ABSTRACT
The lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 μM) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.
INTRODUCTION
The current SARS-CoV-2 pandemic is causing a major crisis in terms of human health and socio-economic losses. Within a period of ~20 years, SARS-CoV-2 is the third zoonotic coronavirus (CoV) to enter the human species, coming after SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome).1 Besides, four human CoVs (i.e. HCoV-229E, -HKU1, -NL63, and -OC43) are endemic in the population and account each year for 15 to 30% of common colds.2 These can evolve into life-threatening lower respiratory tract infections in elderly, children and persons at risk.3, 4 Finally, the Coronaviridae family contains several species causing serious disease in pets and livestock.5
Most young persons infected with SARS-CoV-2 experience no or only mild symptoms of respiratory illness.6 In contrast, in persons with comorbidities or higher age, the viral replication phase is typically followed by a second phase that is characterized by hyperinflammation, acute respiratory distress syndrome and multi-organ failure.7 Hence, management of COVID-19 most likely requires antiviral drugs to suppress initial virus replication, plus anti-inflammatory medication, like corticosteroids, to treat severe cases.8 Several CoV proteins may be suitable drug targets,9, 10 but, at the moment, only two drug classes are far advanced in clinical testing, i.e. anti-spike antibodies11 and the nucleotide analogue remdesivir, which inhibits the CoV polymerase.12-14 Though less explored, the CoV nsp15 endoribonuclease (EndoU) is a highly attractive drug target, since it has no cellular counterpart, its catalytic site is conserved among CoVs, and it is amenable to structure-based design based on available protein structures.15-19 Nsp15 is one of the non-structural proteins (nsps) associated with the replication-transcription complexes (RTCs), the site where CoV RNA synthesis occurs.5, 20 Although the functions of nsp15 are not entirely understood, its EndoU function is known to regulate viral RNA synthesis, limit the recognition of viral dsRNA by cellular sensors and prevent the dsRNA-activated antiviral host cell response.21-25 The concept to inhibit nsp15 is thus unique, since it combines a direct antiviral effect with the potential to revert viral evasion from host cell immunity.
We here report identification of a class of coronavirus nsp15 inhibitors with 1,2,3-triazolo fused betulonic acid structure. We describe their synthesis, structure-activity relationship and the mechanistic findings, in particular resistance data, that corroborate nsp15 as their antiviral target. These biological data accord with the binding model that we obtained by compound docking in the hexameric nsp15 protein structure. The model also explains why the current lead is active against human coronavirus 229E, but not other coronaviruses like SARS-CoV-2. In short, our study validates the nsp15 protein, and particularly the interface where the lead compound binds, as a druggable and pertinent target for developing CoV inhibitors.
RESULTS AND DISCUSSION
Compound synthesis and structure-activity relationship
The 1,2,3-triazolo fused betulonic acid derivatives (Scheme 1) were designed and synthesized in the context of a pharmacological hit discovery program. Betulonic acid bears a pentacyclic triterpenoid core, present in a wide variety of agents with potential pharmacological use,26-28 like the HIV maturation inhibitor bevirimat.29, 30 We decided to fuse betulonic acid with a 1,2,3-triazole moiety, which has the unique property to both accept and donate hydrogen bonds.31-34 These derivatives were synthesized by our recently developed and convenient “triazolization” method to prepare 1,2,3-triazoles from primary amines and ketones.35-38 First, Jones oxidation was performed to convert betulin 1 into betulonic acid 2 (Scheme 1).39 Betulin 1, a natural compound isolated from the bark of Betula species, is commercially available.40-42 Next, the triazolization method was applied to betulonic acid 2 as the ketone source, using primary amines 3 and 4-nitrophenyl azide 4, and the previously reported reaction conditions.13 This yielded a series of sixteen 1,2,3-triazolo fused betulonic acids 5, most of which were isolated at high yield (~80%; Table 1). Diverse primary amines 3 were attached to the 1,2,3-triazole ring to introduce a variety of aromatic or aliphatic moieties.
We next evaluated the compounds in cell-based assays with diverse DNA- and RNA-viruses, and observed strong activity in human embryonic lung (HEL) fibroblast cells infected with HCoV-229E. We used a viral cytopathic effect (CPE) reduction assay, in which protection against CPE (expressed as the antiviral EC50 value) was monitored by the MTS cell viability assay and verified by microscopy. The MTS assay was also used to quantify compound cytotoxicity (expressed as the CC50 value) in mock-infected cells. Whereas the starting compounds betulin 1 and betulonic acid 2 were virtually inactive, all 1,2,3-triazolo fused betulonic acid derivatives proved to be highly effective CoV inhibitors (Table 1). The one exception was 5i (EC50 value: 13 μM), which bears a non-aromatic cyclohexanemethyl substituent. Apparently, introducing this bulky group caused a considerable reduction in antiviral activity and selectivity. Several compounds in the series had EC50 values below 1 μM, which makes them superior to two known CoV inhibitors which we used as reference compounds, i.e. K2243 and GS-441524, the nucleoside form of remdesivir.14 Three analogues stood out for having superior selectivity, i.e. 5n, 5g and 5h, having a selectivity index (ratio of CC50 to EC50) of 27, 31 and 76, respectively. The capacity of 5h to fully suppress HCoV-229E replication at non-toxic concentrations is evident from the microscopic images in Fig. 1A and the dose-response curves in Fig. 1B. Also, 5h fully prevented the formation of dsRNA intermediates of CoV RNA synthesis, as demonstrated by immunofluorescence staining of dsRNA in HCoV-229E-infected human bronchial epithelial 16HBE cells (Fig. 1C).
To conduct a SAR exploration around lead compound 5h (Scheme 2 and Table 2), we first investigated the contribution of the α-methyl-phenylene moiety. Compound 5q, in which this entire moiety is missing, had ~6-fold lower antiviral activity than 5h. When only the α-methyl was missing (5r), the activity was not affected. Compound 5s, which is the epimer at the 1,2,3-triazole substituent, displayed almost the same EC50 value as 5h, indicating that isomerism does not alter the activity. Cytotoxicity was however slightly decreased, resulting in an even better selectivity index (≥ 90) than that of 5h. In order to elucidate the role of the isopropenyl side chain, we reduced this moiety by hydrogenation, yielding compound 5t which was 10- to 20-fold less active. Replacement of the carboxylic acid by a methyl group (5u) proved deleterious.
We next evaluated 5h in cell culture assays with a panel of other CoVs. The compound had no inhibitory effect on mouse hepatitis virus A59 (MHV-A59) and feline infectious peritonitis virus (FIPV), in CPE reduction assays in, respectively, murine fibroblast L2 cells and Crandell-Rees Feline Kidney cells (data not shown). HCoV-229E and FIPV belong to the alpha genus, while MHV-A59 belongs to the beta genus comprising also the highly pathogenic species SARS-CoV-1, MERS-CoV and SARS-CoV-2.44, 45 In VeroE6-eGFP cells infected with SARS-CoV-2, 5h and 5t were inactive [see reference46 for assay description]. Hence, though nicely active against HCoV-229E, 5h appeared, unfortunately, to be confined to this CoV species. Besides, when tested against a broad panel of DNA and RNA viruses, the 1,2,3-triazolo fused betulonic acid derivatives proved inactive against HIV, herpes simplex virus, vaccinia virus, adenovirus, vesicular stomatitis virus, Coxsackie B4 virus, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, Sindbis virus, Punta Toro virus, yellow fever virus and influenza virus (data not shown).
Mechanistic studies establishing nsp15 as the target of 5h
Given the robust activity of the betulonic acid derivatives against HCoV-229E, we used this virus to reveal their mechanism of action and appreciate how their anti-CoV activity spectrum may be expanded. A time-of-addition experiment indicated that 5h acts post-entry at an early stage in viral RNA synthesis, since the molecule started to have reduced activity when added at 6 h p.i. (Fig. 2). For comparison, the action point of the entry inhibitor bafilomycin, an inhibitor of endosomal acidification, was situated before 2 h p.i. K22 was still effective when added as late as 8 h p.i. This CoV inhibitor targets nsp6-dependent anchorage of the viral replication-transcription complexes (RTCs) to host cell-derived double-membrane vesicles.43
Next, we performed two independent virus passaging experiments to select 5h-resistant viruses and identify the viral target. After three cell culture passages under increasing concentrations (up to 40 μM) of 5h, HCoV-229E acquired resistance. Whole virus genome sequencing revealed that this was attributed to two substitutions in nsp15, K60R (first selection) and T66I (second selection), located in the N-terminal part of this protein. For both mutants, 5h exhibited an antiviral EC99 value (= concentration producing 100-fold reduction in virus yield) of >40 μM, which is at least 14-fold higher than the EC99 value of 2.9 μM measured for wild-type virus (Fig. 3A). Both mutant viruses remained fully sensitive to GS-441524. The conclusion that 5h targets nsp15 was corroborated by determining its activity against a reverse-engineered EndoU-deficient H250A-mutant HCoV-229E, which lacks the catalytic His250 residue in the EndoU active site.22 5h proved dramatically less active against this mutant (Fig. 3B), producing a maximal reduction in virus yield of 23-fold compared to 1479-fold for wild-type (WT) virus. Again, GS-441524 proved equally active against nsp15-mutant and wild-type virus. To conclude, we established 5h as an inhibitor of nsp15, and showed that its activity is linked to residues Lys60 and Thr66 in the N-terminal part, plus His250 in the EndoU catalytic site of nsp15. This inhibition of nsp15 accords with the time-of-addition profile of 5h (see above), showing that the compound interferes with an early stage in viral RNA synthesis.
Binding model of 5h in hexameric nsp15 protein
To predict the possible binding site of 5h, the compound was docked into the X-ray structure of hexameric nsp15 protein from HCoV-229E (PDB code: 4RS4). This hexameric structure formed by two trimers is the functional form of nsp15.15 First, a few substitutions (see Experimental section for all details) were introduced to render the protein sequence identical to that of the HCoV-229E virus used in the biological assays. By using a pocket-detection protocol implemented on the Site Finder module of Molecular Operating Environment (MOE)19 software, we identified a druggable binding pocket at the nsp15 dimer interface, surrounded by the catalytic residue His250 in the EndoU active site of one nsp15 monomer and residues Lys60 and Thr66 in the N-terminal domain of the other monomer (Fig. 4A). Next, ligand 5h was placed inside the pocket and docked by using both MOE and GOLD softwares and the common top scoring binding mode was further analyzed.20 This docked result indicates that the carboxylic acid of 5h forms hydrogen bonds with the backbone of residues Cys294 and Thr295 (Fig. 4B). The importance of this interaction is supported by the observation that 5u, the 5h analogue bearing a methyl instead of carboxylic acid group, lacks antiviral activity. Furthermore, the 1,2,3-triazole group of 5h engages in hydrogen-bonding interactions with Thr245, explaining why the parent compounds 1 and 2 are not active against HCoV-229E. At the other side of the pocket, the aromatic ring of 5h makes hydrophobic contacts with Val63 and Leu65. This may explain why nearby mutations K60R and T66I yield resistance to the compound, since these substitutions may negatively affect the interactions of Val63 and Leu65, or disturb the conformation of the loop flanked by both residues.
Analysis of the nsp15 sequence similarity between HCoV-229E and SARS-CoV-2 showed that a few residues in the pocket are not conserved. This explains why docking 5h into the SARS-CoV-2 nsp15 hexamer was unable to identify a similar pose within the top solutions. The largest influence seems attributed to the residue at position 244/245, since substituting Thr245 (present in HCoV-229E) by Gln244 (the corresponding residue in SARS-CoV-2) abrogates a hydrogen-bond interaction with the 1,2,3-triazole group of 5h (Fig. 5). Additionally, the loop between Val/Ile63 and Leu/Pro65 has a slightly different orientation in these two CoV nsp15 proteins. Both factors may explain why 5h is active against HCoV-229E, but not SARS-CoV-2. Still, most of the pocket residues are conserved, underscoring the relevance of this nsp15 interface pocket for drug design. The role of this protein region in forming inter-monomer interactions is evident from reports that nsp15 exists as a monomer when key interactions in this region (i.e. Arg61-Glu266 in SARS-CoV nsp15 and Tyr58-Glu263 in MERS-CoV nsp15) are eliminated by mutation.19, 47 When nsp15 is unable to hexamerize, the EndoU catalytic site undergoes important structural changes that abolish RNA binding and enzymatic activity.48
To conclude, the nsp15 binding mode of 5h, predicted by docking, nicely accords with the biological findings. Namely, the binding model rationalizes the requirement of the 1,2,3-triazolo function and carboxylate substituent; 5h resistance of the nsp15-K60R, -T66I and -H250A mutant viruses; and lack of activity against SARS-CoV-2.
CONCLUSIONS
To conclude, we present the first prototype of CoV nsp15 inhibitors, having a 1,2,3-triazolo fused betulonic acid structure. The SAR analysis, resistance data and docking model provide strong evidence that lead molecule 5h binds to an inter-monomer nsp15 interface lying adjacent to the EndoU catalytic core. This provides an excellent basis to modify the substituents or betulonic acid scaffold, and expand the activity spectrum beyond HCoV-229E. Since 5h appears to interact with the catalytic His250 residue in the EndoU domain of nsp15, the molecule plausibly interferes with the role of EndoU in regulating viral dsRNA synthesis.24 To complement the findings in this report, obtained in non-immune cells, we are currently evaluating the antiviral and immunomodulatory effects of 5h in HCoV-229E-infected human macrophages. This may validate the intriguing concept that nsp15 inhibition could have a dual outcome, by inhibiting CoV replication and promoting host cell antiviral immunity.
EXPERIMENTAL SECTION
Chemistry
1H and 13C NMR spectra were measured on commercial instruments (Bruker Avance 300 MHz, Bruker AMX 400 MHz and 600 MHz). Chemical shifts (δ) are reported in parts per million (ppm) referenced to tetramethylsilane (1H), or the internal (NMR) solvent signal (13C). Melting points were determined using a Reichert Thermovar apparatus. For column chromatography, 70-230 mesh silica 60 (E. M. Merck) was used as the stationary phase. Chemicals received from commercial sources were used without further purification. Reaction dry solvents (toluene, DMF, THF) were used as received from commercial sources. TLC was carried out on Kieselgel 60 F254 plates (Merck).
Exact mass was acquired on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3 μL/min and spectra were obtained in positive (or: negative) ionization mode with a resolution of 15000 (FWHM) using leucine enkephalin as lock mass.
All Liquid Chromatography (LC-MS) data were acquired on Agilent 1100 HPLC with quaternary pump, autosampler, UV-DAD detector and a thermostatic column (25 °C) module coupled to Agilent 6110 single-quadrupole electrospray ionization mass spectrometry (capillary voltage = +3500V or −3000V), detector was set to 210 nm for detection. Injection volume was 10 or 20 μL of a dilution of 100 μg/mL (sample in mobile phase). The column was a Grace Prevail RP-C18 3μm 150mm × 2.1mm. Data collection and analysis was done with Agilent LC/MSD Chemstation software. All tested compounds showed a purity >95.0%.
3-Oxo-lup-20(29)-en-28-oic acid (betulonic acid, 2)
To a solution of betulin (50.0 g, 113 mmol; purchased from Eburon Organics BVBA) in acetone (1500 mL, use ultra-sonic bath to dissolve) was added freshly prepared Jones reagent [Na2Cr2O7, (66.5 g, 226 mmol) and H2SO4 (60 mL) in water (500 mL)] during 1 h in an ice bath. The reaction mixture was allowed to warm to room temperature and stirring was continued for 6 h fellow with TLC. First, MeOH was added and then water to the reaction mixture. Precipitate was filtered off and washed with water (500 mL). The crude product was dried in a vacuum oven, dissolved to Et2O (600 mL) and washed with water (300 mL), 7.5 % hydrochloric acid (200 mL), water (200 mL), saturated aqueous NaHCO3 solution (200 mL) and water (200 mL). The crude reaction mixture was purified by column chromatography (silica gel) whereas eluent was used a mixture of heptane and ethyl acetate 70:30 to afford betulonic acid 23 g 45% yield. Spectroscopic data for betulonic acid was consistent with previously reported data for this compound.25
3-Oxo-lupan-28-oic acid(dihydro-betulonic acid)
Betulonic acid (180 mg, 0.396 mmol, 1.0 equiv.) was dissolved in a mixture of MeOH/THF (2/6 mL). 10% Pd(OH)2 (30 mg) was added under N2 atmosphere. This atmosphere was replaced by H2 atmosphere. The reaction was stirred under H2 atmosphere for 78 h, then filtered through celite and washed with CHCl3 to afford a white solid. The residue obtained was purified by silica gel column chromatography using 100% chloroform as eluent to afford dihydro-betulonic acid (quantitative yield) as a white amorphous powder. The spectra proved the identity of the compound by comparing the data with the literature.26
General Procedure
To a dried screw-capped reaction tube equipped with a magnetic stirring bar was added betulonic acid, amine, 4-nitrophenyl azide, 4 Å molecular sieves (50 mg). The mixture was dissolved in the proper solvent (toluene, DMF) and stirred at 100 °C for 12-72 hours. The reaction was monitored using TLC with the plate first developed with DCM then different ratios petroleum ether/ethyl acetate 7:3, 6:4 were used depending on the substrates. For visualization of TLC plates was used 5% H2SO4 in ethanol, for more sensitive detection was used cerium-ammonium-molybdate after heating to 150–200 °C. The crude reaction mixture was then directly purified by column chromatography (silica gel), at first with CH2Cl2 as an eluent to remove all 4-nitroaniline formed during the reaction, followed by using a mixture of petroleum ether and ethyl acetate as eluent to afford the betulonic acid 1,2,3-triazole derivatives.
1’-(3,4,5-Trimethoxybenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5a)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 3,4,5-trimethoxybenzylamine (56 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5a (122 mg, 84% yield) as off white crystals. m.p. 152 °C. 1H NMR (400 MHz, CDCl3) δ 6.25 (s, 2H), 5.57 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.81 (s, 3H), 3.77 (s, 6H), 3.04 (m, 1H), 2.96 (d, J = 15.4 Hz, 1H), 2.32 – 2.15 (m, 3H), 2.05 – 1.94 (m, 2H), 1.77 – 1.64 (m, 5H), 1.59 – 1.40 (m, 11H), 1.20 (s, 7H), 1.03 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.9, 153.5, 150.2, 141.9, 138.0, 137.4, 132.1, 109.8, 103.5, 60.8, 56.3, 56.1, 54.6, 52.8, 49.3, 49.1, 46.8, 42.4, 40.5, 38.9, 38.5, 37.0, 33.7, 33.3, 32.0, 30.5, 29.8, 29.7, 28.7, 25.5, 21.3, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C40H57N3O5H [M+H]+: 660.4370, found 660.4384.
1’-(3,5-Dimethoxybenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5b)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 3,5-dimethoxybenzylamine (48 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5b (126 mg, 92% yield) as off white crystals. m.p. 159 °C. 1H NMR (400 MHz, CDCl3) δ 6.34 (s, 1H), 6.16 (s, 2H), 5.57 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.71 (s, 6H), 3.08 – 2.99 (m, 1H), 2.95 (d, J = 15.4 Hz, 1H), 2.32 – 2.14 (m, 3H), 2.05 – 1.94 (m, 2H), 1.79 – 1.62 (m, 5H), 1.60 – 1.33 (m, 11H), 1.29 – 1.10 (m, 7H), 1.03 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.6, 161.1, 150.2, 141.8, 138.9, 138.0, 109.8, 104.4, 99.6, 56.4, 55.3, 54.5, 52.8, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.6, 29.8, 28.7, 25.5, 21.3, 21.2, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C39H55N3O4H [M+H]+: 630.4265, found 630.4274.
1’-(Pyridin-4-ylmethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5c)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-(aminomethyl)pyridine (31 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5c (97 mg, 78% yield) as off white crystals. m.p 172 °C. 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 5.6 Hz, 2H), 6.93 (d, J = 5.6 Hz, 2H), 5.66 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.06 (m, 1H), 2.97 (d, J = 15.4 Hz, 1H), 2.36 – 2.15 (m, 3H), 2.06 – 1.95 (m, 2H), 1.83 – 1.64 (m, 5H), 1.60 – 1.37 (m, 11H), 1.32 – 1.21 (m, 7H), 1.16 (s, 3H), 1.01 (m, 3H), 1.00 (m, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.6, 150.4, 149.8, 146.1, 142.3, 138.3, 121.2, 109.7, 56.3, 54.4, 51.6, 49.2, 49.2, 46.9, 42.4, 40.5, 39.0, 38.4, 38.2, 37.0, 33.6, 33.2, 32.1, 30.6, 29.8, 29.6, 28.8, 25.4, 21.5, 21.4, 19.4, 18.8, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C36H50N4O2H [M+H]+: 571.4006, found 571.4013.
1’-(4-Methylbenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5d)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-methylbenzylamine (35 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5d (115 mg, 90% yield) as off white crystals. m.p. 310 °C. 1H NMR (600 MHz, CDCl3) δ 7.09 (d, J = 7.9 Hz, 2H), 6.92 (d, J = 7.9 Hz, 2H), 5.59 (s, br, 2H), 4.75 (s, 1H), 4.63 (s, 1H), 3.03 (m, 1H), 2.95 (d, J = 15.4 Hz, 1H), 2.32 – 2.23 (m, 5H), 2.17 (d, J = 15.4 Hz, 1H), 2.05 – 1.94 (m, 2H), 1.80 – 1.62 (m, 5H), 1.50 (m 11H), 1.22 – 1.09 (m, 7H), 1.03 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.8, 150.2, 141.7, 138.0, 137.4, 133.3, 129.3, 126.3, 109.8, 56.4, 54.5, 52.6, 49.2, 49.1, 46.8, 42.4, 40.8, 40.5, 38.9, 38.4, 38.2, 37.0, 33.7, 33.3, 32.0, 30.5, 29.7, 28.7, 28.4, 25.4, 23.8, 21.3, 21.3, 21.0, 20.8, 19.4, 18.9, 17.5, 17.2, 16.0, 15.6, 14.6. HRMS (ESI+): m/z calculated for C38H53N3O2H [M+H]+: 584.4210, found 584.4217.
1’-(4-Fluorobenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5e)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-fluorobenzylamine (36 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5e (110 mg, 85% yield) as off white crystals. m.p. 309 °C. 1H NMR (400 MHz, CDCl3) δ 7.00 (m, 4H), 5.60 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.08 – 2.99 (m, 1H), 2.95 (d, J = 15.3 Hz, 1H), 2.33 – 2.14 (m, 3H), 2.06 – 1.93 (m, 2H), 1.82 – 1.62 (m, 5H), 1.61 – 1.31 (m, 11H), 1.30 – 1.10 (m, 7H), 1.08 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.1, 163.5, 161.0, 150.2, 142.0, 137.9, 132.2, 132.2, 128.2, 128.1, 115.8, 115.5, 109.8, 56.3, 54.5, 52.1, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.5, 29.8, 28.7, 25.4, 21.3, 21.3, 19.4, 18.8, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C37H50FN3O2H [M+H]+: 588.3959, found 588.3969.
1’-(4-Trifluoromethylbenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5f)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-(trifluoromethyl)benzylamine (50 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5f (112 mg, 80% yield) as off white crystals. m.p. 315 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.69 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.02 (m, 2H), 2.34 – 2.16 (m, 3H), 2.07 – 1.94 (m, 2H), 1.82 – 1.64 (m, 5H), 1.59 – 1.38 (m, 11H), 1.18 (m, 7H), 1.02 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.78 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.4, 150.2, 142.1, 140.5, 138.2, 126.6, 125.7, 125.7, 109.8, 56.4, 54.5, 52.2, 49.3, 49.1, 46.9, 42.4, 40.8, 40.5, 38.9, 38.5, 38.2, 37.0, 33.8, 33.6, 33.3, 32.0, 30.5, 29.8, 29.7, 28.8, 28.4, 25.4, 23.8, 21.4, 21.3, 20.8, 20.5, 19.4, 18.8, 17.5, 17.3, 16.1, 15.7, 14.6, 7.9. HRMS (ESI+): m/z calculated for C38H50F3N3O2H [M+H]+: 638.3927, found 638.3939.
1’-(4-Dimethylaminobenzyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5g)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-(dimethylamino)benzylamine (43 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5g (112 mg, 80% yield) as off white crystals. m.p. 190 °C. 1H NMR (400 MHz, CDCl3) δ 6.97 (d, J = 8.3 Hz, 2H), 6.64 (d, J = 8.3 Hz, 2H), 5.52 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.10 – 2.84 (m, 9H), 2.33 – 2.11 (m, 3H), 1.99 (m, 2H), 1.81 – 1.62 (m, 5H), 1.56 – 1.34 (m, 11H), 1.25 – 1.15 (m, 7H), 1.06 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.76 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.4, 150.2, 150.1, 141.6, 137.7, 127.6, 123.9, 112.5, 109.8, 56.4, 54.6, 52.6, 49.2, 49.1, 46.8, 42.4, 40.5, 40.5, 38.9, 38.9, 38.4, 38.3, 37.0, 33.7, 33.3, 32.1, 30.5, 29.8, 28.7, 25.5, 23.8, 21.3, 21.3, 20.8, 19.4, 18.9, 16.0, 15.6, 14.6. HRMS (ESI+): m/z calculated for C39H56N4O2H [M+H]+: 613.4475, found 613.4480.
1’-(1-Phenylethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5h)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), (S)-(−)-α-methylbenzylamine (35 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5h (107 mg, 84% yield) as off white crystals. m.p. 327 °C. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.21 (m, 3H), 7.13 (d, J = 7.2 Hz, 2H), 5.73 (m, 1H), 4.76 (s, 1H), 4.64 (s, 1H), 3.00 (m, 2H), 2.33 – 2.12 (m, 3H), 2.07 – 1.92 (m, 5H), 1.81 – 1.62 (m, 5H), 1.58 – 1.37 (m, 11H), 1.14 (m, 7H), 1.00 (s, 6H), 0.96 (s, 3H), 0.72 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.6, 150.2, 141.8, 141.1, 137.6, 128.6, 127.5, 126.1, 109.8, 59.1, 56.3, 54.8, 49.3, 49.1, 46.8, 42.4, 40.5, 38.8, 38.4, 37.0, 33.6, 33.3, 32.0, 30.5, 29.7, 29.7, 28.6, 25.4, 23.3, 21.4, 21.3, 19.4, 19.0, 15.9, 15.6, 14.6. HRMS (ESI+): m/z calculated for C38H53N3O2H [M+H]+: 584.4210, found 584.4218.
1’-(Cyclohexylmethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5i)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), cyclohexanemethylamine (33 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5i (103 mg, 82% yield) as off white crystals. m.p. 338 °C. 1H NMR (600 MHz, CDCl3) δ 4.76 (s, 1H), 4.64 (s, 1H), 4.09 (m, 2H), 3.04 (m, 1H), 2.92 (m, 1H), 2.33 – 2.21 (m, 2H), 2.13 (d, J = 15.3 Hz, 1H), 2.07 – 1.95 (m, 2H), 1.79 – 1.64 (m, 10H), 1.61 – 1.34 (m, 11H), 1.32 – 1.19 (m, 10H), 1.18 – 1.08 (m, 5H), 1.00 (s, 3H), 0.99 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.7, 150.2, 140.9, 137.8, 109.8, 56.4, 55.6, 54.7, 49.2, 49.2, 46.9, 42.4, 40.5, 38.8, 38.6, 38.5, 38.2, 37.0, 33.7, 33.3, 32.1, 31.0, 30.6, 29.8, 28.9, 26.3, 25.7, 25.4, 21.5, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C37H57N3O2H [M+H]+: 576.4523, found 576.4529.
1’-(Benzo[d][1,3]dioxol-5-ylmethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5j)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), piperonylamine (43 mg, 1.3 equiv., 0.286 mmol), nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether: EtOAc 9:1 → 6:4) affording 5j (105 mg, 78% yield) as off white crystals. m.p. 313 °C. 1H NMR (300 MHz, CDCl3) δ 6.72 (d, J = 7.9 Hz, 1H), 6.59 – 6.48 (m, 2H), 5.93 (d, J = 0.9 Hz, 2H), 5.53 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.10 – 2.89 (m, 2H), 2.35 – 2.11 (m, 3H), 1.99 (m, 2H), 1.83 – 1.62 (m, 5H), 1.61 – 1.33 (m, 11H), 1.33 – 1.08 (m, 7H), 1.05 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.3, 150.2, 148.1, 147.2, 141.8, 137.9, 130.1, 119.9, 109.8, 108.3, 107.2, 101.1, 56.4, 54.5, 52.6, 50.8, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.5, 29.8, 28.7, 25.4, 21.3, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C38H51N3O4H [M+H]+: 614.3952, found 614.3951.
1’-(Furan-2-ylmethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5k)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), furfurylamine (28 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5k (66 mg, 53% yield) as a brown crystals. m.p. 227 °C. 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 1H), 6.34 – 6.31 (m, 1H), 6.23 (d, J = 3.1 Hz, 1H), 5.55 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.07 – 2.99 (m, 1H), 2.93 (d, J = 15.3 Hz, 1H), 2.30 – 2.11 (m, 3H), 1.99 (d, J = 7.5 Hz, 2H), 1.78 – 1.66 (m, 5H), 1.60 – 1.40 (m, 11H), 1.28 (t, J = 10.2 Hz, 7H), 1.15 (s, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 179.9, 150.2, 148.8, 142.5, 141.6, 137.7, 110.7, 109.9, 109.0, 56.3, 54.6, 49.3, 49.2, 46.8, 46.4, 42.4, 40.6, 39.0, 38.4, 38.3, 37.0, 33.6, 33.3, 32.0, 30.5, 29.8, 29.7, 28.6, 19.4, 18.9, 16.1, 15.7, 14.6. HRMS (ESI+): m/z calculated for C35H49N3O3H [M+H]+: 560.3846, found 560.3857.
1’-((1H-Indol-3-yl)methyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5l)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), tryptamine (46 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5l (115 mg, 84% yield) as off white crystals. m.p. 196 °C. 1H NMR (400 MHz, DMSO) δ 10.87 (s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.10 – 7.02 (m, 2H), 6.96 (m, 1H), 4.72 (s, 1H), 4.72 (s, 1H), 4.59 (s, 1H), 4.54 (s, 2H), 3.17 (s, 1H), 3.03 – 2.92 (m, 1H), 2.70 (d, J = 15.2 Hz, 1H), 2.29 (m, 1H), 2.14 – 2.06 (m, 2H), 1.81 (d, J = 6.9 Hz, 2H), 1.63 (d, J = 29.9 Hz, 5H), 1.57 – 1.24 (m, 11H), 1.25 – 1.01 (m, 7H), 0.96 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.63 (s, 3H). 13C NMR (101 MHz, DMSO) δ 177.7, 150.7, 140.3, 137.8, 136.5, 127.4, 123.7, 121.4, 118.9, 118.3, 111.8, 110.7, 110.1, 55.9, 54.4, 50.1, 49.0, 48.9, 47.0, 42.5, 38.8, 38.1, 36.7, 33.5, 33.3, 32.0, 31.1, 30.5, 29.7, 28.6, 26.9, 25.5, 21.4, 21.1, 19.4, 18.8, 16.1, 15.7, 14.8. HRMS (ESI+): m/z calculated for C40H54N4O2H [M+H]+: 623.4319, found 623.4317.
1’-(2-(1H-Indol-3-yl)ethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5m)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 5-methoxytryptamine (54 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5m (126 mg, 88% yield) as off white crystals. m.p. 240 °C. 1H NMR (400 MHz, DMSO) δ 10.69 (s, 1H), 7.19 (d, J = 8.7 Hz, 1H), 7.05 (s, 1H), 6.76 (s, 1H), 6.67 (d, J = 8.7 Hz, 1H), 4.72 (s, 1H), 4.59 (s, 1H), 4.57 – 4.45 (m, 2H), 3.71 (s, 2H), 3.03 – 2.92 (m, 1H), 2.69 (d, J = 15.3 Hz, 1H), 2.28 (m, 1H), 2.09 (m, 2H), 1.81 (d, J = 6.9 Hz, 2H), 1.73 – 1.55 (m, 5H), 1.32 (m, 11H), 1.19 – 1.02 (m, 7H), 0.96 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.58 (s, 3H). 13C NMR (101 MHz, DMSO) δ 177.7, 153.5, 150.7, 140.3, 137.9, 131.5, 127.8, 124.3, 112.4, 111.7, 110.7, 110.1, 99.9, 55.9, 55.5, 54.4, 50.2, 48.9, 47.0, 42.5, 38.8, 38.1, 36.7, 33.5, 33.3, 32.0, 30.5, 29.7, 28.5, 26.9, 25.5, 22.5, 21.4, 20.9, 19.4, 18.8, 16.0, 15.7, 14.8. HRMS (ESI+): m/z calculated for C41H56N4O3H [M+H]+: 653.4424, found 653.4418.
1’-((1H-Indol-4-yl)methyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5n)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 4-(aminomethyl) indole (42 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5n (98 mg, 73% yield) as off white crystals. m.p. 260 °C. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.22 (m, 1H), 7.07 (m, 1H), 6.52 – 6.40 (m, 2H), 5.93 (m, 2H), 4.76 (s, 1H), 4.65 (s, 1H), 3.07 – 2.93 (m, 2H), 2.31 – 2.16 (m, 3H), 2.05 – 1.93 (m, 2H), 1.77 – 1.64 (m, 5H), 1.59 – 1.35 (m, 11H), 1.34 – 1.15 (m, 7H), 1.12 (d, J = 14.6 Hz, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.78 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.4, 150.2, 141.9, 138.3, 135.7, 128.2, 125.2, 124.5, 122.1, 117.5, 110.6, 109.8, 100.0, 56.3, 54.6, 51.4, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 37.0, 33.8, 33.3, 32.0, 30.5, 29.7, 29.7, 28.4, 25.5, 22.7, 21.3, 20.9, 19.4, 18.9, 16.0, 15.6, 14.6. HRMS (ESI+): m/z calculated for C39H52N4O2H [M+H]+: 609.4162, found 609.4174.
1’-(2-Hydroxyethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5o)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), ethanolamine (17 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by CH2Cl2 : MeOH 95:5) affording 5o (70 mg, 62% yield) as off white crystals. m.p. 236 °C. 1H NMR (400 MHz, DMSO) δ 4.72 (s, 1H), 4.59 (s, 1H), 4.36 (m, 2H), 3.86 (m, 2H), 2.98 (m, 1H), 2.70 (d, J = 15.2 Hz, 1H), 2.30 (m, 1H), 2.11 (m, 2H), 1.82 (d, J = 6.9 Hz, 2H), 1.64 (s,br, 5H), 1.58 – 1.33 (m, 11H), 1.33 – 1.22 (m, 7H), 1.17 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.71 (s, 3H). 13C NMR (101 MHz, DMSO) δ 177.7, 150.7, 140.2, 138.0, 110.1, 79.6, 60.7, 55.9, 54.5, 51.4, 48.9, 47.0, 42.5, 38.9, 38.1, 36.7, 33.6, 33.3, 32.0, 30.5, 29.8, 28.7, 25.5, 21.4, 19.4, 18.8, 16.2, 15.8, 14.8. HRMS (ESI+): m/z calculated for C32H49N3O3H [M+H]+: 524.3846, found 524.3853.
1’-Heptyl-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5p)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), 1-heptylamine (33 mg, 1.3 equiv, 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv, 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5p (72 mg, 57% yield) as off white crystals. m.p. 273 °C. 1H NMR (600 MHz, CDCl3) δ 4.76 (s, 1H), 4.64 (s, 1H), 4.33 – 4.23 (m, 2H), 3.04 (m, 1H), 2.91 (d, J = 15.3 Hz, 1H), 2.33 – 2.22 (m, 2H), 2.13 (d, J = 15.3 Hz, 1H), 2.00 – 1.96 (m, 2H), 1.79 – 1.64 (m, 5H), 1.61 – 1.35 (m, 13H), 1.35 – 1.19 (m, 14H), 1.17 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.88 (m, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.7, 150.2, 141.0, 137.3, 109.8, 56.4, 54.6, 49.6, 49.2, 49.2, 46.9, 42.4, 40.5, 38.9, 38.5, 38.2, 37.0, 33.6, 33.3, 32.1, 31.6, 30.8, 30.6, 29.8, 29.7, 28.8, 28.6, 26.9, 25.4, 22.5, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6, 14.0. HRMS (ESI+): m/z calculated for C37H59N3O2H [M+H]+: 578.4679, found 578.4687.
1H’-Lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5q)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), ammonium acetate (85 mg, 5 equiv., 1.100 mmol), 4-nitrophenyl azide (51 mg, 1.4 equiv., 0.308 mmol), 4 Å molecular sieves (50 mg) and DMF (0.8 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by CH2Cl2 : MeOH 95:5) affording 5q (90 mg, 86% yield) as off white crystals. m.p. 158 °C. Spectroscopic data for compound 5q was consistent with previously reported data for this compound.27 1H NMR (400 MHz, CDCl3) δ 4.77 (s, 1H), 4.64 (s, 1H), 3.03 (d, J = 10.1 Hz, 1H), 2.90 (d, J = 15.5 Hz, 1H), 2.37 – 2.22 (m, 2H), 2.12 (d, J = 15.5 Hz, 1H), 2.00 (dd, J = 19.9, 11.7 Hz, 2H), 1.83 – 1.66 (m, 5H), 1.57 (dd, J = 38.5, 24.6 Hz, 12H), 1.36 – 1.24 (m, 7H), 1.21 (d, J = 7.0 Hz, 4H), 1.01 (s, 3H), 1.00 (s, 3H) 0.78 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.0, 150.3, 150.1, 140.5, 109.8, 56.3, 53.4, 49.2, 49.0, 46.9, 42.5, 40.7, 39.0, 38.4, 37.3, 37.0, 33.3, 33.3, 32.1, 31.0, 30.6, 29.8, 25.5, 23.7, 21.4, 19.4, 19.1, 16.2, 15.6, 14.6. HRMS (ESI+): m/z calculated for C30H45N3O2H [M+H]+: 480.3584, found 480.3585.
1’-Benzyl-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5r)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), benzylamine (31 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5r (115 mg, 92% yield) as off white crystals. m.p. 290 °C. 1H NMR (400 MHz, CDCl3) δ 7.33 – 7.24 (m, 3H), 7.02 (d, J = 7.2 Hz, 2H), 5.64 (s, 2H), 4.76 (s, 1H), 4.64 (s, 1H), 3.09 – 3.00 (m, 1H), 2.96 (d, J = 15.4 Hz, 1H), 2.33 – 2.13 (m, 3H), 2.06 – 1.93 (m, 2H), 1.82 – 1.63 (m, 5H), 1.61 – 1.37 (m, 11H), 1.27 – 1.15 (m, 7H), 1.04 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.5, 150.2, 141.8, 138.0, 136.4, 128.7, 127.7, 126.3, 109.8, 56.4, 54.5, 52.8, 49.2, 49.1, 46.9, 42.4, 40.5, 38.9, 38.5, 38.3, 37.0, 33.7, 33.3, 32.0, 30.5, 29.7, 28.7, 25.4, 23.8, 21.3, 21.3, 19.4, 18.9, 16.0, 15.7, 14.6. HRMS (ESI+): m/z calculated for C37H51N3O2H [M+H]+: 570.4053, found 570.4064.
1’-((S)-1-Phenylethyl)-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole-28-oic acid (5s)
Betulonic acid (100 mg, 1 equiv., 0.220 mmol), (R)-(+)-α-methylbenzylamine (35 mg, 1.3 equiv., 0.286 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.220 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5s (88 mg, 69% yield) as off white crystals. m.p. 327 °C. 1H NMR (600 MHz, CDCl3) δ 7.30 – 7.20 (m, 3H), 7.15 – 7.10 (m, 2H), 5.73 (m, 1H), 4.75 (s, br, 1H), 4.64 (s,br, 1H), 3.02 (m, 1H), 2.95 (d, J = 15.4 Hz, 1H), 2.32 – 2.22 (m, 2H), 2.17 (d, J = 15.4 Hz, 1H), 2.05 – 1.95 (m, 5H), 1.80 – 1.64 (m, 5H), 1.64 – 1.39 (m, 11H), 1.38 – 1.25 (m, 7H), 1.00 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H) 0.72 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.5, 150.2, 141.7, 141.1, 137.6, 128.6, 127.5, 126.1, 109.8, 59.1, 56.4, 54.8, 49.3, 49.1, 46.8, 42.4, 40.5, 38.8, 38.4, 38.3, 37.0, 33.6, 33.3, 32.0, 30.5, 29.7, 28.6, 25.4, 23.3, 21.4, 21.3, 19.4, 19.0, 15.9, 15.6, 14.6. HRMS (ESI+): m/z calculated for C38H53N3O2H [M+H]+: 584.4210, found 584.4214.
1’-((S)-1-Phenylethyl)-1H’-lupano-[2,3-d]-[1,2,3]-triazole-28-oic acid (5t)
Dihydrobetulonic acid (100 mg, 1 equiv., 0.219 mmol), (S)-(−)-α-methylbenzylamine (34 mg, 1.3 equiv., 0.285 mmol), 4-nitrophenyl azide (36 mg, 1 equiv., 0.219 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5t (115 mg, 90% yield) as off white crystals. m.p. 173 °C. 1H NMR (400 MHz, CDCl3) δ 7.33 – 7.27 (m, 2H), 7.22 (m, 3H), 5.73 (m, 1H), 2.98 (d, J = 15.2 Hz, 1H), 2.32 – 2.22 (m, 3H), 2.17 (d, J = 15.3 Hz, 1H), 2.03 (m, 3H), 1.96 – 1.63 (m, 5H), 1.45 (m, 11H), 1.29 – 1.22 (m, 7H), 1.10 (s, 3H), 0.97 (m, 6H), 0.87 (s, 3H), 0.82 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 181.5, 141.7, 141.0, 137.5, 128.6, 127.5, 126.2, 59.2, 56.8, 54.7, 49.1, 48.7, 44.1, 42.6, 40.6, 38.8, 38.3, 38.3, 37.4, 33.8, 33.4, 31.9, 29.8, 29.7, 28.8, 26.8, 23.7, 23.0, 22.7, 21.3, 21.3, 18.9, 16.2, 15.7, 14.7, 14.5. HRMS (ESI+): m/z calculated for C38H55N3O2H [M+H]+: 586.4366, found 586.4370.
1’-((S)-1-Phenylethyl)-28-methyl-1H’-lup-2-eno-[2,3-d]-[1,2,3]-triazole (5u)
Lupenone (100 mg, 1 equiv., 0.235 mmol; provided by Milan Urban), (R)-(+)-α-methylbenzylamine (37 mg, 1.3 equiv., 0.306 mmol), 4-nitrophenyl azide (39 mg, 1 equiv., 0.235 mmol), 4 Å molecular sieves (50 mg) and toluene (0.5 mL). Reaction time is overnight. The product was purified by flash column chromatography (first washed with CH2Cl2 followed by petroleum ether : EtOAc 9:1 → 6:4) affording 5u (107 mg, 83% yield) as off white solid. m.p. 283 °C. 1H NMR (600 MHz, CDCl3) δ 7.29 (t, J = 7.7 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 5.75 (m, 1H), 4.71 (s, 1H), 4.60 (s, 1H), 2.95 (d, J = 15.3 Hz, 1H), 2.39 (m, 1H), 2.17 (d, J = 15.4 Hz, 1H), 2.03 (d, J = 7.0 Hz, 3H), 1.93 (m, 1H), 1.74 – 1.68 (m, 5H), 1.68 – 1.40 (m, 11H), 1.40 – 1.27 (m, 9H), 1.06 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 150.7, 141.9, 141.2, 137.6, 128.6, 127.5, 126.1, 109.5, 77.2, 77.0, 76.8, 59.1, 54.9, 49.2, 48.2, 47.9, 43.0, 42.8, 40.7, 39.9, 38.8, 38.5, 38.2, 35.5, 33.6, 33.3, 29.8, 28.6, 27.5, 25.1, 23.4, 21.5, 19.3, 19.0, 18.0, 15.9, 15.6, 14.5. HRMS (ESI+): m/z calculated for C38H55N3H [M+H]+: 554.4468, found 554.4462.
Biology
Anti-coronavirus evaluation in cell culture
HCoV-229E was purchased from ATCC (VR-740) and expanded in human embryonic lung fibroblast cells (HEL; ATCC® CCL-137). The titers of virus stocks were determined in HEL cells and expressed as TCID50 (50% tissue culture infective dose).49 The cytopathic effect (CPE) reduction assay was performed in 96-well plates containing confluent HEL cell cultures, as previously described.50 Serial compound dilutions were added together with HCoV-229E at an MOI of 100. In parallel, the compounds were added to a mock-infected plate to assess cytotoxicity. Besides the test compounds, two references were included, i.e. K22 [(Z)-N-[3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-3-oxo-1-phenylprop-1-en-2-yl]benzamide;16 from ChemDiv] and GS-441524 (the nucleoside form of remdesivir; from Carbosynth). After five days incubation at 35°C, microscopy was performed to score virus-induced CPE. To next perform the colorimetric MTS cell viability assay, the reagent (CellTiter 96® AQueous MTS Reagent from Promega) was added to the wells, and 24 h later, absorbance at 490 nm was measured in a plate reader. Antiviral activity was calculated from three independent experiments and expressed as EC50 or concentration showing 50% efficacy in the MTS or microscopic assay (see reference51 for calculation details). Cytotoxicity was expressed as 50% cytotoxic concentration (CC50) in the MTS assay.
Immunofluorescence detection of viral dsRNA
Semiconfluent 16HBE cell cultures in 8-well chamber slides (Ibidi) were infected with HCoV-229E (MOI: 1000) in the presence of 12 μM of 5h or GS-441524. After 4 h incubation at 35°C, the inoculum was removed, the compound was added again and the slides were further incubated. At 24 h p.i., the cells were subjected to immunostaining for dsRNA (all incubations at room temperature). After cell fixation with 3.7% formaldehyde in PBS for 15 min, and permeabilization with 0.2% Triton X-100 in PBS for 10 min, unspecific binding sites were blocked with 1% BSA in PBS for 30 min. Next, 1 h incubation was done with mouse monoclonal anti-dsRNA antibody (J2, SCICONS English & Scientific Consulting Kft; diluted 1:1000 in PBS with 1% BSA,), followed by 1 h incubation with goat anti-mouse AlexaFluor488 (A21131, Invitrogen; 1:1000 in PBS with 1% BSA). Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Invitrogen) in PBS for 20 min at RT. Microscopic images were acquired using the Leica TCS SP5 confocal microscope (Leica Microsystems) with a HCX PL APO 63x (NA 1.2) water immersion objective. DAPI and AlexaFluor488 were detected with excitation lines at 405 nm (blue) and 488 nm (green), and emission lines of 410-480 nm (blue) and 495–565 nm (green).
Time-of-addition assay
Confluent HEL cells were infected with HCoV-229E (MOI: 100) in a 96-well plate, and the compounds [5h, bafilomycin A1 (from Cayman) or K22] were added at −0.5, 0.5, 2, 4, 6 or 8 h post-infection (p.i.). At 16 h p.i., the supernatant was discarded and each well was washed twice with ice-cold PBS. The cells were lysed on ice for 10 min with 22 μL lysis mix, consisting of lysis enhancer and resuspension buffer at a 1:10 ratio (both from the CellsDirect One-Step RT-qPCR kit; Invitrogen). Next, the lysates were incubated for 10 min at 75°C and treated with DNase (Invitrogen) to remove interfering cellular DNA. The number of viral RNA copies in each sample was determined by one-step RT-qPCR. Five μL lysate was transferred to a qPCR plate containing 9.75 μL of RT-qPCR mix (CellsDirect One-Step RT-qPCR) and 0.25 μL Superscript III RT/Platinum Taq enzyme, and HCoV-229E N-gene specific primers and probe.52 The RT-qPCR protocol consisted of 15 min at 50°C; 2 min at 95°C; and 40 cycles of 15 s at 95°C and 45 s at 60°C. An N-gene plasmid standard was included to allow absolute quantification of viral RNA genome copies. The data from two independent experiments were expressed as the number of viral RNA copies at 16 h p.i. relative to the virus control receiving no compound.
Selection of resistant coronavirus mutants
HEL cells were infected with HCoV-229E virus (MOI: 25) and 5h was added at different concentrations. After five days incubation at 35°C, the CPE was scored microscopically to estimate the EC50 value. From the highest compound concentration conditions showing virus-induced CPE, the supernatants plus cells were frozen at −80°C. These harvests were further passaged in HEL cells under gradually increasing compound concentrations, until a manifest increase in EC50 was observed. A no compound control condition was passaged in parallel. The final virus passages were submitted to RNA extraction; reverse transcription; high-fidelity PCR; and cycle sequencing on the entire viral genome using a set of 39 primers (sequences available upon request). After sequence assembly with CLC Main Workbench 7.9.1 (Qiagen), the sequences of the viruses passaged in the absence and presence of 5h were aligned in order to identify the 5h resistance sites in the HCoV-229E genome.
Virus yield assay
The virus yield assay was performed in 96-well plates with semiconfluent cultures of human bronchial epithelial 16HBE cells (a gift from P. Hoet, Leuven, Belgium) or confluent HEL cells. Serial dilutions of compound 5h were added and the cells were infected (MOI: 100) with wild-type HCoV-229E (229E-WT), EndoU-deficient HCoV-229E (229E-H250Ansp15)22 or the mutant viruses obtained by passaging under 5h (229E-K60Rnsp15 or 229E-T66Insp15). After 4 h incubation at 35°C, the inoculum was removed, the compound dilutions were added again and the plates were further incubated. At three days p.i., the supernatants were collected, and 2 μL of each supernatant was lysed on ice by adding 11 μL lysis mix containing lysis enhancer and resuspension buffer at a 1:10 ratio. The lysates were incubated for 10 min at 75°C and the viral RNA copy number was determined by RT-qPCR as described above for the time-of-addition assay. The data were collected in three independent experiments and expressed as the fold reduction in viral RNA compared to the virus control receiving no compound.
Computational work
Starting from the published X-ray structure of hexameric nsp15 protein from HCoV-229E (PDB code: 4RS4), we first introduced a series of mutations, i.e. S10Q, S17G, A142T, M219I and S252L, to obtain the nsp15 protein sequence identical to that of the HCoV-229E virus used in the biological experiments. The structures of HCoV-229E nsp15 and SARS-CoV-2 nsp 15 (PDB code: 7K1O) were prepared using MOE (Chemical Computing Group, Montreal, Canada). Hydrogen addition and optimisation of protonation state and rotamers of the mutations were conducted using the AMBER-EHT force field, and identification of the potential binding sites in the multimeric complex was performed using MOE. Docking of betulonic acid derivatives was carried out by means of both MOE and GOLD with default settings, where GBVI/WSA score and Goldscore functions were used, respectively. The common top scoring solution was selected for further research.
ANCILLARY INFORMATION
SUPPORTING INFORMATION
1H NMR and 13C NMR spectra of the synthesized compounds.
CURRENT AUTHOR ADDRESS
Joice Thomas, Arcus Biosciences, Hayward, CA 94545, United States.
AUTHOR INFORMATION
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
B.K. and A.S. contributed equally. B.K. and J.T. performed compound synthesis. A.S. and L.N. designed, performed and interpreted the biological experiments. B.V.L., J.V. and D.J. performed antiviral experiments. T.N. and A.V. performed and interpreted the in silico study. V.T. and R.D. provided materials. B.K., A.S., W.D., A.V. and L.N. co-wrote the manuscript. All authors gave approval to the final version of the manuscript.
ACKNOWLEDGMENT
B.K. acknowledges receipt of an Erasmus Mundus Western Balkan Action 2 and doctoral fellowship from KU Leuven. B.V.L. holds an SB-PhD fellowship from the FWO Research Foundation Flanders (project: 1S66321N). Mass spectrometry was made possible by the support of the Hercules Foundation of the Flemish Government (grant: 20100225-7). We acknowledge the support of NVIDIA Corporation for donating the Titan Xp GPU used for this study. This research work was supported by funding from the KU Leuven (IDO/12/020 and project C14/19/78); the European Union’s Innovative Medicines Initiative (IMI) under Grant Agreement 101005077 [Corona Accelerated R&D in Europe (CARE) project]; and Fundació La Marató de TV3, Spain (Project No. 201832-30). We thank Amy Swinnen and Leentje Persoons for dedicated technical assistance, and Els Vanstreels for providing confocal microscopy training. We also thank Milan Urban for providing lupenone.
ABBREVIATIONS USED
- CC50
- 50% cytotoxic concentration
- CoV
- coronavirus
- COVID-19
- coronavirus disease 2019
- CPE
- cytopathic effect
- DAPI
- 4′,6-diamidino-2-phenylindole
- EndoU
- endoribonuclease
- FIPV
- feline infectious peritonitis virus
- HEL
- human embryonic lung
- MERS
- Middle East respiratory syndrome
- MHV-A59
- mouse hepatitis virus A59
- MTS
- 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
- nsp
- non-structural protein
- p.i.
- post-infection
- RTC
- replication-transcription complex
- SARS
- severe acute respiratory syndrome
- SARS-CoV-2
- severe acute respiratory syndrome coronavirus 2
- TCID50
- 50% tissue culture infective dose