Piperazine skeleton in the structural modification of natural products: a review

Abstract Piperazine moiety is a cyclic molecule containing two nitrogen atoms in positions 1 and 4, as well as four carbon atoms. Piperazine is one of the most sought heterocyclics for the development of new drug candidates with a wide range of applications. Over 100 molecules with a broad range of bioactivities, including antitumor, antibacterial, anti-inflammatory, antioxidant, and other activities, were reviewed. This article reviewed investigations regarding piperazine groups for the modification of natural product derivatives in the last decade, highlighting parameters that affect their biological activity.


Introduction
The piperazine ring, an important class of N-heterocyclic bioactive natural products, is extensively present in biologically active compounds 1 . The piperazine scaffold has been recognised as a privileged structure in drug discovery and is widely distributed in biologically active compounds employed in several different therapeutic fields, including antitumor 2 , antibacterial 3 , anti-inflammatory 4 , antipsychotic 5,6 , anti-Alzheimer 7,8 , antifungal 9 , and antidiabetics 10 . The N-4 nitrogen of piperazine can be used as a basic amine, while the N-1 nitrogen can easily introduce hydrogen bond acceptors and hydrophobic groups through other heterocyclics without necessitating the addition of a stereocenter 11,12 .
Piperazine core has two primary nitrogen atoms which exert the improvement in pharmacokinetic features of drug candidates because of their appropriate pKa 13 . These nitrogen sites lead to the essential increase in water solubility of the drug-like molecules thereby playing a crucial role in the bioavailability. Maintaining a balance between pharmacodynamic and pharmacokinetic profiles of drug-like molecules is the important factor in designing and developing of new drugs, thus, one of the goals in the drug discovery process is to design molecules with a high affinity for their targets and appropriate physicochemical properties. For this purpose, the characteristics of the piperazine template make this molecular subunit a useful and well positioned system in the rationale design of drugs 14 .
Natural products are closely related to human development. Through the process of metabolic evolution, bacteria, fungi, and plants have co-evolved along with animals, greatly exploring the chemical space, and producing a large number of natural products that can alter the senses and behaviour of animals 15 . Designing hybrid molecules based on natural products has become an effective strategy, including combining two or more biologically active compounds from natural sources, and is expected to deliver these compounds to target organs simultaneously. Reportedly, this combination strategy has lower toxicity when compared with traditional single-target molecule co-administration methods. Additionally, hybrid molecules improved dose compliance, reduced drug interactions, and the cost of preclinical evaluation 16,17 . Natural products have always been an alternative source of chemical substances for exploring biological systems, as well as biological pre-verification prototypes for chemical probes and drug discovery, which are considered sources of new drug research and development owing to their unique chemical diversity 18,19 . A significant number of compounds have been developed from plants, microbial metabolites, and marine organisms. Between 1981 and 2019, approximately 23.5% of the new drugs approved globally, 33.6% of the approved small molecule drugs, and 75.6% of the drugs in the anti-infective field were derived from natural products and their derivatives 20 . Given the importance and significant biological activity of the piperazine skeleton in natural products, in recent years, an increasing number of scientific articles, books, and patents have been based on piperazine ring modifications of natural products. A growing wealth of literature reflects the research interests and importance of this field, as well as the potential for developing drugs based on piperazine groups [21][22][23] . Therefore, this article combined the latest literature reports, especially from the perspective of biological activity, to introduce the research progress in this field. We anticipate that this review will provide novel concepts and research directions for the modification of natural products. relationships (SARs) shows that the presence of the phenyl group at C-2 does make a difference for the anti-K562 activity whereas none of the compounds having a methyl substituent at C-2 showed any activity against the K562 cells lines, more compounds of the flavone series (where there is a phenyl group linked to C-2) and only a few of the 2-methyl series displayed activity, and leaving the piperazine ring unsubstituted is better for the anti-K562 activity while for breast cancer, the anti-T47D activity resembles that of the K562 activity. Among them, compound 1 (Figure 1) was reportedly a potential antineoplastic agent against T47D cells (breast cancer cell line), with an IC 50 value of 1.42 mM. Unfortunately, its activity is not as effective as the positive control drug doxorubicin (IC 50 ¼ 0.33 mM).
Anticancer properties of chrysin-based sulfonylpiperazines were reported by Pecere et al. 25 SARs show that the presence of halogen atom(s) was found essential to get better potency against SK-OV-3, HeLa, and A-549 cell lines. Derivative 2 ( Figure 2) with dual fluorine atom substitution on the sulfonylpiperazine ring exhibited the best activity against SK-OV-3 with least 12.67 ± 0.74 lg/ml of IC 50 and was observed as potent as gefitinib (IC 50 ¼ 12.31 ± 0.33 lg/ml). Furthermore, in case of inhibition of HeLa cell line, again derivative with 2,4-difluoro substituent demonstrated remarkable 4.67 ± 1.42 lg/mL of IC 50 , which was better than gefitinib (IC 50 ¼ 17.92 ± 1.50 lg/ml). Patel et al. documented the synthesis of chrysin-piperazine conjugates and evaluated the in vitro anticancer activity against HeLa, CaSki, and SKOV-3 cancer cell lines. The findings revealed that the nature and position of the functional groups on the piperazine core may contribute to the expected antioxidant and anticancer effects from the perspective of structure-activity 26 .
Oroxylin A (5,7-dihydroxy-6-methoxyflavone) is a naturally occurring monoflavonoid isolated from the root of Scutellaria baicalensis Georgi and exhibits potent anticancer activities in vitro and in vivo. Fu et al. synthesised three series of oroxylin derivatives by connecting a nitrogen-containing hydrophilic, heterocyclic ring to the C7-OH via a varying length of the carbon chain. Compounds (3ca-3cc) ( Figure 3) containing a 4-carbon spacer were particularly potent with respect to cellular inhibition of all the three cell lines, with IC 50 value ranging from 1.42 to 9.52 mM, approximately 5-to 20-fold more potent than oroxylin A, except for 3cd and 3ce. Compound 3cb and 3cc were found to be the most potent, with IC 50 of 1.42 mM (HCT116) and 2.98 mM (HepG2), respectively. The results indicate that the length of the chain between the terminal heterocyclic substitutes and oroxylin A is an important factor for their potency. However, with a morpholinyl or N-methyl piperazinyl group at the terminal of 7-O-alkyl oroxylin A, compound 3cd and 3ce (with IC 50 at 12.5-50.6 mM) displayed moderate levels of inhibition, which was similar to that of oroxylin A but over 4-25-fold (HepG2), 2-30-fold (HCT116), 3-6-fold (BCG823) less potent than 3ca-3cc. The morpholinyl or N-methyl piperazinyl substitution in the compounds 3ad-3ae (with IC 50 at 15.4-67.6 mM) and 3bd-3be (with IC 50 at 30.4-74.8 mM) also exhibited lower activities than substitutions with N, N-diethylamino (3aa and 3ba), pyrrolyl (3ab and 3bb) or a piperidyl (3ac and 3bc) group, respectively, regardless to the length of the carbon chain. It may be attributed to the volume of the side chain. Annexin V/PI staining experiments proved that compound 3cc has the highest in vitro efficacy on HepG2 cells among the derivatives. The difference of 3cc between the inhibition of cell proliferation and the apoptotic induction indicated that 3cc was more likely to be a necrosis-inducing agent or both apoptosis/necrosis inducer. Thus, the novel 7-O-alkylamino derivative of oroxylin A, compound 3cc, could be a promising antitumor candidate, and further in vitro and in vivo biological character evaluations are warranted 27 .
Novel hybrid compound 4 ( Figure 4) between chalcone and piperazine demonstrated potent antitumor activity against A549 (a human lung cancer cell line), Hela and SGC7901 cells (IC 50 ¼ 5.24, 0.19, and 0.41 mM, respectively), which was better than positive control drug cisplatin (IC 50 ¼ 11.54 20.52, and 12.44 mM, respectively) 28 . In addition, chalcone-piperazine derivative 5 ( Figure 4) exhibited inhibitory activity against the A549 cell line, with an IC 50 value of 0.19 mM, which was better than positive control drug cisplatin (IC 50 ¼ 11.54 mM) 29 . Mannich bases 6a, 6b, 6c ( Figure 4) were derivatives of chalcone. Though compounds showed a low inhibition potency towards hCA I (25-43%) and hCA II (6-25%) isoforms at an inhibitor concentration of 10 mM, they were more selective (1.5-5.2 times) towards hCA I isoenzyme. Compared with piperidine and N-methylpiperazine, Mannich bases 6b containing morpholine (substituted by fluorine, chlorine or bromine on the benzene ring) had the highest average SI value. The experimental results showed that the chalcone-piperazine hybrid compounds containing acetophenone had good anti-tumour activity. Among them, the substitution of halogen atoms on the benzene ring had a great impact on the anti-tumour activity, and the compounds substituted by fluorine atoms on the benzene ring showed the best activity 30 . A series of chalcone-piperazine derivatives as mono Mannich bases were designed and synthesised by Tugrak and coworkers. The amine part was altered to N-phenylpiperazine, Nbenzylpiperazine, 1-(2-fluorophenyl)piperazine, 1-(4-fluorophenyl) piperazine, and 1-(2-methoxyphenyl)piperazine. All synthesised    The research showed cytotoxicity of some compounds containing piperazine in chalcone-Mannich base derivatives is stronger than other compounds containing different secondary amines (such as morpholine, piperidine, pyrrolidine, dimethylamine, diethylamine, dipropylamine, dibenzylamine) 31,32 . Novel chalcone-dithiocarbamate hybrids were designed, synthesised, and evaluated for their antiproliferative activity against select cancer cell lines (MGC803, MCF7, and PC3). Investigations evaluating modifications and structure-activity relationship (SAR) revealed that substituents on the piperazine unit are important for their inhibitory activity. Among these analogs, 8 showed the best inhibitory activity against PC3 cells (IC 50 ¼ 1.05 mM), which was better than positive control drug 5-FU (IC 50 ¼ 29.31 mM). Cellular mechanism studies elucidated that 8 ( Figure 4) could inhibit colony formation, arrest the cell cycle at the G2/M phase, and induce DNA damage against PC3 cells. On replacing the piperazine ring with the morpholine or pyrrolidine group, the activity showed a particularly noticeable decrease 33 . The discovery of new chromen-4-one derivatives as telomerase inhibitors has been reported. Compound 9 ( Figure 4)  And the substituent R showed a large effect for the activity, heterocycle is better than that benzene ring (compound 9). Further, the substituent number of benzene rings showed great influence on anticancer. Preliminary investigations revealed that compound 9 inhibited telomerase activity by decreasing the expression of dyskerin. Furthermore, the docking results showed that the target compound could strongly interact with residues LYS189 and Asp254 after introducing the piperazinphenyl methanone structure. In vivo experiments, compound 9 could evidently abate pathological changes of hepatic lobules in DEN-induced rats. The hepatic cells showed markedly reduced atypia, full cytoplasm and high differentiation 34 . Reportedly, 4-amino-2H-benzo[h] chromen-2-one analogs were synthesised and evaluated for their anticancer activity. Compound 10 ( Figure 4) displayed potent androgen receptor binding affinity, as well as strong cytotoxic activity against LNCaP-hr cells (IC 50 ¼ 0.52 ± 0.11 mM), which was better than positive control drug finasteride (IC 50 ¼ 14.5 mM). The SAR results showed that the 4-position substituent of the piperazine ring and the ortho-substituted phenyl analogs had relatively good inhibitory activity 35 . Novel resveratrol-chalcone-piperazine amide derivative 11 (R¼-CH ¼ CH 2 ) ( Figure 5) showed superior cytotoxic activity against A549 and HeLa cells (IC 50 ¼ 0.26 and 7.35 mmol/L, respectively), which was better than positive control drug cisplatin (IC 50 ¼ 11.54 and 20.52 mmol/L, respectively). And fluorescence-activated cell sorter (FACS) analysis showed that the compound effectively induced apoptosis in A549 cells. The structure of the compound had a significant effect on anticancer activity. The preliminary structure-activity relationship shows that the anticancer activity was related to the number and category of substituents on the benzene ring, but had nothing to do with the position of  substituents, and the lack of electronic benzene ring led to positive activities 36 .
A gambogic acid derivative 12 ( Figure 6) containing a methylpiperazine substituent was found to possess a most potent inhibitory activity against A549 and BGC-823 cells among all compounds tested. In A549 cell line, compound 12 exhibited the highest potency with an IC 50 value of 0.12 mM, which was 10-fold more potent than gambogic acid. In BGC-823 cell line, compound 12 displayed the best potency with an IC 50 value of 0.57 mM, which was almost 5-fold more potent than gambogic acid. The SARs at the C-37 site of gambogic acid shows that the removal of hydrophobic prenyl tail and introduction of hydrophilic carboxyl group slightly reduced the anti-tumour activity. Further introduction of hydrophobic motifs such as methyl, isoprenyl and tertbutyl groups had little effect on improving the potency. Inspiringly, substituted by aliphatic amines showed obviously increased activities. Meanwhile, the aliphatic amines also could improve the solubility and drug-like properties of the derivatives. Notably, compound 12 containing methylpiperazine substituents was the most excellent derivative with the IC 50 values ranging from 0.12 to 3.10 mM against the five cell lines. Phenylamine groups were not a good moiety for improving the activity at the C-37 site. In addition, although compounds with the flexible amino acid side chains ought to have greatly improved solubility, they were found to be less active or even mostly inactive against all the five cell lines. The introduction of glycine group to C-37 site could increase the selectivity against HCT-116 and HepG2 cells. This study found that compounds with aliphatic amines had higher anti-tumour activity than aniline, improving the solubility and drug-like properties of derivatives 37 .
Li and co-workers revealed that wogonin-piperazine derivative 13 ( Figure 7) possessed the highest potency against HepG2, A549, and BCG-823, with IC 50 values of 1.07 mM, 1.74 mM, and 0.98 mM, respectively, which was better than positive control drug 5-FU (IC 50 ¼ 17.2, 16.1 and 10.1 mM, respectively) and parent compound wogonin (IC 50 ¼ 19.0, 15.8 and 16.5 mM, respectively). The results showed that the anti-tumour activity was enhanced when the 7position of wogonin was occupied by aliphatic amines, in contrast, wogonin derivatives substituted at 8-position or B-ring were generally less potent than the control 5-fluorouracil 38 . In another study, wogonin derivative 14 ( Figure 7) showed excellent activity against MV4-11 (leukaemia cell line) (IC 50 ¼ 20 nM) and CDK9 (IC 50 ¼ 19.9 nM) cell growth. In addition, compound 14 showed much improved physicochemical properties, such as water solubility, compared with the parent compound wogonin. The follow-up studies showed that the compound 14 is selective towards CDK9overexpressing cancer cells over normal cells. Preliminary mechanism studies on the anticancer effect indicated that 14 inhibited the proliferation of MV4-11 cells via caspase-dependent apoptosis. In addition, highlighted compound 14 showed significant antitumor activity in mouse acute myeloid leukaemia (AML) models without producing apparent toxic effects in vivo. Additionally, among the synthesised compounds, the piperazine and methylpiperazine substituted derivatives exhibited higher antiproliferative potency against the HepG2 cell line, while morpholine and pyrrolidine substituted analogs showed dramatically lower antiproliferative activity 39 .
In an investigation targeting prostate cancer cells, 6 of the 48 quercetin derivatives synthesised contained piperazine moieties. Moreover, quercetin derivative 15 ( Figure 8) demonstrated promising efficacy against the PC-3 cell line, with an IC 50 value of 4.47 ± 0.92 mM, being over 2-13 times more potent than quercetin. The experimental results indicated that modification of the hydroxyl groups at positions 3 and 5 significantly improved their inhibitory activity against prostate cancer cells, besides, the 48 derivatives are generally more effective in inhibiting the proliferation of PC-3 and LNCaP cells than DU145 cells 40 .
A series of novel bile acid (including ursodeoxycholic acid, cholic acid, chenodeoxycholic acid) hybrid compounds comprising piperazine were designed for anticancer activity. Among them, compound 16 ( Figure 9) as the most potent against KMS-11, a multiple myeloma cell line, with an LD 50 value of 8.5 ± 0.5 mM.    Concerning the structure-activity relationship, the best activity was obtained with compound 16 having cinnamylpiperazinyl group in the side chain and hydroxyl group at C-7 of the steroid skeleton. These results prompt us in a future study to incorporate other amino-substituted cinnamylpiperazinyl groups in the side chain of chenodeoxycholic acid 41,42 .
Currently, an ursolic acid (UA) derivative, obtained from the leaves and berries of many natural plants, is under development as a lead anticancer agent, which has also been modified by introducing piperazine rings to develop novel compounds. In 2015, Shao and co-workers revealed that substituted piperazine coupled to UA could induce cancer cell apoptosis and arrest cell cycle at the G0/G1 stage in HT-29, HepG2, and RL95-2 cells 43 . In the same year, UA derivative 17 ( Figure 10) with piperazine was reported as a potential anticancer agent, inducing cell apoptosis by G1 cell cycle arrest. Compound 17 displayed the best proliferation among this series of compounds, with IC 50 values of 9.82 mM, 18.97 mM, 13.64 mM, 5.40 mM and 11.06 mM against MGC-803, HCT-116, T24, HepG2 and A549 cancer cell lines, respectively. And compound 17 showed better potent antiproliferative activity than UA (as well as 5-FU) against these five cancer lines, with IC 50 values of 27.08 mM, 38.78 mM, 29.29 mM, 30.21 mM and 35.79 mM. SARs indicated that the synchronous introduction of piperazine and thiourea at C-28 and an acyl group at C-3 may improve the antitumor activity of UA, and solely introduction of an acyl piperazine thiourea at C-28 may decrease the antitumor activity. Moreover, it could be concluded that para-substituents in the phenyl ring were important than meta-substituents to their antiproliferative activities. Western blot and qRT-PCR (quantitative real-time PCR) experiments demonstrated that compound 17 may induce apoptosis through both intrinsic and extrinsic apoptosis pathways. Moreover, 17 has shown a pronounced increase in Fas and caspase-8 activity. Therefore, these studies indicated that the target compound may induce apoptosis, arrest cell cycle progression at the S phase and increase the activity of caspase-3 to inhibit cell growth. It is worth noting that piperazine was used as a linker in this UA modification 44 . UA derivatives targeting hypoxia-inducible factor-1a (HIF-1a) by introducing a 1,3,4-oxadiazole moiety, triazolone, and piperazine ring were designed and synthesised. Compound 18 ( Figure 10) demonstrated excellent activity inhibiting the expression of HIF-1a, with IC 50 ¼ 38.1 mM, possessing a lower appreciable cytotoxic activity (IC 50 > 100 mmol/L) than that of UA (IC 50 ¼ 23.8 mmol/L). SAR indicating that the triazolone moiety was favourable for HIF-1a inhibition in the UA derivatives, and introducing a piperazine ring to the oxadiazole structure can also enhance the biological activity of the compounds 45 46,47 . In 2019, some UA derivatives containing a piperazine moiety presented significant antitumor activities against the cancer cell lines evaluated. The structure-activity relationship studies revealed that the introduction of thiazole on A ring and triazole or tetrazole moiety on C-28 has little help to improve antitumor activities. The introduction of piperazine or homopiperazine can significantly improve the antitumor activity. And 4-fluorobenzyl and piperazine moieties are the crucial anticancer functional groups of these compounds. Among them, compound 20 ( Figure 10) was found most effective, demonstrating IC 50 values of 2.6 mM and 2.1 mM against the HeLa and MKN45 cell lines, respectively. And compound 20 was better than positive control drug cisplatin (IC 50 ¼ 15.1 and 2.8 mM, respectively) and was better than parent compound UA (IC 50 ¼ 15.1 and 16.7 mM, respectively). Compound 20 decreased the apoptosis regulator (BCL2/BAX) ratio, which subsequently disrupted the mitochondrial potential and induced apoptosis and significantly suppressed the growth of Hela xenografts in nude mice 48 . In 2012, UA derivative 22 ( Figure 10) was described as a potent antitumor agent that induced cell apoptosis and inhibited growth against MGC-803 cells (gastric cancer cell), with an IC 50 value of 2.50 ± 0.25 mM, which was better than positive control drug hydroxycamptothecin (IC 50 > 20 mM) and parent compound UA (IC 50 ¼ 24.32 ± 0.57 mM). The SAR studies revealed that incorporation of an acyl piperazine moiety at C-28 while retaining the polar group at C-3 significantly improved the antitumor bioactivities of the compounds 49 . In 2015, Zhao et al. synthesised and investigated a series of piperazinecontaining pentacyclic triterpene derivatives. OA derivative 25 ( Figure 11) and asiatic acid derivative 26 ( Figure 12) was reported as the most promising antitumor agents, with IC 50 values in the range of 7.05-13.13 mM, when evaluated against MCF-7, HeLa, and A549 cell lines. The pronounced influence of the incorporation of piperazine on antiproliferative effects in the series of synthesised compounds could be explained on the capacity for the formation of hydrogen bonds, improvement of water-solubility, and adjustment of molecular physicochemical properties. Furthermore, 25 and 26 can induce cell cycle arrest in all of the three cells 50 .
A series of C-28 amide derivatives of hederagenin were synthesised, with or without acetyl groups at the 3 and 23 positions of ring A, to develop effective cytotoxic agents. The amide derivatives were cytotoxic for a variety of human tumour cell lines. In general, the hydroxylated derivatives were less active than the acetylated derivatives. Among them, compound 28 ( Figure 13) carrying a piperazinyl presented good cytotoxic activity against A2780 cells (EC 50 ¼ 1.9 mM), which was better than parent compound hederagenin (IC 50 > 30 mM) 52 .
Li et al. synthesised a series of acetyl-11-keto-b-boswellic acid (AKBA) derivatives and evaluated their anticancer activity. Indicating that the cytotoxic activity of AKBA was improved effectively through modification of ring A and C-24. Introduction of 2cyano-3-oxo-1-en system in ring A of boswellic acids increased cytotoxicity. The esterification of free carboxyl acid at C-24 resulted in a sharp decrease in the activity. AKBA derivative 29 ( Figure 14) with 2-cyano-3-oxo-1-en in ring A and piperazine    amide at C-24 was reported as the most active in inhibiting the growth of prostate cancer PC-3 (IC 50 ¼ 0.04 mM) and LNCaP (IC 50 ¼ 0.27 mM) cell lines, with 796-fold and 102-fold more potent than AKBA. The mechanism of action of 29 indicated that cell cycle arrest and apoptosis induction were related to its anti-proliferative effects. 29 could regulate the expression levels of cell cycle-and apoptosis-related proteins, such as p21, cyclin D1, Mcl-1 and NOXA, in a concentration-dependent manner. AKBA had a weak ability to inhibit Pin1 activity, which was improved by the modification on ring A 53 .
Zhao and co-workers designed and synthesised several series of dehydroabietic acid piperazine derivatives. The results indicated that compound 30 ( Figure 15) with piperazine moiety showed selectivity for HepG-2 over MCF-7, with IC 50 values of 23.56 and 62.55 mM, respectively 54 . In another study, 1H-dibenzo[a, c]carbazole derivatives of dehydroabietic acid bearing different N-(piperazin-1-yl)alkyl side chains were designed, synthesised, and evaluated for their in vitro anticancer activities against three human hepatocarcinoma cell lines. SAR indicated that methyl, methoxyl, ethoxyl and chloro groups anchored on the indole moiety were more beneficial to the anticancer activity. In addition, the derivatives containing 12-Me and 12-OEt generally showed greater cytotoxic activities than their analogs with the same substituents at C-10, while compound with 10-OMe substituent was relatively more active than compound with 12-OMe. Especially, Compound 31 ( Figure 15) with 12-OEt substituent showed strong cytotoxic activity against SMMC-7721, HepG2, and Hep3B cell lines, with IC 50 values of 1.39 ± 0.13, 0.51 ± 0.09, and 0.73 ± 0.08 mM, respectively. Compared with lead compound and the positive control doxorubicin, it exhibited considerably more potent anticancer activities against three cancer cells and lower cytotoxicity to normal hepatocyte cell line QSG-7701 (IC 50 : 12.52 ± 0.58 mM). Compound 31 could significantly inhibit MEK1 kinase activity with IC 50 of 0.11 ± 0.02 mM. Furthermore, compound 31 increased the level of intracellular reactive oxygen species (ROS), reduced the mitochondrial membrane potential, destroyed the integrity of the cell membrane, ultimately resulting in the proliferation and apoptosis of HepG2 cells. Therefore, it could be a potent MEK inhibitor and a potential anticancer drug 55 .
To explore the SAR improve drug-like properties of celastrol derivatives, 23 derivatives targeting the Hsp90-Cdc37 interaction were synthesised by Jiang et al. SAR indicated that the derivatives that incorporated polar groups on the C29 carboxyl showed promising Hsp90-Cdc37 disruption activities and compound 32 ( Figure 16) containing the piperazine moiety exhibited the most significant Hsp90-Cdc37 activity disruption, with an IC 50 value of 4.71 ± 0.14 mM, which was better than parent compound celastrol (IC 50 ¼ 6.49 ± 0.27 mM). Compound 32 improved solubility and permeability. Compound 32 also exhibited potent antitumor activities, with IC 50 values of 1.01-1.12 mM against MCF-7, Panc-1 and A549, better than parent compound celastrol (IC 50 ¼ 2.02-2.48 mM). Compound 32 could induce the degradation of clients Akt and Cdk4 dose-dependently, suggesting that Compound 32 exerted their antitumor activities through Hsp90 inhibition in part. Moreover, Compound 32 induced cell cycle arrest at G0/G1 phase and physiological apoptosis in Panc-1 cells. Compound 32, with improved Hsp90-Cdc37 disruption activity, cytotoxicity and good druglike 56 . A series of 3-carbamate and 29-ester celastrol derivatives were synthesised by Shan et al. SAR indicated that the activity of derivatives with piperazine was better than that with aniline, Piperidine and morpholine. The substitution of piperazine and morpholine by piperidine and aniline weakened the activity, which might indicate that nitrogen atom and oxygen atom here are favourable as hydrogen bond acceptor but the alkyl  substituents and hydrophobic groups not. The nitrogen atom is more suitable than the oxygen atom here and the groups on it make difference to the activity. The activity gets lower when the substituents get bigger. Meanwhile, we observed a significant loss of cytotoxicity when the size of the substituent group on C-3 hydroxyl become as big as 8-10 carbons length. However, when the size was smaller than that, it did not cause an obvious change in the activity. It indicated a threshold about the size of this position, which is located in front of C-3 hydroxyl. It might affect the activity and pharmacokinetic property of celastrol, and the stereo structure here may improve the selectivity. The carbamates were introduced nitrogen and oxygen atoms as new hydrogen bond acceptors and hydrophilic centre. The antitumor activity of compound 33 ( Figure 16) against several cancer cell lines, with IC 50 values of 5.18 and 1.02 mmol/L against A549 and Bel7402 cell lines. It is slightly less active than the lead compound celastrol (IC 50 ¼ 2.12 mmol/L and 0.44 mmol/L), but compound 33 revealed a dosedependent inhibition and better inhibitory activity, with 70.96% inhibition of A549 cells at a dose of 12 mg/kg when compared with celastrol at 12 mg/kg (p < .05) in vivo 57 . Piperazine-containing celastrol derivative 34 ( Figure 16) was revealed a potential telomerase inhibitor against the MGC-803 cell line, presenting an IC 50 value of 1.00 ± 0.17 mM, which was better than positive control drug 5-FU (IC 50 ¼ 3.58 ± 0.40 mM), AMD (IC 50 ¼ 1.19 ± 0.07 mM) and parent compound celastrol (IC 50 ¼ 1.55 ± 0.25 mM) 58 . In 2017, a series of triterpenoic acid derivatives containing the piperazine moiety exhibited low EC 50 values against several human tumour cell lines 59 .
Reportedly, several artemisone-piperazine-sulphonamide derivatives have been synthesised. The antiproliferative activity of the new compounds was evaluated against human hepatoma cells. Compound 35 ( Figure 17) showed activity against SMMC-7721 cell lines (IC 50 ¼ 0.09 mmol/mL), which was better than parent compound artemisone (IC 50 ¼ 0.44 mmol/mL) 60 . In 2017, a series of novel artemisinin derivatives were designed and synthesised by Sun et al. and were evaluated using the MTT assay against ten cell lines. These derivatives were generally more effective in inhibiting cancer cell growth than artemisinin (IC 50 > 40 mM). Among them, compound 36 ( Figure 17) which bore an N-ethoxycarbonylpiperazine group was the most active against HepG2 and PLC-PRF-5 cells (the IC 50 values were 4.1 and 5.7 mM, respectively) and presented no cytotoxicity on L-02 cells, and 36 might be a valuable potential antitumor drug against HCC. SAR found that introducing the piperazine group into the C10 position of artemisinin could enhance the antiproliferative effect on certain cancer cells to some degree. The structure of compounds 36, which showed the highest activity against hepatocellular carcinoma cells, inspired to improve the oil solubility of the piperazine group into the C10 Figure 17. Chemical structures of artemisinin and its derivatives.
position. Furthermore, this substrate could induce cell cycle arrest in the G2/M phase and apoptosis in HepG2 cells 61 . Reportedly, Yu et al. synthesised twelve artemisinin-piperazine-dithiocarbamate derivatives, all of which had stronger in vitro antitumor activity than dihydroartemisinin. In particular, artemisinin derivative 37 (R ¼ 4-cyanobenzyl) ( Figure 17) showed promising inhibitory activity against the SMMC-7721 cell line, with an IC 50 value of 0.0025 ± 0.04 mM, which was better than positive control drug vincristine (IC 50 ¼ 0.0103 ± 0.03 mM), cytosine arabinoside (IC 50 ¼ 0.0271 ± 0.04 mM) and dihydroartemisinin (IC 50 ) 0.1000 mM) and lower toxicity against LO2 cell lines, presenting an IC 50 of 0.18 ± 0.04 mM 62 . In another study, artemisinin derivatives containing fluorine atoms synthesised by Li et al. were evaluated for their antiproliferative potencies against U87MG, SHSY5Y, MCF-7, MDA-MB-231, A549, and A375 cancer cell lines. Compound 38 ( Figure  17), the most active derivative containing the piperazine moiety, presented an IC 50 value of 2.1 mM against the human breast cancer MCF-7 cells by inducing apoptosis and G1-phase arrest and showed lower cytotoxicity (IC 50 ¼ 6.7 mM) than doxorubicin (IC 50 ¼ 0.9 mM) against L02. SAR showed that the structural modification of artemisinin, i.e. introducing the group of piperazine with attached amide bond into C10 position of artemisinin, led to a dramatic increase of the inhibitory effect on certain cancer cells to some degree 63 .
Narender et al. generated a variety of emodin derivatives and found that the piperazine-containing derivative 39 ( Figure 18) strongly inhibited the proliferation of HepG2 and MDA-MB-231 cancer cell line with an IC 50 of 10.44 mM and 5.027 mM, respectively, which is comparable to marketed drug epirubicin (IC 50 of 4.6 mM and 7.7 mM, respectively). SAR indicated that O-alkylation and C-alkylation of emodin improve the activity in both cell lines, whereas esterification only improves the activity in the MDA-MB-231 cell line. The derivatives 39 was capable of arresting the cell cycle at the G1/S phase, demonstrating caspase-dependent apoptosis in the HepG2 cell line, as well as DNA intercalating activity 64 .
A series of bergenin-1,2,3-triazole hybrids were synthesised and evaluated for their potent activity against cancer cell lines. Among them, piperazine-containing compound 40 ( Figure 19) exhibited potent activity against HeLa and A-549 cells, with IC 50 values of 1.33 mM and 1.86 mM, respectively, and was reportedly equipotent to doxorubicin (IC 50 values 1.98 mM and 1.34 mM respectively). Preliminary structure-activity relationship indicates that the presence of a substituent such as a chlorine atom or a cyano, floro and CF 3 group on aromatic triazole partner enhanced activity with IC 50 values ranging from 1.33 to 9.9 mM on A459 cell as well as HeLa cell lines within the studied series. In addition, flow cytometry analysis had demonstrated that compound 40 triggered cell cycle arrest in the G2/M phase and induced apoptosis in a dose and time-dependent manner. Taken together, compound 40 effectively inhibited tubulin polymerisation, disrupted intracellular tubulin-microtubule balance, resulting in prolonged G2/M cell cycle arrest. Docking studies also indicated a strong hydrophobic interaction with tubulin, thus leading to stable binding, consequently leading to apoptosis of cancer cells 65 .
Yan et al. revealed that glycyrrhetic acid-piperazine derivative 41 ( Figure 20) exhibited the best inhibitory activity against MCF-7 cells, with an IC 50 value of 1.08 mM, which was better than the positive control vandetanib (IC 50 ¼ 1.40 mM). SAR can be concluded that a fragment of a phenylpiperazine skeleton plays a significant role in the antiproliferative effect in vitro. Among the thirteen glycyrrhetic acid derivatives containing phenylpiperazine skeleton, compound 41, which has smaller steric hindrance showed better inhibitory capability than the others, and compounds that contain alkylpiperazine had the same trend. Compound 41 also showed potent inhibitory activity against vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase, with an IC 50 value of 0.35 mM. Docking simulations were performed to discover the binding mode, and the results indicated that compound 41 could bind well to VEGFR2 at the active site 66 .
Combretastatin-A4 linked sulphonyl piperazine derivative 42 ( Figure 21) exhibited remarkable cytotoxicity in all cancerous cell lines investigated. Compound 42 displayed an IC 50 of 0.36 ± 0.02 mM against A549 cells, which was more active than the lead compound Combretastatin-A4 (IC 50 ¼ 0.43 ± 0.02 mM). Further investigations revealed that it could significantly inhibit tubulin assembly, effectively acting at the colchicine binding pocket of tubulin. The SARs of these derivatives indicated that the 4-chloro substitution on the benzene ring C4 of the sulphonamide showed effective activity, and the activity was significantly reduced when sulphonamide ring is substituted by other substituents such as hydrogen, methyl, methoxy and tert-butyl. In addition, the selectivity of compound 42 to A549 cells is almost 3 times that of normal HaCaT cells 67 . O'Boyle et al. reported the synthesis and anticancer activities of combretastatin-A4 piperazine conjugates. The SAR research pointed out that the impact on the viability of MCF-7 breast cancer cells was related to the substituents on the piperazine ring, and the general trend in potency was as follows: phenylpiperazine > acetylpiperazine % cinnamylpiperazine > benzylpiperazine. Amino-substituted p-tolylpiperazine 43    Unfortunately, the activity of 44 is not as high as the lead compound colchicine (IC 50 ¼ 0.032 mM) 69 .
Sarsasapogenin-piperazine derivatives 45, 46a, and 46 b ( Figure 23) displayed significant inhibitory effects against several tumour cell lines. Derivatives 45 revealing potent toxicity against the A549 cancer cell line, with an IC 50 value of 1.70 mM, which was more active than positive control timosaponin A-III (IC 50 ¼ 11.92 mM) and 5-fluorouracil (IC 50 ¼ 42.15 mM). Compounds 46a and 46b exhibited significant cytotoxic activities against the six cell lines, being more potent than their parent compound sarsasapogenin. Furthermore, the p-fluorobenzyloxy series of compounds generally exhibited stronger cytotoxicities against all the tested cancer cells compared with the benzyloxy and p-methoxybenzyloxy series, and the substitution of pyrrolidinyl and piperazinyl groups at the C26 position was the preferred option for these compounds to display antitumor activities. Compound 46b exhibited excellent cytotoxic activity against MCF-7 cell line (IC 50 ¼ 2.95 mM), and was 16.7-fold more potent than sarsasapogenin. Further studies of the cellular mechanism of 46b showed that it arrested MCF-7 cells at the G2/M phase and induced apoptosis and necrosis 70,71 .
In recent years, several berberine derivatives were synthesised by Mistry et al., exerting anticancer activity against various human cancer cell lines. Conversely, a series of derivatives were synthesised by introducing various piperazine ring systems into 12-position of berberine. Mistry and co-workers reported that the berberine-piperazine derivative 47a ( Figure 24) demonstrated potent anticancer effects against HeLa cells, with an IC 50 of 7.327 ± 0.08 mM, and was more potent than the lead compound berberine (IC 50 ¼ 13.42 ± 0.23 mM). The analysis of the results suggested that the derivatives may enhance their ability to bind to the target of the drug mainly through hydrophobic interaction, conjugation and hydrogen bonding on the 9-hydroxyl group. On further preparation, analog 47b (Figure 24), demonstrating a substitution at the 3-position of the phenyl ring of piperazine moiety, was found to be the most effective, with an IC 50 value of 3.983 ± 0.07 mg/mL against HeLa cells, and was more potent than the lead compound berberine (IC 50 ¼ 4.499 ± 0.08 mg/mL). In another report, compound 47c ( Figure 24) with a 3,4-dichlorophenyl piperazine entity was found to be the most active among derivatives tested, with an IC 50 of 5.782 ± 0.55 mg/mL and CC 50 of 320.7 ± 1.04 mg/mL, and the highest TI of 55.47 against HeLa cell lines. The potency of this compound was double that of the parent berberine which showed a TI of 28.16. The results clearly showed that the piperazine-based berberine analogues had stronger anti-tumour activity on the Caski cell line than Hela. It appeared that introduction of electron-withdrawing substituents (e.g. chloro-or fluoro-substituent) on the piperazine-linked benzene ring enhanced anti-tumour activity [72][73][74] . In 2016, berberine derivative 48 ( Figure 24) with a furoyl piperazine substituent exhibited the highest anticancer activity against the CaSki cell line, with an IC 50 value of 6.11 mM, CC 50 value of 344.5 mM, and selectivity index of 56.39. The potency of this compound was double that of the parent berberine which showed a TI of 29.15. Conversely, a series of derivatives were synthesised by introducing various piperazine ring systems into the 9-position of berberine. Concerning the activity regarding the functional group attached to the piperazine ring, the order falls in the way alkyl > chloro >fluoro > nitro 75 . Analog 49 ( Figure 24) with a 4-methylpiperazine substituent showed excellent anticancer property, with a therapeutic index (TI) of 58.53 (HeLa) and 48.76 (CaSki), better than that of a parent compound berberine with 27.41 and 25.84 of TI, and IC 50 values of 5.595 ± 0.02 mg/mL and 6.716 ± 0.05 mg/mL, respectively 76 .
Chen reported the synthesis of oestrone derivatives containing the piperazine moiety and evaluated their antitumor activities against several classical prostate cancer cell lines, including PC3, LNCaP, and DU145. Derivative 50 (R ¼ 3,4-di-Cl) ( Figure 25) showed significant cytotoxic actions against the LNCaP cell line (IC 50 ¼ 0.78 ± 0.34 mM), better than that of positive control finasteride (IC 50 ¼ 14.53 mM). and exhibited better a 1 -adrenergic receptor (AR) subtype selectivity over a 1B (a 1B /a 1A ratio ¼ 14.7). The SAR results showed that the ortho-substituted phenyl derivatives       Further experiments also found that compound 52 may interfere with cellular ATP reserves by affecting cell kinase activity 79 . A series of purine derivatives were synthesised and evaluated against liver Huh7, breast T47D, and colon HCT116 carcinoma cells. Compound 53 ( Figure 27) containing a piperazine moiety had better cytotoxic activities (IC 50 1 mM) than the nucleobase 5-FU and nucleosides fludarabine, cladribine, and pentostatine 80 .
Piperazine carbamate was used to link camptothecin derivatives to prevent rapid hydrolysis. Compound 57 (Figure 29) demonstrated a potent affinity to integrin receptors and a potent ability to inhibit A2780 cells, with an IC 50 value of 0.033 mM. Importantly, a significant increase in t 1/2 for 57 vs irinotecan in rat plasma (13 h vs 45 min, respectively) 84  . SAR study suggested that both aromatic and aliphatic substituents on the sulphonyl-piperazinyl side chain at C-7 can promote potency to some extent, while selected variations of these substituents can greatly affect the activity. This study revealed that the integration of the sulfonylpiperazine modify into the 7-position of camptothecin is a potent strategy for discovering effective cytotoxic camptothecin derivatives 86 .
New analogs of benzo-a-pyrone containing piperazine derivatives have been synthesised. Compound 61 (Figure 30) displayed the greatest activity against the WM266-4 (BRAFV600E) cell line, with an IC 50 value of 1.35 mM. Unfortunately, the activity of 61 was not as good as that of the positive control vemurafenib (IC 50 ¼ 0.07 mM). Following the biological evaluation assays indicated that 61 could result in cellular apoptosis, marked DNA fragmentation, and G0/G1 phase arrest in melanoma cells. Moreover, docking simulation showed that compound 61 could bind well with the crystal structure of BRAFV600E at the active site. Compared  with the compound in which piperazine is connected to the alkyl substituent, the aromatic-substituted compound inhibited BRAFV600 more strongly. Studies pointed out that the change in the position of the substituents on the piperazine-linked benzene ring may have little effect on the inhibitory activity, besides, the compounds substituted by the electron-donating groups on the   benzene ring showed better inhibitory activity than the electronwithdrawing groups 87 .
Myricetin piperazine derivative 62 ( Figure 31) exerted a moderate inhibitory effect against MDA-MB-231 cells (human breast adenoma cell) (IC 50 ¼ 3.87 ± 0.21 mM), was slightly lower than the positive control doxorubicin (IC 50 ¼ 2.01 ± 0.17 mM). The SAR indicated that all examined myricetin derivatives showed good activity against human breast cancer cell lines (MDA-MB-231 and Bcap-37) but poor activity against human gastric cancer cells (SGC-7901 and MGC-803 cell lines) 88 . Ruan et al. reported that myricetin derivative 63 ( Figure 31) containing a piperazine moiety showed potent inhibitory activity against a human breast cancer cell line (MDA-MB-231) (Inhibition rate¼ 86.7% in 10 mmol/L), which was close to epirubicin (94.6%), In addition, through the analysis of the morphological effect on the target tumour, it can be seen that although the cytotoxicity of myricetin is small, its inhibitory effect on the tumour cells is not obvious; and the myricetin derivative after the introduction of piperazine amide has low cytotoxicity and can effectively inhibit the proliferation of tumour cells, which has good anti-tumour research value 89 .
Reportedly, derivative 64 ( Figure 32) (1-p-nitrophenylpiperazinyl substituted derivative of deoxypodophyllotoxin) demonstrated the most active cytotoxicity against A-549, HeLa, and SiHa cells (IC 50 ¼ 0.102, 0.180, and 0.0195 mM, respectively), better than that of parent compound deoxypodophyllotoxin (IC 50 ¼ 0.470, 36.0, and 10.2 mM, respectively). Moreover, flow cytometric analysis indicated that the compound could arrest the cell cycle at the G1 phase in A-549 cells 90 . In 2013, demethylepipodophyllotoxins carbamate derivative 65 (Figure 32) was reported as the most promising compound synthesised in their study, resulting in cell cycle arrest in the G2/M phase and apoptosis in HeLa cells, presenting an IC 50 value of 0.074 mM, better than that of positive control etoposide (IC 50 ¼ 2.91 mM). SAR indicated that the substituent of the amines markedly affected the activity profiles of this compound class, and the compounds with piperazines incorporated appear to be more potent than those with alkyl amines. Second, the compounds with amino acids incorporated appear to be less potent than those with p-nitrobenzylpiperzine directly and the compounds with L-amino acids incorporated appear to be more potent than those with D-amino acid. Lastly, compounds that contain only one substituent at the C-4 position showed superior activities in comparison with compounds simultaneously substituted at the C-4 and C-4 0 position of 4 0 -demethylepipodophyllotoxin 91 . Five bis-epipodophyllotoxin compounds were designed using a structure-based design approach, showing strong evidence that these compounds targeted topoisomerase II in a variety of assays. Compound 66 ( Figure 32) containing a linker with two piperazine rings exhibited the highest activity (IC 50  methylenes. The IC 50 values then coordinately increased when the linker length was increased to ten and twelve methylenes. These results suggested that an optimum chain length was in the order of eight methylene groups, and thus may be the optimum chain length needed for the bis-epipodophyllotoxins to achieve bisintercalation in the DNA within the topoisomerase IIbDNA ternary structure 92 . In 2017, Sun reported that podophyllotoxin piperazine derivative 67 ( Figure 32) manifested prominent cytotoxicity against the MCF-7 cell line (IC 50 ¼ 2.78 ± 0.15 mM). Furthermore, compound 67 inhibited tubulin assembly without causing damage to non-cancerous cells, inducing apoptosis and cell cycle arrest at the G0/M phase in MCF-7 cells. It can be seen from the experimental data that almost all synthesised compounds showed anticancer activity, the substituents on the aromatic ring had a significant effect on the activity. According to the SAR, it was clearly found that compounds with substitution on the phenyl ring of the phenylpiperazine group in general exhibited inhibitory activities, electron-withdrawing groups (F, Cl, NO 2 , CF 3 ) > electrondonating groups (OMe, Me), however, the modification of the 2 or 4 positions of the benzene ring resulted in a decrease in potency 93 .
Chen and co-workers synthesised a series of anthraquinone derivatives to determine their potential telomerase inhibitory activities. It was found that compound 69 ( Figure 34) with benzylpiperazine was found to show the best selectivity towards repressing hTERT expression in H1299 cells among these tested compounds. And compound 69 showed moderate potency against PC-3 with IC 50 ¼ 16.0 mM. Furthermore, SAR analysis indicated that the piperazine linker might be a significant moiety for telomerase inhibition or hTERT repression 95 .
Piperazinylquinolone 70 (Figure 35) was considered a starting point for the design and development of new anticancer agents. Chemical modification of N-4 in the piperazine ring and the carboxylic acid at C-3 could present sufficient flexibility 96 .
Pterins are a class of fused N-heterocyclic compounds containing pyrimidine and pyrazine rings, which widely existed in natural products and bioactive molecules 97 . In a study presented by Huang et al., a piperazine linker was identified as the best hybrid approach for glycyrrhetinic acid and hydroxamic acid. Conjugate 71 ( Figure 36    Vindoline conjugated with N-methylpiperazine (compound 73, Figure 38) demonstrated a significant antiproliferative effect, with IC 50 values of 9.36 mM, 14.10 mM and 25.49 mM, respectively, against HeLa, MCF-7 and MDA-MB-231 cell lines, although the monomer vindoline was inactive (IC 50 > 30 mM). In addition, the experimental results suggested that the compound of ventolin and amino acid, tryptophan methyl piperazine and N-methyl piperazine have significant anti-proliferative effects. Concerning structure-activity relationships, it seems that the 1,2,3-triazole and morpholine moieties could not improve the efficacy of vindoline at all 101 .
A series of indirubin derivatives were designed to improve water solubility and antitumor activity. Among them, compounds containing a piperazine moiety exhibited significant inhibitory activity against human large cell lung cancer cells (LXFL529L cells). Compound 74 (Figure 39) carrying a methylpiperazino-phenylamino substituent attached to 5-carboxyindirubin, with a water solubility of 520 mg/L, displayed the most potent antitumor activities, with an IC 50 value of 0.54 mM, better than that of Positive control 5-carboxyindirubin (IC 50 ¼ 7 mM). When the N-methylpiperazinyl substituent on the benzene ring was changed to dimethylamino, although the water solubility increased, the activity of inhibiting cell proliferation decreased. In addition, the phenyl moiety of 74 replaced by an aliphatic chain also produced a similar effect 102 .
Several baicalein-piperazine derivatives showed moderate antiangiogenic activity, with weak toxicity on normal tissues. Notably, compound 75 ( Figure 40), with the piperazine acetamide group at 6-position, was the most active compound on human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation in vitro. Moreover, consistent with the anti-angiogenic activity, compound 75 significantly inhibited the growth of A549 cells (IC 50 ¼ 4.73 mM). The data indicated the introduction of an     alkyl alcohol substituent at the 6 positions could increase the antiangiogenic activity, and shortening the chain length reduced toxicity. In addition, regarding the 6-O-acetamide substituted scaffold, the substitution of a benzyl group for the phenolic hydroxyl group did not significantly improve the effect on inhibiting angiogenesis, while the compound 75 with a piperazine group showed the strongest anti-angiogenic activity 103 .
Liu et al. reported that apigenin-piperazine derivative 76 ( Figure 41) possessed active antiproliferative activity with the lowest IC 50 values of 16 mg/mL, 11 mg/mL, 25 mg/mL and 32 mg/mL against A549, HeLa, HepG2, and MCF-7 cancer cells, more potent than parent compound apigenin (IC 50 ¼ 1740 mg/mL, 450 mg/mL, 460 mg/mL and > 2000 mg/mL). SAR analysis indicated that compared with the compounds with 2-carbon spacer, 3-carbon-spacercontaining compounds, showed a slight increase in antiproliferative activity against the four cancer cell lines. This result may have been caused by the elongation of the alkyl side chain from two to three C-atoms that retained the modulating activity, with C3bridge derivatives being the most active. The compounds containing N-heterocyclic-ring amino side chains displayed better activities against the four cancer cell lines than those containing alkyl amino side chains. This result may be attributed to the increasing lipophilicity of these compounds, which could influence their ability to reach the target via transmembrane diffusion 104 .
Twenty Pulsatilla saponin A (PSA) derivatives with C ring, C-28, or C-3 modifications were synthesised and evaluated for cytotoxic activities against A549, MCF-7, MDA-MB-231, KB, SMMC-7721, BGC-823, and KB-VIN cell lines. Compound 77 (Figure 42) with the piperazine ring at the 28-COOH position exhibited better cytotoxic activity (IC 50 ¼ 4.68 mM) and lower haemolytic toxicity (HD 50 > 500 mM) against the A549 cell line when compared with PSA (IC 50 ¼ 22.87 mM, HD 50 ¼3.86 mM). The data indicated that the carbon ring (especially the C-12/C-13 double bond) might play an important role in the cytotoxicity and haemolytic toxicity of prostate-specific antigen. The 28-COOH may cause haemolysis of red blood cells. In addition, the fully acetylated sugar chain at position 1 showed weaker cytotoxicity than PSA, indicating that the sugar hydroxyl group is important for the cytotoxicity of PSA. Further molecular biology studies showed that compound 77 might cause G1 cell cycle arrest 105 .

Antibacterial activity
Piperazine derivatives developed by Chue et al. were assessed for their antibacterial potential, demonstrating the synthesis of betulonic acid amides with N-derivatives of piperazine and their antibacterial activities. Compounds 78a and 78b ( Figure 43) inhibited bacterial growth in all tested cultures. The diameter of the growth inhibition zone was in the range of 13 À 16 mm for five of the studied test cultures, which is better than the positive control gentamicin (16 À 21 mm) 106 .
Wang synthesised coumarin derivatives containing a piperazine skeleton as potential antibacterial agents.     Bacillus subtilis; MIC ¼ 0.355 mg/mL for Staphylococcus aureus] compared with the positive control Penicillin G (MIC ¼ 0.752 mg/ mL and 1.685 mg/mL). What's more, it showed the most potent activity against SaFabI with IC 50 of 0.57 mM. The SAR revealed the activity of compounds with substituents on the benzene ring is better than that of unsubstituted compounds, moreover, the parasubstituted compounds showed stronger antibacterial activity than the meta-and ortho-substituted compounds 107 .
Flavone derivatives 80a-80e ( Figure 45) with piperazine rings were found to demonstrate more potent antibacterial activity, showing even 2 to 2.5-fold more potency than that of standard ciprofloxacin and miconazole at the same MIC value of 10 mg/mL. It was observed that the presence of amino alkyl, cyano or alkenyl alkyl group on piperazine ring seemed to be favourable for the high antimicrobial activity, however, the mere presence of electron donor group on the piperazine ring had very little or nothing to do with antimicrobial activity 108 .
In recent years, several pleuromutilin derivatives containing piperazine moieties were designed and synthesised. Pleuromutilin analog 81 (Figure 46) was designed and synthesised with a purine ring as a polar and water solubilising group. In further investigation, analog 81 with piperazine displayed superior in vitro and in vivo antibacterial activity against some Gram-positive strains, including drug-resistant pathogens. Analog 81 with good solubility in water showed remarkable antibacterial activity against all Gram-positive pathogens, including methicillin-resistant S. aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE), except for Enterococcus  faecalis. Importantly, analog 81 exhibited the most potent antibacterial activity against susceptible and resistant S. pneumoniae, with a MIC of 0.063 mg/mL, appeared as a possible alternative to the weak marketed antibacterial agents macrolide erythromycin (MIC > 128 mg/mL), tetracycline minocycline (MIC ¼ 8 mg/mL), and quinolone levofloxacin (MIC ¼ 8 mg/mL). Interestingly, 81 and glycopeptide vancomycin (VCM) exhibited superior ED 50 values (about 1-2 mg/kg) when given intravenously regardless of the frequency of dosing (once or twice), indicating the potential of 81 as an antibacterial agent with efficacy equal to that of VCM 109 . A series of novel pleuromutilin derivatives having a piperazine ring were synthesised under mild conditions. In the ortho-, meta-and paraposition of the benzene ring, electron withdrawing and donating groups were introduced to explore SAR. The ortho-NO 2 substituted derivative 82 (Figure 46) was found to be the most active compound against S. aureus (MIC ¼ 0.0625 mg/mL) among a series of pleuromutilin derivatives, better than the positive control tiamulin (MIC ¼ 0. 25 mg/mL). The results revealed ortho-substituted benzene derivatives have the greatest impact on activity compared with para and meta substitutions, for example, replacement of the nitro group in compound 82 with chlorine group at the same position, which resulted in dramatic decrease in antibacterial activity. The meta-substituted benzene derivatives with CH 3 O-, CH 3 -, and NO 2substituents showed enhanced antibacterial activities, all the para-substituted benzene derivatives bearing methoxyl, methyl, hydroxyl, nitro, and chlorine substituents exhibited moderate antibacterial activities. The compound-bearing nitro group in the para-position showed higher activity compared to other para-position substituted derivatives. The para-position appeared to have less influence on the antibacterial activity of these pleuromutilin derivatives bearing a phenyl piperazine 110 . In 2015, derivative 83 ( Figure 46) was revealed remarkable activity against S. aureus (ATCC25923; MIC of 0.125 mg/mL), which is equal to the control compound tiamulin. The antibacterial activities of 83 to Streptococcus suis (MIC of 2 mg/mL), Streptococcus agalactiae (MIC of 0.5 mg/mL), and Streptococcus dysgalactiae (MIC of 0.5 mg/ mL) were also excellent compared with the control drug erythromycin (MIC of > 128 mg/mL). Further, the results preliminary confirmed that the activity of secondary amine is better than that of a tertiary amine. A molecular docking study indicated that 83 could bind to the active pocket of the ribosome with seven hydrogen bonds 111 . In 2018, pleuromutilin-piperazine derivative 84a ( Figure 46) exhibited moderate antibacterial activity against methicillin-sensitive S. aureus (MSSA) with MIC of 0.125 mg/mL, that even more active than the comparator valnemulin (MIC ¼ 0.25 mg/mL). However, the activity against P. multocida was also examined, and the most efficient compound 84b proffered identical MIC (16 mg/mL) as valnemulin. In addition, the results provided by the dose-response study demonstrated 84b could supply infected mice with a 70% survival rate at a dose of 40 mg/kg via intragastric (i.g.) administration 112 . Furthermore, in 2020, pleuromutilin derivative 85 (Figure 46) was revealed as the most potent derivative against S. aureus and Staphylococcus epidermidis, with a MIC value of 0.0625 mg/mL, showed an almost two-fold increase in activity in comparison with that of reference drugs, and an eightfold increase in activity against Enterococcus faecalis compared to that of tiamulin. Furthermore, the docking study revealed that derivative 85 could interact with the ribosome pocket with more bonds when compared with tiamulin 113 .
Reportedly, a new series of piperazinyl-substituted chrysin derivatives have been designed and synthesised. Among the synthesised compounds, analogs accommodating morpholine, piperidine, and electron-donating groups at the benzene ring showed better antibacterial activities than those that possess electronwithdrawing substituents. The chrysin containing morpholine compound 86 ( Figure 47) displayed the most potent inhibitory activity against Escherichia coli FabH (IC 50 ¼ 5.78 ± 0.24 mM), comparing to the control positive drugs penicillin G (7.56 ± 0.30 mM). In the antibacterial activity assay, 86 was found to be the most active against S. aureus and E. coli (MIC ¼ 1.25 ± 0.01 and 1.15 ± 0.12 mg/mL, respectively), compared with the control positive drugs (MIC ¼ 1.39 ± 0.02 and 1.82 ± 0.47 mg/mL, respectively) 114 .
A series of novel 1,5-diphenyl-2-penten-1-one analogs with a piperazine moiety were synthesised and evaluated for their antifungal and larvicidal activities. Among them, the most potent compound 87 ( Figure 48) demonstrated 100% mortality against mosquito larvae at a concentration of 1 mg/L. Compound 88 (Figure 48), with a 3-position chlorine substituted phenyl group, exhibited the highest activity with an EC 50 value of 0.075 mM against Pythium aphanidermatum, superior to lead compound 1,5-Diphenyl-2-penten-1-one (0.39 mM). Initial SAR analysis showed that a methyl group had significant effects on the biological activities of these compounds, the compounds with N'-unsubstituted piperazine exhibited much better antifungal activities and larvicidal activity against mosquitoes than the compounds with N'methylated piperazine. In addition, the larvicidal activity against mosquitoes had a sharp decline when the substituent on the benzene ring was changed from 4-position to 2 or 3-position 115 .
Eight novel curcumin derivatives were synthesised using the Mannich reaction and evaluated for their antibacterial activities; two derivatives containing a piperazine ring showed remarkable mildew resistance (Figure 49    Xiang et al. synthesised a series of novel 18b-glycyrrhetinic piperazine amides to evaluate in vitro and in vivo antibacterial activities against phytopathogens Xanthomonas oryzae pv. oryzae (Xoo) and X. axonopodis pv. citri (Xac), as well as induced apoptotic behaviours. Screening results revealed that all designed compounds were bioactive against Xoo and Xac. Among them, compound 90 ( Figure 50) bearing an isopropyl tail presented the most potential antibacterial efficiency, with EC 50 values of 2.28 and 1.42 mg/mL against Xoo and Xac, respectively, which is far better than lead compound 18b-glycyrrhetinic acid (EC 50 > 400 mg/mL), positive control bismerthiazol (EC 50 ¼ 92.6 mg/mL and NT) and thiodiazole copper (EC 50 ¼ 121.8 mg/mL and 77.0 mg/mL). Pharmacophore studies suggested that the synergistic effect of glycyrrhetinic acid skeleton and N-containing scaffolds was essential for the antibacterial activity. Further studies revealed that compound 90 could induce the generation of excessive ROS in tested pathogens 118 .
In another study, piperazine-tailored ursolic acid hybrids 91a ( Figure 51) exhibited remarkable antimicrobial activity against Xoo and Xac (EC 50 values were 0.37 and 1.08 mg/mL, respectively), which is far better than lead compound ursolic acid (EC 50 > 400 mg/mL), positive control bismerthiazol (EC 50 ¼ 92.6 mg/mL and NT) and thiodiazole copper (EC 50 ¼ 121.8 mg/mL and 77.0 mg/mL). Studies have shown that the hydroxyl group in the derivative structure had a positive role in promoting antibacterial action, on the contrary, the elimination of the N-ethyl piperazinyl or N-isopropyl piperazinyl group dramatically reduced antibacterial activity. Importantly, compounds 91a and 91b ( Figure 51) could manage bacterial blight in vivo in pot experiments. A preliminary finding revealed that the synthesised compounds could disrupt the equilibrium of the redox system in tested pathogens, leading to excessive ROS accumulation, which induced the apoptosis-like effect in tested pathogens 119 .  Amongst a series of novel 2H-chromen-2-one derivatives bearing 1,2,3-triazole moiety, compound 92 ( Figure 52) containing Nacetylpiperazine moiety was found to be most effective against Pseudomonas aeruginosa (zone of inhibition in 17.1 mm), which is equivalent to the standard (zone of inhibition in 18 mm). Based on, in silico pharmacokinetic studies, compounds 92 was identified as lead compounds for future investigation due to their lower toxicity, high drug score values and good oral bioavailability as per OECD guidelines 120 .

Anti-inflammatory activity
Inflammation is a non-specific immune response to infection, tissue stress, or some other form of injury 121 . Almost all modern human diseases are related to chronic inflammation 122 . Moreover, glucocorticoids and non-steroidal anti-inflammatory drugs currently in clinical use have serious gastrointestinal side effects 123,124 . Therefore, researchers have developed a series of anti-inflammatory piperazine derivatives based on natural products.
A series of flavone piperazine derivatives were synthesised and evaluated for their in vitro anti-inflammatory activity. Among all the compounds screened, compounds 93a-93b ( Figure 53) showed promising TNF-a and IL-6 inhibitory activity. Furthermore, Compound 93ba ( Figure 53) exhibited excellent inhibition against tumour necrosis factor-a (TNF-a) and interleukin (IL)-6 (up to 65-87% TNF-a and 70-93% IL-6 inhibitory activity) at a concentration of 10 mM with reference to standard dexamethasone (71% TNF-a and 84% IL-6 inhibitory activities at 1 mM. Notably, the presence of highly electron-rich groups such as methoxy, pyrimidyl, morpholine on piperazine as well as homologation of chromone and piperazine moiety had strong relevance to the anti-inflammatory activity 108 . Compound 94 (Figure 54) demonstrated significant activity against b-glucuronidase (inhibition rate ¼ 75.43% at 1 mM concentration), which was better than that of natural chalcone (NC, (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-phenylprop-2-eb-1-one, inhibition rate ¼ 28.42% at 1 mM concentration). Further results indicating that chalcones with N-methyl piperazine methyl and piperidine methyl substitutions appear to be crucial for b-glucuronidase inhibition, and N-methyl piperazine group substitution at 3 positions of terminal benzene ring was found to be more suitable for the reduction of DPPH. Compound 94 was revealed the highest antioxidant activity in the DPPH free radical scavenging assay at 1 mM concentration (DPPH reduction rate ¼ 37%) in the synthesised compound, far exceeding NC (DPPH reduction rate ¼ 16.67%) but still weaker than quercetin (DPPH reduction rate ¼ 86.30%) 125 .
Novel chalcone-biscoumarin hybrids were synthesised by exploiting the anti-inflammatory potential of natural chalcone derivatives. All hybrid molecules were evaluated for in vivo antiinflammatory activity using the in vivo para-xylene-induced mice ear-swelling model. Particularly, hybrid 95 ( Figure 55) showed the most active anti-inflammatory activity (inhibition rate, 75.46%), which exceeded that of aspirin (inhibition rate, 64.91%) and was equal to that of celecoxib (inhibition rate, 77.03%). Preliminary SAR analysis indicated that substitution of aryl-piperazine or arylsulphonyl-piperazine with an electron-withdrawing group demonstrated an advantage over the electron-donating group, improving the anti-inflammatory activity 126 .
In a study performed by Li et al., compound 96 ( Figure 56) with a piperazine unit exhibited the most potent nitric oxide (NO) and IL-6 inhibitory activity (IC 50 ¼ 13.3 mM) in the tested unsaturated glycyrrhetic acid derivatives. Moreover, compound 96 significantly reduced lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, as well as IL-6 production, through the MAPK and NF-jB signalling pathways 127 .

Antioxidant activity
In 2019, smilagenin derivative 97 ( Figure 57) was evaluated for its potential neuroprotective effects, and revealed improved cell viability against H 2 O 2 -induced damage, inhibited NO production, and demonstrated strong antioxidant activity. The protective effect of compound 100 at a concentration of 10 mM (Cell viability ¼ 40.5 ± 2.3%) against the oxidative stress triggered by H 2 O 2 in SH-SY5Y cells was more effective than that of positive control N-acetyl-L-cysteine at 1 mM (Cell viability ¼ 37.1 ± 2.5%). The activity results revealed benzyl-substituted piperazine formate derivatives showed the potent neuroprotective activity 128 .
b-elemene piperazine derivatives were synthesised and evaluated for their inhibitory abilities. The data results indicated that these derivatives induced apoptosis through ROS production and reduced c-FLIP levels, activating both death receptor-mediated and mitochondrial-mediated apoptotic pathways 129 .
Coumarin derivative 98 ( Figure 58) exhibited significant activity (IC 50 ¼ 2.1 mM) and good selectivity for AChE with respect to butyrylcholinesterase. Structure-activity relationship studies have shown that the anti-acetylcholinesterase activity of the compound was affected by the type of cyclic amine on the 2-oxo-or 4-oxoalkoxycoumarinbackbone 130 . Coumarin piperazine derivative 99 ( Figure 58) was found to be a potent AChE inhibitor, with an IC 50 value of 4.9 mmol/L against human AChE enzyme (hAChE), while the IC 50 values of reference drug ensaculin and doneprazil were 0.36 ± 0.01 mmol/L and 0.11 ± 0.01 mmol/L, respectively. Among the newly synthesised analogs, the most effective compound was 102, having a methoxy group at the meta position. If the methyl group is attached in the ortho and para positions, the activity was further reduced. In addition, when the ortho-substituted methyl group of the compound was shifted to the para and meta positions, the inhibitory activity declined. Compounds showed better inhibitory activity when nitro was substituted at the meta position, or remarkably decreased if nitro shifted to the ortho and para positions. Subsequently, a SAR study using the molecular field method revealed that the inhibitory mechanism may be attributed to the hydrophobic field and positive charge centre conferred by the coumarin and piperazine moieties 131 .
In the previous section, it was stated that a series of berberine derivatives were successively reported by Mistry et al, simultaneously presenting the anticancer and antioxidant activities. Compound 100 (Figure 59) showed remarkable antioxidant ability in the ABTS assay, with an IC 50 of 4.644 mg/mL. In another report, analog 101 ( Figure 59) exhibited the highest scavenger activity in the DPPH free radical scavenging assay, with an IC 50 of 17.25 mg/ mL. Furthermore, compound 102 ( Figure 59), demonstrating two electron-withdrawing chlorine atoms on the piperazine entity attached to the berberine ring, revealed remarkable radical scavenging potential in the DPPH assay, with an IC 50 of 19.80 ± 0.94 mM, comparable to that of control drug ascorbic acid at 61.03 ± 0.66 mM and far better than berberine at 89.18 ± 1.60 mM. Compound 103 (Figure 59), with a naphthyl piperazine substituent, demonstrated the notable potency in the ABTS þ analysis, with an IC 50 value of 8.94 mM while the parent scaffold berberine with IC 50 ¼ 232.1 ± 2.11 mM and ascorbic acid with IC 50 ¼ 27.02 ± 0.05 mM. Compound 50 ( Figure 25) showed significant antioxidant activity with an IC 50 value of 11.08 and 4.76 mg/mL in DPPH and ABTS bioassays, respectively, while the parent scaffold berberine with IC 50 ¼ 34.29 mg/mL and 82.17 mg/ mL and ascorbic acid with IC 50 ¼ 10.75 mg/mL and 5.528 mg/mL in the DPPH and ABTS bioassays, respectively 72,[74][75][76] .
A series of 2-(2-hydroxyphenyl) pyrimidine/benzothiazole piperazinyl-substituted flavones were synthesised and evaluated for their in vivo biological properties. The results indicated that all tested compounds containing flavonoid-piperazine moieties were found to possess free radical scavenging potential. The SAR study showed that the antioxidant activity was decreased with methoxy group substitution on the flavone unit. Furthermore, the analogue 104 ( Figure 60) benzothiazole substituted piperazine ring showed better antioxidant activity. Hydroxyl free radical scavenging capacity of analogue 104 at 1.25 mM (scavenging ratio ¼ 48.0%) was comparable to positive control thiourea (scavenging ratio ¼ 46.0%), and higher than a-tocopherol (scavenging ratio ¼ 18.7%) 132 .

Nervous system activity
In 2011, by introducing N-methylpiperazine on aromatic compounds, it was expected that the hydrogen bond substitution and additional electrostatic interaction would significantly enhance the binding affinity with the Ab peptide. Curcumin analog 105 ( Figure  61) containing a piperazine ring reportedly demonstrated the most significant activity, with an IC 50 value of 2.5 ± 1.2 mM and 90.2 ± 3.2% inhibition of Ab aggregation at a concentration of 50 mM, a much higher activity than curcumin (IC 50 value of 12.1 ± 1.2 mM and 71.3 ± 1. 3% inhibition of Ab aggregation at a concentration of 50 mM) 133 .
In 2013, tacrine-flavonoid hybrids were connected via piperazine side-arm alkyl groups. Among the series of compounds, compound 106 ( Figure 62) displayed balanced inhibitory effects against acetylcholinesterase (AChE) and self-induced Ab 1À42 aggregation (IC 50 ¼ 133 nM for AChE, IC 50 ¼ 6.5 mM for aggregation), better than tacrine (IC 50 ¼ 260 ± 8 nM for AChE) and curcumin (IC 50 ¼ 20.3 ± 1.2 mM for aggregation). Furthermore, it exhibited moderate metal chelating activity and low cell toxicity. A molecular docking study indicated that the piperazine in the structure could bind to the AChE binding site in the middle of the canyon through a cation-p interaction (5.45 Å) between its protonated nitrogen atom and Tyr334 in the middle of the canyon 134 .    In 2018, a series of multifunctional 3-piperazinecarboxylate sarsasapogenin derivatives were designed and synthesised against AD. The protective effect of compound 107 ( Figure 63) at a concentration of 10 mM (Cell viability ¼ 43.9 ± 2.8%) against the oxidative stress triggered by H 2 O 2 in PC12 cells was stronger than that of positive control N-acetyl-L-cysteine at 1 mM (Cell viability ¼ 35.3 ± 2.5%). Further SAR analysis indicated that benzyl, electrondonating groups, and intramolecular hydrogen bonding may help increase its neuroprotective activity. The report indicated that Nsubstituted piperazine carboxylate can be used as a promising structural unit for designing anti-AD drugs. Significantly, compound 107 protected PC12 cells from Ab-induced damage and improved learning and memory impairments in Ab-injected mice with low-dose administration at 6 mg/kg 135 .
In 2019, genipin analog 108 ( Figure 64) with a benzylpiperazine modification at C-8 position showed a higher inhibition rate (%Inhibition ¼ 22.29) on protecting PC12 cells against injured by Ab 1 -42 than the positive reference donepezil (%Inhibition ¼ 17.65). For compounds containing methyl substituents, replacement at 3-position of the terminal benzene ring was more beneficial to the enzymatic activity than at 4-position substitution. In addition, the methoxy substitution at the terminal phenyl moiety was more advantageous than the methyl group. When an electron-withdrawing groups were introduced at the end of the phenyl ring, the increase in activity was consistent with the enhancement of the electron-withdrawing performance of the substituent. In the entire series, genipin derivatives with ligustrazine as the terminal aryl group were identified as the two most effective compounds. In terms of neuroprotective activity, compounds with benzene as the terminal aryl group revealed better neuroprotective effects than those with ligustrazine as the terminal aryl group 136 .
However, in 2020, Yang et al. found that the introduction of piperazine reduced the anti-AD effect of diosgenin carbamate derivatives 137 .
Coumarin derivative 109 ( Figure 65) demonstrated clear neuroprotection in the middle cerebral artery occlusion rat model by reducing the infarct size and brain-water content, improving neurological function, and suppressing neuronal loss and neuropathological changes in the cortex and hippocampus. Compound 109 at doses of 3.0 and 6.0 mg/kg significantly reduced the percentage of the infarct area within the ipsilateral hemispheres compared with the vehicle control group (20.36 ± 7.9% and 18.89 ± 5.83%, respectively). The same result was observed for Edaravone at 6.0 mg/kg, with which a significant reduction of brain-water content also occurred. Further pharmacokinetic studies have shown that compound 109 could penetrate the blood-brain barrier of rats 138 . Ostrowska and co-workers designed 7-hydroxy-4-methyl coumarin derivatives to increase the affinity to 5-hydroxytryptamine (5-HT) receptors. Compound 110 and 111 ( Figure 65) showed higher affinities to 5-HT 1A receptors (K i value of 0.8 ± 0.009 and 0.50 ± 0.05 nM, respectively) than serotonin (1.3 ± 0.1 nM). Reportedly, substituents at ortho or meta positions in the phenyl group of piperazine play a decisive role in the affinity to 5-HT 1A receptors 139,140 .
The synthesis and evaluation of novel, potential antipsychotic coumarin derivatives were undertaken by Yin Chen et al. Compound 112 ( Figure 66) displayed a high affinity for 5-HT 1A , 5-HT 2A , and dopamine (D 2 ) receptors (K i ¼ 0.2, 3.9 and 5.6 nM, respectively). Based on the results, compound 112 exhibited a higher affinity for the 5-HT 1A receptor when compared with risperidone (K i ¼ 190.2 nM). Moreover, compound 112 demonstrated lower affinity for H 1 receptors (K i ¼ 699.1 nM) when compared with risperidone (K i ¼ 22.9 nM) and clozapine (K i ¼ 6.8 nM) 141 . Chen et al. reported a series of coumarin piperazine (piperidine) derivatives as potential multireceptor atypical antipsychotics. The SAR has shown that several factors affected the binding affinity for the D2, 5-HT 1 A, and 5-HT 2 A receptors of these compounds. (1) Bearing a 6-fluorobenzo[d]isoxazol-3-yl)piperidine moiety exhibited higher affinities to three receptors than those with other amine moieties, (2) the substitution on the 4-position (R 1 ) with methyl as the favoured substituent, (3) the substitution on the 3position (R 2 ) with unsubstituted or substitution (methyl) preferred, and (4) the substitution on the 8-position (R 3 ) with electron-withdrawing (chloro) as the preferred substituent; (5) the substitution on the 5-position (R 5 ) or 6-position (R 4 ) did lower the affinity for the three receptors, (6) the importance of the double bond for affinity to D2, 5-HT 1 A and 5-HT 2 A receptors, and (7) straight fourcarbon chain alkyl was superior to another alkyl linker. Notably, piperazine derivative 113 ( Figure 66) was considered among the most promising derivatives investigated, displaying the strongest affinity for 5-HT 1A receptors. According to the results, compound 113 exhibited a higher affinity for D 2 and 5-HT 1A receptors (D 2 , K i ¼1.4 nM; 5-HT 1A , K i ¼ 0.012 nM) when compared with risperidone (D 2 , K i ¼ 3.7 nM; 5-HT 1A , K i ¼ 180 nM) 142 .
Generally, the introduction of the piperazine group improved the neuroprotective activity when compared with tropolone according to a study reported by Koufaki et al. Compound 114 (Figure 67) showed the most potent protective activity against glutamate-challenged HT22 cells, with an EC 50 value of 0.08 ± 0.02 mM (the EC 50 value of positive control drug tropolone >10 mM, b-thujaplicin ¼ 4.80 ± 0.42 mM) 143 .
A series of flavone derivatives were synthesised and evaluated for their H 3 R inhibitory activity. Among them, a compound containing piperazine ring 115a (Figure 68) exhibited good inhibitory activity against H 3 R. The H 3 R inhibition rate treated with  compound 115a at doses of 25 mmol/ml was 81.32 ± 4.3% while positive control thiazole amide at 5 mmol/mL was 60.92 ± 3.5%. The results revealed that the hydroxyl at C6 also showed a significant effect by comparing compound 115a with 115 b (H 3 R inhibition rate ¼ 12.31 ± 4.5% at doses of 25 mmol/mL) (Figure 68) 144 .
Waszkielewicz et al. reported that a xanthone derivative with piperazine moiety 116 ( Figure 69) showed excellent anticonvulsant activity, with an ED 50 value of 105 mg/kg in the maximal electroshock (MES) seizure assay. In the anticonvulsant test, the mice were administered 0.5 or 4 h before the test, and the doses of the drug were 30, 100 and 300 mg/kg, respectively. Finally, derivative 116 proved to have better performance because it showed no neurotoxicity at both test time points, but both 100 mg/kg and 300 mg/kg were active in MES at 4 h after administration. In another study, xanthone derivative 117a-117d ( Figure 69) containing a piperazine moiety was evaluated for interactions with major classes of G-protein coupled receptors (GPCRs), uptake systems, and ion channels, revealing that it binds to 5-HT 2A , 5-HT 2B receptors, and sodium channels. And all derivatives synthesised were assessed using the SwissADME platform, the results revealed most of the compounds fulfilled Lipinski's rule of five along with good BBB (blood-brain barrier) penetration. In the for-plate test compound 117a-117d significantly increased the number of punished crossings at dose 5 mg/kg by 66.6% and at the dose 10 mg/ kg by 50.1%, respectively. Furthermore, derivative 117a-117d were evaluated by measuring mean plasma and brain concentrations after intragastric administration in mice at a dose of 5 mg/ kg. Among the molecules, compound 117b had the longest halflife, ca. 3 h compared to compound 117a (ca. 1 h). The compound 117d showed the largest apparent volume of distribution (41.3 l/ kg) that indicated the ability for penetration to the deep compartment and binding to tissues. After i.p. administration, all compounds quickly, within 15 min achieve maximum concentration (C max ) in the blood. The molecules were characterised by a high permeability to the brain, with the highest brain to plasma ratio (31.6) for compound 117d. As for the SAR, the partial substitution of piperazine at the 4 positions of the heteroanthrone structure is more conducive to enhancing the antidepressant and anti-anxiety activities in vivo than the 2 positions. Besides, the presence of methoxy or chloro substituent at 6 or 7 positions had a favourable

Cardiovascular activity
A series of xanthone derivatives with piperazine were synthesised and evaluated for their cardiovascular activity. Compound 118 ( Figure 70) exhibited significant antiarrhythmic activity, with an ED 50 value of 0.69 mg/kg in adrenaline-induced arrhythmia, while ED 50 values of Propranolol, Urapidil, Carvedilol were 1.05, 1.26, 0.25 mg/kg, respectively. From the structure-activity relationship, it can be considered that the most active compound contained a 2methoxyphenylpiperazine moiety. Compounds containing 4methoxyphenylpiperazine moieties are less effective in in vivo tests than their 2-substituted analogs. In vivo experiments showed that the binding affinity of all test compounds to the a 2 -receptors and b 1 -adrenoceptors were within the mM-range, and revealed the structural relationship between them. The introduction of chlorine substituents on the xanthone ring also reduced the binding affinity of the a 1 -adrenergic receptors, indicating that the increase in lipophilicity is undesirable 147 .
In a series of polyphenolic natural product analogs bearing an arylpiperazine moiety developed by Xie et al., compound 119 ( Figure 71) acted as a potential antihypertensive agent in spontaneously hypertensive rats (SHRs). In biological investigations, compound 119 reduced the systolic and diastolic blood pressure in SHRs comparable to naftopidil. The studies have shown that the mean arterial pressure (MAP) of 119 on SHR was reduced by nearly 23.7% at 4 h, which is better than the 16.1% of naftopidil at 2 h, demonstrating compound 119 may possess no effects on the basal heart rate. The SAR analysis showed that the substitution position of the linker had an effect on vasodilation activity. In general, most analogs with C6 substitution are more effective than corresponding analogues with C7 and C8 substitution 148 .

Other biological activity
16 semisynthetic C-10 pyrrole Mannich artemisinin derivatives were first evaluated in vitro against the chloroquine-sensitive 3D7   strain of P. falciparum, several of which are more than three times more effective than the natural product artemisinin. According to the analysis of the optimal Mannich side-chain substitution for in vitro and in vivo activity, the morpholine and N-methylpiperazine in the Mannich side-chain substitution 120 ( Figure 72) displayed the best activity among these derivatives. According to the SAR analysis, morpholine, N-methylpiperazine, and sulfonylmorpholine heterocyclics could provide molecules with increased potency both in vitro and in vivo. Furthermore, compound 120 had significantly better antimalarial activity in vivo versus the P. Berghei N strain (ED 50 ¼ 1.99 mg/kg, ED 90 ¼ 5.34 mg/kg, respectively) than water-soluble sodium artesunate (ED 50 ¼ 3.23 mg/kg, ED 90 > 10 mg/kg, respectively) 149 .
To improve solubility and maintain some rigidity, pentacyclic triterpene echinocystic acid dimer 121 (Figure 73) was designed and synthesised by replacing the phenyl group of the linker with piperazine, demonstrating significant anti-hepatitis C (HCV) entry activity, with an IC 50 value of 2.87 nmol/L, two orders of magnitudes more potent than EA (1.4 mmol/L). In addition, the haemolysis of the synthesised compound is eliminated under the concentration gradient of 0, 6.25, 12.5, 25 and 50 mmol/L, while EA had obvious haemolytic side effects when the CC 50 was 15 mmol/L 150 .
Li et al. synthesised a series of berberrubine derivatives by introducing various aminomethyl groups into 12-position of berberrubine and evaluated their anti-diabetic activity against type 2 diabetes mellitus. Compound 122 ( Figure 74) with an N-methyl piperazine-4-methyl group at C-12 showed the most potent anti-diabetic activity and the sensitisation reached 1.26 fold of rosiglitazone. Even if the concentration dropped to 1 mmol/ml, its activity was still comparable to the positive control rosiglitazone. The research suggested compound 122 had a certain effect on promoting glucose uptake on insulin-resistant 3T3-L1 adipocytes and L6 myotubes 151 .
Based on the relationship between results of tyrosinase inhibition and molecular docking, kojic acid derivative 123 ( Figure 75) was found to be a promising antityrosinase agent, and the hydroxymethyl group in the structure provided a metal complex of copper ions and chlorine atoms, allowing conventional hydrogen bonds to interact with tyrosinase, with an IC 50 value of 86.2 mM. Compound 123 showed the antidiphenolase activity of mushroom tyrosinase to be almost 5-fold higher than that of control agent kojic acid (IC 50 ¼ 418.2 mM) 152 .
Nie et al. synthesised a series of a-glucosidase inhibitors based on OA. The piperazine fragment was introduced to link the cinnamic amide unit with OA at C-28. Compound 124 ( Figure 76) with 3, 28-disubstituted OA exhibited potent inhibitory activity, with an IC 50 value of 1.90 mM. Interestingly, the different substituents on the benzene ring of the cinnamic amide unit had a significant effect on the activity. For example, fluorine atoms can reduce the activity, and the expansion of the hydrophobic surface could also enhance the inhibitory activity of a-glucosidase. However, compared with oleanolic acid (IC 50 ¼ 98.5 mM), the introduction of cinnamamide at position C-28 did not result in a significant increase in a-glucosidase inhibitory activity. The activity of 3-OH derivatives had stronger effects than that of 3-OAc derivatives, possibly due to its good steric binding to enzymes. The 3,28disubstituted oleanolic acid derivatives significantly improved the inhibitory activity of a-glucosidase than oleanolic acid 153 .
Reportedly, adenosine analog with piperazine 125 ( Figure 77) exhibited better inhibitory activity against Coxsackievirus B3 (CVB3), with a lower IC 50 value of 5.1 ± 2.3 mg/L and a higher selectivity index (TI ¼ 41.0) than ribavirin (IC 50 ¼ 36.8 ± 9.6 mg/L, TI ¼ 29.1, TI, selectivity index represented by the TC 50 /IC 50 ratio)). The studies have shown that in this series of compounds, the phenyl substitution of piperazine moiety may be more effective than pyridine substitution to enhance antiviral activity. This may be due to the lone pair of electrons in the aromatic ring. In addition,    the calculated log p values of 125 were 0.8 and met the log P criteria, and the acute toxicity level was within 50 LD 50 < 500 mg/ kg, with no carcinogenic and mutagenic toxicity risk 154 .

Conclusion
Piperazine is a part of numerous natural and synthetic molecules, with broad therapeutic potential. In addition to being a pharmacophore, piperazine is used as a linker/bridge between natural products and active structural molecules. This review summarises the research progress in the synthesis of natural product-piperazine derivatives in the past ten years, aiming to discover natural product piperazine hybrid compounds that may possess abundant biological activities. This will aid the scientific community in rationally design and develop novel, targeted, optimised, and diversified natural products-piperazine drugs for the treatment of multifactorial diseases. In conclusion, the piperazine group is widely used in for drug synthesis, attracting great attention from researchers worldwide, and its role cannot be ignored.