Tricyclic Aza-Andrographolide Derivatives from Late-Stage Hydroamination and Their Anti-human Coronavirus (Anti-HCoV) Activity

A late-stage functionalization (LSF) of the natural product andrographolide for the efficient assembly of a range of structurally interesting and diverse tricyclic-aza derivatives was developed. The key to the diversification is a photo-catalyzed intramolecular hydroamination reaction, and acridinium derivatives were demonstrated to be the optimal catalysts. Additionally, the synthesized tricyclic aza-andrographolide derivatives were found to inhibit human coronavirus with high potency.


■ INTRODUCTION
Natural products and their derivatives play a highly essential role in combating numerous diseases, especially cancer and infectious diseases. 1 Data have shown that, from 1981 until 2019, 41% of anticancer and 66% of anti-infective small molecule drugs were derived from or inspired by natural sources. 2 Compared with the traditional synthetic compounds, natural products have enormous structural complexity and scaffold diversity, which make them more likely to interact with biological macromolecules in a selective and specific manner. 3 Driven by modern high-throughput drug discovery, 4 there is an urgent and increasing demand for a large collection of bioactive natural-product-derived compounds with significant structural diversity and complexity. Developing facile and practical methods or strategies to construct them, however, remains a considerable interest in the field. Several strategies for the rapid and highly efficient generation of these structurally diverse natural-product-inspired libraries have been developed, including diversity-oriented synthesis from simple starting materials using convergent strategies such as multicomponent reaction, 5 the use of natural products with high abundance as a starting point for the generation of complex natural product analogues, 6 and solid-phase combinatorial synthesis using the above-mentioned strategies. 7 The diterpene natural product andrographolide, isolated from Andrographis paniculata, possesses versatile biological activities, including anti-inflammatory, 8 antimicrobial, 9 immu-nomodulatory, 10 antidiabetic, 11 cardioprotective, 12 hepatoprotective, 13 and neuroprotective effects. 14 Andrographolide and its derivatives were also found to exert anticancer effects on almost all types of cell lines with varied mechanisms of action. 15 Moreover, it exerts broad-spectrum antivirus properties toward different virus infections. 16 The outbreak of COVID-19, caused by SARS-CoV-2, has become a global pandemic, which has infected almost 519 million people and claimed over 6.2 million lives worldwide. 17 Searching for effective antivirus treatments for COVID-19 from nature is needed, and thus, we are interested in creating a natural-product-like library with potential antivirus activity. 18 Recent studies revealed that andrographolide is a potential inhibitor of the main protease of SARS-CoV-2 (Mpro) in an in silico approach, and it docks successfully in the binding site of SARS-CoV-2 Mpro. 19 Considering its potent antiviral activity and potential interaction with the binding site of SARS-CoV-2, we chose andrographolide with high abundance as a template for analogue generation. Here we present a late-stage functionalization (LSF) 20 approach to tricyclic aza-androgra-pholide derivatives using photo-catalyzed hydroamination as the key reaction, and their anti-HCoV activity.

■ RESULTS AND DISCUSSION
The structure modifications of andrographolide were mostly focused on the functionalization of the 3,14,19-hydroxyls, 8,17double bond, C12−C13 double bonds, and α,β-unsaturated γbutyrolactone moiety (Figure 1a). 21 Modifications at these positions have shown improvement of antivirus or anticancer activity. For example, Golakoti and co-workers found that C-12-substituted amino 14-deoxy-andrographolide derivatives exhibited improved cytotoxic activity compared to andrographolide ( Figure 1b). 22 8,17-Epoxy andrographolide derivatives exhibited significant inhibitory activity toward colon cancer, breast cancer, etc. (Figure 1c,d). 23,24 Andrographolide analogues with 8,17-alkene exo-to-endo isomerization ( Figure  1e) displayed a potent anti-influenza virus activity, and it was approximately 1.5-fold more potent than Lianbizhi, an andrographolide analogue used clinically in China. 25 Since the N-functionalization plays an important role in the activity and PK profiles of natural products, we proposed to incorporate nitrogen atom into the andrographolide skeleton to further enlarge its chemical space, providing the possibility of a novel structure of tricyclic aza-andrographolides ( Figure  1f).
The synthetic pathways toward tricyclic aza-andrographolides mainly include two steps (Scheme 1). The acylated andrographolide 2 was aminated through a base-promoted Michael addition and elimination process, generating C-12aminated-14-deoxy andrographolides 3, 22 followed by photopromoted intramolecular hydroamination of the 8,17-terminal double bond to yield the tricyclic aza-andrographolide derivatives. Based on previous reports, 26 we initially conducted the intramolecular hydroamination of C-12-aminated-14-deoxy andrographolides with the catalysis of the Ir(III) photocatalyst (I), which was indicated to be effective for both intermolecular and intramolecular hydroamination of simple unactivated alkenes. However, the reaction proceeded with quite low yield, and some of the C-12-aminated-14-deoxy andrographolides failed to undergo Ir(III)-catalyzed hydroamination. To address this limitation, we then devoted ourselves to search for more effective catalysts.
We subsequently set out to explore the scope and potential of the photocatalyst-promoted intramolecular hydroamination. As shown in Table 2, a wide range of substrates 3 were surveyed, including those with arylmethyl, alkyl, and cycloalkyl groups. Pleasingly, most of the substrates examined underwent the cyclization smoothly to give the diverse tricyclic azaandrographolides 4 in moderate to good yields. Electronic variation on the aryl ring, including electron-withdrawing, neutral, and electron-donating effects, did not obviously affect the efficiency of the reactions. Among these, some functional groups were well tolerated, such as nitro and hydroxyl, which could serve as handles for further synthetic manipulations. In addition, heterocycles including furan and thiophene were also compatible with the current reaction, providing the desired products in moderate yields. Lastly, a series of substrates bearing aliphatic substituents were investigated, and it was found that acyclic and cyclic amines gave diminished yields. The structures of synthesized compounds 4 were all characterized by 1 H NMR, 13 C NMR, and HRMS, and the stereochemistry was further confirmed by single crystal X-ray diffraction analysis of 4u (see the Supporting Information).
After exploring the scope of the substrates based on the 3,19-diacetate andrographolide, we next investigated other 3,19-substituted substrates, such as 3,19-ketal andrographolides, which were found to have interesting biological activity. 27 To this end, two types of substrates, 7 and 8 (Scheme 2), were synthesized and subjected to the intramolecular hydroamination, leading to the rapid generation of structurally diverse aza-andrographolide derivatives 9a−p and 10a−d, as listed in Table 3.
When compounds 7 and 8 bearing arylmethyl groups served as substrates for cyclization, the desired cyclized products were produced in moderate to good yields (9a−m and 10a−d), while the substrates with branched aliphatic chains furnished the products in low yields (9o and 9p). Considering the aqueous solubility of the cyclized products, we intended to make 3,19-diol derivatives. Accordingly, the selected compound 9 was treated with acetic acid in THF and water to yield the desired product 11 in good yield ( Table 3, 11a−e).
The collection of synthesized aza-andrographolide derivatives 4, 9, 10, and 11 was evaluated in the cytopathic effect (CPE) assays for their antiviral activity against the human coronavirus (HCoV) 229E. All the tested compounds were assayed at 25 μM in the MRC5 cells, and their antiviral activity was calculated based on the protection of the virus-induced CPE normalized by the virus-only control. Additionally, the cytotoxicity of compounds toward MRC5 cells was also assessed under the same conditions, but without virus infection, in parallel. The antiviral activity of compounds is expressed as % inhibition normalized by the cytotoxicity, and selected examples are depicted in Table 4 (entries 1−20). From these assessments, four types of compounds displayed a distinct inhibition profile toward HCoV 229E, with compounds 4a−y and 9a−p being potent and compounds 10a−d and 11a−e being weak. This result indicated that structural moiety at the 3,19-position is also important for activity. We found that compounds 4d, 4h, and 4l−n with halo-or CF 3substituted benzyl exhibited good inhibitory activity (∼100% inhibition) at 25 μM, whereas 4o and 4p with N-furylmethyl or N-thiophenylmethyl showed a weak inhibition effect. Bulky Nsubstituents (4q, 4v, and 4y) gave more favorable results compared to compounds 4r and 4w. As for compounds 9, only substituents at the benzyl-4-position with methyl (9f), tertbutyl (9 g), and nitro (9 h) demonstrated good inhibition. Noteworthily, all the tested compounds demonstrated little toxicity against the MRC5 cells (see the Supporting Information for details).
Four compounds�4l, 4m, 4y, and 9f�were then chosen to assess the anti-HCoV 229E activity through full dose− response curves. These compounds exhibited great potency with a half-maximal effective concentration (EC 50 ) ranging from 10.1 to 23.1 μM (Table 4, entries 21−24), and compound 4y was identified as the most promising one, with an EC 50 of 10.1 μM. The four tested compounds displayed no signs of cytotoxicity, with a half-maximal cytotoxic concentration (CC 50 ) of >100 μM. This result indicated that our synthesized compounds have a potential as anti-HCoV hits for further development.

■ CONCLUSIONS
In conclusion, we have developed a concise approach to the structurally diverse and bioactive tricyclic aza-andrographolide derivatives through photo-redox catalyzed intramolecular hydroamination, in which the acridinium catalyst VI was proven to be the most effective. We anticipate that this method or strategy will find a variety of applications in the late-stage functionalization of natural products and drugs. Preliminary screening of the generated library of aza-andrographolide derivatives led to the identification of small molecules with anti-HCoV activity, and they can serve as promising anti-HCoV hits for further drug development. General Information. Unless noted otherwise, all reactions were performed under a nitrogen atmosphere, and materials obtained from commercial suppliers were used without further purification. The purification of products was conducted by flash column chromatography on a silica gel (200−300 mesh). 1 H NMR spectra were recorded on 300− 500 MHz spectrometers using the residual solvent (δ (CDCl 3 ) = 7.26) as internal standard. All the coupling constants are reported in hertz. 13 C NMR spectra were recorded on the same instruments, and chemical shifts were measured relative to solvent resonances (δ (CDCl 3 ) = 77.0). High-resolution mass spectra were obtained on a quadrupole time-of-flight (QqTOF) mass spectrometer utilizing the electrospray ionization (ESI) method. Reaction conditions: 3 (0.05 mmol, 1 equiv), PC-VI (6 mol %), 2,4,6-triisopropylbezene-1-thiol (TRIP thiol) (0.5 equiv), toluene (2 mL), blue LED (40 W), under N 2 , room temperature, 12 h. Yields shown are the yields of the isolated product.