Ferrocene-Based Compounds with Antimalaria/Anticancer Activity

molecules

Review Ferrocene-Based Compounds with Antimalaria/Anticancer Activity

Sijongesonke Peter and Blessing Atim Aderibigbe * Department of Chemistry, University of Fort Hare, Alice campus, Eastern Cape 5700, South Africa; [email protected] * Correspondence: [email protected]; Tel.: +27-40602-2266

Academic Editors: Carlo Santini and Maura Pellei Received: 11 July 2019; Accepted: 21 August 2019; Published: 7 October 2019 Abstract: Malaria and cancer are chronic diseases. The challenge with drugs available for the treatment of these diseases is drug toxicity and resistance. Ferrocene is a potent organometallic which have been hybridized with other compounds resulting in compounds with enhanced biological activity such as antimalarial and anticancer. Drugs such as ferroquine were developed from ferrocene and chloroquine. It was tested in the 1990s as an antimalarial and is still an eﬀective antimalarial. Many researchers have reported ferrocene compounds as potent compounds useful as anticancer and antimalarial agents when hybridized with other pharmaceutical scaﬀolds. This review will be focused on compounds with ferrocene moieties that exhibit either an anticancer or antimalarial activity.

Keywords: organometallics; malaria; cancer; ferrocene compounds; ferroquine

1. Introduction Malaria is a lethal disease and it is caused by a parasitic protozoan of genus Plasmodium. There are four Plasmodium parasites causing malaria in humans: P. falciparum, P. vivax, P. malariae, and P. ovale. Among the aforementioned parasites, P. falciparum is the most dangerous and it is responsible for over 95% malaria infections worldwide [1,2]. According to a WHO report, over 200 million cases and over 400,000 deaths were reported in 2016 [3]. Young children who are less than five years old and pregnant women were the major victims of malaria infections [1,2]. The most common symptoms that are associated with malaria are fever, fatigue, headaches, vomiting. In serious cases of malaria, symptoms such as yellow skin, seizures, and death have been reported [1–3]. Cancer is also a life-threatening disease involving a rare cell growth which can spread to body organs. Many cancers are caused by genetic mutation (90–95%) and some are caused by inherited genetics (5–10%) [4]. Cancer was accountable for more than 9 million deaths in 2018 especially in Africa and in Asia, according to the World Health Organization [5]. Cancer can affect any body tissue and it is named according to the body organs it affects e.g., lung, liver, breast, colorectal, and stomach cancer [6]. Among the aforementioned cancer types, lung cancer is accountable for the highest number of deaths (1.8 million) because of limited prognosis, followed by colorectal (881,000), stomach (783,000), and liver (782,000), while breast cancer (627,000) is ranked the fifth since its prognosis is favorable [5]. In the treatment of cancer and malaria, the challenges with the currently used drugs are drug toxicity, high cost, and drug resistance [7,8]. Ferrocene compounds have been described as potential compounds with unique antimalarial and anticancer activity [7,8]. In this review, the efficacy of compounds containing ferrocene moiety in vitro and in vivo are reported.

2. Ferrocene (Biological Activity) Ferrocene was ﬁrst discovered accidentally in the early 1950s by two researchers called Kealy and Pauson at Duquesne University while reacting C5H5BrMg with FeCl3 [9]. Ferrocene is useful in the

Molecules 2019, 24, 3604; doi:10.3390/molecules24193604 www.mdpi.com/journal/molecules Molecules 2019, 24, 3604 2 of 27 modern organometallic chemistry industry due to its versatile applications in catalysis, material sciences, medicinal chemistry, and diagnostic applications [10,11]. It has been proven that the combination of organometallic compounds with known antimalarial drugs can result in potent antimalarials. Ferrocene is a good pharmacophore that displays physicochemical properties that have favorable effects on living matter [2,12]. Presently, some researchers have designed hybrid compounds containing ferrocene with several biological activities [2,12]. Antimalarial, anticancer, antitumor, antifungal, and antileishmanial activities are some biological activities exhibited by ferrocene derivatives [2,12]. Hybrid compounds in which ferrocene derivatives are linked to other compounds via selected linkers have been found to increase the efficacy of the compounds hybridized with ferrocene derivatives, resulting from its properties such as good stability to water and air, low toxicity, unfamiliar redox activity, similarities in aromaticity, and chemical versatility [13,14]. The introduction of substituent on the cyclopentadiene ring of ferrocene contributes to its biological activity [15]. The replacement of an aryl or heteroaryl core with a ferrocene core in a bioactive compound contributes to significant changes in the properties of the compounds, such as solubility, hydrophobicity, and lipophilicity [10]. Ferrocene derivatives have been reported to exhibit antiproliferative activity against several cancer cell lines with low toxic effects when compared to known anticancer agents. Its complex also exhibits distinct oxidation-reduction behavior whereby it converts readily to its one-electron oxidation product, the ferricenium ion, a radical cation of eminent stability. The aforementioned reaction is reversible with the inherent electron transfer useful for biological reactions. Its electron transfer and free radical reactions are useful in biological processes. Some of the important biological reactions of ferrocene analogues are ferricenium cation reduction by NADH and metalloproteins; enzymatically oxidation of ferrocene by hydrogen peroxide; recombination of the ferricenium system with an attack on free radicals leading to substituted ferrocene upon proton elimination [16]. Ferricenium cation react with biologically important superoxide anion radicals, resulting in the regeneration of ferrocene and dioxygen. The oxidation of the ferrocene complex to its ferricenium counterpart is feasible whereby ferrocenylcarboxylates interact with the aggressive hydroxyl radical, transforming it into harmless hydroxyl anion. In cancer etiology, free-radical chemistry plays a vital role in the different phases of growth and control of neoplasia [16]. The superoxide and the free radical-scavenging reaction of ferrocene are useful in the inhibition of cancer growth. Furthermore, the deactivating recombination of ferrocene in its oxidized state with the free-radical form of ribonucleotide reductase, an important enzymatic link in DNA synthesis, makes ferrocene a useful scaffold for the development of potent compounds [16]. The aforementioned factors have prompted research on the development of ferrocene derivatives with enhanced biological activity. Köpf-Maier et al. reported ferricenium salts with high biological activity influenced by their good water solubility. The water insoluble ferricenium salts did not exhibit any biological activity [17] Neuse further reported ferrocene-based anticancer drugs which were highly water soluble with excellent anticancer activity [17,18].

3. Ferrocene-Based Compounds with Antimalaria Activity Each antimalarial drug is characterized by a unique mechanism of action [19]. Antimalarial drugs are classified according to the stages of the malarial life cycle in which they act, or based on their structures [20]. They are classified according to the stages of malaria life cycle such as tissue schizontocidal drugs e.g., pyrimethamine (i.e., they act on the erythrocytic stage); hyponozoitocidal drugs e.g., primaquine (i.e., those that act at the exo-erythrocytic stage); blood schizontocidal drugs e.g., quinine; and gametocytocides e.g., chloroquine [19]. They are classified based on their structures such as aryl amino alcohols e.g., halofantrine, quinidine, quinine, mefloquine; 4-aminoquinolines e.g., amodiaquine, chloroquine; folate synthesis inhibitors e.g., sulfonamides, sulfones, proguanil, chloroproguanil; diaminopyrimidine, pyrimethamine; 8-aminoquinolines e.g., primaquine; antimicrobials e.g., fluoroquinolones, doxycycline, clindamycin, tetracycline, azithromycin; Molecules 2019, 24, 3604 3 of 27

Molecules 2019, 24, x 3 of 27 peroxides e.g., artemisinin derivatives; naphthoquinones e.g., atovaquone; and iron chelating agents e.g.,peroxides desferrioxamine e.g., artemisinin [21]. However, derivatives; the naphthoquinones use of a single antimalarial e.g., atovaquone drug; for and the iron treatment chelating of agents malaria ise.g. hampered, desferrioxamine by drug resistance[21]. However, [22]. the Malaria use of a issingle treated antimalarial by combination drug for the therapy treatment in which of malaria two or moreis hampered antimalarials by drug are resistance combined [22]. together. Malaria However, is treatedthe by combinat aforementionedion therapy approach in which also two su orffers more from challengesantimalarials such are as drug–drugcombined together. interaction, However, drug resistance the aforementioned indicating approach the serious also need suffer tos developfrom effchallengesective antimalarials such as drug [23–].drug Some interaction, researchers drug have resistance reported indicating the effi cacy the serious of hybridizing need to develop ferrocene derivativeseffective antimalarials with known antimalarials[23]. Some researchers resulting havein potent reported antimalarials. the efficacy of hybridizing ferrocene derivatives with known antimalarials resulting in potent antimalarials. 3.1. Ferrocene-Quinoline Derivatives 3.1. Ferrocene-Quinoline Derivatives The strategy of hybridizing ferrocene moieties with active antimalarial agents such as chloroquine was firstThe reported strategy in ofthe hybridizing early 1990s and ferrocene there are moieties more studies with active which antimalarial are ongoing. agents Ferroquine such as was chloroquine was first reported in the early 1990s and there are more studies which are ongoing. derived from chloroquine in 1994 by Biot and co-workers at Lille University. It was reported to be Ferroquine was derived from chloroquine in 1994 by Biot and co-workers at Lille University. It was more effective against chloroquine resistance P. falciparum and non-toxic in vivo [18]. Ferroquine was reported to be more effective against chloroquine resistance P. falciparum and non-toxic in vivo [18]. synthesized by the incorporation of ferrocene moiety into chloroquine whereby the methylene group Ferroquine was synthesized by the incorporation of ferrocene moiety into chloroquine whereby the of chloroquine was replaced by a ferrocene moiety (Figure1)[18]. methylene group of chloroquine was replaced by a ferrocene moiety (Figure 1) [18].

NX2 NX2 HN

Fe Fe

Cl N

X = CH 3 FigureFigure 1. 1.Synthesis Synthesis of ferroquine by by Biot Biot and and co co-partners.-partners.

InIn vitro vitroevaluation evaluation of of ferroquine ferroquine againstagainst P. falciparumfalciparum strainsstrains revealed revealed no no correlation correlation between between ferroquineferroquine and and chloroquine chloroquine responses using using standardized standardized initial initial parasitaemina parasitaemina during during the assays the assays.. In Invivo vivo evaluation reported reported by by Long Long et et al. al. on on PlasmodiumPlasmodium-infected-infected mice mice revealed revealed that that a dose a dose of of ferroquineferroquine (10 (10 mg mg/kg/d/kg/d for for four four days) days) waswas suitablesuitable for effective effective treatment treatment [18,24 [18,24]. ].Ferroquine Ferroquine exhibit exhibitss similarsimilar properties properties as its as parent its parent drug, drug, chloroquine. chloroquine. However, How theever, basicity the basicity and lipophilicity and lipophilicity of ferroquine of areferroquine different fromare different that of chloroquine.from that of chloroquine. The protonation The protonation of chloroquine of chloroquine and ferroquine and atferroquine food vacuole at pHfood resulted vacuole in pH lipophilicity resulted in of lipophilicity log D = 1.2of log and D =0.77, −1.2 respectivelyand −0.77, respectively which was which close. was However, close. − − protonationHowever, protonat of chloroquineion of chloroquine and ferroquine and ferroquine at pH 7.4 resultedat pH 7.4in resulted lipophilicity in lipophilicity of log D of= log0.85 D and = 0.85 2.95, respectively.and 2.95, respectively. Ferroquine exhibited Ferroquine lower exhibited pKa values lower when pKa values compared when to chloroquinecompared to suggesting chloroquine low vascularsuggesting accumulation low vascular of ferroquineaccumulation when of ferroquine compared when to chloroquine. compared However,to chloroquine the electron. However, donating the electron donating property of ferrocene and the strong hydrogen bond between the terminal nitrogen property of ferrocene and the strong hydrogen bond between the terminal nitrogen atom and the atom and the 4-amino group may have contributed to its low pKa values. Although ferroquine has 4-amino group may have contributed to its low pKa values. Although ferroquine has low pKa values, low pKa values, it exhibits a significant inhibition effect against beta-haematin formation when it exhibits a significant inhibition effect against beta-haematin formation when compared to chloroquine compared to chloroquine and it is preferentially localized at the lipid–water interface, making it an and it is preferentially localized at the lipid–water interface, making it an effective antimalarial agent effective antimalarial agent when compared to chloroquine [18,25]. when comparedFerroquine to enhanced chloroquine antimalarial [18,25]. activity in vitro and in vivo against P. falciparum has promptedFerroquine several enhanced researchers antimalarial to develop activity antimalarialin vitro drugsand in containing vivo against ferroceneP. falciparum moiety.has They prompted have severalused researchers several strategies to develop to antimalarial develop ferroquine drugs containing derivatives ferrocene such moiety. as hydroxyferroquine, They have used severaltrioxaferroquine strategies to, and develop chloroquine ferroquine-bridged derivatives ferrocenophane such as hydroxyferroquine,with the hope of increasing trioxaferroquine, the efficacy and chloroquine-bridgedof ferroquine. Biot ferrocenophaneet al. synthesized with 3-hydroxyferroquine the hope of increasing derivatives the effi (cacy1a–c) of which ferroquine. inhibited Biot the et al. synthesizedgrowth of 3-hydroxyferroquineP. falciparum in vitro when derivatives compared (1a– cto) whichchloroquine inhibited (Figure the growth2). How ofever,P. falciparum they werein less vitro whenactive compared when compared to chloroquine to ferroquine. (Figure 2In). vitro However, evaluation they were was lessperformed active whenagainst compared 3D7 and to2W ferroquine. strains Inof vitro P. falciparumevaluation for was compounds performed 1a– againstc as shown 3D7 in and Table 2W 1 [18,26 strains]. Derivatives of P. falciparum of trioxaferroquinesfor compounds (21a– –c as5 shown) were reported in Table 1by[ Bellot18,26 ].et al. Derivatives (Figure 3). ofThey trioxaferroquines were effective against (2–5) werechloroquine reported-resistant by Bellot strains et al. (Figure(FcB13 ). and They FcM29) were with e ff ICective50 values against between chloroquine-resistant 16–43 nM [18,27]. strains Compound (FcB1 2 andexhibited FcM29) significant with IC 50 antimalarial activity with IC50 values 17 and 29 nM against FcB1 and FcM29, respectively. In vivo values between 16–43 nM [18,27]. Compound 2 exhibited significant antimalarial activity with IC50 valuesstudies 17 on and P. 29vinkei nM pentteri against infected FcB1 and mice FcM29, at a daily respectively. oral dosage ofIn 10 vivo andstudies 25 mg/kg/d on P. over vinkei a periodpentteri

Molecules 2019, 24, 3604 4 of 27 Molecules 2019, 24, x 4 of 27 Molecules 2019, 24, x 4 of 27 of 30 days were evaluated. The mice administered 10 mg/kg/d exhibited parasitemia below detectable infectedof 30 days mice were at a e dailyvaluated oral. The dosage mice ofadministered 10 and 25 mg10 mg/kg/d/kg/d over exhibited a period parasitemia of 30 days below were detectable evaluated. levels with no recurrence. Mice administered 25 mg/kg/d exhibited parasitemia below detectable Thelevels mice with administered no recurrence. 10 mg Mice/kg/d administered exhibited parasitemia 25 mg/kg/d below exhibited detectable parasitemia levels with below no detectable recurrence. level between 1–18 days but displayed some recurrence between day 17 and 21. Trioxaferroquine Micelevel administered between 1–18 25 days mg/ kgbut/d displayed exhibited some parasitemia recurrence below between detectable day 17 level and between 21. Trioxaferr 1–18oquine days but was very effective in vivo at low dosages (10 mg/kg/d) when compared to the high dosage (25 displayedwas very some effective recurrence in vivo between at low day dosages 17 and (10 21. mg/kg/d) Trioxaferroquine when compared was very to e theffective high in dosage vivo at (25 low mg/kg/d). No significant curative effect with the absence of recurrence was not achieved at 25 dosagesmg/kg/d). (10 mg No/ kg significant/d) when curativecompared effect to the with high the dosage absence (25 of mg recurrence/kg/d). No was significant not achieved curative at e 25ffect mg/kg/d dosage [18,27]. withmg/kg/d the absence dosage of [18,27 recurrence]. was not achieved at 25 mg/kg/d dosage [18,27].

R R

HN X HN X

Fe Fe Cl N Cl N R = CH CH OH, 1a (X= H), 1b (X= CH ), 1c (X= CH CH ) R = CH2CH2OH, 1a (X= H), 1b (X= CH3), 1c (X= CH2CH3) R = CH2CH2OH, 1a (X= H), 1b (X= CH3), 1c (X= CH2CH3)

1a-c 1a-c Figure 2. Chemical structure of compound hydroxyferroquine derivatives (1a–c). FigureFigure 2. 2.Chemical Chemical structure structure ofof compoundcompound hydroxyferroquine derivatives derivatives (1a (1a–c–).c ).

O O O O QC N QC N QC N QC NH O O H O O O O O O Fe Fe Fe Fe NH 3 NH 2 3 2 CQ = CQ = Cl O O Cl O O QC QC QC NH QC NH O O QC NH O O O Fe O Fe N Fe 4 N Fe 4 O O O O 5 5 FigureFigure 3. 3.Chemical Chemical structuresstructures of trioxaferroquine derivatives derivatives (2 (–25–).5 ). Figure 3. Chemical structures of trioxaferroquine derivatives (2–5). SalasSalas et et al. al. synthesized synthesized ferroquine ferroquine derivativesderivatives (chloroquine-bridged(chloroquine-bridged ferrocenophane) ferrocenophane) with with two two Salas et al. synthesized ferroquine derivatives (chloroquine-bridged ferrocenophane) with two ferroceneferrocene rings rings bridging bridging the the terminal terminalnitrogen nitrogen atomsatoms of chloroquine ( (66––1010) )(Figure (Figure 44).). Comparison Comparison ferrocene rings bridging the terminal nitrogen atoms of chloroquine (6–10) (Figure 4). Comparison studiesstudies between between the the chloroquine-bridged chloroquine-bridged ferrocenyl ferrocenyl derivatives and and monosubstituted monosubstituted ferrocenyl ferrocenyl studies between the chloroquine-bridged ferrocenyl derivatives and monosubstituted ferrocenyl analoguesanalogues which which indicated indicated that that thethe monosubstitutedmonosubstituted ferrocenyl analogues analogues capacity capacity to toretain retain their their analogues which indicated that the monosubstituted ferrocenyl analogues capacity to retain their antimalarialantimalarial activity activity against against the the drug-resistant drug-resistant strainsstrains was significant. signiﬁcant. Their Their enhanced enhanced antimalarial antimalarial antimalarial activity against the drug-resistant strains was significant. Their enhanced antimalarial activityactivity was was attributed attributed to the to presence the presence of an of intramolecular an intramolecular hydrogen hydrogen bonding. bondi However,ng. However, the structure the activity was attributed to the presence of an intramolecular hydrogen bonding. However, the andstructure a balance and between a balance the between lipophilicity the lipophilicity and hydrophilicity and hydrophilicity of the bridged of the compounds bridged compounds also contributed also structure and a balance between the lipophilicity and hydrophilicity of the bridged compounds also contributed to their unique structural capability to escape the mechanisms of resistance [18,28]. to theircontributed unique to structural their unique capability structural to escapecapability the to mechanisms escape the mechanisms of resistance of [ 18resistance,28]. [18,28].

1 R1 R1 QC ( ) Fe QC ( )n N Fe ( )n N Fe R R R R

1 1 1 6: n =0 , R1 = H 7: n = 1, R=R1 = H 8: n = 2, R=R1 = H 6: n =0 , R = H 7: n = 1, R=R = H 8: n = 2, R=R1 = H 1 9: n =0 , R1 = CH 1 9: n =0 , R1 = CH3 10: n= 1, R= CH3, R1= H 9: n =0 , R = CH3 10: n= 1, R= CH3, R = H 3

Figure 4. Hybrid compounds 6–10. Molecules 2019, 24, 3604 5 of 27

Biot et al. also reported a ferrocene-triazacyclononane quinolone conjugate (11) (Figure5) with potent antiplasmodial activity against chloroquine strain Dd2 of P. falciparum in vitro [20,29]. Dormale et al. synthesized a ferrocene derivative from ferrocene and 4-aminoquinolines. In vitro, the compound prepared by modification with tartaric acid was the most potent compound. Its antimalarial activity was significant at low concentrations when compared to chloroquine against chloroquine-susceptible strain SGE2 and the chloroquine-resistant strains FCM6 and FCM17. The compound was able to inhibit the resistance of parasites. The compound’s capability to inhibit the efflux mechanism which is common with chloroquine may have contributed to its enhanced antimalarial activity when compared to chloroquine [19,20,30]. A class of quinoline–ferrocene hybrids were reported by N’Da et al. Hybrids with flexible linkers were effective when compared to compounds with rigid linkers which were ineffective against D10 and Dd2 strains of P. falciparum. The compound with the 3-aminopropyl methylamine linker (12) exhibited good antiplasmodial activity (19-fold) in vitro when compared to chloroquine with IC50 values of 0.008 versus 0.148 µM against the Dd2 strain of P. falciparum [20,31].

Table 1. In vitro activities of compound 1a–c against P. falciparum strains (3D7 and W2).

IC50 Values (nM) Compound 3D7 W2 1a 15.4 133.2 1b 21.5 30 1c 11.7 20.4 Ferroquine 7.8 9.7 Chloroquine 10.6 138.9

Biot et al. reported chimeras of ferroquine and thiosemicarbazones (13). In vitro studies on strains of P. falciparum and parasitic cysteine proteas, falciparum-2 revealed that the antimalarial activity of the compounds was significant because of the presence of aminoquinoline which made the compound easily conveyed to the digestive vacuole of the parasite [20,32]. David et al. synthesized quinolone ferrocene ester and evaluated it against chloroquine strains of P. falciparum (Dd2 and D10). The esters prepared from ferrocenylformic acid exhibited antimalarial activity against Dd2 and D10 strains of the P. falciparum. However, their antiplasmodial activity against D10 strain was not high when compared to the chloroquine-sensitive Dd2 strain. The ester with a butyl branch linked to the ferrocene moiety was very effective against all the strains with an IC50 of 0.13 µM on the resistant strain and 2.5-fold higher activity when compared to chloroquine with an IC50 of 0.34 µM. The compounds were highly selective towards P. falciparum [15,33]. Herrmann et al. prepared two derivatives of ferrocene hybrids containing 1,2,3,5-(diisopropylidene)- α-d-glycofuranose moiety combined with chloroquine derivative (14) with good antimalarial activity when tested against two strains of P. falciparum (Dd2 and K1) (Figure5) [ 20,34]. The second class of Herrmann et al. hybrids was prepared by conjugation of ferrocene scaffolds with 7-chloroquinoline via an ether linker followed by the attachment of diisopropylidene protected 6-amino-6-deoxyglucofuranose or 6-amino-deoxygalactopyronose using reductive amination resulting in the isolation of compound 15a and 15b. Both compounds exhibited good antimalarial activity with an IC50 value of 0.77 µM resulting from the presence of the carbohydrate moiety. The resistance indices for the compounds were less than 1, indicating a high activity against the Dd2 strain when compared to the D10 strain [20,35]. Chavain et al. conjugated ferroquine analogues with glutathione reductase inhibitor via a cleavable amide bond for targeting different pathways of the malaria parasite. The analogues antimalarial activity in vitro against NF54 (sensitive) and K1 (resistant) strains of P. falciparum was not significant, suggesting that their mode of action differs from ferroquine and chloroquine modes of action. Their poor antimalarial activity is also attributed to the cleavage of the amide bond and the side chain when metabolized in the digestive vacuole of the parasite. The aforementioned findings reveal how the design of molecules can influence their antimalarial activity [20,36]. Molecules 2019, 24, x 6 of 27 Molecules 2019, 24, x 6 of 27

Moleculesside chain2019 when, 24, 3604 metabolized in the digestive vacuole of the parasite. The aforementioned findings6 of 27 side chain when metabolized in the digestive vacuole of the parasite. The aforementioned findings reveal how the design of molecules can influence their antimalarial activity [20,36]. reveal how the design of molecules can influence their antimalarial activity [20,36].

Q S Q S N CQ N CQ H N CQ HN CQ H N Z H N NN Z N NH N H N Fe H H N Fe N FFee Q Fe 1313 Q 1111 Fe 1122

N* HN* H CQCQ CCQQ Y X Y CCQQ ==

FeFe FFee CCl l * * 15 QQ = = 1414 15 CCl l

Figure 5. Chemical structures of quinolone-ferrocene hybrids (11–15). 13-Z = CH3, 14-Y = 1,2,3,5- FigureFigure 5. 5. ChemicalChemical structures structures of quinolone of quinolone-ferrocene-ferrocene hybrids hybrids (11–15 ()11. 13–15-Z). = 13 CH-Z3, =14-CHY =3 , 1,2,3,514-Y- diisopropylidene glucofuranose moiety, =diisopropylidene1,2,3,5-diisopropylidene glucofuranose glucofuranose moiety, moiety, 15a-X = diisopropylidene-protected 6-amino- 15a-X = diisopropylidene-protected 6-amino-deoxyglucofuranose deoxyglucofuranose,15a-X = diisopropylidene15b-X-protected= 6-amino-6-deoxygalactopyranose. 6-amino-deoxyglucofuranose 15b-X = 6-amino-6-deoxygalactopyranose. 15b-X = 6-amino-6-deoxygalactopyranose. 3.2. Artemisinin-Ferrocene Derivative 3.2. Artemisinin-Ferrocene Derivative 3.2. ArtemisininArtemisinin-based-Ferrocene derivatives Derivative16a –d (artemisinin, artesunate, artemether, and dihydrortemisinin, Artemisinin-based derivatives 16a–d (artemisinin, artesunate, artemether, and respectively)Artemisinin are- characterizedbased derivatives by fast action16a–d and (artemisinin, a short half-life artesunate, (Figure6). Artemisinin-based artemether, and dihydrortemisinin, respectively) are characterized by fast action and a short half-life (Figure 6). combination therapies (ACT) are promising therapeutics eﬀective in controlling P. falciparum drug dihydrortemisinin,Artemisinin-based respectively)combination therapies are characterized (ACT) are bypromising fast action therapeutics and a s horteffective half in-life controlling (Figure 6). resistance. Synthesizing hybrid drugs containing artemisinin derivatives and other eﬀective drugs such ArtemisininP. falciparum-based drug combinationresistance. Synthesizing therapies (ACT)hybrid aredrugs promising containing therapeutics artemisinin effectivederivatives in and controlling other as ferroquine and chloroquine is a good approach to develop potent antimalarials that can overcome P. effectivefalciparum drugs drug resistance. such as ferro Synthesizingquine and hybrid chloroquine drugs containing is a good artemisinin approach derivatives to develop and potent other drug resistance. Ferroquine combined with artesunate has been reported to be a promising antimalarial effectiveantimalarials drugs that such can as overcome ferroquine drug and resistance. chloroquine Ferroquine is a combined good approach with artesunate to develop has been potent therapeuticantimalarialsreported to which be that a promising was can safe overcome at antimalarial all doses drug tested resistance.therapeutic in vivo Ferroquinewhich [37,38 was]. safe combined at all doses with tested artesunate in vivo [37,38has been]. reported to be a promising antimalarial therapeutic which was safe at all doses tested in vivo [37,38]. CH3 CH3 CH H H H 3 CH3 CH CH H H 3 H 3 CH3 CH O O H H 3 O O O O O O OO O O O O H O H O O OO H O O H O OH H H O H O H O O O H H O H O OH H H O O O O H O H OH O O OH O O O 16c 16OdH 1O6a 16b O 16c 16d 16a 16b FigureFigure 6. 6.Artemisinin Artemisinin and its derivatives derivatives..

ReiterReiter et et al. al. reported reported an artemisinin Figure 6. Artemisinin derivative derivative andcontaining containing its derivatives ferrocene ferrocene. moiety moiety and andegonol egonol (17). In (17 ). In vitro studiestudiess indicated indicated that that the the derivative derivative 17 was17 thewas only the one only exhibiting one exhibiting antimalarial antimalarial activity among activity Reiter et al. reported an artemisinin derivative containing ferrocene moiety and egonol (17). In amongthe seven the sevenartemisinin artemisinin–ferrocenyl–ferrocenyl derivatives derivatives studied while studied others while exhibited others anticancer exhibited activity. anticancer Its vitro studies indicated that the derivative 17 was the only one exhibiting antimalarial activity among activity.antimalarial Its antimalarial activity was activity enhanced was when enhanced compared when to comparedthe parent todrug, the egonol parent, with drug, an egonol, inhibition with the seven artemisinin–ferrocenyl derivatives studied while others exhibited anticancer activity. Its anva inhibitionlue of 88 valuenM (Figure of 88 7) nM [37,39 (Figure]. Reiter7)[ et37 al.,39 ].also Reiter reported et al.a second also reported generation a secondof 1,2,4- generationtrioxane- antimalarial activity was enhanced when compared to the parent drug, egonol, with an inhibition of 1,2,4-trioxane-ferroceneferrocene derivatives (18–22 derivatives), and evaluated (18–22 their), and antiplasmodial evaluated their activity antiplasmodial against the 3D7 activity strain against of P. value of 88 nM (Figure 7) [37,39]. Reiter et al. also reported a second generation of 1,2,4-trioxane- thefalciparum 3D7 strain using of chloroquineP. falciparum andusing dihydroartemisinin chloroquine and as controls dihydroartemisinin (Figure 8). These as trioxane controls–ferrocene (Figure8 ). ferrocenecompound derivatives antiplasmodial (18–22 ),activities and evaluated were significant their antiplasmodial with IC50 values activity in the against range the of 3D77.2– 30strain.2 nM. of P. These trioxane–ferrocene compound antiplasmodial activities were signiﬁcant with IC50 values in falciparumCompound using 20 chloroquineIC50 value was and 7.2 dihydroartemisinin nM and promising when as controls compared (Figure to ch 8).loroquine. These trioxane Compound–ferrocene 18 the range of 7.2–30.2 nM. Compound 20 IC50 value was 7.2 nM and promising when compared to compoundand 19 IC 50antiplasmodial values were 8.6 activities and 13.4 nM,were respectively significant [37]. with IC50 values in the range of 7.2–30.2 nM. chloroquine. Compound 18 and 19 IC50 values were 8.6 and 13.4 nM, respectively [37]. Compound 20 IC50 value was 7.2 nM and promising when compared to chloroquine. Compound 18 and 19 IC50 values were 8.6 and 13.4 nM, respectively [37].

Molecules 2019, 24, 3604 7 of 27 Molecules 2019,, 24,, xx 7 of 27

O O O O O

O O Eg = O

O O O 17 17 Fe Fe Eg O O FigureFigure 7.7. HybridHybrid compound 1717.. .

H H O O O O O O O Fc O O Fc Fc = Fe O O Fc = O O 19 18

O Fc H H H H O O O O O o o O H O O O H O O O O O O O O O O O O 20 21 20

O O O O H O O O O Fc O O O O O O 22

FigureFigure 8. 8.Chemical Chemical structures structures of of second second generationgeneration dihydroartemisinin ferrocene ferrocene derivatives derivatives ((18 (18–22–22)).. ).

Amino-artemisinin-ferroceneAmino--artemisinin--ferroceneferrocene 2323 inin whichwhich which ferroceneferrocene ferrocene waswas was incorporatedincorporated incorporated viavia viaaa piperazinepiperazine a piperazine linkerlinker linker toto todihydroarteminisinin dihydroarteminisinin derivatives derivatives was was testedtested tested in in in vitro vitro vitro against againstagainst chloroquine chloroquine chloroquine-sensitive--sensitive NF54 NF54 and and chloroquine-resistantchloroquineine--resistant K1 K1 and and W2 strains W2 strains of Plasmodium of Plasmodiumfalciparum falciparumfalciparum parasites parasites (Figure 9 ().Figure The compound 9).). The The compound exhibited good activity against the asexual parasites with IC5050 values of 2.79 and 3.2 nM exhibited good activity against the asexual parasites with IC50 values of 2.79 and 3.2 nM against K1 and against K1 and W2 strains of Plasmodium falciparum parasites, respectively. The resistance indices W2against strains K1 of andPlasmodium W2 strainsfalciparum of Plasmodium parasites, falciparum respectively. parasites, The respectively. resistance indices The resistance further indicatedindices furtherfurther indicatedindicated thethe compoundcompound reducedreduced capabilitycapability forfor cross resistance [40]. the compound reduced capability for cross resistance [40].

Molecules 2019, 24, 3604 8 of 27 Molecules 2019, 24, x 8 of 27 Molecules 2019, 24, x 8 of 27

H ToTlouleuneene OO H O OO N N Fc O O DDMMSSOO H Na(CH COO) BH N N Fc O H Na(CH33COO)2B2 H O O H H O O OO O (COCl) , rt, 2h THF, rt O O O (COC2l)2, rt, 2h THF, rt 2323 WW OO X X HH FFcc 1616 =NN WW= X==OH ; ; X OH ;; NN H H FigureFigure 9. 9. SynthesisSynthesis Synthesis of ofof amino amino–artemisinin–ferroceneamino–artemisinin––ferroceneferrocene compound compound (23 ( 23). ))..

Delhaes et al. synthesized ferrocene–artemisinin derivatives (24–27) from ferrocene derivatives Delhaes et al. synthesized ferrocene ferrocene–artemisinin–artemisinin derivatives ( 2244–27)) from ferrocene derivatives and dihydroartemisinin in good yield in the range of 60–80% (Figure 10). In vitro studies using HB3 and dihydroartemisinin in good yield in the range of 6 60–80%0–80% (Figure(Figure 1010).). InIn vitro studies using HB3 and SGE2 strains (sensitive) and Dd2 strain (resistant) of P. falciparum showed that compound 26 was and SGE2 SGE2 strains strains (sensitive) (sensitive) and and Dd2 Dd2 strain strain (resistant) (resistant) of P. of falciparumP. falciparum showedshowed that thatcompound compound 26 was26 wasthe the most most effective effective antimalarial. antimalarial. In In vitro vitro evaluationevaluation showed showed that that its its IC50 IC werewere similar similar to tothose those of of the most effective antimalarial. In vitro evaluation showed that its IC5050 were similar to those of artemisinin (HB3 = 12 nM, SGE2 = 11 nM, and 14 nM against Dd2) [41]. The compound’s capacity to artemisinin (HB3 (HB3 == 1212 nM, nM, SGE2 SGE2 == 1111 nM, nM, and and 14 14 nM nM against against Dd2) Dd2) [41]. [41 The]. The compound compound’s’s capacity capacity to tobind bind with with ferroprotoporphyrin ferroprotoporphyrin IX IX was was significant. significant. These These effect effect ofof adducts adducts between between artemisinin artemisinin bindderivatives with ferroprotoporphyrin on heme in the generation IX was of artemisinin significant. radicals These effectwas significant. of adducts between artemisinin derivatives on heme in the generation of artemisinin radicals was significant.significant.

O O O O O O O O O O O O O Fc H H O O O N H Fc Fc H O H O H H O H N Fc O O H Fc O H O O 26O Fc 24 25 O 26 24 25

O H O O O O O O O O O O 27 Fc O 27 Figure 10. Chemical structures of dihydroartemisininFc ferrocene derivatives (24–27). 3.3. FerroceneFigure–Novobiocin 10. ChemicalChemical Derivatives structures structures of dihydroartemisinin ferrocene derivatives ( 2424–27).).

3.3. Ferrocene Ferrocene–NovobiocinNovobiocin–Novobiocin is an antibiotic Derivatives that is produced by bacteria called Streptomyces, its inhibitor of the chaperone, heat shock protein 90 (Hsp90) which is responsible for the stabilization of proteins and it Novobiocin is an antibiotic that is produced by bacteria called Streptomyces, its inhibitor of the is foundNovobiocin to be one is anof theantibiotic antibiotics that that is producedare biological by activebacteria with called anticancer Streptomyces, and antimalarial its inhibitor activity of the chaperone,when it is heatheat incorporated shockshock proteinprotein together 9090 (Hsp90) (Hsp90)with other which which active is is responsiblemolecules responsible such for for theas the ferrocene stabilization stabilization [42–44 of of]. proteins proteins and and it isit isfound found toNovobiocin to be be one one of of derivativesthe the antibiotics antibiotics containing that that are are ferrocene biological biological moieties active active withwith were anticancer anticancer synthesized andand and antimalarialantimalarial documented activity by whenMbaba it is et incorporated al. and they togetherwere tested with against other the active 3D7 molecules strain (sensitive) such asas of ferroceneferrocene P. falciparum [[4242–– 4444in]. ].vitro (Figure 11).Novobiocin Compounds derivatives 28a and 28b containing exhibited ferrocene moderate moieties antimalarial were activity synthesized when comparedand documendocumented to otherted by Mbabasynthesized et al.al. andand compounds theythey werewere which testedtested exhibited against againstthe poorthe 3D7 3D7 antimalarial strain strain (sensitive) (sensitive) activity of when ofP. falciparumP. testedfalciparum againstin vitroin vitrothe(Figure 3D7 (Figure P. 11 ). 11).Compoundsfalciparum Compounds strain28a and 28awith28b and anexhibited inhibition 28b exhibited moderate value of moderate 9 antimalarial nM in vitro antimalarial [43]. activity The when incorporation activity compared when of toferrocenyl compared other synthesized moiety to other synthesizedcompoundson the benzamide compounds which exhibited side which of novobiocin poor exhibited antimalarial resulted poor antimalarial activityin compounds when activity tested with when enhanced against tested the biological against 3D7 P. the activity. falciparum 3D7 P. falciparumstrainFurthermore, with strain an inhibitionthe with cell an viability inhibition value ofstudies 9value nM indicatedin of vitro9 nM[ 43inthat ].vitro Thethe [43]. incorporationinhibition The incorporation growth of ferrocenylof P. of falciparum ferrocenyl moiety by moiety onthe the benzamide side of novobiocin resulted in compounds with enhanced biological activity. Furthermore, on the benzamide side of novobiocin resulted in compounds with enhanced biological activity. Furthermore, the cell viability studies indicated that the inhibition growth of P. falciparum by the

Molecules 2019, 24, 3604 9 of 27

theMolecules cell viability 2019, 24, studies x indicated that the inhibition growth of P. falciparum by the compound9 did of 27 not exhibit toxic effects, which was significant with HeLa cell viability which was greater than 70%. It also indicatedcompound that did the compoundsnot exhibit toxic were effect highlys, which selective was towards significant the with parasitic HeLa cells cell whenviability compared which was to the humangreater cells than [43 70%.]. It also indicated that the compounds were highly selective towards the parasitic cellsMbaba when et compared al. reported to the another human seriescells [43]. of ferrocene–novobiocin derivatives and evaluated them for antiplasmodialMbaba et al. reported activity andanother human series cytotoxicity of ferrocene– usingnovobiocin the 3D7 derivatives strain of andP. falciparum evaluated themand HeLa for cells,antiplasmodial respectively, activityin vitro and. Compound human cytotoxicity29a–c which using contain the 3D7 thestrainN-methyl of P. falciparum group inand the He piperidineLa cells, respectively, in vitro. Compound 29a–c which contain the N-methyl group in the piperidine ring ring exhibited better antimalarial activity with IC50 values that were below 10 µM but compound 29c exhibited better antimalarial activity with IC50 values that were below 10 μM but compound 29c was was the most potent compound with an IC50 value of 0.889 µM[44]. It was reported that these three the most potent compound with an IC50 value of 0.889 μM [44]. It was reported that these three compounds were selective towards the P. falciparum parasite and their HeLa cell viability was below compounds were selective towards the P. falciparum parasite and their HeLa cell viability was below 25% with no toxic effects [44]. Novobiocin and other derivatives from this series of compounds were 25% with no toxic effects [44]. Novobiocin and other derivatives from this series of compounds were inactive with HeLa cell viability that was greater than 75%, which means N-methyl substituents play inactive with HeLa cell viability that was greater than 75%, which means N-methyl substituents play anan important important role role in in the the antiplasmodial antiplasmodial activity activity ofof thesethese compounds and and also also these these compounds compounds were were foundfound to to be be independent independent of of Hsp90 Hsp90 [ 44[44].].

H R2 N Fc

O R3 O O O 1 28a-b R

1 2 3 1 2 3 28a: R = H; R = Bu; R = H 28b: R = H; R = OMe; R = H

H N Fc R2 R3 O O O O 1 29a-c R

1 2 3 1 2 3 1 2 3 29a: R = H; R = NMe; R = CH2 29b: R = H; R = CH2; R = NMe 29c:R = Me; R = NMe; R = CH2

FigureFigure 11. 11.Ferrocenyl–novobiocin Ferrocenyl–novobiocin derivatives ( (28a28a––bb andand 29a29a––c)c.).

3.4.3.4. Ferrocene- Ferrocene Pyrrole- Pyrrole Derivatives Derivatives GuillonGuillon et et al. al. reported reported the the synthesis synthesis of of ferrocenicferrocenic pyrrolo[1-2-a]quinoxalinepyrrolo[1-2-a]quinoxaline derivatives derivatives from from nitroanilinesnitroanilines using using di differentﬀerent strategies strategies (30a (30a––bb andand 31a31a––bb)) (Figure (Figure 12 12).). Compounds Compounds 30a30a–b– bwerewere synthesizedsynthesized via via regio-selective regio-selective palladium palladium catalyzed catalyzed monoamination and and compo compoundund 31a31a–b– bwaswas preparedprepared by by reductive reductive amination amination strategy. strategy. TheseThese derivativesderivatives ( (30a30a––bb andand 31a31a––bb) )were were tested tested against against FcB1FcB1 and and PFB PFB (resistant) (resistant) and and F32 (sensitive)F32 (sensitive) strains strains of P. of falciparum P. falciparumin vitro in [vitro45]. The[45]. antimalarial The antimalarial activity of theactivity compounds of the was compounds dependent was on the dependent nature of on the the substituent nature on of the the bis(3-aminopropyl)piperazine substituent on the bis(3- aminopropyl)piperazine linker. The pyrrolo[1,2-a]quinoxalines with nitro substitutions were the linker. The pyrrolo[1,2-a]quinoxalines with nitro substitutions were the most active compounds against most active compounds against the CQ-sensible F32 strain (IC50 0.038–0.085 mM). The presence of a the CQ-sensible F32 strain (IC 0.038–0.085 mM). The presence of a 4-methoxy substituent on the 4-methoxy substituent on the50 benzyl terminal group also enhanced the antimalarial activity against benzyl terminal group also enhanced the antimalarial activity against F32 strain with IC50 0.045 mM. F32 strain with IC50 0.045 mM. The compound with a 5-nitro-2-hydroxybenzyl substituent on the The compound with a 5-nitro-2-hydroxybenzyl substituent on the bis(3-aminopropyl)piperazine chain bis(3-aminopropyl)piperazine chain exhibited the most potent antimalarial activity with IC50 of 0.048 exhibited the most potent antimalarial activity with IC of 0.048 and 0.060 mM, respectively, against and 0.060 mM, respectively, against CQ-resistant FcB150 and K1 strains. Compounds with NO2 CQ-resistantsubstituents FcB1 inhibited and K1 β- strains.hematin Compoundsformation significantly with NO2 whensubstituents compared inhibited to chloroquineβ-hematin [45]. formation signiﬁcantly when compared to chloroquine [45].

Molecules 2019, 24, x 10 of 27 Molecules 2019, 24, 3604 10 of 27 Molecules 2019, 24, x 10 of 27 N

Fc R1 NN N N H N N Fc Fc R1 N N N H 31a-b N N Fc 1 1 31a: R = H, 31b: R = CH3O 31a-b Figure 12. Structure of synthesized ferrocenyl pyrrolo[1,21 -a]quinoxaline1 derivatives (30a–b and 31a– 31a: R = H, 31b: R = CH3O b). Figure 12. Structure ofof synthesizedsynthesized ferrocenylferrocenyl pyrrolo[1,2-a]quinoxaline pyrrolo[1,2-a]quinoxaline derivatives derivatives (30a (30a–b–andb and31a 31a–b).– 3.5. Isatinb). –Ferrocene Conjugates 3.5. Isatin–Ferrocene Conjugates Isatin is a heterocyclic scaffold that is isolated from the leaves and roots of Couroupita guianesis 3.5. Isatin–Ferrocene Conjugates and CalantheIsatin is adiscolour, heterocyclic Isatis sca tinctoriaffold that and is it isolated is found from to be the versatile leaves and in organic roots of synthesisCouroupita because guianesis ofand the CalanthemanyIsatin possibilities discolour, is a heterocyclic Isatis of modification tinctoria scaffoldand itatthat is C found -is3, isolatedC-5 to, and be versatilefrom N-1 thewith inleaves organicvarious and synthesisactivities roots of becauseCouroupitasuch as ofanticancer, theguianesis many possibilitiesandantimalarial, Calanthe of discolour,and modification antifungal Isatis attinctoria [46 C-3,–48 C-5, ].and Kumar and it is N-1 found et with al. to synthesizedvarious be versatile activities several in organic such isatin as synthesis anticancer,–ferrocene because antimalarial, conjugates of the manyandtethered antifungal possibilities with 1H [46-1,2,3 –of48 ].modification-triazole Kumar derivatives et al. at synthesizedC-3, (C32a-5,– andh) severalvia N -copper1 with isatin–ferrocene- promotedvarious activities azide conjugates–alkyne such as tetheredcycloaddition anticancer, with 1antimalarial,reactionH-1,2,3-triazole and testedand derivativesantifungal them against ([4632a– –chloroquine48h)]. via Kumar copper-promoted - etresistant al. synthesized strains azide–alkyne (3D7 several and W2) isatin cycloaddition of–ferrocene P. falciparum reaction conjugates (Figure and tetheredtested13) [47]. them Thewith presence against 1H-1,2,3 chloroquine-resistant of-triazole a halogen derivatives substitue (32ant strains on–h the) via (3D7 C5 copper position and-promoted W2) of the of P. isatin azide falciparum ring–alkyne and(Figure a cycloaddition propyl 13 )[linker47]. Thereactioninfluence presence and the tested ofantimalarial a halogen them againstsubstituent activity chloroquine of the on thecompounds C5-resistant position [47]. strains of the isatin (3D7 ringand andW2) a of propyl P. falciparum linker influence (Figure 13the) [47]. antimalarialCompound The presence activity32a– dof with a of halogen the n = compounds 2 (ethyl) substitue as a [nt47 linker ].on the did C5 not position exhibit of antimalarial the isatin ring activity and a even propyl at higherlinker influenceconcentrations.Compound the antimalarial With32a–d compoundwith activityn = 2 32e (ethyl)of –theh wcompounds asith a linkern = 3 (propyl) did [47]. not exhibit as a linker antimalarial, antimalarial activity activity even at showed higher concentrations.improvementCompound with With32a IC–d compound50 with values n = 2ranging (ethyl)32e–h withbetweenas a linkern = 33.76 did (propyl) and not exhibit16.20 as a linker,μM antimalarial indicating antimalarial activity that activitythe even chain at showed higherlength concentrations.improvementhas an effect on with With the IC activity 50compoundvalues of rangingthese 32e –compounds.h between with n = 3.76 3 When (propyl) and 16.20compared as µaM linker indicating with, antimalarial ferroquine that the (2.activity chain1–13 length.4 showed nM), has in improvementanvitro eff ectantimalarial on the with activity activityIC50 ofvalues these of ranging compound compounds. between 32e When–h 3.76 was compared and lower. 16.20 with FromμM ferroquine indicating the series (2.1–13.4that of the isatin nM),chain–ferrocenein length vitro haantimalarialderivatives,s an effect only activityon the compounds activity of compound of (these32f–32eh compounds.) with–h was the lower. electron When From comparedwithdrawing the series with of groups isatin–ferroceneferroquine and propyl (2.1–13 derivatives, as.4 nM),a chain in vonlylinkeritro compoundsantimalarial displayed good (32f activity–h antimalarial) with of the compound electron activity withdrawing 32ewith–h IC was50 values groupslower. of 3.76 and From and propyl the 4.58 asseries μM a chain against of isatinlinker chloroquine– displayedferrocene- derivatives,goodresistant antimalarial strains only compounds (3D7 activity and with W2) (32f IC 50– ofh values)P. with falciparum the of 3.76 electron. and Further 4.58 withdrawingµ evaluationM against groups chloroquine-resistant of compounds and propyl (32as fa– strains hchain) for (3D7linkercytotoxicity and displayed W2) against of P.good falciparum HeLa antimalarial mammalian. Further activity evaluation cell withs revealed IC of50 compounds values their of non 3.76 (32-cytotoxic andf–h) 4.58 for cytotoxicity effectμM against and their againstchloroquine selective HeLa- resistantmammalianinhibition strains of cellsP. falciparum revealed(3D7 and [47]. their W2) non-cytotoxic of P. falciparum effect. and Further their evaluation selective inhibition of compounds of P. falciparum (32f–h)[ 47 for]. cytotoxicity against HeLa mammalian cells revealed their non-cytotoxic effect and their selective inhibition of P. falciparum [47]. O O Fc O N O Fc ( ) N N n N N32a-h R ( ) N N n N 32a: R= H; 32b: R= F; 32ac-:h R= CH3; 32d: R= Cl n = 2 32e: R= H; R32f: R= F; 32g: R= CH ; 32h: R= Cl n = 3 3 32a: R= H; 32b: R= F; 32c: R= CH3; 32d: R= Cl n = 2 Figure 3 13.2e Chemical: R= H; 3 structure2f: R= F of; 3 isatin–ferrocene-basedi2sating: R–=ferrocene CH ; 3-2basedh: R= derivativederivative Cl n = 3 ((32a32a––hh).). 3 3.6. Ferrocene–Pyrimidine Conjugates 3.6. Ferrocene–PyrimidineFigure 13. Conjugates Chemical structure of isatin–ferrocene-based derivative (32a–h). Recently, researchersresearchers reportedreported thatthat pyrimidinepyrimidine moietymoiety linkedlinked withwith ferroceneferrocene resultedresulted in hybridhybrid 3.6. Ferrocene–Pyrimidine Conjugates compoundscompounds with good antiplasmodial activity against chloroquine strains of P. falciparumfalciparum.. In vitrovitro antiplasmodialRecently, researchers analysis analysis showed showedreported that thatthat compounds pyrimidine compounds33a– moiety f 33awere–f activelinkedwere against with activ eferrocene the against chloroquine-susceptible resultedthe chloroquine in hybrid- compoundsNF54susceptible strain NF54 (Figurewith straingood 14). antiplasmodial( TheFigure pyrimidine 14). The activity pyrimidine ring substituition against ring chloroquine substituition of methyl esterstrains of methyl group of P. ester at falciparum C-5 group of compound. atIn C vitro-5 of antiplasmodial33acompoundwith an 33a ethyl with analysis or Iso-propyl an ethyl showed or group Iso that-propyl to compounds yield group compound to33a yield–f 33bwere compoundand activ33ce 33bincreased against and 33cthe the increasedchloroquine lipophilicity the- susceptibleoflipophilicity the compounds. NF54 of the strain compounds. Increasing (Figure 14 theIncr). The lipophilicityeasing pyrimidine the lipophilicity enhanced ring substituition theenhanced antiplasmodial of the methyl antiplasmodial ester activity group of activitythe at C drug.-5 of compoundForthe drug. compound For 33a compound 33cwith–e , an the ethyl33c antiplasmodial–e , or the Iso antiplasmodial-propyl activity group decreased activity to yield decreased in compound the order in the 33b33c order >and33d 33c33c> > 33e increased33ddue > 33e to due the decrease in lipophilicity. Compounds substituted with different functional groups (R and P) at lipophilicityto the decrease of thein lipophilicity. compounds. CompoundsIncreasing the substituted lipophilicity with enhanced different the functional antiplasmodial groups1 activity(R1 and ofP) positionthe drug. C-4 For and compound C-5 of pyrimidine 33c–e, the antiplasmodial exhibited good antimalarialactivity decreased activity in withthe order the lipophilicity 33c > 33d > 33e playing due to the decrease in lipophilicity. Compounds substituted with different functional groups (R1 and P)

Molecules 2019, 24, x 11 of 27 MoleculesMolecules2019 2019, 24, 24, 3604, x 11 of11 27 of 27 at position C-4 and C-5 of pyrimidine exhibited good antimalarial activity with the lipophilicity playingat position a major C-4 role, and and C-5 the of pyrimidinereversible oxidation exhibited goodbehavior antimalarial of these hybrid activity compounds with the lipophilicity was similar ato major playingthat of role, ferrocenea major and therole, [49]. reversible and the reversible oxidation oxidation behavior behavior of these hybridof these compounds hybrid compounds was similar was similar to that of ferroceneto that of [49 ferrocene]. [49]. * W(33a-c) = * P W(33a-c) = P N O2N * * 1 N * R O N O2N * 1 N W(33d) = W(33e) = R O N O S W(33d) = W(33e) = N N O N N S W N Fc W N N Fc NO 33a-f H W(33f) = CH3 2 N N NO 33a-f H W(33f) = CH3 2

1 1 1 a (R =1 CH3OO, P= C6H5); b (R 1= C2H5OO, P= C6H5); c (R1 = i-C3H7OO, P= C6H5); a (R = CH3OO, P= C6H5); b (R = C2H5OO, P= C6H5); c (R = i-C3H7OO, P= C6H5); 1 1 1 d (R =1 i-C3H7OO, P = O-C6H4NO2); e (R 1= i-C3H7OO, P = p-C6H4NO2); f (R1 = C2H5OO, P = CH3) d (R = i-C3H7OO, P = O-C6H4NO2); e (R = i-C3H7OO, P = p-C6H4NO2); f (R = C2H5OO, P = CH3) FigureFigure 14. 14. SSchematic chematicSchematic route route showing showing the the synthesis ofof ferroceneferrocene ferrocene-pyrimidine-pyrimidine-pyrimidine conjugates conjugates (33a (33a–f)–. f).).

3.7.3.7. Ferrocenyl Ferrocenyl Chalcones Chalcones ChalconesChalcones are are prepared prepared from from anan aromaticaromatic ketone group group and and they they exhibit exhibit exhibit several several biological biological activitiesactivities such such as as antimalarial antimalarial and and antitumor antitumor activity.activity. Licochalcone Licochalcone from from the the licorice licorice root root in inChina China has hash as beenbeen reported reported to to be be aa a promisingpromising promising antimalarial antimalarialantimalarial agent.agent. Synthesis Synthesis of of ferrocenyl ferrocenyl chalcones chalcones was was obtained obtained byby replacing replacing the the aromatic aromatic aromatic ring ring ring with with with a a a ferrocenyl ferrocenyl ferrocenyl moiety moiety moiety [[50[5050––5353].].]. Researchers Researchers Researchers synthesized synthesized synthesized several several several ferrocenylferrocenylferrocenyl chalcone chalcone chalcone derivatives derivatives derivatives by by by base-catalyzed base base--catalyzed Claisen–Schmidt Claisen Claisen––SchmidtSchmidt condensation. condensation. condensation. However, However, However, only only only two compoundstwotwo compounds compounds (34 and(34 (34 and35 and) 35 were 35) )were were reported reported reported to beto be eﬀ effectiveective antimalarial antimalarial antimalarialagents, agents, agents, with with low low low IC IC IC5050 values50 valuesvalues of of 4.54.5 µµMM µM (A)(A) (A) andand and 5.15.1 5.1µ µMM µM (B) (B) (B) against against against chloroquine-resistant chloroquinechloroquine-resistant strains strainsstrains of of P.of P. falciparumP. falciparum falciparumin in vitro in vitro vitro(Figure (Figure (Figure 15 15))[ 5215) ]. The[52].[52]. positionThe The position position of the of of substitution the the substitution substitution of nitro ofof nitronitro and pyridineand pyridinepyridine rings rings rings inﬂuenced influenced influenced the the biologicalthe biological biological activity activity activity of of the of compounds.thethe compounds. compounds. Compound Compound Compound35 exhibited 35 35 exhibitedexhibited higher higher selective selective indices indicesindices when when when compared compared compared to compound to tocompound compound34 when 34 34 testedwhenwhen tested on tested KB on and on KB KB MDCK and and MDCK MDCK cells ofcells cellsP. falciparum ofof P.P. falciparum(SI = (SI(SI37 = and= 37 37 and SIand= SI SI14), = = 14), 14), respectively. respectively. respectively. For For For compound compound compound 34, 34, the selective indices were between 5 and 9 [52]. the34, the selective selective indices indices were were between between 5 and 5 and 9 [52 9]. [52].

O O O KOH, EtOH, rt Fc O O Y CH KOH, EtOH, rt Fc 3 + O Y Y CH Y 3 + H Fc 34 H Fc 34 O O KOH, EtOH, rt Z O Fc Z O H KOH, EtOH, rt H C Fc Z Fc 3 + H O H3C Fc + Z 35 O 35 Y = 3-Pyridinyl, Z= 4- nitrophenyl Y = 3-Pyridinyl, Z= 4- nitrophenyl Figure 15. Ferrocenyl chalcones derivatives (34–35). Figure 15. Ferrocenyl chalcones derivatives (3434–35).). Kumar et al. reported the synthesis of 1H-1,2,3-triazole tethered 4-aminoquinoline- ferrocenylchalconeKumar et al. derivatives reported the(compounds synthesis 37 , of38 , and1H-1,2,3-triazole- 1,2,339 with-triazole compound tethered 36 acting 44-aminoquinoline--aminoquinoline as a linker) - ferrocenylchalcone(Figure 16) and tested derivatives them for ( (compoundsantimalarialcompounds activity 3737,, 3838, , against and and 3939 P. with falciparum compound chloroquine 3636 acting-resistant as a (W2) linker) (Figure(Figurestrains 1616) in) andvitro tested. Different them strategies for antimalarial were used activity in the against synthesis P. falciparumof compounds chloroquine-resistantchloroquine 37, 38, and-resistant 39. They (W2) were prepared by Cu-promoted azide–alkyne cycloaddition for both compound 37 and 38. strains inin vitrovitro.. DiDifferentﬀerent strategies strategies were were used used in thein the synthesis synthesis of compounds of compounds37, 38 37,, and 38, 39and. They 39. They were Compound 39 was prepared by Huisgen’s azide–alkyne cycloaddition. These derivatives 37–39 were preparedwere prepared by Cu-promoted by Cu-promoted azide–alkyne azide cycloaddition–alkyne cycloaddition for both compound for both 37compoundand 38. Compound 37 and 3839. tested in vitro for antimalarial activity against the W2 (resistant) strain of P. falciparum and they wasCompound prepared 39 by was Huisgen’s prepared azide–alkyne by Huisgen’s cycloaddition. azide–alkyne cycloaddition. These derivatives These37– derivatives39 were tested 37–39in were vitro exhibited good antiplasmodial activity with inhibition values ranging between 0.37–5.08 µM. The fortested antimalarial in vitro for activity antimalarial against activity the W2 against (resistant) the strainW2 (resistant) of P. falciparum strain ofand P. they falciparum exhibited and good they antiplasmodialexhibited good activityantiplasmodial with inhibition activity values with inhibition ranging between values 0.37–5.08ranging betweenµM. The 0.3 alkyl7–5 chain.08 µM. length The inﬂuenced the antimalarial activity of the compounds [52]. In compounds 37a–f with a piperazine

Molecules 2019, 24, x 12 of 27 Molecules 2019, 24, 3604 12 of 27 alkyl chain length influenced the antimalarial activity of the compounds [52]. In compounds 37a–f with a piperazine ring, the length of the chain had no effect on compounds’ antimalarial activity. The ring, the length of the chain had no eﬀect on compounds’ antimalarial activity. The IC values of IC50 values of compounds 37a–f ranged between 2.55–5.08 µM. In compound 38, the piperazine50 ring wascompounds replaced37a by– f4ranged-aminophenol between and 2.55–5.08 the lengthµM. of In compoundthe chain played38, the no piperazine vital role ring in the was biological replaced activityby 4-aminophenol of the compounds. and the length However, of the (38e chain–38f) played exhibited no vital improved role in antimalarial the biological activity activity when of the compounds. However, (38e–38f) exhibited improved antimalarial activity when compared to compared to compounds 37a–f with inhibition values of 38e (n = 6, IC50 = 2.40 µM) and 38f (n = 8, IC50 =compounds 1.16 µM). In37a compound–f with inhibition 39, the chain values length of 38e displayed(n = 6, IC different50 = 2.40 µtrendsM) and for38f derivatives(n = 8, IC 5039a=–1.16i andµ theM). introductionIn compound 39 of, the amino chain length alcohols displayed (amino di-ethanolﬀerent trends and for amino derivatives-propanol)39a– i and substituents the introduction showed of improvementamino alcohols on (amino-ethanol the activity and of compounds. amino-propanol) Increasing substituents the alkyl showed chain improvement length (C on-2 the to Cactivity-5) in compoundsof compounds. 39a Increasing–f with amino the alkyl-ethanol chain as length a substituent (C-2 to C-5) decreased in compounds the activi39a–tyf with of the amino-ethanol compound. However,as a substituent increasing decreased the alkyl the activitychain length of the (C compound.-6 to C-7) improved However, their increasing activity the in alkylwhich chain compound length (C-6 to C-7) improved their activity in which compound 39a (n = 2, IC = 0.95 µM), compound 39a (n = 2, IC50 = 0.95 µM), compound 39d (n = 5, IC50 = 2.92 µM), compound50 39e (n = 6, IC50 = 0.66 µM) 39d (n = 5, IC = 2.92 µM), compound 39e (n = 6, IC = 0.66 µM) and 39f (n = 8, IC = 0.69 µM), and 39f (n = 8,50 IC50 = 0.69 µM), respectively. Compounds50 39g–j with amino-propanol50 substituents exhibitedrespectively. good Compounds antiplasmodial39g –activityj with amino-propanoland the chain length substituents linker did exhibited not increase good nor antiplasmodial decrease their antimalariaactivity and activity. the chain All length of these linker derivatives did not increase 37–39 exhibited nor decrease antimalarial their antimalaria activity activity. but 39g– Allj were of these the derivatives 37–39 exhibited antimalarial activity but 39g–j were the most active compounds with IC most active compounds with IC50 values ranging between 0.37–1.78 µM against W2 (resistant) strains50 valuesof P. falciparum, rangingbetween in vitro [52]. 0.37–1.78 µM against W2 (resistant) strains of P. falciparum, in vitro [52].

N O ( )n N N3 O N N ( )n Fc O N O 37 36 X Fc O HN N X N N O Fc ( ) HN n ( )m O N O 38 O X ( )n N N X = 39 Cl O

Fc Figure 16. ChemicalChemical structures structures of of OO--alkylalkyl azide azide ferrocenylchalcones ferrocenylchalcones ( (36));; piperazine piperazine ( (3737),), phenyl phenyl ( (3838),), amino alcohol (39) –linked–linked 7-chloroquinoline-ferrocenylchalcone7-chloroquinoline-ferrocenylchalcone derivatives.derivatives.

4. Ferrocene Ferrocene-Based-Based Compounds with Anticancer Activity. Activity Cancer is a diseasedisease characterized characterized by by uncontrolled uncontrolled cell cell division division [[54,54,5555].]. It It is is a chronic and complicated disease and it is the cause of thethe highhigh deathdeath raterate globallyglobally [[54,5554,55].]. The most fatalfatal and common cancers are lung, breast, colorectal, stomachstomach,, and liver cancer which are responsible for almost 92% of death cases [[5].5]. More More than than 200 200 anticancer drugs drugs are are available but less than 5% has reached the the market market and and the the drawbacks drawbacks are are caused caused by lack by lackof clinical of clinical trials trialson these on synthesized these synthesized drugs. Somedrugs. anticance Some anticancerr drugs enter drugs phase enter I and phase phase I and II phasebut fail II to but advance fail to through advance phase through III phaseof a clinical III of triala clinical [55]. trialDrug [55 resistance]. Drug resistance is common is commonwith most with anticancer most anticancer drugs and drugs this and has thiscontributed has contributed to a high to deatha high rate death among rate amongcancer cancerpatients. patients. Anticancer Anticancer drugs are drugs grouped are grouped according according to their tomode their of mode actions of [56actions–58].[ 56–58].

Molecules 2019, 24, 3604 13 of 27 Molecules 2019, 24, x 13 of 27

4.1.4.1. Ferrocene–Indole Ferrocene–Indole Hybrids Hybrids IndolesIndoles are are cheap cheap and and have have unique unique properties properties with with anticancer anticancer activity. activity. Ferrocene, Ferrocene, on on the the other other hand,hand, has has unique unique chemical chemical and and pharmacological pharmacological properties. properties. Combining Combining ferrocene ferrocene and and indoles indoles derivativesderivative intos into hybrid hybrid compounds compounds is is a potentiala potential route route suitable suitable for for the the development development of of eff effectiveective anticanceranticancer drugs drugs [55 [55–57–].57]. Quirante Quirante et et al. al. synthesized synthesized ferrocenyl–indole ferrocenyl–indole derivatives derivatives with with ferrocene ferrocene moietymoiety attached attached on on C-3 C- of3 of the the 2-phenylindole 2-phenylindole skeleton skeleton (40 (4–046–46) and) and tested teste ford for anticancer anticancer activity activity using using a A549a A549 human human lung lung carcinoma carcinoma cell cell line line [59 ].[59]. The The synthesized synthesized ferrocenyl–indole ferrocenyl–indole derivatives derivatives are ( 47are–53 (4)7– (Figure53) (Figure 17). Compounds 17). Compounds50–52 5exhibited0–52 exhibited potent potent cytotoxic cytotoxic effect againsteffect against the cancer the cancer cell lines cell with lines IC with50 valuesIC50 values of 5, 7, of and 5, 107, µandM, respectively.10 µM, respectively. Compound Compound50 displayed 50 displayed potent cytotoxic potent cytotoxic activity with activity an IC with50 valuean IC of50 5valueµM. of All 5 µM. the ferrocenyl–indoleAll the ferrocenyl– derivativesindole derivatives (47–53 )(4 were7–53) more were activemore active when when compared compared to theirto their parent parent drugs drugs (40– 46(40).–46 The). The nature nature of the of the substituents substituents (R1 )(R on1) on C-5 C of-5 theof the indole indole ring ring and and halogen halogen substituentssubstituents on on the the para-position para-position (R2 )(R of2) arylof aryl ring ring played played a significant a significant role onrole the on cytotoxic the cytotoxic effect effect of the of hybridthe hybrid molecules molecules [59]. [59]. TheThe order order of of potency potency for for R1 Ron1 on position position 5 was: 5 was: Un-substituted Un-substituted compounds compounds (R1 (R=1 H)= H)> 5-OMe> 5-OMe (50 (50) ) > 5-NO> 5-NO2 (251 (51) >) >5-Cl 5-Cl ( 52(52)) and and for for halogen halogen substituents substituents for for R R22,, thethe potencypotency was:was: p-Hp-H (47) > pp-Cl-Cl ( (4848)) >> p- p-FF ((4949),), respectively.respectively. CompoundCompound47 47was was two-fold two-fold more more active active than than compound compound49 49, compounds, compounds48 48, 49, 49, , andand51 51were were 2-fold 2-fold more more active active than than their their comparable comparable derivatives derivatives (41 (41, 48, 48, and, and44 44), derivative), derivative47 47and and 5353were were 3-fold 3-fold and and 4-fold 4-fold more more active active than than comparable comparable derivatives derivatives40 40and and46 46, respectively., respectively The. The most most activeactive ferrocenyl–indole ferrocenyl–indole compound compound was compound was compound51 and 51 it wasand 25-fold it was more 25-fold active more when active compared when tocompared the indole to compound the indole43 compound [59]. 43 [59].

Fc R1 R1

R2 R2

N Y N Y H H

40-46 47-53 Fc

Fc N N H H

54 55

40–53 (R1) = 41= 42= 43= 47= 48= 49= 50= 54= H, 44= 51= OCH3, 45= 52= NO2, 6= 13= Cl 40–53 (y) = 41= 42= 43= 44= 45= 46= 48= 49= 50= 51= 52= 53= CH, 47= 54= N 40–53 (R2) = 1= 4= 5= 6= 7= 48= 51= 52= 53= 54 = H, 2= 9= Cl, 3= 10= F.

FigureFigure 17. 17.Chemical Chemical structure structure of of indole indole (40 (–4460–)46 and) and ferrocene–indole ferrocene–indole derivatives derivatives (47 (–4557–).55).

Radulovic’Radulovic et’ et al. al. preparedprepared 2-(3-ferrocenylphenyl)-12-(3-ferrocenylphenyl)-1HH--indoleindole (54 (54) )and and 2- 2-(4-ferrocenylphenyl)-(4-ferrocenylphenyl)-1H- 1Hindole-indole (55 (55) )by by substituting substituting ferrocenyl ferrocenyl moiety moiety into a indole derivativederivative atat position position 3 3 and and 4, 4 respectively., respectively. InIn vivo vivostudies studies of the of compounds the compounds (54 and (5455 )and on rat 55 peritoneal) on rat peritoneal macrophages ma forcrophages cell viability for cell evaluation viability revealedevaluation that therevealed macrophage that the viability macrophage was reduced viability by wasmore reduced than 50% by using more low than concentrations 50% using of low theseconcentrations compounds. of The these cytotoxic compounds. eﬀect of The the cytotoxic compounds effect was of dose-dependent the compounds and was a dose high- concentrationdependent and ofa the high compounds concentration reduced of the the compounds viability of redu peritonealced the macrophages viability of peritoneal [60]. macrophages [60]. 4.2. 1,2,4-Trioxane–Ferrocene Hybrids 4.2. 1,2,4-Trioxane–Ferrocene Hybrids 1,2,4-trioxane ferrocene derivatives were reported by Reiter et al. as potential drugs for various 1,2,4-trioxane ferrocene derivatives were reported by Reiter et al. as potential drugs for various biological activities such as antimalarial, anticancer. A 1,2,4-trioxane-ferrocene hybrid, 23, was reported biological activities such as antimalarial, anticancer. A 1,2,4-trioxane-ferrocene hybrid, 23, was reported to be the most active compound from this class against leukemia cells with IC50 values of

Molecules 2019, 24, 3604 14 of 27

Molecules 2019, 24, x 14 of 27 to be the most active compound from this class against leukemia cells with IC50 values of 0.25 µM against0.25 µM CCRF-CEM against CCRF cells-CEM and 0.57cellsµ andM against 0.57 µM CEM against/ADR5000 CEM/ADR5000 cell lines, respectively. cell lines, respectively. Since compound Since 23compoundexhibited 23 good exhibited anticancer good activity,anticancer the activity, second the generation second generation of these compounds of these compounds from this classfrom (1,2,4-trioxane-ferrocene)this class (1,2,4-trioxane-ferrocene) were synthesized were synthesized (24–27). In (2 vitro4–27).studies In vitro against studies leukemia against leukemia cells revealed cells significantrevealed significant cytotoxic e ffect cytotoxic with an effect IC50 value with of an 0.13 ICµM50 whenvalue compared of 0.13 to µM dihydroartemisinin when compared with to andihydroartemisinin IC50 value of 0.48 withµM[ an39 IC,6150 ].value The of compounds 0.48 µM [39,61 with]. The two compounds trioxane moieties with two such trioxane as compound moieties 23such, 24 as, 25 compound, and 27 exhibited 23, 24, 25 cytotoxic, and 27 exhibited effects with cytotoxic IC50 of effect 0.25,s 0.13, with 0.07, IC50 andof 0.25, 0.08 0.13,µM, 0.07 respectively,, and 0.08 againstµM, respectively CCRF-CEM, against cells. CCRF The most-CEM potent cells. The compound most potent against compound CCRF-CEM against cells CCRF was-CEM compound cells was26 withcompound an IC50 26value with of an 0.01 IC50µ valueM. The of hybrids, 0.01 µM.24 The–27 ,hybrids, were more 24– active27, were when more compared active when to their compared parent drugsto their (artemisinin parent drugs and (artemisinin dihydroartemisinin) and dihydroartemisinin) with 3- to 50-fold morewith 3 cytotoxic- to 50-fold effects more towards cytotoxic multidrug effects resistanttowards multidrug cell lines. resistant The molecular cell lines. weight The andmolecular number weight of 1.2,4-trioxane and number moietiesof 1.2,4-trioxane present moieties in these compoundspresent in these played compounds an important played role an important in their cytotoxic role in their effects cytotoxic against effects CEM against/ADR5000 CEM/ADR5000 cell in vitro. Compoundcell in vitro. 25Compoundand 27 with 25 and two 27 moieties with two and moieties a molecular and a weight molecular of 800 weight g/mol of exhibited800 g/mol enhancedexhibited cytotoxicenhanced e cytotoxicffects when effect compareds when tocompared compound to compound23 and 24 with 23 and one 24 moiety with one and moiety a molecular and a molecular weight of 500weight g/mol of [50039, 61g/mol]. [39,61].

4.3.4.3. Ferrociphenols FerrociphenolsFerrociphenols are are reported reported as the as themost most active active antiproliferative antiproliferative agents agentswhen compared when compared to cisplatin to andcisplatin tamoxifen and tamoxifen cancer drugs cancer [56 drugs]. They [56]. have They rich have diverse rich modes diverse of mode actions of which action are which caused are by caused their capabilityby their capability to produce to produce a redox patterna redox (ferrocenyl-ene-phenol)pattern (ferrocenyl-ene-phenol) in the cancer in the cellcancer [56 ].cell Their [56]. targetTheir istarget the mitochondrialis the mitochondrial system system or redox or redox proteins proteins in the in the cancer cancer cells cells [62 [62–65–].65 Pigeon]. Pigeon et et al. al. preparedprepared ferrociphenolferrociphenol derivativesderivatives56a 56aand and56b 56b (Figure(Figure 18 18).). IncreasingIncreasing thethe lengthlength ofof thethe non-polarnon-polar carboncarbon chainchain onon the the north-west north-west side side of the of molecule the molecule aﬀected affected the steric the hindrance steric hindrance and lipophilicity, and lipophilicity thereby decreasing, thereby thedecreasing anticancer the activity anticancer of the activity compounds of the signiﬁcantly.compounds significantly. The presence The of succinimido presence of orsuccinimido phthalimido or substituentsphthalimido in substituents some of the inderivatives some of enhanced the derivatives their antitumoral enhanced activitytheir ant againstitumoral ovarian activity cancer against cell µ linesovarian with cancer low IC cell50 valueslines with which low were IC50 values below 0.08whichM[ wer64e]. below 0.08 µM [64].

OH OH

O O

N N Fc Fc O O OH OH

56b 56a FigureFigure 18.18. HybridsHybrids 56a56a andand 56b56b..

ZanellatoZanellato etet al.al. evaluatedevaluated the anticanceranticancer activity of compoundscompounds 57a andand 57b57b againstagainst malignantmalignant pleuralpleural mesotheliomamesothelioma (MPM)(MPM) cellcell lineslines (Figure(Figure 1919).). Both compounds were eeffectiveffective inin thethe inhibitioninhibition ofof cell proliferation [66 [66].]. Vassieries Vassieries et et al. al. reported reported the thesynthesis synthesis of compound of compound 57a 57aand and57c [6757c]. [The67]. Theantiproliferative antiproliferative effects eff ofects both of compounds both compounds were evaluated were evaluated at a concentration at a concentration of 1 µM on of hormone 1 µM on- hormone-dependentdependent (MCF7) and (MCF7) hormone and-independent hormone-independent (MDA-MB231) (MDA-MB231) breast cancer breast cell lines. cancer Compound cell lines. Compound57c’s antiproliferative57c’s antiproliferative effect was not eff ectsignificant was not when significant compared when to compared 57a which to 57aexhibitedwhich significant exhibited significantantiproliferative antiproliferative activity with activity IC50 with = 0.7 IC µM.50 = 0.7 Theµ M. result The suggest result suggestss that the that cytotoxic the cytotoxic activity activity of the of theferrocenyl ferrocenyl group group is higher is higher than than the the estrogenic estrogenic proliferative proliferative effect effect of of the the diphenol diphenol moiety. moiety. The poor proliferativeproliferative eeffectffect of of57c 57con on MCF7 MCF7 (ER (ER positive) positive) cells cells indicate indicate that that the the presence presence of of a ferrocenea ferrocene group group is notis not suffi sufficientcient for the for generation the generation of antiproliferative of antiproliferative effects and effects the andposition the of position the oxidizable of the oxidizableferrocenyl groupferrocenyl plays group a vital plays role ina vital the antiproliferativerole in the antiproliferati activity ofve the activity compounds of the compounds [67]. [67].

Molecules 2019, 24, 3604 15 of 27 Molecules 2019,, 24,, xx 15 of 27

OH HO

Fcc Fe X

OH 57a--b 57c

57a:: X = OH ;; 57b:: X = O((CH )) N((CH )) 22 33 33 22 FigureFigure 19.19. Chemical structures ofof ferrociphenolferrociphenol compoundcompound ((57a57a––cc).))..

WangWang et al. al. reported reported a series a series of ferrociphenol of ferrociphenol derivatives derivatives with withhydroxypropyl hydroxypropyl (58–60 ()58) andand–60 testedtested) and testedthemthem onon them thethe on tripletriple the negative triplenegative negative breastbreast breast cancercancer cancer lineline MDAMDA line MDA-MB-231--MB--231 with IC with5050 values IC50 values ranging ranging between between 0.26– 0.26–13.3113.31.31 ΜmΜm (( MmFigure (Figure 20). The 20). introduction The introduction of a bulky of a aromatic bulky aromatic group at group the terminal at the terminal hydroxyl hydroxyl position positionresulted resultedin compounds in compounds with poor with cytotoxic poor cytotoxic activity. activity. The replacement The replacement of terminal of terminal hydroxyl hydroxyl with a withchlorine a chlorine or propan or propan-2-one--2--one oxime resulted oxime resulted in compounds in compounds that exhibited that exhibited moderate moderate cytotoxic cytotoxic activity activityagainst the against MDA the--MB MDA-MB-231--231 cells. The cells. introduction The introduction of benzylated of benzylated substituents substituents at the terminal at the hydroxyl terminal hydroxylposition of position compound of compound 60 resulted60 resulted in significant in significant cytotoxic cytotoxic effects e againstffects against the MDA the MDA-MB-231--MB--231 cellsls,, cells,resulting resulting in a inhigh a high lipophilic lipophilic nature nature of the of the compound. compound. Compound Compound 5959 andand 6060 exhibitedexhibited significant significant cytotoxiccytotoxic eeffectffect againstagainst thethe cancercancer cellcell lineslines suggestingsuggesting thatthat thethe presencepresence of an ester linkerlinker makesmakes thethe compoundscompounds proneprone toto hydrolysis,hydrolysis,, resulting resulting inin thethe generationgeneration ofof thethe parent parent drug, drug,58 58 [ [6363[63].].].

Fc OH 58--60

58:: X = OH 59:: X = O--C((=O))-- 60: X = O-C(=O)Ph 60: X = O-C(=O)Ph FigureFigure 20.20. Chemical structures ofof hydroxypropyl-ferrocephenolhydroxypropyl--ferrocephenolferrocephenol derivativesderivativesderivatives (((5858––6060).))..

PlazukPlazuk etet al.al. reported reported the the synthesis synthesis of of ferrocenyl ferrocenyl compounds compounds 6161–64–64 withwith ferrociphenols ferrociphenols and and a a1H 1H--1,2,3-1,2,3-triazolyl--triazolyltriazolyl moietymoiety moiety actingacting acting asas as aa a linkerlinker linker andand and theirtheir their cytotoxiccytotoxic cytotoxic activitiesactivities activities againstagainst against humanhuman breast breast cancercancer cellscells (HCC38 (HCC38 and and MCF-7) MCF--7) ininin vitro vitro vitro (Figure ((Figure 21 21).). SomeSome of of the the compounds compounds exhibited exhibited good good cytotoxic cytotoxic activityactivity againstagainst thethe hormone-independenthormone--independentindependent HCC38HCC38HCC38 breastbreastbreast cancer cancercancer cell cell line.line. The most activeactive compound waswas 6161 with with ICIC505050 == 15.3 μMµM andand itit containedcontained aa para-hydroxyphenylpara--hydroxyphenyl group.group. TheThe introductionintroduction ofof twotwo para-hydroxyphenylpara--hydroxyphenyl moietymoiety inin compoundcompound 63 resultedresulted inin aa decreasedecrease inin thethe anticanceranticancer activityactivity ofof thethe compoundcompound withwith anan ICICIC505050 valuvaluee of 30.6 µμM.M. TheThe introductionintroduction ofof aa 3,5-dihydroxyphenyl3,5--dihydroxyphenyl inin compoundcompound 6464 ledledled tototo aaa significantsignificantsignificant decreasedecreasedecrease ininin thethethe anticanceranticanceranticancer activity.activity.activity. CompoundCompoundCompound 6262 exhibitedexhibited cytotoxiccytotoxic activityactivity againstagainst thethe hormone-dependenthormone--dependent MCF-7MCF--7 cancercancer cellcell lineslines atat highhigh concentrationsconcentrations ofof ICIC505050 == 48.948.9 [68 [68].]. PigeonPigeon etet al.al. synthesizedsynthesized ferrociphenolferrociphenol derivativesderivatives (6565––6767)) withwith enhancedenhancedenhanced cytotoxiccytotoxiccytotoxic activityactivity byby replacingreplacing thethethe phenolphenol groupgroup withwith anan anilineaniline oror acetanilideacetanilide groupgroup andand thesethese derivativesderivatives werewere testedtested againstagainst MCF-7MCF--7 andand MDA-MB-231MDA--MB--231 breastbreast cancercancer cellcell lineslineslines (Figure(Figure(Figure 2222)22))... TheThe eeffectsffects ofof compoundcompound 6565––6767 againstagainst hormone-dependenthormone--dependent breastbreast cancercancer cellscells MDA-MB-231MDA--MB--231 waswas significantsignificant atat ICIC505050 values 0.8, 0.65 0.65,,, and 1.13 μM, respectively.ly. Compound 65’s’s proliferative effect was significant when compared to compound 66 and 67.. The effect of 65 and 66 on MCF--7 cells revealed a lowlow cytotoxiccytotoxic effecteffect atat aa lowlow

Molecules 2019, 24, 3604 16 of 27

µ andMoleculesMolecules 1.13 20192019M,,, 2424, respectively., xx Compound 65’s proliferative effect was significant when compared1616 ofof to2727 compound 66 and 67. The effect of 65 and 66 on MCF-7 cells revealed a low cytotoxic effect at a low concentrationconcentration of of 0.1 0.1 µμM.μM.M. However,However, 6565 exhibitedexhibexhibitedited enhancedenhanced proliferativeproliferative eeffecteffectffectsss whenwhen comparedcompared toto compoundcompound 66.66.66. At AtAt a aa high highhigh concentration concentrationconcentration of of 10of 10µ10M, μM,μM, compound compoundcompound65 exhibited 6565 exhibitedexhibited no significant nono significantsignificant cytotoxic cytotoxiccytotoxic effect buteffecteffect compound butbut compoundcompound66 exhibited 6666 exhibitedexhibited a moderate aa modermoder antiproliferativeateate antiproliferativeantiproliferative effect [ effect69effect]. [69[69]]..

OOHH OOHH

FFcc NN N NN N N OH 62 FFcc N OH 62 N NN 6611 N OOHH

OOHH HHOO Fc Fc N NN N N NN N NN OH OH NN FFcc 64 64 63 63 FigureFigure 21. 21. ChemicalChemical structures structures of of hydroxyphenyl hydroxyphenyl–ferrocephenolhydroxyphenyl––ferrocephenolferrocephenol derivativesderivatives ((616611––6464).))..

NH NH22 OOHH

HH NN

Fc Fc OO FFcc FFcc

65 66 67 65 66 67 FigureFigure 22. 22. CChemicalChemicalhemical structures structures of of 2 2-ferrocenyl-1-1-diphenyl-but-1-ene2--ferrocenylferrocenyl--11--11--diphenyldiphenyl--butbut--11--eneene derivativesderivatives ((656655––6767).)).. 4.4. Ferrocenyl Derivatives of Clotrimazole Drug 4.4.4.4. FerrocenylFerrocenyl DerivativesDerivatives ofof ClotrimazoleClotrimazole DrugDrug Pedotti et al. reported the synthesis of two ferrocenyl–clotrimazole derivatives 68 and 69 by PedottiPedotti et et al.al. reported reported thethe synthesis synthesis of of twotwo ferrocenylferrocenyl––clotrimazoleclotrimazole derivatives derivatives 6868 andand 6969 byby replacing one of the phenyl rings in the clotrimazole structure with a ferrocene moiety and studied their replacingreplacing oneone ofof thethe phenylphenyl ringsrings inin thethe clotrimazoleclotrimazole structurestructure withwith aa ferroceneferrocene moietymoiety andand studiedstudied biological activity against two human cancer cell lines (HT29 and MCF-7) (Figure 23). In compound theirtheir biological biological a activityctivity against against two two human human cancercancer cell cell lines lines (HT29 (HT29 and and MCF MCF--7)7) ( (FigureFigure 23 23).). In In 68, the chlorine was on the ortho-position whereas for compound 69 it was on the para-position. compoundcompound 6868,, thethe chlorinechlorine waswas onon thethe orthoortho--positionposition whereaswhereas forfor compoundcompound 6969 itit waswas onon thethe parapara-- Compound 68 and 69 growth inhibition effects were studied on colorectal cancer cells (HT29) and breast position.position. Compound Compound 6868 andand 6969 growthgrowth inhibition inhibition effects effects w wereere studiedstudied on on colorectal colorectal ca cancerncer cellscells cancer cells (MCF-7) and ferrocenyl–clotrimazole derivatives were more active against breast cancer (HT29)(HT29) andand breastbreast cancercancer cellscells (MCF(MCF--7)7) andand ferrocenylferrocenyl––clotrimazoleclotrimazole derivativesderivatives werewere moremore activeactive cell MCF-7 when compared to the colorectal cancer cell line HT29 with GI50 values of HT29 = 27.51 µM, againstagainst breastbreast cancercancer cellcell MCFMCF--77 whenwhen comparedcompared toto thethe colorectalcolorectal cancercancer cellcell lineline HT29HT29 withwith GIGI5050 MCF-7 = 23.84 µM (68) and HT29 = 28.13 µM, MCF-7 = 20.44 µM(69), respectively. These two valuesvalues ofof HT29HT29 == 27.5127.51 µM,µM, MCFMCF--77 == 23.8423.84 µMµM ((6868)) andand HT29HT29 == 28.1328.13 µM,µM, MCFMCF--77 == 20.4420.44 µMµM ((6969),), compounds (68 and 69) were more active than their parent drug (clotrimazole, GI HT29 = 64.19 µM respectively.respectively. These These two two compounds compounds (68(68 andand 6969)) were were more more active active than than 50 their their parent parent drug drug and MCF-7 = 21.44 µM) but less active when compared to 5-fluorouracil (GI50, HT29 = 5.71 µM and (clotrimazole,(clotrimazole, GIGI5050 HT29HT29 == 64.1964.19 µMµM andand MCFMCF--77 == 21.4421.44 µM)µM) butbut lessless activeactive whenwhen comparedcompared toto 55-- MCF-7 = 2.98 µM). The modification of the structure with ferrocene enhanced the cytotoxic activity fluorouracilfluorouracil (GI(GI5050,, HT29HT29 == 5.715.71 µMµM andand MCFMCF--77 == 2.982.98 µM).µM). TheThe modificationmodification ofof thethe structurestructure withwith of the compounds when compared to the parent drug in HT29 cells. The aforementioned enhanced ferroceneferrocene enhancedenhanced thethe cytotoxiccytotoxic activityactivity ofof thethe compoundscompounds whenwhen comparedcompared toto thethe parentparent drugdrug inin effect is attributed to the redox properties of iron in the ferrocene moiety and also its ability to generate HT29HT29 cells.cells. TheThe aforementionedaforementioned enhancedenhanced effecteffect isis attributedattributed toto thethe redoxredox propertiesproperties ofof ironiron inin thethe cytotoxic reactive oxygen species [14]. ferroceneferrocene moietymoiety andand alsoalso itsits abilityability toto generategenerate cytotoxiccytotoxic reactivereactive oxygenoxygen speciesspecies [14].[14].

Molecules 2019, 24, x 17 of 27 Molecules 2019, 24, 3604 17 of 27 Molecules 2019, 24, x 17 of 27

N N N N Cl N Fc N Fc N Cl N Cl Fc 69Fc 68Cl 69 Figure 23. Chemical 6structures8 of ferrocenyl- clotrimazole derivatives (68–69). Figure 23. Chemical structures of ferrocenyl- clotrimazole derivatives (68–69). 4.5. Ferrocenyl ChalcogenoFigure 23. Chemical Triazole Conjugatesstructures of ferrocenyl- clotrimazole derivatives (68–69). 4.5. Ferrocenyl Chalcogeno Triazole Conjugates 4.5. FerrocenylPanaka et Chalcogeno al. documented Triazole the Conjugates synthesis of ferrocenyl–chalcogeno derivatives (70 and 71a–c) and testedPanaka their cytotoxic et al. documented activity against the synthesis five different of ferrocenyl–chalcogeno cancer cell lines (A549, derivatives MDA (70-MBand-231,71a MCF–c) and-7, Panaka et al. documented the synthesis of ferrocenyl–chalcogeno derivatives (70 and 71a–c) and HeLa,tested theirand HEK cytotoxic-239T) activity with IC against50 values five ranging different between cancer cell2.9 linesand 20 (A549, µM for MDA-MB-231, the first four MCF-7, cancer HeLa,cells, tested their cytotoxic activity against five different cancer cell lines (A549, MDA-MB-231, MCF-7, and HEK-239T) none of the with compounds IC50 values being ranging effective between against 2.9 and 20 HEKµM-239T for the (Figure first four 24). cancer Compound cells, and 70a none–c HeLa, and HEK-239T) with IC50 values ranging between 2.9 and 20 µM for the first four cancer cells, containedof the compounds sulfur and being compound effective 71a against–c contained HEK-239T selenium. (Figure The 24). derivatives Compound with70a –seleniumc contained exhibited sulfur and none of the compounds being effective against HEK-239T (Figure 24). Compound 70a–c higherand compound cytotoxicit71ay– c withcontained IC50 values selenium. ranging The derivatives between 2.9 with and selenium 18.3 µM exhibited compared higher to cytotoxicity the sulfur contained sulfur and compound 71a–c contained selenium. The derivatives with selenium exhibited withderivatives IC50 values with ranginginhibition between values 2.9between and 18.3 4.46µ–M18.9 compared µM, respectively to the sulfur [70]. derivatives Compound with 70a inhibitionexhibited higher cytotoxicity with IC50 values ranging between 2.9 and 18.3 µM compared to the sulfur valuesgood cytotoxic between activity 4.46–18.9 againstµM, two respectively cancer cells [70 ].(A549 Compound and HeLa)70a withexhibited IC50 values good o cytotoxicf 11.6 µM activity (A549) derivatives with inhibition values between 4.46–18.9 µM, respectively [70]. Compound 70a exhibited andagainst 14.8 two µM cancer (HeLa), cells 70c (A549 and 70b and exhibited HeLa) with good IC 50cytotoxicvalues ofactivity 11.6 µ Magainst (A549) MDA and- 14.8MB-µ231M cancer (HeLa), cells70c good cytotoxic activity against two cancer cells (A549 and HeLa) with IC50 values of 11.6 µM (A549) onlyand 70b withexhibited inhibition good values cytotoxic of 9.7 activity and 18.9 against µM, MDA-MB-231 respectively. cancer Compound cells only 71a with displayed inhibition cytotoxic values and 14.8 µM (HeLa), 70c and 70b exhibited good cytotoxic activity against MDA-MB-231 cancer cells activityof 9.7 and against 18.9 µ threeM, respectively. cancer cells Compound with IC50 values71a displayed of 4.56 µM cytotoxic (MDA- activityMB-231), against 4.46 µM three (MCF cancer-7), cellsand only with inhibition values of 9.7 and 18.9 µM, respectively. Compound 71a displayed cytotoxic with10.9 µM IC50 (HeLa)values; compound of 4.56 µM (MDA-MB-231),71b which was the 4.46 mostµM cytotoxic (MCF-7), active and 10.9 compoundµM (HeLa); against compound four cancer71b activity against three cancer cells with IC50 values of 4.56 µM (MDA-MB-231), 4.46 µM (MCF-7), and whichcells with was IC the50 mostvalues cytotoxic of 2.9 µM active (A549), compound 3.35 µM against (MDA four-MB cancer-231), 5.58 cells µM with (MCF IC50-values7), and of 11.6 2.9 µMµM 10.9 µM (HeLa); compound 71b which was the most cytotoxic active compound against four cancer (A549),(HeLa). 3.35CompoundµM (MDA-MB-231), 71c exhibited 5.58highµ cytotoxicM (MCF-7), activity and 11.6on twoµM cancer (HeLa). cells Compound with IC50 values71c exhibited of 3.71 cells with IC50 values of 2.9 µM (A549), 3.35 µM (MDA-MB-231), 5.58 µM (MCF-7), and 11.6 µM µMhigh (A549) cytotoxic and activity18.3 µM on (HeLa), two cancer respectively cells with [70]. IC The50 values selenium of 3.71compoundsµM (A549) ability and to 18.3 preventµM (HeLa), cancer (HeLa). Compound 71c exhibited high cytotoxic activity on two cancer cells with IC50 values of 3.71 isrespectively attributed [to70 its]. Thecapability selenium to inhibit compounds tumor ability cell growth, to prevent its antioxida cancer isnt attributed property to, and its capabilityits ability to µM (A549) and 18.3 µM (HeLa), respectively [70]. The selenium compounds ability to prevent cancer modulateinhibit tumor carcinogen cell growth, metabolism. its antioxidant property, and its ability to modulate carcinogen metabolism. is attributed to its capability to inhibit tumor cell growth, its antioxidant property, and its ability to modulate carcinogen metabolism. N N N N N N Z Z N N Se N N S Fc Fc Z N Z N Se S Fc Fc O HO O O O Z = HO O O Z = O O O O O O a b c

Figure 24. ChemicalChemicala structures structures of ferrocenyl ferrocenyl–chalcogeno–triazoleb –chalcogeno–triazolec conjugates (7070–71).).

4.6. Ferrocenyl–Olefin Derivatives 4.6. FerrocenylFigure–Olefin 24. DerivativesChemical structures of ferrocenyl–chalcogeno–triazole conjugates (70–71). Oliveira et al. documented the antiproliferative effect of tetrasubstituted olefins–ferrocenyl compounds.4.6. FerrocenylOliveira In– etOlefin vitro al. documented Derivativesstudies were the performed antiproliferative on different effect human of tetrasubstituted cancer cells such olefins as MDA-MB-435–ferrocenyl (humancompounds. melanoma), In vitro SF-295studies (human were performed glioblastoma), on different HCT-8 human (human cancer colon cancer),cells such and as HL-60MDA- (humanMB-435 Oliveira et al. documented the antiproliferative effect of tetrasubstituted olefins–ferrocenyl promyelocytic(human melanoma), leukemia). SF-295 Compound(human glioblastoma),72a and 72b HCTdisplayed-8 (human IC 50colonvalues cancer) of 16, andµM HL and-60 14.2 (humanµM, compounds. In vitro studies were performed on different human cancer cells such as MDA-MB-435 promyerespectively,locytic and leukemia). they were Compound found to exhibit72a and moderate 72b displayed antiproliferative IC50 values activityof 16 µM when and compared 14.2 µM, respectivelyto(human other melanoma), compounds, and they SF withwere-295 similar(humanfound to geometryglioblastoma), exhibit moderate when HCT tested antiproliferative-8 (human against colon human activitycancer) breast, whenand cancer HL compared-60 cell (human lines to otherMDA-MB-231promye compoundslocytic (Figure leukemia). with 24similar)[ Compound71]. geometry MDA-MB-435 72a when and tested72b cell displayed lines against was human foundIC50 value breast to bes of highlycan 16cer µM cell sensitive and lines 14.2 MDA to µM, the- respectively, and they were found to exhibit moderate antiproliferative activity when compared to other compounds with similar geometry when tested against human breast cancer cell lines MDA-

Molecules 20192019,, 2424,, 3604x 1818 of 27

MB-231 (Figure 24) [71]. MDA-MB-435 cell lines was found to be highly sensitive to the compounds compoundswith amine side with chains amine at side low chains micromolar at low micromolar activity. The activity. amine Theside aminechains side contributed chains contributed to estrogen to– estrogen–receptorreceptor interactions. interactions. The introduction of an aromatic substituent did not play an importantimportant role in thethe cytotoxicitycytotoxicity activity of the compounds against MDA-MB-231MDA-MB-231 cells.cells. The presence of the less bulky substituent such as acetyl when compared to a pivaloylpivaloyl substituent resulted in the enhanced cytotoxic eeffectﬀect of the compounds against the selectedselected cancercancer cellcell lines.lines. Compounds 73a–i exhibited good antiproliferative activity with IC50 valuesvalues that that are are not not greater greater than than 2 2 µM,µM, compound 73a,, 73d, 73g, 73h, 73i were thethe most active compounds with inhibition values between 1 1–2.9–2.9 µMµM for SF SF-295,-295, 0.8 0.82–1.42–1.4 µMµM for HCT HCT-8,-8, 0.83–160.83–16 µµMM forfor MDA-MB-435,MDA-MB-435, and 0.52–1.90.52–1.9 µµMM forfor HL-60,HL-60, respectivelyrespectively (Figure(Figure 2525).). CompoundCompound 73h exhibited ICIC5050 valuesvalues rangingranging between between 0.52 0.52 and and 1.9 1.9µM µM for for all all human human cancer cancer cell cell lines. lines. Compound Compound73c, 73e73c, 73f73e,were 73f were slightly slightly active active against against all human all human cancer cancer cell lines cell except lines except MDA-MB-435 MDA-MB with-435 IC with50 values IC50 rangingvalues ranging between between 1 and 2.6 1 andµM 2.6 for µM SF-295, for SF 1.0–3.0-295, 1.µM0–3 for.0 µM HCT-8, for HCT and- 0.90–1.68, and 0.9µM0–1 for.6 µM HL-60 for cancerHL-60 cellcancer lines. cell Compound lines. Compound74 was one74 was of the one most of the antiproliferative most antiproliferative compounds compo againstunds all against cancer all cell cancer lines exceptcell lines MDA-MB-435 except MDA with-MB- inhibition435 with inhibition values between values 0.12–1.0betweenµ 0.12M, respectively–1.0 µM, respectively [71]. [71]. JadhavJadhav etet al.al. synthesized a series of ferrocenyl chalcone and amidinesamidines compoundscompounds (75a–j) viavia catalyzed cyclocondensation andand thesethese compoundscompounds werewere testedtested inin vitrovitro for anticancer activity against human breast cancer cell line MDA-MB-435MDA-MB-435 using the sulforhodamine B assay method ( (FigureFigure 26).). Compounds (75a(75a––gg)) exhibited exhibited good good anticancer anticancer activity activity when when compared compared to doxorubicin to doxorubicin (GI50 =(GI18.450= µ18.4M) againstµM) against MDA-MB-435 MDA-MB cancer-435 cancer cell lines cell lines with with GI50 valuesGI50 values between between 16.85–63.2 16.85–µ63.2M withµM with compound compound75b, 75c75b,, and75c, 75fandexhibiting 75f exhibiting low low GI50 GIvalues50 values of 18,of 18, 17.4, 17.4, and and 16.85 16.85µM, µM respectively., respectively. Compounds Compounds (75h (75h–j–) exhibitedj) exhibited poor poor anticancer anticancer activity activity when when compared compared to to doxorubicin doxorubicin with with GI GI5050 valuesvalues rangingranging between 125.5125.5 andand 152.9152.9µ µMM[ 72[72].]. Compound Compound75b 75band and75c 75cexhibited exhibited high high anticancer anticancer activity activity and and the the presence presence of aof nitro a nitro functional functional group group contributed contributed to theirto their anticancer anticancer activity. activity.

1 Y1 Y

Y2 Y2

Fc Y3 Fc Fe Y3 72a-b 73a-i

74 74: R = NH 2 Compounds Y1 Y2 Y3 72a OH H OH 72b H OH OH 73a OH H O(CH2)3NMe2 73b H OH OH 73c NH2 H H 73d H H H 73e OC(O)CH3 H OH 73f OC(O)CH3 H OC(O)CH3 73g O(CH2)3NMe2 H O(CH2)3NMe2 73h O(CH2)3NMe2 H AMINO CITRATE 73i OH H AMINO CITRATE

Figure 25. Chemical structures of ferrocenyl–oleﬁnsferrocenyl–olefins (7272––7474).).

Molecules 2019, 24, 3604 19 of 27 Molecules 2019, 24, x 19 of 27

R Cl

R R N N R N N N N N N Fc Fc Fc Fc Cl

NO NO2 2 d a b c

R Cl R N N N N N N R N N Fc Fc Fc Fc Fc Fc Fc Cl g h e f Cl

R N N * N R R = N N Fc

Fc Fc

j i FigureFigure 26. 26.Chemical Chemical structures structures ofof ferroceneferrocene with 1,4 1,4-dihydropyrimidine-dihydropyrimidine derivatives derivatives (75a (75a–j)–. j).

4.7.4.7. Ferrocene–Carboxylate Ferrocene–Carboxylate Derivatives Derivatives PerezPerez et et al. al. reported reported the the synthesis synthesis ofof ferroceneferrocene derivatives ( (7766––7777) )containing containing carboxylate carboxylate and and acetylateacetylate with with anticancer anticancer activity activity and and their their antiproliferative antiproliferative activity activityin in vitro vitroagainst against MCF-7, MCF-7, MCF-10A, MCF- and10A HT-29, and cancerHT-29 cancer cell lines cell using lines using an MTT an MTT assay assay was alsowas also evaluated evaluated (Figure (Figure 27). 27). Compound Compound76a 76aand 76bandexhibited 76b exhibited moderate moderate antiproliferative antiproliferative activity activity with with IC50 ICvalues50 values of of 45.5 45.5 and and 57 57µ µM.M. Compound Compound 77 exhibited77 exhibited low antiproliferativelow antiproliferative activity activity with with an an IC IC5050value value ofof 103 µMµM against against MCF MCF-7-7 breast breast cancer cancer cellcell lines. lines. The The compounds compounds did did not not exhibitexhibit any significant signiﬁcant antiproliferative antiproliferative activity activity against against HT HT-29-29 and and MCF-10A with inhibition values between 121–298 µM [73]. MCF-10AMolecules 2019 with, 24 inhibition, x values between 121–298 µM[73]. 20 of 28

O O O O O R O R R O R R O R O * O Fe O Fe Fe O Fe Fe R = Fe R = OH O OH R O N R N O O O O 7777 76a 776b6b 76a Figure 27. Cont. R OH

X O Fc Fc N O Fc HO 78a-b 79 80

80: R = CH , 78a: X = Br, 78b: X = I 3 Figure 27. Chemical structures of ferrocene–carboxylate derivatives (76–80).

Vera et al. reported ferrocene–carboxylate compounds with phenyl and halogen (F, I, Cl, and Br). Compound 78a exhibited good antiproliferative activity against MCF-7 cancer cells with an IC50 value of 9.2 µM and compound 78b exhibited high antiproliferative activity against MCF-10A cancer cells with an IC50 value of 7 µM [74,75] (Figure 27). Compound 79 anticancer activity was significant against both MCF-7 and MCF-10A cancer cells with IC50 values 1.4 and 1.6 µM respectively [73,74]. Compound 78a and 80 showed moderate antiproliferative activity against HT-29 cancer cells with IC50 values of 24.4 and 24.0 µM, respectively [73,74] (Figure 28). The in vitro anti-proliferative studies indicated that the presence of 4-fluorophenyl substituent in the compounds resulted in compounds with no cytotoxic effect against the cancer cell lines. However, the presence of a phenyl group enhanced the proliferative effects of the compound against MCF-7 but nullified the proliferative effect of the compound against the MCF-10A cell line.

4.8. Ferrocene Incorporated Selenourea Derivatives Hussain et al. reported ferrocene incorporated with selenourea derivatives (81a–q) (Figure 28) and tested them for their anticancer activity against liver cancer (Hepa 1c1c7), breast cancer (MCF-7), and neuroblastoma (MYCN2 and SK-N-SH), in vitro. Only four compounds (81c, 81h, 81j, and 81m) exhibited moderate anticancer activity against the aforementioned cells, compound 81c displayed moderate cytotoxicity activity against MYCN-2 and MCF-7 with inhibition values of 26.5 and 27 µM, compound 81h exhibited better anticancer activity against MYCN-2 with an inhibition value of 12.2 µM, compound 81j was slightly cytotoxic with an IC50 value of 18.9 µM against MYCN-2 and compound 81m showed low anticancer activity against MYCN-2 with inhibition value of 38.9 µM [75]. The ortho substitution on the phenyl ring attached with the carbonyl carbon influenced the cytotoxic effect of the compounds significantly. The aromatase inhibition and quinone reductase induction activities of the compounds further revealed their capability to inhibit the cancer initiation and propagation.

Molecules 2019, 24, x 20 of 28

O O O R R R O O * Fe O Fe Fe R =

O OH MoleculesR 2019, 24, x N20 of 27 O Molecules 2019O , 24, 3604 77 20 of 27 76b OH 76a R

X R OH O Fc Fc N X O Fc O Fc Fc N HO 78a-b 79 O Fc 80 HO 78a-b 79 80: R = CH , 78a: X = Br, 78b: X = I 80 3 80: R = CH , 78a: X = Br, 78b: X = I Figure 27. Chemical structures 3of ferrocene–carboxylate derivatives (76–80). FigureFigure 27. 27.Chemical Chemical structuresstructures ofof ferrocene–carboxylate derivatives derivatives (76 (76–80–80). ). Vera et al. reported ferrocene–carboxylate compounds with phenyl and halogen (F, I, Cl, and Br). CompoundVeraVera et et al. al. reported78a reported exhibited ferrocene–carboxylate ferrocene–carboxylate good antiproliferative comp compounds activityounds withagainst with phenyl phenyl MCF and- and7 cancer halogen halogen cell (F,s (F, withI, I,Cl, Cl,an and andIC 50 valueBr).Br). Compound of Compound 9.2 µM and78a 78a compoundexhibited exhibited good good78b exhibited antiproliferative antiproliferative high antiproliferative activity against against activityMCF-7 MCF-7 cancer against cancer cells cellsMCF with with-10A an ancancer IC IC50 50 cellsvaluevalue with of of 9.2 an 9.2µ ICM µM50 and valueand compound compound of 7 µM 78b[74,75 78bexhibited exhibited] (Figure highhigh 27). antiproliferative Compound 79 anticancer activity activity against against activity MCF-10A MCF-10A was significant cancer cancer againstcellscells with withboth an an MCF IC IC50 50value- 7value and of MCFof 7 7µ µMM[-10A 74[74,75] ,cancer75] (Figure(Figure cells 2727).with). Compound Compound IC50 values 7979 1.4 anticanceranticancer and 1.6 activityµM activity respectively was was significant significant [73,74 ]. Cagainstompoundagainst both both 78a MCF-7 MCF-7 and and80 and showed MCF-10A MCF-10A moderate cancercancer cellsantiproliferativecells with IC 5050 valuesvalues activity 1.4 1.4 and against and 1.6 1.6 µM HTµM respectively-29 respectively cancer cell[73,74]. [s73 with,74 ]. 50 ICCompoundCompound values of78a 24.478aand and80 8024.0showed showed µM, moderate respectively moderate antiproliferative antiproliferative [73,74] (Figure activity 28activity). against against HT-29 HT-29 cancer cancer cells cells with with IC 50 valuesICThe50 values of in 24.4 vitro of and 24.4 anti 24.0 and-proliferativeµ 24.0M, respectively µM, re studiesspectively [73 indicated,74 [73,74]] (Figure (Figure that 28 the). 28). presence of 4-fluorophenyl substituent in theThe compoundsThein in vitro vitroanti-proliferative anti-proliferative resulted in compounds studies studies indicated indicated with no that that cytotoxic the the presence presence effect of of against 4-fluorophenyl 4-fluorophenyl the cancer substituentsubstituent cell lines. in However,thein compounds the compounds the presence resulted resulted of in a compoundsphenyl in compounds group with enhanced nowith cytotoxic no the cytotoxic proliferative effect againsteffect effects against the cancer of thethe cellcompoundcancer lines. cell However, lines.against MCFtheHowever, presence-7 but nullified the of apresence phenyl the proliferative groupof a phenyl enhanced group effect the enhanced of proliferative the compound the proliferative effects against of the effects the compound MCF of the-10A compound against cell line. MCF-7 against but nullifiedMCF-7 thebut proliferativenullified the proliferative effect of the compoundeffect of the againstcompound the against MCF-10A the cellMCF-10A line. cell line. 4.8. Ferrocene Incorporated Selenourea Derivatives 4.8.4.8. Ferrocene Ferrocene Incorporated Incorporated Selenourea Selenourea Derivatives Derivatives Hussain et al. reported ferrocene incorporated with selenourea derivatives (81a–q) (Figure 28) and testedHussainHussain them et et al.for al. reportedtheir reported anticancer ferrocene ferrocene activity incorporatedincorporated against liver with with cancer selenourea selenourea (Hepa derivatives derivatives 1c1c7), breast (81a (81a– cancerq–)q (Figure) (Figure (MCF 28) -287) ,) and tested them for their anticancer activity against liver cancer (Hepa 1c1c7), breast cancer (MCF-7), and neuroblastoma tested them for (MYCN2 their anticancer and SK activity-N-SH), against in vitro liver. Only cancer four (Hepacompounds 1c1c7), (81c breast, 81h cancer, 81j, and (MCF-7), 81m) and neuroblastoma (MYCN2 and SK-N-SH), in vitro. Only four compounds (81c, 81h, 81j, and 81m) exhibitedand neuroblastoma moderate (MYCN2anticancer and activity SK-N-SH), againstin the vitro aforementioned. Only four compounds cells, compound (81c, 81h ,81c81j ,displayed and 81m) exhibited moderate anticancer activity against the aforementioned cells, compound 81c displayed moderateexhibited cytotoxicity moderate anticancer activity against activity MYCN against-2 and the MCF aforementioned-7 with inhibition cells, compoundvalues of 26.581c anddisplayed 27 µM, moderate cytotoxicity activity against MYCN-2 and MCF-7 with inhibition values of 26.5 and 27 µM, compoundmoderate cytotoxicity 81h exhibited activity better against anticancer MYCN-2 activity and against MCF-7 MY withCN inhibition-2 with an values inhibition of 26.5 value and of 27 12.2µM, compound 81h exhibited better anticancer activity against MYCN-2 with an inhibition value of 12.2 µM,compound compound81h exhibited 81j was better slightly anticancer cytotoxic activity with against an IC50 MYCN-2 value of with 18.9 an µM inhibition against value MYCN of 12.2-2 andµM, µM, compound 81j was slightly cytotoxic with an IC50 value of 18.9 µM against MYCN-2 and compoundcompound 81j81m 81mwas showed showed slightly low low cytotoxic anticancer anticancer with activityactivity an IC50 againstagainstvalue ofMYCN-2MYCN 18.9 µ-M2 withwith against inhibition inhibition MYCN-2 value value and of of compound38.9 38.9 µM µM [7581m[75].]. showed The The ortho ortho low substitution anticancersubstitution activity on on the the against pphenylhenyl MYCN-2 ringring attachedattached with inhibitionwith the the carbonyl carbonyl value of carbon 38.9 carbonµM[ influenced influenced75]. The the ortho the cytotoxicsubstitutioncytotoxic effect effect on the of of phenylthe the compounds compounds ring attached significantly.significantly. with the carbonyl TheThe aromatase carbon influencedinhibition inhibition and the and cytotoxicquinone quinone reductase e ff reductaseect of the inductioncompoundsinduction activities activities significantly. of of the the compounds The compounds aromatase further further inhibition revealedrevealed and their quinone capabilitycapability reductase to to inhibit inhibit induction the the cancer cancer activities initiation initia oftion the andcompoundsand propagation. propagation. further revealed their capability to inhibit the cancer initiation and propagation.

81c(R) = * 81h(R) = * Br

Fc NH Cl CH3 NH Se 81j(R) = 81m(R) = * 81a-q R * O FigureFigure 28. ChemicalChemical structures structures of of ferrocene ferrocene incorporated selenoure selenoureaa derivatives ( 81a–q).).

4.9. Ferrocene Ferrocene–Steroid–Steroid Conjugates Pita et al. reported reported the the synthesis synthesis of of ferrocene ferrocene–steroid–steroid conjugates conjugates ( (82a82a––cc)) ( (FigureFigure 29)) and evaluated their their anticancer anticancer activity activity against against colon colon cancer cancer HT HT-29-29 and and breast breast cancer cancer MCF MCF-7-7 cell cell lines. lines. Compounds (82a–c) exhibited good antiproliferative activity with IC values less than 30 µM against Compounds (82a–c) exhibited good antiproliferative activity with IC5050 values less than 30 µM against the MCF-7 breast cancer cell line when compared to tamoxifen with an IC of 47 µM and 3-estradiol the MCF-7 breast cancer cell line when compared to tamoxifen with an IC5050 of 47 µM and 3-estradiol ferrocenecarboxylate with an IC50 of 9 µM at low concentration. Compound 82a showed high

MoleculesMolecules 20192019,, 2424,, 3604x 2121 of 2727 Molecules 2019, 24, x 21 of 27 Molecules 2019, 24, x 21 of 27 ferrocenecarboxylate with an IC50 of 9 µM at low concentration. Compound 82a showed high antiproliferativeantiproliferativeferrocenecarboxylate activity with exceeding exceeding an IC50 that thatof 9of of µM cisplatin cisplatin at low against against concentration. the the HT HT-29- 29 Compound colon colon cancer cancer 82a cellshowed cell line line with high with an ferrocenecarboxylate with an IC50 of 9 µM at low concentration. Compound 82a showed high aninhibantiproliferative inhibitionition value value of activity of1.2 1.2 µMµ exceedingM[ [11].11 ].The The that presence presence of cisplatin of of ferrocene ferrocene against themoiety moiety HT- 29in in colonthe the compounds compounds cancer cell line enhanced with an thethe antiproliferative activity exceeding that of cisplatin against the HT-29 colon cancer cell line with an anti-proliferativeantiinhib-proliferativeition value activityactivity of 1.2 µM ofof the the[11]. compoundcompound The presence againstagainst of ferrocene thethe HT-29HT- 29moiety coloncolon in cancercancer the compounds cellcell line.line. Estrogen enhanced receptor the inhibition value of 1.2 µM [11]. The presence of ferrocene moiety in the compounds enhanced the betabetaanti playplay-proliferative aa vitalvital rolerole activity inin thethe of anti-proliferativeanti the-proliferative compound against activityactivity the onon HT thethe-29 HT-29HT colon-29 cellcancer cell line.line. cell line. Estrogen receptor anti-proliferative activity of the compound against the HT-29 colon cancer cell line. Estrogen receptor beta play a vital role in the anti-proliferative activity on the HT-29 cell line. beta play a vital role in the anti-proliferative activity on the HT-29 cell line. O O R O R O R O R O R O R HR R O R H R R O R H R H R H Fc Fc R H R H R H H H Fc R H Fc H H Fc Fc Fc HO H H H H FcHO H H H H H H Fc H H HO HO H H HO HO HO HO 82c HO 82b R= CH 82c 82a 82b 3 82c 82a 82b R= CH3 82a R= CH3

FigureFigure 29.29. Hybrid compounds 82a82a––cc.. Figure 29. Hybrid compounds 82a–c. Figure 29. Hybrid compounds 82a–c. Jaouen and co-workers synthesized ferrocenyl compounds (83a–c) (Figure 30) and tested them for JaouenJaouen and and co co-workers-workers synthesized synthesized ferrocenylferrocenyl compoundscompounds ( 83a(83a–c–)c )( Figure(Figure 30) 30) and and tested tested them them Jaouen and co-workers synthesized ferrocenyl compounds (83a–c) (Figure 30) and tested them theirforfor their anticancertheir anticancer anticancer activity activity activityin vitro in in vitro vitroagainst against against two two human human breast breastbreast cancer cancer cancer cell cell cell lines lines lines MCF-7 MCF MCF- and7 -and7 and MDA-MB-231. MDA MDA-MB-MB- - for their anticancer activity in vitro against two human breast cancer cell lines MCF-7 and MDA-MB- Compounds231.231. Compound Compound83as– c83as had83a–c– nochad had e ﬀno ectno effect effect on MDA-MB-231 onon MDAMDA-MB- and-231 and wereand were were estrogenic estrogenic estrogenic on theon on the MCF-7 the MCF MCF cancer-7 -cancer7 cancer cell cell linecell 231. Compounds 83a–c had no effect on MDA-MB-231 and were estrogenic on the MCF-7 cancer cell atline 0.1–1line at at0.µ 10.M–11– concentration,µM1 µM concentration, concentration, compound compound compound83a 83aand and83c 83c83cexhibited exhibitedexhibited cytotoxic cytotoxic cytotoxic activity activity activity on on on MDA-MB-231 MDA MDA-MB-MB-231-231 at line at 0.1–1 µM concentration, compound 83a and 83c exhibited cytotoxic activity on MDA-MB-231 highat highat high concentrations concentrations concentrations with with with IC IC50 IC5values05 0values values of ofof 13.4 13.413.4 and and 18.8 18.8 µ µMµMM,, ,respectively,respectively respectively, while, while while compound compoundcompound 83b 83b83b remain remainremain at high concentrations with IC50 values of 13.4 and 18.8 µM, respectively, while compound 83b remain inactiveinactiveinactive [[6262 [62].]. ]. inactive [62].

OHOH OH OOHH OOHH OH OH FFcc FFcc Fc Fc HOHO S S Fc Fc HO HO S Fc HO HHOO HOHO 838c3c 83b 83c 83a 833bb 838a3a

FigureFigure 30.30. HybridHybrid compounds 83a83a––cc. . FigureFigure 30.30. Hybrid compoundscompounds 83a 83a––c.c . JaouenJaouen et et al. al. also also reported reported the the synthesis synthesis of steroid steroid vectorized vectorized ferrocene ferrocene derivatives derivatives (84a (84a–c)– c) JaouenJaouen et et al. al. also also reported reported the the synthesis synthesis of steroid steroid vectorized vectorized ferrocene ferrocene derivatives derivatives (84a (84a–c)– c) (Figure(Figure 31 )31) and and evaluated evaluated their their antiproliferative antiproliferative eeffectﬀect against prostate prostate cancer cancer cells cells LNCaP LNCaP and and PC PC-3-3 (Figure(Figure 31) 31) and and evaluated evaluated their their antiproliferative antiproliferative effect againstagainst prostate prostate cancer cancer cells cells LNCaP LNCaP and and PC PC-3 -3 withwith IC 50IC50values values ranging ranging between between 4.74.7 andand 8.3 µµM.M. At high concentra concentrationstions (10 (10 µM)µM) compound compound 84a84a–c –c withwith IC IC50 50values values ranging ranging between between 4.74.7 andand 8.3 µM. AtAt highhigh concentra concentrationstions (10 (10 µM) µM) compound compound 84a 84a–c –c exhibitedexhibited good good antiproliferative antiproliferative activity activity butbut poorpoor activity at at low low concentrations concentrations (1 (1 µM)µM) against against PC PC-3-3 exhibitedexhibited good good antiproliferative antiproliferative activity activity butbut poor activityactivity at at low low concentrations concentrations (1 (1 µM) µM) against against PC PC-3 -3 cancercancer cells. cells. Compound Compound84a 84aand and84b 84bdisplayed displayed high antiproliferative activity activity at at high high concentration concentrationss cancercancer cells. cells. Compound Compound 84a 84a and and 84b 84b displayeddisplayed high antiproliferativeantiproliferative activity activity at at high high concentration concentrations s (10(10µM), µM) with, with no no activity activity at at low low concentrations concentrations againstagainst LNCaP cancer cells cells [62 [62].]. (10(10 µM) µM), with, with no no activity activity at at low low concentration concentrations against LNCaPLNCaP cancer cancer cells cells [62 [62]. ]. OH OH OH OH OH OH OH OH Fc OH Fc Fc Fc Fc Fc Fc Fc Fc HO O HO O O HO O O 84c O 84b 84c 84a 84b 84a 84c 84b 84a Figure 31. Hybrid compounds 84a–c. FigureFigure 31.31. HybridHybrid compounds 84a84a––cc. . Figure 31. Hybrid compounds 84a–c. Manosroi and co-partners synthesized ferrocenic–steroid derivatives (85a–e) (Figure 32). Firstly, ManosroiManosroi and and co-partners co-partners synthesized synthesized ferrocenic–steroidferrocenic–steroid derivatives derivatives (85a (85a–e–)e ()Figure (Figure 32 32). Firstly,). Firstly, in vitro cytotoxicity evaluation of the parent drug (doxorubicin) against cell line HeLa revealed (GI50 in vitroin vitroManosroicytotoxicity cytotoxicity and co evaluationevaluation-partners synthesized of of the the parent parent ferrocenic drug drug (doxorubicin) (doxorubicin)–steroid derivatives against against cell ( 85aline cell– HeLae line) (Figure revealed HeLa 32 revealed). (GIFirstly,50 (iGIn vitro50 value cytotoxicity= 0.250 µ gevaluation/mL) for comparison of the parent use. drug Compound (doxorubicin)85a againstand 85b cellexhibited line HeLa antiproliferative revealed (GI50

Molecules 2019, 24, x 22 of 27 Molecules 20192019,, 2424,, 3604x 2222 of 27 value= 0.250 µg/mL) for comparison use. Compound 85a and 85b exhibited antiproliferative activity value= 0.250 µg/mL) for comparison use. Compound 85a and 85b exhibited antiproliferative activity that was comparable with doxorubicin with GI50 values of 0.223 and 0.271 µg/mL.µ Compound 85d activitythat was that comparable was comparable with doxorubicin with doxorubicin with GI with50 values GI50 values of 0.223 of 0.223and 0.271 and 0.271µg/mL.g /CompoundmL. Compound 85d and 85e displayed moderate antiproliferative activity with GI50 values of 0.405 and 0.505 µg/mL while 85dand and85e displayed85e displayed moderate moderate antiproliferative antiproliferative activity activity with withGI50 values GI50 values of 0.405 of and 0.405 0.505 and µg/mL 0.505 µ whileg/mL their parent drugs exhibited low antiproliferative activity on HeLa cancer cells [76]. whiletheir parent their parent drugs drugsexhibited exhibited low antiproliferative low antiproliferative activity activity on HeLa on HeLacancer cancer cells [76 cells]. [76]. O OH OH O OH OH Fc Fc H H H H Fc Fc H H Fc H H Fc H H H H H H H H HO O O HO O O 85c 85a Fc 85c 85a 85b 85b Fc Fc Fc O O O O OH Fc OH Fc

HO HO HO HO 85e 85d 85e 85d Figure 32. Chemical structures of ferrocene–steroids derivatives (85a–e). Figure 32. Chemical structures of ferrocene–steroidsferrocene–steroids derivatives (85a85a––ee).). 4.10. Aminoferrocene Aminoferrocene-Based-Based Derivatives 4.10. Aminoferrocene-Based Derivatives Schikora etet al.al. reportedreported anticanceranticancer activity activity of of aminoferrocene-based aminoferrocene-based compounds compounds (86a (86a–b)– (Figureb) (Figure 33) Schikora et al. reported anticancer activity of aminoferrocene-based compounds (86a–b) (Figure 33against) against LNCaP LNC andaP and DU-145 DU-145 prostate prostate cancer cancer cell cell lines. lines. At At high high concentrations, concentrations, compoundcompound 86b was 33) against LNCaP and DU-145 prostate cancer cell lines. At high concentrations, compound 86b was found to to be be non-toxic non-toxic against against DU-145 DU-145 and LNCaPand LNCaP cancer cancer cells while cells compound while compound86a exhibited 86a exhibited moderate found to be non-toxic against DU-145 and LNCaP cancer cells while compound 86a exhibited moderatecytotoxic activity cytotoxic against activity both against DU-145 both and LNCaP DU-145 cancer and LNCaP cells in cancervitro with cells inhibition in vitro valueswith inhibition that were moderate cytotoxic activity against both DU-145 and LNCaP cancer cells in vitro with inhibition valuesbetween that 18–27 wereµ Mbetween and 11–17 18–27µ M,µM respectively. and 11–17 µMIn, vivorespectively.studiesfurther In vivo revealedstudies further that compound revealed that86a values that were between 18–27 µM and 11–17 µM, respectively. In vivo studies further revealed that wascompound non-toxic 86a against was non human-toxic prostate against adenocarcinoma human prostate xenografts adenocarcinoma in a CBA xenografts mice model in and a CBA exhibited mice compound 86a was non-toxic against human prostate adenocarcinoma xenografts in a CBA mice modelrestricted and antitumor exhibited activity restricted [77 ].antitumorN-benzyl-substituted activity [77]. aminoferrocene-basedN-benzyl-substituted aminoferrocene prodrug 86a exhibited-based model and exhibited restricted antitumor activity [77]. N-benzyl-substituted aminoferrocene-based prodrugsigniﬁcant 86a anticancer exhibited activity significant against anticancer androgen-sensitive activity against human androgen prostate-sensitive adenocarcinoma human prostate cell line prodrug 86a exhibited significant anticancer activity against androgen-sensitive human prostate adenocarcinomaLNCaP which is attributedcell line LNCaP to the increasedwhich is attributed amount of to intracellular the increased reactive amount oxygen of intracellular species during reactive the adenocarcinoma cell line LNCaP which is attributed to the increased amount of intracellular reactive oxygentreatment species of the during cells with the the treatment drug and of its the enhanced cells with cell-membrane the drug and permeability its enhanced when cell-membrane compared oxygen species during the treatment of the cells with the drug and its enhanced cell-membrane permeabilityto 86b. when compared to 86b. permeability when compared to 86b. HN Fc HN Fc O O O O O O B O O Fc B O B B O Fc N O O O N O O O 86b 86a 86b 86a

Figure 33. CChemicalhemical structure of aminoferrocene aminoferrocene-based-based derivatives (86a–b).). Figure 33. Chemical structure of aminoferrocene-based derivatives (86a–b). 4.11. Ferrocene Ferrocene–Lawsone–Lawsone Mannich Derivatives . 4.11. Ferrocene–Lawsone Mannich Derivatives. Ferrocenyl compounds ((87a87a–d) ( (FigureFigure 34)34) modiﬁedmodified by Lawsone Mannich bases were reported Ferrocenyl compounds (87a–d) (Figure 34) modified by Lawsone Mannich bases were reported by Ahmad et al. Their Their antiproliferative antiproliferative studies studies against prostate (BxPC (BxPC-3),-3), breast (MDA (MDA-MB-231),-MB-231), and by Ahmad et al. Their antiproliferative studies against prostate (BxPC-3), breast (MDA-MB-231), and pancreatic (PC-3)(PC-3) cancer cells indicated that compound 87a was the most antiproliferative compound pancreatic (PC-3) cancer cells indicated that compound 87a was the most antiproliferative compound in threethree cancercancer cells cells (87c (87c< 87d< 87d< 87b< 87b< 87a < 87a), exhibiting), exhibiting high high activity activity when when compared compared to the to well-known the well- Lawsonein three cancer Mannich cells bases (87c in< 87d PC-3 < 8 cancer7b < 87a cells.), exhibitingIn vitro studieshigh activity of compound when compared87a on threeto the cancer well-

Molecules 2019, 24, 3604 23 of 27 Molecules 2019, 24, x 23 of 27

known Lawsone Mannich bases in PC-3 cancer cells. In vitro studies of compound 87a on three cancer cells (518A2 melanoma, HT-29 colon carcinoma, HCT-116 colon carcinoma, and vinblastine-resistant cells (518A2 melanoma, HT-29 colon carcinoma, HCT-116 colon carcinoma, and vinblastine-resistant KB-V1/Vb1 cervix carcinoma) revealed good activity with inhibition values of 2.60, 3.58, 4.29, and KB-V1/Vb1 cervix carcinoma) revealed good activity with inhibition values of 2.60, 3.58, 4.29, and µ 0.190.19M, µM, respectively respectively [78 [78].]. The The combination combination ofof Lawsone with with a a 2 2-pyridyl-pyridyl moiety moiety and and a ferrocene a ferrocene-1-yl-1-yl methylaminemethylamine sca scaffoldffold in compound in compound87a 87acontributed contributed to its to anticancer its anticancer activity. activity. The e ff Theect of effect the ferrocene of the inferrocene compound in 87acompoundwas significant. 87a was significant.

O O OH OH H H N N Fc O Fc O X

Y R2 R1 87a-c 87d

1 2 1 2 1 2 87a: X= N; Y= C; R = R = H 87b: X= CH; Y-R = N; R = H 87c: X= CH; Y= C; R = R = F

FigureFigure 34. 34.Chemical Chemical structures structures ofof ferrocene–Lawsoneferrocene–Lawsone Mannich Mannich base base derivatives derivatives (87a (87a–d–).d ).

5.5. Conclusions Conclusion ResearchersResearchers are are currently currently employing employing diff erent different strategies strategies to develop to develop effective effective drugs fordrugs the treatmentfor the oftreatment malaria and of cancer,malaria two and chronic cancer, conditions. two chronic The conditions. aim of the The development aim of the development of new antimalarial of new or anticancerantimalarial drugs or isanticancer to overcome drugs drug is to resistance, overcome toxicity drug resistance, etc. Most oftoxicity the drugs etc. Most are developed of the drugs as hybrid are moleculesdeveloped which as hybrid is a promising molecules approachwhich is a to promising develop compoundsapproach to todevelop overcome compounds drug resistance. to overcome drugAntimalarial-based resistance. hybrid compounds of ferrocene have been extensively studied. Ferroquine, a hybridAntimalarial molecule prepared-based hybrid from compounds chloroquine of and ferrocene ferrocene have exhibit been extensively enhanced inhibition studied. Ferroquine, effect against beta-haematina hybrid molecule formation prepared which from makes chloroquine it more eff ective and ferr thanocene chloroquine. exhibit enhanced Other quinoline–ferrocene inhibition effect against beta-haematin formation which makes it more effective than chloroquine. Other quinoline– hybrid molecules were prepared and some of the molecules inhibited the efflux mechanism which is ferrocene hybrid molecules were prepared and some of the molecules inhibited the efflux mechanism common with chloroquine. However, factors such as rigid linkers and the length of the linkers between which is common with chloroquine. However, factors such as rigid linkers and the length of the the parent drugs influenced the antimalarial activity of some of the hybrid molecules significantly. linkers between the parent drugs influenced the antimalarial activity of some of the hybrid molecules The quinoline moiety was found to influence the transportation of the hybrid molecules to the digestive significantly. The quinoline moiety was found to influence the transportation of the hybrid molecules vacuole of the parasite. However, ferroquine analogues with the presence of a cleavable amide bond to the digestive vacuole of the parasite. However, ferroquine analogues with the presence of a didcleavable not exhibit amide significant bond did antimalarial not exhibit significant activity. Ferrocene antimalarial hybrid activity. molecules Ferrocene containing hybrid molecules artemisinin werecontaining very active artemisinin with the were capability very active to improve with the the capability bioavailability to improve of molecules the bioavailability when compared of withmolecules artemisinin when which compared is hampered with artemisinin by its poor which bioavailability. is hampered by Other its poor ferrocene bioavailability. hybrid molecules Other containingferrocene eitherhybrid novobiocin, molecules containing pyrrole, isatin,either novobiocin, pyrimidine, pyrrole, and chalcones isatin, pyrimidine derivatives, and also chalcones exhibited goodderivati antimalarialves also exhibited activity. Factorsgood antimalarial such as the activity. chain length,Factors nature,such as andthe chain position length, of the nature substituent, and influencedposition of the the biological substituent activity influenced of the the compounds biological inactivity vitro. of the compounds in vitro. InIn vitro vitroand andin in vivo vivoevaluation evaluation of ofthe the ferroceneferrocene hybrids against against several several cancer cancer cell cell lines lines further further revealedrevealed the the e ffiefficacycacy ofof ferrroceneferrrocene hybrid hybrid molecules. molecules. The The length length of the of non the-polar non-polar carbon carbon chain, chain,the theposition position of of the the ferrocenyl ferrocenyl group, group, the the nature nature and and position position of ofthe the substituents substituents in inthe the hybrid hybrid molecules molecules,, andand the the dose dose of of the the compounds compounds used used inin thethe cytotoxicitycytotoxicity evaluation evaluation were were factors factors that that influenced influenced the the cytotoxiccytotoxic eff effectect of of the the compounds. compounds. SeveralSeveral research research reports reports indicateindicate the efficacy efficacy of of ferrocene ferrocene-based-based hybrids. hybrids. However, However, there there is a is a pressingpressing needneed for these molecules obtained obtained so so far far to to be be investigated investigated inin vivo vivo in inorder order to toconfirm confirm the the resultsresults obtained obtainedin in vitro vitro.. However, However, itit isis importantimportant to develop develop hybrid hybrid compounds compounds that that are are structurally structurally simpler, synthetically accessible, and less toxic, with enhanced efficacy and progress through all simpler, synthetically accessible, and less toxic, with enhanced efficacy and progress through all phases phases of clinical practice, to become available and affordable. of clinical practice, to become available and affordable.

Molecules 2019, 24, 3604 24 of 27

Funding: The financial assistance of the Medical Research Council, National Research Foundation and Govan Mbeki Research and Development Center, South Africa towards this research are hereby acknowledged. The views and opinions expressed in this manuscript are those of the authors and not of MRC or NRF. Conflicts of Interest: The authors declare no conflicts of interest.

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