Ferrocene-containing hybrids as potential anticancer agents: Current developments, mechanisms of action and structure-activity relationships

Journal Pre-proof Ferrocene-containing hybrids as potential anticancer agents: Current developments, mechanisms of action and structure-activity relationships Ruo Wang, Huahong Chen, Weitao Yan, Mingwen Zheng, Tesen Zhang, Yaohuan Zhang PII:

S0223-5234(20)30076-3

DOI:

https://doi.org/10.1016/j.ejmech.2020.112109

Reference:

EJMECH 112109

To appear in:

European Journal of Medicinal Chemistry

Received Date: 12 January 2020 Revised Date:

29 January 2020

Accepted Date: 29 January 2020

Please cite this article as: R. Wang, H. Chen, W. Yan, M. Zheng, T. Zhang, Y. Zhang, Ferrocenecontaining hybrids as potential anticancer agents: Current developments, mechanisms of action and structure-activity relationships, European Journal of Medicinal Chemistry (2020), doi: https:// doi.org/10.1016/j.ejmech.2020.112109. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Graphical Abstract

Ferrocene-containing hybrids as potential anticancer agents: Current developments, mechanisms of action and structure-activity relationships Ruo Wang*, Huahong Chen, Weitao Yan, Mingwen Zheng, Tesen Zhang, Yaohuan Zhang College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China

This review covers the ferrocene hybrids with potential in vitro and in vivo anticancer activity which were developed in recent 10 years. The structure-activity relationships and mechanisms of action are also discussed to set up the direction for the design and development of ferrocene hybrids with high efficiency and low toxicity.

Ferrocene-containing hybrids as potential anticancer agents: Current developments, mechanisms of action and structure-activity relationships Ruo Wang*, Huahong Chen, Weitao Yan, Mingwen Zheng, Tesen Zhang, Yaohuan Zhang College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China

Abstract: Cancer is one of the most fatal threatens to human health throughout the world. The major challenges in the control and eradication of cancers are the continuous emergency of drug-resistant cancer and the low specificity of anticancer agents, creating an urgent need to develop novel anticancer agents. Organometallic compounds especially ferrocene derivatives possess remarkable structural and mechanistic diversity, inherent stability towards air, heat and light, low toxicity, low cost, reversible redox, ligand exchange, and catalytic properties, making them promising drug candidates for cancer therapy. Ferrocifen, a ferrocene-phenol hybrid, has demonstrated promising anticancer properties on drug-resistant cancers. Currently, Ferrocifen is in pre-clinical trial against cancers. Obviously, ferrocene moiety is a useful template for the development of novel anticancer agents. This review will provide an overview of ferrocene-containing hybrids with potential application in the treatment of cancers covering articles published between 2010 and 2020. The mechanisms of action, the critical aspects of design and structure-activity relationships are also discussed. Keywords:

ferrocene;

hybrid

compounds;

anticancer;

drug-resistant;

structure-activity

relationship

1. Introduction Organometallic compounds and their unique physic-chemical properties typically used in homogenous catalysis are now being translated as potential candidates for medical purposes [1]. Ferrocene (Figure 1, characterized by a sandwich-like structure) derivatives as the most prominent examples of this class of compounds possess remarkable structural and mechanistic diversity, inherent stability towards air, heat and light, low toxicity, low cost, reversible redox, ligand exchange, and catalytic *

Corresponding author: [email protected] 1

properties [2-4]. Ferrocene derivatives constitute versatile and interesting scaffolds for the drug discovery since this kind of compounds exhibit a variety of pharmacological properties including antibacterial [5,6], antimalarial [7,8], antifungal [9,10], antiviral [11,12], antitubercular [13,14], and anticancer [15,16] activities. In particular, Ferrocifen (Figure 1), a ferrocene-phenol hybrid, had a different mode of action to the platinum anticancer drugs, with the target not only DNA but also proteins and various enzymes such as thioredoxin reductases, demonstrating its promising anticancer properties on drug-resistant cancers [17,18]. Currently, Ferrocifen is in pre-clinical trial against cancers. Obviously, ferrocene moiety is a useful template for the development of novel anticancer agents.

Figure 1. Chemical structures of ferrocene moiety, Ferrocifen, and ferrocene-containing hybrids

This review covers the recent advances of ferrocene-containing hybrids (Figure 1) as potential anticancer agents covering articles published between 2010 and 2020, and the mechanisms of action as well as the structure-activity relationships (SARs) are also discussed to provide an insight for rational designs of more effective candidates.

2. Ferrocene-artemisinin hybrids Artemisinin derivatives could form highly reactive free radicals in the presence of ferrous ion (FeII) and possess potential anticancer activities [19-21]. Moreover, Artemisinin derivatives could promote the calcium-p38 apoptotic pathway, induce iron-dependent endoplasmic reticulum stress in cancer cells [22,23]. Therefore, 2

hybridization of ferrocene and Artemisinin may provide novel anticancer candidates with multiply mechanisms of action. The ferrocene-artemisinin hybrid 1a (Figure 2, half maximal inhibitory concentration/IC50: 130 and 530 nM) exhibited excellent activity against wild-type CCRF-CEM and P-glycoprotein over-expressing multidrug-resistant CEM/ADR5000 leukemic cells with IC50 values in nanomolar level [24]. The SAR indicated that introduction of alkyl linker between ferrocene and Artemisinin moieties had little influence on the activity since hybrid 1b (IC50: 250 and 570 nM) was highly active against the tested two leukemic cell lines [25]. Further incorporation of egonol fragment into ferrocene skeleton (1c, IC50: 70 and 147,270 nM) could enhance the activity against wild-type CCRF-CEM cells, but reduced the activity against multidrug-resistant CEM/ADR5000 leukemic cells significantly [25]. Particularly, hybrid 1a was 3.6 and 1.2 folds more potent than Dihydroartemisinin (IC50: 480 and 680 nM) against CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemic cells respectively, and 43.9 times better than Doxorubicin (IC50: 23.27 µM) against multidrug-resistant CEM/ADR5000 leukemic cells. Moreover, this hybrid also demonstrated great activity against human cytomegalovirus (HCMV, IC50: 130 nM) and Plasmodium falciparum 3D7 strain (P. falciparum, IC50: 12.4 nM), indicating hybrid 1a could serve as a lead compound for the discovery of novel anticancer, antiviral and antimalarial agents.

3

Figure 2. Chemical structures of ferrocene-artemisinin hybrids 1 and 2

Further study indicated that the ferrocene-bis-artemisinin hybrid 2a (Figure 2, IC50: 70 and 1,800 nM) also showed considerable activity against wild-type CCRF-CEM and P-glycoprotein over-expressing multidrug-resistant CEM/ADR5000 leukemic cells [24]. The activity of hybrid 2b (IC50: 80 and 8,200 nM) was lower than that of hybrid 2a, implying introduction of alkyl linker between ferrocene and Artemisinin moieties

was

detrimental

to

the

activity.

The

mono-ester

tethered

ferrocene-bis-artemisinin hybrid 2c (IC50: 10 and 1,960 nM) not only retained the activity against multidrug-resistant CEM/ADR5000 leukemic cells, but also improved the activity against wild-type CCRF-CEM cells when compared with hybrid 2a.

3.

Ferrocene-amino acid/peptide hybrids

Drug-amino

acid/peptide

hybrids

can

overcome

multi-drug

resistance

in

chemotherapy and bind to specific receptors expressed on cancer cells [26]. Therefore, ferrocene-amino acid/peptide hybrids, which retain activities, may provide means to identify novel anticancer candidates with enhanced activity against both drug-sensitive cancer cells and drug-resistant cancer cells. The potency of ferrocenylmethyl-ʟ-tyrosine/tryptophan/methionine hybrids 3-5 (Figure 3) as photocytotoxic agents against HeLa and MCF-7 cancer cell lines were evaluated [27], and the SAR revealed that curcumin ligand was critical for the high activity, while replacement of curcumin fragment by acetylacetonate led to loss of activity (IC50: >100 µM). The hybrids 3a, 4a and 5a (IC50: 2.9-15.7 µM, photo-exposure to visible light 400-700 nm) showed significant activity with respect to their photocytotoxic property in light, and the activity was superior to that of the reference Cisplatin (IC50: 68.7 µM) against HeLa and MCF-7 cancer cell lines. The 4

mechanistic study revealed that these hybrids could induce cell death through the apoptotic

pathway.

Further

study

proved

that

2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline, 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline,

and

dipyrido-[3,2-a:2’,3’-c]phenazine also can be used as ligands, and hybrid 6 (IC50: 0.7 and 0.26 µM, photo-exposure to visible light 400-700 nm) showed remarkable photo-induced activity against HeLa and MCF-7 cancer cell lines [28,29]. The ferrocence-glycine/ʟ-alanine hybrids 7 (IC50: 2.84-46.5 µM) displayed considerable activity against MCF-7 cells, and the activity of hybrids 7a,b and 7e (IC50: 2.84-11.1 µM) was higher than that of the reference Cisplatin (IC50: 16.3 µM) [30,31]. The SAR indicated that hybrids with ʟ-alanine moiety were generally more potent than the corresponding glycine analogs, and introduction of pentafluoro into phenyl ring preferred. The mechanistic study showed that the most active hybrid 7b (IC50: 2.84 µM) which was 5.7-fold more potent than Cisplatin against MCF-7 cells was capable of generating oxidative damage via a reactive oxygen species (ROS)-mediated mechanism. The anticancer SAR of phosphinoferrocene-glycine hybrids 8 (IC50: 4.1-35.4 µM) against human ovarian cancer cell lines A2780 and A2780cisR (acquired resistance to Cisplatin) suggested that the methyl ester at R1 position was much better than amide, and methyl at R2 position was benefit for the activity [32]. Among them, hybrids 8a,b (IC50: 4.1-8.7 µM) were found to be most potent against both drug-sensitive A2780 and drug-resistant A2780cisR human ovarian cancer cell lines. Thus, both of them can be considered as useful starting points in the development of anticancer agents. The alkynyl-containing ferrocene-amino acid hybrids also showed potential activity against H1299 non-small cell lung cancer cell line, and the SAR demonstrated that replacement of amino acids by dipeptides could not improve the activity greatly [33,34]. Particularly, the activity of ferrocene-γ-aminobutyric acid 9 (IC50: 7.2 µM) 5

was in the same level with that of Cisplatin (IC50: 1.5 µM).

Curcumin was critical for the high activity Ferrocene moiety R

R

R

O O Cu N O

Fe

R

R

O O Cu HN O

Fe

Fe

O

Pentafluoro preferred

R

N

O O Cu HN O

O

O

SO

NH

Fe

N N Cu HN O

Amino acid motif has little impact on the activity

OH

N

N H

Fe

4

-Me > -H

7a: R1 = Me, R2 = 3,4,5-triF; 7b: R1 = Me, R2 = pentafluoro; 7c: R1 = H, R2 = 4-F; 7d: R1 = H, R2 = 3,5-diF; 7e: R1 = H, R2 = pentafluoro.

5 6

a: R =

R2 O

7

SO

3

H N R1

; b: R = Me. HO O

R2

Replaced by dipeptides couldn't improve the activity greatly

-Me favored

Could improve the activity H N

X

Fe

Ph Ru P X Methyl ester enhanced Ph the activity O H N R1 O

O

O Fe HN

Fe

H N

N H

NH2

NH O H N

O

O

N H

COOMe COOMe

OEt O 10a 9

8 8a: X = Cl, R1 = OMe, R2 = hexamethyl; 8b: X = MeCN, R1 = OMe, R2 = hexamethyl.

H N O N H

Fe

H N

NH O N H

O

Fe

COOMe COOMe

OEt

O

R2 -H > alkyl, -Bn

NH2

HN

NH3

O

H N

O N H

H N O NH

Fe

NH3 N H

O NH2 H2N

N H

H N

O N H

H2N

H N O NH N H

O N H

H2N

H N O NH

NH O

NH N H

NH2 O

N H

12a

11 11a: R1 = Me, R2 = H; 11a: R1 = Et, R2 = H.

NH

NH2

O

Short alkyl side chain preferred

HN

H N

NH

O

R1

10b

H2N

O

H N

NH2

NH2 O

NH

Fe

N H

O

H N

N H

O NH

O NH2

N H

O NH

12b

Figure 3. Chemical structures of ferrocene-amino acid/peptide hybrids 3-12

The activity of ferrocene-peptide hybrid 10a (Figure 3, IC50: 5.2 µM) was superior to that of analog 10b (IC50: 19.6 µM) against B16 cancer cells, implying the amide linker between ferrocene and peptide motifs could boost up the activity [35]. The mechanistic study demonstrated that these two hybrids mainly led to cell cycle arrest at the G1 phase. Moreover, hybrid 10a also could induce cell cycle arrest in S phase. 6

Accordingly, hybrid 10a may act as a potential candidate for therapeutic treatment of primary B16 murine melanoma cancer. The anticancer SAR of ferrocene-peptide hybrids 11 (IC50: 2.6-20.1 µM) against human lung carcinoma cell line H1299 revealed that short alkyl side chain at ferrocene skeleton (R1 position) was favorable to the activity, while alkyl and benzyl groups at R2 position were harmful to the activity when compared with the unsubstituted analogs [36,37]. Among them, hybrids 11a,b (IC50: 2.6 and 6.1 µM) were as potent as Cisplatin (IC50: 1.5 µM) against H1299 cells, so both of them could serve as lead compounds for further exploitations. Some other ferrocene-peptide hybrids also displayed certain anticancer activity [38-40], and among them, hybrids 12a,b (IC50: 4.3-4.7 µM) showed potential activity against MCF-7 and HT29 cancer cell lines [38]. Moreover, these two hybrids (IC50: 31.3 and 32.5 µM) also showed relatively low cytotoxicity towards normal fibroblast cells, and the selective index (SI) was 6.6-7.5, indicating that there was a therapeutic window.

4.

Ferrocene-azole hybrids

Azole derivatives which could interact with various enzymes and receptors in organisms through diverse noncovalent interactions, possess excellent in vitro and in vivo anticancer activity [41,42]. Therefore, hybridization of ferrocene with azole is a promising strategy to develop novel anticancer candidates.

4.1 Ferrocene-imidazole hybrids Imidazole and its derivatives are the pharmacological significant scaffolds with the broad spectrum of biological properties including anticancer activity [43,44]. Imidazole derivatives can inhibit vascular endothelial growth factor (VEGF), topoisomerase I and II, mitotic spindle microtubules, histone deacetylases, receptor tyrosine kinases and CYP26A1 enzyme. Several imidazole derivatives such as Dacarbazine, Temzolomide, Zoledronic acid, Mercaptopurine, Nilotinib, and 7

Tipifarnib are being used in clinics to cure various cancers currently [45,46]. Hence, hybridization of ferrocene with imidazole may open a door for the opportunities on the development of novel anticancer agents. The ferrocene-clotrimazole derivatives 13a,b (Figure 4, GI50: 20.44-27.51 µM) showed considerable activity against HT29 and MCF-7 cancer cell lines, and the activity was no inferior to that of the parent Clotrimazole (GI50: 64.19 and 21.44 µM) [47]. Some other ferrocene-imidazole hybrids also demonstrated certain anticancer activity, and the most emblematic examples were hybrids 14a,b (GI50: 0.06-1.6 µM) which possessed broad-spectrum activity against 518A2 melanoma, Panc-1 pancreatic ductular adenocarcinoma, MCF-7/Topo breast adenocarcinoma, KB-V1/Vbl cervix carcinoma (optionally pretreated with 24 mm verapamil for 24 h), HCT-116 colon carcinoma, HT-29 and DLD-1 colorectal adenocarcinoma [48-50]. The mechanistic study indicated that hybrids 14a,b could generate the ROS through a genuine ferrocene effect, inhibiting the thioredoxin reductase [48]. Further study revealed that these two hybrids could increase a reorganization of the F-actin cytoskeleton in endothelial and melanoma cells, which was associated with a G1 phase cell cycle arrest and a retarded cell migration. In a Balb/c mouse xenografted with invasive B16-F10 melanoma, hybrid 14b (7.5 mg/kg, b.i.d., intraperitoneally) led to a volume reduction of xenografted tumors by up to 80%, and was well tolerated by mice. The potential in vitro and in vivo anticancer activity as well as low toxicity towards mice make this hybrid deserves further investigation.

8

2BF4

N N R

Fe

N

Fe

Fe

BF4

N

N Fe

Au•PPh3

14a

13

N

HN

N Au

14b

N NH

Fe

CN 15

13a: R = 2-Cl; 13b: R = 4-Cl.

N N

Br N Fe

Ph Ph P Pd Fe S P Ph Ph S

N

O

Au S

n N N 16 16a: n = 6; 16b: n = 8; 16c: n = 10; 16d: n = 12.

17

Figure 4. Chemical structures of ferrocene-imidazole hybrids 13-17

The ferrocene-benzoimidazole hybrid 15 (Figure 4, IC50: 842.6 and 16.5 µM) displayed weak to moderate activity against PC-3 and A549 cancer cell lines, and docking study showed that this hybrid and Erlotinib had similarities in targeting the EGFR [51]. The hybrids 16 (GI50: 16-40 nM) were highly active against MCF-7 cells, and the activity was 3.6-9.1 folds higher than that of the reference Doxorubicin (GI50: 147 nM), implying the potential application of these hybrids as breast cancer therapeutic agents [52]. The hybrid 17 (IC50: 80-940 nM) exhibited excellent activity against HT29, HCT-15, MDA-MB-231, MCF-7, HL-60, human ovarian 2008 and cisplatin-resistant cancer cells, and the activity was 15.3-263.3 times higher than that of Cisplatin (IC50: 4.73-90.12 µM) against all tested cancer cell lines [53]. Moreover, the cytotoxicity of compound 16 (IC50: 36.21 µM) was low against HEK293 non-malignant fibroblasts, and the SI was >38. The mechanistic study demonstrated that this hybrid could inhibit the enzymes thioredoxin reductase (TrxR), glutathione reductase (GR), and increase the production of ROS in HCT-15 cells. In addition, this hybrid also induced cancer cell death by apoptosis.

4.2 Ferrocene-pyrazole hybrids

9

Pyrazole derivatives could interact with various enzymes and receptors such as EGFR, aurora kinase, cyclin dependent kinase, cyclo-oxygenase, heat shock protein, and lypo-oxygenase, and thereby the compounds bearing a pyrazole moiety may possess the anticancer activity [54,55]. Hybridization of ferrocene with pyrazole pharmacophore represents a promising strategy to provide novel anticancer candidates. The activity of ferrocene-pyrazole hybrid 18a (Figure 5, IC50: 27.03 and 34.51 µM) was higher than that of derivative 18b (IC50: 42.24 and 85.51 µM) against MCF-7 and MDA-MB-231 cancer cell lines, suggesting introduction of phenyl group into pyrazole fragment decreased the activity [56]. Further study showed that these hybrids can suppress cell viability and trigger either apoptosis or necrosis in human breast cancer cells, with low cytotoxicity to healthy breast epithelial cells. Moreover, both of them could suppress cell viability of breast cancer cells via the inhibition of PI3K/Akt and ERK1/2 signaling pathways. Incorporation

of

trifluoromethyl

into

pyrazole

was

also

tolerated,

and

ferrocene-pyrazole hybrids 19 (IC50: 4.44-32.34 µM) as well as their regio-isomers 20 (IC50: 5.88-35.06 µM) displayed potential activity against A549, HepG2 and MDA-MB-45 cancer cell lines [57]. The representative compounds 19a (IC50: 6.98-8.43 µM) and 20a (IC50: 6.83-8.96 µM) were in the same level with the reference 5-Fluorouracil (IC50: 2.80 µM) against MDA-MB-45 cells, and more potent than 5-Fluorouracil (IC50: 16.80 and 17.60 µM) against A549 and HepG2 cancer cell lines. The hybrids 21 (IC50: 9.84-18.82 µM) also possess considerable activity against Smmc7721 and SGC7901 cancer cell lines, and all of them were more active than the reference 5-Fluorouracil (IC50: 19.25 and 16.03 µM) [58]. The SAR proved that phenyl ring at R position was more favorable than alkyl group, while introduction of electron-withdrawing halogen atoms on the phenyl ring greatly decreased the activity against Smmc7721 cells, and electron-donating groups reduced the activity against SGC7901 cells. The activity of R-configuration ferrocene-pyrazole hybrids 22 (IC50: 25.3-63.6 µM) was superior to that of the corresponding S-configurated analogs 23 (IC50: 28.1-77.7 µM) against A549 and H322 cancer cell lines, implying the chiral center had some 10

influence on the activity [59,60]. Among them, compound 22a (IC50: 25.5 and 57.8 µM) was found to be most potent against the tested two cancer cell lines, and the activity was comparable to that of Cisplatin (IC50: 20.1 and 28.2 µM) and 5-Fluorouracil (IC50: 11.5 and 33.1 µM). The mechanistic study revealed that these hybrids might exert their anticancer activity through cell cycle arrest. The ferrocene-pyrazole hybrids 24 (IC50: 1.25-17.09 and 21.07-47.85 µM, respectively) and 25 (IC50: 0.28-2.03 and 29.09-41.05 µM, respectively) showed higher activity against cyclooxygenase(COX)-2 than against COX-1, so they can act as potential COX-2 inhibitors [61,62]. The majority of these hybrids (IC50: 6.12-30.28 and 0.34-15.57 µM, respectively) also possessed promising activity against A549, F10, MCF-7 and HeLa cancer cell lines, and the activity was comparable to or better than that of the reference Celecoxib (IC50: 7.55-25.87 µM, respectively). The SAR demonstrated that introduction of sulfonamide into phenyl ring at N-1 position of pyrazole moiety was beneficial to the activity since hybrids 25 were more potent than derivatives 24. For hybrids 25, ester was better than amide at R position, and linear side chain was more favorable than branched side chain for both ester and amide. Hybrids 25a-c (IC50: 0.34-5.78 µM) not only exhibited the highest activity against all tested cancer cell lines, but also displayed low cytotoxicity (IC50: 171.70-280.42 µM) towards normal 239t cells. The further mechanistic studies revealed that hybrid 25b could induce apoptosis of HeLa cells by mitochondrial depolarization, and the antiproliferative activity of this compound was positively correlated with the levels of intracellular NO release in HeLa cells. In HeLa cells xenografted Balb/C female nude mice model, the tumor volume in vehicle control group was rapidly developed, whereas the hybrid 25b (20 mg/kg, intraperitoneally) treated groups showed a dramatic reduce in the tumor weight, and the average weight of Celecoxib (10 mg/kg) treated group tumors was more 4-fold to that of hybrid 25b-treated group. Moreover, no dramatic weight changes were observed in hybrid 25b-treated group. The excellent in vitro and in vivo anticancer potency as well as low toxicity towards mice made this hybrid deserves further investigation.

11

Figure 5. Chemical structures of ferrocene-pyrazole hybrids 18-27

The imine tethered ferrocene-pyrazole hybrid 26 (Figure 5, IC50: 0.09-8.48 nM) was highly active against cervical carcinoma (KB), ovarian carcinoma (SK OV-3), CNS (SF-268), lung (NCI H460), colon adenocarcinoma (RKOP27), leukemia (HL60, U937, and K562), melanoma (SK-MEL-28), neuroblastoma (GOTO, NB-1), cervical (HeLa), fibrosarcoma (HT1080), and liver (HepG2) cancer cell lines, and the activity was no inferior to that of the references Doxorubicin (IC50: 1.13-6.66 nM) and Tamoxifen (IC50: 0.11-1.31 nM) [63]. And thus, hybrid 26 could act as a lead compound for the discovery of novel anticancer agents. The ferrocene-pyrazolone and ferrocene-pyrazoline hybrids also displayed certain anticancer activity, and hybrid 27 which exhibited potent RalA inhibition (IC50: 1.20 µM), also showed considerable activity against PANC-1 and HPAF-II cancer cell 12

lines with IC50 values of 1.6 and 4.8 µM [64,65]. The mechanistic study indicated that this hybrid led to accumulation of ROS in cancer cells, and molecular docking studies of hybrid 27 onto RalA allosteric site suggested that this hybrid bound the site similarly as a C3 exoenzyme substrate peptide.

4.3 Ferrocene-triazole/thiazole hybrids Triazole moiety has the potential to improve pharmacological, pharmacokinetic, and physicochemical profiles of molecules [66], and many triazole-containing compounds such as Cefatrizine and Carboxyamidotriazole have demonstrated excellent anticancer activity. Thus, hybridization of ferrocene with triazole/thiazole moieties may provide valuable therapeutic intervention for the treatment of cancers. The ferrocene-1,2,3-triazole [67-69] and 1,2,4-triazole hybrids [70,71] usually possessed weak to moderate anticancer activity, for example, ferrocene-1,2,3-triazole hybrids 28 (Figure 6, IC50: 38-67 µM against MCF-7 and HT-29 cancer cell lines) and ferrocene-1,2,4-triazole hybrids 29 (IC50: 15.4-209.5 µM against hTERT-BJ and HT1080 cells) showed moderate anticancer activity. Hybrids 30 caused neither apoptosis nor necrosis of A549 cells, but it could exert anticancer effect via inducing G1-phase arrest and senescence through ROS/p38 MAP-kinase pathway [71]. The imine tethered ferrocene-thiazole hybrids 31 (IC50: 4.5-16.5 µM) showed potential activity against MCF-7 cells, and the majority of them were more potent than the ligand (IC50: 15.5 µM), suggesting the metal ions could enhance the activity to some extent [72,73]. Among them, three hybrids 31a-c (IC50: 4.5-9.0 µM) were as active as Cisplatin (IC50: 4.0 µM). The anticancer activity of the hybrids was accompanied by significant increase in the activity of superoxide dismutase, with a parallel decrease in the activities of catalase and glutathione peroxidase, as well as glutathione level. Accordingly, the overproduction of free radicals allowed ROS-mediated cancer cell death.

13

Figure 6. Chemical structures of ferrocene-triazole/thiazole hybrids 28-32

The ferrocene-thiadiazole hybrids 32 (Figure 6) also possessed certain anticancer activity, and the SAR suggested that electron-withdrawing group at R position was more favorable than the electron-donating group [74,75]. Among them, hybrids 32a,b (IC50: 15.51-45.34 µM) showed highest activity against EC-9706 and Eca-109 cells, but the activity was lower than that of the reference Doxorubicin (IC50: 8.56 and 6.52 µM).

5.

Ferrocene-boronic ester hybrids

Boronic esters have the potential to increase intracellular concentration of borate activates borate transporters, resulting in growth inhibition and apoptosis of cancer cells [76-79]. Therefore, hybridization of ferrocene with boronic esters is prone to provide attractive scaffolds for the development of novel cancer agents. The ferrocene-boronic esters hybrids 33 (Figure 7, IC50: 8-44 µM) displayed considerable activity against BL-2, A2780 and Jurkat cancer cell lines, and among them, hybrid 33c (IC50: 15-31 µM) with the highest activity was also non-cytotoxic towards HDFa cells (IC50: >50 µM) [80]. The mechanistic study indicated that these hybrids could dramatically increase the generation of ROS in cancer cells. In the Wistar rats with Guerin's carcinoma (T8), hybrid 33c was found to strongly suppress the growth of Guerin's carcinoma (T8) in Wistar rats at the dose of 30 mg/kg through intraperitoneal route. Moreover, this hybrid (ED50: >240 µM) showed good in vivo safety profile. Therefore, this hybrid can potentially serve as a lead compound for the development of novel anticancer agents. The ferrocene-boronic esters hybrids 34 (IC50: 9-55 µM) displayed potential activity 14

against human promyelocytic leukemia HL-60, and the SAR indicated that introduction of alkyl and benzyl groups into R1 position could enhance the activity [81]. Electron-donating group at phenyl ring (R2 position) was beneficial for the activity, while electron-withdrawing group was detrimental to the activity. Hybrid 34e (IC50: 9 and 25 µM) was found to be most active compound against human glioblastoma-astrocytoma U373 and HL-60 cells, and it was also non-toxic (IC50: >100 µM) towards fibroblasts.

Figure 7. Chemical structures of ferrocene-boronic esters hybrids 33-37

The majority of ferrocene-boronic esters hybrids 35 (Figure 7, IC50: 9-37 µM) were sensitive to HL-60, RAJI, BL-2 and JVM-2 cancer cell lines, and the SAR revealed that the phenyl ring can be replaced by pyridinyl group [82,83]. Further study showed that

ferrocene-bis-boronic

esters

hybrids

36

(IC50:

1.4-20

µM)

and

bis-ferrocene-bis-boronic esters hybrids 37 (IC50: ≥50 µM) also displayed certain anticancer activity, and the SAR suggested that introduction of the second boronic ester moiety could enhance the activity to some extent [82,83]. In particular, hybrid 35a (IC50: 11-35 µM) not only possessed broad-spectrum activity against HL-60, 15

RAJI, BL-2 and JVM-2 cancer cell lines, but also exhibited potential in vivo efficiency (daily 6 times at a dose of 26 µg/kg, survival of the mice was extended from 13.7 days in the control group to 17.5 days in the treated group) in BDF1 mice (carrying L1210 leukemia) model [82]. Moreover, no significant difference in mean body weights between control and hybrid 35a-treated group (>6 mg/kg) in BDF-1 mice model. The potential in vitro and in vivo anticancer activity as well as low toxicity towards mice made this hybrid deserves further investigation.

6.

Ferrocene-chalcone hybrids

Chalcone derivatives can act on a variety of enzymes and receptors in cancer cells such as aromatase, breast cancer resistance protein, P-glycoprotein (P-gp), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tubulin, and VEGF [84-86], and thus, chalcone derivatives exhibited promising in vitro and in vitro activity against both drug-susceptible and drug-resistant even multidrug-resistant cancers. Obviously, ferrocene-chalcone hybrids are potential prototypes for the discovery of novel anticancer candidates. The furan-containing ferrocene-chalcone hybrids 38 (Figure 8, IC50: 1.8-6.0 µM) and their piperazine-containing analogs 39 (IC50: 2.4-3.8 µM) showed promising activity against TK-10, UACC-62 and MCF-7 cancer cell lines, but also displayed relatively high cytotoxicity (IC50: 3.0-55.4 µM) against normal WI-38 HFLF cells [87,88]. The SAR indicated that linear alkyl linkers between ferrocene and chalcone moieties were better than the branched alkyl linkers, and the alkyl linker can be replaced by alkylamino linker. Among them, hybrid 38b (IC50: 1.8-3.6 µM) was 2.2-4.1 folds more potent than Parthenolide (IC50: 5.8-15.0 µM) against TK-10, UACC-62 and MCF-7 cancer cell lines. The majority of pyrazole-containing ferrocene-chalcone hybrids 40 (IC50: 5.42-87.14 µg/mL) were sensitive to HeLa, Fem-x and K562 cancer cell lines, and the activity of compounds 40a-c (IC50: 5.42-9.27 µg/mL) was no inferior to that of Cisplatin (IC50: 4.70 and 5.90 µg/mL) against Fem-x and K562 cancer cell lines [89,90].

16

Figure 8. Chemical structures of ferrocene-chalcone hybrids 38-42

The anticancer SAR of ferrocene-chalcone hybrids 41 (Figure 8) revealed that electron-donating group at phenyl ring was benefit to the activity against MDA-MB-231 cells, while electron-withdrawing halogen atoms reduced the activity [91-93]. Among them, five hybrids 41a-e (IC50: 1.1-4.1 µM) showed promising activity against MDA-MB-231 cells, implying the potential application of these hybrids as breast cancer therapeutic agents. Extension of the five-membered ring to the six-membered ring was also tolerated, but introduction of the second chalcone or ferrocene moiety decreased the activity [94,95]. In particular, hybrid 42 (IC50: 2.97-71.2 µM) which was susceptible to the tested Jurkat, HeLa, MCF-7, A549, and MDA cancer cell lines, was worthy of further investigation.

7.

Ferrocene-coumarin/flavonoid hybrids

Coumarin derivatives and flavonoid derivatives have the potential to exert diverse anticancer mechanisms such as induce cell cycle arrest, angiogenesis inhibition, and kinase

inhibition

[96,97].

Therefore,

hybridization

of

chalcone

with

coumarin/flavonoid may give attractive scaffolds for development of novel cancer agents. The ferrocene-coumarin hybrids 43 (Figure 9, IC50: 11.7-182.9 µM) showed 17

considerable activity against MDA-MB-231 cells, and the SAR indicated that hybrids with hydroxyl at C-7 position of coumarin moiety were more potent than the corresponding benzyloxy analogs [98,99]. Similar SAR results were also observed for piperidine-containing hybrids 44 (IC50: 11.8-51.4 µM) and 45 (IC50: 13.8-108.9 µM) [98]. Hybrids 43a and 44d (IC50: 11.7 and 11.8 µM) were found to be most active against MDA-MB-231 cells, and the activity was around 11 times higher than that of Novobiocin (IC50: 205.1 µM), but far less potent than that of Paclitaxel (IC50: 0.029 µM). The ferrocene-coumarin hybrids 46 (IC50: 1.09-62.57 µM) exhibited potent and broad-spectrum activity against MCF-7, BIU-87, SGC-7901, EC-9706, Eca-109 and Jurkat cancer cell lines, but most of them were less potent than the reference Doxorubicin (IC50: 4.50-8.56 µM) [100]. The SAR revealed that introduction of carbonyl group at X position reduced the activity, and replacement of the ester (Y = O) by amide (Y = NH) could not enhance the activity apparently. Among them, hybrid 46a (IC50: 5.24 and 7.46 µM) which was as potent as Doxorubicin (IC50: 6.09 and 5.44 µM) against BIU-87 and SGC-7901, could serve as a lead compound for further exploitation.

Figure 9. Chemical structures of ferrocene-coumarin/flavonoid hybrids 43-50

The ferrocene-flavonoid hybrid 47 (Figure 9, IC50: 23.0-35.0 µM) was sensitive to HepG2, MCF-7, and CCRF-CEM cancer cell lines, and the mechanistic study 18

suggested that this hybrid could induce oxidative stress, apoptosis, and generate damage to DNA and arrest the cell cycle in G2/M phase [101,102]. The hybrids 48 (IC50: 6.3->100 µM) showed considerable activity against wide-type BHK21, NCI-H69, and drug-resistant BHK21-MRP1 as well as H69AR (overexpress multidrug resistance-associated protein 1/MRP1) cancer cells, and the activity was no inferior to that of Verapamil (IC50: 6.7->100 µM) [103]. The SAR indicated that introduction of hydroxyl group into C-3 position of flavonoid motif was harmful to the activity, and hybrids 49 (IC50: >100 µM) were devoid of activity against B16 cells [104]. It is worth notice that these hybrids were more active against drug-resistant BHK21-MRP1 and H69AR cells (IC50: 6.3-25 µM) than against wide-type BHK21 and NCI-H69 cells (IC50: 43.8->100 µM), demonstrating their potential for fighting against drug-resistant cancers. The ferrocene-flavonoid hybrids 50 (IC50: 18.7-43.5 µM) possessed potential activity against MCF-7, MDA-MB-231, HT-29 and RC-124 cancer cell lines, and the activity was higher than that of Coumestrol (IC50: 48.6->100 µM), but lower than that of Cisplatin (IC50: 1.6-7.7 µM) [105].

8.

Ferrocene-hydroxamic acid hybrids

Hydroxamic acid derivatives as potential histone deacetylase (HDAC) inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cancer cell death [106,107], and the hydroxamic acid-containing agent Vorinostat has already been approved to treat cutaneous manifestations in patients with cutaneous T-cell lymphoma.

Therefore,

hybridization

of

ferrocene

with

hydroxamic

acid

pharmacophore represents a promising strategy to provide novel anticancer candidates. The ferrocene-hydroxamic acid hybrids 51 (Figure 10, IC50: 0.00009-6.07 µM) which exhibited excellent inhibition activity against HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8, also displayed potential activity (IC50: 1.90-5.08 µM) against MCF-7 and MDA-MB-231 cancer cell lines [108-111]. Biological assays showed that exposure of MDA-MB-231 cells to these hybrids resulted in cell cycle perturbation with an alteration of S phase entry and a delay at G2/M transition and in an early ROS production followed by mitochondrial membrane potential dissipation and autophagy 19

inhibition.

Figure 10. Chemical structures of ferrocene-hydroxamic acid hybrids 51 and 52

The ferrocene-hydroxamic acid hybrids 52a,b (Figure 10, IC50: 0.70-2.01 µM) showed great potency against MDA-MB-231 and MCF-7 cancer cell lines, and the activity was no inferior to that of Vorinostat (IC50: 3.64 and 1.04 µM) [112-114]. These results suggested a potential anticancer activity vested in this new class of ferrocene-hydroxamic acid scaffolds.

9.

Ferrocene-indole hybrids

Indole is the common pharmacophore in the development of new anticancer agents since its derivatives can act on various targets such as histone deacetylases, sirtuins, carbonic anhydrase, tyrosine kinase, tubulin, PIM kinases, DNA topoisomerases, P-glycoprotein, multidrug resistance associated protein 1 (MRP1), MRP2 and σ receptors [115,116]. Many indole-containing drugs such as Semaxanib and Sunitinib have already been used in clinics for fighting against various cancers, so hybridization of ferrocene with indole pharmacophore may provide a new strategy to develop anticancer agents. The anticancer SAR of ferrocene-indole hybrids 53 (Figure 11, IC50: 5-27 µM) against A549 cells indicated that introduction of either electron-donating or electron-withdrawing groups into C-5 position of indole moiety could boost up the 20

activity [117]. The phenyl ring at C-2 position of indole fragment was more favorable than pyridinyl ring, while halogen atom at para-position of phenyl ring decreased the activity. The activity of hybrids 53a,b (IC50: 5 and 7 µM) was in the same level with that of 5-Fluorouracil (IC50: <5 µM) against A549 cells, so both of them could potentially serve as lead compounds for the development of novel anticancer chemotherapeutic agents. The majority of ferrocene-bis-indole hybrids 54 (IC50: 1.0-57.9 µM) showed considerable activity against 3,3’-diindolylmethane (DIM)-resistant cancer cell lines (518 A2, KB-V1/Vbl, HT-29), while DIM (IC50: >100 µM) was devoid of activity [118]. The hybrid 54a (IC50: 1.0-6.3 µM) possessed not only excellent activity against DIM-resistant 518 A2, KB-V1/Vbl, and HT-29 cancer cell lines, but also high activity against PC-3, BcPC-3 and MDA-MB-231 cancer cell lines.

Figure 11. Chemical structures of ferrocene-indole hybrids 53-56

Besides the ferrocene-indole hybrids mentioned above, some other derivatives such as E-configurated ferrocenyl-oxindole hybrids 55 (Figure 11, IC50: 0.49->2.0 µM) and their Z-configurated analogs 56 (IC50: 1.17->2.0 µM) also displayed certain anticancer activity [119-121]. The SAR revealed that the E-configurated hybrids 55 were more potent than the corresponding Z-configurated analogs 56 against MDA-MB-231 cells [121]. In particular, hybrids 55a-c (IC50: 0.49-0.89 µM) were comparable to the reference Evodiamine (IC50: 0.28 µM) against MDA-MB-231 cells, so these hybrids represent new potential lead compounds for further development in cancer therapy.

10. Ferrocene-phenol hybrids 21

The ferrocene-phenol hybrid Ferrocifen could not only target DNA, but also act on various proteins and enzymes [15]. Ferrocifen showed promising activity against both drug-sensitive and drug-resistant cancer cells [16], revealing the potential of ferrocene-phenol hybrids as putative anticancer agents. The ferrocene-phenol hybrids 57 (Figure 12, IC50: 0.02-0.14 µM), 58 (IC50: 0.42-1.54 µM) and 59 (IC50: 1.23-2.7 µM) showed potential activity against MDA-MB-231 and PC-3 cancer cell lines, and SAR indicated that replacement of iron by osmium or ruthenium was detrimental to the activity [122-130]. Further study indicated that these hybrids could act as DNA alkylating agents or DNA antimetabolites [122]. Particularly, hybrid 57a (IC50: 48-580 nM) also possessed broad-spectrum activity against a panel of 60 human cancer cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast [122]. This hybrid also showed acceptable acute toxicity in mice, and the maximum tolerated dose was 100 mg/kg. The high and broad-spectrum anticancer activity as well as low toxicity made this hybrid a useful starting point in drug development to combat various types of cancers. The ferrocene-phenol hybrids 60 (IC50: 7-26 µM), 61 (IC50: 0.94-32 µM), 62 (IC50: 6-13 µM) and 63 (IC50: 8.4-27 µM) showed potential activity against SF-295, HCT-8, MDA-MB-435 and HL-60 cancer cell lines [131]. Among them, hybrid 61c (IC50: 0.94-1 µM) which was highly active against SF-295, HCT-8, and HL-60 cancer cell lines, could serve as a lead compound for further exploitation. The activity of hybrids 64 (IC50: 0.14-3.5 µM) was no inferior to that of Ferrocifen (IC50: 1.13 µM) against MDA-MB-231 cells, and the SAR revealed that introduction of hydroxyl into ethyl group or replacement of phenyl ring at R1 position by alkyl side chain could boost up the activity [132-134]. Amongst them, four hybrids 64a-d (IC50: 0.11-0.36 µM) were 3.1-10.2 folds more potent than the reference Ferrocifen against MDA-MB-231 cells.

22

HO

OH

HO

n

OH

Fe

R

OH

Fe Fe

Fe

Fe R

OH

57

58

57a: R = OH; 57b: R = NH2; 57c: R = NHAc; 57d: R = OAc.

58a: R = H; 58b: R = NH2; 58c: R = NHAc; 58d: R = OAc.

R

R

59

61

60

59a: n = 1; 59b: n = 2.

61a: R = H; 61b: R = OH; 61c: R = OAc.

60a: R = OMe; 60b: R = OH.

-CH2OH > -Me OH

OH

OH

R

R2 O Fe

R1 Fe

OH

Fe

Fe

Fe

OH

Alkyl > phenyl ring OH

65

62

64

63

O 64a: R1 = Me, R2 = CH2OH; 64b: R1 = Et, R2 = CH2OH; 64c: R1 = n-Pr, R2 = CH2OH; 64d: R1 = 4-OHPh, R2 = CH2CH2OH.

65a: R =

N O

HO Fe

R 66

66a: R = Ph; 66b: R = Me; 66c: R = Et.

Fe

N n N N

R

N

N

R

N OH

Fe 68

67

68a: R = H; 68b: R = 3-OMe; 68c: R = 5-Cl.

67a: n = 0, R = 3,5-diOH.

Figure 12. Chemical structures of ferrocene-phenol hybrids 57-68

The ferrocene-phenol hybrids 65 (Figure 12, IC50: 0.035-12.9 µM) showed considerable activity against ovarian cancer lines (SK-OV3, A2780, and cisplatin-resistant A2780-cis), and the majority of them were more potent against cisplatin-resistant A2780-cis than against A2780 cancer cells [135,136]. The representative hybrid 65a (IC50: 35-300 nM) was highly active against the tested three cancer cell lines, and the activity was 34.2- and 234.6-fold higher than that of Cisplatin (IC50: 1.2 and 11.5 µM) against A2780 and A2780-cis cancer cell lines. Further study indicated that this hybrid (IC50: 35-140 nM) also exhibited excellent activity against breast

(MDA-MB-231), colorectal

(HCT 116),

pancreatic

(Mia-PaCa-2), and Leukemia (K562) cell lines, but relatively low activity (IC50: 4.1 µM) against doxorubicin-resistant K562R cells, implying this hybrid was partial substrate of P-glycoprotein [135]. The mechanistic study proved that this hybrid generally exerted the anticancer activity via apoptotic and senescence pathways. 23

The ferrocene-phenol hybrids 66 (IC50: 1.66-5.96 µM) also displayed considerable activity against A2780 and A549 cancer cell lines, suggesting the double-bond between ferrocene and phenol moieties was not indispensable for the high activity [137,138]. The 1,2,3-triazole tethered ferrocene-phenol hybrids 67 (IC50: 15.3->100 µM) showed weak to moderate activity against HCC38 and MCF-7 cancer cell lines, and the most active compound 67a (IC50: 22.9 and 84.0 µM) was less potent than the reference Tamoxifen (IC50: 14.9 and 13.1 µM), suggesting the 1,2,3-triazole fragment was not a preferable linker between ferrocene and phenol motifs [139]. Similar results were also observed for azine tethered ferrocene-phenol hybrids 68 [140].

11. Ferrocene-pyrimidine hybrids Cancer cells are reliant on the pyrimidine biosynthesis pathway to survive, and many anticancer agents like Ceritinib and Uramustine contain a pyrimidine moiety [141,142]. Therefore, hybridization of ferrocene with pyrimidine represents a promising strategy to develop novel anticancer candidates. The majority of ferrocene-pyrimidine hybrids 69 (Figure 13, IC50: 1.9-46.8 µM) and 70 (IC50: 1.2-48.9 µM) were sensitive to B16-F10 and A549 cancer cell lines, and the SAR demonstrated that introduction of electron-donating group into phenyl ring was beneficial for the activity, and hybrids with substituent at para-position were more potent than the ortho-position analogs [143]. Among them, two hybrids 69a (IC50: 1.9 and 16.5 µM) and 70a (IC50: 18.0 and 1.2 µM) displayed the most potent inhibitory activity against B16-F10 and A549 cancer cell lines, and the activity was comparable to or better than that of the reference Celecoxib (IC50: 15.22 and 16.03 µM). The mechanistic study proved that hybrid 70a could induce apoptosis in A549 cells, suggesting this hybrid can be developed as a promising anticancer agent in the future. The hybrid 71 (IC50: 0.55-10.63 µM) showed potential activity against BT-549, MDA-MB-231, SK-BR-3, MOLM-13 and MV-4-11 cancer cell lines, and the SAR revealed that the phenyl ring was crucial for the high activity, and removal of the phenyl led to great loss of activity [144]. This hybrid may target a negative regulator of cell migration, and the level of ribosomal protein S6 phosphorylation increased 24

upon incubation with this hybrid, indicating a possible role of hybrid 71 in the inhibition of protein phosphatases. The hybrid 72 (IC50: 0.35-1.1 µM) demonstrated promising activity against L1210, CEM and HeLa cancer cell lines, and the activity was no inferior to that of Cisplatin (IC50: 0.9-1.2 µM) and 5-Fluorouracil (IC50: 0.33-18 µM) [145]. The SAR indicated that the hydroxyalkyl was critical for the high activity, and the hybrid (IC50: 26-94 µM) with hydroxyalkyl only showed weak to moderate activity.

Figure 13. Chemical structures of ferrocene-pyrimidine hybrids 69-74

Some ferrocene-pyrmidinone hybrids also possessed certain anticancer activity, and the activity of compound 73 (Figure 13, IC50: 4.5-23.2 µM) was comparable to that of Cisplatin (IC50: 3.6-36.5 µM) against HT-29, MCF-7, MDA-MB-231, HL-60, and MonoMac6 cancer cell lines [146-150]. This hybrid had potential apoptosis inducing properties on tumor cultures [150], while the hybrids 74a,b could suppress A549 lung cancer cell growth through cell cycle arrest [151].

12. Ferrocene-quinoline hybrids 25

DNA topoisomerases are enzymes that catalyze the alteration of DNA topology with transiently induced DNA strand breakage, so topoisomerases are validated cancer chemotherapy targets [152,153]. Topoisomerase-targeting anticancer drugs such as quinoline derivatives Cabozantinib, Voreloxin, and Quarfloxin could act through topoisomerase poisoning, leading to replication fork arrest and double-strand break formation [154,155]. Thus, hybridization of ferrocene with quinoline may provide valuable therapeutic intervention in the cancer control. The ferrocene-quinoline hybrids 75a,b (Figure 14, IC50: 310 and 740 nM) showed excellent activity against WHCO1 oesophageal cancer cells, and the activity was 17.5-55.8 times higher than that of the references Cisplatin and Ferroquine (IC50: 13.0 and 17.3 µM, respectively) [156]. Further study indicated that hybrid 75b (GI50: 0.6-2.4 µM) also showed potential activity against MCF-7, TK10 and UACC62 cancer cell lines, and the activity was no inferior to that of Etoposide (GI50: 0.8-5.9 µM) [157]. The ferrocene-quinoline hybrid 76a (IC50: 590 and 530 nM) and its bis-ferrocene analog 76b (IC50: 280 and 330 nM) exhibited promising activity against HTB-129 and Caco-2 cancer cell lines, and the activity was far more potent than that of Cisplatin (IC50: 50.50 and 395.80 µM) and Ferroquine (IC50: 8.7 and 101 µM) [158]. The SAR revealed that ferrocene motif was crucial for the activity, and removal of the ferrocene moiety resulted in significant loss of activity. Hybrid 76b was more potent than compound 76a, suggesting incorporation of the second ferrocene moiety could enhance the activity.

26

Figure 14. Chemical structures of ferrocene-quinoline hybrids 75-77

Besides the compounds mentioned above, many other ferrocene-quinoline hybrids which are exemplified by hybrids 77 (Figure 14, IC50: 1.60-10.71 µM) also showed potential

activity

against

drug-sensitive

DLD1,

U87,

NCI-H460,

and

multidrug-resistant DLD1-TxR, U87-TxR, NCI-H460/R cancer cell lines [159-162]. Notably, the activity of hybrids 77 against multidrug-resistant cancer cells (IC50: 1.60-10.14 µM) was higher than that against drug-sensitive counterparts (IC50: 1.75-10.71 µM), suggesting these hybrids were not substrates for P-glycoprotein which was indicated as a major mechanism for multidrug-resistance [159]. Hybrid 77c (IC50: 1.60-3.00 µM) was found to be most active against all tested cancer cell lines, and this hybrid had the potential to increase ROS generation and induce mitochondrial damage in multidrug-resistant cancer cells. It is worth noting that simultaneous treatment of this hybrid with Paclitaxel increased sensitivity of multidrug-resistant cancer cells.

27

13. Ferrocene-steroid hybrids Steroids can act as signaling molecules, and steroid moiety has been a highly privileged motif for the target-based design and development of anticancer agents due to its biodiversity and versatility [163,164]. Moreover, Aromasin, Galeterone and Fulvestrant are the anticancer agents emerged on steroidal pharmacophores. Thus, ferrocene-steroid hybrids are potential prototypes for the discovery of novel anticancer candidates. Five ferrocene-steroid hybrids were screened for their antiproliferative activity against HeLa cells by Manosroi et al., and the activity of hybrids 78a,b (Figure 15, GI50: 223 and 271 nM) was in the same level with that of the reference drug Doxorubicin (GI50: 250 nM) [165]. Further study indicated that incorporation of ester linker between ferrocene and steroid moieties could boost up the activity, and hybrid 79 (IC50: 9 and 24.4 nM) showed potential activity against MCF-7 and HT-29 cancer cell lines [166].

Figure 15. Chemical structures of ferrocene-steroid hybrids 78-81

The ferrocene-steroid hybrids 80a,b (Figure 15, IC50: 8-41 µM) demonstrated considerable activity against MCF-7, T47D and MDA-MB-231 cancer cell lines, and they could serve as vectors and can be recognized by ERα, being delivered into the cell [167,168]. The hybrids 81a,b (IC50: 1.2-20 µM) also showed potential activity against MCF-7 and HT-29 cancer cell lines, and the activity was higher than that of 28

the references Tamoxifen and Cisplatin (IC50: 47-66 µM) [169].

14. Ferrocene-sugar hybrids Sugars are involved in cancer cell dissociation and invasion, cell-matrix interactions, tumor angiogenesis, immune modulation and metastasis formation [170,171], so sugars as pharmacologically significant scaffolds have been of great interest in recent years. Thus, hybridization of the pharmacophore moiety of ferrocene with sugar opens a door for the opportunities on the development of novel anticancer agents. The ferrocene-α-ᴅ-xylofuranose hybrids 82a,b (Figure 16, IC50: 5.5-39.74 µM), ferrocene-α-ᴅ-ribofuranoside

82c

(IC50:

6.51-23.89

µM)

and

ferrocene-α-ᴅ-galactopyranose 82d (IC50: 5.47-29.82 µM) possessed considerable activity against A549, Neuro2a, HeLa, MCF-7 and MDA-MB-231 cancer cell lines, but none of them were superior to the references Doxorubicin (IC50: <1-1.2 µM) and Cisplatin (IC50: ≤1 µM) [172]. Further study revealed that incorporation of the second sugar moiety could not enhance the activity, and hybrids 83a,b (IC50: 38.3 and >100 µM) were less potent than Cisplatin (IC50:12.9 µM) against A2780 cells [173,174]. Replacement of amide linker by 1,2,3-triazole was also tolerated as evidenced by that hybrids 84 (IC50: 5.3-18.05 µM) were comparable to hybrids 82 against A549, Neuro2a, HeLa, MCF-7 and MDA-MB-231 cancer cell lines [175]. Incorporation 1,2,3-triazole between amide and α-ᴅ-xylofuranose, such as compounds 85, was beneficial for the activity against MCF-7 and MDA-MB-237 breast cancer cell lines (IC50: 2.82-11.31 µM), but reduced the activity against HeLa cells (IC50: >100 µM) [176]. Replacement of amide between ferrocene and 1,2,3-triazole by ether or thioether (X = S, 86a,b and 87a,b, IC50: 9.0->200 µM) was harmful to the activity, while selenoether (X = Se, 86c and 87c, IC50: 2.9 and 3.71 µM) could improve the activity

against

A549

cells

[177,178].

The

pyridine-containing

ferrocene-α-ᴅ-xylofuranose complex 88 (IC50: 13.18 µM) was sensitive to A549 cells, but devoid of activity (IC50: >100 µM) against HeLa, MCF-7 and MDA-MB-237 cancer cell lines [179].

29

Figure 16. Chemical structures of ferrocene-sugar hybrids 82-90

The ferrocene-6-deoxy-6-fluoroglucopyranosyl hybrid 89 (Figure 16, IC50: 20-30 µM) exhibited considerable activity against HL-60, MCF-7, DU-145, PC-3 and AR42J cancer cell lines, and could act as a platform for the further exploration [180]. Some of ferrocene-iminosugar hybrids also possessed promising anticancer activity, and hybrids 90 (IC50: 1.2-1.6 µM) showed potential activity against MCF-7 cells, indicating they could be useful templates for the design of novel and potential anticancer agents [181,182].

15. Miscellaneous ferrocene hybrids Pyridine derivatives could target CDK, EGFR, PI3K, RGGT, and hedgehog/GLI signaling, making such compounds as attractive scaffolds for discovery of novel drugs [183,184]. Some pyridine-containing compounds such as Vismodegib and Sonidegib have already used in clinical practice or under clinical trials for the treatment of various cancers, so pyridine moiety is a useful template for the development of novel anticancer agents. The ferrocene-pyridine hybrid 91a (Figure 17) was devoid of 30

activity against MCF-7 and A549 cancer cell lines, while hybrid 91b (IC50: 11 and 5.5 µM) was >5.4-fold more potent than Cisplatin (IC50: 64.1 and >30 µM) against the two cancer cell lines, implying the alkyl linker between ferrocene and 1,2,3-triazole enhanced the activity [185]. The mechanistic study indicated that hybrid 91b could cause DNA degradation and cell apoptosis. However, this hybrid was also toxic towards normal HEK293 cells (IC50: 3 µM). Some other ferrocene-pyridine hybrids also possessed certain anticancer activity, but the activity was generally lower than that of the references [186-192]. The activity of ferrocene-thiourea hybrids 92 (IC50: 1.72-5.56 µM) was comparable to that of Cisplatin (IC50: 1.97 and µM) against THP-1 and MCF-7 cancer cell lines, and the SAR revealed that substituents at phenyl ring had little impact on the activity [193,194]. Further study indicated that replacement of thiourea by selenourea could not improve the activity [195,196]. The

ferrocene-paclitaxel

hybrids

(IC50:

93

<0.005-4.663

µM)

possessed

broad-spectrum activity against SW620, A549, Colo 205, HCT116, HepG2, MCF-7, and multidrug-resistant SW620C, SW620D, SW620E, SW620M as well as SW620V cancer cell lines, and all hybrids except 93f were more potent than the reference 2’,3’-epi-paclitaxel (IC50: 0.379-40.36 µM) [197]. The SAR indicated that introduction of phenyl between amide and ferrocene moieties or replacement of hydroxyl group by phenyl group was harmful to the activity [197,198]. Interestingly, the redox properties of these ferrocenyl derivatives appeared to play a less important role in their mode of action, as there was no correlation between intracellular redox activity and the anticancer activity. In particular, hybrid 93a (IC50: <5-366 nM) was found to be highly active against all tested drug-sensitive and multidrug-resistant cancer cell lines, and the activity was >33.1-fold more potent than that of 2’,3’-epi-paclitaxel against all tested cancer cell lines. Accordingly, this hybrid can be considered as a preclinical candidate for fighting against both drug-sensitive and multidrug-resistant cancers. The ferrocenyl-podophyllotoxin hybrids (IC50: 0.43-39.75 µM) displayed considerable activity against MCF-7 and MDA-MB-231 cancer cell lines, and compound 94 (IC50: 930 and 430 nM) was found to be most active against the tested cancer cell lines, but 31

still far less potent than the reference Podophyllotoxin (IC50: 10 nM) [199]. The ferrocenyl-naphthalimide hybrid 95 (IC50: 4.33-10.52 µM) possessed considerable activity against EC109, BGC823, SGC7901 and HepG2 cancer cell lines, and the activity was 5.1-12.2 times higher than that of the reference Amonafide (IC50: 34.64-129.00 µM) [200]. Removal of the ferrocenyl moiety led to great loss of activity (IC50: 31.00-120.00 µM), demonstrating ferrocenyl fragment was crucial for the activity. The mechanistic study revealed that the cytotoxicity of hybrid 95 was related to the DNA damage in cancer cells.

Harmful to the activity

Extension of the carbon spacer preferred O

O

Fe

N N N

OH O

S

O n

Fe

O

OH O

NH

N

N

O

O

O

O HN

R

NH

N

O

Fe O

S

OAc BzlO

92

91

O

O

OH

OH

Fe

O HN OAc BzlO

O OH

OH

93a 91a: n = 0; 91b: n = 2.

R = F, Me, OMe.

93b-d Replaced by phenyl ring reduced the activity 93b: ortho-; 93c: meta-; 93d: para-. O

O OH O

O

O

OH O O

O

OAc BzlO

HN

O OH

OH

HN

O

OAc BzlO

N

O

O O

O

Crucial for the activity O

O

O

O

Fe

Fe

O

OH

OH

O

O

O O Fe

Fe

MeO

N

N H

N O

OMe OMe

93e

93f 94

NH NH N

linker Fe

Cl

Pt S O

Cl

Fe

O

HO

N 5

OH X

X

102

101a: X = CH2; 101b: X = CH2CH2; 101c: X = CH2O; 101d: X = CH2S.

X Fe

99

O

N N O Cl M Cl N O N

Fe N

O S O O

99a: n = 5, X = CO.

Fe

O

O

100

O

98

101 HO

n O

a: R = NEt2; b: R = pyrrolidinyl; c: R = morpholinyl; d: R = piperidinyl; e: R = 4-methylpiperazin-1-yl; f: R = 4-phenylpiperazin-1-yl.

H N

O S O O

H N

HO

Ph Ph

97

96

O

Ph Ph PF6P S R Fe Pd P S

Ph Ph

96a: without linker; 96b: linker = par a-phenyl

Fe

Ph Ph PF6P S R Pt P S

95

102a: M = Co2+; 102b: M = Ni2+; 102c: M = Zn2+; 102d: M = Cu2+.

O OH

N

N H

Fe

103

Figure 17. Chemical structures of ferrocene hybrids 91-103

The platinum(II) ferrocenyl-guanidine hybrids 96 (Figure 17, IC50: 1.5-2.6 µM) were comparable to Cisplatin (IC50: 1.9-3.0 µM) against HBL-100, HeLa and SW1573 32

cancer cell lines, and 5.7-13.6 folds more potent than Cisplatin (IC50: 15 and 26 µM) against T-47D and WiDr cancer cell lines [201]. Both of them did not show significant changes of the cell cycle, but induced considerable cell death in HBL-100 cells. Moreover, subtle accumulation of cells in S or G2/M phase occurred in HeLa, SW1573 and T47D cells, implying these hybrids could induce growth inhibition in cancer cells by a mechanism which was different from that of Cisplatin. Many

phosphinoferrocene-(dithio)carboxamide

hybrids

also

showed

certain

anticancer activity, and most of hybrids 97 (IC50: 1.6-9.9 µM) and 98 (IC50: 3.0-19.6 µM) demonstrated potential activity against SKOV-3, HepG2, PC12 and A549 cancer cell lines [202-207]. The majority of hybrids 97 were comparable to or better than Cisplatin (IC50: 1.26-9.83 µM) against the tested cancer cell lines, implying the potential application of these hybrids as breast cancer therapeutic agents [202]. The hybrids 99 (IC50: 0.094-3.77 µM and 0.13-4.5 µM against ER (α and β) and HDAC (1 and 6), respectively) and 100 (IC50: 0.03-1.31 µM against ER and HDAC) were screened for their potential as dual-acting ER and HDAC inhibitors by Li et al., and most of them (IC50: 14.8-49.1 µM) were sensitive to MCF-7, MDA-MB-231, and DU-145 cancer cell lines [208]. Moreover, all hybrids (IC50: >50 µM) were non-toxic towards VERO cells. Among them, hybrids 99a (IC50: 14.8-25.3 µM) and 100 (IC50: 15.6-21.2 µM) were found to be most active against MCF-7, MDA-MB-231, and DU-145 cancer cell lines, representing efficient dual-acting agents for treatment of breast cancer. The ferrocenyl-heterocycles also possessed

potential activity against both

drug-sensitive and drug-resistant cancer cell lines, and the activity of hybrids 101 (IC50: 2.2-37 µM) was no inferior to that of Cisplatin (IC50: 1.7-10 µM) against drug-sensitive A2780, SK-OV-3 and drug-resistant A2780cis cancer cell lines [209-212]. Further study indicated that conversion of the free forms to hydrochloride salts was beneficial for the activity (IC50: 0.7-20.1 µM) [209]. The ferrocene-furan hybrids 102 (IC50: 0.0101-50.5 µM) possessed broad-spectrum activity against a panel of cervical carcinoma (KB), ovarian carcinoma (SKOV-3), CNS cancer (SF-268), non-small lung cancer (NCl H460), colon adenocarcinoma (RKOP 27), leukemia (HL60, U937, K562), melanoma (G361, SKMEL- 28), 33

neuroblastoma (GOTO, NB-1), cervical cancer (HeLa), breast cancer (MCF-7), lung fibrosarcoma (HT1080) and hepatocellular liver carcinoma (HepG2) cells, and the metal complexes were more potent than the ligand [213,214]. The activity of the hybrids was no inferior to that of the reference Doxorubicin (IC50: 1.13-6.66 µM) against most of the tested cancer cell lines, suggesting ferrocene-furan hybrids are promising leads for future development of new potential cancer therapeutics. The ferrocene-quinone hybrids such as compound 103 (IC50: 0.5-4.7 µM) showed considerable activity against 518A2, HCT-116, and multi-drug resistant KB-V1/Vbl cancer cell lines, and the activity was superior to that of the references Plumbagin (IC50: 1.1-26.2 µM) and Cisplatin (IC50: 4.1-15.4 µM) [215,216]. Moreover, this hybrid (IC50: >50 µM) was non-toxic towards normal CHF cells. Further study indicated that this hybrid could increase death of cancer cells (sub-G0/G1 fraction) in a dose- and time-dependent manner, and ROS levels were significantly increased in cancer cells. Besides the chalcone hybrids mentioned above, ferrocene-biotin [217,218], ferrocene-carborane [219], ferrocene-glycidyl ether [220,221], ferrocene-imine [222-225],

ferrocene-pyrrole

[226,227],

ferrocene-retinoic

acid

[228],

and

ferrocene-(thio)semicarbazone [229,230] hybrids also showed certain anticancer activity, but the majority of them were no superior to the references. In spite of that, the enriched SAR may point out the direction for further rationale design and modification of these hybrids.

16. Conclusions Cancer leads to millions of deaths annually, but the morbidity and mortality of cancer will continue to grow for a long period. Anticancer agents are a crucial and effective strategy for the treatment of cancers, but the emergency of drug-resistance and the low specificity of currently used anticancer agents create an urgent need to explore novel anticancer drugs with high specificity and great potency against drug-resistant cancers. Ferrocene derivatives constitute versatile and interesting scaffolds for the drug 34

development, and ferrocene-phenol hybrid Ferrocifen which could target not only DNA but also proteins and various enzymes had a different mode of action to the platinum anticancer drugs, revealing the potential of ferrocene hybrids for the treatment of various cancers even multidrug-resistant cancers. Plenty of ferrocene hybrids were developed in the past 10 years, and some of them possessed promising in vitro and in vivo activity against both drug-sensitive and drug-resistant even multidrug-resistant cancers. In particular, hybrids 1, 2, 17, 48, 54, 57a, 65, 77, 93 and 102 possessed broad-spectrum anticancer activity drug-sensitive and/or drug-resistant even multidrug-resistant cancer cells. Conjugates 1a,b, 16 and 26 with IC50 values in nanomolar level were highly active against various cancer cell lines, whereas compounds 14b, 25b and 35a not only exhibited great in vitro and in vivo potency, but also showed favorable in vivo safety profile. Based on these, ferrocene hybrids are potential agents for clinical application in the control and eradication of cancers. This review covers the ferrocene hybrids with potential in vitro and in vivo anticancer activity which were developed in recent 10 years. The structure-activity relationships and mechanisms of action are also discussed to set up the direction for the design and development of ferrocene hybrids with high efficiency and low toxicity.

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51

1.

Ferrocene hybrids constitute versatile and interesting scaffolds for anticancer

drug discovery 2.

Ferrocene-phenol hybrid Ferrocifen is in pre-clinical trial against cancers

3.

The structure-activity relationship is enriched

Conflict of interests We confirm that there is no conflict of interests regarding our manuscript entitled “Ferrocene-containing

hybrids as potential anticancer agents:

Current developments, mechanisms of action and structure-activity relationships”.

Ruo Wang