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. 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  Newman ade 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” , 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 , have al tracers .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 , anti-baThe 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.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 chiral centers, rigid skeleton, diverse functionalities, and the consequent various promising biological activities. Moreover, some of the terpenoids are readily available from nature, thereby can serve as inexhaustible starting materials for the synthesis of bioactive natural products or natural product-like drug screening libraries . By archGardenia jasminoides Ellis, has been reported to possess various biological activities such as anticancer and antioxidant. The introduction of triazole moiety into genipin has been demonstrated to generate derivatives with improved cytotoxicity. For example, C10 genipin-triazole a1  and a2  could exert more potent inhibitory activity than the parental genipin against tumor cells  .Genipin , an irid> 20 μM) . Molecul genipin , Asn-β-2Magnolia grandiflora, could exert moderate inhibitory activity against Bel7402, HCT-116, PANC1, A549, and U87 cancer cell lines (IC50 = 4.93–10.86 μM). Whereas, C13 triazole containing derivative a3 could exert improved inhibitory activity (IC50 = 0.43–1.54 μM), . Preliminary mechanistic studies indicated that a3 could induce apoptosis and inhibit the proliferation and migration of the tested HCT-116 cells. Crooks et al. reported that melampomagnolide B-triazole a4 could exert low micromolar to submicromolar (GI50 = 0.02–1.86 μM) inhibitory activity against the growth of a panel of 60 cancer cell lines derived from different organs including lung, CNS, leukemia, colon, renal, melanoma, ovary, prostate and breast cancer cells (50 = 0.4–0.7 μm) inhibitory activity against two clinical AML specimens. In addition, salt a5, a water-soluble prodrug of a4, could also exert comparable inhibitory activity as a4 in primary AML cells. Whereas, the C13 melampomagnolide B-triazole derivatives generally showed reduced inhibitory activity, with only a6 (GI50: 46 – 76 μM) showed marginal inhibitory activity against the tested cancer cell lines , implying the a-methylene-c-lactone motif may play a key role in retaining the potent cytotoxicity , , and esper cells , and esptoxicity .α-methylene-c-lactone motif of costunolide and dehydrocostus by sodium azide, followed by reaction with various terminal alkynes under CuAAC. In vitro data indicate that costunolide-triazole-coumarin a7 (IC50 = 0.12 μM) could exert more potent cytotoxic activity than the precursor costunolide (IC50 = 0.56 μM) in MDA-MB-231 cells (50 = 0.16 μM) could exert more potent cytotoxic activity than dehydrocostus-lactone (IC50 = 0.56 μM) in MDA-MB-231 cells, while derivative a9 (IC50 = 0.68 μM) showed more potent cytotoxic activity than dehydrocostus-lactone (IC50 = 4.1 μM) in IMR-32 cells  could exert slightly better inhibitory activity than clinical drug isoniazid (MIC = 2.9 μM) against Mycobacterium tuberculosis (Mtb)  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 acid-triazole hybrid a12 could exert selective anti-Toxoplasma gondii activity with a good selectivity index , 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) (Usnic acid is a dibenzofuran secondary metabolite that is isolated from lichen genera. Usnic acid-triazole-saccharin hybrid a10 (MIC = 2.5 is (Mtb) , but fai = 0.96) .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  could exert more potent inhibitory activity than b2  and b3   , indicat= 16 μM) .50: 2.5–4.7 μM) could exert more potent inhibitory activity than its regio-isomer b5 (IC50: 13.5–19.9 μM) and dihydro artemisinin  against a panel of cancer cell lines including P388, MCF-7, HL-60, and LU-1 cells, indicating the position of the amide substituent on the benzene was critical to the potent inhibitory activity (50 = 4.06–36.65 μM) by arresting cell cycle (G2/M phase) and inducing apoptosis in lung and skin cancer cells  and b8  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 HCT-166  and MDA-MB-231 cells , 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 (IC50 = 16.5 μM) could exert more potent antiproliferative activity than artesunate (IC50 = 78.5 μM) against KB cancer cells , C2   could exert more potent anti-proliferative activities than that of oridonin (IC50 = 16.28–24.80 μM)  and C5 (IC50 = 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 (50 = 0.6–5.0 μM) could exert potent anti-proliferative activities on several cancer cell lines  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  (Oridonin is an 3.83 μM)  (Shen et3.83 μM) , C3 (PC-4.80 μM) . In addi03 cells . Later, 21 cells . Due to 0.48 μM) , implyin50 = 0.7–1.2 μM) (50 = 5.90 μM) could exert comparable inhibitory activity to the reference drug cisplatin against HepG2 cells (IC50 = 6.42 μM) (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas fluorescens) strains (MIC = 1.6–3.1 mg/ml) with good drug-like properties and low cytotoxicity in noncancerous mammalian cells   (Hou et –1.2 μM) . Mechani6.42 μM) , and deran cells .γ-lactone-triazole D4 could exert moderate inhibitory activity on MRC-5 (IC50 = 45.1 μM) and AGS (IC50 = 39.2 μM) cancer cells (μg/ml) could inhibit the growth of C. neoformans at the concentration of 250 μg/ml . 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  could exert slightly better inhibitory activity than cisplatin (IC50 = 3.906 μM) against HCT-116 cells  (Kaurenoic acid (KA) is a kaurene diterpene that can be isolated from the fruits of = 53 μM) , togetheE)-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 IC50 values of 8 μM.. (E)-labda is isolated from fresh rhizomes of Curcuma amada. Somappa et al. reported that labdane triazole derivatives D11a (IC50 = 0.75 μM) and D11b (IC50 = 0.77 μM) could exert excellent pancreatic lipase (PL) inhibitory activity, slightly better than the reference drug orlistat (IC50 = 0.8 μM) (Both andrographolide and ( of 8 μM.. In sili 0.8 μM) .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.Tripterygium wilfordii and/or Triptergium regelii. SAR studies indicated that its quinone methide moiety plays a key role in exerting various promising biological activities, thereby its structural modification was mainly conducted at C28 carboxyl group (50 = 0.97 μM), HCT-116 (IC50 = 0.78 μM) and BEL-7402 (IC50 = 0.63 μM) cancer cell lines  and exert low micromolar inhibitory activity against TNF-α-induced NF-κB activation (IC50 = 0.14 μM) (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  by at least partial inhibition of the activity of NF-κB, as well as cell apoptosis and migration.Asiatic acid (AA) is a pentacyclic triterpenoid that was identified from the tropical herb medicine 0.14 μM) . Moleculh PHE307 . NotablyOlea europea L.). It is reported that the introduction of triazole into maslinic acid could promote its anti-inflammatory activity  is more potent than E4 . Regarding the bistriazole derivatives, bis-3,28-disubstituted triazoles were more potent than the bis-2,28-disubstituted triazoles, with derivative E5 showed the highest inhibitory activity . While 2,3,28-trisubstituted hybrids were the most potent series of all the triazole derivatives. Among them, E6 showed the highest potency . These data indicated that both the number and the position of the triazole were critical to the promising anti-inflammatory activity.Maslinic acid is a pentacyclic triterpenoid that is isolated from pomace olive  could inhibit the growth of Leishmania infantum with a higher selectivity index as compared to the commercial drug potassium antimonyl tartrate trihydrate   (In vitro antiproliferative activity data indicated that E9a (IC50 = 3.2–4.0 μM), E9b (IC50 = 3.1–4.0 μM), and E9c (IC50 = 3.2–4.1 μM) could exert potent inhibitory activity against a panel of human cancer cell lines including 518A2, A2780, A549, HT-29, MCF-7, and 8505C, and are at least eight times more potent than the parental hederagenin, while E9d could exert selective inhibitory activity against HT-29 cells (IC50 = 1.6 μM) with low toxicity in normal NIH 3T3 cells.Hederagenin is an oleane-type pentacyclic triterpenoid that can be isolated in large quantities from I = 0.1) , highligI = 178) . C28 HedI = 178) . In vitr50 = 4.97 μM) and HT1080 (IC50 = 3.51 μM) cancer cells. Meanwhile, its C3 saponin triazole relative, C28 hederacolchiside-triazole E11 (IC50 = 0.54–2.66 μM) could exert more potent inhibitory activity than 5-fluorouracil (IC50 = 8.45–69.07 μM) against a panel of cancer cell lines including PC3, HT29, HepG2, A549, HL60, and U937  could exert more potent inhibitory activity than betulinic acid and 5-fluorouracil in HT-29 cells  (50 = 1.3 μM) could exert more potent antiproliferative than betulinic acid (IC50 = 11.5 μM) in HL-60 cells (leukaemia) (50 = 0.38 μM) and HepG2 (IC50 = 1.32 μM) cancer cells (50 > 400 μM for both cell lines), implying the added value of merging betulin and AZT into a single hybrid.Betulinic acid is a pentacyclic lupane-type triterpenoid that is isolated from the stem bark of 29 cells . Mechani = 7 μM) . Mechani = 7 μM) . Shi et ukaemia) . Kiem eter cells . ClearlyCommiphora mukul. By modification of C2 methene and C3 keto, Uppuluri et al. synthesized 27 myrrhanone C-triazoles (In vitro data indicated that E17 could selectively inhibit the growth of DU-145 (IC50 = 13.8 μM) and HepG2 (IC50 = 9.332 μM) cancer cells, while E18 (IC50 = 6.16–9.59 μM) could exert pan inhibitory activity against a panel of cancer cell lines . Both of them were more potent than the precursor myrrhanone C (IC50 = 12.02–26.61 μM). Mechanistic studies indicated that E18 could arrest cell cycle (G2/M phase) and induce cell apoptosis.Myrrhanone C is a natural bicyclic triterpene that is isolated from the gum resin of riazoles . In vitrAlkaloids 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.Sophora flavescens Ait , 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 (50 = 5.01–7.31 μM) could exert broad-spectrum anticancer activities against a panel of cancer cell lines . Notably, F1 is more potent than the combination of matrine and chalcone (IC50 > 50 μM), and also 5-fluotouracil (IC50 = 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 A549-xenografted nude mouse model (10 mg/kg) with no apparent cytotoxicity.Matrine is a quinolizidine alkaloid that is isolated from the root of er cells   could exert more potent inhibitory activity than rucaparib (IC50 = 4.91–13.51 μM) and harringtonine (IC50: 10.55–11.71 μM) in A549, HCT-116, and MCF-7 cancer cells (50 = 0.23–1.13 μM) could exert more potent inhibitory activity than rucaparib (IC50 = 2.58–13.82 μM) in a panel of cancer cell lines   . Mechanicinchona 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   showed the highest potential in MC-4-11 cells, while, F4b (IC50 = 1.2 μM) showed the highest potential in HT-29 cells.Quinine is one of the most abundant natural  and F6) . In vitr50 = 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 (50 = 27 nM) could exert more potent in vitro antimalarial activity than quinine (IC50 = 58 nM) against P. falciparum strain 3D7  and antileishmanial activities  with no apparent adverse effects -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 (IC50 = 30 nM) could exert more potent inhibitory than 20(S)-camptothecin (IC50 = 170 nM) against A549 cancer cells in a Topo I-dependent manner  could exert more potent inhibitory activity than colchicine (IC50 = 13.2–20.4 nM) in a panel of cancer cell lines  (Colchicine is a well-known antimitotic agent that is isolated from nd MES1) . In addiBerberis plants. Berberine-triazoles could not only exert anticancer activity but also exhibit antimalarial activity. Sun et al. reported that F13 could inhibit the growth of SW-1990 (IC50 = 22.2 μM) and SMMC-7721 (IC50 = 14.9 μM) cancer cells (50 = 0.142 μM) could exert antimalarial activity against P. falciparum (3D7) strain with no apparent cytotoxicity in human PC-3 cells (IC50 > 200 μg/ml)  .50 = 8 μM, CC50 = 168 μM, SI = 20) .Hybridization of two different natural products by CuAAC is an efficient strategy to generate novel functional compounds. For example, cytisine-triazole-camphor F15 could exert antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) with low toxicity and good selectivity index (ICSI = 20) . Notably50 values in the range of 1.2–2.3 μM (50 = 1.89–4.89 μm)  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).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 IC2–2.3 μM . In sili4.89 μm) , while hPhenylpropanoids, 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 , implyin50 = 14.9–56.8 μM) could exert potent antioxidant activity in DPPH (IC50 = 30.6 μM) and ABTS+ (IC50 = 9.1 μM) assays (50 = 290.9 μM). Yang et al. reported that chrysin-triazole hybrids G2 (IC50 = 1.02–7.30 μM) could exert more potent inhibitory activity than the parental chrysin (IC50 = 16.30–73.02 μM) in several cancer cell lines  (50 = 17.9 μM) (50 = 10 μM) (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  via the suppression of oxidative damage  assays  (Mistry ) assays , and modGC-9701) . Rao et 17.9 μM) . Chen et= 10 μM) . Mechanind MCF-7 . Mechanie damage .. 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, anti-inflammatory and antiviral activities, etc. Thus, hybridization of chalcone scaffold with another pharmacophore by click chemistry may generate derivatives with valuable therapeutic functions.Chalcones, also known as chalconoids, are a series of natural polyphenols50 values in the range of 4–11 μM , in silico docking studies indicated that H4 has a similar binding pattern to the known antidiabetic drug acarbose with α-glucosidase  and H6 (IC50 = 1.53–2.73 μM) could exert low micromolar antiproliferative activity against SK-N-SH, HepG-2, and MGC-803 cancer cells (50 = 7.22–10.32 μM). Mechanistic studies indicated that H6 could arrest cell cycle (G1 phase) and induce apoptosis of SK-N-SH cancer cells.Liu et al. reported that hybrid H5 (ICer cells . Notably50 = 0.02 μM) and MDA-MB-231 (IC50 = 0.31 μM) with low toxicity in non-tumorigenic MCF-10a epithelial cells (IC50 = 139.29 μM)  .β-carboline-chalcone derivative H8 (IC50 = 21.99 μM) could inhibit the growth of MDA-MB-231 cells (50 = 75 μM), while H9 (IC50 = 10.33 μM) could inhibit the growth of MCF-3 cells and was five times more potent than tamoxifen. Molecular docking results indicated that they could fit well into the ERα ligand binding domain. The analysis of the H9- ERα complex indicated that both direct and water-mediated hydrogen bond interactions of triazole and tetrahydro-β-carboline with residues  crucial to estrogenic inhibitory activity might play a key role in exerting the potent activity  and Gram-negative  bacterial strains at concentrations of 75 and 100 μ/ml  could exert potent antiplasmodial activity against the erythrocytic stages of P. falciparum (3D7 strain) with no apparent cytotoxicity  (μg/ml) could exert comparable antibacterial activity to ciprofloxacin  against a panel of bacterial strains  with low cytotoxicity  (μg/ml) could exert potent antifungal activity with low cytotoxicity   , while b0 mg/ml) , and H130 mg/ml) .in vitro antiplasmodial activity against Plasmodium falciparum  with low cytotoxicity   and an excellent selectivity index , indicating the α, β-unsaturated ketone functionality was not critical for antiplasmodial activity.By merging chloroquine (CQ) pharmacophore and chalcone by a triazole linker, Kumar et al. synthesized a potent hybrid derivative H14, which showed potent I = 311) . Further50 = 13.3 μM; COX-2, IC50 = 0.037 μM; SI = 359.46) as well as good 15-LOX inhibitory activity (IC50 = 1.95 μM)  . SAR stuIn vitro antibacterial and antifungal data indicated that H17a could exert more potent antibacterial activity in both Gram-positive  and Gram-negative  bacterial strains than the reference drug ciprofloxacin , while derivative H17b  could exert more potent antifungal activity than the reference drug fluconazole .Lal et al. synthesized a series of dehydroacetic acid chalcone-triazole hybrids, in which the A ring was replaced by dehydroacetic acid (DHA) . In vitrCoumarins 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, anti-inflammatory, antibacterial, and antifungal activities, etc.50 = 0.9 μM) with low toxicity  (Enterococcus species (MIC = 16 μg/ml). Moreover, it could also inhibit the growth of clinically derived vancomycin-resistant Enterococcus faecium (MIC = 64 μg/ml), whereas the reference antibiotics ceftazidime (CAZ) and ciprofloxacin (CIP) were inactive (MIC >256 μg/ml) (50 = 9.7–41.6 μM)    . Mechani6 μg/ml) . The sam41.6 μM) .hypoxia50, = 0.03 μM; ICnormoxia50, = 1.34; SIhypoxia/normoxia = 46.31) (50 = 0.6 μM) and cisplatin (IC50 = 4.68 μM), respectively, whereas the parental 4-hydroxycoumarin was inactive at the concentration of 100 μM.Guo et al. reported that the ether tethered triazole-coumarin I4 could potently inhibit the growth of MDA-MB-231 cells under hypoxia conditions (IC= 46.31) , and it 50 = 4.96–36.84 μM), and that was comparable to or more potent than that of 5-fluorouracil (IC50 = 7.01–27.07 μM)  .50 = 8.5–29 μM). A subtle change of the position of the trifluoromethyl from meta-to para-position (I6b) led to the inactivity of the compound against Capan-1 and PANC-1 cells (IC50 > 100 μM), but slightly increased cytotoxic activity against MIA PaCa-1 cells (IC50 = 9.6 μM), implying the trifluoromethyl may play a key role in exerting the selective inhibitory activity against MIA PaCa-1 cells  could exert more potent inhibitory activity than colchicine  against PC3 and MGC803 cancer cells (μM) could inhibit tubulin polymerization by interacting with the colchicine binding site.Zhang et al. reported that C7 modified hybrid I7  and MCF-7 (IC50 = 9.83 μM) cancer cells with good selectivity.By using the SPAAC click chemistry of cyclooctyne (DBCO) and azide-coumarin, Paira et al. synthesized I8a and its regio-isomer I8b with the aim of theranostic application . In vitrAmmi majus L. Quan et al. reported that xanthotoxin-triazole I9 could exert potent antiproliferative activity against AGS cells (IC50 = 7.5 μM) with low toxicity  (50 > 100 μM) and the reference drug 5-fluorouracil (IC50 = 29.6 μM).Xanthotoxin is a furanocoumarin identified from the traditional Egyptian medicinal plant  100 μM) , and tha50 values in the range of 8–10 μM  could exert comparable antioxidant activity to ascorbic acid (IC50 = 1.46 μM) in DPPH assays (In vitro anti-inflammatory data indicated that I12 (IC50 = 15.90 μM/ml) could exert more potent activity than the reference drug diclofenac (IC50 = 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 Gram-negative (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  at the concentration of 50 μg/ml, and it was comparable to the reference drug clotrimazole.Dharavath et al. reported that I12  with low cytotoxicity (human hepatoma cell (huh7), CC50 > 100 μg/ml)  . MechaniN-glycoside-coumarin I14 could exert low micromolar (IC50 = 10.97 μM) selective inhibitory activity against breast MCF-7 cancer cells  could exert comparable antibacterial activity to the reference drug ciprofloxacin (MIC = 1 μg/ml) in the tested Gram-positive (Staphylococcus aureus) and the Gram-negative (Pseudomonas aeruginosa) bacterial strains  and I18 (MIC = 3.8 μM) could exert more potent antibacterial activity than the reference drugs pyrazinamide (MIC = 25.2 μM), streptomycin (MIC = 10.7 μM), and ciprofloxacin (MIC = 9.4 μM) against M. tuberculosis (H37Rv)  .S. aureus 209p, MIC = 0.16 μM; S. aureus C-18, MIC = 0.65 μM) could exert 6- and 10-fold more potent antibacterial activity than the reference drug ceftriaxone  against Staphylococcus aureus 209p and C-18 bacterial strains (μM) could exert a 5-fold more potent antibacterial activity than ceftriaxone (MIC = 1.03 μM) against Staphylococcus aureus “Viotko” bacterial strain.Shults et al. reported that I19  by inhibiting the polymerization of the tubulin (IC05= 1.06 μM) (50 = 0.82–4.68 μM) by inhibiting the polymerization of the tubulin (IC50= 1.55 μM)  . The sam1.55 μM) .β-secretase (BACE1) inhibitors   could exert selective BuChE inhibitory activity (50 = 9.4 μM). SAR studies indicated that its selectivity (AchE/BuChE) might result from neither decursinol nor triazole, but the hybrid derivative I24. β-Secretase (BACE1), a transmembrane aspartic protease, is an attractive target for the development of AD therapeutic drugs as it is critical in trigging the amyloidogenic pathway. Miri et al. reported that iminochromene-triazole I25 (β-secretase (BACE1) inhibitor, could exert promising BACE1 inhibitory activity (IC50 = 2.2 μM) with no apparent cytotoxicity. Notably, it could exert a 10-fold more potent neuro protective effect (IC50 = 7.9 μM) than the reference caffeic acid (IC50 = 75.8 μM) in Aβ induced toxicity in pC12 neurons. Foroumadi et al. synthesized a dual AChE (IC50 = 3.4 μM) and BuChE (IC50 = 1.1 μM) inhibitor I26 . Considering that indirectly enhancing endocannabinoid signaling by FAAH inhibitors might be preventing or slowing the progression of neurodegenerative disorders such as Alzheimer’s disease, I27 may be a valuable candidate for AD treatment.For a long time, the great potential of coumarin-triazole hybrids in anti-Alzheimer’s disease drug discovery has been well demonstrated by the target-based screening of acetylcholinesterase (AChE) , butyrylhibitors . For exa 100 μM) . And alsactivity , and thazole I25 , a non-pitor I26 , which cized I27 , a dual 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  with IC50 values in the range of 19.6–42.9 nM (50 = 0.70–4.11 μM) could exert potent inhibitory activity against a panel of cancer cell lines  with weak cytotoxic activity in normal NIH/3T3 cells (IC50 = 89.04 μM) (J3 (IC50 = 4.9–17.5 μM) could exert more potent inhibitory activity than the etoposide (IC50 = 10.5–25.6 μM) in a panel of cancer cell lines including A549, HepG2, HeLa, and LoVo  and inhibit Topo-II β.Podophyllotoxin is a natural lignin that is isolated from the roots of –42.9 nM . Moreove–42.9 nM . Kamal e9.04 μM) . Mechaniand LoVo . Mechaniα-D-(+)-galactosyl residue could exert promising inhibitory activity against a panel of cancer cell lines  with IC50 values in the range of 0.49–6.70 μM,  could exert more potent inhibitory activity than cisplatin (IC50 = 1.67–10.85 μM) in several cancer cell lines including HL-60, SMMC-7721, A549, MCF-7 and SW480 with low toxicity   .50 = 24 nM)  (IC= 24 nM)  (Aneja e= 24 nM) . Further50 values in the range of 15.31–23.51 μM (50 = 7.4 μM) (50 = 1.6 μM) without interfering with the viability of macrophages . Notably, it was more potent than clinical drugs glucantime and pentamidine. De Souza et al. reported that dihydroeugenol-triazole K4 (IC50 = 42.8 μM) could exert comparable trypanocidal activity to the reference drug benznidazole against the epimastigote forms of Trypanosoma cruzi  with low toxicity  could reduce more than 50% of the parasitemia in T. cruzi infected mice.Eugenol is the principal active component of clove oil. Eugenol-triazoles possess several biological activities such as anticancer and anti-parasitic activities. Morjani et al. reported that eugenol-triazole K2 could exert broad-spectrum antiproliferative activity against a panel of cancer cell lines including HT1080, A549, MCF-7, and MDA-MB-231 with IC23.51 μM . Teixeir 7.4 μM) , but alstoxicity . In vivo50 = 1.86 μM) and HeLa (IC50 = 1.33 μM) cells (50 = 6.2–17.6 μM) could exert more potent inhibitory activity than the parental bergenin against EC9706, B16 and MGC803 cancer cells (50 = 6.2 μM) could exert comparable inhibitory activity to 5-fluorouridine (IC50 = 6.3 μM) in EC9706 cancer cells.Bergenin, a dihydroisocoumarin, has been reported to have various biological activities such as anti-HIV, neuroprotective, and anticancer activities . Babu etM) cells , and thaer cells . In addiArctium lappa. Quan et al. reported that arctigenin-triazole K7 could exert more potent and selective anti-Toxoplasma gondii activity  than both of the lead arctigenin  than the reference drug spiramycin .(in vitro data indicated that these compounds could exert good anti-inflammatory activity.Arctigenin is a lignan that is isolated from the dry ripe fruit of  = 0.72).. By usin = 0.72)., and pre50 = 0.24–12.8 μM). Particularly, it could exert submicromolar inhibitory activity against SIAH cells (IC50 = 0.24 μM), (50 = 51.2–66.8 μM). Mechanistic studies indicated that it could stall cell cycle (G2/M phase) and promote tubulin polymerization in the tested HeLa cells.Babu et al. reported that dibenzoclooctene type natural product gomisin B-triazole K10 could exert broad-spectrum antiproliferative activity against a panel of cancer cell lines including A549, DU-145, MDA-MB-231, PANC1, IMR32, and SIHA cells (IC.24 μM), , and it Magnolia denudate. Replacement of the tetrahydrofuran ring of machilin with 1,2,3-triazole, the resulting mimic K11 (IC50 = 1.1 μM) could exert 8-fold more potent inhibitory activity than the recommended drug pentamidine (IC50 = 8.9 μM) against promastigote form of L. amazonensis  showed a higher selectivity index than that of pentamidine , while derivative K12 could exert selective inhibitory activity against Trypanosoma cruzi trypomastigotes with an IC50 value of 28.6 μM and SI value of 29.6.Machilin G is a natural lignan that is isolated from zonensis . MoreovePterocarpus 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-resistant Staphylococcus aureus (MRSA) with an MIC value of 1.2–2.4 μg/ml and a minimum bactericidal concentration (MBC) of 19.5–39 μg/ml, , which could exert 70-fold more potent inhibitory activity than the parental CA-4 against drug in HT-29 cells (IC50 = 8.9 μM) (Combretastatin A-4 (CA-4) is a stilbenoid phenolic natural product that is isolated from the African willow tree,  caffrum . It can  8.9 μM) .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.50 = 4.06 μM) to the reference drug 5-fluorouracil (IC50 = 3.26 μM) in SH-SY5Y cancer cells. Mechanistic studies indicated that L1 could potently inhibit LSD1 (C50 = 3.18 μM), decrease mitochondrial membrane potential, arrest cell cycle (G2/M phase), and induce cell apoptosis. Notably, L1 is the first steroid-based lysine-specific demethylase (LSD1) inactivator. Quan et al. reported that C16 dehydroepiandrosterone-triazole L2 could inhibit the growth of HepG-2 (IC50 = 9.18 μM) and MCF-7 (IC50 = 9.18 μM) cancer cells by arresting cell cycle (G2 phase) and inducing cell apoptosis (α-estrone-triazole L3 (IC50 = 2.6–6.5 μM) could exert broad-spectrum antiproliferative activities against a panel of cancer cell lines including HeLa, MCF-7, A431, A2780, T47D , MDA-MB-231 (expressing HER2 and estrogen receptor) and triple-negative MDA-MB-361 (50 = 1.3–19.1 μM). Mechanistic studies indicated that L3 could induce apoptosis by the intrinsic pathway.Dehydroepiandrosterone is a unique active substance found in sweet potato and yam. Dehydroepiandrosterone derivatives containing triazole at the C3 and/or C16 could exert antiproliferative effects. By molecular hybridization of dehydroepiandrosterone and isatin, Liu et al. synthesized hybrid L1  Yu et a, which cpoptosis . MernyákA-MB-361 , and it Rhizoma anemarrhenae. Song et al. reported that sarsasapogenin-triazole L4 could inhibit the aggregation of Aβ1-42 (IC50 = 5.84 μM)  could significantly ameliorate cognitive impairments in behavioral tests, and it was comparable to or better than the reference drug cisplatin.Sarsasapogenin is one of the active ingredients that is isolated from 5.84 μM) . MoreoveDioscorea deltoidei. Structurally, it is similar to cholesterol and other steroids. Ara et al. reported that diosgenin-triazole hybrids L5a (IC50 = 5.54–10.33 μM), L5b (IC50 = 5.77–8.67 μM) and L5c (IC50 = 6.33–9.44 μM), bearing simple phenyl moiety at the C4’ of the triazole moiety, could inhibit the growth of A549, HCT-116, HT-29, and HBL-100 cancer cell lines  could exert potent inhibitory activity in a panel of cancer cell lines including A549, HeLa, HCT116, K562, K562-Tax (Paclitaxel resistant), CCRF-CEM (childhood T acute lymphoblastic leukemia), U2OS, and HCT116p53/(null p53 gene) by inhibiting tubulin polymerization  (50 = 4.8–5.5 nM) could exert more potent inhibitory activity than fulvestrant (IC50 = 7.7–14.8 nM). Notably, its inhibitory activity was ER– dependent . The biological activities of the other SuFEx click chemistry steroid derivatives are yet to report.Taking advantage of the emerging SuFEx click chemistry, several steroid derivatives such as fluorosulfate L7 , iminosuEA, DBU) . Among t50 = 32.4 μM) (50 = 0.31–3.79 μM) could exert sustained cytotoxicity against a panel of cancer cell lines  and two drug resistant cancer cell lines (Taxol-resistant or cisplatin-resistant A549 cells) with improved aqueous solubility and permeability  . Western32.4 μM) . Zhang eeability . NotablyQuinone 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.genus Lawsonia. Alves et al. reported that Lawsone-glycosyl triazole M3 could exert promising inhibitory against SKBR-3 cells (IC50 = 0.78 μM) with good selectivity index  (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.Lawsone is a natural bioactive quinone that is isolated from  = 22.6) . It is m50 = 8.02–26.12 μM), (50 = 10.87–18.63 μM). Mechanistic studies implied that M4 could arrest cell cycle (G0/G1 phase) and induce apoptosis in MCF-7 cancer cells. While, naphthoquinone-triazole M5 (IC50 = 6.8–10.4 μM) could exert broad-spectrum inhibitory activity against HT-29, MOLT-4, and MCF-7 cancer cell lines , , and thall lines , and cou50 = 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 (IC50 = 9.8 μM)  . SAR stu50: 81.81–99.56 μM) and its regioisomer 1,2-furanaphthoquinone-triazole hybrid M8 (IC50: 23.04–41.10 μM) could only exhibit moderate inhibitory activity against MDA-MB-231 and CaCo-2 cancer cells (50: 0.74–1.77 μM) could exert superior cytotoxic activity against HCT-116 and MCF-7 cancer cells (50: 0.07–0.29 μM) and M11 (IC50: 0.07–0.38 μM) showed high activity against HL-60, PC3, HCT-116, SF295, OVCAR-8, and MDA-MB-435 cancer cell lines (50 = 0.41–1.59 mM) bearing an pyran fragment also showed good inhibitory potency against PC3, HL-60, SF-295, HCT-116 and MDA-MB-435 cancer cells  and N2 (IC50 = 0.69 μM), both bearing a zinc chelating hydroxamate moiety, could exert potent antiproliferative activity against MCF-7 cancer cells, while hybrid N2, which contains a para zinc chelating hydroxamate moiety, could exert potent anti-inflammatory activity .Peptidic macrocyclic histone deacetylases inhibitors (HDACi) contain diverse cap groups that are capable of producing the optimal interactions with amino acid residues that surround the entrance of the HDAC active site, thereby modulating the biological activities of these HDACis. Although they usually could exert nanomolar inhibitory activity against HDACs, their further clinical development has been hindered greatly due to the difficulty in the synthesis of cyclic peptide frameworks for SAR studies. With the aim to solve these drawbacks, Oyelere et al. chose 14-membered macrolide clarithromycin to mimic the peptidic framework of macrocyclic HDACis’. The results indicated that macrolide is a good mimic of the peptide framework. For example, hybrids N1, N2, and N3, could exert moderate HDAC-6 inhibitory activity with IC 6.99 μM   could exert more potent inhibitory against H1299, MGC-803, H460, and Caski cancer cells than that of rapamycin (IC50 = 18.74–35.13 μM)  could also exert more potent inhibitory against A549, 769-P, ECA-109, and Caski cancer cells than rapamycin (IC50 = 12.3–24.5 μM)  . Mechani24.5 μM) . MechaniStreptomyces ambofaciens. Spiramycin-triazole-N-acetylsaccharide N6 could exert potent antibacterial activity against a panel of bacterial strains  with MIC values in the range of 1–4 μg/ml (50 = 0.29 μM) and Caco2 (IC50 = 0.44 μM) cancer cells (50 = 0.60 μM) (50 = 0.32–12.99 μM).Spiramycin is a natural antibiotic that is produced by –4 μg/ml . Salinomer cells , and the0.60 μM) . Notably1) 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 2) As we can see from 3) As most of the natural product-triazole derivatives were screened by phenotypic screening 91%, , therefo4) Beyond CuAAC click chemistry, which generates 1,2,3-triazole 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.nDEL), have already shown their power in the screening of some challenge protein targets (5) Notably, another powerful hit screening technology DNA-encoded library (DEL), and especially the natural product DNA-encoded library  Because most of the natural product click chemistry derivatives were only tested in chondria .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.


N-(benzo[d]thiazolyl) substituent, different combinations of N—H⋯O, N—H⋯N, C—H⋯O and C—H⋯N hydrogen bonds and Br⋯Br interactions lead to supramolecular assemblies in one, two and three dimensions.In three amides, each containing a  N-benzo[d]thiazole substituents. In N-(benzo[d]thiazol-6-yl)-3-bromobenzamide, C14H9BrN2OS, where the two ring systems are nearly parallel to one another [dihedral angle = 5.8 (2)°], the molecules are linked by N—H⋯O and C—H⋯N hydrogen bonds to form ribbons of R33(19) rings, which are linked into sheets by short Br⋯Br interactions [3.5812 (6) Å]. N-(6-Methoxybenzo[d]thiazol-2-yl)-2-nitrobenzamide, C15H11N3O4S, crystallizes with Z′ = 2 in space group Pna21: the dihedral angles between the ring systems [46.43 (15) and 66.35 (13)°] are significantly different in the independent molecules and a combination of two N—H⋯N and five C—H⋯O hydrogen bonds links the molecules into a three-dimensional network. The molecules of 5-cyclopropyl-N-(6-methoxybenzo[d]thiazol-2-yl)isoxazole-3-carboxamide, C15H13N3O3S, exhibit two forms of disorder, in the methoxy group and in the cyclopropylisoxazole unit; symmetry-related pairs of molecules are linked into dimers by pairwise N—H⋯N hydrogen bonds. Comparisons are made with the structures of some related compounds.Crystal structures are reported for three amides containing  The different conformations of the two independent molecules es Fig. 2 confirm x, y, z) is only 1.840 (8) Å from the corresponding site in the molecule at : hence, only one of these sites can be occupied and this, in turn, limits this site occupancy in each molecule to a maximum value of 0.5. Similarly, the atomic site C19 at  is only 1.921 (9) Å from the corresponding site in the molecule at , again limiting the site occupancy to a maximum value of 0.5. Hence the site occupancy for each orientation of the methoxy group must each be exactly 0.5.The molecule of compound (III)ca 10%, as is generally found in planar, or nearly planar, alkoxyarenes C(4)  are linked by C—H⋯N hydrogen bonds to form a C(6) chain, also running parallel to the [010] direction. The combination of these two chain motifs generates a ribbon of 1 screw axis along . Also present in the structure of compound (I)x, y, z) makes contacts with the corresponding atoms at  and , with Br⋯Br distances of 3.5812 (6) Å in each case; however, the C—Br⋯Br angles are 92.64 (18) and 166.44 (10)°, respectively 0] Fig. 4. Also ruly Fig. 5, which ane Fig. 6.et al., 1998abet al., 2000x < 1.0. A second sheet of the type, related to the first by the 21 screw axes, lies in the domain 0 < x < 0.5, and sheets of this type are linked by the C—H⋯O hydrogen bond in involving atom C117, so forming a three-dimensional network: indeed, it is possible to identify a complex chain running parallel to the [100] direction, which defines the linkage of the (100) sheets on Fig. 7; this chon Fig. 7. In the on Fig. 8. The comts Fig. 9.A and N2A in the major disorder form act as hydrogen bond acceptors, but atoms O1B and N2B in the minor disorder form do not. As in (II)A and N2A as the acceptors: if these sites had full occupancy, this interaction would generate a chain of rings running parallel to the [101] direction n Fig. 10. HoweverN-(Benzo[d]thiazol-2-yl)-3-bromobenzamide (IV) thiazole  and 2-nitrobenzoyl chloride . Yield 87%, m.p. 468–470 K; IR (cm−1) 3150 (N—H), 1681 (C=O), 1615 (C=N), 1560 and 1346 (nitro); NMR (CDCl3) δ(1H) 3.80 , 7.2–8.6 , 8.10 ; MS (70 eV) m/z 330 (M+ + 1). Compound (III)d]thiazole  and 5-cyclopropylisoxazole-3-carboxylchloride . Yield 88%, m.p. 453 K: IR (cm−1) 3120 (N—H), 1676 (C=O), 1625 (C=N); NMR (DMSO-d6) δ(1H) 0.2–2.1 , 3.83 , 6.90 , 7.20  and 7.46  ((H-14 and H-15), 7.80 ; MS (70 eV) m/z 316 (M+ + 1).All reagents were obtained commercially and all were used as received. For the synthesis of compound (I)3), 0.97 Å (CH2) or 0.98 Å  and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were allowed to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving the N—H distances shown in Table 1x parameter , calculated  − (I−)]/[(I+) + (I−)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021003637/hb7974sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989021003637/hb7974Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989021003637/hb7974IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989021003637/hb7974IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989021003637/hb7974Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021003637/hb7974IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021003637/hb7974IIIsup7.cmlSupporting information file. DOI: 2075271, 2075270, 2075269CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structural characterization by single-crystal X-ray diffraction revealed that the compound crystallizes in the triclinic space group PThe title compound,  The angles between the equatorial Mg atoms, the central magnesium atom and the axial magnesium atom are between θmin = 90.68° (Mg1iii—Mg1—Mg1iv) and θmax = 99.17° (Mg1ii—Mg1—Mg1iv).The molecules of the title compound are well separated from each other in the crystal structure, with one magnesocene molecule per unit cell. Each molecule has eight neighboring molecules, forming a distorted hexagonal bipyramidal coordination geometry Fig. 3a and 3b3Cp2Mg moiety has eight neighboring molecules within the bc and ac planes , but only six neighboring molecules within the ab plane, forming an almost hexagonal layer (γ = 63.00°), but with the layers being congruent to each other .Each es Fig. 4a and 4ber Fig. 4c.CSD, Version 5.42, update of September 2021; Groom et al., 20165R5)2Mg had previously been reported. In this search, any type of donor complexes of magnesocenes of the form (C5R5)2Mg·nD are not counted. The Mg⋯Cpcentroid bonding distances in these structures lie between 1.9562 (1) and 2.0628 (11) Å and the dihedral angles between the Cp planes are between 0° (co-planar geometry) and 17.892°. Thus, the bond distances and angles in the title compound are within normal ranges of known magnesocenes.A search in the Cambridge Structural Database  bromide. However, in this work, we utilized dibutylmagnesium as a strong base to deprotonate the triisopropylcyclopentadiene . The light-yellow reaction solution was stirred at 333 K overnight. Subsequently, all volatiles were removed in vacuo and a yellow oil was obtained, from which the title compound crystallized over the course of one day at ambient temperature. The crystallized material was washed with small portions of hexane and dried in vacuo to obtain the title compound as a pure, colorless, crystalline solid in 43% yield .To a solution of 4.00 g (20.8 mmol) of an isomeric mixture of triisopropylcyclopentadiene in 100 mL of hexane were added 15.0 mL of a 0.7 1H and 13C NMR spectroscopy: 1H NMR : δ (in ppm) = 1.07 , 1.28 , 1.36 , 2.82–2.92 , 5.77 ; 1H NMR : δ (in ppm) = 1.06 , 2.68 , 2.76 , 4.94 ; 13C{1H} NMR : δ (in ppm) = 24.0 (iPr), 24.4 (iPr), 26.4 (iPr), 26.6 (iPr), 28.7 (iPr), 98.7 (Cp), 125.3 (Cp), 128.6 (Cp); 13C{1H} NMR : δ (in ppm) = 25.9 (iPr), 26.8 (iPr), 27.0 (iPr), 29.1 (iPr), 94.6 (Cp), 119.4 (Cp), 120.9 (Cp).In addition to a structural characterization by single-crystal X-ray diffraction, the title compound was also characterized by 3), and with Uiso(H) = kUeq(C), where k = 1.2 for CH and 1.5 for CH3.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989022001189/pk2661sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022001189/pk2661Isup2.hklStructure factors: contains datablock(s) I. DOI: 2149608CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Measurement of T1 slope (T1S) can be difficult due to the anatomical positioning of the shoulders. And thoracic inlet angle (TIA) was a morphological parameter and not changed by the position. We proposed a new parameter, TIA minus C0-7 angle (TIA-C07), to evaluate C2-7 SVA in order to overcome the T1S imperfection.This was a retrospective radiological analysis of symptomatic subjects. The following cervical parameters were measured: Cervical lordosis angle (CL), C0-7 angle (C0-7), occiput-C2 lordosis angle (O-C2), C2-7 sagittal vertical axis (C2–7 SVA), TIA and TIA-C07. The Pearson correlation test was calculated, and the stepwise multiple regression analysis was conducted to determine the best predictor for C2-7 SVA. A paired sample t test was used to compare the predicted and measured C2-7 SVA.R = 0.645, R2 = 0.416). There was no statistical difference between the predicted and the measured C2-7 SVA .The mean age of 113 patients was 60.02 ± 9.67. The average O-C2, CL, C0-7, TIA, TIA-C07 and C2-C7 SVA was 29.24 ± 8.48°, 13.67 ± 11.22°, 42.91 ± 11.44°, 76.07 ± 9.54°, 33.16 ± 13.18° and 21.34 ± 11.42 mm. The predictive formula was founded: C2-7 SVA = 2.80 + 0.56 * (TIA—C07) (TIA and C0-7 mismatch may significantly impact cervical alignment, and a greater T1A-C07 was related to a greater degree of C2-7 SVA. TIA-C07 may be a more important predictor for C2-7 SVA.The online version contains supplementary material available at 10.1186/s12891-022-05301-0. Cervical spondylosis is a common cause of spinal cord dysfunction and imposes a serious social burden . CervicaAs one measure of symptom and disability status for prognostic purposes, Neck Disability Index (NDI) was widely used to assess self-rated disability in patients with neck pain . StudiesIn 2012, Lee et al. reported that thoracic inlet angle (TIA) was a morphological parameter and not changed by the position . SubsequCombined with the accuracy and invariance of TIA, we proposed a new parameter, TIA minus C0-7 angle, to evaluate C2-7 SVA in order to overcome the T1S imperfection. The purpose of our study was to assess the relationship between TIA minus C0-7 angle and C2-7 SVA.We retrospectively evaluated consecutive patients who presented with cervical spondylosis between January 2019 and June 2021. Inclusion criteria was: Patients with neck pain, radiculopathy symptoms, and/or myelopathy, patients that received both a cervical MRI and a cervical radiograph during a single visit and aged 18 years or older. Exclusion criteria was: 1) previous surgery on the cervical spine. 2) cervical spine deformity resulting from fracture, tumor, infection, or congenital abnormality. 3) neuromuscular disease, or inflammatory arthritis including ankylosing spondylitis and rheumatoid arthritis. Evaluators were blinded to patient demographic and clinical characteristics. Another recruited 26 asymptomatic cases who had cervical MRI and cervical radiograph were used to verify regression equation. This study was approved by the ethical review board at our institution. Informed consent was obtained from all subjects.The cervical sagittal alignment parameters were measured by PACS system. The following parameters were measured through lateral X-ray: (1) occiput-C2 lordosis angle ; (2) C2-7 angle defined as the CL angle ; (3) C0-7 angle measured by the angle subtended between McGregor’s line  and along the inferior endplate of C7; (4) C2-7 SVA defined as the distance between the C2 plumb line and the posterior C7 upper endplate.As a constant morphological parameter, not influenced by the posture, we defined TIA as an angle formed by a line from the center of the T1 upper end plate (T1UEP) vertical to the T1UEP and a line connecting the center of the T1UEP and the upper end of the sternum. TIA was measured through T2-weighted sagittal MRI. Measurements of parameters were shown in Fig. P < 0.05 was considered significant.The data was statistically treated with SPSS 24.0 software . Measurement data was expressed in terms of mean ± SD. The correlations between the parameters were analyzed with the Pearson correlation coefficient. Stepwise multiple linear regression was used to analyze independent variable to affect C2-7 SVA and calculate regression equation. Paired t-test was used to compare measured values of C2-7 SVA with predicted values by regression equation in 26 cases. In this retrospective study, we initially analyzed 118 consecutive patients, of whom 2 were excluded due to cervical spine infection, and 3 were excluded due to previous cervical spine surgery. 113 cases were included for the final analysis. The mean age was 60.02 ± 9.67 . The average values were as follows: O-C2, 29.24 ± 8.48°, CL, 13.67 ± 11.22°, C0-7, 42.91 ± 11.44°, TIA, 76.07 ± 9.54°, TIA-C07, 33.16 ± 13.18°, C2-7 SVA, 21.34 ± 11.42 mm (as shown in Table P < 0.05). With regard to the focus of the research, C2-7 SVA was significantly correlated with O-C2 , CL , C0-7 , TIA  and TIA-C07 . which were included in next multiple regression analysis. There was no correlation between C2-7 SVA and age. All data was shown in Table To explore the correlation between each pair of demographic and radiographic parameters, we performed a correlation coefficient test. We found no correlation between age and other parameters. CL was significantly correlated with O-C2, C0-7, TIA, TIA-C07 and C2-7 SVA .Multiple linear regression analysis was used to model the relationship between C2-7 SVA and potential factors by fitting a linear equation to the data. In our study, we regarded C2-7 SVA as a dependent variable and took O-C2, CL, C0-7, TIA and TIA-C07 as independent variables. The results were shown in Tables According to the multiple linear regression analysis, only one model was formed. In the collinearity analysis, the tolerance was greater than 0.1 and the variance inflation factor (VIF) was less than 5, which supported the independence of factors contributing to the regression equation (as shown in Table P = 0.933) between the predicted C2-7 SVA and the measured C2-7 SVA . Some studies have clarified the correlation between O-C2 and C2-7 SVA [Some studies proved that CL was an important parameter to affect C2-7 SVA , 21, 24.C2-7 SVA , 21, 22.C2-7 SVA , 26. TheC2-7 SVA . When C2Based on the above analysis, we introduced TIA-C07 as an independent variable. In Pearson correlation analysis, we found correlations between C2-7 SVA and O-C2, CL, C0-7, TIA and TIA-C07. Moreover, TIA-C07 had a stronger relation with C2-7 SVA than others. By multiple linear regression analysis, only TIA-C07 was reserved. We got a regression equation: C2-7 SVA = 2.80 + 0.56 * (TIA-C07). By the verification of statistics and 26 cases, we affirmed the rationalization of the equation. It meant that TIA and C07 mismatch may significantly impact cervical alignment Fig. . The preIn this study, the sample size was small. There was no case of severe cervical deformity such as ankylosing spondylitis. The average age was relatively older. The analyzed 113 subjects came from symptomatic patients. The next study in younger and asymptomatic people may be needed. Even so, we firstly founded the relationship between TIA-C07 and C2-7 SVA.The correlation between C2-7 SVA and TIA-C07 was firstly founded. TIA and C0-7 mismatch may significantly impact cervical alignment, and a greater TIA-C07 was related to a greater degree of C2-7 SVA. An individual with large TIA required large C0-7 to preserve physiologic sagittal balance of the cervical spine. TIA-C07 may be a more important predictor for C2-7 SVA. TIA-C07 can be used as a reference for estimating the normal value of C2-7 SVA. The results of this study may provide a useful reference for further studies.Additional file 1.


In which, flavumolines A − D (1 − 4) were four new ones, and flavumoline E (5) was reported as natural compound for the first time. Their chemical structures were elucidated by the analysis of extensive spectroscopic data. The inhibitory activities of these isolates on Cav3.1 low voltage-gated Ca2+ channel, NO production in LPS-activated RAW264.7cells, five human tumor cell lines, as well as acetylcholinesterase (AChE) were tested.Sixteen diterpenoid alkaloids (DAs), including six aconitine-type alkaloids (The online version contains supplementary material available at 10.1007/s13659-021-00302-3. Aconitum species represent a large genus in the Ranunclaceae family . Me. MeAconireported .Aconitum flavum Hand.-Mazz, known as a perennial herb, is mainly distributed in Qinghai, Gansu, and other northwest places in China + . The presence of hydroxyl (3431 cm−1) and carbonyl (1672 cm−1) units was deduced from the IR spectrum. The 13C NMR and DEPT spectra displayed 29 carbons signals, which were divided into five methylenes, nine methines, and five quaternary carbons as well as the signals for a benzoyl and three methoxy groups. Detailly, the characterized signals for a franchetine-type C19-DA core could be distinguished as follows: one representative 6,17-epoxy unit , two quaternary carbons at δC 48.9 (C-4) and 51.0 (C-11), and three methines at δC 46.5 (C-5), 44.3 (C-9), and 47.1 (C-10), together with the typical C-7/C-8 trisubstituted double bond  + in the HRESIMS , which indicated 10 degrees of unsaturation. The 13C NMR and DEPT data showed six methylenes, nine methines, four quaternary carbons, as well as the signals for a benzoyl group, three methoxy groups, and a typical N-ethyl group. Side by side, comparison of its NMR data with those of 6, a known franchetine-type DA isolated previously, indicated that the structure of 4 was similar with that of 6 + , in combination with NMR spectroscopic data. The IR spectrum of 5 showed absorptions due to the hydroxyl (3487 cm−1) and carbonyl (1676 cm−1) functionalities. The 13C NMR and DEPT spectra of 5 displayed 33 carbon resonances including five quaternary carbons , eleven methines, three methylenes, and 14 other signals attributable to a benzoyl, a typical N-ethyl group, an acetoxyl, and three methoxy groups. By carefully analyzing the characteristic resonances of a N-ethyl group at δC 49.2, t (C-21) and δC 13.2, q (C-22), a nitrogen-bearing methine and methylene at δC 60.8, d (C-17) and δC 51.0, t (C-19), an α,β-unsaturated ketone moiety at δH 6.27, 6.46  together with the C-atom signals at δC 132.3, 147.3, and 200.5 were clearly observed. The 1H NMR spectrum also verified resonances assignable to a nitrogen-bearing methine and methylene at δH 2.76 , δH 2.62  and 2.38 , as well as a representative N-ethyl group at δH 2.39, 2.69  and δH 1.00 . These evidences suggested that 5 should be a typical aconitine-type C19-DA . Silica gel , and MCI gel  were used for column chromatography. Fractions were monitored by TLC , and spots were visualized under a UV lamp at 254 nm or by spraying the Dragendorff’ reagent and heating silica gel plates sprayed with 10% H2SO4 in EtOH.Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shmadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. All 1D and 2D NMR spectra were recorded on Bruker DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts  was deposited in the Kunming Institute of Botany.The plants of A. flavum (45 kg) was extracted three times (3 × 150 L) with MeOH at room temperature and then concentrated to 3.88 kg under reduce pressure. The crude extract was suspended in 1% HCl followed by basification with 10% aqueous NH4OH (pH 9 − 10) and subsequently, extracted with ethyl acetate to afford crude alkaloids (808 g). The total alkaloids fraction was separated on a silica gel column  to yield eight fractions (Fr. 1 − 8). Then Fr. 1 (35 g) was separated on a silica gel column  to yield six sub-fractions (Fr. 1a − 1f). Fr. 1a (2.3 g) was separated on a silica gel column  and then semi-preparative HPLC  using MeOH/H2O  as the mobile phase afforded 6 (57 mg), 7 (10 mg) and 8 (23 mg). Fr. 1b (2.7 g) was separated on a silica gel column  followed by preparative HPLC using MeOH/H2O  to yield 4 (128 mg), 14 (15 mg), 15 (9.7 mg) and 16 (6 mg). Fr. 1c (3 g) was separated on a silica gel column , followed by semi-preparative HPLC using MeOH/H2O  as the mobile phase to afford 10 (8 mg), 11 (3 mg) and then yield 9 (230 mg) by recrystallizaiton. Fr. 1d (7 g) was separated on a silica gel column  yielded 12 (50 mg) and then on semi-preparative HPLC using MeOH/H2O  as the mobile phase to afford 13 (10 mg). Fr. 1e (5.5 g) yielded 2 (7.3 mg) and 3 (20.1 mg) by preparative HPLC using MeOH/H2O  as mobile phase, subsequently, afforded 5 (4.7 mg) and 1 (2 mg) isolated by Sephadex II column using acetone.Air-dried and powdered plant material  of α]D19 −4.9 ; IR (KBr) νmax: 3431, 2975, 2933, 2828, 1718, 1672, 1282, 1099 cm−1; For 13C NMR data .White powder; [α]D18 −12.5 ; IR (KBr) νmax: 3447, 2938, 2882, 2828, 1717, 1670, 1280, 1099 cm−1; For 13C NMR data .White powder; [α]D18 −9.7 ; IR (KBr) νmax: 3534, 3402, 2974, 2824, 1717, 1280, 1103 cm−1; For 13C NMR data .White powder; [α]D1919 −3.9 ; IR (KBr) νmax: 3437, 2973, 2932, 2826, 1719, 1279, 1101 cm−1; For 13C NMR data .Colorless oily liquid; [α]D18 + 4.4 ; IR (KBr) νmax: 3487, 2973, 2936, 1721, 1676, 1280 cm−1; For 13C NMR data .White powder; [4.4 c 0.12, MeOH; 2, 2 CaCl2, 10 Glucose and 10 HEPES (pH 7.4 adjusted with CsOH). The intracellular solutions contained (in mM) 127 Cs-methanesulphonate, 2MgCl2, 2Na2ATP, 10 HEPES and 11 EGTA (pH 7.4 adjusted with CsOH). The tested compounds (30 μM) were added.All experiments were performed at room temperature (~ 22 °C). Pipettes were fabricated from borosilicate glass (World Precision Instru-ments) using a micropipette puller , and were fire-polished to resistances of 2 ~ 4 M for whole-cell recording. Whole-cell currents were elicited by 150 ms depolarization to − 40 mV at 4 s intervals from a holding potential (HP) of − 100 mV. Currents were amplified by Axopatch 200B and digitized by Digidata 1440A (Molecular Devices). Currents were low-pass filtered at 2 kHz and sampled at 10 kHz. pCLAMP 10 software (Molecular Devices) was used for data acquisition and analysis. The extracellular solutions contained (in mM) 142 CsCl, 1 MgCl5 cells/well) and treated with serial dilutions of the compounds with a maximum concentration of 50 μM in triplicate, followed by stimulation with 1 μg/mL LPS (Sigma) for 18 h. NO production in the supernatant was assessed by Griess reagents (Sigma). The absorbance at 570 nm was measured with a microplate reader . NG-Methyl-L-arginine acetate salt  was used as a positive control [The murine macrophage cell line RAW264.7 was obtained from Cell Bank of Chinese Academy of Sciences. RAW264.7 cells were seeded in 96-well cell culture plates -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)  assay [50 value of each compound was calculated with Reed and Muench’s method [The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were obtained from ATCC . Cells were cultured in RMPI-1640 or DMEM medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified atmosphere with 5% COA) assay . Brieflys method .S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′-dithio-bis-(2-nitrobenzoic) acid , acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture  containing phosphate buffer (pH 8.0), test compound (50 μM), and acetyl cholinesterase (0.02U/mL), was incubated for 20 min (37 °C). Then, the reaction was initiated by the addition of 40 μL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for one hour. Tacrine was used as positive control with final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: (%) inhibition = (E—S)/E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).Acetylcholinesterase (AChE) inhibitory activity of the compounds isolated was assayed by the spectrophotometric method developed by Ellman et al. with sliSupplementary file1 (doc 9284 kb)Below is the link to the electronic supplementary material.


As cancer is one of the main leading causes of mortality, a series of monotherapies such as chemotherapy, gene therapy and radiotherapy have been developed to overcome this thorny problem. However, a single treatment approach could not achieve satisfactory effect in many experimental explorations.4-iron oxide nanoparticle (Au4-IO NP-cRGD) based on aggregation-induced emission (AIE) as a multifunctional theranostic system. Besides Au4 cluster-based fluorescence imaging and enhanced radiotherapy, iron oxide (IO) nanocluster could realize magnetic resonance (MR) imaging and Fenton reaction-based chemotherapy. Abundant toxic reactive oxygen species generated from X-ray irradiation and in situ tumor-specific Fenton reaction under acidic microenvironment leads to the apoptotic and necrotic death of cancer cells. In vivo studies demonstrated good biocompatibility of Au4-IO NP-cRGD and a high tumor suppression rate of 81.1% in the synergistic therapy group.In this study, we report the fabrication of cyclic RGD peptide (cRGD) modified AuThe successful dual-modal imaging and combined tumor therapy demonstrated AIE as a promising strategy for constructing multifunctional cancer theranostic platform.The online version contains supplementary material available at 10.1186/s12951-021-01191-x. It has been widely used for treatment of local solid tumors such as breast, colorectal, esophageal, lung and brain tumors –3. RT har Au NPs . Most reotherapy .2•−), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (•OH) [2O2 (∼100 μM) inside the tumor cells [2O2 to produce •OH have been developed [2+/Fe3+) under acidic conditions and move into cytosol to further affect ROS homeostasis inside cells [2O2 concentration and ion release rate. Accordingly, developing combination therapies based on several treatment methods is in great demand.Reactive oxygen species (ROS) describes the chemical species formed upon incomplete reduction of oxygen, which mainly includes superoxide anion  . ROS bural (•OH) –24. It hor cells –28. Thereveloped –32. For de cells . However14 NC, which was non-luminescent in dichloromethane (good solvent) but displayed red emission which was further enhanced with the increase of n-hexane (poor solvent) fraction [4-IO NP assemblies based on AIE management in which non-fluorescent Au4 cluster solution assembled with IO NC in PBS buffer due to aurophilicity  imaging of tumors in vivo. The simple Au4-IO NP-cRGD assemblies based on AIE exhibited effective targeted Fenton reaction assisted enhanced radiotherapy.As an efficient way to achieve bright emission from none-emissive single molecules, aggregation-induced emission (AIE) has embodied its superiority in bioimaging and cancer theranostics in the last decade –38. Aparfraction . The efffraction , 45. Her4 cluster (2 mM) was dissolved in DMF and then diluted with ethanol to 125 μM. 50 μM PEG modified-IO NC in PBS was added into ethanol solution of Au4 cluster with a volume ratio (IO NC: Au4 cluster) of 6:4 for uniform oscillation. For the conjugation of cRGD on the surface of Au4-IO NP, cRGD  was added into aqueous suspension of Au4-IO NP with uniform oscillation. Then, aqueous suspension was centrifuged (8000 rpm for 5 min) to remove free gold clusters and IO NC for further utilization.Au4 cells per well and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2. After cell attachment, the cell medium was then replaced with 100 μL fresh medium containing different concentrations of Au4-IO NP/Au4-IO NP-cRGD . The detailed dilution procedure: 2 mM Au4 cluster stock solution in DMF was firstly diluted with ethanol to 125 μM, and then 50 μM PEG modified-IO NC in cell culture medium was added into the above prepared Au4 cluster solution with a volume ratio (IO NC:Au4 cluster) of 6:4 to produce Au4-IO NP. After cRGD functionalization, Au4-IO NP-cRGD was diluted with cell culture medium for cytotoxicity assay. The control group was cell culture medium containing 0.04% DMF and 1.56% ethanol. After 24 h, CCK-8 reagent (10 μL per well) was added to the wells and incubated for 1 h. The absorbance at 450 nm was measured using a SpectraMax absorbance reader. The relative cell viability (%) was calculated as follows: (Atest/Acontrol) × 100.4T1 cells were seeded into 96-well plates at a density of approximately 104 cells/dish. 12 h later, the cell medium was replaced with fresh medium containing Au4-IO NP-cRGD or Au4-IO NP  for incubation. Confocal images of the cells were acquired with a Leica TCS SP8 confocal fluorescence microscope (excited at 405 nm).4T1 cells were cultured on glass-bottomed Petri dishes at an initial density of 2 × 104-IO NP/Au4-IO NP-cRGD). 6 h later, the cells were treated with different doses of X-ray  irradiation, and macroscopic cell colonies formed after 2 weeks. The cell colonies were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet for counting. Each group was analyzed in triplicate and counted by Image J software. The cell survival fraction was calculated as the ratio of the number of colonies formed in treated wells to the number of colonies formed in untreated well . The cell survival curve was estimated by a multi-target single-hit model (S = 1 − (1 − e−D/D0)n and then D0 and n were calculated by the Sigma Plot 12.0 software. The SER was expressed with the following formulae: SER = Dq(control group)/Dq(treated group) and Dq = ln(n) × D0, where Dq is the quasi-threshold dose. The control group was cell culture medium containing 0.04% DMF and 1.56% ethanol.4T1 cells were seeded in cell culture dishes (35 mm × 12 mm) and incubated overnight for cell attachment. Then, the cells were divided into control group  and treatment groups .4T1 cells were cultured on glass-bottomed Petri dishes with an initial density of 4 × 104 cells/dish overnight and incubated with nanomaterials Au4-IO NP/Au4-IO NP-cRGD of 2 μM for 6 h followed by irradiation at 0 and 4 Gy X-ray. After 24 h, the cells were stained with the CellEvent™ Caspase-3/7 Green Reagent. Confocal images were taken with a Leica TCS SP8 confocal fluorescence microscope (excitation: 488 nm).4T1 cells were seeded in glass-bottomed Petri dishes at an initial density of 2 × 105 cells/well were first cultured in 6-well plates overnight. After complete attachment, the cells were incubated with Au4-IO NP/Au4-IO NP-cRGD (2 μM) for 6 h. Then, the cells were irradiated by X-ray (0 and 4 Gy) and further cultured for 24 h before washing and collection. Annexin V-FITC/PI double staining was used for apoptosis analysis using flow cytometry.4T1 cells with a density of 2 × 104T1 cells were cultured on glass-bottomed petri dishes for incubation of materials and X-ray treatment. 24 h later, the cells were fixed with 4% paraformaldehyde and permeated with 0.25% Triton X-100. Then, the cells were blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with γ-H2AX antibody  overnight at 4 °C. Ultimately, the secondary antibody Alexa Fluor™ 647 tag goat anti-rabbit IgG (H + L)  was added for 1 ~ 2 h, and DAPI was used to stain cell nuclei. The cells were imaged using Leica TCS SP8 confocal fluorescence microscope at excitation wavelengths of 405 and 647 nm.4-IO NP/Au4-IO NP-cRGD (2 μM). After 6 h, the medium was removed, and then the prepared test solution (OH580 Stain Working solution) was added and incubated for 1 h at 37 °C. Then these groups were treated with or without X-ray (4 Gy). Finally, the cells were stained with DAPI, and imaged using Leica TCS SP8 confocal fluorescence microscope at excitation wavelengths of 405 and 540 nm.4T1 cells were cultured on glass-bottomed petri dishes and treated with or without Au6 4T1 cells. When the tumor reached a volume of ∼150 mm3, the mice were used for biodistribution and cancer therapy studies.All animal procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Zhengzhou University. BALB/c SPF mice  were housed in a controlled environment with a 12 h/12 h light/dark cycle. A maximum of five animals were housed together and provided with food and water ad libitum. The mouse breast cancer cell line 4T1 is widely used as a syngeneic tumor model of breast cancer. 4T1 tumors were established by subcutaneously injecting approximately 5 × 104-IO NP-cRGD (10 mg/kg). The mice were euthanized at 6, 24, 48 h, and 7 days after injection of materials, and the tumors and major organs were harvested. Then, the tumors and major organs were dispersed in 5 mL HNO3 overnight and digested with 5 mL of 30% H2O2 under boiling at 200 °C. The amounts of Au in each sample were measured using ICP-MS.4T1 tumor-bearing mice were randomly divided into four groups followed by an intravenous injection of 100 μL of Au4-IO NP-cRGD (10 mg/kg). Fluorescence imaging was conducted at 6 and 48 h post-injection using a PerkinElmer IVIS Spectrum in vivo imaging system. Imaging parameters were as follows: binning factor = 8, aperture = 2, exposure time = 2 s,excitation filter = 430, emission filter = 520. For in vivo MR imaging, tumor-bearing mice were anesthetized by isoflurane (3%) in oxygen and placed in an animal-specific body coil. MR images were acquired at 0, 6, 24, and 48 h after intravenous injection of Au4-IO NP-cRGD (10 mg/kg) or saline (as control) using a MR SOLUTIONS MRS-4717 system. Imaging parameters were as follows: echo spacing = 17, echo train = 7, slices = 18, slice thickness = 1 mm, FOV ratio = 1, FOV = 18 × 35 mm2.Tumor-bearing mice were intravenous injected with Au4-IO NP-cRGD, (3) 4 Gy, (4) Au4-IO NP + 4 Gy, and (5) Au4-IO NP-cRGD + 4 Gy. Then, 100 μL of saline was intravenous injected into the mice in group 1 and 3, while the mice in group 2 and 5 or group 4 were intravenous injected with 100 μL Au4-IO NP-cRGD or Au4-IO NP (10 mg/kg) every 3 days for 14 days, respectively. Afterwards, the mice were subjected to X-ray irradiation 4 Gy at 48 h after injection. The mice were photographed and their body weights were measured every other day for 14 days. Also, the length (L) and width (W) of the tumors were measured to calculate the tumor volume (V) according to the following equation: V = L × W2/2.Murine breast cancer cell line 4T1 was widely used as a syngeneic tumor model of breast cancer, which was the tumor entity with the highest incidence in the world. 4T1 tumor-bearing mice were weighed and randomly divided into five groups: (1) Control, (2) AuAt day 15 after the treatment, all mice were killed, and their peripheral blood and organs were collected for serum biochemistry analysis and pathological investigation respectively. The hearts, livers, spleens, lungs, kidneys, and tumors were fixed in 10% neutral formalin solution overnight. Then, the samples were embedded in paraffin blocks, sectioned into 5 μm slices, and mounted onto glass slides. After H&E staining, images were acquired with a Leica DMi1 inverted microscope.t test. Significant differences were noted with asterisks as P < 0.05 (*) or P < 0.01 (**).All data were collected in at least triplicate and represented as means ± SD. Differences between two groups were examined by the two-tailed Student's 4(C6H10S2N)4 cluster and PEG modified iron oxide nanocluster (IO NC) were synthesized at a high yield according to published methods [4 cluster was confirmed by powder X-ray diffraction (PXRD)  [4-IO nanoparticles (NPs) were acquired by mixing Au4 cluster in organic phase and IO NC in aqueous solution, then cRGD was linked to PEG ligand of IO NC for cancer cell targeting  rose to 40%  is stable at about 100 nm, even if the assemblies were further diluted with cell medium . After conjugation of cRGD peptide, the zeta potential of NP increased from − 12.03 mV (Au4-IO NP) to 3.83 mV (Au4-IO NP-cRGD)  also confirmed the successful modification of cRGD peptide . DLS measurements of Au4-IO NP-cRGD in serum confirmed a relatively narrow size distribution.Au methods . The pha4C6H0S2N4 clu4C6H0S2N4 cluon (AIE) . Au4-IO 4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4C6H0S2N4 clu4-IO NP-cRGD as a radiosensitizer, we measured the relative cell viabilities of mouse breast cancer cells (4T1) with and without X-ray irradiation through a CCK-8 assay kit. As depicted in Fig. 4-IO NP and Au4-IO NP-cRGD displayed a dose-dependent cytotoxicity against 4T1 cells, and Au4-IO NP-cRGD was slightly more toxic due to the targeting effect of cRGD. Additional X-ray irradiation caused enhanced proliferation inhibition of cells treated with Au4-IO NP/Au4-IO NP-cRGD  showed brighter fluorescence than that of Au4-IO NP treated cells  from IO NC in this study. Methylene blue (MB) was employed to quantify the •OH production efficiency [4-IO NP-cRGD + H2O2 group (decreased to 93.1%) and Au4-IO NP-cRGD group at pH 6.5 (decreased to 93.7%), the UV absorbance of MB in the Au4-IO NP-cRGD + H2O2 group at pH 6.5 decreased to 78.6% , substantial cytotoxicity was observed due to Fenton reaction based •OH generation /Dq (Treated group)) [4-IO NP-cRGD for 4T1 cells was calculated to be 1.573 ) , 55 of A4-IO NP-cRGD based RT, the cellular •OH generation after treatment with different NPs was first detected by a mitochondrial •OH detection assay kit [4-IO NP-cRGD + X-ray treated group was slightly higher than that of Au4-IO NP + X-ray group, achieving 4.4 and 7.1 times of fluorescence intensity of 4 Gy and Au4-IO NP-cRGD group, respectively. Meanwhile, DNA damage of 4T1 cells after treating with Au4-IO NP-cRGD and X-ray was tested by immunofluorescence staining of γ-H2AX, a marker of double-stranded DNA damage [4-IO NP-cRGD, and X-ray groups, representing the ignorable DNA break. Notably, 4T1 cells treated with Au4-IO NP-cRGD + X-ray exhibited the brightest red fluorescence signals, indicating a higher DNA damage level than Au4-IO NP + X-ray group  activity could also be a possible mechanism for the synergetic treatment effect [To further explore the therapeutic mechanism of Aussay kit . As showA damage , 58. Vert effect , 59.4-IO NP-cRGD in tumor imaging and therapy was explored. Firstly, the biodistribution of Au4-IO NP-cRGD in 4T1 tumor-bearing mice was monitored by inductively coupled plasma mass spectrometry (ICP-MS) via determination of Au. As shown in Additional file 4-IO NP/Au4-IO NP-cRGD accumulation in the tumor site achieved maximum at 48 h post-injection (10 mg/kg). After intravenous injection of Au4-IO NP-cRGD or saline, MR imaging data were acquired at 0, 6, 24 and 48 h time points with a 4.7 T system. T2-weighted images displayed targeted signal from Au4-IO NP-cRGD in tumor , playing a supplementary role in Au based enhanced RT. 4T1 tumor-bearing mice were treated with an experimental procedure as follows: mice were intravenous injected with Au4-IO NP-cRGD every three days and exposed to X-ray (4 Gy) at 48 h post injection . As shown in Fig. 4-IO NP-cRGD and X-ray irradiation were significantly suppressed. Compared with the control group, the inhibition rates of tumor volume and tumor weight in synergistic therapy group reached 79.7% and 81.1%, respectively  resulted lower accumulation efficiency in tumors and relatively poor in vivo radiotherapy effect  staining of the main organ of mice were performed after 14 days treatment. Blood biochemical tests showed that the levels of cholesterol (CHOL), albumin (ALB), aminotransferase (AST), urea nitrogen (BUN), creatinine (CREA), and lactate dehydrogenase (LDH-L) in the Au4-IO NP-cRGD + X-ray group were similar to those in control group mice  PEG modified IO NC and Au4 cluster in DMF with (b) 0% PBS, (c) 20% PBS, (d) 40% PBS, and (e) 60% PBS, (f) 80% PBS. Fig. S4. DLS spectra of (a) Au4-IO NP in DMF/ethanol with 60% medium and (b) 1000 times diluted Au4-IO NP using cell medium. Fig. S5. Emission spectra and (b) DLS spectra of Au4-IO NP-cRGD in serum at day 0, 3, 5, and 7. Fig. S6. Zeta potential of Au4-IO NP and Au4-IO NP-cRGD. Fig. S7.1H-NMR spectra of cRGD-PEG-COOH. The characteristic peak of cRGD and PEG confirm the structure of cRGD-PEG-COOH. Fig. S8.1H-NMR spectrum and DOSY spectrum of cRGD-PEG-COOH. Fig. S9. Flow cytometric assay of 4T1 cells after incubation with PBS, Au4-IO NP and Au4-IO NP-RGD. Fig. S10. In vitro cytotoxicity against 4T1 cells of Au4-IO NP and Au4-IO NP-cRGD with 4 Gy X-ray irradiation. Fig. S11. Confocal imaging of 4T1 cells after incubation with Au4 and IO NC. Scale bar: 50 μm. Fig. S12. Viability of 4T1 cells in different treatment groups: (1) Au4-IO NP-cRGD, (2) pH 6.5, (3) H2O2, (4) Au4-IO NP-cRGD pH 6.5, (5) Au4-IO NP-cRGD + H2O2, and (6) Au4-IO NP-cRGD + H2O2 pH 6.5. Fig. S13. Live/dead imaging of 4T1 cells after receiving different treatments . Green: live; red: dead. Scale bar: 50 μm. Fig. S14. Flow cytometric assay of 4T1 cells . Fig. S15. Flow cytometric assay of 4T1 cells with different treatments (batch 1). Fig. S16. Flow cytometric assay of 4T1 cells with different treatments (batch 2). Fig. S17. Flow cytometric assay of 4T1 cells with different treatments (batch 3). Fig. S18. Representative images of the colony formation assay of 4T1 cells with different treatments. Fig. S19. Survival curve of 4T1 cells received Au4-IO NP. Fig. S20. The fluorescence intensity of hydroxyl radical imaging of 4T1 cells (λex = 540 nm) at 6 h after administering different treatments. (1) Control, (2) Au4-IO NP-cRGD, (3) 4 Gy, (4) Au4-IO NP + 4 Gy, and (5) Au4-IO NP-cRGD + 4 Gy. Fig. S21. Confocal images of γ-H2AX expression in 4T1 cells after receiving various treatments. Scale bar: 50 μm. Fig. S22. The fluorescence intensity of γ-H2AX imaging of 4T1 cells (λex = 647 nm) at 6 h after administering different treatments. (1) Control, (2) Au4-IO NP-cRGD, (3) 4 Gy, (4) Au4-IO NP + 4 Gy, and (5) Au4-IO NP-cRGD + 4 Gy. Fig. S23. The fluorescence intensity of Caspase-3/7 imaging of 4T1 cells (λex = 488 nm) at 6 h after administering different treatments. (1) Control, (2) Au4-IO NP-cRGD, (3) 4 Gy, (4) Au4-IO NP + 4 Gy, and (5) Au4-IO NP-cRGD + 4 Gy. Fig. S24. The biodistribution of (a) Au4-IO NP and (b) Au4-IO NP-cRGD at 6 h, 24 h, 48 h, 72 h, and 7 d after intravenous injection. Fig. S25. (a) Images of dissected tumors in control, Au4-IO NP-cRGD, and Au4-IO NP-cRGD + 4 Gy. (b) Images of dissected tumors in 4 Gy group. Fig. S26. (a) Representative images of mice of Au4-IO NP + 4 Gy group at days 0 and 14. (b) Tumor volume curve of the mice. (c) Mouse body growth curve. (d) Image of dissected tumors in Au4-IO + 4 Gy.


DACH-ZYC-Phos-C1 as the best ligand. The synthetic potential of the C-allenylation products is demonstrated. Furthermore, the enantioselectivity observed with DACH-ZYC-Phos-C1 is rationalized by density functional theory studies.Pyrazolones are a vital class of heterocycles possessing various biological properties and much attention is paid to the diversified synthesis of enantiopure pyrazolone derivatives. We describe here the development of diphenylphosphinoalkanoic acid based chiral bisphosphine ligands, which are successfully applied to the palladium-catalyzed asymmetric allenylation of racemic pyrazol-5-ones. The reaction affords C-allenylation products, optically active pyrazol-5-ones bearing an allene unit, in high chemo- and enantioselectivity, with  Chiral pyrazolone derivatives show promising biological activity in commercial drugs. Here, the authors report an enantioselective allenylation of pyrazolones by fine tuning of Trost’s ligands, which leads to a chiral pocket featuring high efficiency and asymmetric induction in the catalytic process. Of particular interest are the pyrazolone derivatives with a chiral center -3aa in 73% yield and 50% ee; interestingly, Trost ligand L11 could give (S)-3aa in 84% yield and 75% ee. With L11 as the ligand, subsequent investigation on the solvent effect showed that toluene was the best one, affording 85% yield (S)-3aa with 84% ee -3aa in 92% yield and 85% ee, together with a trace amount of 4aa at 60 °C -3aa in 85% ee, albeit with a low conversion (14% yield with 67% recovery of 1a) (Table S)-3aa in 75 and 80% yields, respectively, with a similar ee together with 4% of the N-allenylation product 4aa (Table S)-3aa was improved to 88% when the concentration of the reaction was 0.02 M. The ee value of (S)-3aa was similar with 5.0 mol% of the ligand -3aa in 79% yield and 90% ee as the chiral ligand with 2% of N-allenylation product 4aa. Solvent screening (Table S)-3aa in 100% yield and 95% ee. Using toluene as solvent, DACH-ZYC-Phos-C2 and C3 were re-tested , Pd2(dba)3•CHCl3 (2.5 mol%), and DACH-ZYC-Phos-C1 (5.0 mol%) in toluene (0.02 M) at 60 °C -3fa dropped to 78% with 86% yield, indicating a serious steric effect (Table 2 was then examined with benzyl 2-(n-butyl)buta-2,3-dienyl carbonate 1a. The reaction of 4-pyrazol-5-ones 2b–2d with 1a afforded (R)-3ab~(S)-3ad in similar yields and enantioselectivity -3ae~(S)-3ak in 86–97% yields and 94–98% ee (Table S)-3ak -3am was obtained in 95% yield and 94% ee by using ent-DACH-ZYC-Phos-C1 as the ligand (Table R)-3ao and (R)-3ap suggested that coordination of the hydroxyl oxygen may affect the enantioselectivity -3at was only 36% with 62% yield -3bu in 84% yield with 95% ee while the reaction of N-benzyl pyrazol-5-one 2 v and 1b afforded (S)-3bv in 82% yield with 90% ee -3am in a similar yield and enantioselectivity -3am was established in 55% yield and 99% ee to afford tricyclic product -5am containing a bicyclic motif with anti-tumor activity46 -3da with o-(propen-2-yl)phenol could easily construct a seven-membered heterocycle in product -6da 47. The allene unit in (S)-3da also could be transformed into a 2-propynyl side chain in (S)-7da upon treatment with TiCl4.The reaction of ity Fig. . Subsequ50. To better understand the superior enatioselectivity and the advantage of DACH-ZYC-Phos-C1 in the allenylation of pyrazolone, we have conducted X-ray single crystal diffraction studies: firstly, Pd(II)-DACH-ZYC-Phos-C1 complex (III) was prepared by the reaction of PdCl2 and DACH-ZYC-Phos-C1 with excess amount of bases in toluene  was obtained from the reaction of Pd(OAc)2 with DACH-Phenyl Trost ligand with excess amount of bases in THF51. Both complexes were then recrystalized from CHCl3/n-hexane to afford single crystals suitable for the X-ray diffraction study. It was obvious that the angle (P1-Pd1-P2) and the distance between P1 and P2 in III are greater than those in IV . However, this complex III failed to catalyze the enantioselective allenylation under the standard conditions or with AcOH as the protic additive -ive Fig. , indicat1b with pyrazol-5-one 2a catalyzed by the DACH-ZYC-Phos-C1-ligated palladium catalyst . These transition structures are denoted as TS_left_Si_a/b, TS_left_Re_a/b, TS_right_Si, and TS_right_Re, separately . Among them, TS_left_Si_a with the Si-face attack of 2a anion, is found to be the most favorable one, which is consistent with the dominant formation of S-products observed in the experimental studies.Figure 2a anion. The structures of TS_left_Si_a and TS_left_Si_b, leading to the formation of the allenylation product with S configuration, are stabilized by the hydrogen bonding forming between the ligand’s N-H with the carbonyl oxygen and the hydrazine nitrogen, separately. In the structure of TS_left_Si_a, the H···O distance is 1.81 Å, and the bond angle of N − H···O is 175.0°, which are consistent with the common hydrogen bonding parameters53. Based on the electron density at the bond critical point, the hydrogen binding energies (BE) were calculated to analyze the strength of the hydrogen bonding54. The bond energy of the hydrogen bonding in TS_left_Si_a was estimated to be about 8.1 kcal/mol. Although the influence of the π/π interactions between the aromatic rings of the ligand and N-Ph pyrazolones on the enantioselectivity could be excluded based on the results of (S)-3bu and (S)-3bv, we have also tried to analyze the non-covalent interactions (NCI) in TS_left_Si_a using the Multiwfn56 and VMD programs57 and no obvious π–π interactions are found so far from the analysis (see TS_left_Si_a).The ligand’s right-side amide N-H is available for hydrogen bonding to either the carbonyl oxygen or the hydrazine nitrogen of the approaching TS_left_Si_b features a longer H···N distance of 2.18 Å and a smaller N − H···O bond angle of 163.1°, suggesting a weaker interaction, which estimated to be 4.8 kcal/mol. Similarly, hydrogen-bonding interactions also exist in the structure of TS_left_Re_a and TS_left_Re_b, which would provide the allenylation product with R configuration. TS_left_Re_a, is stabilized by the hydrogen bonding formed between the ligand’s N-H and the carbonyl oxygen of 2a anion, which was estimated to be about 6.3 kcal/mol. Moreover, TS_left_Re_a suffers the unfavorable interaction between 2a anion and the methylene-π-allyl moiety, which have been clearly shown in the Newman projection along the forming C–C bond in Fig. TS_left_Re_a as compared to TS_left_Si_a by 4.5 kcal/mol. The bond energy of the hydrogen bonding in TS_left_Re_b was estimated to be about 5.4 kcal/mol, which is around 2.7 kcal/mol weaker than the one in TS_left_Si_a , thus, accounting for the preference of TS_left_Si_a over TS_left_Re_b by 2.3 kcal/mol. Furthermore, TS_right_Si is also stabilized by the hydrogen bonding formed between the ligand’s N-H and the carbonyl oxygen of 2a anion. No corresponding TS is able to be located with hydrogen bonding formed between the ligand’s N-H and the hydrazine nitrogen. The bond energy of the hydrogen bonding in TS_right_Si was estimated to be about 5.8 kcal/mol. What’s more, the steric repulsions between 2a anion and the methylene-π-allyl moiety destabilize TS_right_Si. Both factors lead to the less stability of TS_right_Si than TS_left_Si_a by 3.3 kcal/mol. In addition, there is no hydrogen bonding existing in TS_right_Re. And unfavorable steric interactions between the two phenyl groups of 2a anion and the DACH-ZYC-Phos-C1 ligand further destabilize TS_right_Re, making it the least stable transition state . The calculated enantioselectivity, basing on the energy difference between TS_left_Si_a and TS_left_Re_b of 2.3 kcal/mol, is 94%, which is in perfect agreement with the experimental ee value of 93% -DACH-ZYC-Phos-C1 complex and DFT calculations. In addition, these types of stretchable chiral pockets may also provide flexiblity and show great potential applications in catalytic enantioselective allylation, allenylation, and other reactions. Such studies are being actively pursued in our laboratory with very promising results and will be reported in due courses.In summary, we have developed a DPPAA-based ligand for the highly chemo- and enantioselective allenylation of pyrazol-5-ones with benzyl 2,3-dienyl carbonates (up to 97% ee). Many synthetically useful functionalities were tolerated under the catalysis of Pd/2(dba)3•CHCl3 , -DACH-ZYC-Phos-C1 , toluene (5 mL), 1a /toluene (11.7 mL), and 2a /toluene (8.3 mL) sequentially under nitrogen atmosphere at room temperature. The flask was put into a pre-heated oil bath and the reaction was complete after being stirred at 60 °C for 12 h as monitored by TLC (eluent: petroleum ether/ethyl acetate = 20/1). The resulting mixture was filtrated through a short column of silica gel eluted with ethyl acetate (15 mL × 3). After evaporation, the crude residue was purified by chromatography on silica gel (eluent: petroleum ether (60–90 °C)/ethyl acetate = 60/1) to afford a pure part of (S)-3aa and the impure part was further purified by chromatography on silica gel (eluent: petroleum ether (60–90 °C)/ethyl acetate = 40/1). Two-round chromatography afforded (S)-3aa  as a liquid: 95% ee  = 5.7 min, tR (minor) = 8.9 min); [α]D20 = −8.2 ; 1H NMR  δ 7.55 , 7.30 , 7.22–7.02 , 4.64–4.49 , 3.18 , 2.89 , 2.69 , 2.39 , 2.14 , 1.93–1.80 , 1.43–1.17 , 0.83 ; 13C NMR  δ 205.3, 174.6, 161.4, 137.6, 134.1, 129.1, 128.5, 128.1, 127.2, 124.8, 119.3, 98.3, 77.7, 60.1, 42.8, 36.1, 32.3, 29.4, 22.1, 14.7, 13.8; IR (neat) ν (cm−1) 3063, 3031, 2956, 2927, 2859, 1954, 1712, 1597, 1500, 1455, 1440, 1402, 1366, 1123; MS (EI): m/z (%) 372 , 186 (100); HRMS calcd. for C25H28N2O [M]+: 372.2202; Found: 372.2202.To a flame-dried Schlenk flask were added PdSupplementary InformationPeer Review File