5.8 Hz, 1H), 7.87 (d, J = 8.5 Hz, 2H), 8.03 (s, 1H), 8.11 (s, 1H), 9.59 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.4, 22.3, 33.4, 35.3, 36.8, 45.4, 48.9 (2C), 51.4, 53.6, 63.4, 78.4, 79.2, 105.3, 118.5 (2C), 120.5, 120.9, 121.2, 124.4, 126.0 (d, J = 3.4 Hz, 2C), 127.0, 129.7, 133.5, 140.9, 144.8, 145.2, 145.6, 153.5, 157.8, 158.0; 19F-NMR (470 MHz, DMSO-d6): δ (ppm): −59.79; HRMS (ESI): m/z calcd. for C32H34ClF3N7O3 [M + H]+: 656.2363. Found: 656.2347.

(7S,8S,9aR,10R)-10-((4-(((5-Fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (30)

The reaction was accomplished starting from 5 and alkyne 12 in tert-BuOH/H2O (2:1). Purification was conducted with column chromatography on silica gel with DCM/MeOH (15:1) increased to 100% MeOH. Yield: 0.121 g, 69%; white crystals; m.p.: 200–205 °C; [α]D20 = +5.42 (c 0.29 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.93 (d, J = 10.5 Hz, 1H), 1.19 (d, J = 10.7 Hz, 1H), 1.37 (d, J = 14.8 Hz, 1H), 1.58–1.48 (1H, m), 1.90–1.78 (2H, m), 2.30–2.22 (1H, m), 2.38 (s, 4H), 2.64 (dd, J = 5.5, 11.9 Hz, 1H), 3.55 (d, J = 11.4 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 3.81 (dd, J = 4.9, 11.6 Hz, 1H), 3.85 (s, 3H), 4.61 (dd, J = 11.8, 13.9 Hz, 1H), 4.80 (dt, J = 6.0, 16.1 Hz, 2H), 5.39 (dd, J = 4.8, 14.1 Hz, 1H), 5.77 (t, J = 6.1 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.98 (s, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.69 (s, 1H), 7.78–7.75 (2H, m). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.4, 22.0, 29.6, 32.7, 34.7, 36.3, 39.1, 45.4, 49.5 (2C), 51.7, 53.1, 64.3, 78.6, 79.9, 120.5, 121.1, 123.5, 123.8, 127.3, 130.0, 133.4, 139.1, 139.2 (d, J = 19.4 Hz), 141.2 (d, J = 243.2 Hz) 144.7, 145.4, 152.5 (d, J = 12.0 Hz), 155.8. 19F-NMR (470 MHz, CDCl3) δ (ppm): −170.0; HRMS (ESI): m/z calcd. for C29H35FN9O3 [M + H]+: 576.2847. Found: 576.2832.

(7S,8S,9aR,10R)-10-((4-(((5-Chloro-2-((1-methyl-1′H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (31)

The reaction was accomplished starting from 5 and alkyne 13 in tert-BuOH/H2O (2:1). Column chromatography on silica gel with DCM/MeOH (6:1) gave compound 31. Yield: 0.17 g, 95%; white crystals; m.p.: 166–170 °C; [α]D20 = +9.9 (c 0.19 MeOH); 1H NMR (500 MHz, CDCl3) δ (ppm): 0.95 (d, J = 11.1 Hz, 1H), 1.18 (d, J = 11.1 Hz, 1H), 1.39–1.32 (1H, m), 1.58–1.47 (1H, m), 1.90–1.77 (3H, m), 2.30–2.22 (1H, m), 2.37 (s, 3H), 2.64 (dd, J = 5.5, 11.9 Hz, 1H), 3.55 (d, J = 11.5 Hz, 1H), 3.73 (d, J = 11.5 Hz, 1H), 3.84–3.79 (1H, m), 3.85 (s, 3H), 4.61 (t, J = 13.0 Hz, 1H), 4.81 (d, J = 5.7 Hz, 2H), 5.39 (dd, J = 4.8, 14.0 Hz, 1H), 6.05 (s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.38–7.29 (1H, m), 7.55 (s, 1H), 7.68 (s, 1H), 7.76 (s, 1H), 7.87 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm): 21.4, 22.0, 32.8, 34.7, 36.8, 39.2, 45.4, 49.5 (2C), 51.7, 53.1, 64.3, 78.7, 79.9, 120.5 (2C), 121.4, 122.9, 123.9, 127.3, 130.0, 131.1 (2C), 133.4, 139.2, 144.7 (2C), 157.9 (2C); HRMS (ESI): m/z calcd. for C29H35ClN9O3 [M + H]+: 592.2551. Found: 592.2540.

Methyl 4-((4-(((1-(((7S,8S,9aR,10R)-7,8-Dihydroxy-8-(hydroxymethyl)-1-methyl-4b,6,7,8,9,10-hexahydro-5H-7,9a-methanobenzo[a]azulen-10-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-5-fluoropyrimidin-2-yl)amino)benzoate (32)

The reaction was accomplished starting from 5 and alkyne 14 in THF/H2O (2:1). Purification was conducted with column chromatography on silica gel with DCM/MeOH (19:1). Yield: 0.084 g, 44%; white crystals; m.p.: 251–253 °C; [α]D20 = +11.35 (c 0.13 DMSO); 1H NMR (500 MHz, DMSO) δ (ppm): 1.19–1.12 (m, 2H), 1.53–1.41 (m, 2H), 1.58 (d, J = 10.3 Hz, 1H), 1.73–1.63 (m, 2H), 2.07 (s, 3H), 2.19–2.11 (m, 1H), 2.60 (dd, J = 5.4, 11.9 Hz, 1H), 3.36 (d, J = 5.7 Hz, 2H), 3.71 (s, 1H), 3.83–3.78 (m, 4H), 4.07 (s, 1H), 4.22 (t, J = 5.7 Hz, 1H), 4.53 (dd, J = 7.4, 14.4 Hz, 1H), 4.70 (dq, J = 5.9, 14.0 Hz, 2H), 5.10 (dd, J = 6.3, 14.4 Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 7.4 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 7.80 (s, 4H), 7.96 (d, J = 3.3 Hz, 1H), 8.03 (t, J = 5.8 Hz, 1H), 8.12 (s, 1H), 9.50 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.5, 22.3, 33.5, 35.3, 36.2, 45.4, 48.9 (2C), 51.4, 52.0, 53.6, 63.4, 78.4, 79.3, 117.5 (2C), 120.5, 121.3, 124.5, 127.0, 129.7, 130.4 (2C), 133.6, 139.3 (d, J = 18.9 Hz), 141.0, 141.8 (d, J = 246.5 Hz), 145.1, 145.6, 146.2, 152.4 (d, J = 12.6 Hz), 155.6 (d, J = 2.8 Hz), 166.5; 19F-NMR (470 MHz, DMSO-d6) δ (ppm): −165.6; HRMS (ESI): m/z calcd. for C33H37FN7O5 [M + H]+: 630.2840. Found: 630.2825.

Methyl 4-((5-Chloro-4-(((1-(((7S,8S,9aR,10R)-7,8-dihydroxy-8-(hydroxymethyl)-1-methyl-4b,6,7,8,9,10-hexahydro-5H-7,9a-methanobenzo[a]azulen-10-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)amino)pyrimidin-2-yl)amino)benzoate (33)

The reaction was accomplished starting from 5 and alkyne 15 in tert-BuOH/H2O (2:1). Product 33 precipitated in EtOH was purified by filtration. Yield: 0.084 g, 43%; white crystals; m.p.: 283–285 °C; [α]D20 = +54.1 (c 0.13 DMSO); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.20–1.12 (m, 2H), 1.53–1.41 (m, 2H), 1.60 (d, J = 10.4 Hz, 1H), 1.72–1.65 (m, 2H), 2.05 (s, 3H), 2.19–2.11 (m, 1H), 2.59 (dd, J = 5.4, 11.6 Hz, 1H), 3.36 (d, J = 5.6 Hz, 2H), 3.70 (s, 1H), 3.79 (s, 4H), 4.07 (s, 1H), 4.22 (t, J = 5.5 Hz, 1H), 4.52 (dd, J = 7.2, 14.2 Hz, 1H), 4.72 (dq, J = 5.8, 14.1 Hz, 2H), 5.08 (dd, J = 6.4, 14.4 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.88 (d, J = 7.4 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 7.83–7.74 (m, 5H), 8.04 (s, 1H), 8.09 (s, 1H), 9.63 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.4, 22.4, 33.5, 35.3, 36.8, 45.4, 48.8 (2C), 51.4, 52.1, 53.6, 63.5, 78.4, 79.3, 105.3, 118.1 (2C), 120.5, 121.8, 124.4, 127.0, 129.7, 130.4 (2C), 133.6, 141.0, 145.2, 145.6, 145.8, 153.5, 157.8, 157.9, 166.5; HRMS (ESI): m/z calcd. for C33H37ClN7O5 [M + H]+: 646.2545. Found: 642.2534.

(7S,8S,9aR,10R)-10-((4-(((5-Fluoro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-8-(hydroxymethyl)-1-methyl-4b,5,6,8,9,10-hexahydro-7H-7,9a-methanobenzo[a]azulene-7,8-diol (34)

The reaction was accomplished starting from 5 and alkyne 16 in tert-BuOH/H2O (2:1). The product was isolated after purification by column chromatography on silica gel with DCM/MeOH (19:1 increased to 9:1). Yield: 0.18 g, 92%; pale yellow crystals; m.p.: 121–124 °C; [α]D20 = –3.87 (c 0.18 MeOH); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.20–1.13 (m, 2H), 1.52–1.42 (m, 2H), 1.57 (d, J = 10.3 Hz, 1H), 1.72–1.62 (m, 2H), 2.10 (s, 3H), 2.20–2.12 (m, 1H), 2.62 (dd, J = 5.2, 11.8 Hz, 1H), 2.98–2.94 (m, 4H), 3.36 (d, J = 5.8 Hz, 2H), 3.71–3.67 (m, 5H), 3.80 (t, J = 6.8 Hz, 1H), 4.08 (s, 1H), 4.22 (t, J = 5.8 Hz, 1H), 4.53 (dd, J = 7.5, 14.3 Hz, 1H), 4.65 (dq, J = 5.8, 14.0 Hz, 2H), 5.09 (dd, J = 6.3, 14.3 Hz, 1H), 6.80 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 7.3 Hz, 1H), 6.90 (d, J = 7.3 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.51 (d, J = 8.7 Hz, 2H), 7.77 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 3.5 Hz, 1H), 8.06 (s, 1H), 8.72 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.5, 22.4, 33.5, 35.3, 36.0, 45.4, 48.8 (2C), 49.9 (2C), 53.6, 63.5, 66.6 (2C), 78.5, 79.3, 116.1 (2C), 120.2, 120.5 (2C), 124.6, 127.1, 129.8, 133.6, 134.3, 139.4 (d, J = 18.3 Hz), 141.0, 141,2 (d, J = 243.6 Hz), 145.2, 145.7 (2C), 146.1, 152.2 (d, J = 12.2 Hz), 156.5 (d, J = 2.8). 19F-NMR (470 MHz, DMSO-d6) δ (ppm): −168.5; HRMS (ESI): m/z calcd. for C35H42FN8O4 [M + H]+: 657.3313. Found: 657.3294.

3.4. Antimicrobial Analyses

The synthesized compounds were dissolved in DMSO and diluted with H2O to reach concentration levels of up to 100 μg/mL and 10 μg/mL with a final DMSO content of 1%. Then, the resulting solutions were investigated in a microdilution assay using bacterial and yeast strains including Bacillus subtilis SZMC 0209 (Gram-positive), Staphylococcus aureus SZMC 14,611 (Gram-positive), Escherichia coli SZMC 6271 (Gram-negative), and Pseudomonas aeruginosa SZMC 23,290 (Gram-negative) as well as Candida albicans SZMC 1533 and C. krusei SZMC 1352, respectively, according to the M07-A10 CLSI guideline [39] and our previous work [47,48]. The microbe suspensions were prepared from fresh cultures cultivated overnight in different fermentation broths for both bacteria (10 g/L peptone, 5 g/L NaCl, 5 g/L yeast extract) and yeast (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) at 37 °C, and their concentrations were set to 4 × 105 cells/mL with sterile media. After that, 100 μL of suspension containing the bacterial or yeast cells and 100 μL of the test solutions were dispensed into each well of 96-well plates and incubated for 24 h at 37 °C and 32 °C for bacteria and yeasts, respectively. The blank sample was a mixture of 100 μL broth and 100 μL of a test solution used for background correction, while 100 μL of microbial suspension supplemented with 100 μL of 1% DMSO was applied as a negative control. For the positive control, ampicillin (Sigma, Kawasaki, Japan) or nystatin (Sigma) was applied for bacteria or fungi, respectively, at two final concentration levels (50 μg/mL and 5 μg/mL). The inhibitory effects were calculated as the percentage of the negative control after blank correction using the following formula: inhibitory effect (%) = 100 − ((ODtest − ODblank)/ODnegative) × 100). Here, ODtest represents the OD values of the test solution, ODnegative represents the OD values of the negative control, and ODblank represents the OD values of blank samples.

3.5. General Procedure for Molecular Docking

Protein structures were obtained from the Protein Data Bank (PDB code: 6M1S, resolution 2.25 Å). Protein preparation steps, including the removal of water molecules, addition of polar hydrogens, and assignment of Kollman charges, were carried out using AutoDock Tools 1.5.7 [49]. The energy minimization of the protein structures was performed using Chimera 1.18. The grid box was centered on the co-crystallized native ligand’s coordinates (X = 22.036, Y = 1.330, Z = 41.041) with dimensions of 30 × 30 × 30 Å. Ligand structures were drawn in ChemDraw 16.0, optimized, and prepared using AutoDock Tools. Docking simulations were conducted using AutoDock Vina [40] with the protein kept rigid and the ligand flexible. Validation involved redocking the native co-crystallized ligand, achieving RMSD values below 3.0 Å for the top 5 scored compounds [50]. The docking results were visualized and analyzed using Discovery Studio (version 16.0, BIOVIA, San Diego, CA, USA, Dassault Systèmes), which also generated 3D and 2D interaction diagrams.

4. Conclusions

A series of gibberellic acid-based 2,4-diaminopyrimidines was designed and synthesized from commercially available gibberellic acid. The antimicrobial activity of the prepared compounds was explored in B. subtilis, S. aureus, E. coli, and P. aeruginosa bacteria, as well as in C. krusei and C. albicans fungi. The best diaminopyrimidine derivative 29 with 5-chloro and 2-((4-(trifluoromethyl)phenyl)amino substituents showed reliable antimicrobial activities against both P. aeruginosa and C. krusei and was proven to be an excellent lead molecule for further investigation. Similarly, compound 20 expressed a selective antibacterial effect against P. aeruginosa, while compound 28 was effectively selective against fungus. The P. aeruginosa DNA gyrase inhibitor activity of compounds 20 and 29, as a possible pathway, was examined by molecular interactions between the active compounds and their potential targets using molecular docking studies, which were conducted using the AutoDock Vina program.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ph18020168/s1: Figures S1–S131: 1H, 13C, 19F NMR, COSY, NOESY, HSQC, and HMBC spectra of new compounds; Figures S132–S134 and Tables S1–S4: docking study; Figure S135: 2D-diagram showing interactions between the best pose of compound 30 and P. aeruginosa DNA gyrase (PDB code: 6M1S); Figure S136: 3D presentation showing the binding location and accumulated poses predicted by the software of the original ligand and P. aeruginosa DNA gyrase (PDB code: 6M1S); Figure S137: 3D presentation showing the binding location and accumulated poses predicted by the software of compounds 20 and P. aeruginosa DNA gyrase (PDB code: 6M1S); Figure S138: 3D presentation showing the binding location and accumulated poses predicted by the software of compounds 29 and P. aeruginosa DNA gyrase (PDB code: 6M1S); Figure S139: 3D presentation showing the binding location and accumulated poses predicted by the software of compounds 30 and P. aeruginosa DNA gyrase (PDB code: 6M1S).

Author Contributions

Z.S. and A.S. conceived and designed the experiments; D.D. and N.R.S. performed the experiments, analyzed the data, and wrote the experimental part; Z.S., D.D. and A.S. discussed the results and contributed to writing this paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by financial support from the Hungarian Research Foundation (NKFI K138871). Project no. TKP2021-EGA-32 has been implemented with support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA funding scheme.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We are grateful for financial support from the Hungarian Research Foundation and the Ministry of Innovation and Technology of Hungary. The high-resolution mass spectrometric analysis was performed by Robert Berkecz.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of this manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
MDPIMultidisciplinary Digital Publishing Institute
DOAJDirectory of open access journals
TLAThree-letter acronym
LDLinear dichroism
CuAAC reactionCopper-catalyzed azide–alkyne cycloaddition
DCMDichloromethane
DIPEAN,N-Diisopropylethylamine
DMAP4-(Dimethylamino)pyridine
DMSODimethyl sulfoxide
MW Microwave
NMO4-Methylmorpholine N-oxide
NOENuclear Overhauser effect
SZMCSzeged Microbiology Collection
TEATriethylamine

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Pharmaceuticals 18 00168 sch001
Scheme 1. Synthesis of allo-gibberic acid azides. (i) HCl, 1.2 M, 65 °C, 3 h, 70%. (ii) CH3I, Cs2CO3, MeCN, reflux, 1 h, 92%. (iii) LiAlH4, dry THF, 40 °C, 3 h, 81%. (iv) TsCl, DIPEA, DMAP (cat.), DCM, 40 °C, 24 h, 75%. (v) NaN3, DMF, 80 °C, 5 h, 78%. (vi) OsO4, NMO, acetone, RT, 24 h, 98%.
Scheme 1. Synthesis of allo-gibberic acid azides. (i) HCl, 1.2 M, 65 °C, 3 h, 70%. (ii) CH3I, Cs2CO3, MeCN, reflux, 1 h, 92%. (iii) LiAlH4, dry THF, 40 °C, 3 h, 81%. (iv) TsCl, DIPEA, DMAP (cat.), DCM, 40 °C, 24 h, 75%. (v) NaN3, DMF, 80 °C, 5 h, 78%. (vi) OsO4, NMO, acetone, RT, 24 h, 98%.
Pharmaceuticals 18 00168 sch001
Pharmaceuticals 18 00168 sch002
Scheme 2. Synthesis of 2,4-diaminopyrimidine-derived alkynes. (i) Propargylamine (2 eq.), TEA, MeCN, 25 °C, 24 h, 8 (65%), 9 (88%). (ii) (a) Trifluoromethylaniline (1.1 eq.), EtOH, MW, 150 °C, 200 W, 19 bar, 2 h, 10 (58%), 11 (52%); (b) 1-methyl-1H-pyrazol-4-amine (1.1 eq.), EtOH, MW, 150 °C, 200 W, 19 bar, 2 h, 12 (91%), 13 (50%); (c) methyl 4-aminobenzoate (1 eq.), EtOH, MW, 100 °C, 200 W, 17 bar, 2 h, 14 (28%), 15 (81%); (d) 4-morpholinoaniline (1 eq.), EtOH, MW, 100 °C, 200 W, 17 bar, 2 h, 16 (52%).
Scheme 2. Synthesis of 2,4-diaminopyrimidine-derived alkynes. (i) Propargylamine (2 eq.), TEA, MeCN, 25 °C, 24 h, 8 (65%), 9 (88%). (ii) (a) Trifluoromethylaniline (1.1 eq.), EtOH, MW, 150 °C, 200 W, 19 bar, 2 h, 10 (58%), 11 (52%); (b) 1-methyl-1H-pyrazol-4-amine (1.1 eq.), EtOH, MW, 150 °C, 200 W, 19 bar, 2 h, 12 (91%), 13 (50%); (c) methyl 4-aminobenzoate (1 eq.), EtOH, MW, 100 °C, 200 W, 17 bar, 2 h, 14 (28%), 15 (81%); (d) 4-morpholinoaniline (1 eq.), EtOH, MW, 100 °C, 200 W, 17 bar, 2 h, 16 (52%).
Pharmaceuticals 18 00168 sch002
Pharmaceuticals 18 00168 sch003
Scheme 3. Synthesis of allo-gibberic acid-based monoamino- and diaminopyrimidines. (i) Cu(OAc)2.H2O (5 mol%), Na ascorbate (10 mol%), alkyne (1.1 eq.), 45 °C, 48 h; solvent mixture: tert-BuOH/H2O (2:1) for 17 (42%), 18 (33%), 19 (30%), 20 (59%), 21 (45%), 22 (47%), 24 (36%), 25 (91%), 26 (51%), 27 (34%), 28 (52%), 29 (36%), 30 (69%), 31 (95%), and 34 (92%), THF: H2O (2:1) for 23 (79%), 32 (44%), and 33 (43%), and i-PrOH:H2O (2:1) for 24 (36%).
Scheme 3. Synthesis of allo-gibberic acid-based monoamino- and diaminopyrimidines. (i) Cu(OAc)2.H2O (5 mol%), Na ascorbate (10 mol%), alkyne (1.1 eq.), 45 °C, 48 h; solvent mixture: tert-BuOH/H2O (2:1) for 17 (42%), 18 (33%), 19 (30%), 20 (59%), 21 (45%), 22 (47%), 24 (36%), 25 (91%), 26 (51%), 27 (34%), 28 (52%), 29 (36%), 30 (69%), 31 (95%), and 34 (92%), THF: H2O (2:1) for 23 (79%), 32 (44%), and 33 (43%), and i-PrOH:H2O (2:1) for 24 (36%).
Pharmaceuticals 18 00168 sch003
Pharmaceuticals 18 00168 g001
Figure 1. Relative configuration of azide 5 determined by NOESY experiment.
Figure 1. Relative configuration of azide 5 determined by NOESY experiment.
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Pharmaceuticals 18 00168 g002
Figure 2. (A) Three-dimensional presentation of the complex between a known inhibitor and P. aeruginosa DNA gyrase (PDB code: 6M1S). (B) Two-dimensional presentation of the interactions between a known inhibitor and P. aeruginosa DNA gyrase (PDB code: 6M1S).
Figure 2. (A) Three-dimensional presentation of the complex between a known inhibitor and P. aeruginosa DNA gyrase (PDB code: 6M1S). (B) Two-dimensional presentation of the interactions between a known inhibitor and P. aeruginosa DNA gyrase (PDB code: 6M1S).
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Figure 3. Three-dimensional presentation of (A) interactions between compound 20 and DNA gyrase of P. aeruginosa (PDB code: 6M1S) and (B) interactions between compound 29 and DNA gyrase of P. aeruginosa (PDB code: 6M1S).
Figure 3. Three-dimensional presentation of (A) interactions between compound 20 and DNA gyrase of P. aeruginosa (PDB code: 6M1S) and (B) interactions between compound 29 and DNA gyrase of P. aeruginosa (PDB code: 6M1S).
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Figure 4. Three-dimensional presentation of the binding locations of the original ligand (yellow), compound 20 (green), compound 29 (cyan), and compound 30 (purple).
Figure 4. Three-dimensional presentation of the binding locations of the original ligand (yellow), compound 20 (green), compound 29 (cyan), and compound 30 (purple).
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Table 1. Antimicrobial activities of the synthesized compounds.
Table 1. Antimicrobial activities of the synthesized compounds.
Inhibitory Effect (%) ± RSD (%)
Gram-PositiveGram-NegativeYeast
CompoundConc. (μg/mL)B. subtilisS. aureusE. coliP. aeruginosaC. kruseiC. albicans
Ampicillin5099.7 ± 2.299.1 ± 1.299.1 ± 1.299.1 ± 1.2--
5100.3 ± 0.2100.0 ± 2.1100.0 ± 2.1100.0 ± 2.1--
Nystatine50----100.0 ± 4.6100.0 ± 1.0
5----77.5 ± 6.4100.0 ± 2.0
450100.0 ± 5.9100.0 ± 1.9100.0 ± 2.644.5 ± 1.686.6 ± 22.048.3 ± 5.5
528.7 ± 9.419.8 ± 7.9-18.3 ± 3.895.0 ± 6.332.5 ± 2.5
550-3.2 ± 3.015.4 ± 1.215.8 ± 1.9-4.6 ± 7.1
515.6 ± 5.38.5 ± 4.4-14.9 ± 3.962.1 ± 2.23.9 ± 2.2
175058.7 ± 3.112.3 ± 6.082.6 ± 2.122.8 ± 5.559.3 ± 4.67.7 ± 2.2
569.3 ± 9.445.1 ± 6.742.4 ± 4.850.0 ± 7.741.8 ± 4.0-
1850-43.4 ± 5.3-31.0 ± 5.058.8 ± 9.313.8 ± 3.1
511.7 ± 7.214.2 ± 4.0-49.4 ± 3.050.9 ± 17.810.6 ± 2.2
195082.2 ± 4.850.6 ± 3.318.8 ± 2.319.8 ± 3.846.0 ± 8.5-
5-17.9 ± 1.913.3 ± 1.418.5 ± 2.0--
205023.4 ± 3.726.1 ± 4.617.2 ± 4.981.6 ± 4.212.4 ± 3.04.9 ± 2.9
5-15.8 ± 4.09.5 ± 2.2100.0 ± 1.412.9 ± 2.83.2 ± 3.3
2150-23.6 ± 2.827.2 ± 5.67.6 ± 1.279.2 ± 1.671.8 ± 1.1
5-17.0 ± 4.5-12.0 ± 4.139.6 ± 8.537.3 ± 1.1
2250-9.5 ± 1.932.1 ± 2.4-58.6 ± 3.247.1 ± 0.6
5-2.4 ± 2.5-9.3 ± 8.729.2 ± 10.332.1 ± 0.6
2350--48.1 ± 2.5-57.4 ± 5.749.8 ± 1.1
5-20.1 ± 6.8-4.6 ± 1.920.2 ± 2.321.4 ± 1.3
2450----56.8 ± 9.058.1 ± 1.1
5-5.9 ± 1.1-10.6 ± 1.7-35.3 ± 0.5
2550---10.1 ± 2.242.3 ± 0.727.0 ± 0.3
5-24.1 ± 5.4-3.4 ± 0.7-22.5 ± 0.3
2650--17.5 ± 6.515.5 ± 3.118.5 ± 13.65.2 ± 1.3
5-16.7 ± 6.4-42.3 ± 12.9--
2750--22.7 ± 1.330.2 ± 2.561.3 ± 10.8-
539.5 ± 7.719.8 ± 6.6-21.9 ± 5.5100.0 ± 21.356.3 ± 2.0
285096.5 ± 5.294.7 ± 1.697.5 ± 1.433.6 ± 3.926.7 ± 8.17.6 ± 1.8
5---21.9 ± 2.5100.0 ± 3.056.1 ± 0.7
2950100.7 ± 1.850.1 ± 5.532.4 ± 12.793.2 ± 3.484.0 ± 5.219.1 ± 0.6
5--5.7 ± 5.1100.0 ± 1.443.7 ± 18.9-
3050-17.0 ± 8.3--41.7 ± 2.748.1 ± 0.9
5----17.7 ± 6.534.5 ± 2.3
3150-100.0 ± 8.161.2 ± 3.349.3 ± 2.554.5 ± 4.460.2 ± 0.4
5-17.0 ± 2.827.5 ± 13.9-27.0 ± 4.218.9 ± 0.5
3250-45.9 ± 6.140.1 ± 7.815.9 ± 0.653.8 ± 2.760.1 ± 2.1
5-2.7 ± 2.212.3 ± 2.17.4 ± 4.929.2 ± 10.328.6 ± 0.8
3350-100.0 ± 2.860.7 ± 1838.4 ± 0.857.0 ± 6.967.3 ± 0.9
5-11.1 ± 4.022.3 ± 1.312.4 ± 3.1-27.0 ± 0.6
3450-21.7 ± 1.418.4 ± 2.1-50.7 ± 3.229.6 ± 0.3
513.1 ± 7.625.8 ± 3.0--26.1 ± 9.431.6 ± 0.3
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Depp, D.; Sebők, N.R.; Szekeres, A.; Szakonyi, Z. Stereoselective Synthesis and Antimicrobial Studies of Allo-Gibberic Acid-Based 2,4-Diaminopyrimidine Chimeras. Pharmaceuticals 2025, 18, 168. https://doi.org/10.3390/ph18020168

AMA Style

Depp D, Sebők NR, Szekeres A, Szakonyi Z. Stereoselective Synthesis and Antimicrobial Studies of Allo-Gibberic Acid-Based 2,4-Diaminopyrimidine Chimeras. Pharmaceuticals. 2025; 18(2):168. https://doi.org/10.3390/ph18020168

Chicago/Turabian Style

Depp, Dima, Noémi Regina Sebők, András Szekeres, and Zsolt Szakonyi. 2025. "Stereoselective Synthesis and Antimicrobial Studies of Allo-Gibberic Acid-Based 2,4-Diaminopyrimidine Chimeras" Pharmaceuticals 18, no. 2: 168. https://doi.org/10.3390/ph18020168

APA Style

Depp, D., Sebők, N. R., Szekeres, A., & Szakonyi, Z. (2025). Stereoselective Synthesis and Antimicrobial Studies of Allo-Gibberic Acid-Based 2,4-Diaminopyrimidine Chimeras. Pharmaceuticals, 18(2), 168. https://doi.org/10.3390/ph18020168

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