Click Chemistry in Natural Product Modification

Click chemistry is perhaps the most powerful synthetic toolbox that can efficiently access the molecular diversity and unique functions of complex natural products up to now. It enables the ready synthesis of diverse sets of natural product derivatives either for the optimization of their drawbacks or for the construction of natural product-like drug screening libraries. This paper showcases the state-of-the-art development of click chemistry in natural product modification and summarizes the pharmacological activities of the active derivatives as well as the mechanism of action. The aim of this paper is to gain a deep understanding of the fruitful achievements and to provide perspectives, trends, and directions regarding further research in natural product medicinal chemistry.


INTRODUCTION
Natural products (NPs) are secondary metabolites that are produced by the evolutionary optimization of nature. They usually possess diverse and complex architectures and are endowed with versatile pharmacological activities, offering an abundant source for therapeutic drug discovery (Hunter, 2008;Jiménez, 2018) Newman and Cragg, 2020;Rodrigues et al., 2016;Li and Vederas, 2011). Natural product-based drug discovery can date back to the isolation of morphine, the first pharmacologically active pure natural product which was purified by Friedrich Sertürner more than 200 years ago. From then on, considerable works have been devoted to the synthesis of natural derivatives Foley et al., 2020), and/or natural product-like screening libraries with the aim of therapeutic drug discovery (Huigens et al., 2013;Crane and Gademann, 2016;Ma et al., 2019;Wilson et al., 2020;Xie et al., 2020). These efforts have led to the discovery of various important clinical drugs, such as anticancer agents (e.g., taxol and doxorubicin), immunosuppressants (e.g., cyclosporine and doxorubicin), antimalarial agents (e.g., quinine and artemisinin) and lipid regulate drugs (e.g., lovastatin and relatives). Even today, natural products still serve as a fundamental source of diverse biological functions, facilitating the development of chemical biology and drug discovery.
As natural products are usually complex molecules with various stereo centers, sp3 carbon, and labile functionalities, the de novo synthesis of natural products or their derivatives always need complicated synthetic strategies, and accomplished in time-consuming multistep syntheses with low quantity and a limited number of derivatives. Therefore, chemistries that can be used for the late-stage functionalization and diversification of natural products are highly desirable, and should meet the following criteria: 1) reliable, selective, orthogonality to other functionalities; 2) modular, broad substrate scope; 3) high yield; 4) operational simplicity. In 2001, these criteria were codified by Professor K. Barry Sharpless, who termed such ideal chemistries as "click chemistry" (Kolb et al., 2001). From then on, click chemistry reactions, especially the Cu(I)catalyzed Huisgen 1,3-dipolar cycloaddition between alkynes and azides (CuAAC) was quickly recognized as versatile players in the modification of various molecules, especially complex natural products, providing enhanced properties or new functions for chemical biology and drug discovery ( Figure 1).
Despite the success of CuAAC, the search for other click chemistries has never stopped. Today several elegant click chemistries have been well developed, such as strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demanded Diels-Alder (IEED-DA), and Sulfur (VI) Fluoride Exchange (SuFEx) chemistry. These chemistries have played a key in chemical biology and drug discovery, particularly the emerging SuFEx chemistry, another ideal click reaction proposed by Professor Sharpless in 2014 (Dong et al., 2014;Barrow et al., 2019), have already gained wide application in the synthesis of drug screening libraries (Kitamura et al., 2020;Smedley et al., 2020), late-stage modification of drugs and natural products (Li S. et al., 2017;Liu et al., 2018), DNA-encoded library synthesis Xu H. et al., 2019;Zhang et al., 2021), and the synthesis of 18 F radio tracers (Zheng et al., 2021).
Indeed, now that natural products have met with click chemistry, a new era of natural product-based drug discovery has come. Previously, several elegant review papers have summarized the CuAAC reaction in medicinal chemistry, mainly focused on the synthesis of 1,2,3-trizaoles for various properties such as anti-cancer (Xu Z. et al., 2019;Liang et al., 2021), anti-bacterial, etc., (Kacprzak et al., 2016;Rani et al., 2020;Guo et al., 2021;Kumar et al., 2021;Serafini et al., 2021). Especially, this review focuses on the late-stage modification of natural products by using not only the CuAAC reaction but also other click chemistries such as SPAAC and especially the emerging SuFEx chemistry ( Table 1) (Dong et al., 2014), and thus encompasses a much wider variety of natural product derivatives and the corresponding pharmacological activities. These natural product derivatives are classified according to their structural features, covering a time span mainly of the last decade.
The aim of this paper is to showcase the state-of-the-art development of click chemistry in natural product modification, thereby gain a deep understanding of the fruitful achievements, and provide perspectives, trends, and directions regarding further research in natural product medicinal chemistry.

CLICK CHEMISTRY-BASED MODIFICATION OF TERPENOIDS
Terpenoids, also known as isoprenoids, are the largest class of plant secondary metabolites, representing 60% of the known natural products. Terpenoids derive from 5-carbon isoprene and usually have oxygen-containing functionalities. Many diterpenoids, especially cyclic sesqui-, di-and triterpenoids are endowed with bewildering structural features such as multiple
Usnic acid is a dibenzofuran secondary metabolite that is isolated from lichen genera. Usnic acid-triazole-saccharin hybrid a10 (MIC 2.5 μM) could exert slightly better inhibitory activity than clinical drug isoniazid (MIC 2.9 μM) against Mycobacterium tuberculosis (Mtb) (Bangalore et al., 2020), but failed to show any antibacterial activity against Bacillus subtilis, while hybrid a11 (MIC 40.9 μM) could exert good antibacterial activity against Bacillus subtilis. Molecular docking studies indicated that the usnic acid moiety of a10 could occupy the active site of Mtb enzyme enoyl reductase (InhA), while the oxygen of sulfamide in saccharin could engage a hydrogen bond with GLN100, and the triazole moiety could have π−π stacking interaction with PHE97. Guo et al. reported that usnic acidtriazole hybrid a12 could exert selective anti-Toxoplasma gondii activity with a good selectivity index (IC 50 261 μM, SI 1.34), which is slightly better than the reference drugs sulfadiazine (SI 1.15), pyrimethamine (SI 0.89), and spiramycin (SI 0.72) and also the parental (+)-usnic acid (SI 0.96) .
Gossypol is a natural yellow pigment bi-sesquiterpene that acts as a plant defense system against insects and fungi. Pyta et al. reported that gossypol-triazole a13 (MIC 16 μg/ml) could exert comparable inhibitory as miconazole against Fusarium oxysporum (MIC 16 μg/ml). Mechanistic studies indicated that a13 might inhibit biosynthesis of ergosterol, thereby exerting its anti-fungal activity .
Hybridization of dihydroartemisinin with other natural products or drug molecules is another good strategy to obtain potential anticancer compounds. Artemisinin-coumarin hybrid b7, b8, and b9 could only exert moderate cytotoxic activity against MDA-MB-231, HCT-116, and HT-29 cancer cells under normoxic conditions (Tian et al., 2016;Yu et al., 2018). Whereas, under anoxic conditions, hybrid b7 (anoxic, IC 50 0.05 μM; normoxic, IC 50 17.7 μM) and b8 (anoxic, IC 50 0.01 μM; normoxic, IC 50 1.5 μM) showed 334-fold and 150-fold more potent than that under normoxic conditions in HT-29 cells, which is probably associated with the high expression of CA IX on the membrane of HT-29 cells. While, hybrid b9 showed a 41.38-fold and 20.03-fold higher activity than that under normoxic conditions in IC 50 0.43 μM; normoxic, IC 50 17.96 μM) and MDA-MB-231 cells (anoxic,IC 50 3.62 μM; normoxic, IC 50 72.5 μM), respectively. Structure activity relationship (SAR) studies indicated that the spacer between triazole-artiartemisinin and triazole-coumarin as well as the substituents on the coumarin were critical to the selective inhibitory activity. Artemisinin-azidothymidine hybrid b10 (IC 50 16.5 μM) could exert more potent antiproliferative activity than artesunate (IC 50 78.5 μM) against KB cancer cells (Tien et al., 2016), indicating the azidothymidine moiety plays a key role in the increased activity. In addition, SAR studies showed that hybrids with ester triazole-linker could exert more potent antiproliferative activity than hybrids with amide triazole-linker.
Oridonin is an ent-kaurene diterpenoid that was initially isolated from various Isodon species, which was widely used as home remedy herb medicine in China and Japan. Oridonintriazoles generally showed broad-spectrum anticancer activity. For example, C14 oridonin-triazole C1 (HTC116: IC 50 6.89 μM; MCF7: IC 50 6.81 μM), C2 (HTC116: IC 50 1.94 μM; MCF7: IC 50 3.83 μM) ( Figure 4) , C3 (PC-3: IC 50 3.1 μM; LNCaP: IC 50 4.1 μM) could exert more potent anti-proliferative activities than that of oridonin (IC 50 16.28-24.80 μM) . In addition, they could effectively overcome drug resistance and showed weak cytotoxicity on non-cancer cells. SAR studies indicated that the phenyl 1,2,3-triazole moiety and the linker between oridonin and triazole play a key role in improving antiproliferative activity. Preliminary mechanistic studies indicated that C3 could arrest cell cycle (G2/M phase) and induce apoptosis of PC-3 cells. Through the introduction of azide or alkyne linkers at the C20 hydroxyl group, Liu et al. synthesized a focused library of Jiyuan oridonin A-triazoles, in vitro data indicated that all the triazole derivatives could exert good anti-proliferative activities. Among them, C4 (IC 50 4.26-8.95 μM) and C5 (IC 50 2.70-5.04 μM) could exert broad-spectrum inhibitory activity against a panel of cancer cell lines including Eca109, EC9706, SMMC7721, and MCF7. Mechanistic studies indicated that C5 could promote intracellular ROS level, arrest cell cycle (G2/M phase) and significantly induce cell apoptosis in the tested MGC-803 cells (Ke et al., 2018b). Later, the same group reported that C6 (IC 50 0.6-5.0 μM) could exert potent anti-proliferative activities on several cancer cell lines (Eca109, EC9706, SMMC7721, and MCF7) with good selectivity towards normal cells. Mechanistic studies indicated that C6 could inhibit cell migration with the Wnt signaling pathway involved, arrest cell cycle (G1 phase), and induce cell apoptosis in the tested SMMC-7721 cells (Ke et al., 2018a). Due to the challenge of the introduction of azide at C1 of oridonin, there are only limited C1 triazole derivatives have been reported, and according to the reported data, derivative C7 could exert submicromolar inhibitory activity on the tested cancer cells Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 774977 6 (Ding et al., 2013), implying the introduction of triazole at C1 of oridonin was tolerated.
Abietane-type diterpenes are a series of tricyclic terpenoids that possess various biological activities. For example, dehydroabietic acid and abietic acid are readily available diterpenoids that can be isolated from disproportionated rosin. They have been widely used as starting materials for the synthesis of natural products or natural product-like drug screening libraries (Xu et al., 2014b;Xu et al., 2017). With the installation of azide or alkyne functionalities at C14 or C18, several series of dehydroabietic acid-triazole derivatives were synthesized. The screening of antiproliferative and antibacterial activities indicated that D1 could inhibit the growth of several cancer cell lines (IC 50 0.7-1.2 μM) ( Figure 5) (Hou et al., 2017a). Mechanistic studies indicated that D1 could induce apoptosis of MDA-MB-231 cells. In addition, the C18 triazole derivative D3 (IC 50 5.90 μM) could exert comparable inhibitory activity to the reference drug cisplatin against HepG2 cells (IC 50 6.42 μM) , and derivative D2 could exert antibacterial activities against both Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gramnegative bacteria (Escherichia coli and Pseudomonas fluorescens) strains (MIC 1.6-3.1 mg/ml) with good druglike properties and low cytotoxicity in noncancerous mammalian cells (Hou et al., 2017b).
Triptolide is an abietane-type diterpene that was identified from Tripterygium wilfordii Hook.f (TWHF). It has the unique structural features of three successive epoxides and an unsaturated α, β-lactone. Due to its various promising biological activities, considerable work has been devoted to its total synthesis, structural modification, and targeted delivery with the aim to reduce its toxicity (Hou et al., 2019b;Xu and Liu, 2019;Zhang X. et al., 2019). With the introduction of azidomethyl at C14 of triptolide, Li et al. synthesized a series of C14 triazole substituted epi-triptolide derivatives D5a, D5b, and D5c (Xu et al., 2014a). In vitro data indicated that these derivatives only showed weak cytotoxic activity as compared to triptolide. However, considering various promising biological activities of triptolide (Hou et al., 2019b), further evaluation of their biological activates such as neuroprotective and anti-inflammatory activities are highly expected.
Isosteviol is a tetracyclic ent-beyerane diterpene that is endowed with multifarious bioactivities and can be readily isolated from stevia plant. Diversification of isosteviol by click chemistry at C19 or C15 could generate derivatives with potential anticancer activity. Tao et al. reported that C15 isosteviol-triazole D7 (IC 50 2.987 μM) could exert slightly better inhibitory activity than cisplatin (IC 50 3.906 μM) against HCT-116 cells . Quan et al. reported that C19 isosteviol-triazole D8 could inhibit the growth of several cancer cell lines and HepG2) with IC 50 values in the range of 5.38-15.91 μM, and that was 1.3-to 4.6-fold more potent than the reference drug 5fluorouracil, and 6.3-to 16.8-fold more potent than the parental isosteviol (Luan et al., 2019). Mechanism studies indicated that D8 could inhibit colony formation and arrest cell cycle (S phase) in HCT-116 cells.
Kaurenoic acid (KA) is a kaurene diterpene that can be isolated from the fruits of X. aethiopica. Oliveira et al. reported that C19 kaurenoic acid-triazole D9 could exert moderate antimalarial activity (IC 50 53 μM) (De Santos et al., 2016), together with good selectivity (selective index (SI) 774). Both andrographolide and (E)-labda are labdane diterpenes. Andrographolide is one of the principal biological active compounds of andrographis paniculate, a traditional herb medicine widely used in China and India for the treatment of multiple diseases such as inflammation and cancer. Chinthala et al. reported that andrographolide triazole derivative D10 could exert selective inhibitory activity against K562 cancer cells with IC 50 values of 8 μM. (Chinthala et al., 2016). In silico docking studies indicated that D10 could bind with transient receptor potential vanilloid 1 (RPV1). (E)-labda is isolated from fresh rhizomes of Curcuma amada. Somappa et al. reported that labdane triazole derivatives D11a (IC 50 0.75 μM) and D11b (IC 50 0.77 μM) could exert excellent pancreatic lipase (PL) inhibitory activity, slightly better than the reference drug orlistat (IC 50 0.8 μM) (Jalaja et al., 2018).
Triterpenes represent an important class of natural terpenoids that are composed of three terpene units. They are endowed with the abilities to balance hormones, blood pressure, circulation, and digestion, and they also have been documented with anti-viral and anti-inflammatory activities.
Asiatic acid (AA) is a pentacyclic triterpenoid that was identified from the tropical herb medicine Centella asiatica. It has been reported to possess various biological activities such as antiinflammation, antidiabetics, and antitumor. Huang et al. reported that AA-triazole E2 could bind to NF-κB (KD 0.36 μM) and exert low micromolar inhibitory activity against TNF-α-induced NF-κB activation (IC 50 0.14 μM) . Molecular docking studies indicated that E2 could fit well in the active site of NF-κB. The fluorine of benzene could form one hydrogen-bonding interaction with DNA chain (DA6), while the benzene could engage π−π interactions with PHE307 . Notably, 1,2,3-triazole as a hydrogen acceptor could establish four hydrogen bonds with amino hydrogen of LYS272 and DA5, indicating that the triazole moiety was crucial for the improved potency of E2. C-2 and C-3 hydroxy groups of AA could form two hydrogen bonds with the DNA backbone P O of DG2. Also, C-23 hydroxy of AA formed two hydrogen bonds with LYS249. Moreover, the pentacyclic skeleton of AA moiety was surrounded by LYS241, PRO243, SER246, ASN247, LYS249, ASP271, and LYS272 via the hydrophobic interaction. Further, mechanistic studies indicated that E2 could inhibit NF-κB DNA binding, nuclear translocation, and IκBα phosphorylation. In vitro data showed that E2 could inhibit the growth of A549 cells (IC 50 2.67 μM) by at least partial inhibition of the activity of NF-κB, as well as cell apoptosis and migration.

CLICK CHEMISTRY-BASED MODIFICATION OF ALKALOIDS
Alkaloids are a series of nitrogen-containing compounds of plant origin, they usually possess various pronounced physiological effects on humans and other animals, such as morphine, quinine, strychnine, nicotine, and ephedrine. Matrine is a quinolizidine alkaloid that is isolated from the root of Sophora flavescens Ait (also known as Kushen), which is a traditional Chinese herb medicine that has been used for the treatment of liver diseases for thousands of years. Zhao et al. reported that matrine-triazol-chalcone hybrids could inhibit the growth of a panel of cancer cells (Figure 7) (Zhao et al., 2015). Among them, F1 (IC 50 5.01-7.31 μM) could exert broadspectrum anticancer activities against a panel of cancer cell lines (A549, Bel-7402, Hela and MCF-7). Notably, F1 is more potent than the combination of matrine and chalcone (IC 50 > 50 μM), and also 5-fluotouracil (IC 50 8.93-40.38 μM). SAR studies indicated that the α, β-unsaturated ketone moiety and the triazole together might play a key in determining the promoted inhibitory activity. Further studies indicated that F1 could induce apoptosis in A549 cells, and suppress tumor growth in A549xenografted nude mouse model (10 mg/kg) with no apparent cytotoxicity.
Quinine is one of the most abundant natural cinchona alkaloids, and also is the mainstay of antimalarial drugs. With the introduction of azide at C9, C2' and C6' of quinine, Boratyński et al. synthesized a focused library of triazole containing chinchona alkaloids (F4a, F4b, F5, and F6) (Boratyński et al., 2018). In vitro data indicated that nearly all the derivatives could exert moderated antiproliferative activities. Among them, F4a (IC 50 0.53 μM) showed the highest potential in MC-4-11 cells, while, F4b (IC 50 1.2 μM) showed the highest potential in HT-29 cells.
The conjugation of small molecules with ferrocene, a unit that showed tunable redox characteristics, can usually generate new molecules with unexpected properties. Pešić et al. reported that ferrocene-quinine conjugate F8 (IC 50 2.34-2.13 μm) could not only inhibit the growth of drug-sensitive NCI-H460 cancer cells, but also multi-drug resistant (MDR) NCI-H460/R cancer cells (Podolski-Renić et al., 2017). Mechanistic studies indicated that F8 could increase ROS production and induce mitochondrial damage in MDR cancer cells, highlighting the importance of the ferrocene moiety. Panda et al. reported that F7 (IC 50 27 nM) could exert more potent in vitro antimalarial activity than quinine (IC 50 58 nM) against P. falciparum strain 3D7 (Faidallah et al., 2016), and the reason is probably due to the introduction of the hydrophobic alkyl chain at C9, thereby increasing the penetration ability of the parental scaffold. Sahu et al. reported that C19 quinine-triazole derivative F9 could exert potent antimalarial (P. falciparum, IC 50 0.25 μM) and antileishmanial activities (L. donavani, IC 50 1.78 μM) with no apparent adverse effects (Sahu et al., 2019). The structural toxicological activity relationship studies indicated that the introduction of the triazole moiety to quinine would result in decreased toxicity. 20(S)-Camptothecin is a potent DNA topoisomerase I inhibitor that isolated from Camptotheca acuminata in 1966. With the installation of alkyne at C10 of homocamptothecin, followed by reactions with various azides under CuAAC, Xu et al. synthesized a series of C10 homocamptothecin-triazole derivatives. Among them, derivative F10 (IC 50 30 nM) could exert more potent inhibitory than 20(S)-camptothecin (IC 50 170 nM) against A549 cancer cells in a Topo I-dependent manner . Mechanistic studies indicated that F10 could arrest cell cycle at the G2 and S phases.
Colchicine is a well-known antimitotic agent that is isolated from Colchicum autumnale. By utilization of the fast and efficient CuAAC derivatization strategy, Schmalz et al. synthesized C7 colchicine-triazole F11, which (IC 50 3.5-5.52 nM) could exert more potent inhibitory activity than colchicine (IC 50 13.2-20.4 nM) in a panel of cancer cell lines (THP-1, Jurkat, Hela, A549 and MES1) (Thomopoulou et al., 2016). In addition, one of the derivative F12 could not only distort the microtubule morphology but also exert a significant centrosomedeclustering effect on MDA-MB-231 cells and H1975 cells.
Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 774977 Theophylline, a naturally occurring purine base, is a bronchodilator drug that is used for the treatment of various respiratory diseases such as chronic pulmonary obstructive disease and asthma. Murugulla et al. reported that theophylline-triazole F16 could exert potent cytotoxicity on a panel of cancer cells with IC 50 values in the range of 1.2-2.3 μM (Ruddarraju et al., 2017). In silico docking results indicated that F16 might bind to human epidermal growth factor receptor 2 (EGFR II). Triazole-tethered theophylline-nucleoside hybrid F17 could inhibit the growth of A549, HT-29, MCF-7 and A375 cancer cells (IC 50 1.89-4.89 μm) (Ruddarraju et al., 2016), while hybrid F18 could exert potent antibacterial activities against both Gram-positive (Staphylococcus aureus, Bacillus cereus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains with MIC values (MIC 0.03125-0.125 μg/ml), which are comparable to or more potent than that of the clinical drug ciprofloxacin (MIC 0.0156-0.0625 μg/ml).

CLICK CHEMISTRY-BASED MODIFICATION OF PHENYLPROPANOIDS
Phenylpropanoids, also known as cinnamic acids, are a series of secondary metabolites that are synthesized by plants from phenylalanine and tyrosine. It mainly includes flavonoids, chalcones, isoflavonoids, lignols, coumarins, stilbenes, aurones, catechin, and phenolic acids.
Flavones are a series of privileged polyphenolic natural products that possess broad-spectrum pharmacological activities. Their synthetic derivatives have been reported to have antitumor, antioxidant, anti-inflammatory, and antiviral activities, etc. Some flavones such as luteolin are under clinical trials for the treatment of cancer (Maggioni et al., 2015), implying the potential of flavones or their derivatives in innovative drug discovery.
Through  (Qi et al., 2018). Mechanistic studies indicated that G4 could promote the level of cellular reactive oxygen species (ROS) and reduce the mitochondrial membrane potential, thereby inducing the apoptosis of SKOV-3 cells. It could also modulate the expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax). Sangwan et al. reported that bavachinin-triazole G5 (IC 50 30.5-36.3 μM) could exert comparable antiproliferative activity to bavachinin against a panel of cancer cell lines including A549, HCT-116, PC-3, and MCF-7 . Mechanistic studies indicated that G5 could induce apoptotic cell death via PARP cleavage and loss of MMP, and it could also inhibit cell migration and colony formation in A549 cells. Baicalein is isolated from Scutellaria baicalensis. Niue et al. reported that baicalein-triazole G6 could prevent respiratory tract infection by respiratory syncytial viruses (RSV) via the suppression of oxidative damage .
Chalcones, also known as chalconoids, are a series of natural polyphenols. Structurally, they are α, β-unsaturated ketones, consisting of two aromatic rings conjugated by an α, β-unsaturated carbonyl system. Chalcones and their derivatives possess various biological functions such as anticancer, antiinflammatory and antiviral activities, etc. Thus, hybridization of chalcone scaffold with another pharmacophore by click  (Figure 9) . Mechanistic studies indicated that H1 could arrest cell cycle (G2/S phase) and induce apoptosis of MIA-Pa-Ca-2 cells by reducing mitochondrial potential and activating PARP-1 and caspase-3.
Kamal et al. reported that triazole-chalcone hybrids H2 could exert low micromolar inhibitory activity against Hela, DU145, HepG2, and A549 cancer cells with IC 50 values in the range of 1.5-7.7 μM (Hussaini et al., 2016). SAR studies indicated that the α, β-unsaturated ketone of the chalcone skeleton was critical to the potent cytotoxicity, as the replacement of the α, β-unsaturated ketone moiety would result in significant loss of cytotoxicity. Kumar et al. reported that chalcone-triazole hybrid H3 could inhibit the growth of MCF-7, DU-145, and 1MR-32 cancer cells with IC 50 values in the range of 17.1-29.9 μM (Chinthala et al., 2015), in silico docking studies indicated that H3 might bind to DNA topoisomerase IIα. While hybrid H4 could exert α-glucosidase inhibitory activity (IC 50 67.77 μM), in silico docking studies indicated that H4 has a similar binding pattern to the known antidiabetic drug acarbose with α-glucosidase (Chinthala et al., 2015). Notably, the 1,2,3triazole ring might serve as a hydrogen bond acceptor to form two hydrogen bonds with Arg526, and it might also have π−π stacking interaction with Trp406.
By merging chloroquine (CQ) pharmacophore and chalcone by a triazole linker, Kumar et al. synthesized a potent hybrid derivative H14, which showed potent in vitro antiplasmodial activity against Plasmodium falciparum (CQR W2 strain, IC 50 114.1 nM) with low cytotoxicity (Hela cells, IC 50 36.5 μM; SI 311) . Further, with the trapping of the α, β-unsaturated ketone functionality of the hybrid by hydrazine hydrate, followed by acetylation with acetic acid, they have synthesized N-acetylpyrazoline derivative H15, which showed increased activity against Plasmodium falciparum (CQR W2 strain, IC 50 53.7 nM) and an excellent selectivity index (Hela cells, IC 50 42.7 μM; SI 795), indicating the α, β-unsaturated ketone functionality was not critical for antiplasmodial activity.
Coumarins are privileged natural products that possess a fascinating array of biological activities. Their synthetic derivatives have also been reported to exert various pharmacological activities such as anticancer, antiinflammatory, antibacterial, and antifungal activities, etc.
By using the SPAAC click chemistry of cyclooctyne (DBCO) and azide-coumarin, Paira et al. synthesized I8a and its regioisomer I8b with the aim of theranostic application (Sinha et al., 2016). In vitro data indicated that both I8a and I8b could exhibit maximum quantum yields and good uptake by MCF-7 cells, implying their potential for cancer diagnosis. Moreover, they could inhibit the growth of Hela (IC 50 17.5 μM) and MCF-7 (IC 50 9.83 μM) cancer cells with good selectivity.
Shults et al. reported that reoselone-triazole I10 could inhibit the growth of CEM-13, U-937, and MT-4 cancer cells with IC 50 values in the range of 8-10 μM (Lipeeva et al., 2015). Molecular docking studies indicated that I10 might bind to the active site of phosphodiesterase (PDE-4B) and showed good interactions with amino acid residues of PDE4B. Both the aryl-substituted triazole and the dihydrofurocoumarin were involved in the π−π stacking interaction with Phe446 (Lipeeva et al., 2015). Notably, the triazole ring of I10 might involve in the binding with sulfurs of Met347 and Met431 by forming π−sulfur interactions. Bahulayan et al. reported that some coumarin-containing macrocyclic derivatives, like I11, could also exert inhibitory activity against cancer cells (Raj and Bahulayan, 2017). Dharavath et al. reported that I12 (IC 50 1.29 μM) could exert comparable antioxidant activity to ascorbic acid (IC 50 1.46 μM) in DPPH assays (Dharavath et al., 2020). In vitro antiinflammatory data indicated that I12 (IC 50 15.90 μM/ml) could exert more potent activity than the reference drug diclofenac (IC 50 17.52 μM) in heat-induced hemolytic assays. The antibacterial activity evaluation results indicated that I12 could inhibit the growth of the tested Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gramnegative (Escherichia coli and Klebsiella pneumonia) bacterial strains at the concentrations of 10 or 20 μg/ml. Moreover, I12 could also exert antifungal activity against three fungal strains (Aspergillus niger, Aspergillus favus, and Fusariumoxy sporum) at the concentration of 50 μg/ml, and it was comparable to the reference drug clotrimazole.
Sagar et al. reported that triazole-N-glycoside-coumarin I14 could exert low micromolar (IC 50 10.97 μM) selective inhibitory activity against breast MCF-7 cancer cells (Kumari et al., 2019). Mechanistic studies indicated that I14 could promote the level of cellular reactive oxygen species (ROS), thereby inducing the generation of toxic products in MCF-7 cancer cells.
Podophyllotoxin is a natural lignin that is isolated from the roots of Podophyllum hexandrum. Podophyllotoxin and its derivatives could exert antiproliferative activity by inhibiting tubulin polymerization, while epipodophyllotoxins and its derivatives could inhibit topoisomerase II. So far, some podophyllotoxin-/epipodophyllotoxin-derived therapeutic agents such as teniposide and etoposide have already entered into clinical use for the treatment of cancer. Diversification of podophyllotoxin by click chemistry has been reported to generate derivatives with increasing inhibitory activity whereas reducing toxicity. For example, 4α-podophyllotoxin-triazole J1 could exert broad-spectrum inhibitory activity against a panel of cancer cell lines (A549, PC-3, MCF-7, U251, SKBR-3, and LNCaP) with IC 50 values in the range of 19.6-42.9 nM ( Figure 11). Moreover, it could effectively overcome drug resistance, and showed weak cytotoxicity in non-cancer cells. Preliminary mechanistic studies implied that J1 could interact with microtubule, arrest cell cycle (G2/M phase) and induce cell apoptosis in PC-3 cells (Hou et al., 2019c). Kamal et al. reported that epipodophyllotoxin-triazole J2 (IC 50 0.70-4.11 μM) could exert potent inhibitory activity against a panel of cancer cell lines (A549, MCF-7, DU-145, Hela, HepG2, and HT-29) with weak cytotoxic activity in normal NIH/3T3 cells (IC 50 89.04 μM) (Reddy et al., 2018). Mechanistic studies indicated that J2 could inhibit topoisomerase II, arrest cell cycle (G2/M phase), and effectively induce apoptosis of the tested DU-145 cells. Hui et al. reported that podophyllotoxin-triazole-coumarin hybrid J3 (IC 50 4.9-17.5 μM) could exert more potent inhibitory activity than the etoposide (IC 50 10.5-25.6 μM) in a panel of cancer cell lines including A549, HepG2, HeLa, and LoVo (Hao et al., 2019). Mechanistic studies indicated that J3 could bind to CT DNA, disrupt microtubules, arrest cell cycle (G1 phase) and inhibit Topo-II β.
Ferulic acid is an abundant phenolic phytochemical found in plant cell walls. Abid et al. reported that ferulic acid-triazole K1 could exert selective inhibitory activity against carbonic anhydrase IX (CA IX) (IC 50 24 nM) ( Figure 12) (Aneja et al., 2020). Further studies indicated that K1 could inhibit colony formation and cell migration, downregulate CA IX expression, decrease epithelial to mesenchymal transition (EMT), and induce apoptosis in HepG2 cancer cells.
Eugenol is the principal active component of clove oil. Eugenoltriazoles possess several biological activities such as anticancer and anti-parasitic activities. Morjani et al. reported that eugenoltriazole K2 could exert broad-spectrum antiproliferative activity against a panel of cancer cell lines including HT1080, A549, MCF-7, and MDA-MB-231 with IC 50 values in the range of 15.31-23.51 μM (Taia et al., 2020). Teixeira et al. reported that eugenol-triazole K3 could not only exert extracellular leishmanicidal activities (IC 50 7.4 μM) (Teixeira et al., 2018), but also intracellular leishmanicidal activities against leishmania parasites inside peritoneal macrophages (IC 50 1.6 μM) without interfering with the viability of macrophages (IC 50 211.9 μM; SI 132.5). Notably, it was more potent than clinical drugs glucantime and pentamidine. De Souza et al. reported that dihydroeugenoltriazole K4 (IC 50 42.8 μM) could exert comparable trypanocidal activity to the reference drug benznidazole against the epimastigote forms of Trypanosoma cruzi (T. cruzi., Y strain) with low toxicity (Souza et al., 2020). In vivo data showed that K4 (100 mg/kg, p. o.) could reduce more than 50% of the parasitemia in T. cruzi infected mice.
Bergenin, a dihydroisocoumarin, has been reported to have various biological activities such as anti-HIV, neuroprotective, and anticancer activities (Bajracharya, 2015). Babu et al. reported that derivative K5 could inhibit the growth of A549 (IC 50 1.86 μM) and HeLa (IC 50 1.33 μM) cells (Pavan Kumar et al., 2019), and that was comparable to doxorubicin. Mechanistic studies indicated K5 could arrest cell cycle (G2/M phase) and induce apoptosis in Hela cells. Moreover, it could inhibit the polymerization of tubulin and disrupt the balance of intracellular tubulin-microtubule. Yang et al. reported that K6 (IC 50 6.2-17.6 μM) could exert more potent inhibitory activity than the parental bergenin against EC9706, B16 and MGC803 cancer cells . In addition, it (IC 50 6.2 μM) could exert comparable inhibitory activity to 5-fluorouridine (IC 50 6.3 μM) in EC9706 cancer cells.
Arctigenin is a lignan that is isolated from the dry ripe fruit of Arctium lappa.  (Zhang H.-b. et al., 2018). By using sulfur (VI) exchange chemistry, Zhang et al. synthesized derivatives K8 and K9 (Zhang S. et al., 2019), and preliminary in vitro data indicated that these compounds could exert good anti-inflammatory activity.
Pterostilbene is a bioactive natural stilbenoid that is isolated from blueberries and Pterocarpus marsupium heartwood. Structurally, it is very similar to resveratrol, a healthy benefiting compound that is rich in red wine. Diversification of pterostilbene by CuAAC could generate derivatives with improved antibacterial activity. For example, derivative K13 could exert potent antibacterial activity against methicillin- .5-39 μg/ml, (Tang et al., 2019), while the MIC value of pterostilbene is 41-161.5 μg/ml. Mechanistic studies indicated that it could inhibit DNA polymerase, but not the bacterial cell membrane and cell wall. Combretastatin A-4 (CA-4) is a stilbenoid phenolic natural product that is isolated from the African willow tree, Combretum caffrum (Shan et al., 2011). It can exert potent reversible inhibitory activity in the polymerization of tubulin. Structural modifications of CA-4 have yielded several novel CA-4 derivatives with potent tubulin inhibitory activity. Taking advantage of the powerful sulfur (IV) fluoride exchange (SuFEX) click chemistry, Wu et al. synthesized the fluorine sulfonate CA-4 K14 (IC 50 8.9 μM), which could exert 70fold more potent inhibitory activity than the parental CA-4 against drug in HT-29 cells (IC 50 8.9 μM) .

CLICK CHEMISTRY-BASED MODIFICATION OF STEROIDS
Steroids are series of naturally occurring compounds that are ubiquitously distributed in animals, plants and fungi, etc. They can act as signaling molecules or as key components of cell membranes. Derivatization of steroids by click chemistry can quickly generate novel molecules with new functions for drug discovery.
Sarsasapogenin is one of the active ingredients that is isolated from Rhizoma anemarrhenae. Song et al. reported that sarsasapogenin-triazole L4 could inhibit the aggregation of Aβ 1-42 (IC 50 5.84 μM) (Wang W. et al., 2018). Moreover, in vitro data indicated that L4 could exert moderate neuroprotective effects against H 2 O 2 -induced neurotoxicity in SH-SY5Y cells. Further in vivo studies showed that L4 (17.5 mg/kg, p. o.) could significantly ameliorate cognitive impairments in behavioral tests, and it was comparable to or better than the reference drug cisplatin.

CLICK CHEMISTRY-BASED MODIFICATION OF XANTHONES AND QUINONES
Xanthones is a series of bioactive substance that can be readily obtained from plants and/or microorganisms. The key structural feature of these compounds is a biphenyl pyranone containing a planar three-ring system. Yu et al. reported that M1 could exert inhibitory activity against A549 cells (IC 50 32.4 μM) . Western blotting data indicated that M1 could significantly upregulate protein levels of caspase 3, Bax, c-Jun N-terminal kinase, and also p53 in A549 cells (Figure 14). Zhang et al. reported that gambogic acid-triazole M2 (IC 50 0.31-3.79 μM) could exert sustained cytotoxicity against a panel of cancer cell lines (U2OS, HepG2, A549, and HCT116) and two drug resistant cancer cell lines (Taxol-resistant or cisplatin-resistant A549 cells) with improved aqueous solubility and permeability . Notably, it could exert in vivo antitumor activity in A549-transplanted mice models. Quinone is a privileged pharmacophore that presents in many bioactive natural products, such as mitomycin, saintopin, and doxorubicin. Its various promising biological activities might attribute to i) its ability to generate ROS, which usually leads to the damage of DNA, and ii) its ability to electrophilic arylation of critical cellular nucleophiles. Thus, the derivatization of quinone by using click chemistry would quickly generate molecules with desirable functions.
Lawsone is a natural bioactive quinone that is isolated from genus Lawsonia. Alves et al. reported that Lawsone-glycosyl triazole M3 could exert promising inhibitory against SKBR-3 cells (IC 50 0.78 μM) with good selectivity index (normal HGF cell, IC 50 17.65 μM, SI 22.6) (Ottoni et al., 2020). It is more potent than lawsone (IC 50 > 50 μM), which could be ascribed to the introduction of the peracetylated D-glucose to the hybrid M3, thereby generating a more favorable lipophilic-hydrophilic balance and being absorbed by tumor cells more easily.
Anthraquinone has the structural core of anthracycline. Meng et al. reported that anthraquinone-triazole derivative M6 (IC 50 0.6 μM) could exert more potent inhibitory against xanthine oxidase_ a well-known target for the treatment of hyperuricemia and gout, than the reference allopurinol (IC 50 9.8 μM) . SAR studies revealed that the benzaldehyde moiety might play a more important role than the anthraquinone moiety in its inhibitory potency.
Due to the complex structure of rapamycin, CuAAC appears to be the best strategy for the generation of novel rapamycin derivatives. For example, C42 rapamycin-triazole N4 (IC 50 6.05-25.88 μM) could exert more potent inhibitory against H1299, MGC-803, H460, and Caski cancer cells than that of rapamycin (IC 50 18.74-35.13 μM) (Xie et al., 2016). Mechanism studies indicated that N4 could cause the change of cell morphological, and induce apoptosis of the tested Caski cells. Moreover, it could inhibit the mTOR signaling by downregulating mTOR phosphorylation and its downstream key proteins, P70S6K1 and S6. Thus, N4 may have the potential to serve as a new mTOR inhibitor. C28 rapamycin-triazole N5 (IC 50 12.8-14.8 μM) could also exert more potent inhibitory against A549, 769-P, ECA-109, and Caski cancer cells than rapamycin (IC 50 12.3-24.5 μM) (Huang Q. et al., 2018). Mechanistic studies indicated that N5 could inhibit the mTOR signaling by downregulating mTOR phosphorylation and its downstream key proteins such as P70S6K1 and 4EBP1.

CONCLUSION REMARKS AND FUTURE PERSPECTIVES
As natural products are usually complex molecules with little modification space and some of them even contain labile functionalities, the structural modification of natural products with the aim to optimize their drawbacks or the construction of natural product-like drug screening libraries are the most fascinating challenges in organic synthesis. Therefore, the development of synthetic toolboxes that facilitates efficient access to the molecular diversity and unique functions of natural products is highly desirable. One such, perhaps the most successful toolbox is click chemistry, which enables the ready synthesis of a diverse set of natural product derivatives, especially the 1,2,3-triazole derivatives of terpenoids, alkaloids, steroids etc., in a highly efficient manner. Beyond the optimization of the original biological activity and the improvement of kinetics and drug-like properties, many of these derivatives have been endowed with new functions, and thereby could serve as an inexhaustible source for discoveries in drug development. In addition, click chemistry, especially the CuAAC reaction, have also been widely used in the synthesis of homodimers or heterodimers of natural products even in the presence of labile functionalities, mainly due to their high orthogonality reaction properties as compared to other chemistries such as the synthesis of amides and esters. Nevertheless, to fully utilize the power of click chemistry in natural product-based drug discovery, there remain several issues and new directions for future research in the area. 1) One of the most important merits of click chemistry is modular synthesis, which can quickly generate diverse libraries of large numbers of new compounds. However, as we can see from Figure 1, there are usually only a limited number of click chemistry derivatives that have been synthesized and screened for their functions. Thus, it would be impossible to probe the desired chemical space to generate ideal hit compounds. The reason is probably that most of the click chemistry building blocks are commercially unavailable and must be prepared. Fortunately, a 2019 paper reported a perfect solution for the synthesis of various azides by using fluorosulfuryl azide as an efficient diazo transfer reagent (Meng et al., 2019). In the future, the rational design and synthesis of modular natural product building blocks with functionalities that can react with other click chemistry building blocks in large numbers would be a useful strategy to probe the large chemical space. 2) As we can see from Figure 1, about 68% of click chemistry natural product derivatives have only been selected for anticancer activity, and thus their other functions are missing. In the future, it is important to be aware of the selection of the multiple functions of the natural product click chemistry derivatives against different phenotypes or targets. 3) As most of the natural product-triazole derivatives were screened by phenotypic screening (91%, Figure 1), therefore, their exact molecular targets are ambiguous. In the future, the rational design of target-based selection systems for natural product click chemistry libraries will be an important research area. 4) Beyond CuAAC click chemistry, which generates 1,2,3triazole derivatives, some other emerging click chemistries like SuFEX chemistry have already shown their power in the generation of valuable hit molecules, and thus also could be used in natural product modification in the future. 5) Notably, another powerful hit screening technology DNAencoded library (DEL), and especially the natural product DNA-encoded library (nDEL), have already shown their power in the screening of some challenge protein targets Xie et al., 2020). So, if we can connect natural products with DNA-encoded libraries and diversify them by click chemistry, we could quickly generate a huge natural product derivative library with unprecedented skeleton diversity. In addition, as DEL selection is affinitybased screening, the exert molecular target of the identified hit compounds are clearly after they have been deconvoluted from the screened DEL library. 6) Because most of the natural product click chemistry derivatives were only tested in in vitro assays, their metabolic, pharmacodynamic, and toxicity profiles should be carefully studied in the future. For example, a recent paper reported that 1H-1,2,3-triazole containing anticancer chemotherapeutic might potentially lead to cardiotoxicity by the impairment of mitochondria (Stephenson et al., 2020).

AUTHOR CONTRIBUTIONS
HX, SZ, and BL conceived the research. HX, SZ, and BL designed the structure of the paper. XZ, SZ, and XW drafted the manuscript. HX, SZ, and BL provided critical revision of this article. HX, SZ, and BL supervised the findings of the work and approved the manuscript for submission. All authors agreed with the final version of this manuscript.

FUNDING
Part of this work is supported by the National Natural Science Foundation of China, China (No. 21977070 and U19A2011).