molecules Article Trimethoprim: An Old Antibacterial Drug as a Template to Search for New Targets. Synthesis, Biological Activity and Molecular Modeling Study of Novel Trimethoprim Analogs Agnieszka Wróbel 1,* , Dawid Maliszewski 1, Maciej Baradyn 2 and Danuta Drozdowska 1 1 Department of Organic Chemistry, Medical University of Bialystok, 15222 Bialystok, Poland; [email protected] (D.M.); [email protected] (D.D.) 2 Department of Physical Chemistry, University of Bialystok, Institute of Chemistry, 15245 Bialystok, Poland; [email protected] * Correspondence: [email protected]; Tel.: +50-253-3188 Received: 3 December 2019; Accepted: 24 December 2019; Published: 27 December 2019 Abstract: A new series of trimethoprim (TMP) analogs containing amide bonds (1–6) have been synthesized. Molecular docking, as well as dihydrofolate reductase (DHFR) inhibition assay were used to confirm their affinity to bind dihydrofolate reductase enzyme. Data from the ethidium displacement test showed their DNA-binding capacity. Tests confirming the possibility of DNA binding in a minor groove as well as determination of the association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2 and poly (dG-dC)2. Additionally, the mechanism of action of the new compounds was studied. In conclusion, some of our new analogs inhibited DHFR activity more strongly than TMP did, which confirms, that the addition of amide bonds into the analogs of TMP increases their affinity towards DHFR. Keywords: trimethoprim analogs; DNA-binding agents; dihydrofolate reductase; molecular docking 1. Introduction Folate metabolism has long been recognized as an important and attractive target for the development of therapeutic agents against bacterial, parasitic infections [1], and cancer therapy [2,3]. Dihydrofolate reductase (DHFR) is an essential enzyme, which catalyzes the reduction of dihydrofolate acid (7,8-dihydrofolate, DHF) to tetrahydrofolic acid (5,6,7,8-THF) using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor (Scheme1)[ 4–6]. The crucial role of DHFR is related to biosynthesis pathways of the thymidylate and purines, as well as several other amino acids like glycine, methionine, serine, and N-formyl-methionyl tRNA [7–9]. Inhibition of folate-metabolizing enzymes leads to an imbalance in the pathways involved in active synthesizing thymidylate, disrupts DNA replication, and eventually causes cell death [10]. Therefore, DHFR is a very good example of a well-established molecular target of new active compounds, which could be approved as antibacterial drugs and therapeutic agents against a variety of fatal disorders e.g., cancer [11,12]. Several classes of compounds have been explored for their potential antifolate activity; among the most outstanding are diaminopyrimidine [13,14], diaminoquinazolin [15], diaminopteridine [16], and also diaminotriazines [17]. Over the last decade, a lot of research projects have focused on the search for new compounds active against this enzyme, most often derivatives of methotrexate (MTX), which is confirmed to be an effective inhibitor by extensive literature in this field [18,19]. Molecules 2020, 25, 116; doi:10.3390/molecules25010116 www.mdpi.com/journal/molecules Molecules 2020, 25, 116 2 of 15 Molecules 2020, 25, x FOR PEER REVIEW 2 of 15 H COOH N N H2N CH2 CH N 2 N CH2NH CO NH CH OH COOH Dihydrofolate acid (7,8-dihydrofolate, DHF) NADPH + H+ DHFR NADP+ H COOH H2N N N CH2 CH N 2 N CH2NH CO NH CH H OH COOH Tetrahydrofolic acid (5,6,7,8-THF) Scheme 1. Distinct step in folate metabolism: tetrahydrofolate synthesis pathway [6]. Scheme 1. Distinct step in folate metabolism: tetrahydrofolate synthesis pathway [6]. Our last reviewOur presents last review the presents current state the current of knowledge state of on knowledge the modification on the modification of known DHFR of known DHFR inhibitors as anticancerinhibitors agents,as anticancer and shows agents, that multitargetand shows compounds that multitarget represent compounds a promising represent approach a promising for discoveringapproach new structures for discovering for anticancer new structures therapy for [ 20antica]. Moreover,ncer therapy numerous [20]. Moreover, quotations numerous from quotations literature showfrom the literature variety of show structures the variety bearing of 1,3-thiazolestructures bearing [21], 1,3,4-thiadiazole, 1,3-thiazole [21], or 1,3,4- 1,2,4-triazole thiadiazole, or 1,2,4- moiety in varioustriazole fused moiety heterocyclic in various systems fused [22 heterocyclic–24], as well assystem 1,3,5-triazines [22–24], [25 as– 28well] or as biguanide 1,3,5-triazine and [25–28] or dihydrotriazinebiguanide derivatives and [dihydrotriazine29]. derivatives [29]. In turn, theIn mostturn, successfulthe most successful inhibitor inhibitor against bacterialagainst bacterial DHFR isDHFR trimethoprim is trimethoprim (TMP) (TMP) [2,4- [2,4-diamino-5-(3,4,5-trimethoxybenzyl)diamino-5-(3,4,5-trimethoxybenzyl) pyrimidine], pyrimidine], which which is is a a synthetic, synthetic, broad-spectrum broad-spectrum antimicrobial antimicrobialagent agent [30]. [30]. It It is is mainly mainly used used in in the the treatment treatment of of urinary urinary tract tract infections, infections, both both alone alone and and in combination combination with aa sulfonamide sulfonamide (e.g., (e.g., sulfamethoxazole, sulfamethoxazole, sulfadiazine, sulfadiazine, sulfamoxole) sulfamoxole) [31]. This [31]. antibiotic This antibiotic is a is a pyrimidinepyrimidine antifolate antifolate drug, which drug, selectively which selectively inhibits the inhi bacterialbits the enzyme bacterial dihydrofolate enzyme dihydrofolate reductase reductase (DHFR). The(DHFR). mechanism The of mechanism this inhibition of this consists inhibition in preventing consists in the preventing conversion the of conversion DHF to an of active DHF to an active form, i.e., THFform, [3,32 i.e.,]. One THF of [3,32]. our reviews One of presents our reviews an extensive presents range an extensive of research range literature of research on the firstliterature on the and most recentfirst achievements and most recent in TMPachievements analogs asin DHFRTMP analogs inhibitors as DHFR and underlines inhibitors newand underlines directions innew directions developing andin developing modeling DHFR and modeling inhibitors DHFR [33]. Currently, inhibitors Pedrola[33]. Currently, et al. [34 ]Pedrola showed et group al. [34] of TMPshowed group of analogs displayTMP meaningful analogs display structural meaningful features of thestructural initial drugfeatures together of the with initial relevant drug modifications together with relevant at several points,modifications keeping antibioticat several potencypoints, keeping and showing antibiotic satisfactory potency antimicrobialand showing profilesatisfactory (good antimicrobial activity levelsprofile and reduced (good growthactivity rates), levels especially and reduced against growth methicillin-resistant rates), especiallyStaphylococcus against methicillin-resistant aureus (Figure1). TheStaphylococcus new products aureus may open(Figure new 1). possibilities The new products to fight bacterialmay open infections. new possibilities The literature to fight bacterial analysis confirmedinfections. that The there literature are only analysis few reports confirmed that would that confirmthere are the only biological few reports activity that of would TMP confirm the analogs targetingbiological anticancer activity properties. of TMP analogs Singh ettargeting al. [35] modifiedanticancer the properties. antibacterial Singh agent et al. TMP [35] to modified the compounds antibacterialA and B (Figure agent1) TMP with to promising compounds anticancer A and B applications. (Figure 1) with These promising two compounds anticancer applications. had significantThese tumor two growthcompounds inhibitory had significant activities tumor over 60 growth human inhibitory tumor cell activities lines and over exhibited 60 human tumor cell appreciable interactionslines and exhibited with DHFR appreciable [34]. Algul interactions et al. [36] havewith developedDHFR [34]. a Algul new nonclassical et al. [36] have series developed of a new propargyl-linkednonclassical DHFR inhibitors.series of Itpropargyl-linked was observed that DHFR interactions inhibitors. of propargyl-linked It was observed inhibitors that interactions of with Leu22, Thr56,propargyl-linked Ser59, Ile60 inhibitors could potently with Leu22, inhibit Thr56, human Ser59, DHFR Ile60 (hDHFR), could inpotently contrast inhibit to weak human DHFR inhibition of(hDHFR),DHFR byin TMP.contrast Based to weak on SARs inhibition (structure-activity of hDHFR by relationships), TMP. Based Algul on SARs et al. (structure-activity [36] reported thatrelationships), hydrophobic Algul substitutions et al. [36] at reported C6 and thethat propargylic hydrophobic position substitutions increased at C6 anticancer and the propargylic potency. Significantly,position increased propargyl-linked anticancer compound potency.C (FigureSignificantly,1) exhibited propargyl-linked 3500-fold greater compound potency C (Figure 1) than TMP [36exhibited]. 3500-fold greater potency than TMP [36]. Recent trends in medical chemistry suggest the developing of multitargeting and multifunctional compounds—in addition, it is a worldwide medical research strategy [37]. The term “designed multiple ligands” was coined by Morphy and Rankovic to describe the abovementioned Molecules 2020, 25, x FOR