Cinnamic Acid Derivatives and Their Biological Efficacy

International Journal of Molecular Sciences

Review

Cinnamic Acid Derivatives and Their Biological Efficacy Review NgonidzasheCinnamic Ruwizhi Acid and Blessing Derivatives Atim Aderibigbe and * their Biological DepartmentEfficacy of Chemistry, University of Fort Hare, Alice Campus, Alice 5700, Eastern Cape, South Africa; [email protected] * NgonidzasheCorrespondence: Ruwizhi [email protected] and Blessing Atim Aderibigbe * Received:Department 25 March of Chemistry, 2020; Accepted: University 6 May of2020; Fort Published:Hare, Alice 9 Campus, August 2020 5700 Alice, Eastern Cape, South Africa; [email protected] Abstract:* Correspondence:The role played [email protected] by cinnamic acid derivatives in treating cancer, bacterial infections, diabetesReceived: and 25 neurological March 2020; disorders, Accepted: among 06 May many, 2020; hasPublished: been reported. date Cinnamic acid is obtained from cinnamon bark. Its structure is composed of a benzene ring, an alkene double bond and an acrylic acidAbstract: functional The group role played making by it possiblecinnamic to acid modify derivatives the aforementioned in treating cancer, functionalities bacterial with infections, a variety ofdiabetes compounds and resultingneurological in bioactivedisorders, agents among with many enhanced, has been e ffireported.cacy. The Cinnamic nature of acid the is substituents obtained incorporatedfrom cinnamon into cinnamicbark. Its structure acid has beenis composed found to of play a benzene a huge ring, role inan either alkene enhancing double bond or decreasing and an theacrylic biological acid efunctionalfficacy of thegroup synthesized making it cinnamicpossible to acid modify derivatives. the aforementi Some ofoned the derivativesfunctionalities have with been reporteda variety to beof morecompounds effective resulting when compared in bioactive to the agents standard with drugsenhanced used efficacy. to treat chronicThe nature or infectious of the diseasessubstituentsin vitro incorporated, thus making into themcinnamic very acid promising has been therapeutic found to play agents. a huge Compoundrole in either20 enhancingdisplayed potentor decreasing anti-TB activity, the biological compound efficacy27 exhibited of the synt significanthesized antibacterialcinnamic acid activity derivatives. on S. aureusSome ofstrain the of bacteriaderivatives and compounds have been reported with potent to be antimalarial more effect activityive when are compared35a, 35g ,to35i the, 36i standard, and 36b drugs. Furthermore, used to treat chronic or infectious diseases in vitro, thus making them very promising therapeutic agents. compounds 43d, 44o, 55g–55p, 59e, 59g displayed potent anticancer activity and compounds 86f–h Compound 20 displayed potent anti-TB activity, compound 27 exhibited significant antibacterial were active against both hAChE and hBuChE. This review will expound on the recent advances on activity on S. aureus strain of bacteria and compounds with potent antimalarial activity are 35a, 35g, cinnamic acid derivatives and their biological efficacy. 35i, 36i, and 36b. Furthermore, compounds 43d, 44o, 55g–55p, 59e, 59g displayed potent anticancer activity and compounds 86f–h were active against both hAChE and hBuChE. This review will Keywords: cinnamic acid; antimicrobial; anticancer; antimalarial; Alzheimer’s disease expound on the recent advances on cinnamic acid derivatives and their biological efficacy.

Keywords: cinnamic acid; antimicrobial; anticancer; antimalarial; Alzheimer’s disease

1. Introduction1. Introduction CinnamicCinnamic acid, acid, a a natural natural aromatic aromatic carboxylic carboxylic acidacid [[11] (Figure (Figure 11),), is is a a key key chemical chemical found found in in plants plants suchsuch as Cinnamomumas Cinnamomum cassia cassia(Chinese (Chinese cinnamon) cinnamon) and andPanax Panax ginseng ginseng, fruits,, fruits, whole whole grains, grains, vegetables vegetables and honeyand [honey1]. The [1]. presence The presence of an acrylicof an acrylic acid group acid gr substitutedoup substituted on the on phenyl the phenyl ring ring gives gives cinnamic cinnamic either a ciseither ora a transcis or configurationa trans configuration with the with latter the beinglatter thebeing most the commonmost common of the of two the two [2,3]. [2,3]. Studies Studies have reportedhave reported that cinnamic that acidcinnamic exhibit acid antioxidant, exhibit antioxidant, antimicrobial antimicrobial [4], anticancer [4], [5 ],anticancer neuroprotective, [5], anti-inflammatoryneuroprotective, andanti-inflammatory antidiabetic properties. and antidiabetic [6]. Cinnamic properties. acid [6]. terminates Cinnamic radical acid terminates chain reactions radical by donatingchain reactions electrons by that donating react withelectrons radicals that formingreact with stable radicals products forming [7 ].stable It is products also used [7]. as It a is fragrant also ingredientused as a infragrant toiletries, ingredient flavorings in toiletries, cosmetics flavorings and detergents cosmetics [ 2and]. Cinnamic detergents acid [2]. Cinnamic can be prepared acid can by enzymaticbe prepared deamination by enzymatic of phenylalanine deamination of [8 phenylalanine]. [8].

FigureFigure 1. 1.Chemical Chemical structure of cinnamic cinnamic acid acid (1 (1).). Besides the cinnamic acid derivatives that occur naturally in plants [2], the presence of a benzene Besides the cinnamic acid derivatives that occur naturally in plants [2], the presence of a benzene ringring and and the the acrylic acrylic acid acid group group makes makes itit possiblepossible to modify it it resulting resulting in in synthetic synthetic cinnamic cinnamic acid acid derivatives [9]. Apart from them being intermediates in producing other compounds like stilbenes Int. J. Mol. Sci. 2020, 21, 5712; doi:10.3390/ijms21165712 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x; doi: FOR PEER REVIEW www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5712 2 of 34 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 37 derivativesand styrenes, [9]. Apartcinnamic from acid them derivatives being intermediates exhibit antibacterial, in producing antifungal, other compoundsanti-inflammatory like stilbenes [10], andneuroprotective styrenes, cinnamic [11], anticancer acid derivatives [12] and antidiabetic exhibit antibacterial, activities [13]. antifungal, Cinnamic anti-inflammatory acid derivatives that [10 ], neuroprotectivehave been reported [11], anticancer by researchers [12] and antidiabeticinclude ferulic activities acid, [13curcumin]. Cinnamic [11], acid caffeic derivatives acid, p- that havehydroxycinnamic been reported byacid researchers [14] coumaric include and ferulicchlorogeni acid,c acids curcumin [15], [etc.11], These caffeic cinnamic acid, p-hydroxycinnamic acid analogues acidare [ 14differentiated] coumaric and by chlorogenicthe presence acids of hydroxyl [15], etc. grou Theseps cinnamicon the phenyl acid analogues ring that areare dieitherfferentiated free or by themethoxylated presence of hydroxyl[15]. groups on the phenyl ring that are either free or methoxylated [15]. TheThe biological biological activities activities of of di differentfferent cinnamiccinnamic acid derivatives derivatives have have been been attributed attributed to tothe the nature nature andand position position of of the the substituent substituent groups. groups. DrugDrug resist resistanceance and and the the absence absence of of curative curative therapies therapies with with lowlow adverse adverse side side eff effectect profiles profiles to to control control cancer, cancer, microbial microbial growth, growth, neurological neurological disorders disorders etc. etc. has has led toled research to research on the on development the development of therapeutic of therapeutic compounds compounds based based on on cinnamic cinnamic acid acid [16 [16].]. Due Due to to the the biological efficacy of derivatives of cinnamic acid, this review will report different recently biological efficacy of derivatives of cinnamic acid, this review will report different recently synthesized synthesized derivatives of cinnamic acid and their biological activity in vitro and in vivo. derivatives of cinnamic acid and their biological activity in vitro and in vivo.

2.2. Cinnamic Cinnamic Acid Acid Derivatives Derivatives with with AntimicrobialAntimicrobial Activity The World Health Organization (WHO) recognizes infectious diseases caused by bacteria, The World Health Organization (WHO) recognizes infectious diseases caused by bacteria, viruses and fungi as a global health threat, especially in developing and poor countries. About 3.5 viruses and fungi as a global health threat, especially in developing and poor countries. About million people die from infectious diseases annually [17]. In 2018, the WHO reported 37.9 million 3.5 million people die from infectious diseases annually [17]. In 2018, the WHO reported 37.9 million people to be living with Human Immunodeficiency Virus (HIV) worldwide with 770,000 dying from peopleAIDS-related to be living illnesses with [18]. Human The Immunodeficiency2019 Global tuberculosis Virus (TB) (HIV) reported worldwide an estimated with 770,000 10.0 million dying TB from AIDS-relatedinfections and illnesses 1.2 million [18]. deaths The 2019 in 2018. Global Of the tuberculosis TB cases, 8.6% (TB) were reported people an living estimated with HIV 10.0 [19]. million The TB infectionsappearance and of 1.2 drug-resistant million deaths microbial in 2018. strains Of has the led TB to cases, difficulty 8.6% in were treating people diseases living such with as TB HIV and [19 ]. Themalaria. appearance Cinnamic of drug-resistant acid derivatives microbial are known strains for has their led antimicrobial to difficulty inactivity treating and diseases researchers such are as TB andsearching malaria. for Cinnamic antimicrobial acid derivativesagents which are are known more foreffective their antimicrobialthan the currently activity used and standard researchers drugs are searching[20]. for antimicrobial agents which are more effective than the currently used standard drugs [20]. DengDeng et al.et extractedal. extracted a 4-hydroxycinnamic a 4-hydroxycinnamic acid derivative, acid methyl derivative, 2-{(E)-2-[4-(formyloxy)phenyl] methyl 2-{(E)-2-[4- ethenyl}-4-methyl-3-oxopentanoate(formyloxy)phenyl]ethenyl}-4-methyl-3-oxopentanoate (2) (Figure2) from a ( plant-based2) (Figure 2) endophyticfrom a plant-based fungus endophyticPyronema sp. Thefungus biological Pyronema activity sp. ofThe the biological compound activity was evaluated of the compoundin vitro on was a non-tuberculous evaluated in vitro mycobacterium, on a non- Mycobacteriumtuberculous mycobacterium, marinum, responsible Mycobacterium for skin infections. marinum, The responsible compound for exhibited skin infections. a good inhibitory The effcompoundect with an exhibited IC50 of 64 a µgoodM[21 inhibitory]. effect with an IC50 of 64 µM [21].

O R

H O O 2 R = OCH 3

FigureFigure 2. 2.Chemical Chemical structurestructure of 4-hydroxycinnamic4-hydroxycinnamic acid acid derivative derivative (2 ()2 [21].)[21 ].

KorosecKorosec et et al. al. testedtested antifungalantifungal activity of of cinnamic cinnamic acid acid derivatives, derivatives, most most of ofthem them were were synthesizedsynthesized [ 22[22].]. The The antifungal antifungal activity activity was wasbased based on targeting on targeting CYP53A15, CYP53A15, a unique afungal unique enzyme fungal enzymeknown known to play to playa role a rolein demethyling in demethyling lanosterol, lanosterol, subsequently subsequently promoting promoting fungal fungal growth growth [23]. [23 ]. DerivativesDerivatives ( 3(–3-99)) (Figure(Figure3 )3) inhibited inhibited CYP53A15 CYP53A15 enzymaticenzymatic activityactivity against the the three three fungi fungi (Cochliobolus(Cochliobolus lunatus lunatus,, Aspergillus Aspergillus niger nigerand Pleurotusand Pleurotus ostreatus ostreatus) thereby) thereby revealing revealing the potential the potential antifungal activityantifungal of cinnamic activity acidof cinnamic derivatives acid derivatives [22]. [22].

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Figure 3. Chemical structures of cinnamic acid derivatives with antifungal activity (3–9)[22]. Figure 3. Chemical structures of cinnamic acid derivatives with antifungal activity (3–9) [22]. Based on the Table1, compounds 3 and 4 were the best inhibitors of C. lunatus growth. The percentageBased on the fungal Table growth 1, compounds increased 3 and from 4 were compounds the best 3inhibitorsto 9 revealing of C. lunatus the weak growth. antifungal The percentage fungal growth increased from compounds 3 to 9 revealing the weak antifungal activity of activity of some of the compounds. Compound 4 was antifungal activity was potent on A. niger some of the compounds. Compound 4 was antifungal activity was potent on A. niger inhibitor and inhibitor and compound 6 was not eﬀective in inhibiting P. ostreatus growth. Compound 5 exhibited compound 6 was not effective in inhibiting P. ostreatus growth. Compound 5 exhibited broad broad antifungal activity in all the three fungal species. The presence of an electron-withdrawing antifungal activity in all the three fungal species. The presence of an electron-withdrawing group on group on the phenyl ring enhanced the antifungal activity of the compound. The type of phenyl ring the phenyl ring enhanced the antifungal activity of the compound. The type of phenyl ring substituentsubstituent and and its its position position also also played played an importantan important role [role23]. [23]. 2-nitro 2-nitro or 4-cyano or 4-cyano substitution substitution inhibited fungalinhibited growth fungal to thegrowth optimum to the [optimum20]. Compound [20]. Compound5 also exhibited 5 also exhibited the best inhibitionthe best inhibition on CYP53A15 on activityCYP53A15 making activity it a goodmaking candidate it a good for candidate the further for the development further development of antifungal of antifungal drugs [22 drugs]. [22].

TableTable 1. 1.Comparing Comparing percentagepercentage fungal growth growth of of the the three three fungi fungi when when treated treated with with 0.5 0.5mmol/L mmol of/L of cinnamiccinnamic acid acid derivatives derivatives ((33–9–9)[) [22].22].

%Fungal Growth in 0.5 mmol/L of Inhibitor CompoundsCompounds %Fungal Growth in 0.5 mmol/L of Inhibitor C. lunatus A. niger P. ostreatus 3 C. lunatus22 A.niger 85 P. ostreatus 38 4 25 66 43 3 5 22 3085 2838 29 4 6 25 3466 10943 00 7 5 30 4228 8429 62 8 58 60 67 6 9 34 63109 5000 53 7control 42 9084 10962 55 8 58 60 67 Atmaram Upare 9 et al. synthesized63 cinnamic50 acid derivatives53 (10–26) (Figure4) in which the carboxyl group was replaced with bioisostere 1,2,4-oxadiazole followed by substituting the phenyl control 90 109 55 ring of the cinnamic acid resulting in a series of novel styryl oxadiazoles. The compounds were tested in vitroAtmaramat day 8 Upare for their et anti-tubercularal. synthesized cinnamic activity againstacid derivatives H37Ra strain (10–26 of) Mycobacterium(Figure 4) in which tuberculosis the (Tablecarboxyl2). Compoundgroup was replaced10 was with 20 times bioisostere less active 1,2,4-oxadiazole when compared followed to by cinnamic substituting acid. the Compounds phenyl havingring of electron-donating the cinnamic acid resulting substituents in a series on the of paranovel position styryl oxadiazoles. of the phenyl The ringcompounds (11–13) were were tested inactive in vitro at day 8 for their anti-tubercular activity against H37Ra strain of Mycobacterium tuberculosis with IC50 > 30 µg/mL [24]. (Table 2). Compound 10 was 20 times less active when compared to cinnamic acid. Compounds For the compounds with halogen substitutes on the phenyl ring, the position, number of having electron-donating substituents on the para position of the phenyl ring (11–13) were inactive substituents and the type of halogen affected the biological activity of the compounds. Para-substituted with IC50 ˃30 µg/mL [24]. chloro-compound 14 (IC50 = 4.54 µg/mL) was more active when compared to ortho-substituted compound 15 (IC50 = 9.91 µg/mL), suggesting that the presence of chlorine on the para position enhanced the antibacterial activity of the compound [25]. Compound 17 with a 4-fluoro phenyl ring substitution was the most active of all the compounds containing fluorine with IC50 = 0.36 µg/mL indicating that fluoro substitution favored anti-tuberculosis activity of the synthesized compound. Int. J. Mol. Sci. 2020, 21, 5712 4 of 34

However, compound 24 displayed high IC50 value greater than 30 µg/mL suggesting the nature of the ﬂuorineInt. J. Mol. substituent Sci. 2020, 21, wasx FOR also PEER important REVIEW [24]. 4 of 37

FigureFigure 4. 4.Chemical Chemical structures structures ofof the synthesizedsynthesized novel novel styryl styryl oxadiazoles oxadiazoles [24]. [24 ].

Table 2. In vitro anti-tubercular activity of compounds 10–26 against M.tb H37Ra on day 8 [24]. For the compounds with halogen substitutes on the phenyl ring, the position, number of

substituents and theCompound type of halogen IC 50affected(µg/mL) the biological Cpd. activity IC50 (µ gof/mL) the compounds. Para- substituted chloro-compound 14 (IC50 = 4.54 µg/mL) was more active when compared to ortho- 1 0.06 18 >30 substituted compound 1510 (IC50 = 9.91µg/mL),1.42 suggesting 19that the presence11.01 of chlorine on the para position enhanced the antibacterial11 activity>30 of the compound20 [25]. Compound0.045 17 with a 4-fluoro phenyl ring substitution was12 the most active>30 of all the compounds21 containing0.56 fluorine with IC50 =0.36 µg/mL indicating that fluoro13 substitution>30 favored anti-tuberculosis22 > activity30 of the synthesized compound. However, compound14 24 displayed4.53 high IC50 value23 greater than0.56 30 µg/mL suggesting the nature of the fluorine substituent15 was also9.91 important [24]. 24 >30 16 2.35 25 >30 17 0.36 26 1.56 Table 2. In vitro anti-tubercular activity of compounds 10–26 against M.tb H37Ra on day 8 [24].

The introductionCompound of an electron-withdrawingIC50 (µg/mL) group Cpd. improved IC50 (µg/mL) their antibacterial activity (i.e., compounds 20 and 21). Outstandingly, compound 20 with a carboxylic acid at the para position 1 0.06 18 ˃30 exhibited potent anti-TB activity (IC50 = 0.045 µg/mL) when compared to cinnamic acid. Thus, generally, the presence of an electron-withdrawing group on the para position of the phenyl ring favored significant 10 1.42 19 11.01 anti-TB activity [25]. Compounds with electron-withdrawing groups exhibited improved activity (compound 23 with IC = 0.56 µg/mL was more active than compound 16 with IC = 2.35 µg/mL) while 11 50 ˃30 20 0.045 50 compounds with electron-donating substituents on the phenyl ring did not display a significant anti-TB activity [24]. 12 ˃30 21 0.56 Malheiro et al. synthesized cinnamic acid derivatives by modifying the carboxyl group, the alkene and13 the phenyl ring (27˃30–32 ) (Figure5) and studied22 their˃30 e ﬀects, together with selected phytochemicals, on controlling planktonic Escherichia coli, Staphylococcus aureus and Enterococcus hirae. The phytochemicals14 and derivatives4.53 were also compared23 with the commonly0.56 used biocides such as sodium hypochlorite, triclosan, hydrogen peroxide, etc. Compound 27 appeared to be the most potent on all the three15 bacteria species, with9.91 better eﬃcacy than24 sodium hypochlorite˃30 (Table3)[16].

16 2.35 25 ˃30

17 0.36 26 1.56

The introduction of an electron-withdrawing group improved their antibacterial activity (i.e., compounds 20 and 21). Outstandingly, compound 20 with a carboxylic acid at the para position exhibited potent anti-TB activity (IC50 =0.045 µg/mL) when compared to cinnamic acid. Thus,

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generally, the presence of an electron-withdrawing group on the para position of the phenyl ring favored significant anti-TB activity [25]. Compounds with electron-withdrawing groups exhibited improved activity (compound 23 with IC50 = 0.56 µg/mL was more active than compound 16 with IC50 = 2.35 µg/mL) while compounds with electron-donating substituents on the phenyl ring did not display a significant anti-TB activity [24]. Malheiro et al. synthesized cinnamic acid derivatives by modifying the carboxyl group, the alkene and the phenyl ring (27–32) (Figure 5) and studied their effects, together with selected phytochemicals, on controlling planktonic Escherichia coli, Staphylococcus aureus and Enterococcus hirae. The phytochemicals and derivatives were also compared with the commonly used biocides such as sodium hypochlorite, triclosan, hydrogen peroxide, etc. Compound 27 appeared to be the most Int. J. Mol. Sci. 2020 21 potent on all the, ,three 5712 bacteria species, with better efficacy than sodium hypochlorite (Table 3) [16].5 of 34

Figure 5. Chemical structures of compounds, 27–32 [16]. Figure 5. Chemical structures of compounds, 27–32 [16]. Generally, compounds 28–30 inhibition of bacterial growth on all the three species was not Generally, compounds 28–30 inhibition of bacterial growth on all the three species was not significant when compared to the control (Table3). However, the compound 27 antibacterial effect significant when compared to the control (Table 3). However, the compound 27 antibacterial effect was significant on the S. aureus strain of bacteria studied when compared to the control, sodium was significant on the S. aureus strain of bacteria studied when compared to the control, sodium hypochlorite. The results of the compounds showed that modifications on the alkene group, phenyl hypochlorite. The results of the compounds showed that modifications on the alkene group, phenyl ringring and and carboxylic carboxylic acid acid functional functional groups groups a affectffectthe the biologicalbiological activity of of the the compounds compounds [16]. [16 ].

TableTable 3. Minimum3. Minimum inhibitory inhibitory concentration concentration (MIC) (MIC) of of representative representative biocides biocides andand derivativesderivatives against E. coliE., S. coli, aureus S. aureusand E.and hirae E. hirae[16]. [16]. Compounds E. coli (mM) S. aureus (mM) E. hirae (mM) Compounds E. coli (mM) S. aureus (mM) E. hirae (mM) Sodium hypochlorite 5 8 5 SodiumTriclosan hypochlorite 5 0.003 8 0.003 5 0.02 27 3 3–5 5–8 Triclosan 28 0.00320–25 0.003 >25 0.02 >25 27 29 3 15–203-5 15–20 5–8 20–25 30 15–20 15–25 18–25 28 20–25 ˃25 ˃25 31 >13 >13 >13 29 32 15–20> 25 15–20 >25 20–25 >25 30 15–20 15–25 18–25 Antimicrobial31 activities of chlorogenic˃13 acid (compound˃13 33) (Figure˃136) include antibacterial activity against32 multi-drug resistant bacteria,˃25 antifungal˃25 and anti-viral activities˃25 against HSV-1 and HSV-2, adenovirus, Ebola and HIV. Antifungal effects of compound 33 are known as it is effective againstAntimicrobialCandida albicans activitiesthrough of impactingchlorogenic the acid cell (compound membrane. 33 It) is(Figure effective 6) againstinclude Escherichiaantibacterial coli , Staphylococusactivity against aureus multi-drug, Staphylococcus resistant epidermidis bacteria, antifungaland Klebsiella and pneumonia anti-viral ,activities among otheragainst bacteria. HSV-1 and It has broadInt.HSV-2, J. applicationsMol. adenovirus,Sci. 2020, 21 in, x foodFOREbola PEER due and REVIEW to HIV. its insensitivity Antifungal effects against of probiotic compound bacteria 33 are [known26]. as it is effective6 of 37 against Candida albicans through impacting the cell membrane. It is effective against Escherichia coli, Staphylococus aureus, Staphylococcus epidermidis and Klebsiella pneumoniaR , among other bacteria. It has HO2C broad applications in food due to its insensitivity against probiotic bacteria [26]. O

O R

33 R R R R-OH FigureFigure 6. 6.Chemical Chemical structure of chlorogenic chlorogenic acid acid (33 (33) )[[26].26 ].

WangWang et et al. al. evaluated evaluated anti-Tobacco anti-Tobacco mosaicmosaic virus (TMV) activities activities of of synthesized synthesized cinnamic cinnamic acid acid derivativesderivatives containing containing dithioacetal dithioacetal moieties. moieties. OnlyOnly 34a andand 34b34b (Figure(Figure 7)7) displayed displayed curative curative activities activities ofof 82.6% 82.6% and and 83.5%, 83.5%, respectively respectively whichwhich werewere signiﬁcantsignificant when when compared compared to to ribavirin ribavirin (70.3%) (70.3%) and and xiangcaoliusuobingmixiangcaoliusuobingmi (71.7%), (71.7%), used used as as thethe controlcontrol [[27].27].

Figure 7. Cinnamic acid derivatives with dithioacetal moieties (34a and 34b) [27].

Compounds 34a and 34b antiviral activities were comparable to the activity of ningnanmycin (85.2%) used as the control. Treatment of TMV particles with 34b caused serious damage and fracture. Molecular docking results showed that 34b bonded excellently with amino acid residues GLN34, THR37, ARG90, and ARG46 of TMV coat protein via hydrogen bonds [27]. Despite the several reports on cinnamic acid derivatives, the compounds with potent antimicrobial activities are 3-5 which were effective against fungal strains such as C. lunatus, A. niger and P. ostreatus when compared to cinnamic acid [22]. Furthermore, compounds with high antibacterial activity are 20 which exhibited potent anti-TB activity when compared to cinnamic acid [24] and compound 27 which was effective on E. coli, S. aureus and E. hirae when compared to sodium hypochlorite [16].

3. Cinnamic Acid Derivatives with Anticancer Activity In 2018, WHO reported 18.1 million new cancer cases and 9.6 million deaths making cancer one of the leading causes of death [28]. The high number of people who die from cancer has led to researchers making unremitted efforts to develop safe and efficient antitumor agents [29]. The effectiveness of most of the currently used chemotherapeutic agents is severely limited by drug resistance [30]. Most drugs fail during invasion and metastasis of cancers making patients succumb to the disease [30]. Cinnamic acid derivatives have anticancer effects and are effective against a wide range of cancers such as breast [5] colon, lung, etc. Antiproliferative activity of cinnamic derivatives against tumors has been reported [31]. Cinnamic acid derivatives, such as cinnamaldehyde, use apoptosis, among other ways, to destroy cancerous cells [32]. Perkoic et al. synthesized harmicine and cinnamic acid hybrids (35–37) (Figure 8). In vitro cytotoxicity of the hybrids on a human liver hepatocellular carcinoma cell line (HepG2) depended on

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R HO2C O

O R 33 R R R R-OH Figure 6. Chemical structure of chlorogenic acid (33) [26].

Wang et al. evaluated anti-Tobacco mosaic virus (TMV) activities of synthesized cinnamic acid derivatives containing dithioacetal moieties. Only 34a and 34b (Figure 7) displayed curative activities of 82.6% and 83.5%, respectively which were significant when compared to ribavirin (70.3%) and Int.xiangcaoliusuobingmi J. Mol. Sci. 2020, 21, 5712 (71.7%), used as the control [27]. 6 of 34

Figure 7. Cinnamic acid derivatives with dithioacetal moieties (34a and 34b)[27]. Figure 7. Cinnamic acid derivatives with dithioacetal moieties (34a and 34b) [27]. Compounds 34a and 34b antiviral activities were comparable to the activity of ningnanmycin Compounds 34a and 34b antiviral activities were comparable to the activity of ningnanmycin (85.2%) used as the control. Treatment of TMV particles with 34b caused serious damage and fracture. (85.2%) used as the control. Treatment of TMV particles with 34b caused serious damage and fracture. Molecular docking results showed that 34b bonded excellently with amino acid residues GLN34, Molecular docking results showed that 34b bonded excellently with amino acid residues GLN34, THR37,THR37, ARG90, ARG90, and and ARG46ARG46 ofof TMVTMV coat protein via via hydrogen hydrogen bonds bonds [27]. [27 ]. DespiteDespite the the several several reports reports on cinnamic on cinnamic acid derivatives, acid derivatives, the compounds the compounds with potent with antimicrobial potent activitiesantimicrobial are 3– activities5 which wereare 3-5 eff whichective were against effective fungal against strains fungal suchas strainsC. lunatus such ,asA. C. niger lunatus,and A.P. ostreatusniger whenand comparedP. ostreatus to cinnamicwhen compared acid [22 ].to Furthermore, cinnamic acid compounds [22]. Furthermore, with high antibacterialcompounds activitywith high are 20 whichantibacterial exhibited activity potent are anti-TB 20 which activity exhibited when potent compared anti-TB to cinnamicactivity when acid compared [24] and compound to cinnamic27 acidwhich was[24] e ffandective compound on E. coli 27, S.which aureus wasand effectiveE. hirae onwhen E. coli compared, S. aureus and to sodium E. hirae when hypochlorite compared [16 to]. sodium hypochlorite [16]. 3. Cinnamic Acid Derivatives with Anticancer Activity 3. CinnamicIn 2018, WHO Acid Derivatives reported 18.1 with million Anticancer new cancer Activity cases and 9.6 million deaths making cancer one of the leadingIn 2018, causes WHO of reported death [28 18.1]. The million high new number cancer of peoplecases and who 9.6 die million from deaths cancer making has led cancer to researchers one makingof the leading unremitted causes eff ortsof death to develop [28]. The safe high and nu emberfficient of people antitumor who agents die from [29 ].cancer The has effectiveness led to ofresearchers most of the making currently unremitted used chemotherapeutic efforts to develop agents safe and is severely efficient limited antitumor by drugagents resistance [29]. The [ 30]. Mosteffectiveness drugs fail of during most invasionof the currently and metastasis used chemot of cancersherapeutic making agents patients is severely succumb limited to the diseaseby drug [ 30]. Cinnamicresistance acid [30]. derivatives Most drugs havefail during anticancer invasion effects andand metastasis are eff ectiveof cancers against making a wide patients range succumb of cancers suchto the as disease breast [ 5[30].] colon, Cinnamic lung, etc.acid Antiproliferativederivatives have anticancer activity of effects cinnamic and derivatives are effective against against tumors a wide has beenrange reported of cancers [31 ].such Cinnamic as breast acid [5] derivatives,colon, lung, etc. such Antiproliferativ as cinnamaldehyde,e activity use of apoptosis,cinnamic derivatives among other ways,against to destroytumors canceroushas been reported cells [32 ].[31]. Cinnamic acid derivatives, such as cinnamaldehyde, use apoptosis, among other ways, to destroy cancerous cells [32]. Perkoic et al. synthesized harmicine and cinnamic acid hybrids (35–37) (Figure8). In vitro Perkoic et al. synthesized harmicine and cinnamic acid hybrids (35–37) (Figure 8). In vitro cytotoxicity of the hybrids on a human liver hepatocellular carcinoma cell line (HepG2) depended cytotoxicity of the hybrids on a human liver hepatocellular carcinoma cell line (HepG2) depended on on the harmicine type and substituent in the cinnamic acid derivative. Compounds 36d, 36e and 36f were the most effective compounds against HepG2 cell line with IC50 values of 3.11, 2.19 and 0.74 µM, respectively, when compared to harmine with IC50 greater than 250 µM[33]. The increased cytotoxicity of compounds 36d, 36e and 36f is attributed to them being O-harmicines while the others are N-harmicines and N,O-bis-harmicines. However, no specific structure–activity relationship was reported [33]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 37

the harmicine type and substituent in the cinnamic acid derivative. Compounds 36d, 36e and 36f were the most effective compounds against HepG2 cell line with IC50 values of 3.11, 2.19 and 0.74 µM, respectively, when compared to harmine with IC50 greater than 250 µM[33]. The increased cytotoxicity of compounds 36d, 36e and 36f is attributed to them being O-harmicines while the others Int. J.are Mol. N Sci.-harmicines2020, 21, 5712 and N,O-bis-harmicines. However, no specific structure–activity relationship was 7 of 34 reported [33].

FigureFigure 8. 8.Structures Structures ofof cinnamic acid-harmine acid-harmine hybrids hybrids (35–37 (35)– [33].37)[ 33].

PelleritoPellerito et al.et al. synthesized synthesized ferulate ferulate derivative, tr tributyltin(IV)ibutyltin(IV) ferulate. ferulate. In vitroIn vitro studiesstudies on colon on colon cancercancer cells, cells, HCT116, HCT116, Caco-2 Caco-2 and and HT-29 HT-29 showedshowed that that the the ferulic ferulic acid acid did did not notalter alter the viability the viability of any of any of theof threethe three cell cell lines lines even even up up to to 1 1µ µMM concentrationconcentration while while tributyltin(IV) tributyltin(IV) ferulate ferulate showed showed remarkable remarkable cell viabilitycell viability reduction reduction at at 400 400 nM nM ( (–78.9%78.9% for HCT116, HCT116, –64.1%64.1% for forCaco-2 Caco-2 and and–57.5%57.5% for HT-29). for HT-29). Tributyltin(IV) ferulate induced autophagic− cell death and− arrested the cell cycle thereby− reducing Tributyltin(IV) ferulate induced autophagic cell death and arrested the cell cycle thereby reducing colon cancer cell viability. Thus, apart from it showing remarkable cell viability against the tested cell colon cancer cell viability. Thus, apart from it showing remarkable cell viability against the tested cell lines, it also showed a very significant effect at very low doses making the novel ferulic acid lines,derivative it also showed a promising a very colon significant cancer therapy effect at candidate very low [34]. doses making the novel ferulic acid derivative a promisingMatlou colon et al. cancer synthesized therapy zinc candidate and indium [34]. tetra cinnamic acid phthalocyanine (Pc) complexes (Matlou38–40) (Figure et al. synthesized9) which were zinc covalently and indium linked tetrathrough cinnamic an amide acid bond phthalocyanine to amino-functionalized (Pc) complexes (38–40magnetic) (Figure nanoparticles9) which were (AMNPs) covalently [35]. Cinnamic linked acid through was connected an amide to bond the nanoparticles to amino-functionalized due to its magneticantitumor nanoparticles activity [36]. (AMNPs) Compounds [35 38a–c]. Cinnamic were compared acid was with connected compounds to 39a–c the nanoparticles for photodynamic due to its antitumortherapy activity and in vitro [36]. cytotoxicity Compounds against38a–c humaweren comparedbreast adenocarcinoma with compounds cell lines.39a–c Compoundsfor photodynamic 38a, therapy39a, 38b and andin vitro39b tripletcytotoxicity quantum against yield improved human afte breastr linking adenocarcinoma to AMNPs but celldecreased lines. for Compounds 39c (0.65) 38a, and 38c (0.73) due to photoisomerization on the alkene double bond of cinnamic acid [37]. The triplet 39a, 38b and 39b triplet quantum yield improved after linking to AMNPs but decreased for 39c (0.65) lifetimes for complex 40 was shortened from 258 µs to 224 µs due to triplet energy lowering and non- and 38c (0.73) due to photoisomerization on the alkene double bond of cinnamic acid [37]. The triplet radiative decays increasing after linking AMNPs to the complex. Singlet oxygen quantum yields also µ µ lifetimesincreased for complexin the presence40 was of AMNPs shortened for 38a from and 258 39a ands to decreased 224 s due for 40 to. triplet energy lowering and non-radiative decays increasing after linking AMNPs to the complex. Singlet oxygen quantum yields also increased in the presence of AMNPs for 38a and 39a and decreased for 40. All the six compounds showed very low in vitro cytocidal effects in the dark with compound 38c showing the least cell viability at 78% at 80 µg/mL. This indicated that the used drug have low dark cytotoxicity. Photodynamic activity (PDT) of compounds 38a–c on MCF-7 cells increased as the drug concentration increased from 5–80 µg/mL and the cell death was evaluated based on the cell confluence of viable cells. PDT activity decreased for compounds 39a–c since phthalocyanine photoactivity decreased upon linking to AMNPs due to phthalocyanine complex aggregation. Compound 38b outperformed 38a and 38c due to spin-orbit coupling and efficient transfer of energy to ground state molecular oxygen, which gives it the significant cytocidal activity against the tumorigenic cells [35]. Lima et al. synthesized cinnamic acid derivatives having 1,2,3-triazolic moiety (41a–m and 42a–m) (Figure 10) and evaluated their antimetastatic activity. At 100 µmol/L, The compounds 41a, 41b, 41f, 41h–j, 41l, 41m, 42a–e, 42g–I and 42k–m significantly suppressed B16-F10 cell migration when compared to the cells treated with 0.4% DMSO. Compound 42a was the most effective with cell migration suppression of 86% when compared to the control. B16-F10 cell proliferation was also significantly impaired by compound 42a. There was a decrease in B16-F10 cell invasion and adhesion upon treatment of compound 42a for 24 h which confirmed the antimetastatic activity of cinnamic acid derivatives against colon carcinoma and human lung adenocarcinoma cells [38]. Int. J. Mol. Sci. 2020, 21, 5712 8 of 34 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 37

Figure 9. Chemical structures of zinc and indium tetra cinnamic acid phthalocyanine (Pc) complexes Figure 9. Chemical structures of zinc and indium tetra cinnamic acid phthalocyanine (Pc) complexes covalently linked to amino-functionalized magnetic nanoparticles (AMNPs) [35]. Int. J.covalently Mol. Sci. 2020 linked, 21, x FORto amino-functionalized PEER REVIEW magnetic nanoparticles (AMNPs) [35]. 9 of 37

All the six compounds showed very low in vitro cytocidal effects in the dark with compound 38c showing the least cell viability at 78% at 80 µg/mL. This indicated that the used drug have low dark cytotoxicity. Photodynamic activity (PDT) of compounds 38a–c on MCF-7 cells increased as the drug concentration increased from 5-80 µg/mL and the cell death was evaluated based on the cell confluence of viable cells. PDT activity decreased for compounds 39a–c since phthalocyanine photoactivity decreased upon linking to AMNPs due to phthalocyanine complex aggregation. Compound 38b outperformed 38a and 38c due to spin-orbit coupling and efficient transfer of energy to ground state molecular oxygen, which gives it the significant cytocidal activity against the tumorigenic cells [35]. FigureFigure 10. 10.Chemical Chemical structures of of compounds compounds 41a–m41a–m andand 42a–m42a–m [38]. [38]. Lima et al. synthesized cinnamic acid derivatives having 1,2,3-triazolic moiety (41a–m and 42a– m)B16-F10 (FigureB16-F10 10) cell cell and colonies colonies evaluated werewere their reduced reduced antimetastatic when when treate treated activity.d with with different At 100 diff erentconcentrationsµmol/L, concentrations The compounds of compound of compound41a, 42a 41b, 42a41f,at 12.5 12.541h-j, µmol/Lµ mol41l,/ L41m,(25.5%), (25.5%), 42a–e, 25 µmol/L 25 42g–Iµmol (20.6%)and/L (20.6%)42k–m and 50 significantly and µmol/L 50 µ (93.1%)mol suppressed/L (93.1%)when compared whenB16-F10 compared to cell the control.migration to theSince when control. Sincecomparedcell cell proliferation, proliferation, to the cellsmigration, migration,treated invasion, with invasion, 0.4% adhesion DMSO. adhesion and Compound colonization and colonization 42a are wasthe central the are most steps the effective central in the in stepswith vivo incell the in vivomigrationmetastasis metastasis suppression process process [39], of [the 3986%], positi the when positiveve resultscompared results exhibited to exhibited the bycontrol. compound by compoundB16-F10 42a cellsuggest42a proliferationsuggest its efficacy its was effi in cacyalso in reducingsignificantlyreducing B16-F10 B16-F10 impaired cells cells metastatic by metastatic compound potential.potential. 42a. There Vero Vero was and and afibroplast decrease fibroplast (NIH3T3) in (NIH3T3)B16-F10 cells cell cells appeared invasion appeared somewhatand adhesion somewhat sensitive to compound 42a at 100 µmol/L and no hemolysis was significant at different sensitiveupon treatment to compound of compound42a at 100 42aµmol for/ L24 and h which no hemolysis confirmed was the significant antimetastatic at di ffactivityerent concentrations. of cinnamic concentrations. Molecular binding prediction indicated that the compound 42a inhibitory ability is Molecularacid derivatives binding against prediction colon indicated carcinoma that and the human compound lung42a adenocarcinomainhibitory ability cells is [38]. due to its interaction due to its interaction with MMP-9 and MMP-2, enzymes involved directly in melanoma progression with[40] MMP-9 The heteroatoms and MMP-2, in enzymescompound involved 42a have directlythe capability in melanoma to form hydrogen progression bonds [40 with] The proteins heteroatoms in compoundthereby inhibiting42a have the the two capability enzymes [10]. to form hydrogen bonds with proteins thereby inhibiting the two enzymesWang [10]. et al. designed and synthesized novel oleanolic acid (OA)-cinnamic acid ester derivatives (43a–dWang and et al. 44a–o designed) and glycyrrhetinic and synthesized acid (GA)-cinnamic novel oleanolic acid acid ester (OA)-cinnamic derivatives (45a–p acid) ester(Figure derivatives 11) (43a–dandand assessed44a–o their) and in vitro glycyrrhetinic cytotoxicity acid against (GA)-cinnamic MCF-7 (breast acid cancer), ester HeLa derivatives (cervical cancer) (45a–p )and (Figure L- 11) andO2 assessed (a normal their hepaticin vitro cell)cytotoxicity [5]. against MCF-7 (breast cancer), HeLa (cervical cancer) and L-O2 (a normal hepatic cell) [5].

Figure 11. Chemical structures of the novel novel oleanolic acid (OA)-cinnamic acid ester derivatives (43a-d and 44a-o) and glycyrrhetinic acid (GA)-cinnamic acid ester derivatives (45a-p) [5].

Based on the Table 4 below with selected compounds, compound 45c exhibited the most significant cytotoxicity on L-O2 cells. The strong inhibitory activity (1.79 µM) against MCF-7 cells shown by compound 44e suggests that linking 4-methylcinnamic acid to the C-OH position of OA- 2-chlorobenzyl ester has the ability to significantly improve the inhibition of MCF-7 cells. Compound

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 37

Figure 10. Chemical structures of compounds 41a–m and 42a–m [38].

B16-F10 cell colonies were reduced when treated with different concentrations of compound 42a at 12.5 µmol/L (25.5%), 25 µmol/L (20.6%) and 50 µmol/L (93.1%) when compared to the control. Since cell proliferation, migration, invasion, adhesion and colonization are the central steps in the in vivo metastasis process [39], the positive results exhibited by compound 42a suggest its efficacy in reducing B16-F10 cells metastatic potential. Vero and fibroplast (NIH3T3) cells appeared somewhat sensitive to compound 42a at 100 µmol/L and no hemolysis was significant at different concentrations. Molecular binding prediction indicated that the compound 42a inhibitory ability is due to its interaction with MMP-9 and MMP-2, enzymes involved directly in melanoma progression [40] The heteroatoms in compound 42a have the capability to form hydrogen bonds with proteins thereby inhibiting the two enzymes [10]. Wang et al. designed and synthesized novel oleanolic acid (OA)-cinnamic acid ester derivatives (43a–d and 44a–o) and glycyrrhetinic acid (GA)-cinnamic acid ester derivatives (45a–p) (Figure 11) Int.and J. Mol. assessed Sci. 2020 their, 21, 5712 in vitro cytotoxicity against MCF-7 (breast cancer), HeLa (cervical cancer) and9 L- of 34 O2 (a normal hepatic cell) [5].

Figure 11. Chemical structures of the novel novel oleanolic acid (OA)-cinnamic acid ester derivatives (43a–dFigureand 11.44a–o Chemical) and structures glycyrrhetinic of the acidnovel (GA)-cinnamic novel oleanolic acid acid ester (OA)-cinnamic derivatives acid (45a–p ester)[ derivatives5]. (43a-d and 44a-o) and glycyrrhetinic acid (GA)-cinnamic acid ester derivatives (45a-p) [5]. Based on the Table4 below with selected compounds, compound 45c exhibited the most significant cytotoxicityBased onon L-O2 the Table cells. 4 The below strong with inhibitory selected activitycompounds, (1.79 compoundµM) against 45c MCF-7 exhibited cells the shown most by compoundsignificant44e cytotoxicitysuggests that on linkingL-O2 cells. 4-methylcinnamic The strong inhibitory acid to activity the C-OH (1.79 position µM) against of OA-2-chlorobenzyl MCF-7 cells shown by compound 44e suggests that linking 4-methylcinnamic acid to the C-OH position of OA- ester has the ability to significantly improve the inhibition of MCF-7 cells. Compound 44o was 2-chlorobenzyl ester has the ability to significantly improve the inhibition of MCF-7 cells. Compound the most potent compound against HeLa cells with IC50 of 1.35 µM, close to that of the control, gefitinib (1.28 µM) [5].

Table 4. IC50 values of oleanolic acid (OA), glycyrrhetinic acid (GA) and their derivatives towards L-O2, MCF-7 and HeLa cells [5].

IC50 (µM) IC50 (µM) Compounds Compounds L-O2 MCF-7 HeLa L-O2 MCF-7 HeLa OA 32.9 >100 >100 44m >100 38.89 56.29 GA >100 64.16 98.99 44o >100 >100 1.35 43d >100 32.49 1.55 45c 10.19 >100 >100 43a >100 >100 >100 45e >100 36.84 >100 43e >100 1.79 >100 45f >100 >100 >100 44h >100 >100 >100 45i >100 46.53 >100

Compounds 44a, 44h and 44f could not inhibit any of the three cell lines. The structure–activity relationships showed no clear trend when the substituent on the benzyl of GA was either electron-donating or electron-withdrawing. However, the electron-donating group (CH3) on the ortho position of the benzyl group on OA increased the inhibitory effect of the compounds on HeLa cells. Compounds 43d and 44o were further evaluated due to their cytotoxic effect against HeLa cells and they were found to induce apoptosis and increased reactive oxygen species levels in a dose-dependent manner. Both compound 43d and 44o induced autophagy in HeLa cells [5]. Endo et al. examined autophagy-inducing activity of cinnamic acid derivatives in Brazilian green propolis: p-coumaric acid (46), caffeic acid (47), chlorogenic acid (33), drupanin (48), 3,4-caffeoylquinic acid (3,4-CQA) (49), artepillin C (ArtC) (50), baccharin (51) and caffeic acid phenethyl ester (CAPE) (52) (Figure 12) by subjecting them to castration-resistant prostate cancer (CRPC) CWR22R1 cells. Compounds 33, 46, 47, and 48 were less cytotoxic but compounds 49–52 significantly decreased prostate cancer [41]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 37

44o was the most potent compound against HeLa cells with IC50 of 1.35 µM, close to that of the control, gefitinib (1.28 µM) [5].

Table 4. IC50 values of oleanolic acid (OA), glycyrrhetinic acid (GA) and their derivatives towards L- O2, MCF-7 and HeLa cells [5].

IC50 (µM) Compounds IC50 (µM) Compounds L-O2 MCF-7 HeLa L-O2 MCF-7 HeLa

OA 32.9 ˃100 ˃100 44m ˃100 38.89 56.29 GA ˃100 64.16 98.99 44o ˃100 ˃100 1.35 43d ˃100 32.49 1.55 45c 10.19 ˃100 ˃100 43a ˃100 ˃100 ˃100 45e ˃100 36.84 ˃100 43e ˃100 1.79 ˃100 45f ˃100 ˃100 ˃100 44h ˃100 ˃100 ˃100 45i ˃100 46.53 ˃100 Compounds 44a, 44h and 44f could not inhibit any of the three cell lines. The structure–activity relationships showed no clear trend when the substituent on the benzyl of GA was either electron- donating or electron-withdrawing. However, the electron-donating group (CH3) on the ortho position of the benzyl group on OA increased the inhibitory effect of the compounds on HeLa cells. Compounds 43d and 44o were further evaluated due to their cytotoxic effect against HeLa cells and they were found to induce apoptosis and increased reactive oxygen species levels in a dose- dependent manner. Both compound 43d and 44o induced autophagy in HeLa cells [5]. Endo et al. examined autophagy-inducing activity of cinnamic acid derivatives in Brazilian green propolis: p-coumaric acid (46), caffeic acid (47), chlorogenic acid (33), drupanin (48), 3,4- caffeoylquinic acid (3,4-CQA) (49), artepillin C (ArtC) (50), baccharin (51) and caffeic acid phenethyl ester (CAPE) (52) (Figure 12) by subjecting them to castration-resistant prostate cancer (CRPC) Int.CWR22R1 J. Mol. Sci. 2020 cells., 21 Compounds, 5712 33, 46, 47, and 48 were less cytotoxic but compounds 49-52 significantly10 of 34 decreased prostate cancer [41].

Figure 12. Chemical structures of the cinnamic acid derivatives in propolis components [41]. Figure 12. Chemical structures of the cinnamic acid derivatives in propolis components [41]. CWR22Rv1 cells viability after 24 h treatment revealed compound 52 to be the most potent, even killing 90% of the cells at 30 µM dosage. Compounds 50 and 51 significantly increased LC3-II levels, showing high autophagy-inducing activity and compound 50 was more potent than 51. Further examinations showed that compound 46 induced autophagy through mechanisms different from lysosomal enzymes inhibition [41]. Choudhari et al. also reported the in vitro potency of Indian stingless bee propolis as anticancer agents [42]. Treatment of A549 cells with compound 52 (50 µM) significantly decreased, in a dose-dependent manner, the expression of claudin-2, a protein highly expressed in most cancer cells [43]. Compound 52 up-regulated lκB levels and inhibited lκB phosphorylation with no effect on Akt, thus decreasing the transcriptional activity of claudin-2 in A549 cells. Compound 52 enhanced the sensitivity of the cells to anticancer agents such as doxorubicin by increasing the paracellular permeability to anticancer agents. Shimizu and Suzuki reported the ability of compound 52 to reduce the inflammation stage in macrophages, supporting the results of Endo and his colleagues [44]. Positive results have been reported for bioavailability and pharmacokinetics of compound 52 in rodents but they still remain unclear in humans [45]. Cotreatment of CWR22Rv1 cells with compound 50 and wortmannin or U0126, which are autophagy inhibitors, greatly decreased the viable cells and significantly increased lactose dehydrogenase release when compared to treating with compound 50 alone indicating the enhancement of the cytotoxic potency of compound 50 by autophagy inhibition. The authors also reported the involvement of necroptosis in the cell death when treated with compound 50 and autophagy inhibitor. Thus based on their findings, compound 50 induces autophagy and apoptosis in CWR22Rv1 cells and most importantly, autophagy inhibition further increases apoptosis and induces necrosis [41]. Ge et al. designed and synthesized novel cinnamic acid-progenone hybrids (53a–o) (Figure 13) and evaluated their biological activity as potential antiproliferative agents against A549, H157, HepG2, MCF-7, and HL-60 cell lines (Table5)[31]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 37

CWR22Rv1 cells viability after 24 h treatment revealed compound 52 to be the most potent, even killing 90% of the cells at 30 µM dosage. Compounds 50 and 51 significantly increased LC3-II levels, showing high autophagy-inducing activity and compound 50 was more potent than 51. Further examinations showed that compound 46 induced autophagy through mechanisms different from lysosomal enzymes inhibition [41]. Choudhari et al. also reported the in vitro potency of Indian stingless bee propolis as anticancer agents [42]. Treatment of A549 cells with compound 52 (50 µM) significantly decreased, in a dose-dependent manner, the expression of claudin-2, a protein highly expressed in most cancer cells [43]. Compound 52 up-regulated lκB levels and inhibited lκB phosphorylation with no effect on Akt, thus decreasing the transcriptional activity of claudin-2 in A549 cells. Compound 52 enhanced the sensitivity of the cells to anticancer agents such as doxorubicin by increasing the paracellular permeability to anticancer agents. Shimizu and Suzuki reported the ability of compound 52 to reduce the inflammation stage in macrophages, supporting the results of Endo and his colleagues [44]. Positive results have been reported for bioavailability and pharmacokinetics of compound 52 in rodents but they still remain unclear in humans [45]. Cotreatment of CWR22Rv1 cells with compound 50 and wortmannin or U0126, which are autophagy inhibitors, greatly decreased the viable cells and significantly increased lactose dehydrogenase release when compared to treating with compound 50 alone indicating the enhancement of the cytotoxic potency of compound 50 by autophagy inhibition. The authors also reported the involvement of necroptosis in the cell death when treated with compound 50 and autophagy inhibitor. Thus based on their findings, compound 50 induces autophagy and apoptosis in CWR22Rv1 cells and most importantly, autophagy inhibition further increases apoptosis and induces necrosis [41]. Ge et al. designed and synthesized novel cinnamic acid-progenone hybrids (53a–o) (Figure 13) and evaluated their biological activity as potential antiproliferative agents against A549, H157, Int.HepG2, J. Mol. Sci. MCF-7,2020, 21 and, 5712 HL-60 cell lines (Table 5) [31]. 11 of 34

FigureFigure 13. 13.Chemical Chemical structures structures of of designeddesigned andand synthesizedsynthesized novel novel cinnamic cinnamic acid-progenone acid-progenone hybrids hybrids (53a–o(53a-o))[31 [31].].

CompoundsCompounds53f 53fand and53g 53gwere werethe the mostmost potentpotent compounds agai againstnst all all the the five ﬁve cancer cancer cell cell lines lines (Table(Table5). 5). Based Based on on the the IC IC5050values values ofof compoundscompounds 53b–53g53b–53g, ,3-OH 3-OH or or 4-OH 4-OH increased increased the the potency potency of of thethe compounds compounds while while methylation methylation of of thethe hydroxylhydroxyl or changing changing its its position position decreased decreased the the compound compound activityactivity especially especially towards towards A549 A549 and andHL-60 HL-60 cancercancer cell lines. lines. Antiproliferative Antiproliferative activity activity also also decreased decreased uponupon introducing introducing another another methoxyl methoxyl groupgroup ((53h53h) or dialkylation of of hydroxyl hydroxyl (53i–53l)(53i–53l) indicatingindicating the the rolerole played played by by 3-OH 3-OH and and 4-OH 4-OH on on the the phenyl phenyl ring ring on on the the compound’s compound’s activity activity whichwhich waswas observedobserved in 53min being53m being more more active active than than53n 53nand and53o 53o[31 ].[31].

Table 5. In vitro antiproliferative activity of compounds 53a–o as IC50 (µM) [31].

Cpd. MCF-7 A549 H157 HepG2 HL-60 Cpd. MCF-7 A549 H157 HepG2 HL-60 53a >50 >50 >50 >50 >50 53i >50 >50 >50 >50 >50 53b >50 >50 3.72 5.46 >50 53j >50 >50 >50 >50 10.19 53c 4.16 >50 24.50 6.55 >50 53k >50 >50 >50 >50 >50 53d 8.85 30.98 21.33 22.59 15.68 53l >50 >50 >50 5.5 >50 53e >50 >50 24.54 3.81 >50 53m 6.55 36.20 30.67 >50 11.26 53f 4.91 3.22 4.28 3.45 6.87 53n >50 >50 >50 >50 >50 53g 6.46 5.16 3.77 8.86 3.51 53o >50 >50 >50 3.10 >50 53h 6.11 >50 17.98 5.22 25.64 Dox 1.51 0.31 0.48 1.82 0.91

At 50 µM, all the compounds exhibited inhibition less than 50% on two non-tumor cell lines, BEAS2B and MCF-10A, thus the hybrids were considered to be selectively toxic towards the cancer cell lines compared to the non-tumor cells. Cell cycle results showed 53f arrested the cell progression in G1 phase and enhanced apoptosis in A549 cells, the regulation of Bax/Bcl-2 expression by 53f contributing to the latter [31]. After reviewing several cinnamamides and their structure–activity relationships in anticancer activity, Gaikwad et al. deduced that the compounds were more active when the ortho group remained unsubstituted but electron-donating groups were tolerated (Scheme1). Electron-withdrawing groups such as NO2, CN and CF3 at the para position were important for potency and selectivity with tert-Bu and OCH3 being exceptions since they are electron-donating groups contributed to the activity of the compounds. The presence of electron-donating groups such as OCH3, vinyl and NH-R at the meta position was preferred for anticancer activity. For the R2 substitution, heterocyclic and aromatic amines signiﬁcantly elevated anticancer activity while aliphatic amines showed relatively low potency [25]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 37

Table 5. In vitro antiproliferative activity of compounds 53a-o as IC50 (µM) [31].

Cpd. MCF-7 A549 H157 HepG2 HL-60 Cpd. MCF-7 A549 H157 HepG2 HL-60

53a ˃50 ˃50 ˃50 ˃50 ˃50 53i ˃50 ˃50 ˃50 ˃50 ˃50

53b ˃50 ˃50 3.72 5.46 ˃50 53j ˃50 ˃50 ˃50 ˃50 10.19

53c 4.16 ˃50 24.50 6.55 ˃50 53k ˃50 ˃50 ˃50 ˃50 ˃50

53d 8.85 30.98 21.33 22.59 15.68 53l ˃50 ˃50 ˃50 5.5 ˃50

53e ˃50 ˃50 24.54 3.81 ˃50 53m 6.55 36.20 30.67 ˃50 11.26

53f 4.91 3.22 4.28 3.45 6.87 53n ˃50 ˃50 ˃50 ˃50 ˃50

53g 6.46 5.16 3.77 8.86 3.51 53o ˃50 ˃50 ˃50 3.10 ˃50

53h 6.11 ˃50 17.98 5.22 25.64 Dox 1.51 0.31 0.48 1.82 0.91

At 50 µM, all the compounds exhibited inhibition less than 50% on two non-tumor cell lines, BEAS2B and MCF-10A, thus the hybrids were considered to be selectively toxic towards the cancer cell lines compared to the non-tumor cells. Cell cycle results showed 53f arrested the cell progression in G1 phase and enhanced apoptosis in A549 cells, the regulation of Bax/Bcl-2 expression by 53f contributing to the latter [31]. After reviewing several cinnamamides and their structure–activity relationships in anticancer activity, Gaikwad et al. deduced that the compounds were more active when the ortho group remained unsubstituted but electron-donating groups were tolerated (Scheme 1). Electron- withdrawing groups such as NO2, CN and CF3 at the para position were important for potency and selectivity with tert-Bu and OCH3 being exceptions since they are electron-donating groups contributed to the activity of the compounds. The presence of electron-donating groups such as OCH3, vinyl and NH-R at the meta position was preferred for anticancer activity. For the R2 Int. J. Mol. Sci. 2020, 21, 5712 12 of 34 substitution, heterocyclic and aromatic amines significantly elevated anticancer activity while aliphatic amines showed relatively low potency [25]. O O R2 OH N H R1 R2 NH2 54

Scheme 1. General synthetic route for cinnamamide. Reagents and conditions: POCl3, TEA, DCM, 0 Scheme 1: General synthetic route for cinnamamide. Reagents and conditions: (a) POCl3, TEA, DCM, ◦C[0o25C ].[25]. Most anticancer agents used to treat hepatocellular carcinoma suﬀer from drug resistance [46]. Most anticancer agents used to treat hepatocellular carcinoma suffer from drug resistance [46]. In order to overcome drug resistance, Ling et al. developed β-carboline and N-hydroxycinnamamide In order to overcome drug resistance, Ling et al. developed β-carboline and N-hydroxycinnamamide compounds (55a–p) (Figure 14)[30]. All the compounds, 55a–p, inhibited histone deacetylase 1 compounds (55a–p) (Figure 14) [30]. All the compounds, 55a–p, inhibited histone deacetylase 1 (HDAC1),(HDAC1), akey a key HDAC HDAC subtype subtype shown shown in cancer in cellcancer diﬀ erentiationcell differentiation and proliferation and proliferation [30]. Compound [30]. 55p displayed the best potency with IC of 1.3 Nm when compared to suberoylanilide hydroxamic acid Compound 55p displayed the best potency50 with IC50 of 1.3 Nm when compared to suberoylanilide (SAHA)hydroxamic (IC50 =acid142 (SAHA) nM), an (IC approved50 = 142 nM), HDAC an approved inhibitor. HDAC The presence inhibitor. of The an presence electron-donating of an electron- group suchdonating as the Rgroup group such such as asthe methoxyl R group such (55g–j as) ormethoxyl more than (55g–j one) or methoxyl more than groups one methoxyl (5m–p) enhancedgroups inhibitoryInt.(5m–p J. Mol.) enhancedSci. potency 2020, 21 compared, inhibitoryx FOR PEER toREVIEWpotency compounds compared with to a hydrogencompounds (55a,b with) a or hydrogen methyl ( 55c,d(55a,b)[)30 or]. methyl 13 of 37 (55c,d) [30].

Figure 14. Chemical structures of compounds 55a–p [30]. Figure 14. Chemical structures of compounds 55a-p [30]. Antiproliferative activities of the compounds against human colon cancer cells (HCT116), humanAntiproliferative hepatocellular carcinomaactivities of (HCC) the compound cells (HepG2s against and human SMMC-7721), colon cancer and human cells (HCT116), lung cancer human hepatocellular carcinoma (HCC) cells (HepG2 and SMMC-7721), and human lung cancer cells cells (H1299) showed that 55e, 55g–I and 55o,p (IC50 = 0.41–4.29 µM) showed better antiproliferative (H1299) showed that 55e, 55g–I and 55o,p (IC50 = 0.41-4.29 µM) showed better antiproliferative potency when compared to SAHA (IC50 = 4.79–7.02 µM) (Table6). Compound 55p displayed the best potency when compared to SAHA (IC50 = 4.79-7.02 µM) (Table 6). Compound 55p displayed the best antiproliferative eﬀect against drug-sensitive Bel7402 and drug-resistant Bel7402/5-FU with IC50 values antiproliferative effect against drug-sensitive Bel7402 and drug-resistant Bel7402/5-FU with IC50 of 0.85 and 2.09 µM, respectively. Compound 55p induced better Bel7402/5-FU cell apoptosis than values of 0.85 and 2.09 µM, respectively. Compound 55p induced better Bel7402/5-FU cell apoptosis SAHAthan SAHA together together with autophagic with autophagic ﬂux activity flux activity in Bel7402 in Bel7402/5-FU/5-FU cells cells by down-regulating by down-regulating p62 p62 and and LC3-1 andLC3-1 up-regulating and up-regulating LC3-II proteins, LC3-ΙΙ proteins, making making it a potential it a potential candidate candidate in developing in developing anticancer anticancer agents to treatagents multi-drug to treat multi-drug resistant human resistant cancer human [30 cancer]. [30].

Table 6. IC values of compounds 55g,h, 55k,l, and 55o,p against drug-sensitive HCC Bel7402 and Table 6. IC5050 values of compounds 55g,h, 55k,l, and 55o,p against drug-sensitive HCC Bel7402 and drug-resistantdrug-resistant HCC HCC Bel7402 Bel7402/5-FU/5-FU cellscells [[30].30].

IC50(µM) Compounds IC50(µM) Compounds Bel7402 Bel7402/5-FU SAHABel7402 4.72 Bel7402/5-FU 9.83 5-FU 15.6 61.7 SAHA 55g 4.72 1.039.83 2.17 5-FU 55h 15.6 2.1561.7 2.90 55k 3.24 4.38 55g 55l 1.03 2.712.17 3.06 55h 55o 2.15 1.972.90 3.66 55p 0.85 2.09 55k 3.24 4.38 55l 2.71 3.06 Substituted cinnamic acyl sulfonamide derivatives (56a–58e) (Scheme2) were synthesized and 55o 1.97 3.66 screened for their antiproliferative activity on MCF-7 human breast cancer cells. Based on Table7, 55p 0.85 2.09 Substituted cinnamic acyl sulfonamide derivatives (56a–58e) (Scheme 2) were synthesized and screened for their antiproliferative activity on MCF-7 human breast cancer cells. Based on Table 7, compound 56a displayed the best activity (IC50 = 0.17 µg/mL) when compared to the control, colchicine [47].

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compound 56a displayed the best activity (IC50 = 0.17 µg/mL) when compared to the control, colchicineInt. J. Mol. Sci. [47 2020]. , 21, x FOR PEER REVIEW 14 of 37

Scheme 2. General synthesis of cinnamic acid sulfonamide derivatives (56a–58e). Reagents and Scheme 2: General synthesis of cinnamic acid sulfonamide derivatives (56a–58e). Reagents and conditions: (i) K2CO3, acetone, reﬂux, 29 h; (ii) piperidine, pyridine, 80–90 ◦C, 24 h; (iii) substituted conditions: (i) K2CO3, acetone, reflux, 29h; (ii) piperidine, pyridine, 80–90 °C, 24h; (iii) substituted benzene-sulfonamides, Dichloromethane (DCM), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide benzene-sulfonamides, Dichloromethane (DCM), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 4-Dimethylaminopyridine (DMAP), overnight [47]. (EDCI), 4-Dimethylaminopyridine (DMAP), overnight [47]. Compound 56a had no substituent on B phenyl ring but had a benzdioxan group on the A ring Compound 56a had no substituent on B phenyl ring but had a benzdioxan group on the A ring meaning the presence of a substituent on the B phenyl ring decreased the antiproliferative activity meaning the presence of a substituent on the B phenyl ring decreased the antiproliferative activity of ofthe the compound. compound. Tubulin Tubulin polymerization polymerization assay assay showed showed that that compound compound 56a56a waswas the themost most potent potent compoundcompound at at inhibiting inhibiting tubulintubulin polymerization inin vitro. vitro .Molecular Molecular docking docking revealed revealed that that compound compound 56a56aformed formed one one hydrogen hydrogen bondbond andand Van der Waals Waals forces forces with with tubulin tubulin [47]. [47 ].

Table 7. AntiproliferativeAntiproliferative activities activities an andd anti-tubulin anti-tubulin activities activities of 56a of 56a-58e– 58eagainstagainst MCF-7 MCF-7 cells cells[47]. [47].

Cpd. n R MCF-7 IC50 (µg/mL) ITP IC50 (µM) Cpd. n R MCF-7 IC50 (µg/mL) ITP IC50 (µM) n R MCF-7 IC50 ITP IC50 Cpd. n R MCF-7 IC50 ITP IC50 56a 1 H 0.17 0.88 57d 2 Cl 51.31 87.92 Cpd.56b 1 CH3 48.95 40.85 57e 2 Br >100 >100 56c 1 F(µg/mL) 74.05(µM) 76.90 58a 3 H(µg/mL) 53.87 (µM) 49.01 56d 1 Cl 47.27 94.72 58b 3 CH3 >100 >100 56e 1 Br 89.01 51.59 58c 3 F >100 >100 56a57a 1 2H H 0.17 2.16 0.88 5.47 57d 58d 32 Cl Cl 51.31 >100 87.92>100 57b 2 CH >100 93.33 58e 3 Br >100 >100 56b 1 CH3 348.95 40.85 57e 2 Br ˃100 ˃100 57c 2 F 52.13 27.46 Colchicine 0.33 1.36 56c 1 F 74.05 76.90 58a 3 H 53.87 49.01 56d Toolabi1 Cl et al. 47.27 synthesized 6-Cinnamoyl-4-arylaminothienopyrimidines 94.72 58b 3 CH3 ˃100 and compounds˃100 59e and 59g56e (Figure1 Br15 ) were 89.01 very effective 51.59 against A549 58c and HeLa cell3 F lines [48˃100]. Compound 59e˃100exhibited in57a vitro antiproliferative2 H 2.16 activity against 5.47 both A549 58d and HeLa3 cellCl lines˃100 with IC50 values˃100 of 0.04 and 0.004 µM, respectively. Compound 59g had an IC50 of 0.033 µM against HeLa cells. Both compounds 57b 2 CH3 ˃100 93.33 58e 3 Br ˃100 ˃100 were better than the reference drug, erlotinib with IC50 values of 3.80 and 7.48 µM against A549 and HeLa 57c 2 F 52.13 27.46 Colchicine 0.33 1.36 cells, respectively. Compound 59e was further found to inhibit EGFR and ERH1/2 phosphorylation in Toolabi et al. synthesized 6-Cinnamoyl-4-arylaminothienopyrimidines and compounds 59e and HeLaInt. cell J. Mol. lines Sci. [202048]., 21, x FOR PEER REVIEW 15 of 37 59g (Figure 15) were very effective against A549 and HeLa cell lines [48]. Compound 59e exhibited in vitro antiproliferative activity against both A549 and HeLa cell lines with IC50 values of 0.04 and 0.004 µM, respectively. Compound 59g had an IC50 of 0.033 µM against HeLa cells. Both compounds were better than the reference drug, erlotinib with IC50 values of 3.80 and 7.48 µM against A549 and HeLa cells, respectively. Compound 59e was further found to inhibit EGFR and ERH1/2 phosphorylation in HeLa cell lines [48].

FigureFigure 15. 15.Structure Structure ofof compounds 59e59e andand 59g59g [48].[48 ].

Yang et al. synthesized a number of cinnamic acid derivatives and found only compound 60 (Figure 16) which exhibited 25.78% cell viability when tested against leukemia (HL-60) cells at 20 µM for 48h [49]. The structure–cytotoxic activity relationship revealed that the presence of a trimethoxyl group on the phenyl ring, an ethylenediamine linker, inserting aryl substitutes at the C-α position and the presence of α,β-unsaturated ketone carbonyl substituent are all beneficial for the cytotoxicity of the compound. Compound 60 showed better uptake in HL-60 cells than in normal cells, with the mitochondria being the main site of accumulation. At 10 and 15 µM, compound 60 mainly induced apoptosis while at 20 µM it was elicited necrosis of the HL-60 cells [49].

Figure 16. Chemical structure of compound 60 [49].

The potent derivatives with anticancer activities which need further studies are 36d, 36e, 36f, 43e, 56a, 43d, 44o, 55e and 55g–p. Cinnamic acid derivatives with potent anticancer activity against liver cancer are compounds 36d, 36e and 36f [33]. Compound 43e and 56a were potent on MCF-7 cell lines in vitro [5,47] and compounds 43d, 44o, 55e and 55g were active against cervical cancer cell lines when compared to other derivatives which have been reported [5,48]. Compounds 55g–55p were effective on human hepatocellular carcinoma cell line drug resistant cancer cell lines [30].

4. Cinnamic Acid Derivatives with Antiparasitic Activity Parasitic diseases such as Psoroptic acariasis, malaria and leishmaniasis remain a major threat to public health especially to millions of people living in poor tropical countries [50]. In 2018, an estimated 405,000 people died of malaria, a parasitic infection calling for more measures to reduce the number of fatalities [51]. Resistance to broad spectrum anthelmintics in livestock helminths is a global threat to livestock such as sheep and goats [50]. The effects caused by these parasitic diseases on humans and livestock has led to the development of cinnamic acid derivatives with antiparasitic activity. Perkovic et al. synthesized hybrids of harmine and cinnamic acid (35a–37i) (Figure 8) for antiplasmodial evaluation against the erythrocytic stage of Plasmodium falciparum (chloroquine- sensitive Pf3D7 and chloroquine-resistant PfDd2 strains) and hepatic stage of P. berghei as well as cytotoxicity against HepG2 cell line. From Table 8, compounds 36c, 36g, 36h and 37b (IC50 = 0.13-0.16 µM) were the most active against Pf3D7 strain with 37b exhibiting the most potency against Pf3D7 and PfDd2. Results on harmicines activity against P. berghei showed that they were more effective with (IC50 0.4–2.62 µM) when compared to the reference drug, primaquine (IC50 = 8.4 µM). Conjugating harmine with cinnamic acid derivatives via triazole linker produced hybrids with

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 37

Int. J. Mol. Sci. 2020, 21, 5712 14 of 34 Figure 15. Structure of compounds 59e and 59g [48]. Yang et al. synthesized a number of cinnamic acid derivatives and found only compound 60 (Figure Yang16) which et al. exhibitedsynthesized 25.78% a number cell viability of cinnami whenc acid tested derivatives against leukemiaand found (HL-60) only compound cells at 20 60µ M (Figure 16) which exhibited 25.78% cell viability when tested against leukemia (HL-60) cells at 20 µM for 48h [49]. The structure–cytotoxic activity relationship revealed that the presence of a trimethoxyl for 48h [49]. The structure–cytotoxic activity relationship revealed that the presence of a trimethoxyl group on the phenyl ring, an ethylenediamine linker, inserting aryl substitutes at the C-α position group on the phenyl ring, an ethylenediamine linker, inserting aryl substitutes at the C-α position and the presence of α,β-unsaturated ketone carbonyl substituent are all beneﬁcial for the cytotoxicity and the presence of α,β-unsaturated ketone carbonyl substituent are all beneficial for the cytotoxicity of the compound. Compound 60 showed better uptake in HL-60 cells than in normal cells, with the of the compound. Compound 60 showed better uptake in HL-60 cells than in normal cells, with the µ mitochondriamitochondria being being the the main main site site of of accumulation.accumulation. At 10 10 and and 15 15 µM,M, compound compound 6060 mainlymainly induced induced apoptosisapoptosis while while at at 20 20µ MµM it it was was elicited elicited necrosis necrosis ofof thethe HL-60 cells [49]. [49].

FigureFigure 16. 16.Chemical Chemical structure of compound compound 6060 [49].[49].

TheThe potent potent derivatives derivatives with with anticancer anticancer activities activities which which need need further further studies studies are are36d 36d, 36e, 36e, 36f, 36f, 43e, , 56a43e, 43d, 56a, 44o, 43d, 55e, 44oand, 55e55g–p and .55g–p Cinnamic. Cinnamic acid derivativesacid derivatives with with potent potent anticancer anticancer activity activity against against liver cancerliver arecancer compounds are compounds36d, 36e 36d,and 36e36f and[33 36f]. Compound[33]. Compound43e and 43e and56a were56a were potent potent on MCF-7on MCF-7 cell cell lines in vitrolines in[5 ,vitro47] and [5,47] compounds and compounds43d, 44o 43d, 55e, 44o,and 55e55g andwere 55g were active active against against cervical cervical cancer cancer cell linescell lines when comparedwhen compared to other derivativesto other derivatives which have whic beenh have reported been reported [5,48]. Compounds[5,48]. Compounds55g–55p 55g–55pwere e ﬀwereective oneffective human hepatocellularon human hepatocellular carcinoma carcinoma cell line drug cell line resistant drug resistant cancer cell cancer lines cell [30 lines]. [30].

4. Cinnamic4. Cinnamic Acid Acid Derivatives Derivatives with with AntiparasiticAntiparasitic Activity ParasiticParasitic diseases diseases such such as asPsoroptic Psoropticacariasis, acariasis, malaria and and leishmaniasis leishmaniasis remain remain a amajor major threat threat to to publicpublic health health especially especially to millionsto millions of people of people living liv ining poor in tropicalpoor tropical countries countries [50]. In [50]. 2018, In an 2018, estimated an 405,000estimated people 405,000 died ofpeople malaria, died a of parasitic malaria, infection a parasiti callingc infection for more calling measures for more to measures reduce the to number reduce of fatalitiesthe number [51]. Resistanceof fatalities to[51]. broad Resistance spectrum to broad anthelmintics spectrum in anthelmintics livestock helminths in livestock is a helminths global threat is a to global threat to livestock such as sheep and goats [50]. The effects caused by these parasitic diseases livestock such as sheep and goats [50]. The eﬀects caused by these parasitic diseases on humans and on humans and livestock has led to the development of cinnamic acid derivatives with antiparasitic livestock has led to the development of cinnamic acid derivatives with antiparasitic activity. activity. Perkovic et al. synthesized hybrids of harmine and cinnamic acid (35a–37i) (Figure8) for Perkovic et al. synthesized hybrids of harmine and cinnamic acid (35a–37i) (Figure 8) for antiplasmodial evaluation against the erythrocytic stage of Plasmodium falciparum (chloroquine-sensitive antiplasmodial evaluation against the erythrocytic stage of Plasmodium falciparum (chloroquine- Pf3D7sensitive and chloroquine-resistantPf3D7 and chloroquine-resistant PfDd2 strains) PfDd2 and strains) hepatic and stage hepatic of P. stage berghei of asP. wellberghei as as cytotoxicity well as against HepG2 cell line. From Table8, compounds 36c, 36g, 36h and 37b (IC = 0.13–0.16 µM) were cytotoxicity against HepG2 cell line. From Table 8, compounds 36c, 36g, 36h and50 37b (IC50 = 0.13-0.16 theµM) most were active the againstmost active Pf3D7 against strain Pf3D7 with strain37b exhibiting with 37b theexhibiting most potency the most against potencyPf3D7 againstand Pf3D7PfDd2 . Resultsand PfDd2 on harmicines. Results on activity harmicines against activityP. berghei againstshowed P. berghei that showed they werethat they more were effective more witheffective (IC 50 0.4–2.62with (ICµM)50 when0.4–2.62 compared µM) when to the compared reference drug,to the primaquine reference drug, (IC50 =primaquine8.4 µM). Conjugating (IC50 = 8.4 harmineµM). withConjugating cinnamic acidharmine derivatives with cinnamic via triazole acid linkerderivatives produced via triazole hybrids linker with produced excellent antiplasmodialhybrids with activity when compared to the parent compounds at the erythrocytic and hepatic stages of Plasmodium infection [33]. Structure–activity relationship showed that the general activity pattern was 36 > 37 > 35 except for the p-OCH3 substituted series which had 37b as the most potent compound. For series 35 and 37, the bulkiness of R2 enhanced their antiplasmodial activity in vitro. Replacing hydrogen with ﬂuorine on the m- and p- positions of the cinnamic acid ring resulted in compounds with enhanced antiplasmodial activity for series 35 and 36 (for example 35e > 35a, 35d > 35a) but not for series 37 [33]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 37

excellent antiplasmodial activity when compared to the parent compounds at the erythrocytic and hepatic stages of Plasmodium infection [33].

Table 8. IC50 (µM) values for compounds 35–37 after in vitro screening against erythrocytic stage of P. falciparum (Pf3D7 and PfDd2 strains) and hepatic stages of P. berghei [33].

Cpd Pf3D7 PfDd2 P. berghei Cpd Pf3D7 PfDd2 P. berghei

35a 1.26 23.76 2.62 36g 0.15 0.69 - 35b 0.61 16.61 - 36h 0.13 0.46 - 35c 0.50 1.57 - 36i 0.33 3.06 0.66 Int.35d J. Mol. Sci. 20200.89, 21 , 5712 2.70 - 37a 0.56 1.59 - 15 of 34 35e 0.87 2.21 - 37b 0.13 0.16 0.4

35f Table 8. IC0.6550 ( µM) values2.55 for compounds- 35–37 after37cin vitro screening0.44 against0.65 erythrocytic - stage of 35g P. falciparum0.44(Pf3D7 and1.8 PfDd27 strains)1.12 and hepatic stages37d of P. berghei2.50[33 ]. 3.23 - 35h Cpd0.56 Pf3D7 2.26 PfDd2 P.1.10 berghei Cpd37e Pf3D70.62 PfDd28.00 P. - berghei 35i 35a 0.771.26 2.82 23.76 - 2.62 37f36g 0.151.07 17.94 0.69 - - 36a 35b 0.260.61 1.09 16.61 - - 3736hg 0.130.25 3.28 0.46 - - 35c 0.50 1.57 - 36i 0.33 3.06 0.66 36b 35d 0.430.89 0.69 2.70 - - 37i37a 0.561.34 4.02 1.59 - - 36c 35e 0.160.87 0.4 2.217 - - C37bQ 0.130.003 0.20 0.16 - 0.4 35f 0.65 2.55 - 37c 0.44 0.65 - 36d 35g 0.200.44 1.19 1.87 - 1.12 Harmine37d 2.508.25 ˃ 3.2327.7 - - 36e 35h 0.200.56 0.84 2.26 - 1.10 Prima37e quinee 0.62- - 8.00 8.4 - 35i 0.77 2.82 - 37f 1.07 17.94 - 36f 36a 0.340.26 1.01 1.09 - - 37g 0.25 3.28 - Structure–activity36b 0.43 relationship 0.69 showed - that the general37i activity1.34 pattern was 4.02 36˃37˃35 except - for 36c 0.16 0.47 - CQ 0.003 0.20 - the p-OCH3 substituted series which had 37b as the most potent compound. For series 35 and 37, the 36d 0.20 1.19 - Harmine 8.25 >27.7 - bulkiness of R2 enhanced their antiplasmodial activity in vitro. Replacing hydrogen with fluorine on 36e 0.20 0.84 - Primaquinee - - 8.4 the m-36f and p-0.34 positions 1.01of the cinnamic - acid ring resulted in compounds with enhanced antiplasmodial activity for series 35 and 36 (for example 35e ˃ 35a, 35d ˃ 35a) but not for series 37 [33]. RodriguesRodrigues etet al.al. synthesized cinnamic cinnamic acid acid derivatives derivatives (61–63 (61–63) (Figure) (Figure 17) 17 and) and tested tested their their leishmanicidalleishmanicidal activity activity against against promastigotepromastigote and and amastigote amastigote forms forms of of LeishmaniaLeishmania braziensis braziensis. At. 10 At µM, 10 µ M, allall the the derivatives derivatives with with anan isobenzofuranoneisobenzofuranone moiety moiety (compounds (compounds 61a–d)61a–d were) were toxic toxic to promastigotes to promastigotes withwith survival survival less less thanthan 75%.75%. Compounds Compounds 62a-–d62a–d werewere not not toxic toxic to to promastigotes promastigotes of L. of braziliensisL. braziliensis. . CompoundsCompounds with with a a triazolic triazolic moiety moiety (63a–d (63a–d) revealed) revealed that that only only compound compound63a 63aand and63c 63cwere were toxic toxic with 80%with and 80% 75% and survival. 75% survival. Compound Compound63b was 63b the was most the activemost active and its and activity its activity was similar was similar to the to positive the control,positive amphotericin control, amphotericin B (3.125 µ Bg (3.125µg/mL)/mL) (Table9)[ (Table3]. 9) [3].

Figure 17. Chemical structures of compounds 61–63 (a–d)[3]. Figure 17. Chemical structures of compounds 61-63 (a–d) [3]. When treated against amastigote forms, compounds 61b, 62b and 62d were the most active with 80.47%, 86.32% and 80.12% toxicity, respectively, which was lower than amphotericin B (98.25%). Compounds 61b and 62b were further assayed because of their non-toxic nature to macrophages and their IC50 values were 7.58 µM and 3.05 µM, respectively, when tested in vitro on the promastigote forms [3].

Table 9. % toxicity of the compounds on Leishmania intracellular amastigotes after 48 h. All compounds were 10 µM[3].

Compounds % Toxicity Cpd. % Toxicity 61a 43.87 62c 59.10 61b 80.47 62d 80.12 61c 59.53 63a 45.10 61d 50.14 63b 67.84 62a 19.91 63c 69.24 62b 86.32 63d 19.67 Int. J. Mol. Sci. 2020, 21, 5712 16 of 34

These findings suggest that the presence of an electron-withdrawing group on the phenyl ring of cinnamic acid enhanced the compound’s anti-leishmanicidal activity. Halogenation of the phenyl ring of the triazole moiety plays an important role on the anti-leishmanicidal activity of the compounds. The results showed that the combination of two different bioactive chemical groups can yield effective novel anti-leishmanicidal drugs [3]. Mancilla-Montelongo et al. evaluated the in vitro anthelmintic (AH) activity of cinnamic acid (1) and its six derivatives (p-coumaric acid (46), caffeic acid (47), trans-ferulic acid (64), trans-sinapic acid (65), 3,4-dimethoxycinnamic acid (66) and chlorogenic acid (51) (Figure 18) against the larvae and eggs of a gastrointestinal nematode, Haemonchus contortus. Only ferulic acid and chlorogenic acid showed notable AH activity >74% and >39% egg inhibition, respectively, at 300 µg/mL. Although compounds 1, 46 and 47 showed no egg hatching inhibition even at 400 µg/mL, they damaged the larvae structurally, leadingInt. J. Mol. to theirSci. 2020 mortality, 21, x FOR [PEER15]. REVIEW 18 of 37

Figure 18. Chemical structures of cinnamic acid with in vitro anthelmintic (AH) activity [15]. Figure 18. Chemical structures of cinnamic acid with in vitro anthelmintic (AH) activity [15]. The authors also made mixtures (M) of the analogues. M1 was a combination of all the seven The authors also made mixtures (M) of the analogues. M1 was a combination of all the seven analogues.analogues. M2 M2 was was a a combination combination ofof compounds 6464 andand 3333 whichwhich blocked blocked larval larval hatching. hatching. M3 M3was wasa a combinationcombination of compounds 1,1 ,46,46 ,64,64 65, 65 andand 3333. M4. M4 was was a combination a combination of compounds of compounds 1, 46 1and, 46 65and 65 and the the combination combination resulted resulted in inthe the larval larval damage. damage. M5, M5,which which is a combination is a combination of compounds of compounds 1 and 1 and46,46 exhibited, exhibited significant significant ovicidal ovicidal activity activity.. M1 M1 exhibited exhibited an an effective effective concentration concentration 50% 50% (EC (EC50)50 of) of 1628.101628.10µg µg/mL,/mL, M4 M4 (1143.75 (1143.75µ gµg/mL)/mL) and and M3 M3 (923.37 (923.37 µµg/mL).g/mL). M2 and M5 M5 showed showed no no significant significant EC EC50 50 valuevalue revealing revealing an an antagonistic antagonistic e ffeffect.ect. The The presencepresence of two free free hydroxyl hydroxyl groups groups in in compound compound 4747 diddid notnot produce produce any any significant significant e ffeffectect of of the the compound compound activeactive against H.H. contortus contortus eggseggs suggesting suggesting that that thethe activity activity of of the the compounds compounds is is based based on on theirtheir abilityability to inhibit inhibit hatching-rel hatching-relatedated processes processes and and their their abilityability to to form form a complexa complex film film by by binding binding to to thethe proteinsproteins [[15].15]. ChenChen et et al. al. synthesized synthesized a series a series of cinnamic of cinnamic acid derivatives acid derivatives (67–79) (and67– 79evaluated) and evaluated their efficacy their effiagainstcacy against PsoroptesPsoroptes cuniculi cuniculi, a non-burrowing, a non-burrowing ear mite ear causing mite causing PsoropticPsoroptic acariasisacariasis (Figure (Figure19). The 19 ). Thesynthesized synthesized cinnamic cinnamic acid acid derivatives derivatives included included thiol thiol esters, esters, esters, esters, ketones, ketones, amides amides and and analogues analogues withwith a heteroaromatica heteroaromatic ring. ring. TheThe structure–activitystructure–activity relationship relationship of of the the derivatives derivatives was was studied. studied. Compounds exhibiting higher activity were further studied for median lethal concentrations (LC50) and median lethal times (LT50) in comparison with the reference drug, ivermectin [52].

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Compounds exhibiting higher activity were further studied for median lethal concentrations (LC50) andInt. median J. Mol. Sci. lethal 2020, 21 times, x FOR (LT PEER50) REVIEW in comparison with the reference drug, ivermectin [52]. 19 of 37

FigureFigure 19. 19.Chemical Chemical structuresstructures of compounds 67–7967–79 [52].[52].

BasedBased on on Table Table 10 10,, compounds compounds67 67-75–75 andand 79 had a mean mortality mortality of of 84% 84% and and above above with with only only compoundscompounds77 77and and78 78exhibiting exhibiting mean mean mortalitymortality below 40% at at 0.5 0.5 mg/mL. mg/mL. These These compounds compounds were were potentpotent when when compared compared to ivermectin,to ivermectin, an acaricidalan acaricidal agent agent due due to factors to factors such such as lower as lower molecular molecular weight andweight simpliﬁed and structure,simplified astructure,ﬀordability affordability and non-toxicity and non-toxicity to mammals to and mammals higher activityand higher [52,53 activity]. All the compounds[52,53]. All displayed the compounds LC50 values displayed higher LC50 than values ivermectin. higher than ivermectin.

TableTable 10. 10.Mean Mean mortality mortality percentages percentages of ofPsoroptes Psoroptes cuniculicuniculi when incubated incubated with with 0.5 0.5 mg/mL mg/mL and and 0.25 0.25 mgmg/mL/mL of of di ﬀdifferenterent compounds compounds [52 [52].].

Mean Mortality % Mean Mortality % LC50 [mM] (24 h) LT50(h) Cpd. LC50 [mM] (24 h) LT50 (h) Cpd. 0.5 mg/mL 0.25 mg/mL 0.5 mg/mL 0.25 mg/mL 67 100.0 78.3 1.07 13.3 68 100.0 98.3 0.56 12.0 67 69 100.0 90.078.3 76.0 1.07 0.87 13.9 13.3 70 84.0 68.0 0.74 15.2 68 71 100.0 100.098.3 100.0 0.56 0.45 9.8 12.0 69 72 90.0 100.076.0 80.0 0.87 0.82 15.6 13.9 73 100.0 84.0 0.64 6.8 70 74 84.0 100.068.0 100.0 0.74 0.36 2.6 15.2 75 98.0 66.0 71 76 100.0 65.0100.0 16.7 0.45 9.8 77 38.3 - 72 78 100.0 26.080.0 - 0.82 15.6 79 94.0 44.0 73 Ivermectin100.0 75.084.0 68.3 0.64 0.28 8.9 6.8 74 100.0 100.0 0.36 2.6

75The cinnamic acid98.0 esters (67–70), the66.0 type of alkyl group, the length of the chain and the degree of branching greatly aﬀected their biological activity. The compounds with potent biological activity were76 compound 68, 7165.0, 73 and 74. Steric16.7 hindrance close to the ester group decreased the activity of the77 compounds. The38.3 conversion of a- carbonyl on the ester group to a methylene group led to a notable decrease in activity as seen when comparing 68 with 78. Activity decreased when ester (68) was78 changed to thiol ester26.0 (79). Replacing- the phenyl group (68) with 2-pyridinyl (71 ) or 2-furanyl 79 94.0 44.0

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Ivermectin 75.0 68.3 0.28 8.9

The cinnamic acid esters (67–70), the type of alkyl group, the length of the chain and the degree of branching greatly affected their biological activity. The compounds with potent biological activity Int. J. Mol. Sci. 2020, 21, 5712 18 of 34 were compound 68, 71, 73 and 74. Steric hindrance close to the ester group decreased the activity of the compounds. The conversion of a carbonyl on the ester group to a methylene group led to a notable (74decrease) increased in theactivity activity as seen [52]. when Based comparing on the diﬀ 68erent with reports 78. Activity on the decreased antiparasitic when activity ester of(68 cinnamic) was derivatives,changed to the thiol potent ester cinnamic(79). Replacing acid derivativesthe phenyl group with ( antimalarial68) with 2-pyridinyl activity ( are71) or36c 2-furanyl, 36g, 36h (74)and 37bincreased[33]. the activity [52]. Based on the different reports on the antiparasitic activity of cinnamic derivatives, the potent cinnamic acid derivatives with antimalarial activity are 36c, 36g, 36h and 37b 5. Cinnamic[33]. Acid Derivatives with Neurological Activity

5. CinnamicThe brain, Acid an organ Derivatives of soft with nervous Neurological tissue is containedActivity in the skull of vertebrates. It functions as the coordinating centre of sensation, intellectual and nervous activity [54]. When the brain is damaged,The itbrain, can an aff ectorgan many of soft diff nervouserent things, tissue is including contained memory, in the skull sensation, of vertebrates. and even It functions personality. as A numberthe coordinating of brain diseasescentre of and sensation, neurological intellectua disordersl andhave nervous been activity found which[54]. When include the brain brain tumor, is damaged, it can affect many different things, including memory, sensation, and even personality. A traumatic brain injury and Alzheimer’s disease, and these need to be treated for the well-functioning number of brain diseases and neurological disorders have been found which include brain tumor, of the brain [55]. The treatment of Alzheimer’s disease is of prime importance since the disease is traumatic brain injury and Alzheimer’s disease, and these need to be treated for the well-functioning characterized by memory loss, cognitive impairment and can be fatal. Treatment methods include of the brain [55]. The treatment of Alzheimer’s disease is of prime importance since the disease is restoring acetylcholine levels [56] and clearing the neurotoxic amyloid-beta protein [1]. Research has characterized by memory loss, cognitive impairment and can be fatal. Treatment methods include provenrestoring that acetylcholine cinnamic acid levels derivatives [56] and have clearing neuroprotective the neurotoxic ability amyloid-beta and scientists protein keep [1]. Research on looking has for neuroprotectiveproven that cinnamic agents acid based derivatives on cinnamic have acid neuropro with bettertective biological ability and activity scientists [6 ].keep on looking for neuroprotectiveLan et al. reacted agentsN -benzylbased on pyridinium cinnamic acid with with cinnamic better biological acids of diactivityfferent [6]. substitutions to obtain cinnamicLan acid et al. derivatives reacted N-benzyl (80a–n) pyridinium (Scheme3) thatwith inhibit cinnamic cholinesterase, acids of different suggesting substitutions potential to obtain e fficacy in treatingcinnamic Alzheimer’s acid derivatives disease. (80a–n Compounds) (Scheme 3)80a–n that inhibitwere highlycholineste selectiverase, suggesting towards acetylcholinesterace potential efficacy fromin electrictreating eel (AChE)Alzheimer’s and butylcholinesteracedisease. Compounds from 80a–n equine serumwere (BuChE).highly selective However, towards they were moreacetylcholinesterace selective towards from AChE electric when eel compared (AChE) and to BuChE.butylcholinesterace Compound from80l was equine effective serum at (BuChE). inhibiting AChEHowever, with ICthey50 of were 12.1 more nM whileselective compound towards AChE80n had when the compared highest inhibitory to BuChE. activity Compound against 80l BuChE was (ICeffective50 = 1.9 nM).at inhibiting The low AChE cinnamic with acid IC50 inhibitoryof 12.1 nM activity while compound of IC50 > 100 80n nM had revealed the highest the requirementinhibitory of theactivity N-benzyl against pyridium BuChE (IC moiety50 = 1.9 for nM). enhanced The low activity cinnamic [11 ].acid inhibitory activity of IC50 ˃ 100 nM revealed the requirement of the N-benzyl pyridium moiety for enhanced activity [11].

SchemeScheme 3. 3:Synthetic Synthetic route route for for compounds compounds 80a–n. 80a–n. Reagents Reagents and and conditions: conditions: (i)(i) DMAPDMAP/EDCl,/EDCl, DCM, rt,

12rt, h; (ii)12h:(ii) CH 3CHCN,3CN, reﬂux, reflux, 1–3 1-3h h [11 [11].].

The methoxy groups present at positions 3 and 4 of the N-benzyl pyridium moiety increased the inhibitory activity of the compound which was significant in compounds 80i–n when compared to 80b–g. Activity decreased upon the introduction of fluorine, methyl and bromine on the meta, para position (for example, compound 80a was more potent than 80g for AChE). Compounds with different substituents (80i–n) had better activity against BuChE which was observed in compound 80h and compounds with a Br substitution showed higher potency (80f, 80g, 80l and 80m) (Table 11). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 21 of 37

The methoxy groups present at positions 3 and 4 of the N-benzyl pyridium moiety increased the inhibitory activity of the compound which was significant in compounds 80i–n when compared to 80b–g. Activity decreased upon the introduction of fluorine, methyl and bromine on the meta, para position (for example, compound 80a was more potent than 80g for AChE). Compounds with different substituents (80i–n) had better activity against BuChE which was observed in compound Int. J. Mol. Sci. 2020, 21, 5712 19 of 34 80h and compounds with a Br substitution showed higher potency (80f, 80g, 80l and 80m) (Table 11). The compounds’ ability to inhibit BuChE was affected more by the substituent size when compared Theto compounds’the electronic ability properties. to inhibit Compound BuChE 80l was had aﬀ aected dual moreinhibitory by the potency substituent and further size when studies compared showed to thethat electronic it exhibited properties. good neuroprotective Compound 80l activityhad aagainst dual inhibitoryAβ1-42 toxicity potency in PC12 and cells further and it studiescan penetrate showed into the brain, which was observed in PAMPA-BBB assay in vitro making compound 80l a leading that it exhibited good neuroprotective activity against Aβ1-42 toxicity in PC12 cells and it can penetrate intonovel the compound brain, which for wasthe treatment observed of in Alzheimer’s PAMPA-BBB disease assay [11].in vitro making compound 80l a leading novel compound for the treatment of Alzheimer’s disease [11]. Table 11. Inhibition concentration (IC50) of ChEs activity in nM [11].

Table 11. Inhibition concentration (IC ) of ChEs activity in nM [11]. Compound eeAChE eqBuChE Cpd.50 eeAChE eqBuChE Compound eeAChE eqBuChE Cpd. eeAChE eqBuChE 80a 54.1 6.3 80i 51.4 2.7 80a 54.1 6.3 80i 51.4 2.7 80b80b 102.6102.6 8.1 8.1 80j 80j 90.7 90.73.8 3.8 80c80c 1263.81263.8 25.0 25.0 80k 80k 29.6 29.63.1 3.1 80d 93.5 7.4 80l 12.1 2.6 80d 93.5 7.4 80l 12.1 2.6 80e 90.8 10.0 80m 92.6 2.5 80f80e 153.690.8 10.0 2.5 80m 80n 92.6 50.32.5 1.9 80g 1450.5 2.1 Cinnamic acid >100,000 >100 80f 153.6 2.5 80n 50.3 1.9 80h 135.2 4.5 Donepezil 40.2 4.5 80g 1450.5 2.1 Cinnamic acid ˃100 000 ˃100 80h 135.2 4.5 Donepezil 40.2 4.5 Ghafary et al. designed cinnamic acid-tryptamide hybrids (81a–l) (Scheme4) and evaluated their inhibitoryGhafary activity et al. against designed acetylcholinesterace cinnamic acid-tryptamide (AChE) hybrids and butylcholinesterace (81a–l) (Scheme 4) (BChE) and evaluated (Table 12their)[56 ]. Theinhibitory presence activity of either against an electron-donating acetylcholinesterace or an(AChE) electron-withdrawing and butylcholinesterace group (BChE) on position (Table 4 of12) the unsubstituted[56]. The presence tryptamine of either derivatives an electron-donating (81a–f) reduced or an electron-withdrawing the AChE inhibitory group activity on position and the 4 same of effectthe wasunsubstituted observed tryptamine in compounds derivatives containing (81a–f) any reduced substituent the AChE on the inhibitory 4-position activity of the pendantand the same benzyl groupeffect in was the observed 5-methoxytryptamine in compounds containing series (81g–l any), su exceptbstituent for on81i the. Replacing4-position of fluorine the pendant with benzyl different electron-withdrawinggroup in the 5-methoxytryptamine groups greatly improvedseries (81g–l), BChE except inhibition for 81i meaning. Replacing inhibition fluorine of BChEwith different potentially electron-withdrawing groups greatly improved BChE inhibition meaning inhibition of BChE depended more on hydrophobic or hydrogen interactions between the chloro or nitro groups with the potentially depended more on hydrophobic or hydrogen interactions between the chloro or nitro BChE active site than on the electron-withdrawing effect [56]. groups with the BChE active site than on the electron-withdrawing effect [56].

Scheme 4. Synthetic route for compounds 81a–l. Reagents and conditions: (i) K2CO3/DMF/80 ◦C, (ii) Scheme 4: Synthetic route for compounds 81a–l. Reagents and conditions: (i) K2CO3/DMF/800C, (ii) pyrrolidine/pyridine/EtOH/reﬂux, (iii) EDCl/HOBt/CH3CN/rt [56]. pyrrolidine/pyridine/EtOH/reflux, (iii) EDCl/HOBt/CH3CN/rt [56].

Table 12. Cholinesterace inhibitory activity IC50 (µM) of compounds 81a–l [56].

Cpd R1 R2 AChE BChE Cpd R1 R2 AChE BChE

81a H H 14.93 2.12 81h F OCH3 29.67 5.89 81b F H 32.17 9.36 81i Cl OCH3 13.42 2.86 81c Cl H 24.7 0.89 81j NO2 OCH3 31.45 4.28 81d NO2 H 34.49 0.55 81k CH3 OCH3 31.85 12.25 81e CH3 H 39.1 7.73 81l OCH3 OCH3 25.11 7.69 81f OCH3 H 34.95 2.51 Donepezil 0.027 7.79 81g H OCH3 14.18 6.63 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 22 of 37 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 22 of 37 For compounds 81g–l, the presence of an electron-withdrawing group (81i; 81j and 81h) enhancedFor compounds the anti-BChE 81g–l, activity the presencewith 81g–j of andan electron-withdrawing81l being more potent group than the(81i; standard 81j and drug, 81h) donepezil.enhanced theCompound anti-BChE 81d activity displayed with excellent 81g–j andneuroprotective 81l being more ability potent on PC12 than neuron the standard cells at 10 drug, µM withdonepezil. cell viability Compound of 62.71. 81d Moleculardisplayed excellentdocking studiesneuroprotective showed that abil ity81d on inhibited PC12 neuron BChE cells via aat mixed- 10 µM typewith modecell viability of inhibition of 62.71. [56]. Molecular docking studies showed that 81d inhibited BChE via a mixed- type mode of inhibition [56].

Table 12. Cholinesterace inhibitory activity IC50 (µM) of compounds 81a–l [56]. Table 12. Cholinesterace inhibitory activity IC50 (µM) of compounds 81a–l [56].

Cpd R1 R2 AChE BChE Cpd R1 R2 AChE BChE Cpd R1 R2 AChE BChE Cpd R1 R2 AChE BChE

81a H H 14.93 2.12 81h F OCH3 29.67 5.89 81a H H 14.93 2.12 81h F OCH3 29.67 5.89 Int. J. Mol.81b Sci. 2020 F, 21, 5712 H 32.17 9.36 81i Cl OCH3 13.42 2.86 20 of 34 81b F H 32.17 9.36 81i Cl OCH3 13.42 2.86 81c Cl H 24.7 0.89 81j NO2 OCH3 31.45 4.28 81c Cl H 24.7 0.89 81j NO2 OCH3 31.45 4.28 For compounds81d NO2 81g–lH , the presence34.49 0.55 of an electron-withdrawing81k CH3 groupOCH3 ( 81i31.85; 81j and 12.2581h ) enhanced 81d NO2 H 34.49 0.55 81k CH3 OCH3 31.85 12.25 the anti-BChE81e CH activity3 H with 81g–j39.1 and7.7381l being81l more potentOCH than3 OCH the3 standard25.11 drug, 7.69 donepezil. 81e CH3 H 39.1 7.73 81l OCH3 OCH3 25.11 7.69 Compound81f 81dOCHdisplayed3 H excellent34.95 neuroprotective2.51 Donep abilityezil on PC12 neuron0.027 cells at7.79 10 µ M with cell viability of81f 62.71. OCH Molecular3 H docking34.95 studies2.51 showedDonep thatezil81d inhibited BChE0.027 via a mixed-type7.79 mode 81g H OCH3 14.18 6.63 of inhibition81g [56H]. OCH3 14.18 6.63 InIn a a recent recent study, study, Medvedeva Medvedeva et et al. al. reported reported the the ability ability of of 3-methoxy-4-hydroxycinnamic 3-methoxy-4-hydroxycinnamic acid acid ( 64), (64), In3,4- a dimethoxycinnamicrecent study, Medvedeva acid ( 66et )al. and reported 3-methoxy-4-acetamidoxycinnamic the ability of 3-methoxy-4-hydroxycinnamic acid (82) (Figure acid 20) 3,4- dimethoxycinnamic acid (66) and 3-methoxy-4-acetamidoxycinnamic acid (82) (Figure 20)[57] to [57](64), to3,4- prevent dimethoxycinnamic alpha-synuclein acid amyloid (66) and transforma 3-methoxy-4-acetamidoxycinnamiction, thus aiding the treatment acid (of82 )Parkinson’s (Figure 20) prevent alpha-synuclein amyloid transformation, thus aiding the treatment of Parkinson’s disease [58]. disease[57] to prevent[58]. The alpha-synuclein IC50 values of the amyloid compounds transforma were tion,251 µMthus for aiding 64, 50 the µM treatment for 82 and of 13 Parkinson’s µM for 2. The IC values of the compounds were 251 µM for 64, 50 µM for 82 and 13 µM for 2. The compounds Thedisease compounds50 [58]. The wereIC50 values also non-toxic of the compounds towards humawere n251 neuroblastoma µM for 64, 50 cellµM linefor 82making and 13 them µM forvery 2. were also non-toxic towards human neuroblastoma cell line making them very promising for treating promisingThe compounds for treating were synucleinopathiesalso non-toxic towards [57]. human neuroblastoma cell line making them very synucleinopathiespromising for treating [57]. synucleinopathies [57].

FigureFigure 20.20. Chemical structures structures of of compounds compounds 6464, 66, 66 andand 82 82[57,58].[57,58 ]. Figure 20. Chemical structures of compounds 64, 66 and 82 [57,58]. ChenChen et et al. al. evaluatedevaluated the ability ability of of new new tacrine-cinn tacrine-cinnamicamic acid acid hybrids hybrids in inhibiting in inhibiting cholinesterase cholinesterase asas potential potentialChen et therapeutics therapeutics al. evaluated forfor the thethe ability treatmenttreatment of new of of tacrine-cinn Alzheimer’s Alzheimer’samic disease. disease. acid hybridsOnly Only 83,83 in 84, inhibiting84 andand 85 85(Figure cholinesterase(Figure 21) 21were) were chosenchosenas potential to to validate validate therapeutics theirtheir inhibitoryinhibitory for the trea activitiestment of onAlzheimer’s on human human cholinesterases cholinesterases disease. Only 83,(AChE (AChE 84 and and and 85 BuChE). (Figure BuChE). 21)TheThe were IC50 IC 50 valuesvalueschosen of of to the the validate three three compounds theircompounds inhibitory on on human humanactivities AChE AChE on human andand BuChE,BuChE, cholinesterases respectively,respectively, (AChE werewere and 10.210.2 BuChE). nMnM and The 6.3 IC nM50 for 83forvalues, 16.5 83, nM16.5of the andnM three 5.7and nMcompounds 5.7 fornM84 forand 84 on and 15.7human 15.7 nM AChEnM and and 8.0 and 8.0 nM BuChE, nM for for85 .respectively,85 Binding. Binding patterns patterns were 10.2 of of compounds nMcompounds and 6.3 83nM 83and 84andforwith 83 84, cholinesterases16.5with nM cholinesterases and 5.7 showednM for showed 84π -andπ stacking π 15.7-π stacking nM interactions, and interactions, 8.0 nM for hydrogen 85 hydr. Bindingogen bonds bondspatterns and and van of vancompounds der der Waals Waalsforces. 83 and 84 with cholinesterases showed π-π stacking interactions, hydrogen bonds and van der Waals Atforces. 25 µM, At the 25 µM, three the compounds three compounds inhibited inhibited Aβ1–42 Aaggregationβ1–42 aggregation with with an inhibitory an inhibitory rate betweenrate between 31.82% forces. At 25 µM, the three compounds inhibited Aβ1–42 aggregation with an inhibitory rate between and31.82% 34.57% and when 34.57% compared when compared to resveratrol to resveratrol (30.36%), (30.36%), a reference a reference compound compound [59]. [59]. 31.82% and 34.57% when compared to resveratrol (30.36%), a reference compound [59].

FigureFigure 21.21. Chemical structures structures of of novel novel tacrine-cinnamic tacrine-cinnamic acid acid hybrids hybrids [59]. [59 ]. Figure 21. Chemical structures of novel tacrine-cinnamic acid hybrids [59].

Behavioural studies revealed that all the three compounds, 83–85 showed enhanced cognitive function in the Institute of Cancer Research (ICR) mice in vivo. Administration of the three compounds caused no remarkable morphological liver changes making them safe and promising compounds for the treatment of Alzheimer’s disease [59]. Lan et al. reported on ferulic acid derivatives (86a–o) (Figure 22) as multi-target inhibitors against Alzheimer’s disease. All the ferulic acid derivatives inhibited eeAChE and eqBuChE with IC50 ranging from 0.055 µM to 42.26 µM (Table 13). When evaluated on human ChE (hChE), compounds 86f–h were potent against both hAChE and hBuChE with inhibitory activity higher than the reference compound, donepezil (Table 13)[60]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 37

Behavioural studies revealed that all the three compounds, 83-85 showed enhanced cognitive function in the Institute of Cancer Research (ICR) mice in vivo. Administration of the three compounds caused no remarkable morphological liver changes making them safe and promising compounds for the treatment of Alzheimer’s disease [59]. Lan et al. reported on ferulic acid derivatives (86a–o) (Figure 22) as multi-target inhibitors against Alzheimer’s disease. All the ferulic acid derivatives inhibited eeAChE and eqBuChE with IC50 ranging from 0.055 µM to 42.26 µM (Table 13). When evaluated on human ChE (hChE), compounds 86f–h were potent against both hAChE and hBuChE with inhibitory activity higher than the reference Int.compound, J. Mol. Sci. 2020 donepezil, 21, 5712 (Table 13) [60]. 21 of 34

Figure 22. Chemical structures of compounds 86a–o [60]. Figure 22. Chemical structures of compounds 86a–o [60]. The presence of the methoxyl and hydroxyl groups favored the inhibition of Aβ aggregation. The presence of the methoxyl and hydroxyl groups favored the inhibition of Aβ aggregation. Kinetic studies using compound 86g showed competition for the binding site between the compounds Kinetic studies using compound 86g showed competition for the binding site between the and acetylcholine [60]. The interactions involved π-π stacking, hydrogen bond and cation-π [61]. compounds and acetylcholine [60]. The interactions involved π-π stacking, hydrogen bond and At concentrations between 6 and 50 µM, compound 86g exhibited neuroprotective eﬀects. Compounds cation-π [61]. At concentrations between 6 and 50 µM, compound 86g exhibited neuroprotective 86f 86g effects.and Compoundscrossed the 86f BBB and thereby 86g crossed reaching the BBB the biologicalthereby reaching targets the in the biological CNS [60 targets]. in the CNS [60]. Table 13. IC50 values of compounds 86f–h on human AChE (hAChE) and BuChE (hBuChE) [60].

Table 13. IC50 values of Cpd.compounds 86f–h hAChE on human (nM) AChE HBuChE (hAChE) (andµM) BuChE (hBuChE) [60]. 86f 16.5 0.76 Cpd. hAChE (nM) HBuChE (µM) 86g 19.7 0.59 86h 13.7 0.66 86f 16.5 0.76 Donepezil 30.2 8.7

86g 19.7 0.59 Zhang et al. synthesized many compounds and found compound 87 (Figure 23) to be the most potent on both the nerve and86h endothelium cells.13.7 Structure–activity0.66 relationship analysis showed that the compounds with three methoxyl groups were the most potent indicating the role played by the Donepezil 30.2 8.7 methoxylInt. J. Mol.group Sci. 2020 in, 21 optimizing, x FOR PEER REVIEW the compound’s activity [9]. 24 of 37 Zhang et al. synthesized many compounds and found compound 87 (Figure 23) to be the most O potent on both the nerve and endothelium cells. Structure–activity relationship analysis showed that O O the compounds with three methoxyl groups were theN most potent indicating the role played by the methoxyl group in optimizing the compound’s activityH [9]. O O R 87 R = OH

FigureFigure 23. 23. ChemicalChemical structure of of compound compound 8787 [9].[9 ].

Compound 87 exhibited high neuroprotective ability against H2O2-induced damage with EC50 Compound 87 exhibited high neuroprotective ability against H2O2-induced damage with EC50 µ µ valuesvalues of of 3.26 3.26 MµM and and 2.41 2.41 µMM in in HBMEC-2 HBMEC-2 andand SH-SY5Y cells, cells, respectively. respectively. Compound Compound 8787 showedshowed potential in inhibiting the apoptosis of HBMEC-2 and SH-SY5Y cells induced by H O , and also potential in inhibiting the apoptosis of HBMEC-2 and SH-SY5Y cells induced by H2O22, 2and also inhibitedinhibited neuronal neuronal apoptosis apoptosis and and promoted promoted angiogenesisangiogenesis [9]. [9]. AdisakwattanaAdisakwattana et et al. al. evaluated evaluated thethe in vitro inhibitoryinhibitory effects eﬀects of of cinnamic cinnamic acid acid derivatives derivatives (1, (27,1, 27, 46,46,47,64,66,88–9547, 64, 66, 88–95) )(Figure (Figure 24) 24 against) against Protein Protein Tyrosine Tyrosine Phosphatase Phosphatase 1B (PTP1B). 1B (PTP1B). Results Results showed showed that thatreplacing replacing the the carboxyl carboxyl group group of ofcinnamic cinnamic acid acid (1) ( 1(16.0%) (16.0% inhibition) inhibition) with with alcohol, alcohol, aldehyde aldehyde and and methylmethyl ester ester groups groups ( 89,(89, 27 27and and92 92)) (12.5–18.0%(12.5–18.0% inhibition) did did not not increase increase the the PTP1B PTP1B inhibition inhibition percentage. Compounds 46 and 94 exhibited the highest inhibition of 39.4% and 35.9%, respectively, showing the role played by the hydroxyl group on the ortho- or para-positions. Introducing two moieties on cinnamic acid resulted in compounds with better inhibition (21.5–24.4%) when compared to cinnamic acid except for compound 64 [13].

Figure 24. Structures of cinnamic acid derivatives effective against Protein Tyrosine Phosphatase 1B (PTP1B) [13].

The most potent compounds with effective neurological activity are compounds 86f–h. They were active against both hAChE and hBuChE when compared to donepezil in vitro [60]. There is a pressing need for these compounds to be evaluated in vivo.

6. Cinnamic Derivatives with Antidiabetic Activity Diabetes mellitus is characterized as a metabolic disorder that results from a defect in insulin action, secretion or both [62]. The International Diabetes Federation estimated the global prevalence of diabetes mellitus to increase to 591 million by 2035 from 382 million reported in 2013. The inability to regulate blood glucose also causes many complications such as angiopathy, retinopathy and

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 24 of 37

O O O N H O O R 87 R = OH

Figure 23. Chemical structure of compound 87 [9].

Compound 87 exhibited high neuroprotective ability against H2O2-induced damage with EC50 values of 3.26 µM and 2.41 µM in HBMEC-2 and SH-SY5Y cells, respectively. Compound 87 showed potential in inhibiting the apoptosis of HBMEC-2 and SH-SY5Y cells induced by H2O2, and also inhibited neuronal apoptosis and promoted angiogenesis [9]. Adisakwattana et al. evaluated the in vitro inhibitory effects of cinnamic acid derivatives (1, 27, Int.46,47,64,66,88–95 J. Mol. Sci. 2020, 21,) 5712(Figure 24) against Protein Tyrosine Phosphatase 1B (PTP1B). Results showed that22 of 34 replacing the carboxyl group of cinnamic acid (1) (16.0% inhibition) with alcohol, aldehyde and methyl ester groups (89, 27 and 92) (12.5–18.0% inhibition) did not increase the PTP1B inhibition percentage.percentage. Compounds Compounds46 46and and94 94 exhibitedexhibited the highest highest inhibition inhibition of of 39.4% 39.4% and and 35.9%, 35.9%, respectively, respectively, showingshowing the the role role played played by by the the hydroxyl hydroxyl groupgroup onon thethe ortho-ortho- or para-positions. Introducing Introducing two two moietiesmoieties on on cinnamic cinnamic acid acid resulted resulted in in compoundscompounds with better better inhibition inhibition (21.5–24.4%) (21.5–24.4%) when when compared compared toto cinnamic cinnamic acid acid except except for for compound compound64 64[ [13]13]..

FigureFigure 24. 24.Structures Structures of of cinnamic cinnamic acidacid derivativesderivatives effect eﬀectiveive against against Protein Protein Tyrosine Tyrosine Phosphatase Phosphatase 1B 1B (PTP1B)(PTP1B) [13 [13].].

TheThe most most potent potent compounds compounds with with eﬀ ectiveeffective neurological neurological activity activity are are compounds compounds86f–h 86f–h.. They They were activewere against active bothagainst hAChE both hAChE and hBuChE and hBuChE when compared when compared to donepezil to donepezilin vitro in[60 vitro]. There [60]. isThere a pressing is a needpressing for these need compounds for these compounds to be evaluated to be evaluated in vivo. in vivo.

6. Cinnamic6. Cinnamic Derivatives Derivatives with with Antidiabetic Antidiabetic Activity DiabetesDiabetes mellitus mellitus is characterizedis characterized as aas metabolic a metabolic disorder disorder that that results results from from a defect a defect in insulin in insulin action, secretionaction, orsecretion both [62 or]. both The International[62]. The International Diabetes FederationDiabetes Federation estimated estimated the global the prevalence global prevalence of diabetes mellitusof diabetes to increase mellitus to to 591 increase million to by591 2035 million from by 382 2035 million from 382 reported million in reported 2013. The in 2013. inability The toinability regulate bloodto regulate glucose blood also causes glucose many also complicationscauses many complications such as angiopathy, such as retinopathy angiopathy, and retinopathy neuropathy and [63 ]. The inhibitory eﬀect of cinnamic acid derivatives on Protein Tyrosine Phosphatase 1B (PTP1B) has been one of the several ways to regulate blood glucose [13]. Amalan et al. also reported the ability of compound 46 in lowering blood glucose and improving insulin levels [64]. Compounds 94 and 89 (Figure 24) inhibited p-NPP hydrolysis catalysed by PTP1B with IC50 values of 137.74 and 168.40 nM. Kinetic studies proved compounds 89 and 94 to be non-competitive inhibitors with the ability of being developed into highly selective PTP1B inhibitors [64], thus cinnamic acid derivatives can be further explored as PTP1B inhibitors for the treatment of diabetes [13]. Hypochlorous acid (HOCl) and H2O2 scavenging assay results showed - compound 95 has the antioxidant capability to capture free radicals and reactive species such as O2· and HOCl/OCl- [64] which causes damage to the organs in the body [65]. Adisakwattana et al. evaluated compounds 1, 46–47, 64, 66, 88–91 and 93–95 (Figure 24) for their inhibitory activity against intestinal α-glucosidase (maltase and sucrose). The most eﬀective compounds (64, 90 and 47) exhibited a non-competitive inhibition, better on sucrase when compared to maltase (Table 14), although these compounds were less potent than acarbose with IC50 values of 2.2 and 4.8 µM for maltase and sucrase, respectively. Combining compounds 64, 90 and 47 with acarbose Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 25 of 37

neuropathy [63]. The inhibitory effect of cinnamic acid derivatives on Protein Tyrosine Phosphatase 1B (PTP1B) has been one of the several ways to regulate blood glucose [13]. Amalan et al. also reported the ability of compound 46 in lowering blood glucose and improving insulin levels [64]. Compounds 94 and 89 (Figure 24) inhibited p-NPP hydrolysis catalysed by PTP1B with IC50 values of 137.74 and 168.40 nM. Kinetic studies proved compounds 89 and 94 to be non- competitive inhibitors with the ability of being developed into highly selective PTP1B inhibitors [64], thus cinnamic acid derivatives can be further explored as PTP1B inhibitors for the treatment of diabetes [13]. Hypochlorous acid (HOCl) and H2O2 scavenging assay results showed compound 95 has the antioxidant capability to capture free radicals and reactive species such as O2·- and HOCl/OCl- [64] which causes damage to the organs in the body [65]. Adisakwattana et al. evaluated compounds 1, 46-47, 64, 66, 88-91 and 93–95 (Figure 24) for their Int. J. Mol. Sci. 2020, 21, 5712 23 of 34 inhibitory activity against intestinal α-glucosidase (maltase and sucrose). The most effective compounds (64, 90 and 47) exhibited a non-competitive inhibition, better on sucrase when compared producedto maltase additive (Table inhibition14), although resulting these compounds in reduced postprandialwere less potent hyperglycemia than acarbose and with hyperinsulinemia IC50 values of in type2.2 and 2 diabetes 4.8 µM [for66 ].maltase and sucrase, respectively. Combining compounds 64, 90 and 47 with acarbose produced additive inhibition resulting in reduced postprandial hyperglycemia and hyperinsulinemia in type 2 diabetes [66]. Table 14. IC50 values for maltase and sucrase inhibition by compounds 47, 64 and 90 [67].

Table 14. IC50 values for maltaseCompounds and sucrase Maltase inhibition Sucrase by compounds 47, 64 and 90 [67].

Compounds47 Maltase0.74 0.49 Sucrase 64 0.79 0.45 4790 0.760.74 0.450.49 64 0.79 0.45 Wang et al. isolated a cinnamic90 acid glycoside0.76 derivative, 0.45 dissectumoside (96), from the roots of H. dissectumWang et al.(Figure isolated 25 a). cinnamic When evaluated acid glycoside for itsderivative,in vitro triglyceridesdissectumoside accumulation (96), from the activityroots of in 3T3-L1H. dissectum adipocytes, (Figure compound 25). When 96evaluatedincreased for activityits in vitro from triglycerides 7.8% at 1 accumulationµM through activity 32.1% atin 103T3-L1µM to 40.1%adipocytes, at 30 µM. compound The authors 96 increased reported thatactivity compound from 7.8%96 atprovided 1 µM through antidiabetic 32.1% e atﬀect 10 [µM67] asto 40.1% previously at reported30 µM. [ 68The] due authors to its reported ability to that eﬀectively compound transport 96 provided glucose antidiabetic from the medium effect [67] into as adipocytes previously and triglyceridesreported [68] [67 due]. to its ability to effectively transport glucose from the medium into adipocytes and triglycerides [67].

FigureFigure 25. 25.Chemical Chemical structurestructure of dissectumoside, 9696 [67].[67 ].

XuXu et et al. al. evaluatedevaluated the the activity activity of of coumarin coumarin derivatives derivatives (97a–i (97a–i and and98a–i98a–i) (Figure) (Figure 26) as 26 α)- as α-glucosidaseglucosidase inhibitors inhibitors with with both both the the 97 97 series series and and the the 98 98 series series deriva derivativestives having having the the same same substituents.substituents. Compounds Compounds98a–i 98a–iwere were lessless potentpotent with 98b98b havinghaving the the highest highest inhibition inhibition of of54.745 54.745 at at 100Int.100µ MJ. µM Mol. and andSci. IC 2020 IC50 50of, 21 of 94.52, x94.52 FORµ PEER µM,M, the REVIEWthe rest rest hadhad inhibitioninhibition less than than 50% 50% and and IC IC5050 ˃100>100 µMµ M[[69].69 ]. 26 of 37

FigureFigure 26. 26.Chemical Chemical structuresstructures ofof coumarin derivatives derivatives (97a–i (97a–i andand 98a–i98a–i) [69].)[69 ].

SinceSince compounds compounds97a–i 97a–iwere werebetter better inhibitorsinhibitors (Table 15)15) than than 98a–i98a–i, ,it it showed showed that that conjugating conjugating cinnamiccinnamic acid acid with with 4-hydroxycoumarin 4-hydroxycoumarin waswas betterbetter than with 7-hydroxycoumarin. 7-hydroxycoumarin. Compound Compound 97b97b exhibitedexhibited the the most most e ﬀeffectiveective inhibition inhibition activityactivity of 89.92% and and IC IC5050 ofof 12.98 12.98 µMµM suggesting suggesting that that the the presencepresence of of a methyla methyl group, group, which which isis anan electron-donatingelectron-donating group, group, favored favored the the withdrawing withdrawing group group (97e–i(97e–i). The). The two two most most potent potent compounds, compounds,97a 97aand and97b 97b,, showed showed that that their theirα-glucosidase α-glucosidase inhibition inhibition was reversible.was reversible. Compound Compound97b ﬁtted 97b fitted well intowellthe into active the active siteof siteα-glucosidase of α-glucosidase forming forming a hydrogen a hydrogen bond withbond the with LYS293 the LYS293 amino acidamino sequences acid sequences [69]. [69].

Table 15. Inhibitory Activity of Compounds 97a–i on α-glucosidase [69].

Compounds 97a 97b 97c 97d 97e 97f 97g 97h 97i

R1 H CH3 OCH3 F Cl Br CF3 OH OH

R2 H H H H H H H H OCH3 % inhibition 83.51 89.92 73.15 68.50 33.10 60.99 41.87 33.19 19.51

IC50 (µM) 19.64 12.98 36.88 41.73 ˃100 69.30 ˃100 ˃100 ˃100

7. Cinnamic Acid Derivatives with Antioxidant and Anti-Inflammatory Activity and in Cosmetics. Biomolecular oxidations of reactive oxygen and nitrogen species create oxidative stress. Free radicals produce damaged membranes, produce DNA nicks and inflict other oxidative injuries. Accumulation of free radicals activates proto oncogenes causing carcinogenesis [70]. There is a need to scavenge free radicals in the body to minimize the damage inflicted by free radicals. Cinnamic acid derivatives are known to have antioxidant properties [4]. Three mechanisms are reported in phenolic compounds during free radical scavenging. The first one involves a hydrogen atom being abstracted from the antioxidant by the radical. The second step involves the formation of a radical cation ArOH+• through the transfer of an electron to the free radical from the antioxidant and the deprotonation yielding ArO• radical and ROH. In the last step, the phenolic compound is deprotonated and there is an electron transfer from the phenoxide anion to RO• to yield phenoxyl radicals [71]. Muhammad et al. isolated a new bioactive compound (99) (Figure 27) from the plant, Viola betonicifolia. At the concentration range of 2-50 µg/mL, compound 99 inhibited DPPH (diphenyl-2- picryl hydrazyl) free radicals with IC50 of 124 µM which was higher than for common antioxidants α-tocopherol (IC50 of 96 µM) and vitamic C (IC50 of 90 µM). As an anti-glycating agent, compound 99 showed moderate activity with IC50 of 355 µM when compared to the IC50 of rutin, a standard drug which was 294 µM. Thus, due to its free radical scavenging ability, compound 99 has the potential to delay and prevent diabetic complications [72].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 26 of 37

Figure 26. Chemical structures of coumarin derivatives (97a–i and 98a–i) [69].

Since compounds 97a–i were better inhibitors (Table 15) than 98a–i, it showed that conjugating cinnamic acid with 4-hydroxycoumarin was better than with 7-hydroxycoumarin. Compound 97b exhibited the most effective inhibition activity of 89.92% and IC50 of 12.98 µM suggesting that the presence of a methyl group, which is an electron-donating group, favored the withdrawing group (97e–i). The two most potent compounds, 97a and 97b, showed that their α-glucosidase inhibition was reversible. Compound 97b fitted well into the active site of α-glucosidase forming a hydrogen bond with the LYS293 amino acid sequences [69].

Table 15. Inhibitory Activity of Compounds 97a–i on α-glucosidase [69]. Int. J. Mol. Sci. 2020, 21, 5712 24 of 34

Compounds 97a 97b 97c 97d 97e 97f 97g 97h 97i Table 15. Inhibitory Activity of Compounds 97a–i on α-glucosidase [69]. R1 H CH3 OCH3 F Cl Br CF3 OH OH Compounds 97a 97b 97c 97d 97e 97f 97g 97h 97i R2 H H H H H H H H OCH3 R1 H CH3 OCH3 F Cl Br CF3 OH OH % Rinhibition2 H 83.51 H89.92 H73.15 H 68.50 33.10 H 60.99 H 41.87 H 33.19 H 19.51 OCH 3 % inhibition 83.51 89.92 73.15 68.50 33.10 60.99 41.87 33.19 19.51 ICIC5050( µ(µM)M) 19.64 19.64 12.9812.98 36.8836.88 41.73 41.73 >˃100100 69.30 69.30 ˃100>100 ˃100>100 ˃100> 100

7. Cinnamic7. Cinnamic Acid Acid Derivatives Derivatives with with Antioxidant Antioxidant andand Anti-InflammatoryAnti-Inflammatory Activity Activity and and in in Cosmetics Cosmetics. Biomolecular oxidations of reactive oxygen and nitrogen species create oxidative stress. Free radicalsBiomolecular produce oxidations damaged of membranes, reactive oxygen produce and DNAnitrogen nicks species and inflictcreate otheroxidative oxidative stress. injuries. Free Accumulationradicals produce of free damaged radicals activatesmembranes, proto produce oncogenes DNA causing nicks carcinogenesisand inflict other [70 oxidative]. There isinjuries. a need to Accumulation of free radicals activates proto oncogenes causing carcinogenesis [70]. There is a need scavenge free radicals in the body to minimize the damage inflicted by free radicals. Cinnamic acid to scavenge free radicals in the body to minimize the damage inflicted by free radicals. Cinnamic acid derivatives are known to have antioxidant properties [4]. Three mechanisms are reported in phenolic derivatives are known to have antioxidant properties [4]. Three mechanisms are reported in phenolic compounds during free radical scavenging. The first one involves a hydrogen atom being abstracted compounds during free radical scavenging. The first one involves a hydrogen atom being abstracted from the antioxidant by the radical. The second step involves the formation of a radical cation ArOH+ from the antioxidant by the radical. The second step involves the formation of a radical cation ArOH+• • throughthrough the the transfer transfer of of an an electron electron to to thethe freefree radicalradical from the antioxidant antioxidant and and the the deprotonation deprotonation yieldingyielding ArO ArO• radical• radical and and ROH. ROH. In In the the last last step, step, the the phenolic phenolic compound compound isis deprotonateddeprotonated and there is anis electron an electron transfer transfer from from the the phenoxide phenoxide anion anion to ROto RO• to• to yield yield phenoxyl phenoxyl radicals radicals [ 71[71].]. MuhammadMuhammad et et al.al. isolated isolated a anew new bioactive bioactive compound compound (99) (Figure (99) (Figure 27) from 27) the from plant, the Viola plant, Violabetonicifolia. betonicifolia At .the At concentration the concentration range of range2-50 µg/mL, of 2–50 compoundµg/mL, 99 compound inhibited DPPH99 inhibited (diphenyl-2- DPPH (diphenyl-2-picrylpicryl hydrazyl) free hydrazyl) radicals free with radicals IC50 of with124 µM IC 50whichof 124 wasµM higher which than was for higher common than antioxidants for common antioxidantsα-tocopherolα-tocopherol (IC50 of 96 µM) (IC 50andof vitamic 96 µM) C and (IC50 vitamic of 90 µM). C (IC As50 anof anti-glycating 90 µM). As an agent, anti-glycating compound agent, 99 compoundshowed moderate99 showed activity moderate with IC activity50 of 355 with µM ICwhen50 of compared 355 µM whento the comparedIC50 of rutin, to a thestandard IC50 of drug rutin, a standardwhich was drug 294 whichµM. Thus, was due 294 µtoM. its Thus,free radical due to scavenging its free radical ability, scavenging compound ability, 99 has compound the potential99 tohas thedelay potential and prevent to delay diabetic and prevent complications diabetic [72]. complications [72].

Figure 27. Structure of compound 99 [72].

Kortenska et al. reported the antioxidant activity of compounds 46, 64, 65, 100–103 (Figure 28) in pure triacylglycerols of sunflower oil (TGSO) at 80 ◦C. Compound 101 showed the highest antioxidant activity at 0.1 mM [73]. The reaction of a phenol antioxidant with lipid radicals produces phenoxyl radicals which are stabilized by unpaired electrons delocalized around the aromatic ring [61]. Replacing hydrogen atoms in the o-position with prenyl groups increased the electron density of the OH-moiety through inductive effects thereby enhancing its reactivity towards lipid radicals [74]. This resulted in compounds 101 and 102 being more active than compound 46 [73]. Transformation of the phenol in compounds, 100 and 102 led to the loss of antioxidant activity as there was no hydrogen donating group left. The antioxidant efficiency was found to decrease in the order: 65 > 101 > 103 > 64 > 46 100 102. Inhibition degree (measure of antioxidant strength), ≈ ≈ that is the number of times the antioxidant shortened the oxidation chain length, followed the sequence α-tocopherol > 101 > 103 > 65 > 64 > 46 100 102. The antioxidant activity, calculated as the ability ≈ ≈ of an inhibitor to terminate the oxidation chain and also the decreased oxidation rate during induction period, followed the sequence: α-tocopherol > 101 > 103 > 65 > 64 > 46 100 102 [73]. ≈ ≈ Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 27 of 37

Figure 27. Structure of compound 99 [72].

Kortenska et al. reported the antioxidant activity of compounds 46, 64, 65, 100–103 (Figure 28) in pure triacylglycerols of sunflower oil (TGSO) at 80 °C. Compound 101 showed the highest antioxidant activity at 0.1 mM. [73]. The reaction of a phenol antioxidant with lipid radicals produces phenoxyl radicals which are stabilized by unpaired electrons delocalized around the aromatic ring [61].Int. J. Replacing Mol. Sci. 2020 hydrogen, 21, x FOR PEER atoms REVIEW in the o-position with prenyl groups increased the electron density 27 of 37 of the OH-moiety through inductive effects thereby enhancing its reactivity towards lipid radicals Figure 27. Structure of compound 99 [72]. [74]. This resulted in compounds 101 and 102 being more active than compound 46 [73].

Kortenska et al. reported the antioxidant activity of compounds 46, 64, 65, 100–103 (Figure 28) in pure triacylglycerols of sunflower oil (TGSO) at 80 °C. Compound 101 showed the highest antioxidant activity at 0.1 mM. [73]. The reaction of a phenol antioxidant with lipid radicals produces phenoxyl radicals which are stabilized by unpaired electrons delocalized around the aromatic ring [61]. Replacing hydrogen atoms in the o-position with prenyl groups increased the electron density of the OH-moiety through inductive effects thereby enhancing its reactivity towards lipid radicals Int. J. Mol. Sci. 2020, 21, 5712 25 of 34 [74]. This resulted in compounds 101 and 102 being more active than compound 46 [73].

Figure 28. Chemical structures of compounds 46, 64, 65, 100-103 [73].

Transformation of the phenol in compounds, 100 and 102 led to the loss of antioxidant activity as there was no hydrogen donating group left. The antioxidant efficiency was found to decrease in the order: 65 ˃ 101 ˃ 103 ˃ 64 ˃ 46 ≈ 100 ≈ 102. Inhibition degree (measure of antioxidant strength), that is the number of times the antioxidant shortened the oxidation chain length, followed the sequence α-tocopherol ˃ 101 ˃ 103 ˃ 65 ˃ 64 ˃ 46 ≈ 100 ≈ 102. The antioxidant activity, calculated as the ability of an inhibitorFigure 28. to terminate the oxidation chain and46 64 also65 the100 decreased103 oxidation rate Figure 28.Chemical Chemical structuresstructures ofof compounds 46,, 64, ,65, ,100-103– [73].[73 ]. during induction period, followed the sequence: α-tocopherol ˃ 101 ˃ 103 ˃ 65 ˃ 64 ˃ 46 ≈ 100 ≈ 102 [73]. 104a–c NazirTransformation et al. synthesized of the phenol cinnamic in acidcompounds, derivatives 100 and ( 102) led (Scheme to the5 loss) with of antioxidant tyrosinase inhibitionactivity Nazir et al. synthesized cinnamic acid derivatives (104a–c) (Scheme 5) withµ tyrosinase inhibition activityas there for was hyperpigmentation no hydrogen donating treatment. group left. Compound The antioxidant104c (IC efficiency50 = 5.7 wasM) found was theto decrease most potent in activity for hyperpigmentation treatment. Compound 104c (IC50 = 5.7 µM) was the most potent compoundthe order: (Table 65 ˃ 101 16) ˃ and 103 kinetic ˃ 64 ˃ studies46 ≈ 100revealed ≈ 102. Inhibition that compound degree (measure104c inhibited of antioxidant tyrosinase strength), [75]. compound (Table 16) and kinetic studies revealed that compound 104c inhibited tyrosinase [75]. that is the number of times the antioxidant shortened the oxidation chain length, followed the sequence α-tocopherol ˃ 101 ˃ 103 ˃ 65 ˃ 64 ˃ 46 ≈ 100 ≈ 102. The antioxidant activity, calculated as the ability of an inhibitor to terminate the oxidation chain and also the decreased oxidation rate during induction period, followed the sequence: α-tocopherol ˃ 101 ˃ 103 ˃ 65 ˃ 64 ˃ 46 ≈ 100 ≈ 102 [73]. Nazir et al. synthesized cinnamic acid derivatives (104a–c) (Scheme 5) with tyrosinase inhibition activity for hyperpigmentation treatment. Compound 104c (IC50 = 5.7 µM) was the most potent compound (Table 16) and kineticScheme studies 5: 5. SynthesisSynthesis revealed of of compounds compounds that compound (104a-c) (104a–c 104c [75].)[75 inhibited ]. tyrosinase [75]. µ TreatmentTreatment of of B16F10 B16F10 melanomamelanoma cells with with compound compound 104c104c (30(30 µg/mL)g/mL) showed showed cell cell viability viability of of higherhigher than than 99%. 99%. Compound Compound104c 104cproved proved a a better better inhibitor inhibitor than than kojic kojic acid acid since since 125 125µ µg/mLg/mL of of kojic kojic acid wasacid needed was needed to achieve to achieve the same the inhibition same inhibi eﬀectedtion effected by 30 µ gby/mL 30 ofµg/mL compound of compound104c. Thus 104c compound. Thus 104ccompoundis a promising 104c is a melanogenic promising melanogenic regulator [75 regulator]. [75].

Table 16. Substituents, mushroom tyrosinase inhibitory activity, IC50 (µM) and kinetics of compounds 104a–c [75]. Scheme 5: Synthesis of compounds (104a-c) [75].

Treatment of B16F10 melanomaCpd cells Rwith compound IC50 (µ M)104c (30 K µg/mL)f (µM) showed cell viability of higher than 99%. Compound104a 104c provedH a better inhibitor 39.4 than kojic -acid since 125 µg/mL of kojic acid was needed to achieve104b the same inhibi2-OHtion effected 172.8 by 30 µg/mL - of compound 104c. Thus compound 104c is a promising104c melanogenic4-OH regulator [75]. 5.7 11 Kojic acid 16.7 n.d.

n.d., not determined. Kf is the kinetic reaction constant.

Gießel et al. synthesized cinnamic acid derivatives (105–112) (Figure 29) for use in flavors and fragrances. When evaluated for their biological activity, the compounds were weak inhibitors of both eeAChE and eqBuAChE with the highest inhibition being 45.5%. Cytotoxic studies against several human tumour cell lines and non-malignant mouse fibroblasts showed the compounds to be non-cytotoxic since they all had EC50 values of more than 30 µM, thus can be safely used as cosmetics and flavoring. The compounds had a typical vanillin smell suggesting their potential use in fragrances and flavor [12]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 28 of 37

Table 16. Substituents, mushroom tyrosinase inhibitory activity, IC50 (µM) and kinetics of compounds 104a–c [75].

Cpd R IC50 (µM) Kf (µM)

104a H 39.4 - 104b 2-OH 172.8 - 104c 4-OH 5.7 11 Kojic acid 16.7 n.d.

n.d., not determined. Kf is the kinetic reaction constant. Gießel et al. synthesized cinnamic acid derivatives (105–112) (Figure 29) for use in flavors and fragrances. When evaluated for their biological activity, the compounds were weak inhibitors of both eeAChE and eqBuAChE with the highest inhibition being 45.5%. Cytotoxic studies against several human tumour cell lines and non-malignant mouse fibroblasts showed the compounds to be non- cytotoxic since they all had EC50 values of more than 30 µM, thus can be safely used as cosmetics and flavoring. The compounds had a typical vanillin smell suggesting their potential use in fragrances Int. J. Mol. Sci. 2020, 21, 5712 26 of 34 and flavor [12].

FigureFigure 29.29. Chemical structures structures of of compounds compounds 105-112105–112 [12].[12 ].

TaofiqTaofiq et et al. al. evaluated evaluated thethe anti-inflammatoryanti-inflammatory and and anti-tyrosinase anti-tyrosinase activities activities of ofp-coumaric p-coumaric acid acid µ (compound(compound46 46)). Its. Its anti-inflammatory anti-inflammatory activity activity was was measured measured to be to EC be50 ECof 15250 of g152/mL µg/mL and anti-tyrosinase and anti- activitytyrosinase of EC activity50 > 2.0 of mgEC50/mL. ˃ 2.0 [76 mg/mL.]. The [76]. ability The of ability compound of compound46 to inhibit 46 to inhibit tyrosinase tyrosinase was credited was tocredited its ability to toits reduceability microphthalmia-associatedto reduce microphthalmia-associated transcription transcription factor (MITF) factor [(MITF)77] and [77] tyrosinase and mRNAtyrosinase expression mRNA asexpression high as 73%as high and as 82%, 73% respectively.and 82%, respectively. The results The showed results thatshowed compound that 46compoundhas the potential 46 has the to potential be used to together be used withtogether other with bioactive other bioactive ingredients ingredients for protection for protection against hyperpigmentationagainst hyperpigmentation [76]. Gunia-Krzyzak [76]. Gunia-Krzyzak et al. reported et al. reported compound compound46-containing 46-containing cream that cream decreased that erythemadecreased and erythema pigmentation and pigmentation when applied when daily applied on the daily foreskin on the armforeskin [78]. arm [78]. LeeLee et et al. al. reported reported thethe ability of hydroxycinnamic hydroxycinnamic acids acids (cinnamic (cinnamic acid acid (1), (1 p-coumaric), p-coumaric acid acid (46), (46 ), cacaffeicffeic acid acid (47 (47)) and and chlorogenic chlorogenic acidacid ( 33)) on on reducing reducing furan furan and and trapping trapping α-dicarbonylα-dicarbonyl compounds, compounds, whichwhich are are possible possible human human carcinogens.carcinogens. [79]. [79]. Addition Addition of of water-soluble water-soluble antioxidants antioxidants such such as caffeic as caff eic acid,acid, chlorogenic chlorogenic acid and and ferulic ferulic acid acid has has been been foun foundd to reduce to reduce levels of levels furan of derivatives furan derivatives [80]. When [ 80]. Whencompound compound 47 was47 addedwas added to canned-coffee to canned-co modelffee modelsystems systems (CCMS) (CCMS)containing containing glyoxal (GO), glyoxal furan (GO), was reduced from 59.76 µg/L to 48.31 µg/L while compound 51 reduced it to 41.38 µg/L. furan was reduced from 59.76 µg/L to 48.31 µg/L while compound 51 reduced it to 41.38 µg/L. Compound 47 reduced furan in methyglyoxal (MGO) from 45.79 µg/L to 35.41 µg/L while Compound 47 reduced furan in methyglyoxal (MGO) from 45.79 µg/L to 35.41 µg/L while compound 33 reduced it to 32.65 µg/L. The results showed the capability of the compounds to compound 33 reduced it to 32.65 µg/L. The results showed the capability of the compounds to suppress furan formation in CCMS. Compounds 47 and 33 showed better GO and MGO trapping efficiency when compared to compounds 1 and 46. This was attributed to the presence of two hydroxyl groups on the aromatic ring trapping the dicarbonyls thereby causing the reduction of furan concentration in CCMS [79]. Garcia-Jimenez et al. reported the catalysis and inhibition of tyrosinase when subjected to different cinnamic acid derivatives (1, 46, 47, 88, 91 and 93–95) (Figure 30). Results of the compounds in the presence of 4-tert-butylcatechol (TBC) showed that compounds 1, 91, 93, 94 and 95 are competitive inhibitors whereas compounds 46, 47 and 88 are substrates of tyrosinase. The inhibition constants were affected by the ability of the substituent group to increase the charge on the benzene ring [81]. The methoxy group, OCH3 is an electron donor and it increased the charge on the benzene ring making the inhibition constant to be in the order 95 > 93 > 91 > 1. Tyrosinase could not hydroxylate compound 94 but was able to hydroxylate compound 88 because compound 94 has a great distance for ligand hydroxylation while compound 88 has a distance feasible for hydroxylation [81]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 29 of 37

suppress furan formation in CCMS. Compounds 47 and 33 showed better GO and MGO trapping efficiency when compared to compounds 1 and 46. This was attributed to the presence of two hydroxyl groups on the aromatic ring trapping the dicarbonyls thereby causing the reduction of furan concentration in CCMS [79]. Garcia-Jimenez et al. reported the catalysis and inhibition of tyrosinase when subjected to different cinnamic acid derivatives (1, 46, 47, 88, 91 and 93–95) (Figure 30). Results of the compounds in the presence of 4-tert-butylcatechol (TBC) showed that compounds 1, 91, 93, 94 and 95 are competitive inhibitors whereas compounds 46, 47 and 88 are substrates of tyrosinase. The inhibition Int. J.constants Mol. Sci. 2020 were, 21 ,affected 5712 by the ability of the substituent group to increase the charge on the benzene27 of 34 ring [81].

FigureFigure 30. 30.Chemical Chemical structures structures of compounds 1,1 46,, 46 47,, 47 88,, 88 91, 91andand 93-9593 [81].–95 [81].

RibeiroThe methoxy et al. evaluated group, OCH the3 is inhibitionan electron donor of cyclooxygenase and it increased enzymes the charge (COX-1 on the benzene and COX-2) ring by synthesizedmaking the cinnamic inhibition acid constant derivatives to be in (113a–h the order, 114a–h 95 ˃ 93, 115a–c˃ 91 ˃ 1,.116a–c Tyrosinaseand could117) (Schemenot hydroxylate6). Table 17 showedcompound the most 94 but important was able structure–activity to hydroxylate compound relationship 88 because for COX-1 compound inhibition 94 has witha great the distance presence of the di-substitutedfor ligand hydroxylation aromatic ringswhile havingcompound one 88 hydroxyl has a distance group feasible and one for methoxyl hydroxylation group [81]. or two hydroxyl groups,Int. J. Mol. anRibeiro Sci. amide 2020 et, group21al., x FORevaluated and PEER the REVIEW the absence in hibition of an ofα ,cyclooxygenaseβ-double bond enzymes [82]. (COX-1 and COX-2) 30by of 37 synthesized cinnamic acid derivatives (113a–h, 114a–h, 115a–c, 116a–c and 117) (Scheme 6). Table 17 showed the most important structure–activity relationship for COX-1 inhibition with the presence of the di-substituted aromatic rings having one hydroxyl group and one methoxyl group or two hydroxyl groups, an amide group and the absence of an α,β-double bond [82].

Scheme 6. Synthesis of compounds 113a–h, 114a–h, 115a–c, 116a–c and 117. Reagents and conditions: Scheme 6: Synthesis of compounds 113a–h, 114a–h, 115a–c, 116a–c and 117. Reagents and conditions: (i)(i) Dimethylformamide Dimethylformamide (DMF),(DMF), TriethylamineTriethylamine (TEA),(TEA), Benzotriazol-1-yloxy)tris (dimethylamino) (dimethylamino) phosphoniumphosphonium hexaﬂuorophosphate(BOP), hexafluorophosphate(BOP), DichloromethaneDichloromethane (DCM), room room temperature temperature (rt) (rt) [82]. [82 ].

TheseThese aforementioned aforementioned features features gave gave compounds compounds114e 114e(IC (IC50 =50 24= 24µM µM and and 65% 65% inhibition), inhibition),114g 114g(IC 50 = 9(ICµ50M = and9 µM 85% andinhibition) 85% inhibition) and and116c 116c(IC 50(IC=50 15= 15µ MµM and and 84% 84% inhibition).inhibition). Compounds Compounds 116a116a andand 117117were were also also good good COX-1 COX-1 inhibitors inhibitors meaningmeaning aa tri-substitutedtri-substituted aromatic aromatic ring ring with with one one hydroxyl hydroxyl groupgroup and and two two methoxyl methoxyl groups groups and and nonoα α,β,β-double-double bond made made compound compound 116a116a effective.eﬀective. The The best best COX-1COX-1 inhibitor inhibitor was was compound compound117 117(IC (IC5050 = 4.34.3 µM)µM) meaning meaning the the 3,5-di- 3,5-di-terttert-butyl-4-hydroxyl-butyl-4-hydroxyl aromatic aromatic substitution,substitution, the the presence presence of of tertiary tertiarydiethylamide diethylamide group and and an an αα,β,β-double-double bond bond contributed contributed to toits its biologicalbiological activity activity [82 [82].].

Table 17. IC50 and inhibition % of PGE2 production via COX-1 and COX-2, in human whole blood [82].

Cpd. COX-1 COX-2 Cpd. COX-1 COX-2

IC50 Inhibition IC50 Inhibition IC50 Inhibition IC50 Inhibition

(µM) (%) (µM) (%) (µM) (%) (µM) (%)

113a N.A. N.A. N.A. N.A. 114g 9 85 21 79 113b N.A. N.A. N.A. N.A. 114h N.A. N.A. N.A. N.A. 113c ˃100 41 N.A. N.A. 115b 97 57 N.A. N.A. 113e ˃100 39 3.0 93 116a 11 82 62 90 114a 24 82 12.7 99.0 116b ˃100 24 76 57 114b 14 81 20 98.3 116c 15 84 13.4 98.0 114c 87 60 30 74 117 4.3 79 1.09 85 114d 45 82 19 91 Ind. - 91 - - 114e 24 65 2.4 99.4 Cel. - - - 71 114f N.A. N.A. N.A. N.A.

Int. J. Mol. Sci. 2020, 21, 5712 28 of 34

Table 17. IC50 and inhibition % of PGE2 production via COX-1 and COX-2, in human whole blood [82].

Cpd. COX-1 COX-2 Cpd. COX-1 COX-2

IC50 (µM) Inhibition (%) IC50 (µM) Inhibition (%) IC50 (µM) Inhibition (%) IC50 (µM) Inhibition (%) 113a N.A. N.A. N.A. N.A. 114g 9 85 21 79 113b N.A. N.A. N.A. N.A. 114h N.A. N.A. N.A. N.A. 113c >100 41 N.A. N.A. 115b 97 57 N.A. N.A. 113e >100 39 3.0 93 116a 11 82 62 90 114a 24 82 12.7 99.0 116b >100 24 76 57 114b 14 81 20 98.3 116c 15 84 13.4 98.0 114c 87 60 30 74 117 4.3 79 1.09 85 114d 45 82 19 91 Ind. - 91 - - 114e 24 65 2.4 99.4 Cel. - - - 71 114f N.A. N.A. N.A. N.A. N.A.—Non active up to highest tested concentration of 100 µM. All the compounds were tested at 100 µM except for compound 117 tested at 12.5 µM for COX-1 and 3.1 µM for COX-2. The two reference drugs were tested at 1 µM. Where Ind. and Cel. represent Indomethacin and Celecoxib, respectively. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 31 of 37

N.A. – Non active up to highest tested concentration of 100 µM. All the compounds were tested at 100 µM except for compound 117 tested at 12.5 µM for COX-1 and 3.1 µM for COX-2. The two Int. J. Mol. Sci. 2020, 21, 5712 29 of 34 reference drugs were tested at 1 µM. Where Ind. and Cel. represent Indomethacin and Celecoxib, respectively.

CompoundsCompounds113e 113e,, 114a,114a, 114e,114e 116c,, 116c and, and 117 appeared117 appeared to be the to best be theCOX-2 best inhibitors COX-2 with inhibitors IC50 withvalues IC50 of values3.0, 12.7, of 2.4, 3.0, 13.4, 12.7, and 2.4, 1.09 13.4,µM, respectively. and 1.09 µ M,The respectively.structure–activity The relationship structure–activity of the relationshipcompounds of therevealed compounds the presence revealed the3,5-di- presencetert-butyl-4-hydroxyl 3,5-di-tert-butyl-4-hydroxyl aromatic substitution, aromatic substitution, tertiary tertiarydiethylamide diethylamide group group and an and α,β an-doubleα,β-double bond (compound bond (compound 132) and132 a )disubstituted and a disubstituted aromatic aromatic ring with ring withtwo two hydroxyl hydroxyl groups, groups, an anamide amide group group and and an α an,β-doubleα,β-double bond bond influenced inﬂuenced the biological the biological activity activity of ofthese compounds. Since Since compounds compounds 113e,113e 114e,, 114e 116d, 116d andand 117117 werewere the the most most potent potent compounds compounds againstagainst COX-2, COX-2, they they are are suggested suggested as as potential potential non-steroidalnon-steroidal anti-inf anti-inﬂammatorylammatory drugs drugs [82]. [82 ]. RuanRuan et et al. al. reported reported the the potency potency ofof a resveratrol-basedresveratrol-based cinnamic cinnamic acid acid derivative, derivative, 118118 (Figure(Figure 31). 31 ). CompoundCompound118 118inhibited inhibited LPS-mediated LPS-mediated expressionexpression of inducible inducible nitric nitric oxide oxide synthase synthase (iNOS) (iNOS) and and COX-2COX-2 in in RAW264.7 RAW264.7 cells cells when when analysed analysed by by WesternWestern blotblot [[83].83].

FigureFigure 31. 31.Chemical Chemical structure of compound compound 118118 [83].[83].

Inflammation-relatedInflammation-related diseasesdiseases areare characterizedcharacterized by by the the expressions expressions of of iNOS iNOS and and COX-2. COX-2. CompoundCompound188 188suppressed suppressed the productionthe production of NO of in LPS-inducedNO in LPS-induced RAW264.7 RAW264.7 cells in vitro cells. Furthermore, in vitro. it inhibitedFurthermore, LPS-induced it inhibited protein LPS-in expressionduced protein and immunofluorescence expression and immunofluorescence study showed that study the compoundshowed slightlythat the lowered compound the activation slightly lowered p65 in the the nuclei. activation The anti-inflammatory p65 in the nuclei. roleThe ofanti-inflammatory the compound is role partially of attributedthe compound to its inhibitory is partially eff attributedect on the to NF- itsκ Binhibitory signaling effect pathway on the LPS-induced NF-κB signaling RAW pathway 264.7 cells. LPS- [83]. induced RAW 264.7 cells. [83]. 8. Conclusions 8. ConclusionsResearch reports have shown that derivatives of cinnamic acid are active in vitro when compared to someResearch of the currently reports usedhave drugsshown such that as derivatives the anticancer, of cinnamic antimicrobial acid drugsare active etc. whichin vitro were when used ascompared control. The to some antimicrobial, of the currently anticancer used and drugs anti-oxidant such as the activity anticancer, of the antimicrobial derivatives drugs was influenced etc. which by thewere nature used and as positioncontrol. The of theantimicrobial, substituent anticancer groups on and the anti-oxidant phenyl ring. activity The lengthof the derivatives of the linker was also influencedinfluenced the by biological the nature activity and position of the of derivatives. the substituent groups on the phenyl ring. The length of the linkerFurthermore, also influenced the toxicological the biological studies activity performed of the derivatives. on some of the derivatives revealed the non-toxic effects ofFurthermore, the compounds the toxicological suggesting studies their suitabilityperformed foron some applications of the derivativesin vivo. However, revealed the the non- major draw-backtoxic effects pertaining of the compounds to the synthesized suggesting derivatives their suitability is that for most applications of them lackin vivo.in vivo However,data. Thus,the addingmajor todraw-back the in vitro pertaininganalyses to that the havesynthesized been carried derivatives on most is that of most the potent of them derivatives, lack in vivo there data. is a needThus, to proceedadding to to thein vivoin vitrostudies. analyses More that research have been on thecarried development on most of of the cinnamic potent derivatives, derivatives there has the potentialis a need to to result proceed in potent to in compoundsvivo studies. thatMore will research successfully on the reachdevelopment clinical trials.of cinnamic derivatives has the potential to result in potent compounds that will successfully reach clinical trials.

Funding:Acknowledgments:This research The received financial no external assistance funding. of the Medical Research Council and National Research Acknowledgments:Foundation, South TheAfrica financial towards assistance this research of the are Medical hereby acknowledged. Research Council The and views National and opinions Research expressed Foundation, in SouththisAfrica manuscript towards arethis those research of the authors are hereby and acknowledged. not of MRC or NRF. 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. Conflicts of Interest: The authors declare no conflicts of interest. Abbreviations

Int. J. Mol. Sci. 2020, 21, 5712 30 of 34

Abbreviations

Cpd. Compound IC50Half maximal inhibitory concentration EC50 Half maximal eﬀective concentration HSV Herpes simplex virus HIV Human immunodeﬁciency virus DMSO Dimethyl sulfoxide TEA Triethylamine DCM Dichloromethane EDCl 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide DMAP 4-Dimethylaminopyridine DMF D imethylformamide HOBt Hydroxybenzotriazole CNS Central Nervous System BBB Blood-Brain Barrier AKT Protein kinase B ERK extracellular signal-regulated kinase

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