Boronic Acids and Their Derivatives in Medicinal Chemistry: Synthesis and Biological Applications

Review Boronic Acids and Their Derivatives in Medicinal Chemistry: Synthesis and Biological Applications

Mariana Pereira Silva 1,2, Lucília Saraiva 2,* , Madalena Pinto 1 and Maria Emília Sousa 1,*

1 CIIMAR, Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n◦ 228, 4050-313 Porto, Portugal; up201909171@ff.up.pt (M.P.S.); madalena@ff.up.pt (M.P.) 2 LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n◦ 228, 4050-313 Porto, Portugal * Correspondence: lucilia.saraiva@ff.up.pt (L.S.); esousa@ff.up.pt (M.E.S.); Tel.: +351-22-0428689 (M.E.S.)

Academic Editor: Diego Muñoz-Torrero Received: 2 September 2020; Accepted: 19 September 2020; Published: 21 September 2020

Abstract: Boron containing compounds have not been widely studied in Medicinal Chemistry, mainly due to the idea that this group could confer some toxicity. Nowadays, this concept has been demystified and, especially after the discovery of the drug bortezomib, the interest for these compounds, mainly boronic acids, has been growing. In this review, several activities of boronic acids, such as anticancer, antibacterial, antiviral activity, and even their application as sensors and delivery systems are addressed. The synthetic processes used to obtain these active compounds are also referred. Noteworthy, the molecular modification by the introduction of boronic acid group to bioactive molecules has shown to modify selectivity, physicochemical, and pharmacokinetic characteristics, with the improvement of the already existing activities. Besides, the preparation of compounds with this chemical group is relatively simple and well known. Taking into consideration these findings, this review reinforces the relevance of extending the studies with boronic acids in Medicinal Chemistry, in order to obtain new promising drugs shortly.

Keywords: boronic acids; biological applications; synthesis of boronic acid derivatives; boron-containing compounds

1. Introduction The use of boron in the design of drugs is fairly recent and most biological activities of these compounds have been reported over the last decade [1]. For a long time, compounds with boron were put aside in studies of Medicinal Chemistry because they were thought to be toxic, mainly because of their use in ant poisoning. Nowadays, this believe is demystified, and boron-containing compounds are usually considered as non-toxic [2]. Boron compounds are found in nature in high concentrations, mainly in vegetables, fruits, and nuts, and the boron natural derivative boric acid is even used as a preservative in eyewash and as a buffer in biological assays [2]. There are many organoboron compounds (Figure1) but, in organic chemistry, boronic acid is the most commonly studied boron compound [2]. Boronic acids were first synthetized in 1860 by Edward Frankland [3] and can be used as building blocks and synthetic intermediates [1,4]. The application of these compounds in synthetic chemistry is due to their versatile reactivity, stability, and low toxicity [5–7] and also because when considering drug design boronic acid is further degraded to boric acid, a “green compound”, being the latter eliminated by the body [2,5,8].

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Figure 1. Organoboron compounds.

Boronic acids were first synthetized in 1860 by Edward Frankland [3] and can be used as building blocks and synthetic intermediates [1,4]. The application of these compounds in synthetic chemistry is due to their versatile reactivity, stability, and low toxicity [5–7] and also because when considering drug design boronic acid is further degraded to boric acid, a “green compound”, being Figure 1. Organoboron compounds. the latter eliminated by the body [2,5,8]. Boronic acids have unique physicochemical and electronic characteristics although boron has Boronic acidsacids havewere unique first synthetized physicochemical in 1860 and by electronicEdward Frankland characteristics [3] and although can be boron used has as interesting similarities with carbon. Besides, boronic acids are also considered bioisosteres of interestingbuilding blocks similarities and synthetic with carbon. intermediates Besides, boronic [1,4]. The acids application are also considered of these bioisosterescompounds of in carboxylic synthetic carboxylic acids [7]. acidschemistry [7]. is due to their versatile reactivity, stability, and low toxicity [5–7] and also because when Boronic acids are considered Lewis acids (Figure 2a), having a pKa value of 4–10 [2,4,5,9]. These consideringBoronic drug acids design are considered boronic acid Lewis is further acids degraded (Figure2a), to havingboric acid, a p a Ka“greenvalue compound”, of 4–10 [2 ,4being,5,9]. values can vary depending on the substituents on the boronic acids derivatives [4,9]; usually aryl Thesethe latter values eliminated can vary by depending the body on[2,5,8]. the substituents on the boronic acids derivatives [4,9]; usually aryl boronic acids are more acidic than alkyl boronic acids [5]. If an electron‐withdrawing group is boronicBoronic acids areacids more have acidic unique than physicochemical alkyl boronic acids and [5 ].electronic If an electron-withdrawing characteristics although group isboron attached has attached to an aromatic boronic acid, the pKa value decreases, while an electron‐donating group tointeresting an aromatic similarities boronic acid, with the carbon. pKa value Besides, decreases, boronic while acids an electron-donatingare also considered group bioisosteres increases the of increases the pKa value [5,10]. pcarboxylicKa value [acids5,10]. [7]. Boronic acids are considered Lewis acids (Figure 2a), having a pKa value of 4–10 [2,4,5,9]. These values can vary depending on the substituents on the boronic acids derivatives [4,9]; usually aryl boronic acids are more acidic than alkyl boronic acids [5]. If an electron‐withdrawing group is attached to an aromatic boronic acid, the pKa value decreases, while an electron‐donating group increases the pKa value [5,10].

Figure 2. Ionization equilibrium of boronic acid in aqueous solutions. ( (aa)) Boronic Boronic acid acid as as Lewis Lewis acid. acid. (b) Boronic acid asas Brønsted–Lowry acid.acid.

At physiological pH, pH, boronic boronic acids acids remain remain at at their their protonated protonated uncharged uncharged trigonal trigonal form form but, but, in in aqueous solutions with pH values higher than pKa, they are converted to anionic tetrahedral aqueous solutions with pH values higher than pKa, they are converted to anionic tetrahedral forms forms[2,4,6,7]. [2 ,Therefore,4,6,7]. Therefore, the equilibrium the equilibrium between betweenthe ionized the and ionized unionized and unionizedform depends form on depends differences on Figure 2. Ionization equilibrium of boronic acid in aqueous solutions. (a) Boronic acid as Lewis acid. dibetweenﬀerences pH between level and pH pKa level value and of p Kathevalue boronic of theacid. boronic Only in acid. rare Only cases, in boronic rare cases, acids boronic are considered acids are (b) Boronic acid as Brønsted–Lowry acid. consideredBrønsted–Lowry Brønsted–Lowry acids (Figure acids 2b); (Figure this2 b);occurs this occurswhen whenthe formation the formation of the of thetetrahedral tetrahedral form form is isunfavorable unfavorable [5]. [5 ]. At physiological pH, boronic acids remain at their protonated uncharged trigonal form but, in As Lewis acids, these compounds can form complexes with Lewis bases like hydroxide anions, aqueous solutions with pH values higher than pKa, they are converted to anionic tetrahedral forms and electron-donatingelectron‐donating groups, like nitrogen or oxygen and and also behave as as electrophiles [1,2,8,10]. [1,2,8,10]. [2,4,6,7]. Therefore, the equilibrium between the ionized and unionized form depends on differences Therefore, theythey cancan form form a a reversible reversible non-ionic non‐ionic bond bond with with nucleophilic nucleophilic biological biological compounds compounds such such as between pH level and pKa value of the boronic acid. Only in rare cases, boronic acids are considered enzymeas enzyme residues, residues, nucleic nucleic acids, acids, or hydroxylor hydroxyl groups groups from from carbohydrates carbohydrates [2]. [2]. These These characteristics characteristics are Brønsted–Lowry acids (Figure 2b); this occurs when the formation of the tetrahedral form is veryare very interesting interesting for the for creation the creation of possible of possible compounds compounds containing containing boronic boronic acid that acid could that be could used forbe unfavorable [5]. therapeuticused for therapeutic applications applications as follows. as follows. As Lewis acids, these compounds can form complexes with Lewis bases like hydroxide anions, Bortezomib (Figure 33a),a), alsoalso knownknown asas Velcade,Velcade, was was the the ﬁrst first boronic boronic acid-containing acid‐containing drug drug and electron‐donating groups, like nitrogen or oxygen and also behave as electrophiles [1,2,8,10]. approved byby Food Food and and Drug Drug Administration Administration (FDA) (FDA) in 2003 in 2003 and byand European by European Medicines Medicines Agency Agency (EMA) Therefore, they can form a reversible non‐ionic bond with nucleophilic biological compounds such in(EMA) 2004. in This 2004. drug This is drug a proteasome is a proteasome inhibitor, inhibitor, used for used treatment for treatment of multiple of multiple myeloma myeloma [2,7,11]. [2,7,11]. Due to as enzyme residues, nucleic acids, or hydroxyl groups from carbohydrates [2]. These characteristics theDue overall to the overall desirable desirable features features of boronic of boronic acids and acids since and the since approval the approval of Velcade, of Velcade, interest forinterest boronic for are very interesting for the creation of possible compounds containing boronic acid that could be acidsboronic in acids Medicinal in Medicinal Chemistry Chemistry has been has arising been throughoutarising throughout the years, the leading years, leading to the discovery to the discovery of two used for therapeutic applications as follows. moreof two medications more medications approved approved by drug authorities—ixazomibby drug authorities—ixazomib and vaborbactam. and vaborbactam. Ixazomib (Figure Ixazomib3b), Bortezomib (Figure 3a), also known as Velcade, was the first boronic acid‐containing drug a N-dipeptidyl boronic acid approved by FDA and EMA in 2015 and 2016, respectively, is also used for approved by Food and Drug Administration (FDA) in 2003 and by European Medicines Agency treatment of multiple myeloma, having the same mechanism of bortezomib [12–14]. Vaborbactam (EMA) in 2004. This drug is a proteasome inhibitor, used for treatment of multiple myeloma [2,7,11]. (Figure3c) was approved by FDA in 2017 and by EMA in 2018. This cyclic boronic acid, a β-lactamase Due to the overall desirable features of boronic acids and since the approval of Velcade, interest for boronic acids in Medicinal Chemistry has been arising throughout the years, leading to the discovery of two more medications approved by drug authorities—ixazomib and vaborbactam. Ixazomib

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(Figure 3b), a N‐dipeptidyl boronic acid approved by FDA and EMA in 2015 and 2016, respectively, Molecules 2020, 25, 4323 3 of 40 is also used for treatment of multiple myeloma, having the same mechanism of bortezomib [12–14]. Vaborbactam (Figure 3c) was approved by FDA in 2017 and by EMA in 2018. This cyclic boronic acid, inhibitor,a β‐lactamase has beeninhibitor, used has in been combination used in combination with antibiotics with for antibiotics the treatment for the of treatment urinary, abdominal,of urinary, andabdominal, lung infections and lung [15 infections–17]. [15–17].

Figure 3. Drugs containing boronic acid approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA).(EMA). ((a)) Bortezomib. (b) Ixazomib. ( c) Vaborbactam.

Since the the approval approval of of these these drugs, drugs, many many biological biological applications applications have have been been reported reported over over the last the lastyears years for different for diﬀ erentboronic boronic acid derivatives. acid derivatives. Several Severalreviews reviewshave addressed have addressed in depth their in depth syntheses their syntheses[18–21] and [18 pharmaceutical–21] and pharmaceutical applications applications [1,2,4,6–8,22]. [1,2 ,4,6–8,22]. The main objective of this review is to provide a broad overview and update on the Medicinal Chemistry ofof boronic boronic acids, acids, as as well well of someof some of their of their derivatives, derivatives, emphasizing emphasizing studies studies of structure–activity of structure– relationshipsactivity relationships and other and applications other applications beyond pharmaceuticals. beyond pharmaceuticals. Furthermore, Furthermore, we expect thatwe expect this review that willthis review increase will the increase interest ofthe medicinal interest of chemists medicinal to thischemists privileged to this motif. privileged motif. 2. Synthesis and Biological Applications of Boronic Acids Derivatives 2. Synthesis and Biological Applications of Boronic Acids Derivatives Boronic acid is a stable and generally a non-toxic group that is easily synthetized and, due to Boronic acid is a stable and generally a non‐toxic group that is easily synthetized and, due to these features, can be used in several synthetic reactions including metal-catalyzed processes like these features, can be used in several synthetic reactions including metal‐catalyzed processes like Suzuki–Miyaura reaction, acid catalysis, asymmetric synthesis of amino acids, and hydroboration. Suzuki–Miyaura reaction, acid catalysis, asymmetric synthesis of amino acids, and hydroboration. Because of this, organoboron compounds are extensively used as building blocks intermediates in Because of this, organoboron compounds are extensively used as building blocks intermediates in organic chemistry reactions [23–27]. One of the most noticeable applications of these compounds is organic chemistry reactions [23–27]. One of the most noticeable applications of these compounds is the cross-coupling metal-catalyzed Suzuki–Miyaura, where they are used in order to form new C-C the cross‐coupling metal‐catalyzed Suzuki–Miyaura, where they are used in order to form new C‐C bonds. This is a complex reaction that involves steps such as oxidative addition, ligand substitution, bonds. This is a complex reaction that involves steps such as oxidative addition, ligand substitution, transmetalation, and reductive elimination [23,24]. transmetalation, and reductive elimination [23,24]. When in drugs, boronic acids are mostly present in the form of aryl boronic acids. Besides this When in drugs, boronic acids are mostly present in the form of aryl boronic acids. Besides this form, heterocycles containing boronic acids, such as pyridinyl, pyrrolyl, and indolyl derivatives, form, heterocycles containing boronic acids, such as pyridinyl, pyrrolyl, and indolyl derivatives, are are also very useful in Medicinal Chemistry and their syntheses is accomplished in a similar fashion also very useful in Medicinal Chemistry and their syntheses is accomplished in a similar fashion of of aryl boronic acids [5]. The process of transmetalation to palladium(II) halides is a crucial step in aryl boronic acids [5]. The process of transmetalation to palladium(II) halides is a crucial step in cross‐ cross-coupling reactions of organoboron compounds [28]. One of the ﬁrst and most common methods coupling reactions of organoboron compounds [28]. One of the first and most common methods to to synthetize phenylboronic compounds was through electrophilic trapping of arylmetal intermediates synthetize phenylboronic compounds was through electrophilic trapping of arylmetal intermediates with borate esters at low temperature (Scheme1a). This reaction usually involves the addition of a with borate esters at low temperature (Scheme 1a). This reaction usually involves the addition of a methylborate to a solution of phenylmagnesium bromide but unfortunately results in low yields [5,29]. methylborate to a solution of phenylmagnesium bromide but unfortunately results in low yields Besides using a Grignard reagent, in this synthetic pathway, boronic acids can also be prepared through [5,29]. Besides using a Grignard reagent, in this synthetic pathway, boronic acids can also be prepared lithium–halogen exchange (Scheme1a) [ 30,31]. Aryl boronic acids can also be obtained through through lithium–halogen exchange (Scheme 1a) [30,31]. Aryl boronic acids can also be obtained transmetallation of aryl silanes and stannanes, transition metal-catalyzed coupling of aryl halides through transmetallation of aryl silanes and stannanes, transition metal‐catalyzed coupling of aryl with diboronic acid reagents and for direct boronylation by transition metal-catalyzed aromatic C–H halides with diboronic acid reagents and for direct boronylation by transition metal‐catalyzed functionalization [5]. aromatic C–H functionalization [5].

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Scheme 1. Synthetic processes to obtain aryl boronic acids. ( a)) Electrophilic trapping of arylmetal intermediates withwith borate borate esters esters from from aryl aryl halides halides using using Grignard Grignard reagents reagents or through or through lithium–halogen lithium– halogenexchange, exchange, respectively. respectively. (b) Coupling (b) ofCoupling aryl halides of aryl with halides diboronic with acid diboronic reagents. acid (c) reagents. Direct boronylation (c) Direct boronylationby transition metal-catalyzedby transition metal aromatic‐catalyzed C–H functionalization. aromatic C–H functionalization. (d) Bromide–lithium (d) exchangeBromide–lithium reaction exchangethrough ﬂow reaction chemistry. through (e) flow Transmetallation chemistry. (e of) Transmetallation aryl silanes and stannanes.of aryl silanes and stannanes.

The coupling of aromatic halides of diboronic acid reagents (Scheme 11b)b) waswas discovereddiscovered byby Miyaura et al., where diboronyl esters like diborylpinacolate (B2pin2) undergo a cross-coupling reaction Miyaura et al., where diboronyl esters like diborylpinacolate (B2pin2) undergo a cross‐coupling withreaction arylbromides, with arylbromides, iodides, andiodides, triflates and under triflates palladium under palladium catalysis. Acatalysis. limitation A oflimitation this reaction of this is reactionthe overwhelming is the overwhelming price of diboronic price of acid diboronic reagents, acid justifying reagents, the justifying use of a catalyst the use like of a palladium catalyst like (II) chloride (PdCl2). Alternatively, the diboronic acid reagent could be replaced by pinacolborane [5,32]. palladium (II) chloride (PdCl2). Alternatively, the diboronic acid reagent could be replaced by pinacolboraneDirect boronylation [5,32]. of Direct phenyl boronylation rings (Scheme of 1phenylc) can be rings used (Scheme for the synthesis1c) can be of used phenylboronic for the synthesis acids ofas anphenylboronic atom-economy acids method. as an In atom this reaction,‐economy diboronyl method. esters In this of dialkoxyboranesreaction, diboronyl can beesters used asof dialkoxyboranesdiboronic acid reagents can be and used a catalyst, as diboronic for instance acid reagents iridium orand rhodium, a catalyst, is needed for instance for producing iridium theor rhodium,desired product is needed [33 for,34 ].producing More recently, the desired it has product been proved [33,34]. that More it is recently, possible it to has synthetize been proved boronic that itacids is possible by using to flowsynthetize chemistry boronic (Scheme acids1 d).by using Through flow bromine–lithium chemistry (Scheme exchange 1d). Through reactions bromine– and by lithiumsuccessfully exchange suppressing reactions the and protonation by successfully and butylation suppressing side reactions the protonation through flow and chemistry, butylation it side was possiblereactions to through prioritize flow the chemistry, borylation it reaction was possible and, therefore, to prioritize the the synthesis borylation of a phenylboronicreaction and, therefore, acid was theaccomplished synthesis of [35 a]. phenylboronic In the case of transmetallationacid was accomplished of aromatic [35]. silanesIn the andcase stannanes of transmetallation (Scheme1 e),of aromatic silanes and stannanes (Scheme 1e), trialkylaryl silanes and stannanes are transmetallated

Molecules 2020, 25, 4323 5 of 40 trialkylaryl silanes and stannanes are transmetallated with a boron halide, resulting in an arylboron dibromide compound that then undergoes acidic hydrolysis, resulting in the formation of relatively simple phenylboronic acids [5,36]. As previously mentioned, over the years, boronic acids have proved to be very interesting compounds in Medicinal Chemistry, due to their overall characteristics and various biological applications and potential molecular targets. In Figure4, the general molecular targets already described for boronic acids are represented [1,4,7,22]. Table1 shows some of the clinical trials that are ongoing or have been already completed with boronic acids and other Boron containing compounds. In this review some of the molecular targets of boronic acid-containing compounds mentioned in Figure4 will be addressed with more detail, being the sections of the review divided according to the Molecules 2020, 25, x FOR PEER REVIEW 10 of 43 type of therapeutic application of boronic acid derivatives.

FigureFigure 4. 4. MolecularMolecular targetstargets ofof boronic boronic acids. acids. SerineSerine ProteasesProteases include:include: DipeptidylDipeptidyl PeptidasePeptidase IVIV (DPPIV),(DPPIV), Factor Factor Xa Xa (fXa) (fXa) and and XIa, XIa, Hepatitis Hepatitis C C Virus Virus (HCV) (HCV) NS3, NS3, Hormone-sensitive Hormone‐sensitive lipaselipase (HSL),(HSL), IgA1IgA1 protease, protease, PancreaticPancreatic cholesterol esterase esterase (CEase), (CEase), Prostate Prostate specific speciﬁc antigen antigen (PSA), (PSA), Thrombin, Thrombin, α‐ αChymotrypsin,-Chymotrypsin, α‐αlytic-lytic protease, protease, β‐β-lactamases.lactamases. Other Other enzymes enzymes include: Aminopeptidases, Arginase,Arginase, humanhuman Carbonic Carbonic anhydrase anhydrase IX IX (hCA (hCA IX), IX), hCA hCA XII), XII), Cysteine Cysteine protease, protease, Fatty Fatty Acid Acid Amide Amide Hydrolase Hydrolase (FAAH),(FAAH), Histone Histone deacetylases deacetylases (HDACs), (HDACs), HIV-1 HIV Protease‐1 Protease (HIV-1 (HIV PR),‐ Leucyl-tRNA1 PR), Leucyl synthetase‐tRNA synthetase (LeuRS), RNA-dependent(LeuRS), RNA‐dependent RNA polymerase RNA NS5Bpolymerase (NS5B), NS5B Tyrosine (NS5B), Kinase Tyrosine DYRK1A Kinase (DYRK1A), DYRK1A Steroid (DYRK1A), sulfatase (STS),Steroidγ-Glutamyl sulfatase transpeptidase(STS), γ‐Glutamyl (γ-GT), transpeptidase 3Clike protease (γ‐ (3CLpro).GT), 3Clike Proteins protease include: (3CLpro). Murine Proteins double minuteinclude: 2 Murine (MDM2), double NorA minute eﬄux 2 pump (MDM2), (NorA), NorA Penicillin efflux pump binding (NorA), protein Penicillin 3 (PBP3), binding PBP4, protein PBP5, 3 Transthyretin(PBP3), PBP4, (TTR), PBP5, 20STransthyretin proteasome. (TTR), Transcription 20S proteasome. factors Transcription include: Hypoxia-inducible factors include: factor Hypoxia 1-α‐ (HIFinducible 1-α). Receptorsfactor 1‐α (HIF include: 1‐α). Chemokine Receptors include: receptors Chemokine 1 (CXCR1), receptors CXCR2, 1 CXCL8, (CXCR1), Epidermal CXCR2, growthCXCL8, factorEpidermal receptor growth tyrosine factor kinase receptor (EGFR-TK), tyrosine Estrogen kinase (EGFR Receptor‐TK),α Estrogen(ERα). Receptor α (ERα).

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Table 1. Some of the completed and ongoing clinical trials with boronic acid compounds and other boron-containing compounds. Source: www.clinicaltrials.gov (accessed on May 2020).

Study Purpose Phase Status To study whether the combination of CHOP 1 with Combination therapy with bortezomib in advanced stage Phase 1 bortezomib to overcome drug resistance induced by Completed (October 2009) aggressive lymphomas Phase 2 pro-apoptotic proteins BAX and BCL-XS 2 To establish a safe and tolerable dose combination of Combination therapy with ixazomib for the treatment selinexor and ixazomib when used together for the Phase 1 Ongoing of sarcoma treatment of certain types of advanced sarcoma Lymphoma study in sombination with bortezomib To determine the safety, tolerability and MTD 3 of AMG-655 Phase 1 Completed (August 2011) or vorinostat 4 when combined with bortezomib or vorinostat Bortezomib in relapsed or refractory AIDS To evaluate the MTD of bortezomib in patients with AIDS Phase 1 Completed (January 2015) 5-related sarcoma 5-related Kaposi sarcoma To determine the MTD and dose-limiting toxicities of Combination therapy with bortezomib in ovarian epithelial intraperitoneal bortezomib when combined with Phase 1 Completed (January 2018) cancer, fallopian tube cancer, or primary peritoneal cancer intraperitoneal carboplatin To identify the effective and tolerable dose of the Combination therapy with bortezomib in acute bortezomib/sorafenib combination in acute myeloid Phase 1 Ongoing myeloid leukemia leukemia; to recommend a dose level for phase 2 and define specific toxicities To determine the RD 6 for phase 2 dose of alisertib and bortezomib when combined with rituximab in patients with Combination therapy with bortezomib in lymphoma Phase 1 Ongoing relapsed/refractory mantle cell and B-cell low grade non-Hodgkin lymphoma To evaluate the safety, tolerability and pharmacokinetics of First time in human safety and pharmacokinetics study of single ascending and repeat oral doses of GSK3036656 in Phase 1 Completed (August 2017) GSK3036656 in healthy subjects healthy adults Intravenous ixazomib in pediatric participants with To determine the MTD and/or phase 2 RD, safety and relapsed or refractory acute lymphoblastic leukemia or Phase 1 Ongoing toxicity, and pharmacokinetics lymphoblastic lymphoma To study the side effects and best dose of pevonedistat Ixazomib and pevonedistat in treating multiple myeloma when combined with ixazomib in multiple myeloma that Phase 1 Ongoing that has come back or does not respond to treatment has come or does not respond to treatment To determine the safety and tolerability of ixazomib in HIV infected patients who are on ART 7 that suppresses HIV The effect of ixazomib on the latent HIV reservoir Phase 1 Ongoing replication; to determine the effect of ixazomib on the size of the HIV reservoir Molecules 2020, 25, 4323 7 of 40

Table 1. Cont.

Study Purpose Phase Status Dose escalation study of nelfinavir and ixazomib in patients To determine the MTD of the combination in advanced Phase 1 Ongoing with advanced solid tumors or lymphoma solid tumors or lymphoma To characterize the safety and tolerability of ixazomib when Safety, tolerability and pharmacokinetics of multiple rising administered as multiple oral doses at escalating dose Phase 1 Completed (January 2018) doses of ixazomib in lupus nephritis levels in participants with lupus nephritis To determine the proper dose and side effects of ixazomib Study of the combination of ixazomib and fulvestrant Phase 1 Completed (June 2018) when combined with fulvestrant Dose-finding, pharmacokinetics, and safety of To determine the dose-finding and evaluate meropenem-vaborbactam in pediatric patients with pharmacokinetics, safety, and tolerability in pediatric Phase 1 Ongoing bacterial infections patients (<18 years) with serious bacterial infections To determine the cumulative irritation potential Cumulative irritation test of tavaborole Phase 1 Completed (February 2007) of tavaborole To estimate observed application site adverse events Study to assess local tolerability of crisaborole 2% ointment following topical applications of crisaborole and vehicle in Phase 1 Completed (April 2019) in healthy participants healthy participants To study side effects and best dose of venetoclax combined Combination therapy with bortezomib with/without Phase 1 with daratumumab, bortezomib, and dexamethasone in Ongoing venetoclax in multiple myeloma Phase 2 relapsed or refractory multiple myeloma To determine the MTD/RD of bortezomib and pegylated Combination therapy with bortezomib in plasma liposomal doxorubicin hydrochloride when in combination Phase 1 Completed (December 2018) cell leukemia with a fixed dose of daratumumab, lenalidomide, Phase 2 and dexamethasone (phase 2) To determine the safety and the MTD of bortezomib when Phase 1 Combination chemotherapy with bortezomib in lymphoma combined with rituximab, methotrexate, and cytarabine Ongoing Phase 2 in lymphoma Ixazomib in the prophylaxis of chronic To evaluate the efficacy of ixazomib at the doses stablished Phase 1 Ongoing graft-versus-host disease in phase 1 Phase 2 To study the effect of systemic treatment with PS-341 8 PS-341 8 followed by removal of prostate in prostate cancer doing correlative scientific markers assessing apoptosis; to Phase 2 Completed (December 2013) evaluate protease protein targets and angiogenesis markers To evaluate the toxicity of the use of vorinostat and Bortezomib and vorinostat in patients with multiple bortezomib as maintenance therapy after Phase 2 Completed (March 2017) myeloma who have undergone stem cell transplant autologous transplant Fulvestrant with/without bortezomib in inoperable locally To determine whether the combination of bortezomib to Phase 2 Ongoing or metastatic estrogen receptor positive breast cancer fulvestrant improves median progression free survival Molecules 2020, 25, 4323 8 of 40

Table 1. Cont.

Study Purpose Phase Status To determine the complete and partial response rate of Bortezomib and lenalidomide in mantle cell lymphoma bortezomib and lenalidomide therapy in relapsed or Phase 2 Completed (April 2019) refractory mantle cell lymphoma To evaluate the complete and near-complete response rate Combination therapy with bortezomib in previously of the bortezomib/pegylated liposomal doxorubicin Phase 2 Completed (September 2019) untreated symptomatic multiple myeloma regimen in previously untreated, symptomatic multiple myeloma To determine the overall response rate of rituximab, Rituximab, venetoclax, and bortezomib in lymphoma venetoclax, and bortezomib in relapsed/refractory diffuse Phase 2 Ongoing large B-cell lymphoma To evaluate the overall hematologic response rate for Combination therapy with/without bortezomib and ibrutinib with bortezomib and dexamethasone added for Phase 2 Completed (April 2019) dexamethasone in immunoglobulin light chain amyloidosis lack of response in amyloid light chain To evaluate the progression-free survival of patients with Combination therapy with bortezomib followed by daratumumab, bortezomib, and dexamethasone treatment Phase 2 Ongoing combination therapy with ixazomib in multiple myeloma followed by daratumumab, ixazomib, and dexamethasone treatment Melphalan and bortezomib as conditioning regimen for To evaluate the effectiveness of bortezomib when combined autologous and allogeneic stem cell transplants in to standard chemotherapy medicine(s) for treatment of Phase 2 Completed (May 2019) multiple myeloma multiple myeloma To find the MTD of the combination of ixazomib and Study of ixazomib and erlotinib in solid tumors erlotinib in advanced solid tumors; to study the safety of Phase 2 Ongoing these drugs Efficacy and safety of ixazomib and dexamethasone To evaluate the safety and efficacy of this combination in Phase 2 Completed (October 2019) refractory autoimmune cytopenia refractory autoimmune cytopenia To study the side effects of ibrutinib citrate when combined Ibrutinib and ixazomib citrate in relapsed or refractory with ixazomib; to determine the effect in refractory Phase 2 Ongoing Waldenstrom macroglobulinemia Waldenstrom macroglobulinemia To study the effect of ixazomib to desensitize highly Ixazomib for Desensitization Phase 2 Ongoing sensitized kidney transplant recipients Absorption and systemic study of tavaborole in patients To determine the absorption, systemic pharmacokinetics Phase 2 Completed (May 2007) with moderate to severe onychomycosis (ADME 9 I) and accumulation in the nail of tavaborole Absorption and systemic study of tavaborole in patients To determine the absorption and systemic Phase 2 Completed (May 2007) with moderate to severe onychomycosis (ADME II) pharmacokinetics of tavaborole Safety and efficacy study of crisaborole ointment to treat To determine the safety and efficacy of crisaborole ointment Phase 2 Completed (March 2008) plaque type psoriasis 5% in the treatment of plaque type psoriasis Molecules 2020, 25, 4323 9 of 40

Table 1. Cont.

Study Purpose Phase Status To characterize the mechanism of action of crisaborole Crisaborole ointment 2% skin biomarker biopsy study in ointment 2% through evaluation of efficacy and changes in Phase 2 Completed (May 2018) atopic dermatitis key skin biomarkers in atopic dermatitis lesions treated with crisaborole ointment 2% To study the effect of lenalidomide, dexamethasone, Combination therapy with/without bortezomib in and bortezomib compared to dexamethasone and Phase 3 Ongoing previously untreated multiple myeloma lenalidomide alone in treating patients with previously untreated multiple myeloma Combination therapy with bortezomib in newly diagnosed To study the effect of bortezomib and sorafenib tosylate in Phase 3 Ongoing acute myeloid leukemia newly diagnosed acute myeloid leukemia Ixazomib citrate, lenalidomide, dexamethasone, To evaluate the impact of combination of ixazomib, and zoledronic acid or zoledronic acid alone after radiation lenalidomide, and dexamethasone to zoledronic acid in Phase 3 Ongoing therapy in solitary plasmacytoma of bone multiple myeloma progression rate To determine if this combination therapy improves Study of dexamethasone plus ixazomib or physician’s hematologic response, heart and kidney deterioration and choice in relapsed or refractory systemic light mortality rate comparing to a physician’s choice of a Phase 3 Ongoing chain amyloidosis chemotherapy regimen in relapsed or refractory systemic light chain amyloidosis To determine the effect of ixazomib maintenance therapy on Study of oral ixazomib maintenance therapy after initial progression-free survival in newly diagnosed multiple therapy in newly diagnosed multiple myeloma not treated myeloma who have had a major or partial response to Phase 3 Ongoing with stem cell transplantation initial therapy and who have not undergone stem-cell transplantation To compare the efficacy, safety and tolerability of Efficacy, safety, tolerability of meropenem-vaborbactam meropenem-vaborbactam with the best available therapy compared to best available therapy in treating serious Phase 3 Completed (July 2017) for infections caused by infections in adults carbapenem-resistant Enterobacteriaceae Efficacy and safety evaluation of tavaborole topical solution To determine whether tavaborole topical solution is a safe Phase 3 Completed (January 2013) to treat onychomycosis of the toenail and effective treatment for onychomycosis of the toenail Evaluation of the safety and pharmacokinetics of To evaluate the safety and pharmacokinetics of tavaborole tavaborole topical solution for the treatment of 5% topical solution in treating distal subungual Phase 4 Completed (July 2017) onychomycosis in children and adolescents onychomycosis of the toenail in children and adolescents 1 CHOP: Cyclophosphamide, doxorubicin, vincristine, prednisolone; 2 BCL-XS: Small B-cell lymphoma; 3 MTD: Maximum tolerated dose; 4 AMG-655: Conatumumab; 5 AIDS: Acquired immune deficiency syndrome; 6 RD: Recommended dose; 7 ART: Antiretroviral therapy; 8 PS-341: bortezomib; 9 ADME: Absorption, Distribution, Metabolism and Elimination (pharmacokinetic parameters). Molecules 2020, 25, 4323 10 of 40

2.1. Anticancer Activity Cellular homeostasis is accomplished through deterioration of intracellular proteins. This is possible due to the action of the essential ubiquitin-proteasome pathway [1,4,37]. This pathway is involved in the regulation of cell-cycle and apoptosis through stabilization of proteins such as the tumor suppressor p53 [1,37], making proteasome a promising target for cancer therapy [1,4,37,38]. As mentioned before, bortezomib is the first in-class proteasome inhibitor used in the treatment of multiple myeloma [1,2,7,37]. Bortezomib drug design was based on the first synthetic peptide proteasome inhibitors, the aldehyde proteasome inhibitors. It was demonstrated that the replacement of the aldehyde moiety with boronic acid led to the resolution of problems associated with these peptide aldehyde inhibitors, for instance, the rapid dissociation from proteasome, the inactivation by oxidation, the lack of specificity for proteasome and the unfavorable pharmacokinetics. Besides, the boronic acid group significantly increased the potency of the proteasome inhibitor [38–40]. The reason why boronic acid was added in the drug design of this compound was due to the known high selectivity and low dissociation rates of this group towards the active site of proteasome. It is known that the boronic acid moiety of bortezomib forms a complex with the nucleophilic N-terminal threonine hydroxyl group, present in the active site of the proteasome, leading to the disruption of the protein complex NF-γB, and ultimately promoting growth inhibition and apoptosis of cancer cells [7,41]. Besides bortezomib, other boronic acid compounds have also shown activity as proteasome inhibitors. Chalcones are a well-established class of anticancer agents that induce apoptosis in many types of cancer cells, including breast cancer. One limitation of chalcones is related to the carboxylic analogues that do not reveal selective toxicity towards malignant epithelial breast cells, also targeting healthy cells [42]. One approach for breast cancer therapy is through inhibition of mouse double minute 2 (MDM2) oncoprotein that is overexpressed in these cancer cells [42]. MDM2 is capable of negatively regulate p53 and is therefore responsible for blocking p53 tumor suppression activity [42,43]. The development of irreversible and selective chalcones is an interesting approach for breast cancer therapy. Since the carboxylic acid group binds near the Lysine51 (Lys51) of MDM2, and being boronic acid bioisosteres, the latter were proposed as promising to be associated with chalcones, which could lead to a selective targeting [42]. The donating amino group of Lys51, present in MDM2, is linked to the boronic acid scaffold, and this binding is possibly stronger than when carboxylic acid is present. At the same time occurs the break of the salt bridge with glutamic acid25 (E25) of MDM2, which results in a stronger selective inhibition of MDM2 in cancer cells and, consequently, growth arrest attributable to the boronic acid group. The replacement of carboxylic acid group by boronic acid (compounds 2, 4, and 5) revealed an increase in selective toxicity towards breast cancer cells (results in Table2) and also that these boronic acid-containing chalcones are more toxic towards these cells than any other known chalcones [42]. In addition to the chalcone-boronic acid derivatives 2, 4, and 5, other researchers [37] have also studied the replacement of carboxylic acid by boronic acid in chalcones. Two compounds, AM 58 (3) and AM 114 (8) were synthetized and, revealed cytotoxic activity against human colon carcinoma cells (HCT116 p53+/+ cells: 3, half maximal inhibitory concentration (IC50) of 3.85 µM; 8, IC50 of 1.49 µM) by inhibiting the 20S proteasome, which consequently resulted in an agglomeration of some ubiquitinated proteins and of p53. It is thought that AM 58 (3) and AM 114 (8) interrupt the MDM2-p53 interaction through connection to the binding site [37]. The synthesis of compounds 4–8 is based on Claisen–Schmidt aldol condensation [37,42]. In the case of compounds 2–5 (Scheme2), the intermediate that is formed ( 1) is treated with pinacol (bromomethyl)boronate and sodium hydride (NaH) in the presence of DMSO (dimethyl sulfoxide). The process of deprotection occurs in alkaline conditions [37]. Compound 8 was prepared in a different way (Scheme3). A solution of methyl-piperidinone ( 6, 1 eq.) was mixed with a formylphenylboronic acid (7, 3 eq.) in presence of EtOH and KOH (6 eq.) [37]. Molecules 2020, 25, x FOR PEER REVIEW 11 of 43 Molecules 2020, 25, x FOR PEER REVIEW 11 of 43 types of cancer cells, including breast cancer. One limitation of chalcones is related to the carboxylic types of cancer cells, including breast cancer. One limitation of chalcones is related to the carboxylic analogues that do not reveal selective toxicity towards malignant epithelial breast cells, also targeting analogues that do not reveal selective toxicity towards malignant epithelial breast cells, also targeting healthy cells [42]. One approach for breast cancer therapy is through inhibition of mouse double healthy cells [42]. One approach for breast cancer therapy is through inhibition of mouse double minute 2 (MDM2) oncoprotein that is overexpressed in these cancer cells [42]. MDM2 is capable of minute 2 (MDM2) oncoprotein that is overexpressed in these cancer cells [42]. MDM2 is capable of negatively regulate p53 and is therefore responsible for blocking p53 tumor suppression activity negatively regulate p53 and is therefore responsible for blocking p53 tumor suppression activity [42,43]. The development of irreversible and selective chalcones is an interesting approach for breast [42,43]. The development of irreversible and selective chalcones is an interesting approach for breast cancer therapy. Since the carboxylic acid group binds near the Lysine51 (Lys51) of MDM2, and being cancer therapy. Since the carboxylic acid group binds near the Lysine51 (Lys51) of MDM2, and being boronic acid bioisosteres, the latter were proposed as promising to be associated with chalcones, boronic acid bioisosteres, the latter were proposed as promising to be associated with chalcones, which could lead to a selective targeting [42]. The donating amino group of Lys51, present in MDM2, which could lead to a selective targeting [42]. The donating amino group of Lys51, present in MDM2, is linked to the boronic acid scaffold, and this binding is possibly stronger than when carboxylic acid is linked to the boronic acid scaffold, and this binding is possibly stronger than when carboxylic acid is present. At the same time occurs the break of the salt bridge with glutamic acid25 (E25) of MDM2, is present. At the same time occurs the break of the salt bridge with glutamic acid25 (E25) of MDM2, which results in a stronger selective inhibition of MDM2 in cancer cells and, consequently, growth which results in a stronger selective inhibition of MDM2 in cancer cells and, consequently, growth arrest attributable to the boronic acid group. The replacement of carboxylic acid group by boronic arrest attributable to the boronic acid group. The replacement of carboxylic acid group by boronic acid (compounds 2, 4, and 5) revealed an increase in selective toxicity towards breast cancer cells acid (compounds 2, 4, and 5) revealed an increase in selective toxicity towards breast cancer cells (results in Table 2) and also that these boronic acid‐containing chalcones are more toxic towards these (results in Table 2) and also that these boronic acid‐containing chalcones are more toxic towards these cells than any other known chalcones [42]. cells thanMolecules any other2020 ,known25, 4323 chalcones [42]. 11 of 40

Table 2. Growth inhibition (IC50 1, μM) effect of chalcone‐boronic acids (2, 4, and 5) and chalcone‐ 1 Table 2. Growth inhibition (IC50 , μM) effect of1 chalcone‐boronic acids (2, 4, and 5) and chalcone‐ carboxylicTable acids 2. (9 andGrowth 10) analogues inhibition in breast (IC50 cancer, µM) cell eﬀ lines.ect of chalcone-boronic acids (2, 4, and 5) and carboxylic acids (9 and 10) analogues in breast cancer cell lines. chalcone-carboxylic acids (9 and 10) analogues in breast cancer cell lines. Compounds MDA‐MB‐435 MDA‐MB‐231 Compounds MDA‐MB‐435 MDA‐MB‐231 Compounds2 MDA-MB-43510 8.8 MDA-MB-231 2 10 8.8 4 16 8.5 2 4 1016 8.5 8.8 4 5 168.8 8.8 8.5 8.8 5 5 8.88.8 8.8

18 44 1818 44 44

1313 18 18 13 18

Molecules 2020, 25, x FOR PEER REVIEW 12 of 43

1 1 IC50: Half maximalIC50: Half inhibitory maximal inhibitory concentration. concentration. Molecules 2020, 25, x FOR1 IC PEER50: Half REVIEW maximal inhibitory concentration. 12 of 43 In addition to the chalcone‐boronic acid derivatives 2, 4, and 5, other researchers [37] have also In addition to the chalcone‐boronic acid derivatives 2, 4, and 5, other researchers [37] have also studied the replacement of carboxylic acid by boronic acid in chalcones. Two compounds, AM 58 (3) studied the replacement of carboxylic acid by boronic acid in chalcones. Two compounds, AM 58 (3) and AM 114 (8) were synthetized and, revealed cytotoxic activity against human colon carcinoma and AM 114 (8) were synthetized and, revealed cytotoxic activity against human colon carcinoma cells (HCT116 p53+/+ cells: 3, half maximal inhibitory concentration (IC50) of 3.85 μM; 8, IC50 of 1.49 cells (HCT116 p53+/+ cells: 3, half maximal inhibitory concentration (IC50) of 3.85 μM; 8, IC50 of 1.49 μM) by inhibiting the 20S proteasome, which consequently resulted in an agglomeration of some μM) by inhibiting the 20S proteasome, which consequently resulted in an agglomeration of some ubiquitinated proteins and of p53. It is thought that AM 58 (3) and AM 114 (8) interrupt the MDM2‐ ubiquitinated proteins and of p53. It is thought that AM 58 (3) and AM 114 (8) interrupt the MDM2‐ p53 interaction through connection to the binding site [37]. p53 interaction through connection to the binding site [37]. The synthesis of compounds 4–8 is based on Claisen–Schmidt aldol condensation [37,42]. In the The synthesis of compounds 4–8 is based on Claisen–Schmidt aldol condensation [37,42]. In the case of compounds 2–5 (Scheme 2), the intermediate that is formed (1) is treated with pinacol case of compounds 2–5 (Scheme 2), the intermediate that is formed (1) is treated with pinacol (bromomethyl)boronate and sodium hydride (NaH) in the presence of DMSO (dimethyl sulfoxide). (bromomethyl)boronate and sodium hydride (NaH) in the presence of DMSO (dimethyl sulfoxide). The process of deprotection occurs in alkaline conditions [37]. Compound 8 was prepared in a The process of deprotection occurs in alkaline conditions [37]. Compound 8 was prepared in a different way (Scheme 3). A solution of methyl‐piperidinone (6, 1 eq.) was mixed with a different way (Scheme 3). A solution of methyl‐piperidinone (6, 1 eq.) was mixed with a formylphenylboronic acid (7, 3 eq.) in presence of EtOH and KOH (6 eq.) [37]. formylphenylboronic acid (7, 3 eq.) in presence of EtOH and KOH (6 eq.) [37]. Scheme 2. Synthesis of the chalcone‐boronic acid compounds 2–5. Reaction conditions: (i) KOH, Scheme 2. Synthesis of the chalcone-boronic acid compounds 2–5. Reaction conditions: (i) KOH, MeOH,Scheme reflux, 2. 0Synthesis °C to room of the temperature chalcone‐boronic (r.t.), 55acid min; compounds (ii) (1) NaH, 2–5. pinacolReaction (bromomethyl)boronate,conditions: (i) KOH, MeOH,MeOH, reﬂux, reflux, 0 ◦C 0 to°C room to room temperature temperature (r.t.), (r.t.), 55 55 min;min; ( iiii)) (1) (1) NaH, NaH, pinacol pinacol (bromomethyl)boronate, (bromomethyl)boronate, THF, 0 °C to r.t., overnight; (2) NaOH, H2O. THF,THF, 0 ◦C to0 °C r.t., to overnight;r.t., overnight; (2) (2) NaOH, NaOH, H H2O.2O.

Scheme 3. Synthesis of the chalcone‐diboronic acid compound 8. Reaction conditions: (i) KOH, EtOH, Scheme 3. Synthesis of the chalcone chalcone-diboronic‐diboronic acid compound 8.. Reaction conditions: (i) KOH, EtOH, H2O, 0 °C to r.t., overnight. H22O, 0 °C◦C to r.t., overnight. DipeptidylDipeptidyl boronic boronic acids acids are are common common groups groups used for for the the design design of of proteasome proteasome inhibitors inhibitors due due Dipeptidylto the bioactivity boronic of bortezomib acids are [1]. common A tridimentional groups used‐quantitative for the structure–activitydesign of proteasome relationship inhibitors (3D‐ due to the bioactivity of bortezomib [1]. A tridimentional-quantitative structure–activity relationship to theQSAR) bioactivity study of of bortezomib bortezomib [1]. allowed A tridimentional the synthesis‐quantitative of compounds structure–activity with promising relationship proteasome (3D‐ (3D-QSAR) study of bortezomib allowed the synthesis of compounds with promising proteasome QSAR)inhibitory study activityof bortezomib [40]. The 3Dallowed‐QSAR studiesthe synthesis revealed thatof compounds there are three with important promising regions proteasome for the inhibitory activity [40]. The 3D-QSAR studies revealed that there are three important regions for the inhibitoryinteraction activity between [40]. Thebortezomib 3D‐QSAR and studiesproteasome: revealed (i) A thatcovalent there site are to three allow important the bonding regions between for the interactionbortezomib between and proteasome bortezomib Thyr1 and residue;proteasome: (ii) an aromatic(i) A covalent ring; (iii) site a pyrazinylto allow asthe an bonding acceptor betweenand bortezomibhydrophobic and proteasome group. Based Thyr1 on these residue; results, (ii) a an series aromatic of dipeptide ring; (iii) boronic a pyrazinyl acid compounds as an acceptor were and synthesized, being compounds 15 and 16 (Scheme 4) the most promising ones. Compound 15 has a hydrophobic group. Based on these results, a series of dipeptide boronic acid compounds were better IC50 value than bortezomib (15, IC50 of 4.60 nM; bortezomib, IC50 of 7.05 nM) and can be synthesized,considered being a lead compounds compound. 15 The and studies 16 (Scheme of the mechanism 4) the most of promisingaction indicated ones. that Compound this dipeptide 15 has a betterboronic IC50 value acid proteasome than bortezomib inhibitor (stops15, IC progression50 of 4.60 ofnM; cell cyclebortezomib, at G2/M phaseIC50 of in U2667.05 nM)cells, leadingand can be consideredto growth a lead inhibition compound. in cancer The cells. studies Pharmacokinetic of the mechanism studies in of rats action revealed indicated that compound that this 15 dipeptide can boronicbe administratedacid proteasome intravenously, inhibitor stopsbut optimization progression is ofrequired cell cycle to improve at G2/M the phase concentration in U266 cells, level leading of to growththe compound inhibition at thein cancer therapeutic cells. target Pharmacokinetic [40]. studies in rats revealed that compound 15 can be administrated intravenously, but optimization is required to improve the concentration level of the compound at the therapeutic target [40].

Molecules 2020, 25, 4323 12 of 40 interaction between bortezomib and proteasome: (i) A covalent site to allow the bonding between bortezomib and proteasome Thyr1 residue; (ii) an aromatic ring; (iii) a pyrazinyl as an acceptor and hydrophobic group. Based on these results, a series of dipeptide boronic acid compounds were synthesized, being compounds 15 and 16 (Scheme4) the most promising ones. Compound 15 has a better IC50 value than bortezomib (15, IC50 of 4.60 nM; bortezomib, IC50 of 7.05 nM) and can be considered a lead compound. The studies of the mechanism of action indicated that this dipeptide boronic acid proteasome inhibitor stops progression of cell cycle at G2/M phase in U266 cells, leading to growth inhibition in cancer cells. Pharmacokinetic studies in rats revealed that compound 15 can be administrated intravenously, but optimization is required to improve the concentration level of the compoundMolecules 2020 at, 25 the, x FOR therapeutic PEER REVIEW target [40]. 13 of 43

Scheme 4. 4. SynthesisSynthesis of the of thedipeptide dipeptide boronic boronic acids acids15–17, 15and–17 of, the and prodrug of the prodrug18. Reaction18. conditions: Reaction conditions:(i) R1COOH, (i)R 1COOH,1‐ethyl‐3 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride hydrochloride (EDCI), (EDCI), 1‐ 1-hydroxybenzotriazolhydroxybenzotriazol (HOBt), (HOBt), N,N N,N-diisopropylethylamine‐diisopropylethylamine (DIPEA), (DIPEA), CH2Cl CH2, −2Cl102 ,°C 10to r.t.;◦C ( toii) r.t.;(1) − (LiOHii) (1)∙H LiOH2O, MeOH,H2O, MeOH,H2O, r.t.; H (2)2O, 1 N r.t.; HCl, (2) EtOAc, 1 N HCl, r.t.; EtOAc, (iii) EDCI, r.t.; HOBt, (iii) EDCI, DIPEA, HOBt, CH2Cl DIPEA,2, −10 °C CH to2 Clr.t.;2, · (iv10) 1◦ CN to HCl, r.t.; MeOH, (iv) 1 N n HCl,‐hexane, MeOH, r.t., 22 n-hexane, h; (v) EtOAc, r.t., 22 diethanolamine h; (v) EtOAc, diethanolamine (DEA), 74 °C to r.t., (DEA), overnight. 74 ◦C to − r.t., overnight. More recently, the same group (Lei et al.) reported another study that revealed the improvement of theseMore compounds recently, the in sameorder groupto be orally (Lei et administrated al.) reported another[44]. For study that, a that prodrug revealed (18, the Scheme improvement 4) based ofon these compound compounds 17 (Scheme in order 4) to was be orally designed. administrated This prodrug [44]. For 18 that,showed a prodrug both in (18 vitro, Scheme and4 )in based vivo on compound 17 (Scheme4) was designed. This prodrug 18 showed both in vitro and in vivo activities activities (17, IC50 of 8.21 nM; 18, IC50 of 6.74 nM) and has good pharmacokinetic properties [44]. (17, ICIn50 theof 8.21synthesis nM; 18 of, ICcompounds50 of 6.74 nM) 15 and and 16 has (Scheme good pharmacokinetic 4), an initial step properties of acidic [ 44saponification]. of the peptideIn the synthesis ester occurs, of compounds forming an 15aminoand 16acid(Scheme 11. The4 ),acid an groups initial step of the of acidicamino saponificationacid then reacted of thewith peptide amino esterboronate occurs, trifluoroacetate forming an amino 12 to give acid dipeptidyl11. The acid boronic groups ester of the 13 amino. The boronic acid then ester reacted then withundergoes amino transesterification boronate trifluoroacetate with isobutyl12 to boronic give dipeptidyl acid 14, producing boronic ester compounds13. The boronic15–17 in ester moderate then undergoesto high yields transesterification [40]. For the with synthesis isobutyl of boronicthe proteasome acid 14, producing inhibitor compounds prodrug 1815 (Scheme–17 in moderate 4), the todipeptidyl high yields boronic [40]. For acid the 17 synthesis reacted ofwith the diethanolamine proteasome inhibitor (DEA), prodrug producing18 (Scheme the desired4), the dipeptidylcompound with a yield of approximately 65% [44]. Although peptide boronic acids are a very important chemical class of proteasome inhibitors, they usually have poor pharmacokinetic properties and rapidly undergo in vivo inactivation. These results are mainly associated with instability of the peptide bonds [45]. Aiming to improve these properties, urea‐containing peptide boronic acids were designed. A common strategy to improve pharmacokinetic and pharmacodynamic proprieties of peptide drugs is to replace amine or amide group by an urea scaffold. This led to the discovery of a potent compound (25) in different cell lines (ChT‐L, IC50 of 0.0002 nM; HepG‐2, IC50 of 19.38 nM; MGC‐803, IC50 of 3.962 nM). Its potency is due to the distance between boronic acid and the urea scaffold; it is thought that low potency may occur when these groups were next to each other, allowing the formation of a cyclic boronic acid derivative (Figure 5), which happens due to the strong electrophilicity of boron atom. In terms of pharmacokinetics, compound 25 proved to have more in vivo stability than bortezomib, being this related to a higher life‐time and larger volume of distribution. This compound also revealed relatively low toxicity [45].

Molecules 2020, 25, 4323 13 of 40 boronic acid 17 reacted with diethanolamine (DEA), producing the desired compound with a yield of approximately 65% [44]. Although peptide boronic acids are a very important chemical class of proteasome inhibitors, they usually have poor pharmacokinetic properties and rapidly undergo in vivo inactivation. These results are mainly associated with instability of the peptide bonds [45]. Aiming to improve these properties, urea-containing peptide boronic acids were designed. A common strategy to improve pharmacokinetic and pharmacodynamic proprieties of peptide drugs is to replace amine or amide group by an urea scaffold. This led to the discovery of a potent compound (25) in different cell lines (ChT-L, IC50 of 0.0002 nM; HepG-2, IC50 of 19.38 nM; MGC-803, IC50 of 3.962 nM). Its potency is due to the distance between boronic acid and the urea scaffold; it is thought that low potency may occur when these groups were next to each other, allowing the formation of a cyclic boronic acid derivative (Figure5), which happens due to the strong electrophilicity of boron atom. In terms of pharmacokinetics, compound 25 proved to have more in vivo stability than bortezomib, being this related to a higher life-time and larger volume of distribution. This compound also revealed relatively lowMolecules toxicity 2020, [2545, x]. FOR PEER REVIEW 14 of 43

Figure 5. Possible formationformation ofof a a cyclic cyclic boronic boronic acid acid derivative, derivative, when when boronic boronic acid acid is locatedis located next next to anto ureaan urea group. group.

For the synthesis of compound 25 (Scheme(Scheme5 5),), an an amine amine derivative derivative ( (19)) andand L-phenylalanineL‐phenylalanine methyl esterester hydrochloride hydrochloride (20 )(20 were) were used used as initial as reagentsinitial reagents to form theto ureaform moiety. the urea The moiety. intermediate The α (intermediate21) was then ( hydrolyzed21) was then with hydrolyzed NaOH. The with -aminoNaOH. boronicThe α‐amino acid pinacol boronic ester acid (pinacol23) was ester later ( coupled23) was α withlater thecoupled carboxylic with the acid carboxylic (represented acid in(represented molecule 22 in), molecule giving the 22),-amino giving boronic the α‐amino acid pinacolboronic ester acid (pinacol24). Finally, ester the(24). urea-containing Finally, the urea peptide‐containing boronic peptide acid ( 25boronic) was obtainedacid (25) bywas elimination obtained by of elimination the pinacol protectingof the pinacol group protecting [45]. group [45]. The majority of boronic acid anticancer agents are proteasome inhibitors. However, besides this mechanism of action, boronic acid compounds that act on cancer targets other than proteasome have also proved to be promising. A study based on the antimitotic agent combretastatin A-4 (CA-4) [46], an agent that binds reversibly to the colchicine binding site of tubulin and prevents cellular division [46,47], was done in order to improve its solubility, without compromising its activity, since it is the low solubility in water that prevented the development of this compound as a drug [46]. Based on the fact that boronic acids could act as bioisosteres of the aromatic hydroxyl group of the C-ring of CA-4 and that at physiological pH levels, this compound remains at its unionized form (pKa 9–10), boronic acid analogues of CA-4 were synthetized. A cis and trans-boronic acid analogues (32 and 33, respectively) were tested and, as predicted by the docking studies, these compounds were more potent inhibitors of tubulin polymerization than CA-4. Interestingly, proliferation is dependent of the geometry of the double bond, being the analogue cis (32, Scheme6) more e ffective (32, IC50 of 1.5 µM; 33, IC50 of 7.8 µM; CA-4, IC50 of 2.0 µM). Besides these effects, no toxicity was revealed, and water solubility was enhanced, being the cis analogue (32) more stable and soluble than CA-4 thus allowing oral delivery. Another interesting aspect is that it is thought that the cis-boronic acid analogue 32 will not have a short plasma half-life, since the carbon–boron bond attached to the aromatic ring does not suffer hydrolysis [46].

Scheme 5. Synthesis of the urea‐peptide boronic acid 25. Reaction conditions: (i) CDI, dimethylformamide (DMF), MeCN, r.t.; (ii) (1) NaOH, 0 °C; (2) HCl, 0 °C; (iii) N,N′‐

dicyclohexylcarbodiimide (DCC), HOBt, DIPEA, CH2Cl2, r.t. (iv) (1) DEA, EtOAc, r.t.; (2) HCl, H2O, EtOAc, r.t., 2 h.

The majority of boronic acid anticancer agents are proteasome inhibitors. However, besides this mechanism of action, boronic acid compounds that act on cancer targets other than proteasome have also proved to be promising. A study based on the antimitotic agent combretastatin A‐4 (CA‐4) [46], an agent that binds reversibly to the colchicine binding site of tubulin and prevents cellular division [46,47], was done in order to improve its solubility, without compromising its activity, since it is the low solubility in water that prevented the development of this compound as a drug [46]. Based on the fact that boronic acids could act as bioisosteres of the aromatic hydroxyl group of the C‐ring of CA‐4 and that at physiological pH levels, this compound remains at its unionized form (pKa 9–10), boronic acid analogues of CA‐4 were synthetized. A cis and trans‐boronic acid analogues (32 and 33, respectively) were tested and, as predicted by the docking studies, these compounds were more potent inhibitors of tubulin polymerization than CA‐4. Interestingly, proliferation is dependent of the geometry of the double bond, being the analogue cis (32, Scheme 6) more effective (32, IC50 of 1.5

Molecules 2020, 25, x FOR PEER REVIEW 14 of 43

Figure 5. Possible formation of a cyclic boronic acid derivative, when boronic acid is located next to an urea group.

For the synthesis of compound 25 (Scheme 5), an amine derivative (19) and L‐phenylalanine methyl ester hydrochloride (20) were used as initial reagents to form the urea moiety. The intermediate (21) was then hydrolyzed with NaOH. The α‐amino boronic acid pinacol ester (23) was later coupled with the carboxylic acid (represented in molecule 22), giving the α‐amino boronic acid Moleculespinacol2020, ester25, 4323 (24). Finally, the urea‐containing peptide boronic acid (25) was obtained by elimination14 of 40 of the pinacol protecting group [45].

Molecules 2020, 25, x FOR PEER REVIEW 15 of 43

μM; 33, IC50 of 7.8 μM; CA‐4, IC50 of 2.0 μM). Besides these effects, no toxicity was revealed, and water SchemesolubilityScheme 5. wasSynthesis 5. enhanced, Synthesis of the being urea-peptideof the the cisurea analogue boronic‐peptide acid (boronic3225) more. Reaction acid stable 25 conditions: and. Reaction soluble ( i) conditions:than CDI, CA dimethylformamide‐4 thus(i) CDI,allowing (DMF),oral delivery.dimethylformamide MeCN, Another r.t.; (ii) (1) interesting (DMF), NaOH, 0MeCN, aspect◦C; (2) isr.t.; HCl, that (ii 0 it)◦ C;is(1) thought ( iiiNaOH,) N,N that0 -dicyclohexylcarbodiimide0 the°C; cis(2)‐ boronicHCl, 0 acid °C; analogue(iii (DCC),) N,N 32′‐ HOBt, will DIPEA,not havedicyclohexylcarbodiimide CH a short2Cl2, r.t.plasma (iv) (1) half DEA,‐ (DCC),life, EtOAc, since HOBt, the r.t.; DIPEA,carbon–boron (2) HCl, CH H2Cl2O,2, bondr.t. EtOAc, (iv attached) (1) r.t., DEA, 2 h. to EtOAc, the aromatic r.t.; (2) HCl, ring H does2O, not sufferEtOAc, hydrolysis r.t., 2 h. [46].

SchemeScheme 6. Synthesis 6. Synthesis of theof the cis cis (32 (32) and) and trans-boronic trans‐boronic acid acid (33 (33) )analogues analogues based based on onthe theantimitotic antimitotic agentagent CA-4. CA Reaction‐4. Reaction conditions: conditions: ( i)) NaBH NaBH4, 4MeOH,, MeOH, H2O, H 2EtOAc,O, EtOAc, r.t., 30 r.t., min; 30 (ii min;) PBr (3,ii CH) PBr2Cl32,, 0 CH °C 2Cl2, 0 ◦Cto to r.t., r.t., overnight; (iii (iii) )PPh PPh3, 3,THF, THF, r.t., r.t., 6 6h; h; ( (iviv) )3 3-bromo-4-methoxybenzaldehyde,‐bromo‐4‐methoxybenzaldehyde, sodium sodium bis(trimethylsilyl)amidebis(trimethylsilyl)amide (NaHMDS), (NaHMDS), THF, THF, −7878 °CC to to r.t., r.t., 6.5 6.5 h; h; (v ()v (1)) (1) 5% 5% EtOAc/n EtOAc‐hexane,/n-hexane, column column − ◦ chromatography;chromatography; (2) n-BuLi,(2) n‐BuLi, B(OMe) B(OMe),3 THF,, THF, −7878 °CC to to r.t., r.t., overnight; overnight; (3) (3) 3N 3N HCl, HCl, r.t., r.t., 30 min. 30 min. 3 − ◦ In theIn synthesis the synthesis of these of compoundsthese compounds (Scheme (Scheme6), 3,4,5-trimethoxy-benzaldehyde 6), 3,4,5‐trimethoxy‐benzaldehyde ( 26 )( was26) was reduced to givereduced the alcohol to give (represented the alcohol (represented in molecule in molecule27) that was 27) that treated was treated with phosphorus with phosphorus tribromide tribromide yielding yielding the corresponding benzyl bromide (represented in molecule 28) that subsequently was the corresponding benzyl bromide (represented in molecule 28) that subsequently was transformed transformed into a phosphonium salt (29). A Wittig coupling allowed the transformation of this salt 29 into ainto phosphonium cis and trans saltstilbenes ( ). ( A30 Wittigand 31) coupling that were allowed separated the through transformation flash column of chromatography. this salt into cis and After this separation, the isomers were lastly converted into the desired cis and trans‐boronic acids [46]. Later, the same group [46,47] developed two chalcone analogues of compound 32 (36 and 38) and their effects on tubulin polymerization and growth inhibition were evaluated, in various human cancer cell lines [47]. From these compounds, 36 was the most potent one. Interestingly, this analogue revealed no inhibition on tubulin polymerization, but proved to have antiproliferative activity against different cell lines at micromolar and nanomolar concentrations (Table 3) and also revealed angiogenesis inhibition at 5 μΜ concentration. In fact, this anti‐angiogenic activity is very promising, since the compounds that target cancer vascular cells usually do not demonstrate drug resistance comparing to cancer epithelial cells [47].

Molecules 2020, 25, 4323 15 of 40 trans stilbenes (30 and 31) that were separated through flash column chromatography. After this separation, the isomers were lastly converted into the desired cis and trans-boronic acids [46]. Later, the same group [46,47] developed two chalcone analogues of compound 32 (36 and 38) and their effects on tubulin polymerization and growth inhibition were evaluated, in various human cancer cell lines [47]. From these compounds, 36 was the most potent one. Interestingly, this analogue revealed no inhibition on tubulin polymerization, but proved to have antiproliferative activity against different cell lines at micromolar and nanomolar concentrations (Table3) and also revealed angiogenesis inhibition at 5 µM concentration. In fact, this anti-angiogenic activity is very promising, since the compounds that target cancer vascular cells usually do not demonstrate drug resistance comparing to cancer epithelial cells [47].

Molecules 2020, 25, x FOR PEER REVIEW 16 of 43 MoleculesTable 2020 3., 25Antiproliferative, x FOR PEER REVIEW activity of chalcone-boronic acid analogue 36 in different cell lines.16 of 43 Table 3. Antiproliferative activity of chalcone‐boronic acid analogue 36 in different cell lines. 1 Table 3. AntiproliferativeCell activity Line of chalcone‐boronic acid analogueGI50 ( µ36M) in different cell lines. Cell line GI50 1 (μM) MCF7 (breast cancer)1 0.034 Cell line GI50 (μM) SNB-19 (CNSMCF72 (breastcancer) cancer) 0.034 0.01 MCF7 (breast 2cancer) 0.034≤ NCI-H460SNB (lung‐19 (CNS cancer) cancer) ≤0.01 0.028 SNB‐19 (CNS 2 cancer) ≤0.01 SNB-19NCI (colon‐H460 cancer) (lung cancer) 0.028 0.01 NCI‐H460 (lung cancer) 0.028≤ UACC-62SNB (melanoma)‐19 (colon cancer) ≤0.01 0.034 ≤ SK-OV-3SNBUACC (ovarian‐19‐ 62(colon cancer) (melanoma) cancer) 0.0340.01 0.01 ≤ DU-145SK (prostateUACC‐OV‐3‐62 (ovarian cancer) (melanoma) cancer) 0.034≤0.01 0.050 SK‐OV‐3 (ovarian cancer) ≤0.01 1 GI : Half maximal cellDU growth‐145 inhibitory(prostate concentration;cancer) 20.050CNS: Central nervous system. 50 DU‐145 (prostate cancer) 0.050 1 GI50: Half maximal cell growth inhibitory concentration; 2 CNS: Central nervous system. 1 GI50: Half maximal cell growth inhibitory concentration; 2 CNS: Central nervous system. Chalcone analogues 36 and 38 were synthetized through Claisen–Schmidt condensation under Chalcone analogues 36 and 38 were synthetized through Claisen–Schmidt condensation under alkaline conditions [47]. For compound 36 (Scheme7), condensation between a ketone ( 34) and a alkalineChalcone conditions analogues [47]. For36 and compound 38 were 36synthetized (Scheme 7),through condensation Claisen–Schmidt between condensationa ketone (34) underand a substitutedalkalinesubstituted aldehyde conditions aldehyde (35 [47]. )(35 occurred. )For occurred. compound In In the the case36 case (Scheme of of compound 7), condensation 3838 (Scheme(Scheme between 8),8 a), Friedel–Crafts a Friedel–Crafts a ketone (34 acylation) and acylation a with 1-bromo-2-methoxybenzenesubstitutedwith 1‐bromo aldehyde‐2‐methoxybenzene (35) occurred. (37 )( 37In occurred) the occurred case firstlyof firstlycompound followed followed 38 (Scheme by by ketone ketone 8), a deprotection deprotectionFriedel–Crafts andand acylation a finalfinal step of Claisen–Schmidtwithstep of1‐ bromoClaisen–Schmidt‐2‐methoxybenzene condensation condensation [47 (37]. ) [47].occurred firstly followed by ketone deprotection and a final step of Claisen–Schmidt condensation [47].

SchemeScheme 7. 7.Synthesis Synthesis of the the chalcone chalcone-boronic‐boronic acid acid analogue analogue 36. Reaction36. Reactionconditions: conditions: (i) (1) 1‐(3,4,5 (‐i) (1) Scheme 7. Synthesis of the chalcone‐boronic acid analogue 36. Reaction conditions: (i) (1) 1‐(3,4,5‐ 1-(3,4,5-trimethoxyphenyl)ethenone,trimethoxyphenyl)ethenone, 5‐formyl 5-formyl-2-methoxyboronic‐2‐methoxyboronic acid, acid,NaOH, NaOH, r.t., overnight; r.t., overnight; (2) H2O, (2) HCl. H2O, HCl. trimethoxyphenyl)ethenone, 5‐formyl‐2‐methoxyboronic acid, NaOH, r.t., overnight; (2) H2O, HCl.

Scheme 8. Synthesis of the chalcone‐boronic acid analogue 38. Reaction conditions: (i) CH2Cl2, AlCl3, Scheme 8. Synthesis of the chalcone-boronic acid analogue 38. Reaction conditions: (i) CH Cl , AlCl , Schemeacetic anhydride, 8. Synthesis r.t. of to the 40 chalcone°C, 1 h; (‐iiboronic) Ethylene acid glycol, analogue benzene, 38. Reaction reflux, conditions: PTSA; (iii) ( (1)i) CH THF,2Cl 2n,2 AlCl‐BuLi,2 3, 3 aceticacetic anhydride, anhydride, r.t. r.t. to to 40 40◦C, °C, 1 1 h; h; ( ii(ii)) Ethylene Ethylene glycol, benzene, benzene, reflux, reﬂux, PTSA; PTSA; (iii) ( (1)iii) THF, (1) THF, n‐BuLi, n-BuLi, B(OMe)3, −78 °C, 90 min; (2) HCl, r.t., 30 min; (iv) (1) 3,4,5‐trimethoxybenzaldehyde, NaOH, r.t., B(OMe)B(OMe), 783, −78C, °C, 90 90 min; min; (2) (2) HCl, HCl, r.t., r.t., 3030 min;min; ( iviv)) (1) (1) 3,4,5 3,4,5-trimethoxybenzaldehyde,‐trimethoxybenzaldehyde, NaOH, NaOH, r.t., r.t., overnight;3 − ◦(2) H2O, HCl. overnight;overnight; (2) H (2)2O, H2 HCl.O, HCl. A boronic acid (43, Scheme 9) based on commercially available fulvestrant was designed and synthetizedA boronic to improve acid (43 ,the Scheme bioavailability 9) based ofon this commercially drug, so it couldavailable be administrated fulvestrant was orally designed and not and by synthetizedintramuscular to improveinjection. the Fulvestrant bioavailability is a selective of this drug, estrogen so it couldreceptor be administrated down‐regulator orally (SERD) and notthat by is intramuscularindicated for estrogeninjection. receptor Fulvestrant positive is a selective(ER+) metastatic estrogen breastreceptor cancer down and‐regulator acts by (SERD) competitively that is indicatedbinding to for estrogen estrogen receptor receptor α (ER positiveα). The (ER+)replacement metastatic of 3‐ OHbreast group cancer of fulvestrant and acts by by competitivelya boronic acid bindingled to a to substantial estrogen receptor reduction α (ER ofα first). The pass replacement metabolism, of 3‐ OHwhich group consequently of fulvestrant led by to a aboronic significant acid ledimprovement to a substantial of bioavailability reduction whileof first maintaining pass metabolism, the pharmacological which consequently proprieties. led Itto is athought significant that improvementboronic acid derivatives of bioavailability reduce while the first maintaining pass metabolism the pharmacological due to their ability proprieties. to bind It iscovalently thought thatand boronicreversibly acid to derivatives1,2‐ and 1,3 ‐ reducediols present the first in pass sugar metabolism molecules dueand toglycoproteins, their ability tomaking bind covalentlythe compound and reversibly to 1,2‐ and 1,3‐ diols present in sugar molecules and glycoproteins, making the compound

Molecules 2020, 25, x FOR PEER REVIEW 17 of 43

Moleculesunavailable2020, 25, 4323 to pass through glucuronidation. Besides improving the bioavailability of fulvestrant,16 of 40 compound 43 as also similar effects on cell growth inhibition in different breast cancer cell line (Table 4) [48]. A boronic acid (43, Scheme9) based on commercially available fulvestrant was designed and synthetizedTable to 4. improve Effects on the growth bioavailability inhibition (IC50 of, μ thisM) of drug, fulvestrant so it and could compound be administrated 43 in different orallybreast and not by intramuscularcancer cell injection.lines. Fulvestrant is a selective estrogen receptor down-regulator (SERD) that is indicated for estrogen receptorCompound positive (ER+MCF) metastatic‐7 breastT47D cancer and acts by competitively binding to estrogen receptorFulvestrantα (ERα). The replacement0.0015 of 3-OH0.0012 group of fulvestrant by a boronic acid led to a substantial reduction43 of first pass metabolism,0.0032 which0.0061 consequently led to a significant improvement of bioavailability while maintaining the pharmacological proprieties. It is thought that The first synthetic step of to obtain this compound (Scheme 9) involves the formation of triflate boronic acid derivatives reduce the first pass metabolism due to their ability to bind covalently and of 17‐acetyl S‐deoxo fulvestrant (40) using as a starting material 17‐acetyl‐S‐deoxo fulvestrant (39), reversiblyfollowed to 1,2- by Miyaura and 1,3- borilyation diols present reaction in sugar with molecules bis(pinacolato)diboron and glycoproteins, in the presence making of the catalytic compound unavailableamount toof palladium(II) pass through acetate. glucuronidation. After the removal Besides of the improving acetyl group the (represented bioavailability in molecule of fulvestrant, 41) compoundunder alkaline43 as also conditions, similar deprotection effects on celloccurs growth through inhibition oxidation in with diff metaerent‐chloroperoxybenzoic breast cancer cell line (Tableacid4)[ 48(mCPBA),]. forming the desired product [48].

SchemeScheme 9. Synthesis 9. Synthesis of of a SERDa SERD boronic boronic acid acid analogueanalogue 4343 basedbased on on the the drug drug fulvestrant. fulvestrant. Reaction Reaction conditions:conditions: (i) Triflic (i) Triflic anhydride, anhydride, pyridine, pyridine, CH CHCl2Cl,2, −10 °C;C; (ii (ii) )BB2pinpin2, palladium(II), palladium(II) acetate acetate (Pd(OAc) (Pd(OAc)2), ), 2 2 − ◦ 2 2 2 tricyclohexylphosphine,tricyclohexylphosphine, MeCN, MeCN, 80 ◦ 80C; °C; (iii )(iii KOH,) KOH, MeOH MeOH/THF,/THF, 0 ◦ 0C °C to to r.t., r.t., 4 h;4 h; (iv ()iv mCPBA,) mCPBA, CH CH2Cl2Cl22,, 0 ◦C. 0 °C. Table 4. Eﬀects on growth inhibition (IC50, µM) of fulvestrant and compound 43 in diﬀerent breast cancerA cellstrategy lines. developed for targeting release of camptothecin, an anticancer compound that binds and subsequently inhibits the enzyme topoisomerase I, which consequently leads to cell death, was Compound MCF-7 T47D accomplished by development of a boronic acid prodrug [49]. The 7‐ethyl‐10‐boronic acid 47 (Scheme 10) was designed basedFulvestrant on the fact that alkyl and0.0015 aryl boronic acids are0.0012 easily oxidized by hydrogen peroxide (H2O2) and that usually43 cancer cells have0.0032 increased levels of 0.0061reactive oxygen species (ROS),

The ﬁrst synthetic step of to obtain this compound (Scheme9) involves the formation of triﬂate of 17-acetyl S-deoxo fulvestrant (40) using as a starting material 17-acetyl-S-deoxo fulvestrant (39), followed by Miyaura borilyation reaction with bis(pinacolato)diboron in the presence of catalytic amount of palladium(II) acetate. After the removal of the acetyl group (represented in molecule 41) Molecules 2020, 25, 4323 17 of 40 under alkaline conditions, deprotection occurs through oxidation with meta-chloroperoxybenzoic acid (mCPBA), forming the desired product [48]. A strategy developed for targeting release of camptothecin, an anticancer compound that binds and subsequently inhibits the enzyme topoisomerase I, which consequently leads to cell death, was accomplished by development of a boronic acid prodrug [49]. The 7-ethyl-10-boronic acid 47 (Scheme 10) was designed based on the fact that alkyl and aryl boronic acids are easily oxidized by hydrogenMolecules 2020 peroxide,, 25,, x FOR (H PEER2O REVIEW2) and that usually cancer cells have increased levels of reactive18 of oxygen 43 species (ROS), comparing to normal cells [49,50]. Another factor that makes boronic acid prodrug 47 interestingcomparing for selective toto normal delivery cells [49,50].[49,50]. of camptothecin Another factorfactor is thatthat that makes the surface boronic of acid cancer prodrug cells 47 is interestinginteresting covered with forfor the selective delivery of camptothecin is that the surface of cancer cells is covered with the polysaccharide polysaccharideselective delivery glycocalyx of camptothecin and boronic is that acids the surface can bind of cancer to the cells 1,2- is and covered 1,3-diols with ofthe glycocalyx polysaccharide forming glycocalyx and boronic acids can bind toto thethe 1,2‐ ‐ and 1,3‐‐diols of glycocalyx formingforming boronate esters, boronate esters, which is known to enhance drug selectivity towards cancer cells [49,51]. Through which isis known toto enhance drug selectivity towardstowards cancer cells [49,51].[49,51]. Through thethe oxidation of 10‐‐ the oxidation of 10-boronic acid to 10-OH by H2O2, which is present in high levels in cancer cells, boronic acid toto 10‐‐OH by H22O22,, which isis present inin high levelslevels inin cancer cells, thethe selective releaserelease of the selectivecamptothecin release directly of camptothecin toto cancer cells directly can be accomplished to cancer cells [49].[49]. can be accomplished [49].

Scheme 10. Synthesis of the camptothecin prodrug with boronic acid moiety (47). Reaction conditionsScheme ( 10.i) Synthesis N,N-bis(trifluoromethylsulfonyl)aniline, of thethe camptothecin prodrug with boronic triethylamine acid moiety (TEA),((47).). Reaction DMF, conditions 60 ◦C, 3 h; (ii) [1,1((ii)) 0N,N-bis(diphenylphosphino)ferrocene]dichloropalladium(II)‐‐bis(trifluoromethylsulfonyl)aniline, triethylaminetriethylamine (TEA),(TEA), DMF, (Pd(dppf)Cl 60 °C,°C, 23), h; KOAc, ((iiii)) [1,1[1,1 B′‐′‐2pin2, 1,4-dioxane,bis(diphenylphosphino)ferro 80 ◦C, 12 h; (iii) NaIOcene]dichloropalladium(II)4, NH4OAc, 1:1 C3H6 O:H(Pd(dppf)Cl(Pd(dppf)Cl2O, r.t.,22), 24), KOAc, h. B22pin22,, 1,4‐‐dioxane, 80 °C,°C, 12 h; ((iiiiii)) NaIO44,, NH44OAc, 1:1 C33H66O:H22O, r.t.,r.t., 24 h. In the synthesis of prodrug 47 (Scheme 10), the camptothecin drug (44) was first converted into triflate (45InIn) and thethe synthesis then occurred of prodrug a borylation 47 (Scheme(Scheme of the 10), intermediate thethe camptothecin that was drug mediated ((44)) was by firstfirst palladium-catalyzed converted intointo triflate (45) and then occurred a borylation of the intermediate that was mediated by palladium‐ Miyauratriflate reaction, (45) and followed then occurred by a finala borylation step of of reduction the intermediate of boronate that was ester mediated (represented by palladium in molecule‐ catalyzed Miyaura reaction,reaction, followedfollowed by a finalfinal step of reductionreduction of boronate ester (represented(represented inin (46)[49]. molecule ((46)) [49].[49]. Similar to this approach, the phenylboronic acid prodrug 49 (Figure6) of methotrexate ( 48, Figure6) Similar toto thisthis approach, thethe phenylboronic acid prodrug 49 (Figure(Figure 6) of methotrexate ((48,, was designedFigure 6) was for designed a more forfor selective a more selective and eff ective and effective treatment treatmenttreatment of rheumatoid of rheumatoidrheumatoid arthritis arthritis (RA), (RA),(RA), since since the presencethethe presence of high of levels high of levelslevels ROS of in ROS inflammatory inin inflammatoryinflammatory cells cells is associated isis associated with with RA RA pathology pathology [[52].[52].52].

Figure 6. Methotrexate and its phenylboronic acid prodrug (48 and 49, respectively). Figure 6. Methotrexate and itsits phenylboronic acid prodrug ((48 and 49,, respectively).respectively). Likewise, a recent study based on the elevated levels of ROS in some cancers showed the development of a boronicLikewise, acid prodrug a recentrecent for thestudy selective based deliveryon thethe elevated of a tyrosine levelslevels kinase of ROS inhibitor inin some drug cancers in cancer showed cells. the Tyrosinethe development of a boronic acid prodrug forfor thethe selective delivery of a tyrosinetyrosine kinase inhibitorinhibitor drug inin cancer cells. Tyrosine kinases are involvedinvolved inin processes of cell differentiation, proliferation, and apoptosis and are usually activated inin an abnormal manner inin cancer cells. One problem of some of thesethese drugs isis thatthat theythey do not have cancer cells specificity, being also toxictoxic toto normal cells. This occurs with crizotinib, revealingrevealing hepatotoxicity thatthat consequently leadsleads toto thethe reductionreduction or

Molecules 2020, 25, 4323 18 of 40 kinases are involved in processes of cell differentiation, proliferation, and apoptosis and are usually activated in an abnormal manner in cancer cells. One problem of some of these drugs is that they do not have cancer cells specificity, being also toxic to normal cells. This occurs with crizotinib, revealing hepatotoxicity that consequentlyMolecules leads 2020 to, 25, the x FOR reduction PEER REVIEW or discontinuation of the treatment with this drug. Crizotinib19 of 43 has a 2-aminopyridine moiety that binds to the amino acids in the ATP-binding pocket of kinases tyrosine-protein kinase Metdiscontinuation (c-MET), ROS of proto-oncogene the treatment with 1 tyrosine-proteinthis drug. Crizotinib kinase has a 2 (ROS1)‐aminopyridine and anaplastic moiety that lymphoma binds kinase to the amino acids in the ATP‐binding pocket of kinases tyrosine‐protein kinase Met (c‐MET), ROS (ALK). Theproto synthesis‐oncogene of boronic1 tyrosine acid‐protein prodrugs kinase (ROS1) of crizotinib and anaplastic (51 and lymphoma52, Scheme kinase 11) proved(ALK). The to be a good approach forsynthesis reverting of boronic the problems acid prodrugs associated of crizotinib with (51 crizotinib. and 52, Scheme When 11) present proved into highbe a good levels, approach which occurs in for reverting the problems associated with crizotinib. When present in high levels, which occurs in malignant cells, H2O2 oxidizes the boronic acid moiety and allows the selective release of crizotinib. It was also provedmalignant that, in casecells, ofH2 prodrugO2 oxidizes51 the, more boronic than acid half moiety of the and prodrug allows the remains selective intact release in of biological crizotinib. conditions, It was also proved that, in case of prodrug 51, more than half of the prodrug remains intact in therefore notbiological releasing conditions, crizotinib therefore [53]. not releasing crizotinib [53].

Scheme 11. Synthesis of the crizotinib prodrugs with boronic acid moiety 51 and 52. Reaction conditions:

(i) Di-tert-butyl dicarbonate, THF,12 h; (ii) (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol, CO, Pd(OAc) , copper(II) acetate monohydrate (Cu(OAc) H O), KI, DMSO, MeCN; (iii) TFA, CH Cl , 2 2· 2 2 2 r.t., 2 h (51) or 30 min (52); (iv) 4-hydroxybenzaldehyde, 2-picoline borane, AcOH/MeOH, overnight. Molecules 2020, 25, x FOR PEER REVIEW 20 of 43

Scheme 11. Synthesis of the crizotinib prodrugs with boronic acid moiety 51 and 52. Reaction conditions: (i) Di‐tert‐butyl dicarbonate, THF, 12 h; (ii) (4‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐

yl)phenyl)methanol, CO, Pd(OAc)2, copper(II) acetate monohydrate (Cu(OAc)2∙H2O), KI, DMSO, MoleculesMeCN;2020, (25iii,) 4323 TFA, CH2Cl2, r.t., 2 h (51) or 30 min (52); (iv) 4‐hydroxybenzaldehyde, 2‐picoline borane,19 of 40 AcOH/MeOH, overnight.

TheThe 2-aminopyridine2‐aminopyridine of of crizotinibcrizotinib ( (5050)) isis modifiedmodified (Scheme (Scheme 11 11):): FirstFirst occurredoccurred aa protectionprotection ofof thethe piperidinepiperidine nitrogennitrogen withwith aa tert-butyloxycarbonyltert‐butyloxycarbonyl (Boc)(Boc) group,group, followedfollowed byby selectiveselective functionalization ofof thethe 2-aminopyridine.2‐aminopyridine. InIn the case of prodrug 5151,, thethe moiety was introducedintroduced viavia carbamate,carbamate, whilewhile inin prodrugprodrug 5252 waswas via via alkylation, alkylation, using using a a reductive reductive amination amination approach approach [ 53[53].]. ItIt isis thoughtthought that that the the autotaxin-lysophosphatidic autotaxin‐lysophosphatidic acid acid axis axis (ATX-LPA (ATX‐LPA axis) axis) has has an an important important role role in tumorin tumor progression progression and and metastasis. metastasis. The activityThe activity of the of extracellular the extracellular enzyme enzyme autotaxin autotaxin (ATX) is(ATX) related is torelated a threonine to a threonine residue inresidue the active in the site active and site the inhibitionand the inhibition of this enzyme of this couldenzyme be could a good be strategy a good forstrategy cancer for therapy. cancer Basedtherapy. on Based the hit on compound the hit compound 2,4-thiazolidinedione 2,4‐thiazolidinedione (IC50 of 2.50 (IC50µ M),of 2.50 a series μM), of a SARseries studies of SAR were studies performed were performed in order to in find order a selective to find a compound selective compound for cancer cells. for cancer A first cells. SAR A study first revealedSAR study that revealed the methoxy that the and methoxy carboxylic and carboxylic acid moieties acid were moieties important were important for the inhibition for the inhibition of ATX. Withof ATX. the objectiveWith the of objective improving of inhibitionimproving and inhibition targeting, and the targeting, carboxylic the acid carboxylic group was acid replaced group by was its bioisosterereplaced by boronic its bioisostere acid 55 [boronic41]. This acid replacement 55 [41]. This was replacement based on the was knowledge based on thatthe knowledge carboxylic acidthat couldcarboxylic bind toacid the could active bind site ofto threoninethe active oxygen site of andthreonine that boronic oxygen acid and moiety that boronic in bortezomib acid moiety binds toin thebortezomib nucleophilic binds oxygen to the lone nucleophilic pair of threonine oxygen [lone41,54 pair]. It wasof threonine proved that [41,54]. the introductionIt was proved of boronicthat the acidintroduction in para position of boronic significantly acid in para enhanced position thesignificantly inhibition enhanced of autotaxin, the inhibition both in in of vitro autotaxin,and in vivoboth assays,in in vitro resulting and in in vivo a 440-fold assays, moreresulting active in inhibitora 440‐fold (IC more50 of active 6 nM) inhibitor [41]. (IC50 of 6 nM) [41]. TheThe synthesissynthesis ofof thisthis compoundcompound ((5555)) waswas performedperformed byby aa convergentconvergent synthesissynthesis (Scheme(Scheme 1212),), wherewhere the the thiazolane thiazolane-2,4-dione‐2,4‐dione (53 ()53 and) and diphenyl diphenyl boronic boronic acid (54 acid) moieties (54) moieties were prepared were separately prepared separately[41,55]. The[41 synthesis,55]. The of synthesis the diphenyl of the boronic diphenyl acid boronic is accomplished acid is accomplished via Suzuki–Miyaura via Suzuki–Miyaura reaction [55]. reactionFinally, [through55]. Finally, Knoevenagel through Knoevenagel condensation, condensation, these two moieties these two are moieties linked to are produce linked the to produce compound the compound55 [41,55]. 55 [41,55].

SchemeScheme 12.12. SynthesisSynthesis ofof thethe boronicboronic acidacid autotaxinautotaxin inhibitorinhibitor 5555.. ReactionReaction conditions:conditions: ( i) NaH, DMF, 1-(chloromethyl)-4-ﬂuorobenzene,1‐(chloromethyl)‐4‐fluorobenzene, r.t., r.t., 22 22 h; (h;ii )B(ii2)pin B2pin2, Pd(dppf)Cl2, Pd(dppf)Cl2, KOAc,2, KOAc, DMF, DMF, 80 ◦ C,80 18 °C, h; 18 (iii h;) THF, (iii) NaIOTHF, 4NaIO, HCl;4, (HCl;iv) EtOH, (iv) EtOH, piperidine, piperidine, 22 h, reﬂux. 22 h, reflux.

AnotherAnother interesting interesting application application of of boronic boronic acid acid derivatives derivatives in cancer in cancer therapy therapy is through is through the design the ofdesign histone of deacetylaseshistone deacetylases (HDAC) (HDAC) inhibitors inhibitors [56,57]. It [56,57]. is known It is that known these that inhibitors these areinhibitors associated are withassociated inhibition with of inhibition cell growth, of cell stimulation growth, stimulation of terminal of di terminalﬀerentiation differentiation in tumor cells, in tumor and cells, can even and preventcan even malignant prevent malignant tumors formation. tumors Theformation. design ofThe these design compounds of these wascompounds based on was their based deacetylation on their mechanism,deacetylation acting mechanism, on lysine acting residue on lysine of these residue epigenetic of these enzymes epigenetic [56 enzymes,57]. The [56,57]. zinc ion The located zinc ion in thelocated active in sitethe ofactive this site enzyme of this coordinates enzyme coordinates the electrophilic the electrophilic carbon of the carbon carbonyl of the group carbonyl present group in a substrate. This attack leads to the formation of a tetrahedral transition state, which is stabilized by the zinc-oxygen interaction. Since the boronic acids can act as electrophiles and be attacked by nucleophilic groups, boronic acids with α-amino acid moiety (59–61) were designed, in which boronic acid substitutes the acetamide of the acetylated lysine substrate. The boronic acid group is attacked Molecules 2020, 25, x FOR PEER REVIEW 21 of 43

present in a substrate. This attack leads to the formation of a tetrahedral transition state, which is stabilized by the zinc‐oxygen interaction. Since the boronic acids can act as electrophiles and be

Moleculesattacked2020 by, 25nucleophilic, 4323 groups, boronic acids with α‐amino acid moiety (59–61) were designed,20 of in 40 which boronic acid substitutes the acetamide of the acetylated lysine substrate. The boronic acid group is attacked by water in the active site, forming a transition state analogue that binds to the zinc byion, water leading in theto the active inhibition site, forming of HDAC a transition that subsequently state analogue lead thatto cancer binds cell to the growth zinc ion,inhibition leading (59 to, theIC50 inhibition of 2.0 μM; of 60 HDAC, IC50 of that 2.0 μ subsequentlyM; 61, IC50 of lead 1.4 μ toM). cancer The cell α‐amino growth acid inhibition moiety (is59 also, IC 50veryof 2.0importantµM; 60, ICfor50 theof inhibitory 2.0 µM; 61 ,activity, IC50 of 1.4sinceµM). its Thereplacementα-amino decreases acid moiety the is potency also very [56]. important for the inhibitory activity,For sincethe preparation its replacement of these decreases compounds, the potency it was [56 ].first necessary to prepare the α‐amino acid derivativeFor the (57 preparation), which was of these synthesized compounds, from it diethyl was first aminomalonate necessary to prepare (56). A the seriesα-amino of transformations acid derivative (such57), whichas protection was synthesized with Boc, hydrolysis, from diethyl decarboxylation, aminomalonate and (56). hydroboration A series of transformations using a cross‐coupling such as protectioncatalyst, catalyst, with Boc, led hydrolysis,to the α‐amino decarboxylation, acid derivative and [56]. hydroboration After that, the using main a synthetic cross-coupling steps to catalyst, obtain catalyst,the desired led to compounds the α-amino (Scheme acid derivative 13) followed [56]. After a that, process the main of deprotection synthetic steps of to obtainthe amine, the desired then compoundscondensation (Scheme with an 13 aryl) followed carboxylic a process acid, ofand deprotection finally hydrolysis of the amine, of the then boronic condensation ester (represented with an aryl in carboxylicmolecule 58 acid,) [56]. and finally hydrolysis of the boronic ester (represented in molecule 58)[56].

Scheme 13.13. Synthesis of thethe boronicboronic acidsacids HDACHDAC inhibitorsinhibitors 5959––6161.. ReactionReaction conditions:conditions: (i) (Boc)(Boc)22O, TEA, THF, r.t.; ( (iiii)) NaOEt, NaOEt, 5 5-Bromopent-1-ene,‐Bromopent‐1‐ene, EtOH, EtOH, reflux; reﬂux; (iii ()iii LiOH) LiOH∙H2O,H O,EtOH, EtOH, H2O, H 0O, °C; 0 (ivC;) · 2 2 ◦ (toluene,iv) toluene, reflux; reﬂux; (v) 3‐ (Biphenylv) 3-Biphenyl-NH‐NH2, EDCI,, EDCI,HOBt∙H HOBt2O, DMF,H O, r.t.; DMF, (vi) r.t.;cyclooctadiene (vi) cyclooctadiene iridium chloride iridium 2 · 2 chloridedimer ([Ir(cod)Cl] dimer ([Ir(cod)Cl]2), bis(diphenylphosphino)methane2), bis(diphenylphosphino)methane (dppm), pinacolborane, (dppm), pinacolborane, CH2Cl2, r.t.; CH (vii2Cl) 2HCl,, r.t.; (EtOAc,vii) HCl, CHCl EtOAc,3, r.t. or CHCl R‐COOH,3, r.t. orEDCI, R-COOH, HOBt, DMF, EDCI, r.t. HOBt, or R‐ DMF,COCl, r.t. TEA, or CH R-COCl,2Cl2, N,N TEA,‐dimethyl CH2Cl‐42‐, N,N-dimethyl-4-aminopyridineaminopyridine (DMAP), r.t.; (viii) (DMAP), NH4OAc, r.t.; NaIO (viii)4, NH acetone,4OAc, H NaIO2O, r.t.,4, acetone, 48 h. H2O, r.t., 48 h.

One problem associated with bortezomib is related to drug resistance and relapse in multiplemultiple myeloma. To To overcome this, a very recent study [[57]57] designed new dual targets that act on HDAC and on proteasome, based on other studies that suggest that overexpression HDAC might related to resistance ofof multiple multiple myeloma myeloma to bortezomib.to bortezomib. Through Through introduction introduction of zinc-binding of zinc‐binding groups groups to peptide to boronatepeptide boronate with diff erentwith aminodifferent acid amino residues, acid various residues, compounds various werecompounds obtained, were being obtained, compound being66 thecompound most promising. 66 the most This promising. compound 66Thisnot compound only proved 66 to not inhibit only both proved proteasome to inhibit (IC both50 of proteasome 1.1 nM) and HDAC(IC50 of (IC 1.150 nM)of 255 and nM), HDAC but also (IC showed50 of 255 antiproliferative nM), but also activityshowed against antiproliferative different multiple activity myeloma against celldifferent lines multiple (RPMI-8226, myeloma IC50 of cell 6.66 lines nM; (RPMI U266,‐8226, IC50 ofIC50 4.31 of 6.66 nM; nM; KM3, U266, IC50 ICof50 10.1 of 4.31 nM) nM; including KM3, IC a50 cell of line10.1 associatednM) including with multiplea cell line myeloma associated drug with resistance multiple to bortezomib myeloma drug (KM3 /resistanceBTZ, IC50 (to66 )bortezomib of 8.98 nM; IC(KM3/BTZ,50(bortezomib) IC50(66 of) of 226 8.98 nM) nM; [57 IC]. 50(bortezomib) of 226 nM) [57]. Compound 66 was synthetized (Scheme 14) through a two-step coupling reaction with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU). First, the intermediates 63 and 65 were synthetized separately: The intermediate 63 was obtained through a step of protection Molecules 2020, 25, x FOR PEER REVIEW 22 of 43

MoleculesCompound2020, 25, 4323 66 was synthetized (Scheme 14) through a two‐step coupling reaction with 221‐(1H of 40‐ benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminium tetrafluoroborate (TBTU). First, the intermediates 63 and 65 were synthetized separately: The intermediate 63 was obtained through a step of protection of 6262with with boc-L-phenylalanine, boc‐L‐phenylalanine, followed followed by aby process a process of deprotection; of deprotection; meanwhile, meanwhile, a coupling a coupling reaction ofreaction 4-(methoxycarbonyl)benzoic of 4‐(methoxycarbonyl)benzoic acid (64) with acid benzene-1,2-diamine (64) with benzene‐ was1,2‐diamine followed was by a followed demethylation by a reaction,demethylation giving reaction, origin to giving intermediate origin to65 .intermediate The last step 65 of. The the synthesislast step of of the compound synthesis66 ofcorresponds compound to66 acorresponds coupling reaction to a coupling of 63 and reaction65 with of TBTU 63 and [57 65]. with TBTU [57].

Scheme 14. SynthesisSynthesis of of the the HDAC HDAC and and proteasome proteasome dual dual target target inhibitor inhibitor 6666. Reaction. Reaction conditions: conditions: (i) ((1)i) (1) Boc Boc-L-Phenylalanine,‐L‐Phenylalanine, DMF, DMF, 0 °C; 0 ◦ C;(2) (2) TBTU, TBTU, N‐ N-methylmorpholinemethylmorpholine (NMM), (NMM), 16 16 h; h;(ii ()ii HCl,) HCl, EtOAc, EtOAc, 2 2h; h; (iii (iii) (1)) (1) Benzene Benzene-1,2-diamine,‐1,2‐diamine, TBTU, TBTU, DMF, DMF, 0 0 °C;◦C; (2) (2) TEA, TEA, 8 8 h; h; ( (iviv)) LiOH, LiOH, MeOH, MeOH, 3 3 h; h; ( (vv)) (1) (1) TBTU, DMF, 00 ◦°C;C; (2)(2) NMM,NMM, 88 h.h. 2.2. Antibacterial Activity 2.2. Antibacterial Activity Currently, antibiotic resistance is among the major concerns in human health. Bacteria resistance Currently, antibiotic resistance is among the major concerns in human health. Bacteria resistance can usually be associated with the overexpression of different classes of β-lactamases by Gram can usually be associated with the overexpression of different classes of β‐lactamases by Gram negative bacteria, and the design of novel potential inhibitors of this enzyme is a very useful strategy. negative bacteria, and the design of novel potential inhibitors of this enzyme is a very useful strategy. A well-known example of a β-lactamase inhibitor is the clavulanic acid [2,58–61]. The first report A well‐known example of a β‐lactamase inhibitor is the clavulanic acid [2,58–61]. The first report that that boronic acids could act as β-lactamase inhibitors was made by a research group from Oxford boronic acids could act as β‐lactamase inhibitors was made by a research group from Oxford University that noticed that borate ions inhibit β-lactamase I [62]. The capacity of boronic acids as University that noticed that borate ions inhibit β‐lactamase I [62]. The capacity of boronic acids as inhibitors of β-lactamase is related with their ability to connect with the serine residue of this enzyme. inhibitors of β‐lactamase is related with their ability to connect with the serine residue of this enzyme. The electrophilic boron group resembles the carbonyl present in β-lactam ring, and the replacement of The electrophilic boron group resembles the carbonyl present in β‐lactam ring, and the replacement the carbonyl with the boron leads to the formation of a tetrahedral adduct with serine that consequently of the carbonyl with the boron leads to the formation of a tetrahedral adduct with serine that inhibits these enzymes in a reversible way. The formation of this complex leads to the inhibition of the consequently inhibits these enzymes in a reversible way. The formation of this complex leads to the synthesis of bacteria cell wall [1,2,7,58,59,63,64]. The boronic acids that form this tetrahedral adduct inhibition of the synthesis of bacteria cell wall [1,2,7,58,59,63,64]. The boronic acids that form this and cause this inhibition can also be referred as boronic acid transition state inhibitors (BATSI), and are tetrahedral adduct and cause this inhibition can also be referred as boronic acid transition state non-lactam transition state analogues. Specific interactions with various β-lactamases can be reached inhibitors (BATSI), and are non‐lactam transition state analogues. Specific interactions with various through introduction of different chemical groups on the carbon that carries the boronic acid group, β‐lactamases can be reached through introduction of different chemical groups on the carbon that providing an inhibitory activity in a precise manner for the different types of these enzymes. Because of carries the boronic acid group, providing an inhibitory activity in a precise manner for the different these characteristics, studies about selective and potent boronic acid compounds are being made types of these enzymes. Because of these characteristics, studies about selective and potent boronic to discover novel compounds that can be administrated in combination with a β-lactam antibiotic agent [1,58,60,64,65]. Molecules 2020, 25, x FOR PEER REVIEW 23 of 43

Molecules 2020, 25, 4323 22 of 40 acid compounds are being made to discover novel compounds that can be administrated in combination with a β‐lactam antibiotic agent [1,58,60,64,65]. A very very recent recent drug, drug, vaborbactam, vaborbactam, was was approved approved by FDA by FDAin 2017 in and 2017 by andEMA by in EMA2018, acting in 2018, as actinga β‐lactamase as a β-lactamase inhibitor [17]. inhibitor Besides [17 ].vaborbactam, Besides vaborbactam, other boronic other acid boronic compounds acid compounds have shown have the shownsame activity. the same Some activity. of these Some β‐lactamase of these β inhibitor-lactamase boronic inhibitor acid boronic derivatives acid are derivatives chiral α‐ areamido chiral‐β‐ αtriazolylethaneboronic-amido-β-triazolylethaneboronic acids 70 and acids 71 (Scheme70 and 71 15).(Scheme The design 15). The of these design compounds of these compounds was based was on basedthe knowledge on the knowledge that boronic that acid boronic is a acid bioisostere is a bioisostere of the carbonyl of the carbonyl present present in β‐lactam in β-lactam [58]. This [58]. Thisknowledge knowledge also lead also the lead creating the creating of a BATSI of a with BATSI higher with affinity. higher These aﬃnity. compounds These compounds bind covalently bind covalentlyand reversibly and to reversibly serine residue to serine S318, residue tyrosine S318, residue tyrosine Y150, residue and Y150,lysine andresidue lysine K67 residue of class K67 C β‐ of classlactamases, C β-lactamases, being the being hydroxyl the hydroxyl group groupof boronic of boronic acid acid responsible responsible for for this this inhibitor inhibitor-enzyme‐enzyme interaction. The The triazole triazole substituent substituent was was also also used used as as a bioisostere a bioisostere of phenyl of phenyl in order in order to facilitate to facilitate the thesynthesis synthesis process process and and cell cell penetration penetration [58]. [58 It]. was It was proved proved that that compounds compounds 7070 andand 7171 werewere very eeffectiveﬀective againstagainst problematic problematic highly highly resistant resistant bacteria bacteria strains strains found found in hospitals in hospitals (70, inhibitory (70, inhibitory constant (Kconstanti) of 0.004 (Ki)µ ofM; 0.00471,K μi M;of 0.00871, Kiµ ofM) 0.008 [58]. μM) [58].

Scheme 15. Synthesis of the α‐α-amido-amido‐β‐β-triazolylethaneboronictriazolylethaneboronic acids 70 and 71. Reaction conditions:

(i) NaNNaN3,,H H2O, EtOAc, tetrabutylammonium iodide (TBAI);(TBAI); ((iiii)) LiCHClLiCHCl2,, −100 °C;C; ((iiiiii)) LiN(SiMeLiN(SiMe3)2,, 3 2 2 − ◦ 3 2 THF, −100 °CC to r.t., overnight; ((iviv)) (1)(1) MeOH,MeOH, THF,THF, −1010 °CC to r.t., 11 h;h; (2)(2) RCOCl,RCOCl, −10 °C,C, 1 h; (v)H) H2O, − ◦ − ◦ − ◦ 2 terttert-BuOH,‐BuOH, CuSO4,, 60 °C,◦C, 18 18 h; h; ( (vivi)) MeCN, MeCN, HCl, n‐-hexane,hexane, r.t., r.t., 3 h.

The initialinitial step step of theof the stereoselective stereoselective synthesis synthesis of the compoundsof the compounds70 and 71 70(Scheme and 71 15 )(Scheme corresponds 15) tocorresponds the production to the of productionα-acylamido- of α‐β-azidoacylamido derivative‐β‐azido67 derivative, followed 67 by, followed a reaction by with a reaction alkynes with68 and,alkynes at last,68 and, the at deprotection last, the deprotection of the boronic of the ester boronic moiety ester (represented moiety (represented in molecule in 69molecule) occurs 69 by) transesterificationoccurs by transesterification with phenylboronic with phenylboronic acid. The acid. formation The formation of the triazole of the istriazole regioselective, is regioselective, and the desiredand the productdesired product is obtained is obtained at quantitative at quantitative yields [58 yields]. [58]. Another studied studied boronic boronic acid acid β‐βlactamase-lactamase inhibitor inhibitor was was compound compound SM23 SM23 (72, Figure (72, Figure 7). This7). Thiscompound compound is also is alsoa BATSI a BATSI and and is considered is considered as asone one of of the the best best inhibitors inhibitors of of class class C ββ‐-lactamaseslactamases reported to date. Its Its design design was was based based on the mimetic approach, in which the didifferentfferent chemical groups with a suitable stereochemistry were arranged with a boronic acid in order to resemble the natural substratesubstrateβ β‐-lactamlactam [60 [60,64].,64]. SAR SAR studies studies revealed revealed that thethat introduction the introduction of a group of a with group negative with chargenegative in charge the side in chain, the suchside aschain, a carboxylate, such as a improves carboxylate, binding improves affinity binding and cell affinity penetration and andcell thatpenetration one of the and hydroxyl that one groups of the hydroxyl of boronic groups acid is of important boronic acid to form is important an interaction to form with an the interaction nitrogen ofwith serine the nitrogen residues of (Ser64 serine and residues Ser315) (Ser64 in the and oxyanion Ser315) holein the of oxyanion the active hole site of of theβ-lactamases active site of [64 β‐]. Alactamases recent publication [64]. A recent revealed publication another revealed interesting another application interesting of compound application72 ofas compound an inhibitor 72 of as the an formationinhibitor of of the biofilms formation by Pseudomonas of biofilms by aeruginosa, Pseudomonas a Gram-negative aeruginosa, a Gram bacteria‐negative that is responsiblebacteria that for is

Molecules 2020,, 25,, 4323x FOR PEER REVIEW 2324 of 4043 responsible for many nosocomial infections in immunosuppressive patients and is very resistant. manyWhen nosocomialexposed to SM23 infections (72), init is immunosuppressive observed inhibition patients in the production and is very of resistant. virulence When factors exposed related to SM23the formation (72), it is of observed biofilms inhibitionof P. aeruginosa in the, like production pyoverdine of virulence and elastase, factors without related co to‐administration the formation of bioﬁlmsan antibiotic of P. [60]. aeruginosa , like pyoverdine and elastase, without co-administration of an antibiotic [60].

Figure 7. Compound SM23 (72), an inhibitor of class C β-lactamases and of biofilms formation. Figure 7. Compound SM23 (72), an inhibitor of class C β‐lactamases and of biofilms formation. The synthesis of 72 was accomplished using the same approach of compounds 70 and 71 The synthesis of 72 was accomplished using the same approach of compounds 70 and 71 (Scheme 15). (Scheme 15). To obtain a β-lactamase inhibitor that could act on a larger spectrum and also to achieve oral To obtain a β‐lactamase inhibitor that could act on a larger spectrum and also to achieve oral administration, a cyclic boronic acid (75) was synthetized. Biological activity studies showed that administration, a cyclic boronic acid (75) was synthetized. Biological activity studies showed that compound 75 has high inhibitory activity against the β-lactamases serine enzyme of classes A, C, compound 75 has high inhibitory activity against the β‐lactamases serine enzyme of classes A, C, and and D, and also against the metalloenzymes [66]. The fact that one single compound can have potent D, and also against the metalloenzymes [66]. The fact that one single compound can have potent activity against serine β-lactamases and metalloenzyme is very attractive (minimum inhibitory activity against serine β‐lactamases and metalloenzyme is very attractive (minimum inhibitory concentrations (MICs) of compound 75 when combined with commercially available β-lactam concentrations (MICs) of compound 75 when combined with commercially available β‐lactam antibiotics are represented in Table5). In serine enzymes, the covalent binding of the boron with the antibiotics are represented in Table 5). In serine enzymes, the covalent binding of the boron with the catalytic serine residue is done in a “slow-binding” kinetics, while in the metalloenzymes covalent catalytic serine residue is done in a “slow‐binding” kinetics, while in the metalloenzymes covalent bond occurs between the boron and the water molecule of the enzyme and in a “fast-on, fast-off” type bond occurs between the boron and the water molecule of the enzyme and in a “fast‐on, fast‐off” type of kinetics. Other interesting characteristics that contribute for the attention given to this compound of kinetics. Other interesting characteristics that contribute for the attention given to this compound 75 are revealed by in vivo studies in rats, showing similar pharmacokinetic properties to β-lactam 75 are revealed by in vivo studies in rats, showing similar pharmacokinetic properties to β‐lactam antibiotics and a good oral bioavailability, which allows it not only to be administrated intravenously antibiotics and a good oral bioavailability, which allows it not only to be administrated intravenously but also orally. Besides, no toxic effects were shown [66]. but also orally. Besides, no toxic effects were shown [66]. Table 5. Minimum inhibitory concentration (MIC, µg/mL) of β-lactam antibiotics alone and when Table 5. Minimum inhibitory concentration (MIC, μg/mL) of β‐lactam antibiotics alone and when combined with compound 75. combined with compound 75. CPM 1 CPM + 75 CTLZN 2 CTLZN + 75 MP 3 MP + 35 CPM 1 CPM + 75 CTLZN 2 CTLZN + 75 MP 3 MP + 35 32 0.06 32 0.125 1 0.015 32 ≤≤ 0.06 32 0.125 1 ≤0.015 ≤ 1 2 3 1 CPM:CPM: cefepime; cefepime; 2 CTLZN:CTLZN: ceftolozane; MP:3 MP: meropenem. meropenem.

To achieveachieve the the synthesis synthesis of compound of compound75 (Scheme 75 (Scheme 16), the key16), stepsthe thatkey neededsteps that to be needed accomplished to be were:accomplished Boc protection were: Boc of phenol protection73, deprotonation, of phenol 73, deprotonation, then acylation then of the acylation aromatic of ring, the Heckaromatic reaction, ring, brominationHeck reaction, (represented bromination in molecule(represented74), borylationin molecule catalyzed 74), borylation by palladium, catalyzed palladium-catalyzed by palladium, cyclopropanation,palladium‐catalyzed and cyclopropanation, finally hydrolysis and of protecting finally hydrolysis groups [ of66 ].protecting groups [66]. In addition toto overexpressionoverexpression of ofβ β‐-lactamaseslactamases in in bacteria, bacteria, another another known known resistance resistance mechanism mechanism is relatedis related to to effl effluxux pumps, pumps, thus thus the the discovery discovery and and design design of novelof novel antibacterial antibacterial drugs drugs that that could could act act by inhibitingby inhibiting the actionthe action of these of these pumps pumps is an excitingis an exciting approach approach [67,68]. [67,68]. It has been It has recognized been recognized that boronic that acidboronic derivatives acid derivatives can be promising can be inhibitorspromising ofinhibitors chromosomal of chromosomal NorA efflux pumpNorA ofeffluxStaphylococcus pump of aureusStaphylococcus. These pumps aureus are. These involved pumps in bacterial are involved resistance in to thebacterial antibacterial resistance fluoroquinolones, to the antibacterial specially tofluoroquinolones, hydrophobic fluoroquinolones specially to hydrophobic such as ciprofloxacin fluoroquinolones [67]. Synthetic such as inhibitors ciprofloxacin of effl [67].ux pumps Synthetic are usuallyinhibitors based of efflux on heterocyclic pumps are compounds usually andbased in pumpon heterocyclic efflux inhibition compounds assays oftenand in use pump the alkaloid efflux reserpineinhibition as assays a positive often control, use the but alkaloid this alkaloid reserpine is not as useda positive in therapy control, due but to neurotoxicity this alkaloid atis therapeuticnot used in concentrationstherapy due to (minimum neurotoxicity modulatory at therapeutic concentration concentrations (MMC) of (minimum 4–8 µg/mL). modulatory Due to the formationconcentration of a stable(MMC) transition of 4–8 μg/mL). complexes Due withto the sugars formation and amino of a stable acids, transition heterocyclic complexes boronic with acid sugars compounds and amino were acids, heterocyclic boronic acid compounds were tested for the inhibition of NorA efflux pump, and

Molecules 2020, 25, 4323 24 of 40 Molecules 2020, 25, x FOR PEER REVIEW 25 of 43 compoundstested for the 6 inhibition‐(3‐phenylpropoxy)pyridine of NorA efflux pump,‐3‐ andboronic compounds acid (76 6-(3-phenylpropoxy)pyridine-3-boronic) and 6‐(4‐phenylbutoxy)pyridine‐3‐ boronicacid (76 )acid and (77 6-(4-phenylbutoxy)pyridine-3-boronic) proved to be efficient. A SAR study proved acid (77 that—(i)) proved the to bebest effi resultscient. were A SAR obtained study provedwhen boronic that—(i) acid the bestis at results the para were position; obtained (ii) when boron boronic atom acid is isessential, at the para since position; the replacement (ii) boron atom by carbonylatedis essential, since and the hydroxymethyl replacement by analogues carbonylated did not and show hydroxymethyl any activity; analogues (iii) esterification did not show of anythe boronicactivity; acid (iii) esterification leads to a decrease of the boronic in activity; acid (iv) leads an to aryl a decrease in C‐6 position in activity; retains (iv) an inhibitory aryl in C-6 activity. position It wasretains also inhibitory shown that activity. these compounds It was also ( shown76 and that77) have these no compounds intrinsic antibacterial (76 and 77 activity) have no and intrinsic can be usedantibacterial to potentiate activity the and activity can be usedof the to quinolone potentiate antibiotics the activity ciprofloxacin of the quinolone and antibiotics norfloxacin ciprofloxacin (Table 6), promotingand norfloxacin the accumulation (Table6), promoting of ethidium the accumulation bromide (a NorA of ethidium pump fluorescent bromide (a NorAsubstrate), pump and fluorescent showing nosubstrate), undesirable and cytotoxicity showing no due undesirable to the selectivity cytotoxicity for bacterial due to the efflux selectivity pumps for(no bacterialeffect on human efflux pumps efflux (nopump eff ectP‐glycoprotein) on human effl [67].ux pump P-glycoprotein) [67].

Scheme 16. SynthesisSynthesis of of the the cyclic cyclic boronic acid 75, inhibitor of serine β‐β-lactamaseslactamases and metallo enzymes. Reaction conditions: (i) Boc O, DMAP, CH Cl , r.t., 30 min; (ii) (1) Lithium diisopropylamide enzymes. Reaction conditions: (i)2 Boc2O, DMAP,2 2 CH2Cl2, r.t., 30 min; (ii) (1) Lithium (LDA), THF, 78 C, 1 h; (2) Boc O, DMAP, CH Cl , r.t., overnight; (iii) (1) TFA, CH Cl , r.t., 16 h; diisopropylamide− ◦ (LDA), THF, −278 °C, 1 h; (2) Boc2 22O, DMAP, CH2Cl2, r.t., overnight;2 (iii2) (1) TFA, (2) acetone, TFA, triﬂuoroacetic anhydride (TFAA), 70 C, overnight; (iv) acrylic acid, Pd(OAc) , CH2Cl2, r.t., 16 h; (2) acetone, TFA, trifluoroacetic anhydride◦ (TFAA), 70 °C, overnight; (iv) acrylic2 tris(o-tolyl)phosphine (P(o-toly) ), TEA, DMF, 100 C, 14 h; (v) (1) Br , CHCl , 0 C, 2 h; (2) TEA, DMF, acid, Pd(OAc)2, tris(o‐tolyl)phosphine3 (P(o‐toly)3),◦ TEA, DMF, 100 °C,2 14 h; 3(v) ◦(1) Br2, CHCl3, 0 °C, 2 0 C to r.t., 8 h; (vi)B ((+)pinanediol) , PdCl (dppf), KOAc, dioxane, 60 C, 2 h; (vii) CH N , Pd(OAc) , h;◦ (2) TEA, DMF, 0 °C2 to r.t., 8 h; (vi)2 B2((+)pinanediol)2 2, PdCl2(dppf), KOAc,◦ dioxane, 602 °C,2 2 h; (vii2) THF, 20 ◦C to r.t., 12 h; (viii) (1) column chromatography; (2) 3 N NaOH, dioxane, r.t., 30 min; (3) TFA, CH2N−2, Pd(OAc)2, THF, −20 °C to r.t., 12 h; (viii) (1) column chromatography; (2) 3 N NaOH, dioxane, triethylsilane (TES), i-BuB(OH)2, 0 ◦C to r.t., 30min. r.t., 30 min; (3) TFA, triethylsilane (TES), i‐BuB(OH)2, 0 °C to r.t., 30min.

Table 6.6. Minimum modulatory concentration (MMC, μµg/mL)g/mL) of ciproﬂoxacinciprofloxacin and norﬂoxacinnorfloxacin when alone or combined with compounds 76 or 77..

CIPR 1 CIPRCIPR 1 CIPR+ 76 + 76 CIPRCIPR+ +77 77 NORNOR 2 2NOR + NOR76 NOR+ 76 + 77 NOR + 77 1616 44 44 64 648 88 8 1 2 CIPR:1 CIPR: ciprofloxacin; ciproﬂoxacin; 2 NOR:NOR: norﬂoxacin. norfloxacin.

The syntheticsynthetic process process of of the the compounds compounds76 and76 and77 (Scheme 77 (Scheme 17) was 17) basedwas based on the on halogen–lithium the halogen– lithiumexchange exchange reaction reaction with subsequent with subsequent boronation boronation [67]. [67]. Another interesting boronic acid derivative that proved to have antibacterial activity is the bis(indolyl)methane boronic acid derivative 80. This compound was discovered in a study with the purpose of testing a series of bis(indolyl)methane compounds that could be used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA), a type of infection that often occurs in hospitals, and that is diﬃcult to treat. In that study [69], a set of bis(indolyl)methane derivatives were synthetized (Scheme 18) where only the substituent attached to the aryl or indole moiety diﬀered. It was discovered that when boronic acid was attached to the aryl moiety, the activity against MRSA was observed (MIC of 7.81 µg/mL), and no toxicity was revealed. Docking analysis also predicted that the action of

Molecules 2020, 25, x FOR PEER REVIEW 25 of 43

compounds 6‐(3‐phenylpropoxy)pyridine‐3‐boronic acid (76) and 6‐(4‐phenylbutoxy)pyridine‐3‐ boronic acid (77) proved to be efficient. A SAR study proved that—(i) the best results were obtained when boronic acid is at the para position; (ii) boron atom is essential, since the replacement by carbonylated and hydroxymethyl analogues did not show any activity; (iii) esterification of the boronic acid leads to a decrease in activity; (iv) an aryl in C‐6 position retains inhibitory activity. It was also shown that these compounds (76 and 77) have no intrinsic antibacterial activity and can be used to potentiate the activity of the quinolone antibiotics ciprofloxacin and norfloxacin (Table 6), promoting the accumulation of ethidium bromide (a NorA pump fluorescent substrate), and showing no undesirable cytotoxicity due to the selectivity for bacterial efflux pumps (no effect on human efflux pump P‐glycoprotein) [67].

Scheme 16. Synthesis of the cyclic boronic acid 75, inhibitor of serine β‐lactamases and metallo enzymes. Reaction conditions: (i) Boc2O, DMAP, CH2Cl2, r.t., 30 min; (ii) (1) Lithium

diisopropylamide (LDA), THF, −78 °C, 1 h; (2) Boc2O, DMAP, CH2Cl2, r.t., overnight; (iii) (1) TFA,

CH2Cl2, r.t., 16 h; (2) acetone, TFA, trifluoroacetic anhydride (TFAA), 70 °C, overnight; (iv) acrylic acid, Pd(OAc)2, tris(o‐tolyl)phosphine (P(o‐toly)3), TEA, DMF, 100 °C, 14 h; (v) (1) Br2, CHCl3, 0 °C, 2

h; (2) TEA, DMF, 0 °C to r.t., 8 h; (vi) B2((+)pinanediol)2, PdCl2(dppf), KOAc, dioxane, 60 °C, 2 h; (vii)

CH2N2, Pd(OAc)2, THF, −20 °C to r.t., 12 h; (viii) (1) column chromatography; (2) 3 N NaOH, dioxane,

r.t., 30 min; (3) TFA, triethylsilane (TES), i‐BuB(OH)2, 0 °C to r.t., 30min.

Table 6. Minimum modulatory concentration (MMC, μg/mL) of ciprofloxacin and norfloxacin when alone or combined with compounds 76 or 77. Molecules 2020, 25, 4323 25 of 40 Molecules 2020, 25, x FORCIPR PEER 1 REVIEWCIPR + 76 CIPR + 77 NOR 2 NOR + 76 NOR + 77 26 of 43 16 4 4 64 8 8 Scheme 17. Synthesis of NorA efflux pump boronic acid inhibitors compounds 76 and 77. Reaction bis(indolyl)methane boronic acid1 CIPR: derivative ciprofloxacin; could 2 be NOR: related norfloxacin. to binding to leucyl-tRNA synthetase, conditions: (i) (1) NaH, anhydrous THF, 0 °C to r.t., 30 min then reflux, 1 h; (2) 5‐bromo‐2‐ an enzyme that plays an important role in bacterial protein synthesis. It was also revealed that the fluoropyridine, reflux, 12 h; (ii) (1) n‐BuLi, anhydrous ether, −78 °C, 1 h; (2) B(OiPr)3, −78 °C to r.t., 1 The synthetic process of the compounds 76 and 77 (Scheme 17) was based on the halogen– actionh; of(3) this3 N HCl. derivative 80 might be bactericidal and that it is more specific against Gram positive bacteria,lithium exchange which might reaction be related with subsequent to the peptidoglycan boronation layer [67]. [ 69]. Another interesting boronic acid derivative that proved to have antibacterial activity is the bis(indolyl)methane boronic acid derivative 80. This compound was discovered in a study with the purpose of testing a series of bis(indolyl)methane compounds that could be used for the treatment of methicillin‐resistant Staphylococcus aureus (MRSA), a type of infection that often occurs in hospitals, and that is difficult to treat. In that study [69], a set of bis(indolyl)methane derivatives were synthetized (Scheme 18) where only the substituent attached to the aryl or indole moiety differed. It was discovered that when boronic acid was attached to the aryl moiety, the activity against MRSA was observed (MIC of 7.81 μg/mL), and no toxicity was revealed. Docking analysis also predicted that the action of bis(indolyl)methane boronic acid derivative could be related to binding to leucyl ‐ tRNAScheme synthetase, 17. Synthesis an enzyme of NorA that e fflplaysux pump an important boronic acid role inhibitors in bacterial compounds protein76 synthesis.and 77. Reaction It was also revealed conditions: that the (i) action (1) NaH, of anhydrous this derivative THF, 0 ◦80C tomight r.t., 30 be min bactericidal then reflux, and 1 h; (2) that 5-bromo-2-fluoropyridine, it is more specific against reflux, 12 h; (ii) (1) n-BuLi, anhydrous ether, 78 C, 1 h; (2) B(OiPr) , 78 C to r.t., 1 h; (3) 3 N HCl. Gram positive bacteria, which might be related− to◦ the peptidoglycan3 − layer◦ [69].

Scheme 18. SynthesisSynthesis of of the the bis(indolyl)methane bis(indolyl)methane boronic acid derivative 80, a leucyl leucyl-tRNA‐tRNA synthetase inhibitor. Reaction conditions: (i) Fe(ox)–Fe O ,H O, reﬂux. inhibitor. Reaction conditions: (i) Fe(ox)–Fe3O44, H2O,2 reflux.

The synthesis of compound 80 was mediated through a Fe(ox)–Fe3O4 promoted condensation The synthesis of compound 80 was mediated through a Fe(ox)–Fe3O4 promoted condensation reaction, reacting aldehyde with a boronic acid (78) substituent with indoles (79) in water [69]. reaction, reacting aldehyde with a boronic acid (78) substituent with indoles (79) in water [69]. 2.3. Antiviral Activity 2.3. Antiviral Activity The capacity of boronic acids to bind to carbohydrates allows their use for the treatment the virus withThe high capacity glycosylated of boronic envelopes, acids such to bind as the to humancarbohydrates immunodeficiency allows their virus use (HIV)for the [7 treatment]. Once again, the theirvirus physicochemicalwith high glycosylated characteristics envelopes, proved such to as be the associated human immunodeficiency with this biological virus application. (HIV) [7]. Once again,One their of physicochemical the diseases considered characteristics a universal proved health to be problem associated is the with acquired this biological immune application. deficiency syndromeOne of (AIDS), the diseases and it isconsidered caused by a the universal human immunodeficiencyhealth problem is virusthe acquired type 1 (HIV-1) immune [70 deficiency]. The Rev responsesyndrome element (AIDS), (RRE) and it is is an caused RNA by sequence the human of HIV-1 immunodeficiency with an important virus role type in the1 (HIV proliferation‐1) [70]. The of thisRev response virus. Through element interaction (RRE) is an with RNA Rev sequence protein, of RREHIV‐ allows1 with an virus important RNA replication role in the contributingproliferation toof this its life virus. cycle. Through Therefore, interaction this Rev-RRE with Rev interaction protein, RRE is considered allows virus as RNA a promising replication drug contributing target for HIVto its treatment life cycle. [Therefore,70,71]. Branched this Rev peptides‐RRE interaction (BPs) are is a goodconsidered targeting as a strategy, promising since drug they target form for strong HIV interactionstreatment [70,71]. with the Branched transactivation peptides response (BPs) are element a good (TAR) targeting of HIV, strategy, that is related since they to the form virus strong RNA, andinteractions also proved with tothe have transactivation no cytotoxicity response associated, element good (TAR) cell of permeability, HIV, that is related and can to bindthe virus to TAR RNA, at submicromolarand also proved levels. to have Based no cytotoxicity on the fact that associated, boronic acidsgood formcell permeability, reversible covalent and can bonds bind with to TAR Lewis at submicromolar levels. Based on the fact that boronic acids form reversible covalent bonds with Lewis bases and that this kind of bases are present in RNA of HIV, for instance 20-and 30-hydroxyl groups, boronicbases and acid that moiety this kind was introducedof bases are to present these BPs in [RNA70]. Thus, of HIV, this for introduction instance 2′‐ couldand 3 enhance′‐hydroxyl the groups, affinity ofboronic branched acid peptide moiety boronic was introduced acids (BPBAs) to these towards BPs RNA. [70]. AThus, series this of BPBAsintroduction were synthesized could enhance through the splitaffinity and of pool branched synthesis, peptide and it wasboronic proved acids that (BPBAs) these compounds towards RNA. increase A theseries affi nityof towardsBPBAs were RRE synthesized through split and pool synthesis, and it was proved that these compounds increase the

Molecules 2020, 25, 4323 26 of 40 Molecules 2020, 25, x FOR PEER REVIEW 27 of 43 and,affinity by towards binding toRRE this and, RNA by sequence, binding to the this interaction RNA sequence, Rev-RRE the is interaction disrupted, Rev consequently‐RRE is disrupted, leading toconsequently inhibition of leading viral replicationto inhibition [70 of, 71viral]. SAR replication studies [70,71]. revealed SAR that studies when revealed boronic that acid when moiety boronic was removed,acid moiety a decrease was removed, in affinity a decrease was shown in affinity and also was that shown the acidity and ofalso boron that is the important acidity forof boron binding is aimportantffinity, since for thebinding introduction affinity, ofsince an electron-withdrawingthe introduction of an group electron placed‐withdrawing in ortho thegroup boronic placed acid in increasedortho the boronic the Lewis acid acidity increased of boron the Lewis and subsequently, acidity of boron their and binding subsequently, affinity their [70]. binding An increase affinity in acidity[70]. An of increase boronic in acids acidity is of associated boronic acids with is the associated increase with of electrophilicity the increase of and electrophilicity thus can form and more thus stablecan form complexes more stable [5,70 complexes]. The best [5,70]. compound The best was compound BPBA3 (81 ,was Figure BPBA38), which (81, Figure showed 8), a which great cellularshowed uptakea great cellular and inhibition uptake ofand p24 inhibition capsid proteinof p24 capsid of HIV protein and HIV of HIV replication and HIVin replication vitro (IC inof vitro5 µ(ICM).50 50 ≈ Theof ≈ binding5 μM). The of this binding compound of this to compound RRE suggests to RRE a conformational suggests a conformational change of the tertiary change structure of the tertiary of the RNA,structure which of mightthe RNA, be more which predisposed might be tomore be cleaved predisposed by RNases, to be the cleaved enzymes by responsibleRNases, the for enzymes defense mechanismresponsible tofor RNA defense virus mechanism [71]. to RNA virus [71].

Figure 8. BPBA compound (81), disruptor of Rev-RRE interaction and inhibitor of HIV-1 replication. Figure 8. BPBA compound (81), disruptor of Rev‐RRE interaction and inhibitor of HIV‐1 replication. Another strategy for HIV treatment is through the design of HIV-1 protease inhibitors. The HIV-1 Another strategy for HIV treatment is through the design of HIV‐1 protease inhibitors. The HIV‐ protease is also critical in viral replication, so inhibitors of this enzyme are a good approach for 1 protease is also critical in viral replication, so inhibitors of this enzyme are a good approach for developing HIV antiviral drugs. The drug darunavir is known to be a HIV-1 protease inhibitor and its developing HIV antiviral drugs. The drug darunavir is known to be a HIV‐1 protease inhibitor and mechanism of action consists in targeting the S2 enzymatic subsite, forming hydrogen bonds with the its mechanism of action consists in targeting the0 S2′ enzymatic subsite, forming hydrogen bonds with amides present in Asp30 [72,73]. An ideal protease inhibitor must have the ability to interact with three the amides present in Asp30 [72,73]. An ideal protease inhibitor must have the ability to interact with local sides; these include the main chain and side chain of Asp30 and a molecule of water coordinated three local sides; these include the main chain and side chain of Asp30 and a molecule of water to the main chain of Gly48. Based on the knowledge that boronic acids can form strong hydrogen bonds coordinated to the main chain of Gly48. Based on the knowledge that boronic acids can form strong and these bonds are essential for this inhibitory activity, a 4-sulfonylphenylboronic acid (compound 84) hydrogen bonds and these bonds are essential for this inhibitory activity, a 4‐sulfonylphenylboronic was designed through replacement of the 4-sulfonylaniline of darunavir, and the effects of this acid (compound 84) was designed through replacement of the 4‐sulfonylaniline of darunavir, and substitution were analyzed [72]. It was observed that the boronic acid 84 acts as a competitive inhibitor the effects of this substitution were analyzed [72]. It was observed that the boronic acid 84 acts as a of HIV-1 protease and that the affinity towards the target was 20-fold greater than darunavir (darunavir, competitive inhibitor of HIV‐1 protease and that the affinity towards the target was 20‐fold greater Ki of 10 pM; boronic acid derivative 84,Ki of 0.5 pM). X-Ray crystallography studies proved that the than darunavir (darunavir, Ki of 10 pM; boronic acid derivative 84, Ki of 0.5 pM). X‐Ray boronic acid moiety interacts with all three locals referenced above, thus contributing to a significantly crystallography studies proved that the boronic acid moiety interacts with all three locals referenced better affinity. Besides these proprieties, this boronic acid (84) is non-toxic to human cells and, unlike above, thus contributing to a significantly better affinity. Besides these proprieties, this boronic acid aniline moiety in darunavir, has no related genotoxicity as a result of in vivo metabolic activation, (84) is non‐toxic to human cells and, unlike aniline moiety in darunavir, has no related genotoxicity since that the product of metabolic phase I activation in boronic acids is an alcohol, later metabolized as a result of in vivo metabolic activation, since that the product of metabolic phase I activation in in phase II in order to be eliminated from the body [72]. boronic acids is an alcohol, later metabolized in phase II in order to be eliminated from the body [72]. The synthesis of 84 (Scheme 19) was accomplished by a palladium-catalyzed coupling The synthesis of 84 (Scheme 19) was accomplished by a palladium‐catalyzed coupling reaction. reaction. The initial step corresponds to a coupling reaction between the starting material 82 and The initial step corresponds to a coupling reaction between the starting material 82 and 4‐ 4-bromobenzenesulfonyl chloride, followed by a palladium-coupling reaction with B2pin2 and bromobenzenesulfonyl chloride, followed by a palladium‐coupling reaction with B2pin2 and further further addition of 2,5-dioxopyrrolidin-1-yl((3R,3S,6R)-hexahydrofuro[2,3b]furan-3-yl) carbonate. addition of 2,5‐dioxopyrrolidin‐1‐yl((3R,3S,6R)‐hexahydrofuro[2,3b]furan‐3‐yl) carbonate. The The hydrolysis of intermediate 83 leads to the final product 84 [72]. hydrolysis of intermediate 83 leads to the final product 84 [72].

Molecules 2020, 25, 4323 27 of 40 Molecules 2020, 25, x FOR PEER REVIEW 28 of 43

Scheme 19.19. SynthesisSynthesis of of HIV HIV-1‐1 protease inhibitorinhibitor 8484 basedbased onon thethe drugdrug darunavir. darunavir. ReactionReaction

conditions:conditions: ( (ii)) TEA, TEA, 4 4-bromobenzenesulfonyl‐bromobenzenesulfonyl chloride, chloride, CH CH2Cl2Cl2, 2dry, dry N2 Natmosphere,2 atmosphere, 0 °C, 0 ◦16C, h; 16 (ii h;) ii iii (KOAc,) KOAc, B2pin B2,pin Pd(dppf)Cl2, Pd(dppf)Cl2–CH22Cl–CH2, 1,42Cl‐dioxane,2, 1,4-dioxane, 80 °C, 24 80 h;◦C, (iii 24) (1) h; HCl, ( ) (1) dioxane, HCl, dioxane, r.t., 4 h; (2) r.t., TEA, 4 h; (2)2,5‐ TEA,dioxopyrrolidin 2,5-dioxopyrrolidin-1-yl‐1‐yl ((3R,3S,6R) ((3R,3S,6R)-hexahydrofuro[2,3b]furan-3-yl)‐hexahydrofuro[2,3b]furan‐3‐yl) carbonate, carbonate, r.t., 16 r.t.,h; 16(iv h;) (iv) acetone/H O, NaIO , NH OAc, r.t., 12 h. acetone/H2O, NaIO2 4, NH4 4OAc,4 r.t., 12 h. The specific binding between influenza A virus (IAV) glycoprotein hemagglutinin (HA) and The specific binding between influenza A virus (IAV) glycoprotein hemagglutinin (HA) and the the glycan receptor of the host is crucial for the infection and transmission of this virus in humans. glycan receptor of the host is crucial for the infection and transmission of this virus in humans. This This virus uses as receptors for endocytosis the glycans that are present in cell surface and also virus uses as receptors for endocytosis the glycans that are present in cell surface and also possesses possesses glycan residues to interact with host cells. Since boronic acids can form stable complexes with glycan residues to interact with host cells. Since boronic acids can form stable complexes with saccharides, compounds in which boronic acids were added to quinolones, were designed (87–92) with saccharides, compounds in which boronic acids were added to quinolones, were designed (87–92) the purpose of disrupting the binding of the virus to glycans in host cells [74]. The boronic acid moiety with the purpose of disrupting the binding of the virus to glycans in host cells [74]. The boronic acid was introduced in quinolones because these derivatives are known to interact with DNA and also to moiety was introduced in quinolones because these derivatives are known to interact with DNA and have anti-viral activities [74,75]. This study suggests that the boronic acid is important to interfere with also to have anti‐viral activities [74,75]. This study suggests that the boronic acid is important to the importation of ribonucleoprotein (RNP) of IAV, complexes that are important for DNA replication interfere with the importation of ribonucleoprotein (RNP) of IAV, complexes that are important for of this virus, and delay the virus access into the nucleus of host cells. These compounds, especially DNA replication of this virus, and delay the virus access into the nucleus of host cells. These compound 88, proved to inhibit mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B compounds, especially compound 88, proved to inhibit mitogen‐activated protein kinase (MAPK) (NF-κB) signalling pathways—it is thought that MAPK and NF-κB can be involved in IAV replication and nuclear factor‐kappa B (NF‐κB) signalling pathways—it is thought that MAPK and NF‐κB can and viral RNA (vRNA) synthesis, respectively. SAR studies demonstrated that the boronic acid is be involved in IAV replication and viral RNA (vRNA) synthesis, respectively. SAR studies critical for anti-IAV activity of these quinolone derivatives and, moreover, it enhances the anti-IAV demonstrated that the boronic acid is critical for anti‐IAV activity of these quinolone derivatives and, activity, both in vitro and in vivo assays (in vitro: 88, IC50 of 2.5 µM; oseltamivir, IC50 of 9.5 µM). moreover, it enhances the anti‐IAV activity, both in vitro and in vivo assays (in vitro: 88, IC50 of 2.5 Another interesting fact is that compound 88 also revealed an inhibitory activity in other virus strains. μM; oseltamivir, IC50 of 9.5 μM). Another interesting fact is that compound 88 also revealed an Moreover, rats treated with this compound displayed a higher survival rate for IAV than the rats inhibitory activity in other virus strains. Moreover, rats treated with this compound displayed a treated with the commercially available drug oseltamivir [74]. higher survival rate for IAV than the rats treated with the commercially available drug oseltamivir For the synthesis of these compounds (Scheme 20), the first step involved a substitution reaction [74]. of 11-chloroquindoline derivative 85 with an aromatic diamine (represented in molecule 86), followed For the synthesis of these compounds (Scheme 20), the first step involved a substitution reaction by amidation with 3- or 4-carboxyphenylboronic acid [74]. of 11‐chloroquindoline derivative 85 with an aromatic diamine (represented in molecule 86), followed by amidation with 3‐ or 4‐carboxyphenylboronic acid [74].

Molecules 2020, 25, 4323 28 of 40 Molecules 2020, 25, x FOR PEER REVIEW 29 of 43 Molecules 2020, 25, x FOR PEER REVIEW 29 of 43

Scheme 20.20. SynthesisSynthesis of of boronic acid derivatives 8787––9292 asas IAVIAV replicationreplication inhibitors.inhibitors. ReactionReaction conditions: ( i) Aromatic diamine, HCl, 2 2-ethoxyethanol,‐ethoxyethanol, 100 ◦°C,C, 2 h; ( ii) 4 4-carboxyphenylboronic‐carboxyphenylboronic acid or 3-carboxyphenylboronic3‐carboxyphenylboronic acid, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholiniumacid, 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methyl‐morpholinium chloride (DMT-MM),chloride (DMT 2-ethoxyethanol,‐MM), 2‐ethoxyethanol, r.t., 18 h. r.t., 18 h.

Hepatitis CC virus virus (HCV) (HCV) is responsibleis responsible for for chronic chronic HCV HCV infection, infection, which which affects affects millions millions of people of worldwide,people worldwide, leading toleading liver cirrhosis to liver and cirrhosis hepatocellular and hepatocellular carcinoma. carcinoma. The design ofThe already design existing of already class ofexisting antiviral class compounds of antiviral usedcompounds for HCV, used the for non-structural HCV, the non protein‐structural 5B (NS5B) protein polymerase 5B (NS5B) polymerase inhibitors, isinhibitors, complicated is complicated due to lack due of homology to lack of with homology the binding with sitesthe binding of different sites genotypes of different of HCVgenotypes and the of quickHCV and rise the of mutants quick rise that of mutants are resistant that toare HCV resistant drug to [ 76HCV]. The drug study [76]. of The the study introduction of the introduction of boronic acidsof boronic in NS5B acids inhibitors in NS5B wasinhibitors done towas investigate done to investigate if it could bringif it could advantages bring advantages for targeting for HCV targeting that areHCV resistant that are to resistant NS35. Boronic to NS35. acids Boronic are known acids are for known reversible for reversible covalent inhibition covalent inhibition of hydroxyl of proteases,hydroxyl andproteases, thus the and study thus of boronicthe study acid of compounds boronic acid as HCV compounds protease inhibitorsas HCV wasprotease accomplished inhibitors [22 was,76]. Inaccomplished the case of mutants [22,76]. resistant In the case to NS5Bof mutants polymerase resistant inhibitors, to NS5B thepolymerase introduction inhibitors, of boronic the acidintroduction group to thisof boronic class of acid antivirals group (to93 this, Figure class9) of revealed antivirals to be(93 essential, Figure 9) for revealed the inhibition to be essential of HCV RNA-dependentfor the inhibition RNAof HCV polymerase RNA‐dependent (93, half RNA maximal polymerase effective (93 concentration, half maximal (EC effective) of 3.2 concentration nM). This phenomenon (EC50) of 3.2 occurs nM). of HCV RNA‐dependent RNA polymerase (93, half maximal effective50 concentration (EC50) of 3.2 nM). inThis the phenomenon initial step ofoccurs RNA in polymerase the initial step replication of RNA cycle. polymerase It is thought replication that cycle. this happensIt is thought through that β stabilizationthis happens ofthrough-flap, stabilization a portion of of NS5B β‐flap, that a is portion important of NS5B in the that regulation is important of polymerase in the regulation initiation of andpolymerase binding initiation of template and RNA, binding in an of inactivated template RNA, state in [76 an]. inactivated state [76].

Figure 9. NS5B polymerase inhibitor for HCV treatment (93) and its metabolites (94 and 95). Figure 9. NS5B polymerase inhibitor for HCV treatment (93) and its metabolites (94 and 95).

Molecules 2020, 25, 4323 29 of 40

Compound 93 entered in clinical trials but, unfortunately, the formation of two metabolites (compoundsMolecules 202094, 25and, x FOR95 PEER, Figure REVIEW9) through an easy benzylic oxidation with significantly30 of higher 43 half-life timesCompound (t1/2(94 93) =entered5 h; t1 /2in(95 clinical) = 45 trials h) led but, to theunfortunately, discontinuation the formation of this clinicalof two trialmetabolites (present in Table(compounds1)[ 77,78]. The 94 and low 95 half-life, Figure time9) through value an in easy the parent benzylic compound oxidation with (93) significantly implies a high higher daily half dose‐ of this druglife times for e(tffi1/2cacy(94) = [ 578 h;]. t1/2 With(95) = the 45 goalh) led of to improving the discontinuation the pharmacokinetic of this clinical trial proprieties (present in to Table avoid the formation1) [77,78]. of these The low metabolites half‐life time and value to maintain in the parent the compound efficacy of (93 this) implies compound, a high daily a second dose generationof this of NS5Bdrug inhibitors for efficacy were [78]. designed With the by goal the of same improving research the group, pharmacokinetic in which aproprieties N-phenyl to derivative avoid the (100) revealedformation to be of the these most metabolites promising and one to maintain [78]. To the obtain efficacy this of this N-phenyl compound, benzoxaborole a second generation100, the of used strategyNS5B was inhibitors based were on the designed elimination by the ofsame the research benzylic group, carbon in which in compound a N‐phenyl93 derivative. Pharmacokinetic (100) revealed to be the most promising one [78]. To obtain this N‐phenyl benzoxaborole 100, the used studies showed that this derivative could be a good candidate for clinical trials, since: (i) Comparing to strategy was based on the elimination of the benzylic carbon in compound 93. Pharmacokinetic 93 compoundstudies showed, clearance that this improved derivative in could mice, be rats, a good dogs candidate and monkeys—lower for clinical trials, since: clearance (i) Comparing can correlate with theto compound elimination 93, ofclearance benzylic improved oxidation; in mice, (ii) no rats, inhibition dogs and of monkeys—lower cytochrome P450 clearance (CYP) can isoform correlate 2C9 was observed—inhibitionwith the elimination of thisof benzylic enzyme oxidation; could lead (ii) tono safety inhibition problems; of cytochrome (iii) no P450 formation (CYP) ofisoform the metabolite 2C9 94 eitherwas inobserved—inhibition vitro or in vivo [78 of]. this It is enzyme thought could that, lead instead to safety of benzylic problems; oxidation, (iii) no formation the benzoxabolore of the goesmetabolite through oxidative-deborylation 94 either in vitro or in [vivo78]. [78]. It is thought that, instead of benzylic oxidation, the Thebenzoxabolore synthesis goes of this through second oxidative generation‐deborylation NS5B [78]. inhibitor (100, Scheme 21) was done through The synthesis of this second generation NS5B inhibitor (100, Scheme 21) was done through nucleophilic aromatic substitution (SNAr) coupling of metabolite 94 with the nitro fluorobenzene substratenucleophilic (96). Thearomatic nitro substitution group of (S theNAr) intermediate coupling of metabolite97 suffered 94 awith reduction the nitro forming fluorobenzene aniline 98. substrate (96). The nitro group of the intermediate 97 suffered a reduction forming aniline 98. This This reaction was followed by a process of chlorination ortho-to aniline group and subsquent reaction was followed by a process of chlorination ortho‐to aniline group and subsquent Sandmeyer Sandmeyer reaction to change the NH of the aniline to a bromine. Compound 99 was produced reaction to change the NH2 of the aniline2 to a bromine. Compound 99 was produced through a throughreduction a reduction reaction reaction with further with protection further protection with chloromethyl with chloromethyl methyl ether methyl (MOM ether‐Cl). Finally, (MOM-Cl). Finally,compound compound 100 was100 synthetizedwas synthetized through through an acid‐ ancleavage acid-cleavage reaction [78]. reaction [78].

SchemeScheme 21. 21.Synthesis Synthesis of of NS5B NS5B inhibitorinhibitor second second generation generation derivative derivative 100. 100Reaction. Reaction conditions: conditions: (i) o (i) MethylMethyl 5-ﬂuoro-2-nitrobenzoate, 5‐fluoro‐2‐nitrobenzoate, Na Na2CO2CO3, DMF,3, DMF, 70 70C, ◦72C, 72h, ( h,ii) (iipalladium) palladium on carbon on carbon (Pd/C), (Pd /C), o THF/THF/EtOH,,EtOH„ H2, H r.t.,2, r.t., 24 24 h; h; (iii (iii)) (1) (1) DMF,DMF, 60 ◦C,C, 5 5min; min; (2) (2) MeCN, MeCN, HBr, HBr, NaNO NaNO2, H2O,2,H 0 °C,2O, 30 0 min;◦C, (3) 30 min; CuBr, 50 °C, 30 min; (4) MeOH, LiBH4/THF, −5 oC, 2 h; (5) THF, DIPEA, chloromethyl methyl ether (3) CuBr, 50 ◦C, 30 min; (4) MeOH, LiBH4/THF, 5 ◦C, 2 h; (5) THF, DIPEA, chloromethyl methyl ether (MOM‐Cl), 50 oC, 18 h; (iv) (1) B2Pin2, Pd(dppf)Cl− 2, 1,4‐dioxane, N2, 108 °C, 22 h; (2) THF, MeOH, HCl, (MOM-Cl), 50 ◦C, 18 h; (iv) (1) B2Pin2, Pd(dppf)Cl2, 1,4-dioxane, N2, 108 ◦C, 22 h; (2) THF, MeOH, HCl, 70 oC, 18 h. 70 ◦C, 18 h.

Molecules 2020, 25, 4323 30 of 40

Flavivirus is responsible for several types of infections like Zika, West Nile, and Dengue virus infections, being the inhibition of flaviviral proteases an interesting approach for the treatment of these infections, particularly, with enzyme inhibitors that could act by covalent binding. Therefore, a researchMolecules group 2020 analyzed, 25, x FOR thePEER e REVIEWffect of the boronic acid moiety in the inhibition of flaviviral31 of 43 proteases, and a series of dipeptide boronic acids (exemplified herein with compound 105) were synthetized [79]. A SAR study showedFlavivirus that is responsible the activity for several is related types to of boronic infections acids like Zika, present West at Nile, the and C-terminus Dengue virus and that the infections, being the inhibition of flaviviral proteases an interesting approach for the treatment of replacementthese of infections, boronic acidsparticularly, with with amides enzyme led inhibitors to almost that inactive could act compounds. by covalent binding. The Therefore, presence a of boronic acids led toresearch a 1000-fold group analyzed higher atheffi nityeffect towardsof the boronic flaviviral acid moiety proteases in the inhibition (Ki values of flaviviral of 105 proteases,in Table 7) and no cytotoxicityand was a series observed. of dipeptide It was boronic also acids proved (exemplified that the herein boronic with acidcompound105 forms 105) were a hydrogen synthetized bond with the oxygen[79]. of theA SAR active study site showed of Ser135 that the residue activity is of related the flaviviral to boronic acids protease present [79 at]. the C‐terminus and that the replacement of boronic acids with amides led to almost inactive compounds. The presence of boronic acids led to a 1000‐fold higher affinity towards flaviviral proteases (Ki values of 105 in µ TableTable 7) 7.andInhibitory no cytotoxicity activity was (K observed.i, M) of It compound was also proved105 in that different the boronic flaviviral acid proteases. 105 forms a hydrogen bond with the oxygen of the active site of Ser135 residue of the flaviviral protease [79]. Dengue Virus Protease West Nile Virus Protease Zika Virus Protease Table0.051 7. Inhibitory activity (Ki, μM) of compound 0.082 105 in different flaviviral proteases. 0.04 Dengue Virus Protease West Nile Virus Protease Zika Virus Protease 0.051 0.082 0.04 The approach to synthesize compound 105 (Scheme 22) starts with a reaction with N-methylmorpholineThe approach (NMM), to synthesize with subsequent compound coupling 105 (Scheme reaction, 22) starts leading with toa thereaction formation with N of‐ bromide intermediatemethylmorpholine101. This intermediate (NMM), with wassubsequent then coupling transformed reaction, into leading azide to the102 formationand, after of bromide hydrogenation, converted intointermediate Boc-protected 101. This guanineintermediate103 was. The then guanidine transformed group into azide formed 102 and, was after then hydrogenation, deprotected. The final converted into Boc‐protected guanine 103. The guanidine group formed was then deprotected. The step correspondsfinal step to corresponds the deprotection to the deprotection of the boronic of the boronic ester ester104 104using using TFA TFA [[79].79].

Scheme 22.SchemeSynthesis 22. Synthesis of the of ﬂaviviralthe flaviviral protease protease inhibitorinhibitor 105105 withwith boronic boronic acid group. acid group.Reaction Reaction conditions:conditions: (i) (1) (i NMM,) (1) NMM, isobutyl isobutyl chloroformatechloroformate (IBCF); (IBCF); (2) (R) (2)‐4‐bromo (R)-4-bromo-1-((3aS,4S,6S,7aR)-3a,‐1‐((3aS,4S,6S,7aR)‐3a,5,5‐ trimethylhexahydro‐4,6‐methanobenzo[d][1,3,2]dioxaborol‐2‐yl)butan‐1‐amine, CH2Cl2, DIPEA, 5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)butan-1-amine, CH2Cl2, DIPEA, THF, –15 °C to r.t., overnight; (ii) (1) NaN3, DMF, 100 °C, 1 h; (2) H2, Pd/C, MeOH, r.t., overnight; (iii) THF, 15 C to r.t., overnight; (ii) (1) NaN , DMF, 100 C, 1 h; (2) H , Pd/C, MeOH, r.t., overnight; − ◦ 3 ◦ 2 (iii) bis-Boc-pyrazole-1-carboxamidine, DMAP, MeOH, r.t., 48 h; (iv) TFA, CH2Cl2, HCl, r.t., overnight; (v) PhB(OH)2,H2O, diethyl ether, HCl (aq.), r.t., overnight. MoleculesMolecules2020, 202025, 4323, 25, x FOR PEER REVIEW 32 of 4331 of 40

bis‐Boc‐pyrazole‐1‐carboxamidine, DMAP, MeOH, r.t., 48 h; (iv) TFA, CH2Cl2, HCl, r.t., overnight; (v) 2.4. SensorsPhB(OH) and Delivery2, H2O, diethyl Systems ether, HCl (aq.), r.t., overnight. Although2.4. Sensors mostand Delivery of the Systems mentioned biological applications of boronic acid derivatives are related with theirAlthough role as enzyme most of inhibitors,the mentioned some biological boronic applications acid derivatives of boronic can beacid used derivatives as sensors are andrelated in drug deliverywith systems.their role as The enzyme ability inhibitors, of boronic some acids boronic to react acid reversibly derivatives andcan be covalently used as sensors with 1,2- and and in 1,3- diolsdrug allows delivery them systems. to be sensor The ability of carbohydrates, of boronic acids such to react as glucose, reversibly and and therefore covalently they with can 1,2‐ beand useful for monitoring1,3‐ diols allows diseases them suchto be sensor as diabetes of carbohydrates, [6,8]. This such sensing as glucose, is possible and therefore because they upon can bindingbe useful with saccharides,for monitoring a change diseases is absorption such as diabetes is observed. [6,8]. ChangesThis sensing in absorptionis possible because spectra upon can be binding related with to steric eﬀectssaccharides, due to the a saccharidechange is absorption interaction is observed. with boronic Changes acids in absorption or it can also spectra be related can be related to hydrogen to steric bond effects due to the saccharide interaction with boronic acids or it can also be related to hydrogen bond loss that occurs when boronic acids are converted to boronic esters upon interaction with glucose. loss that occurs when boronic acids are converted to boronic esters upon interaction with glucose. These changes in absorption can be measured by complexation between a boronic acid with a dye. These changes in absorption can be measured by complexation between a boronic acid with a dye. One exampleOne example is a poly(L- is a poly(L and‐ D-lysine)and D‐lysine) modiﬁed modified with phenylboronicwith phenylboronic acid ( 106acid, Figure(106, Figure 10), considered 10), as opticalconsidered sensor as [ optical6,10,80 sensor]. [6,10,80].

FigureFigure 10. Boronic10. Boronic acid acid derivatives derivatives as as sensors.sensors. 106106: Poly(: Poly(L-L‐ and and D‐lysine) D-lysine) with phenylboronic with phenylboronic acid as acid as aa glucose glucose sensor; sensor; 107107:: Boronic acid acid derivative derivative with with a fluorophore a ﬂuorophore for forsaccharide saccharide sensing; sensing; 108: 108: Poly(anilinePoly(aniline boronic boronic acid) acid) polymer polymer asas a dopamine dopamine sensor; sensor; 109109: Anthracene: Anthracene-based‐based diboronic diboronic acid, an acid, an oligosaccharideoligosaccharide sensor.sensor.

ThereThere are alsoare also some some fluorescence fluorescence quenching-based quenching‐based saccharides saccharides sensors sensors [6,10,80,81]. [6,10,80, 81Examples]. Examples of of this kindthis kind of sensors of sensors are are photoinduced photoinduced electron electron transfertransfer (PET) (PET) materials materials with with boronic boronic acid. acid. Polymers Polymers withwith boronic boronic acids acids and and a fluorophore,a fluorophore, for for example,example, an an amine amine (107 (107, Figure, Figure 10), 10 in), the in thepresence presence of of saccharides produce increased fluorescence, having a higher specificity for glucose. The interaction saccharides produce increased fluorescence, having a higher specificity for glucose. The interaction between this polymer and a saccharide decreases PET, leading increased fluorescence emission between this polymer and a saccharide decreases PET, leading increased fluorescence emission [6,10,80]. [6,10,80]. Although promising, this polymer presents some disadvantages: Poor water solubility, Althoughinterference promising, with external this polymer factors presents such as the some solvent, disadvantages: poor stability, Poor and water is easily solubility, decomposed interference by withlight external [80]. factors such as the solvent, poor stability, and is easily decomposed by light [80]. BesidesBesides sensing sensing saccharides, saccharides, boronic boronic acid acid polymers polymers can can alsoalso bebe usedused in patients with with diabetes diabetes for insulinfor delivery. insulin delivery. The polymers The polymers used are mainlyused are hydrogels mainly hydrogels that disintegrate that disintegrate or swell uponor swell hydrophilicity upon increasehydrophilicity in glucose increase presence, in glucose allowing presence, the release allowing of insulin the release [6]. of Poly(aniline insulin [6]. Poly(aniline boronic acid) boronic polymers (108,acid) Figure polymers 10) can (108 be, used Figure not 10) only can forbe used saccharide not only sensing for saccharide but also sensing in dopamine but also sensingin dopamine [6,10 ,80]. Variations in dopamine concentration are associated with some neurodegenerative diseases such as Parkinson’s disease, and the detection of the concentration of this neurotransmitter can be measured by the oxidation of dopamine to dopamine-o-quinone with electrodes. The use of boronic acids in this method provides a high selectivity of electrodes and therefore a more precise detection [6]. Molecules 2020, 25, x FOR PEER REVIEW 33 of 43

sensing [6,10,80]. Variations in dopamine concentration are associated with some neurodegenerative diseases such as Parkinson’s disease, and the detection of the concentration of this neurotransmitter can be measured by the oxidation of dopamine to dopamine‐o‐quinone with electrodes. The use of Moleculesboronic2020 acids, 25 ,in 4323 this method provides a high selectivity of electrodes and therefore a more precise32 of 40 detection [6]. Recently,Recently, a a sensor sensor withwith thethe abilityability to recognize Lewis oligosaccharides selectively waswas developed andand couldcould bebe usefuluseful inin thethe early detection andand diagnosis ofof cancer. This sensor is an anthracene-basedanthracene‐based diboronicdiboronic acidacid andand itit cancan recognizerecognize HEPG2HEPG2 cellscells ( (109109,, Figure Figure 10 10))[ [80].80].

3.3. Biological Applications of Other Boron ContainingContaining CompoundsCompounds BesidesBesides boronic boronic acids, acids, there there are otherare other boron boron containing containing compounds compounds with interesting with interesting and promising and therapeuticpromising applications.therapeutic applications. This section will This briefly section discuss will the briefly application discuss of boronthe application compounds, of such boron as diazaborines,compounds, such benzoxaboroles, as diazaborines, and boronicbenzoxaboroles, esters. and boronic esters. DiazaborinesDiazaborines werewere oneone ofof thethe firstfirst compoundscompounds withwith boronboron atomatom thatthat werewere investigatedinvestigated forfor theirtheir therapeutictherapeutic proprieties. Diazaborines Diazaborines were were identified identified as asantimicrobial antimicrobial agents agents by Gronowitz by Gronowitz et al. et that al. thatobserved observed that that these these compounds compounds share share some some similarities similarities with with the the antibiotic antibiotic nitrofurantoin nitrofurantoin [82,83]. [82,83]. TheseThese compoundscompounds are are mainly mainly eff ectiveeffective against against Gram-negative Gram‐negative bacteria bacteria like Escherichia like Escherichia coli; for instance,coli; for compoundinstance, compound 110 (Figure 110 11 (Figure) is potent 11) against is potent this against bacterium this bacterium (MIC of 1.25 (MICµg /ofmL) 1.25 [84 μ].g/mL) [84].

Figure 11. 110 111 Figure 11. Examples of bioactivebioactive diazaborines.diazaborines. 110,, compound compound with antibacterial activity; 111: Compound with anticancer activity. Compound with anticancer activity. In addition to this activity,diazaborines also proved to be eﬀective against breast cancer. Compound In addition to this activity, diazaborines also proved to be effective against breast cancer. 111 (Figure 11) mimics estradiol structure and proved to have an antiproliferative activity against Compound 111 (Figure 11) mimics estradiol structure and proved to have an antiproliferative activity human breast cancer cell line MCF-7, with an approximate IC50 value of 5 µmol/L[85]. against human breast cancer cell line MCF‐7, with an approximate IC50 value of 5 μmol/L [85]. Other well-known compounds containing boron and with interesting bioactivity are Other well‐known compounds containing boron and with interesting bioactivity are benzoxaboroles. These compounds are characterized by incorporating boron atom in a heterocycle that benzoxaboroles. These compounds are characterized by incorporating boron atom in a heterocycle is attached to an aromatic ring [80]. Since the discovery of the antifungal benzoxaborole drug tavaborole that is attached to an aromatic ring [80]. Since the discovery of the antifungal benzoxaborole drug (compound 112), the interest of these compounds in Medicinal Chemistry has been increasing [86–88]. tavaborole (compound 112), the interest of these compounds in Medicinal Chemistry has been In comparison to boronic acids, they are usually more soluble compounds [89]. increasing [86–88]. In comparison to boronic acids, they are usually more soluble compounds [89]. They are mainly known for their application as anti-infective and antiparasitic activities. In Table8, They are mainly known for their application as anti‐infective and antiparasitic activities. In Table itMolecules is summarized 2020, 25, x FOR some PEER of REVIEW the most recent reported activities of benzoxaborole compounds. 34 of 43 8, it is summarized some of the most recent reported activities of benzoxaborole compounds.

Table 8. Examples of knownTable biological 8. Examples activities of known of benzoxaborole biological activities compounds. of benzoxaborole compounds.

CompoundCompound Biological ActivityBiological References activity References

Drug for treatment of onychomycosis—leucyl-tRNADrug for treatment of synthetaseonychomycosis—leucyl[88,90] ‐tRNA synthetase inhibitor [88,90] inhibitor

Selective leucyl‐tRNA synthetase inhibitor for Mycobacterium tuberculosis (M. [91] tuberculosis, IC50 1 of 0.2 μM)

Selective inhibitor of human carbonic anhydrases (CAs) (Ki 2 of 69 ‐414 nM) [92]

Selective inhibitor of β‐CA (Cryptococcus neoformans, Ki of 78 nM; Candida glabrata, Ki [93] of 75 nM)

Molecules 2020, 25, x FOR PEER REVIEW 34 of 43 Molecules 2020,, 25,, x FOR PEER REVIEW 34 of 43

Table 8. Examples of known biological activities of benzoxaborole compounds. Table 8. Examples of known biological activities of benzoxaborole compounds. Compound Biological activity References Molecules 2020, 25, 4323 Compound Biological activity33 of 40 References

Drug for treatment of onychomycosis—leucyl‐tRNA synthetase inhibitor [88,90] Table 8. Cont.Drug for treatment of onychomycosis—leucyl‐tRNA synthetase inhibitorinhibitor [88,90]

Compound Biological Activity References

SelectiveSelective leucyl-tRNA leucyl synthetase‐tRNA synthetase inhibitor inhibitor for Mycobacterium tuberculosis (M. forSelectiveMycobacterium leucylleucyl‐ tuberculosistRNA synthetase(M. inhibitorinhibitor[91 ]for Mycobacterium tuberculosis (M. [91] 1 [91] 1 tuberculosis, IC50 1 of 0.2 μM) [91] tuberculosis, IC50 of 0.2 µtuberculosisM) ,, IC5050 1 of 0.2 μ μM)

Selective inhibitor of human carbonic 2 Selective inhibitor of human carbonic anhydrases[92] (CAs) (Ki 22 of 69 ‐414 nM) [92] anhydrasesSelective (CAs) inhibitorinhibitor (Ki 2 of of 69 human -414 nM) carbonic anhydrases (CAs) (Kii of 69 ‐ ‐414 nM) [92]

Selective inhibitor inhibitor of ofβ-CA β‐CA (Cryptococcus (Cryptococcus neoformans, Ki of 78 nM; Candida glabrata, Ki Selective inhibitorinhibitor of β‐ β‐CA (Cryptococcus neoformans,, Kii of 78 nM; Candida glabrata,, Kii [93] neoformans,Ki of 78 nM; Candida glabrata,Ki [93] [93] of 75 nM) of 75 nM) of 75 nM) Molecules 2020, 25, x FOR PEER REVIEW 35 of 43 MoleculesMolecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 3535 ofof 4343

Anti‐Plasmodium3 agent (P. Anti-Plasmodium agent (P. falciparumAntiAnti, ED‐‐Plasmodium90Plasmodium agentagent ((P.P. 3 of 0.85 mg/kg) - promising for the treatmentfalciparum, ED[9094 33 ]of 0.85 [94] falciparumfalciparum,, EDED9090 3 ofof 0.850.85 [94][94] of malariamg/kg) ‐ promising for the treatment of malaria mg/kg)mg/kg) ‐ ‐ promisingpromising forfor thethe treatmenttreatment ofof malariamalaria

Oral ectoparasiticide agent Oral ectoparasiticide agentOralOral ectoparasiticideectoparasiticide agentagent for Dermacentor variabilis forfor DermacentorDermacentor variabilisvariabilis (American dog ticks) and(American(American dogdog[ 95ticks)ticks)] andand [95][95] Ctenocephalides felis (cat Ctenocephalides felis (cat 4 CtenocephalidesCtenocephalides felisfelis (cat(cat ﬂeas) (EC50 of 96.6 µM) 4 fleas) (EC50 44 of 96.6 μM) fleas)fleas) (EC(EC5050 4 ofof 96.696.6 μ μM)M)

Topical agent for treatment Topical agent for treatmentTopicalTopical agentagent forfor treatmenttreatment of cutaneous leishmaniasisofof cutaneouscutaneous leishmaniasisleishmaniasis [96] [96][96] (Leishmania major, EC of(Leishmania 4.26 µM; major, EC50 of 4.26 μM; [96] 50 ((LeishmaniaLeishmania majormajor,, ECEC5050 ofof 4.264.26 μ μM;M; L.tropica, EC50 of 2.01 µM)L.tropica, EC50 of 2.01 μM) L.tropicaL.tropica,, ECEC5050 ofof 2.012.01 μ μM)M)

OHOH O SerineSerine β‐ β‐ NN OO BB Serine β- Serine β‐ H N B lactamase inhibitor (Class A, HH22NN OO lactamase inhibitor (Classlactamaselactamase A, inhibitorinhibitor (Class(Class A,A, 2 S O N SS IC50 < 0.005–0.026 µM; IC5050 ˂ 0.005–0.026 μΜ; NN ICIC50 ˂˂ 0.005–0.0260.005–0.026 μΜ μΜ;; OHOH µ [97][97] OH Class C, IC50 of 0.012–0.067ClassM; C, IC50 of 0.012–0.067[97] μM; [97] ClassClass C,C, ICIC5050 ofof 0.012–0.0670.012–0.067 μ μM;M; Class D, IC50 of 0.088–0.12 µM) of ClassClass D,D, ICIC5050 ofof 0.088–0.120.088–0.12 μ μM)M) ofof pathogenspathogens OO pathogensClass D, IC50 of 0.088–0.12 μM) of pathogens O 5 such as E. coli (MIC 5 of 0.25–1suchsuch asasµg E./E.ml) colicoli (MIC(MIC 55 ofof 0.25–10.25–1 μ μg/ml)g/ml) 119119 such as E. coli (MIC of 0.25–1 μg/ml) 119

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Table 8. Cont. Molecules 2020,, 25,, x FOR PEER REVIEW 36 of 43 Compound Biological Activity References

Antiproliferative and proapoptotic Antiproliferative and proapoptotic agent for ovarian cancer line cell A2780 [98] [98] agent for ovarian cancer line cell A2780 (IC50 of 14.8 μM) (IC50agentof 14.8 forµ M)ovarian cancer line cell A2780 (IC of 14.8 μM)

Topical anti-inﬂammatoryTopical anti‐inflammatory drugdrug for atopic for atopic dermatitis ‐ ‐ phosphodiesterase type 4 dermatitis—phosphodiesterase type 4 [99] [99] (PDE4) inhibitor promising (PDE4) inhibitor promising(PDE4) inhibitor promising for psoriasis for psoriasis

1 2 3 4 11 IC50: Half maximal inhibitory concentration; 22 Ki: Inhibitory constant; 3 3ED90: Minimum effective dose in 90% of patients; 4 EC50: Half maximal effective concentration; IC50:: Half Half maximal maximal inhibitory inhibitory concentration; concentration; KKi:i :Inhibitory Inhibitory constant; constant; EDED90: Minimum: Minimum effective eﬀective dose dose in in 90% 90% of of patients; EC : Half maximal effective concentration; 5 4 5 Moleculespatients; MIC: 2020 Minimum, 25EC, x50 FOR: Half inhibitoryinhibitory PEER maximal REVIEW concentration. eﬀective concentration; MIC: Minimum inhibitory concentration. 37 of 43

Besides boronic acids, some boronic esters also seem to be promising compounds in Medicinal Chemistry, especiallyespecially as anticancer (exemplified(exemplified with 122) and antibacterial agents (exemplified (exemplified with 123). Compound 122 (Figure 1212)) was discovered as aa selectiveselective proteasomeproteasome inhibitorinhibitor (ChT-L,(ChT‐L, ICIC5050 of 2.7 nM; HCT116, IC50 ofof 31.8 31.8 nM), nM), acting acting as as a growth inhibitor through induction of G2/M G2/M cell cycle arrest and, through through p53 p53 accumulation, accumulation, and and proved proved to to also also act act as asa pro a pro-apoptotic‐apoptotic compound compound [100]. [100 As]. Asantibacterial antibacterial agents, agents, compound compound 123 123 (Figure (Figure 12) 12 proved) proved to to be be effective effective against against E.E. coli coli typetype I I signal peptidase (EcLepB), an enzyme that produces a vast number of crucial proteins for survival virulence of this bacteria. Besides Besides inhibiting inhibiting this this enzyme (MIC of 4–16 μµgg/mL),/mL), this moleculemolecule demonstrateddemonstrated eeffectsffects on bacterial type I signal peptidase (SPase I) in other bacteria strains, such as P. aeruginosa (MIC of 32–64 μµgg/mL),/mL), KlebsiellaKlebsiella pneumoniaepneumoniae (MIC(MIC ofof 88 μµgg/mL),/mL), andand S.S. aureusaureus(MIC (MIC of of 2 2µ μgg/mL)/mL) [ [101].101].

Figure 12. BoronicBoronic esters esters as as promising promising compounds in Medicinal Chemistry. 122: Selective Selective proteasome inhibitor and and apoptotic apoptotic agent; agent; 123123: Antibacterial: Antibacterial agent agent against against E. coliE., coli P. ,aeruginosaP. aeruginosa, K. pneumoniae,, K. pneumoniae and, andS. aureusS. aureus. . 4. Conclusions 4. Conclusions Boron compounds in Medicinal Chemistry have been neglected until the discovery of bortezomib, Boron compounds in Medicinal Chemistry have been neglected until the discovery of a peptide boronic acid proteasome inhibitor drug with anticancer activity. After this drug, other boronic bortezomib, a peptide boronic acid proteasome inhibitor drug with anticancer activity. After this acid derivative drugs have been developed, for instance, ixazomib and vaborbactam. The lack of drug, other boronic acid derivative drugs have been developed, for instance, ixazomib and interest in these compounds was mainly due to the stigma that boron atom was associated with toxicity, vaborbactam. The lack of interest in these compounds was mainly due to the stigma that boron atom which has been proven to be a false concept. Due to the chemical characteristics of boronic acids was associated with toxicity, which has been proven to be a false concept. Due to the chemical and the subsequent ability to form reversible tetrahedral bonds with substrates such as enzymes, characteristics of boronic acids and the subsequent ability to form reversible tetrahedral bonds with saccharides, and nucleic acids, the use of this moiety in several compounds for therapeutic application substrates such as enzymes, saccharides, and nucleic acids, the use of this moiety in several compounds for therapeutic application has been arising. Throughout this review, it has been shown the diverse applications of boronic acids in therapy, being the most relevant as anticancer, antibacterial, and antiviral agents and as sensors or delivery systems, as resumed in Figure 13. From a Medicinal Chemist perspective, the following considerations can be taken into account: (i) Introduction of boronic acids can improve various proprieties, including pharmacokinetic characteristics, such as bioavailability and toxicity; (ii) all of these proprieties are due to the physicochemical characteristics of boronic acids; (iii) the synthetic approaches to obtain these compounds are well known and relatively easy to accomplish; (iv) although boronic acid is the most common studied group, other boron containing molecules also proved to have interesting applications. It is therefore important to continue the investigation of the capacities of boron containing compounds in Medicinal Chemistry, in order to improve already known compounds or to obtain new ones.

Molecules 2020, 25, 4323 35 of 40 has been arising. Throughout this review, it has been shown the diverse applications of boronic acids in therapy, being the most relevant as anticancer, antibacterial, and antiviral agents and as sensors or deliveryMolecules 2020 systems,, 25, x FOR as resumed PEER REVIEW in Figure 13. 38 of 43

FigureFigure 13.13. BiologicalBiological applicationsapplications andand respectiverespective activitiesactivities ofof boronic boronic acid acid derivatives. derivatives.

AuthorFrom Contributions: a Medicinal The Chemist manuscript perspective, was conceived the following by M.E.S.; considerations M.P.S. collected can be the taken primary into account:data and (i)compiled Introduction draft manuscripts. of boronic M.E.S. acids and can L.S. improve supervised various the development proprieties, of the including manuscript, pharmacokinetic and M.E.S., L.S., characteristics,and M.P. assisted such in data as interpretation, bioavailability manuscript and toxicity; evaluation, (ii) and all editing. of these All proprieties authors have are read due and to agreed the physicochemicalto the published version characteristics of the manuscript. of boronic acids; (iii) the synthetic approaches to obtain these compounds are well known and relatively easy to accomplish; (iv) although boronic acid is the Funding: This research was supported by national funds through FCT ‐ Foundation for Science and Technology mostwithin common the scope studied of UIDB/04423/2020, group, other UIDB/50006/2020, boron containing UIDP/04423/2020 molecules also (Group proved of Natural to have Products interesting and applications.Medicinal Chemistry), It is therefore and importantunder the project to continue PTDC/SAU the investigation‐PUB/28736/2017 of the (reference capacities POCI of boron‐01‐0145 containing‐FEDER‐ compounds028736), co‐financed in Medicinal by COMPETE Chemistry, 2020, Portugal in order 2020 to improve and the European already Union known through compounds the ERDF or and to obtainby FCT newthrough ones. national funds.

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