Natural and Biomimetic Antitumor Pyrazoles, a Perspective

molecules

Review Natural and Biomimetic Antitumor Pyrazoles, A Perspective

Nádia E. Santos 1,* , Ana R.F. Carreira 2 , Vera L. M. Silva 1 and Susana Santos Braga 1,*

1 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] 2 CICECO–Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] * Correspondence: [email protected] (N.E.S.); [email protected] (S.S.B.)

Received: 14 February 2020; Accepted: 15 March 2020; Published: 17 March 2020

Abstract: The present review presents an overview of antitumor pyrazoles of natural or bioinspired origins. Pyrazole compounds are relatively rare in nature, the first ones having been reported in 1966 and being essentially used as somniferous drugs. Cytotoxic pyrazoles of natural sources were first isolated in 1969, and a few others have been reported since then, most of them in the last decade. This paper presents a perspective on the current knowledge on antitumor natural pyrazoles, organized into two sections. The first focuses on the three known families of cytotoxic pyrazoles that were directly isolated from plants, for which the knowledge of the medicinal properties is in its infancy. The second section describes pyrazole derivatives of natural products, discussing their structure–activity relationships.

Keywords: pyrazole alkaloids; pyrazofurin; dystamycin analogues; curcuminoid pyrazoles

1. Introduction The biochemical machinery of plants and marine life is the source of an immense variety of compounds with medicinal activity. Natural products were, traditionally, the main source of medicinal actives, and today they continue to provide useful new drugs. Examples of natural antitumorals include paclitaxel (taxol) [1] and a few marine drugs such as cytarabine, eribulin, and trabectedin [2–6]. Notably, ﬁve-membered heterocycles are found in a variety of natural antitumor compounds. Figure1 presents some examples of these molecules. The structural diversity of the pentameric heterocycles in such natural compounds is notable, from nitrogen heterocycles like pyrrole and imidazole, to pentameric cycles containing two kinds of heteroatoms like thiazole and oxazole. Distamycin A, obtained from Streptomyces netropsis, features several pyrrole rings; it is an antitumor, antimicrobial, and antiviral agent that acts by altering the conformation of DNA via irreversible binding to its A–T rich domains [7,8]. Leucamide A, isolated from the marine sponge Leucetta microraphis of the Great Barrier Reef of Australia, has an uncommon chemical structure with a mixed methyloxazole and thiazole pair. It has selective inhibitory action against tumor cells (being innocuous on microbes and algae) [9]. Phenylahistin is a metabolite of the fungus Aspergillus ustus that presents an imidazole moiety associated with a diketopiperazine [10]. It has antitumor activity via inhibition of tubulin polimerisation [11]. Topsentin is also a natural imidazole derivative. This compound is isolated from Caribbean deep-sea sponges of the Spongosorites genus, and it features antitumor activity on mouse models that has been associated with binding at the minor groove of DNA [12]. The promising properties of natural molecules such as topsentin, leucamide A, and dystamycin A have made them promising leads for the development of derivatives with improved activity (see, for instance, the family of compounds described in Section 3.2).

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Figure 1. Structures of distamycin A, leucamide A, phenylahistin, and topsentin, four examples of Figure 1. Structures of distamycin A, leucamide A, phenylahistin, and topsentin, four examples of Figurenatural 1anti. Structurestumor compounds of distamycin exhibiting A, leucamide a variety A, of phenylahistin pentameric heterocycles., and topsentin, four examples of natural antitumor compounds exhibiting a variety of pentameric heterocycles. natural antitumor compounds exhibiting a variety of pentameric heterocycles. Within pentameric heterocycles, pyrazoles, comprising comprising two two adjacent nitrogen atoms, are the Within pentameric heterocycles, pyrazoles, comprising two adjacent nitrogen atoms, are the less abundant abundant ones ones in in n natureature and and also also less less known known and and explored explored as as natural natural products. products. The The scarci scarcityty of less abundant ones in nature and also less known and explored as natural products. The scarcity of naturalof natural pyrazoles pyrazoles has hasbeen been attributed attributed to the to difficulty the diﬃculty in the in formation the formation of the ofN– theN bond N–N by bond living by natural pyrazoles has been attributed to the difficulty in the formation of the N–N bond by living organismsliving organisms [13]. Nevertheless, [13]. Nevertheless, pyrazole pyrazole is found is found in the in the structure structure of of a a few few alkaloids, alkaloids, namely namely,, organisms [13]. Nevertheless, pyrazole is found in the structure of a few alkaloids, namely, withasomnine and cinachyrazoles A, B B,, and C (Figure2 2).). WithasomnineWithasomnine isis aa papaverin-likepapaverin-like sedativesedative withasomnine and cinachyrazoles A, B, and C (Figure 2). Withasomnine is a papaverin-like sedative that occurs in the roots of Withania somnifera [1[144], the root bark of Newbouldia laevis [1[155], and in that occurs in the roots of Withania somnifera [14], the root bark of Newbouldia laevis [15], and in Elytraria acaulis [1[166]].. The The cinachyrazoles cinachyrazoles A, A, B B,, and and C are 1,3,5-trimethylpyrazole1,3,5-trimethylpyrazole alkaloids recently Elytraria acaulis [16]. The cinachyrazoles A, B, and C are 1,3,5-trimethylpyrazole alkaloids recently isolated fromfrom seasea sponge sponge species species of ofthe the genus genusCinachyrella Cinachyrella, and, theyand theyhave nohave known no known bioactivity, bioactivity, having isolated from sea sponge species of the genus Cinachyrella, and they have no known bioactivity, havingdisplayed displayed no cytotoxicity no cytotoxicity in tests in with tests cultured with cultured cancer cellcancer lines cell [17 lines]. [17]. having displayed no cytotoxicity in tests with cultured cancer cell lines [17].

Figure 2. Structures of withasomnine and cinachyrazoles A, B, and C, pyrazole alkaloids occurring in Figure 2. Structures of withasomnine and cinachyrazoles A, B, and C, pyrazole alkaloids occurring in plants and sea sponges. Figureplants and2. Structures sea sponges. of withasomnine and cinachyrazoles A, B, and C, pyrazole alkaloids occurring in plants and sea sponges. Notably, the pyrazole ring by itself (i.e., unsubstituted) was demonstrated to have some biological activity.Notably, It features the pyrazole anti-inflammatory ring by itsel propertiesf (i.e., unsubst in carrageenan-inducedituted) was demonstrated mice paw to oedema have some [18], Notably, the pyrazole ring by itself (i.e., unsubstituted) was demonstrated to have some biologicalnephroprotecting activity. action It features in cisplatin-induced anti-inflammatory mice properties kidney damage in carrageenan [19], and- ainduced caspase-mediated mice paw biological activity. It features anti-inflammatory properties in carrageenan-induced mice paw oedemaapoptotic [1 eff8ect], nephroprotecting on lung cancer cells action (A549 iline)n cisplatin [20]. The-induced pyrazole mice core is kidney also found damage in the [1 structure9], and ofa oedema [18], nephroprotecting action in cisplatin-induced mice kidney damage [19], and a caspasea few synthetic-mediated commercial apoptotic drugs,effect suchon lung asthe cancer nonsteroidal cells (A549 anti-inflammatory line) [20]. The pyrazole agents celecoxib core is also and caspase-mediated apoptotic effect on lung cancer cells (A549 line) [20]. The pyrazole core is also foundlonazolac, in andthe two structure antitumor of drugs,a few represented synthetic incommercial Figure3: Encorafenib, drugs, such for as the theoral treatmentnonsteroidal of found in the structure of a few synthetic commercial drugs, such as the nonsteroidal antiskin-inflammatory cancer [21–24], age andnt crizotinibs celecoxib [25 and–27 l],onazolac also taken, and orally two andanti usedtumor in drugs lung, cancer. represented in Figure anti-inflammatory agents celecoxib and lonazolac, and two antitumor drugs, represented in Figure

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Molecules3: Encorafenib 2019, 25, ,1364 for the oral treatment of skin cancer [21–24], and crizotinib [25–27], also taken3 orally of 12 and used in lung cancer. 3: Encorafenib, for the oral treatment of skin cancer [21–24], and crizotinib [25–27], also taken orally Molecules 2020, 25, 1364 3 of 12 and used in lung cancer.

Figure 3. Antitumor pyrazoles available in the market: encorafenib (tradename Braftovi®) and

crizotinib (tradename Xalkori®). Figure 3. Antitumor pyrazoles available in the market: encorafenib (tradename Braftovi®) and Figure 3. Antitumor pyrazoles available in the market: encorafenib (tradename Braftovi®) and crizotinib (tradename Xalkori®). crizotinibThis review (tradename is organi Xalkorized into®). two sections. It starts by presenting natural pyrazoles that have reported cytotoxic activity. The second section looks into the families of pyrazole compounds that This review is organized into two sections. It starts by presenting natural pyrazoles that have deriveThis from review or are is inspiredorganized by into active two natural sections. molecules It starts, identifyingby presenting their natural main targetspyrazoles of actionthat have and reported cytotoxic activity. The second section looks into the families of pyrazole compounds that derive reportedcomparing cytotoxic the activities activity. ofThe compound second sections with looksin the into same the families family toof pyraz discloseole compounds some important that from or are inspired by active natural molecules, identifying their main targets of action and comparing derivestructure from–activity or are relationsinspired hipsby active. Data natural resultin moleculesg from on, theiridentifying safety theiris present mained targets when ofpossible, action andand the activities of compounds within the same family to disclose some important structure–activity comparingthe implications the activitiestowards a ofpossible compound futures medicinalwithin the use same are discussed. family to disclose some important relationships. Data resulting from on their safety is presented when possible, and the implications structure–activity relationships. Data resulting from on their safety is presented when possible, and towards a possible future medicinal use are discussed. the2. A implicationsntitumor Pyrazoles towards from a possible Natural future Sources medicinal use are discussed. 2. Antitumor Pyrazoles from Natural Sources 2.2.1. A ntiPyrazoletumor A Plkaloidsyrazoles Found from in N Waturalatermelon Sources Seeds 2.1. PyrazoleA new Alkaloidspyrrolopyrazole Found in ( Watermelon1) and its g Seedsalactosylated derivative (2), represented in the Figure 4, 2.1. Pyrazole Alkaloids Found in Watermelon Seeds wereA recently new pyrrolopyrazole isolated from (1 ) the and seeds its galactosylated of Citrullus derivative lanatus, a ( species2), represented of watermelon in the Figure [28].4 , These were recentlycompoundsA new isolated pyrrolopyrazolewe fromre theclassified seeds (1 of) Citrullus and as itspyrazole g lanatusalactosylated , aalkaloids species derivative of watermelonand (2 postulated), represented [28]. These to compounds in derive the Figure werefrom 4, wereclassiﬁedL-α-amino recently as-β pyrazole-(pyrazolyl isolated alkaloids - fromN)-propanoic the and seeds postulated acid, of Citrullusa naturally to derive lanatus occurring from, al -α species-amino- amino of βacid -(pyrazolyl- watermelon with a pyrazoleN )-propanoic [28]. These ring, compoundsacid,found a in naturally the juicewe occurringre of watermelonsclassified amino acidas [1 3]. withpyrazole a pyrazole alkaloids ring, foundand in postulated the juice of watermelons to derive [13 from]. L-α-amino-β-(pyrazolyl-N)-propanoic acid, a naturally occurring amino acid with a pyrazole ring, found in the juice of watermelons [13].

Figure 4. Structure of two pyrazole derivatives 1 and 2 isolated from the seeds of Citrullus lanatus Figure 4. Structure of two pyrazole derivatives 1 and 2 isolated from the seeds of Citrullus lanatus watermelon. 1-[2-(5-hydroxymethyl-1H-pyrrole-2-carbaldehyde-1-yl)ethyl]-1H-pyrazole (1) showed nowatermelon. cytotoxicity, 1-[2- while(5-hydroxymethyl 1-({[5-(α-d-galactopyranosyloxy)methyl]-1-1H-pyrrole-2-carbaldehyde-1-yl)ethyl]H-pyrrole-2-carbaldehyde-1-yl}--1H-pyrazole (1) showed -ethyl)-1Figureno cytotoxicity, 4H. Structure-pyrazole whileof (2 )two was 1pyrazole- mildly({[5-(α- cytotoxicD -derivativesgalactopyranosyloxy)methyl] on mouse 1 and melanoma2 isolated from cells.-1H- pyrrolethe seeds-2- carbaldehydeof Citrullus lanatus-1-yl}- watermelon.-ethyl)-1H-pyrazole 1-[2-(5- hydroxymethyl(2) was mildly cytotoxic-1H-pyrrole on- mouse2-carbaldehyde melanoma-1- yl)ethyl]cells. -1H-pyrazole (1) showed noThe cytotoxicity, cytotoxicity while of compounds 1-({[5-(α-D-1galactopyranosyloxy)methyl]and 2 was tested against-1 aH skin-pyrrole cancer-2-carbaldehyde cell line (mouse-1-yl}- B16 melanoma-Theethyl) cytotoxicity- 4A51H-pyrazole cell line). of (2 )compoundsMild was mildly activity cytotoxic 1 was and observed 2 on was mouse tested for melanoma compound against cells a 2skin,. with cancer 70.4% cell growth line inhibition(mouse B16 at amelanoma concentration 4A5 ofcell 100 line).µM, Mild but notactivity for compound was observed1. This for can compound be attributed 2, with to the70.4% higher growth solubility inhibition and incorporationat a concentrationThe cytotoxicity of 2 into of 100of the compounds μ cellsM, but that not results 1 for and compound from 2 was the tested presence 1. This against ofcan a sugarbe a attributedskin moiety cancer into itscell the chemical linehigher (mouse solubility structure. B16 melanomaand incorporation 4A5 cell ofline). 2 into Mild the activity cells that was results observed from for the compound presence of2, awith sugar 70.4% moiety growth in its inhibition chemical at2.2.structure. a concentration Pyrazofurin, A of Natural 100 μM, C-Nucleoside but not for compound 1. This can be attributed to the higher solubility and incorporationPyrazofurin (3of), 2 depicted into the incells Figure that 5results, is a naturally from the occurringpresence of nucleoside a sugar moiety analogue in its produced chemical structure.by Streptomyces candidus. It has been known for decades, but only very recently has its biosynthesis begun to be elucidated, with researchers unveiling the involvement of PyrN, a new enzyme capable

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2.2. Pyrazofurin, A Natural C-Nucleoside Pyrazofurin (3), depicted in Figure 5, is a naturally occurring nucleoside analogue produced by MoleculesStreptomyces2020, 25 candidus, 1364 . It has been known for decades, but only very recently has its biosynthesis4 of 12 begun to be elucidated, with researchers unveiling the involvement of PyrN, a new enzyme capable of forming the N–NN–N bond [2929].]. Since its discovery in 1969, pyrazofurin has been the target ofof muchmuch interest due to to its its antimicrobial, antimicrobial, antiviral, antiviral, and antitumorantitumor properties, and for this reason a patent covering its bioindustrial production processprocess waswas ﬁledfiled onon thethe samesame yearyear (and(and grantedgranted inin 1974)1974) [[3030].].

Figure 5. Pyrazofurin, or 3-β-d-ribofuranosyl-4-hydroxy-1H-pyrazole-5-carboxamide (3), a natural Figure 5. Pyrazofurin, or 3-β-D-ribofuranosyl-4-hydroxy-1H-pyrazole-5-carboxamide (3), a natural pyrazole obtained from Streptomyces candidus strains. pyrazole obtained from Streptomyces candidus strains.

Pyrazofurin works as an antimetabolite, inhibiting orotidine-50-monophosphate decarboxylase Pyrazofurin works as an antimetabolite, inhibiting orotidine-5′-monophosphate decarboxylase and stopping the biosynthesis of pyrimidine [31]. Its antineoplastic activity was demonstrated in and stopping the biosynthesis of pyrimidine [31]. Its antineoplastic activity was demonstrated in rats, and a relatively broad range of tumors were shown to be sensitive to it, including Walker rats, and a relatively broad range of tumors were shown to be sensitive to it, including Walker carcinosarcoma, Ca755 adenocarcinoma, plasma cell myeloma, and various types of lymphosarcoma carcinosarcoma, Ca755 adenocarcinoma, plasma cell myeloma, and various types of lymphosarcoma and of breast carcinoma [32]. Phase I clinical trials were conducted on human patients with disseminated and of breast carcinoma [32]. Phase I clinical trials were conducted on human patients with cancer, but objective tumor regression was not observed in any of the 50 patients deemed suitable for disseminated cancer, but objective tumor regression was not observed in any of the 50 patients response evaluation [33]. Following this study, interest in pyrazofurin as an antitumor drug has faded. deemed suitable for response evaluation [33]. Following this study, interest in pyrazofurin as an Nevertheless, and considering the modern tools and methods available for chemical modification antitumor drug has faded. Nevertheless, and considering the modern tools and methods available procedures, this molecule is worth revisiting as an inspiring model to design derivatives with better for chemical modification procedures, this molecule is worth revisiting as an inspiring model to activity [34]. design derivatives with better activity [34]. 2.3. Pyrazole Derivatives from the Tall-stilted Mangrove Tree 2.3. Pyrazole Derivatives from the Tall-stilted Mangrove Tree The tall-stilted mangrove, Rhizophora apiculata, is a tree of the Indo-West Pacific that grows in intertidalThe tall wetlands-stilted mangrove, in close association Rhizophora with apiculata other, is species a tree ofof thethe mangrove.Indo-West Pacific Used inthat traditional grows in medicine,intertidal wetlands stilt mangrove in close bark association is associated with with other treatment species of of angina, the mangrove boils, and. Used fungal in traditional infections; leavesmedicine, and stilt bark mangrove have been bark used is as associated an antiseptic with and treatment to treat diarrhea,of angina, dysentery, boils, and fever, fungal and infections malaria,; albeitleaves with and unknownbark have ebeenffectiveness used as ratesan antiseptic [35]. and to treat diarrhea, dysentery, fever, and malaria, albeitInvestigation with unknown of effectiveness the main active rates components[35]. in R. Apiculata was conducted by preparing methanolInvestigation extracts of of the the main whole active plant compo and analyzingnents in R. their Apiculata composition was conducted [36]. Characterization by preparing ofmethanol the composition extracts of the of thewhole extract plant hasand analyzing revealed thetheir presence composition of a[36 new]. Characteri pyrazolezation derivative of the (composition4) as well as of several the extract other has compounds, revealed the including presence a of 4,5-dihydropyrazyltriazole a new pyrazole derivative derivative (4) as well and as Nseveral-phenyl-1-pyrazolidinecarboxamide, other compounds, including all depicted a 4,5 in-dihydro the Figurepyraz6y.ltriazole Moreover, derivative the extract and displayedN-phenyl-1 tumor-reducing-pyrazolidinecarboxamide, properties in all mouse depicted melanoma in the Figure models, 6. withMoreover, reduction the ofextract tumor displayed volume, increasedtumor-reducing body weight,properties and in a mouse 50% raise melanoma in the survival models, rate with after reduction 30 days. of Astumor a remark volume, to theincreased report ofbody Prabhu weight et, al.and [36 a ],50% we raise note in that the the sur antitumorvival rate after activity 30 days. reported As a forremark the extract to the report cannot of be Prabhu directly et correlatedal. [36], we withnote that compound the anti4tumor. There activity are multiple reported chemical for the extract entities cannot in the be extract, directly and correlated the activity with maycompound result from4. There one are of multiple them or fromchemical a combined entities in eff theect. extract It would, and thus the beactivity interesting may result to isolate from andone evaluateof them or the from different a combined compounds effect in. It the would extract thus to be allow interesting for the identification to isolate and of evaluate the active the molecules different andcompounds possible synergiesin the extract between to allow them. for the identification of the active molecules and possible synergies between them.

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Figure 6. Structures of three selected compounds from those occuring in the methanol Figure 6. Structures of three selected compounds from those occuring in the methanol extract extract obtained from the whole plant of R. apiculata, which include a pyrazole derivative, obtained from the whole plant of R. apiculata, which include a pyrazole derivative, (5-bromo-2-hydroxy-phenyl)-(1-phenyl-1H-pyrazol-4-yl)ketone (4), a dihydropyrazole derivative, (5-bromo-2-hydroxy-phenyl)-(1-phenyl-1H-pyrazol-4-yl)ketone (4), a dihydropyrazole derivative, and a pyrazolidine derivative. and a pyrazolidine derivative. 3. Pyrazole Analogues of Natural Products 3. Pyrazole Analogues of Natural Products 3.1. Curcuminoid Pyrazoles for Telomerase Inhibition 3.1. Curcuminoid Pyrazoles for Telomerase Inhibition Curcumin, a diaryl heptano-3,5-dione, is the main active compound present in the rhizome of CurcumaCurcumin, longa (turmeric). a diaryl heptano Used- for3,5- centuriesdione, is the as amain traditional active compound medicine, curcuminpresent in is the able rhizome to act onof Curcumamultiple biologicallonga (turmeric) targets,. Used thus havingfor cen aturies varied as seta traditional of activities: medicine, anti-inflammatory, curcumin is antioxidant, able to act and on multipleantitumor. biological The later targets, has, in thus recent having years, a varied gained set growing of activities: recognition anti-inflammatory due to the good, antioxidant results, from and anticlinicaltumor trials. The on later patients has, within recent various years, types gain ofed cancer growing [37]. recognition due to the good results from clinicalA knowntrials on target patients of curcumin with various is telomerase. types of cancer Curcumin [37]. interferes with the expression of the genes that encodeA known hTERT, target an of RNA curcumin component is telomerase of telomerase. Curcumin [38,39], interfer thus increasinges with telomerasethe expression expression of the genesand activity. that encode This enzymehTERT, hasan theRNA function component of repairing of telomerase damage [38,39 to the], ends thus of increasing the DNA telomerase caused by expcontinuousression and replication, activity. beingThis enzyme active in has stem the cellsfunction and dormantof repairing in adultdamage somatic to the cells. ends Reactivationof the DNA causedof telomerase by continuous is a critical replication step in carcinogenesis,, being active asin it stem makes cells neoplasic and dormant cells immortal, in adult that somatic is, able cells to. Rreplicateeactivation indefinitely. of telomerase is a critical step in carcinogenesis, as it makes neoplasic cells immortal, that is, ableCurcuminoid to replicate pyrazolesindefinitely. are a class of curcumin analogues obtained by replacement of the diketone moietyCurcumino with a pyrazoleid pyrazoles ring. Theyare a were class first of curcumin developed analogues as anti-inflammatory obtained by agents replacement [40]. In ofrecent the diketoneyears, with moiety the discovery with a pyrazole of hTERT ring as one. They of thewere targets first ofdeveloped curcumin, as these anti- structuresinflammatory became agent interestings [40]. In recentfor cancer years, therapy. with the In adiscovery first set of of studies, hTERT a libraryas one of thirteenthe targets curcuminoid of curcumin, pyrazoles these structures was prepared became and interestingscreened for for cytotoxicity cancer therapy. using In the a cancerfirst set cell of studies, lines HeLa a library (cervix of carcinoma), thirteen curcumino HT-29 (colonid pyrazoles carcinoma), was preparedand MCF-7 and (breast screened cancer), for and cytotoxicit a nontumory using line, the HEK-293 cancer cell (human lines embryonic HeLa (cervix kidney carcinoma) cells) [41, HT]. The-29 (coloncompounds carcinoma)5–8, represented, and MCF-7 in (breast Figure cancer),7, are highlighted and a nontumor in the presentline, HEK review-293 (human for their embryonic superior kidneyperformance. cells) [4 Notably,1]. The compounds all the studied 5–8 compounds, represented were in Figure able to7, inhibitare highlighted the growth in ofthe HeLa present cells, review with fICor theirvalues superior ranging performance. from 1.7 0.6Notably,µM for all compound the studied5, compounds to 36.1 0.4 wereµM for able the to less inhibit potent the compound growth of 50 ± ± HeLa(not shown). cells, with Against IC50 values the cell ranging lines MCF-7 from 1.7 and ± HT-29,0.6 μM the for most compound active compounds5, to 36.1 ± 0.4 were μM5 andfor the7, withless potentIC50 values compound under (not 15 µ M.shown). The compounds Against the were cell lines further MCF investigated-7 and HT- regarding29, the most their active ability compounds to inhibit werethe expression 5 and 7, with of the IC50 hTERT values gene under using 15 μ quantitativeM. The compounds PCR (RT-qPCR). were further Compounds investigated5, 6, regardingand 8 strongly their abilitydownregulate to inhibit hTERT the expression gene expression of the hTERT to 49%, gene 42%, using and 44%,quantitative respectively, PCR (RT of the-qPCR). value Compounds for nontreated 5, 6cells., and Note8 strongly that these downregulate values are hTERT higher gene than expression the one shown to 49% by, 42 curcumin,%, and 44%, which respectively, reduced hTERTof the valueexpression for non totreated 59%. The cells. safety Note of thethat compounds these values was are evaluated higher than on nontumorthe one shown cells ofby embryoniccurcumin, kidneywhich reducedline HEK-293. hTERT The expression IC50 values to 59% against. The thissafety cell of line thewere compounds quite low, was which evaluated raises on severe nontumor safety cells issues. of embryonicThe most striking kidney case line isHEK that-293. of compound The IC50 values5, more against toxic on this nontumor cell line cell were line quite than low against, which the tumorraises severelines. Insafety fact, issues.5 has an The IC mostof 0.72striking0.35 caseµM is onthat the of embryonic compound kidney 5, more HEK-293 toxic on cells.nontumor Compound cell line7 50 ± than against the tumor lines. In fact, 5 has an IC50 of 0.72 ± 0.35 μM on the embryonic kidney HEK-293

Molecules 2020, 25, 1364 6 of 12 Molecules 2019, 25, 1364 6 of 12 cells.also performed Compound poorly, 7 also with performed an IC ofpoorly 2.9 , 0.6with on an this IC cell50 of line. 2.9 Overall,± 0.6 on allthis the cell compounds line. Overall, are toxicall the to 50 ± compounds are toxic to kidney cells as their IC50 values are below 55 μM. kidney cells as their IC50 values are below 55 µM.

Figure 7. Structures of curcumin and of selected compounds from two families of curcuminoid Figure 7. Structures of curcumin and of selected compounds from two families of curcuminoid pyrazoles: curcumin pyrazole, 5; the hemicurcumin pyrazoles 6–8 [41]; and the 1H-substituted pyrazoles: curcumin pyrazole, 5; the hemicurcumin pyrazoles 6–8 [41]; and the 1H-substituted derivatives 9–19 [42]. derivatives 9–19 [42]. Another family of curcuminoid pyrazoles was developed by inserting, in the 1H-position of the Another family of curcuminoid pyrazoles was developed by inserting, in the 1H-position of the pyrazole, a variety of substituents, with higher predominance of aryl groups [42]. Their cytotoxic pyrazole, a variety of substituents, with higher predominance of aryl groups [42]. Their cytotoxic evaluation was conducted on the HeLa, MCF-7, and A549 (lung adenocarcinoma) cell lines. Figure7 evaluation was conducted on the HeLa, MCF-7, and A549 (lung adenocarcinoma) cell lines. Figure 7 presents a selection of eleven compounds, 9–19, that had the strongest activities. All of these eleven presents a selection of eleven compounds, 9–19, that had the strongest activities. All of these eleven compounds had IC50 values lower than 20 µM against the three tumor cell lines tested. Regarding compounds had IC50 values lower than 20 μM against the three tumor cell lines tested. Regarding growth inhibition of cells of the MCF-7 line, compounds 11 and 12 were the most active ones, with growth inhibition of cells of the MCF-7 line, compounds 11 and 12 were the most active ones, with IC50 values of 6.0 0.7 and 5.8 0.4 µM, respectively. Compound 12 also performed well against IC50 values of 6.0 ±± 0.7 and 5.8 ± 0.4± μM, respectively. Compound 12 also performed well against the the A549 and HeLa cell lines, with IC50 values of 8.0 1.4 and 9.8 0.8 µM, respectively. Studies on A549 and HeLa cell lines, with IC50 values of 8.0 ± 1.4± and 9.8 ± 0.8± μM, respectively. Studies on the the toxicological safety of these compounds on healthy cells were not conducted. Considering the toxicological safety of these compounds on healthy cells were not conducted. Considering the abovementioned toxicity of compound 5 (the parent curcumin pyrazole), studies with nontumor cell abovementioned toxicity of compound 5 (the parent curcumin pyrazole), studies with nontumor cell lines are vital for the realistic assessment of the applicability of these compounds in tumor chemotherapy. lines are vital for the realistic assessment of the applicability of these compounds in tumor chemotherapy.

3.2. Pyrazole-bearing Dystamycin Analogues as DNA Alkylating Agents

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Molecules 2019, 25, 1364 7 of 12 3.2. Pyrazole-bearing Dystamycin Analogues as DNA Alkylating Agents Distamycin A has, as mentioned in the introduction, the ability to recognize and bind to DNA Distamycin A has, as mentioned in the introduction, the ability to recognize and bind to DNA regions rich in A-T pairs, which means that it can be used as a vector for DNA. This concept was regions rich in A-T pairs, which means that it can be used as a vector for DNA. This concept was employed to create a new family of DNA alkylating agents. These compounds exhibited two active employed to create a new family of DNA alkylating agents. These compounds exhibited two active subunits: the first one, meant to act as a vector, comprised pyrazole analogues of distamycin A; the subunits: the ﬁrst one, meant to act as a vector, comprised pyrazole analogues of distamycin A; other one was a DNA-alkylating subunit [43]. Figure 8 shows a selection of the six most active the other one was a DNA-alkylating subunit [43]. Figure8 shows a selection of the six most active compounds, 20–25. They were evaluated for cytotoxicity using three human cell lines associated with compounds, 20–25. They were evaluated for cytotoxicity using three human cell lines associated blood cancer: T-lymphoblast (Molt/4 and CEM cell lines) and B-lymphoblast (Daudi cell line). In with blood cancer: T-lymphoblast (Molt/4 and CEM cell lines) and B-lymphoblast (Daudi cell line). general, compounds 20–22 performed better than compounds 23–25. A special highlight should be In general, compounds 20–22 performed better than compounds 23–25. A special highlight should be given to compound 22, the most active of the entire family, featuring IC50 values in the nanomolar given to compound 22, the most active of the entire family, featuring IC50 values in the nanomolar range (between 7.4 and 71 nM, depending on the cell line). Also noteworthy is the direct correlation range (between 7.4 and 71 nM, depending on the cell line). Also noteworthy is the direct correlation between the increasing number of pyrrolic rings in the 20–22 series and the increase in antiproliferative between the increasing number of pyrrolic rings in the 20–22 series and the increase in antiproliferative activity. In turn, for the series of compounds 23–25, the activity seemed to follow an inverse pattern, activity. In turn, for the series of compounds 23–25, the activity seemed to follow an inverse pattern, with compound 23, having only one pyrrole ring, being the most active. with compound 23, having only one pyrrole ring, being the most active.

Figure 8. The six most active compounds from a family of pyrazole analogues of distamycin A with Figure 8. The six most active compounds from a family of pyrazole analogues of distamycin A with DNA alkylating activity [43]. DNA alkylating activity [43]. A second family of pyrazole analogues of distamycin A agents comprised an α-methylidene-A secondγ -butyrolactone family of pyrazole as the alkylating analogues subunit of distamycin [44]. In this A series, agents some comprised compounds, an suchα-methylidene as 26–28 (Figure-γ-butyrolactone9) had a phenyl as the substituentalkylating subunit at the γ [44].-position In this of series, the lactone, some compounds while a few, such others as (not26–28 shown) (Figure presented 9) had a methylphenyl group substituent in the sameat the position. γ-position The of phenyl-containing the lactone, while compounds, a few others26 –(not28, wereshown) those present withed better a methyl activity group against in the human same lymphoma position. The cell phenyl lines (Molt-containing/4 and CEM8).compounds, They 26 also–28, were those with better activity against human lymphoma cell lines (Molt/4 and CEM8). They also exhibited apoptotic activity on the human leukemia cell line HL-60. Also noteworthy is the fact that the polypyrrolic frame in compounds 26–28 was lengthy (n = 2 or 3). Thus, compounds 26–28 are

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exhibitedMolecules 2019 apoptotic, 25, 1364 activity on the human leukemia cell line HL-60. Also noteworthy is the fact8 of that 12 the polypyrrolic frame in compounds 26–28 was lengthy (n = 2 or 3). Thus, compounds 26–28 are structurally very similar to dystamycin A,A, whichwhich hashas threethree pyrrolespyrroles ((nn == 3).3). This may lay aa structuralstructural rationalerationale for their high cytotoxicity. On the other hand, compound 26,, withwith onlyonly twotwo pyrroles,pyrroles, waswas thethe strongest in inducing apoptosis. apoptosis.

Figure 9. The three most active compounds from a second-generation family of pyrazole analogues of Figure 9. The three most active compounds from a second-generation family of pyrazole analogues distamycin A with DNA alkylating properties and apoptosis-inducing activity [44]. of distamycin A with DNA alkylating properties and apoptosis-inducing activity [44]. 3.3. Phthalazinone-derived Pyrazoles for Aurora Kinase Inhibition 3.3. Phthalazinone-derived Pyrazoles for Aurora Kinase Inhibition The phthalazinone skeleton is an uncommon structure in natural compounds that seems to be associatedThe phthalazinone with the metabolism skeleton of is some an uncommon fungal species. structure It is easily in natural metabolized compounds to phthalazine that seems N-oxide to be byassociatedFusarium with monoliforme the metabolismand Cunninghamella of some fungal elegans species.[45]. A naturalIt is easily derivative metabolized of phthalazinone to phthalazine was isolatedN-oxide in by 2019 Fusarium from lichens monoliforme of the Amycolatopsis and Cunninghamellagenus [46 ]. elegans This new [45 natural]. A natural compound derivative was called of amycophthalazinonephthalazinone was isolated A (Figure in 102019), and from it haslichens shown of fairthe Amycolatopsis antimicrobial activitygenus [46 against]. ThisStaphylococcus new natural aureuscompound, Salmonella was called typhi, and amycophthalazinoneCandida albicans. A (Figure 10), and it has shown fair antimicrobial activityPhthalazinone against Staphylococcus is also an importantaureus, Salmonella pharmacophore typhi, and for Candida antitumor albicans activity.. Several derivatives, includingPhthalazinone the family is of also compounds an important29, in pharmaco which thephore benzene for anti ringtumor of the activity structure. Several of phthalazinone derivatives, isincluding replaced the by family pyrazole of compounds moiety, were 29, patentedin which the for benzene use as adjuvants ring of the in structure cancer chemotherapyof phthalazinone [47 is]. Inreplaced another by patent, pyrazole compounds moiety, we ofre the patented family numberedfor use as genericallyadjuvants in as cancer30 were chemotherapy equally claimed [47].for In useanother as antitumor patent, compounds agents, either of the alone family or numbered in combination generically [48]. as This 30 familywere equally comprised claimed a very for use large as varietyantitumor of pyrazolophthalazinonesagents, either alone or in for combination which the antitumor[48]. This family activity compris was attributeded a very to large Aurora variety kinase of inhibitionpyrazolophthalazinones [48,49]. Aurora for kinases which Athe and anti Btumor are two activity enzymes was attr involvedibuted in to diAuroraﬀerent kinase steps ofinhibition the cell division[48,49]. Aurora process. kinases Their inactivation A and B are allows two stopping enzymes the involved mitosis in process different and, steps subsequently, of the cell inhibiting division tumorprocess growth. Their viainactivation apoptosis. allows stopping the mitosis process and, subsequently, inhibiting tumor growth via apoptosis. A small library of compounds, 31-33, bearing a 5-methylpyrazole attached to the phthalazinone core (Figure 10), was developed and studied in comparison with compounds bearing a nonsubstituted pyrazole (not shown) [50]. Structure–activity relationship studies have demonstrated

Molecules 2019, 25, 1364 9 of 12

that the presence of the 5-methylpyrazole brought higher activity than nonsubstituted pyrazole, which was explained by the fact that the methyl group can easily bind in a largely hydrophobic pocket. Compounds 31–33 were excellent inhibitors of Aurora kinase A, with values in the nanomolar range (IC50 of 31, 65, and 35 nM, respectively); compound 31 was also able to inhibit Aurora kinase B, with an IC50 of 30 μM. In vitro cytotoxicity studies against MCF-7 breast cancer cell Molecules 2020, 25, 1364 9 of 12 line showed IC50 values of 1.6 μM for 31 and 33 and of 12.6 μM for 32.

Figure 10. Phthalazinone, its natural derivative amycophthalazinone A, and its pyrazole derivatives: Figure 10. Phthalazinone, its natural derivative amycophthalazinone A, and its pyrazole derivatives: families 29 [47] and 30 [48] (with high structural diversity of substituents at positions R1,R2,R3, and families 29 [47] and 30 [48] (with high structural diversity of substituents at positions R1, R2, R3, and R4), and compounds 31–33 [50]. R4), and compounds 31–33 [50]. A small library of compounds, 31-33, bearing a 5-methylpyrazole attached to the phthalazinone core (FigureA subsequent 10), was bioavailability developed and evaluation studied in comparisonin mice showed with compoundsthat all three bearing compounds a nonsubstituted displayed pyrazoleplasma concentrations (not shown) [50 above]. Structure–activity the cellular IC relationship50 at the half studies-life and have adequate demonstrated oral bioavailability. that the presence The ofhalf the-life 5-methylpyrazole itself is relatively brought short, higherranging activity between than roughly nonsubstituted two and pyrazole,four hours, which and wasafter explained 24 h the bycompounds the fact that were the completely methyl group eliminated can easily from bind the in body a largely [50]. hydrophobic pocket. Compounds 31–33 were excellent inhibitors of Aurora kinase A, with values in the nanomolar range (IC50 of 31, 65, and 4. Conclusions 35 nM, respectively); compound 31 was also able to inhibit Aurora kinase B, with an IC50 of 30 µM. In vitroPyrazolecytotoxicity compounds studies are against seldom MCF-7 found breast in nature. cancer They cell linewere showed first identified IC50 values in natural of 1.6 µ productsM for 31 andin the33 and 1960 ofs, 12.6 whicµMh for witnessed32. the discovery of withasomnine, a pyrazole alkaloid with sleepA-inducing subsequent properties. bioavailability In the evaluation 1970s, the in mice first showed natural that pyrazole all three with compounds cytotoxic displayed activity, plasmapyrazofurin, concentrations was reported. above Now, the cellularthe number IC50 ofat theknown half-life natural and pyrazoles adequate has oral increased, bioavailability. especially The half-lifein the last itself decade, is relatively and more short, of these ranging compounds between are roughly expected two to and be fourdiscovered hours, in and the after forthcoming 24 h the compoundsyears as a result were completely of the growing eliminated efforts from in naturalthe body product [50]. identification. Very little is known, however, about the ability of these compounds to stop tumor growth. Compound 2 was only tested 4. Conclusions on one tumor line (melanoma), having displayed mild cytotoxicity. Regarding compound 4, the Pyrazole compounds are seldom found in nature. They were first identified in natural products in the 1960s, which witnessed the discovery of withasomnine, a pyrazole alkaloid with sleep-inducing properties. In the 1970s, the first natural pyrazole with cytotoxic activity, pyrazofurin, was reported. Now, the number of known natural pyrazoles has increased, especially in the last decade, and more of these compounds are expected to be discovered in the forthcoming years as a result of the growing Molecules 2020, 25, 1364 10 of 12 efforts in natural product identification. Very little is known, however, about the ability of these compounds to stop tumor growth. Compound 2 was only tested on one tumor line (melanoma), having displayed mild cytotoxicity. Regarding compound 4, the activity still needs to be corroborated as it was tested in tandem with other compounds as part of a multicomponent plant extract. The herein gathered reports of bioinspired pyrazoles show more promising outcomes, albeit some of these families of compounds remain a few steps away from translation into the clinic. Curcuminopyrazoles (5–8) have a good antitumor action, but their structures need further improvement to achieve good toxicological safety profiles. Pyrazole-bearing analogues of dystamycin A, carefully designed to bind irreversibly to DNA, are excellent in inducing apoptosis of leukemia cells (HL-60). However, studies on their safety regarding healthy cells are still unavailable. Finally, pyrazolophthalazinones are gaining recognition as an emerging class of selective antitumor drugs. Several of these compounds (families 29 and 30) are already patented. New families under development include the one of compounds 31–33. These combine very potent activity with adequate bioavailability and a short time of permanence in the body, which helps ensure reduced exposure and thus minimize any potential secondary effects.

Funding: Thanks are due to the University of Aveiro and to FCT/MCTES (Fundação para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior) for general financial support to the QOPNA research unit (reference UID/QUI/00062/2019), to the associated laboratory LAQV-REQUIMTE (project reference UIDB/50006/2020) and to the associated laboratory CICECO (references UIDB/50011/2020 & UIDP/50011/2020). Vera L. M. Silva thanks for the Assistant Professor position within CEE-CINST/2018; since 01/09/2019. Ana Carreira acknowledges FCT for the Ph.D. grant SFRH/BD/143612/2019. Conflicts of Interest: The authors declare no conflict of interest.

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