Aminoalkyl Derivatives of Guanidine Diaromatic Minor GrooveBinders with Antiprotozoal ActivityCaitriona McKeever,† Marcel Kaiser,‡,§ and Isabel Rozas*,†
†School of Chemistry, Trinity Biomedical Sciences Institute, University of Dublin, Trinity College, 152-160 Pearse St., Dublin 2,Ireland‡Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland§University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
*S Supporting Information
ABSTRACT: Considering the strong DNA minor groove bindingobserved for our previous series of diaromatic symmetric and asymmetricguanidinium and 2-aminoimidazolinium derivatives, we report now thesynthesis of new aminoalkyl derivatives of diaromatic guanidines withpotential as DNA minor groove binders and antiprotozoal activity. Thepreparation of these aminoalkyl derivatives (12a−e, 13a−e, 14a−c,e,15a−e, 16a−e) is presented as well as their affinity for DNA which wasevaluated by means of DNA thermal denaturation experiments. Finally,the antiprotozoal activity of most of these aminoalkyl minor groovebinders was evaluated in vitro against Trypanosoma brucei rhodesiense (8compounds) and Plasmodium falciparum (18 compounds). The O-linkedderivatives 13c and 14c showed 100 nM activities against P. falciparum,whereas for T. b. rhodesiense all compounds tested showed micromolaractivity. Some of the derivatives prepared seem to exert the antimalarial activity by binding to the DNA minor groove whereasother sets of compounds could exert this antimalarial activity by inhibiting the parasite dihydrofolate reductase, for example.
■ INTRODUCTION
DNA is a key target for drugs aiming to interfere with normalcellular processes because of its involvement with how genesare expressed and how proteins are created. There are severalways in which drugs can target DNA, and one particular way isby binding in the minor groove of the double helix, which is thepreferred site of interaction between DNA and proteins.1 Thistype of DNA ligand has been shown to exhibit differentbiological activities.2 In particular, it has been found thatcompounds that target the minor groove can be used fortreating tropical diseases because they inhibit transcriptiondirectly or block the actions of DNA-dependent enzymes. Twowell-known examples of minor groove binders (MGBs) arepentamidine and furamidine (Figure 1). Furamidine is a keyaromatic diamidine active against Trypanosoma (T.) species invitro. Pentamidine, an aliphatic analogue of furamidine,3 isorally active and effective in the treatment of human Africantrypanosomiasis (sleeping sickness) which is caused bybloodstream infections with parasitic protozoans of thesubspecies T. brucei (T. b.) rhodesiense or T. b. gambiense.Recent studies show that bis-guanidine and bis-2-amino-imidazoline diphenyl derivatives, related to furamidine, displaypotent antitrypanosomal activity in vitro and in vivo against T.b. rhodesiense. In addition, a correlation between antitrypano-somal activity and DNA binding affinity has been observed,suggesting a possible mechanism of action for thesecompounds.4 The X-ray structure for the complex of
furamidine with the d(CGCGAATTCGCG)2 oligomer hasbeen obtained,5 displaying the snug fit of this compound withthe AATT sequence indicative of minor groove binding. Thiscomplex could be responsible for the inhibition of the microbialtopoisomerase enzyme leading to anti-Pneumocystis cariniiactivity.6 In addition, the X-ray crystal structure of one of thebis-2-aminoimidazoline derivatives has been published, showinga very similar binding mode to that of furamidine.7
A large amount of evidence indicating that DNA minorgroove binding is related to the antiprotozoal activity ofcompounds has been published;8−14 pentamidine has beenshown to linearize kinetoplast DNA from trypanosomes,15
which could play an important role in the compound’smechanism of action. Recently, some evidence has beenpublished of minor groove binders with good antiprotozoalactivity but poor DNA binding.16 Moreover, antimalarial drugscontaining guanidine-like groups such as proguanyl orpyrimethamine were prepared in the past as selective inhibitorsof the parasite dihydrofolate reductase thymidine synthetase(DHFR-TS).17−20 It is clear that further research in this area isrequired to deepen the understanding of the antiprotozoalmechanism of action of these derivatives.We have reported in the past the preparation of symmetric
and asymmetric diaromatic guanidinium/2-aminoimidazoli-
nium derivatives (Figure 1), studying the influence of the linker(X in Figure 1) and cations on minor groove binding by meansof DNA denaturation experiments with both random sequenceDNA (salmon sperm) and AT specific polynucleotides[poly(dA·dT)2 and poly(dA)·(dT)].4,21 In general, theincreases in DNA denaturation temperature (ΔTm) obtainedindicated strong binding to DNA, especially for thosecompounds with a NH or a CO group linking the phenylrings. Moreover, in a different article, we determined the modeof binding and binding constants of these compounds by anumber of biophysical techniques concluding that they stronglybind in the minor groove.22 In addition, we prepared mono-guanidinium-like derivatives, leaving one of the aromatic aminogroups unsubstituted and therefore yielding monocations.These compounds showed very poor affinity toward DNAprobably due to their monocationic nature.Taking this into account and exploiting the binding activity
of our MGBs, we intend to combine the aliphatic linker ofpentamidine and the diaromatic structure of our MGBs byattaching aminoalkyl chains to one of the aromatic rings. Thisaminoalkyl group will be protonated at physiological pH and,moreover, the aliphatic chain will help to displace the spine ofhydration present in the minor groove by hydrophobicinteractions thereby increasing the strength of binding.
Hence, our aim is to explore the influence that such anaminoalkyl chain could have in the minor groove binding andantiprotozoal activity of these diaromatic compounds. Thus, inthis article we present the synthesis, biophysical, andbiochemical study of a series of aminoalkyl diaromatic MGBconjugates (aminoalkyl-MGBs, see Scheme 1) using astemplates the compounds previously developed in ourlaboratory.
■ RESULTS AND DISCUSSION
Synthesis. The preparation of the aminoalkyl-MGBsproposed involves, first, the syntheses of the mono-guanidy-lated diaromatic systems, as previously described;8 this step isthen followed by Boc-protection of the amino terminus ofvarious amino acids of different lengths and, finally, theconjugation of both moieties by means of an amidefunctionality.Different aminoalkyl acids with three, four, seven, ten, and
eleven methylene groups (4-aminobutanoic, 5-aminopentanoic,8-aminooctanoic, 11-aminoundecanoic, and 12-aminododeca-noic acids) were considered. The amino acid must be protectedso that the amino group of the mono-guanidine MGB attacks atthe carbonyl terminus leading to a successful coupling reaction.Different Boc-protection conditions were explored23−25 and,
Figure 1. Furamidine, pentamidine, and symmetric (bis-guanidine, bis-2-aminoimidazoline) and asymmetric (guanidine/2-aminoimidazole)derivatives previously prepared in our laboratory.
Scheme 1. Synthesis of the Hydrochloride Salts of the Aminoalkyl-MGB Conjugates 12a−e, 13a−e, 14a−c,e, 15a−e, and 16a−e
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finally, methanol at 60 °C for 20 h in the presence oftriethylamine was utilized, yielding the corresponding com-pounds 1−5 (Scheme 1).The Boc-protected mono-guanidine diaromatic derivatives
6a−e (Scheme 1) were prepared (as previously published byus8,26) by reaction of the commercial diamines (in excess) with1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, trie-thylamine, and mercury(II) chloride in dichloromethane ordimethylformamide.To join the Boc-protected amino acid with the mono-
guanidine MGB derivatives, different coupling reactions wereexplored. Thus, we tried using the corresponding acid chlorideof the amino acid and the mono-guanidine in acetonitrile in thepresence of triethylamine,27 or coupling reagents such as N,N’-
dicyclohexylcarbodiimide28 or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU).29
This last method, which gave the best results, involveddissolving the Boc-protected amino acid, the Boc-protectedmono-guanidine, TBTU, and N,N-diisopropylethylamine(DIEA) in acetonitrile at room temperature under argon. Inthat way, we prepared in good yields of compounds 7a−e, 8a−e, 9a−e, 10a−e, and 11a−e (Scheme 1) which were fullycharacterized.Finally, different deprotection methods for the Boc-protected
intermediates were tested, and the most successful results wereobtained with 4 M hydrochloric acid/dioxane in 2-propanol(IPA)/dichloromethane, leading to the direct formation of thehydrochloride salts (Scheme 1). Further purification by reverse
Table 1. Overall (Total) Yields and Thermal Denaturation Experiments’ Resultsa of the Aminoalkyl-MGB Conjugates (12a−eto 16a−e) and the Corresponding Bis- and Mono-guanidinium Unconjugated MGBs (17a−e and 18a−e) with Salmon SpermDNA
aMelting temperature of salmon sperm DNA in phosphate buffer (10 mM) is 68 °C. Three measurements were carried out for each compound.bReference 31, calf thymus, phosphate buffer. cReference 32, calf thymus, MES buffer. dReference 33, poly(dA-dT) and calf thymus, Tris HCl buffer.eReference 34, poly(dA-dT), PIPES buffer.
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phase chromatography was required. The total overall yieldsobtained for each product in the coupling and deprotectionsteps are presented in Table 1.DNA Affinity. Biophysical studies were carried out to
determine the affinity of these aminoalkyl-MGB conjugatestoward DNA by means of DNA thermal denaturationexperiments, which provide an easy and effective method toscreen a large number of compounds. A solution of knownconcentration of unspecific wild type salmon sperm DNA (150μM, which gives an absorbance of 1 au) was used, and thechange in the thermal melting temperature (ΔTm) wasmonitored when adding the molecule to be tested at aknown concentration.Thermal denaturation results obtained for the aminoalkyl-
MGBs are presented in Table 1 and Figure 2. Previously, when
studying the unconjugated MGBs, the best results wereachieved for the symmetric bis-guanidinium derivatives (17a−e30 in Table 1); for that reason, the ΔTm values obtained forthese bis-guanidiniums and for the corresponding mono-guanidines (18a−e8) are also presented in Table 1 to allow abetter structural comparison with the present conjugates. Thiswill show the effect of the charged aminoalkyl chain on theaffinity of the diaromatic mono-guanidiniums. It will also showthe optimal length for these aminoalkyl chains in terms of DNAaffinity.In Table 1 it can be observed that the introduction of an
aminoalkyl chain, and in particular short chains, results inincreased Tm. In general, most of the aminoalkyl-MGBconjugates showed moderately increased binding to DNAwith ΔTm between 2 and 6. There are several differencesbetween the amino mono-guanidines and the aminoalkyl-MGBconjugates that may lead to these increases in the DNA affinityof the latter. First, the total length of the aminoalkyl-MGBmolecules is larger than that of the corresponding amino MGB
compounds (see computational results) which could lead to abetter fitting into the minor groove, occupying more space anddisplacing more of the water molecules within. Second, whereasthe amino MGBs are monocations at physiological pH, theaminoalkyl-MGB conjugates are dicationic, which favors theinteractions in the negatively charged environment of the minorgroove. Third, the aminoalkyl chain may better direct theammonium group to establish strong hydrogen bonds (HBs)and ionic interactions with the nucleic bases in the minorgroove of DNA.Looking at the nature of the X linker, NH-linked derivatives
gave the best results in general, but some of the O-, CO-, andCH2CH2-linked compounds (12b, 12d, 13c, 13e, and 14c)displayed very good binding also. The highest affinities (ΔTm =6) were achieved for compounds with O- and NH-linkers andan aminoalkyl chain of four methylene groups (n = 2,compounds 13c and 13e). Furthermore, compound 16e (n =9) shows a ΔTm = 5 probably due to the nature of the NHlinker. However, the corresponding amino MGB derivatives didnot show strong binding to DNA, indicating that theaminoalkyl chain is playing a favorable role in the interactionwith DNA.Regarding the aminoalkyl chain length, those compounds
with four methylene groups (n = 2, compounds 13a−e) gavethe best results and elongating the chain seemed to interferewith the binding except for those compounds with a NH groupas a linker (15e, n = 8, and 16e, n = 9). Compounds 13b, 13c,and 13d showed ΔTm increases of 5, 4, and 5 °C whencompared to the corresponding amino MGB derivatives,respectively. This could indicate that very short alkyl chainscannot contribute extra interactions with DNA to improve thebinding, whereas alkyl chains longer than four CH2 groups aretoo long or too lipophilic to achieve an optimal interaction.Compounds 12b (X = CH2CH2) and 12d (X = CO) showedthe strongest binding for the series where n = 1. This differencecan be attributed to the distance between the cations in 12bbeing larger than that of 12a.8 This could result in theformation of optimum HBs with the DNA base pairs. Also in12d the HB acceptor carbonyl linker could lead to strongerinteractions between the molecule and the minor groove.To have a general idea of the distance between the
guanidinium and the ammonium cations in these aminoalkyl-MGB conjugates, models for the X = NH derivatives with n = 1,2, 5, 8, and 9 aminoalkyl chains were optimized using densityfunctional theory (DFT) methods (B3LYP/6-31+G*,35 mim-icking water solvation with PCM36). Considering the highflexibility that the aminoalkyl chains exhibit, several con-formations would be possible; however, only that with theaminoalkyl chain completely extended (C−C−C−C angles =180°) was considered (see examples in Figure 3).The C(+)−N(+) distances recorded for the five conjugates
are presented in Table 2. As expected, in these “extended”conformations the C−N distance increases with the length of
Figure 2. Graph showing the DNA thermal denaturation results ofcompounds 12b (X = CH2CH2, n = 1), 13c (X = O, n = 2), and 14c(X = O, n = 5).
Figure 3. Examples of optimized “extended” conformations for compounds 13e and 14e at B3LYP/6-31+G* (PCM-water) level of computation(left and right, respectively).
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the aminoalkyl chain, and this is related to the ΔTm values withexception of compound 13e which shows a much better ΔTmthan expected from the chain length. It seems that the optimalintercationic distance for binding should be around 18−19 Å;however, because of the enormous flexibility of the long chainsand the hydrophobic favorable interactions that can beestablished within the minor groove by displacing the waterspine, it cannot be discarded that aminoalkyl chains with sevenmethylene groups could adopt a conformation that would resultin a good binding to DNA.Biological Results. Based on the DNA affinity results, the
activity of several of the aminoalkyl-MGBs was evaluated invitro against T. b. rhodesiense (STIB900 strain37) (compounds12b, 13b−e, 14c, 15e, 16e), and against P. falciparum (NF54strain38) and rat skeletal myoblasts (L639) cells (compounds12a−c,e, 13a−e, 14c, 15a−c,e and 16a,c−e). Viability of T. b.rhodesiense and L6 cells was assessed by cell-mediated reductionof resazurin. The 3H-hypoxanthine incorporation assay wasused to measure the in vitro antimalarial activity. All thecorresponding half-maximal inhibitory concentration (IC50)values obtained are displayed in Table 3.All aminoalkyl-MGBs tested display IC50 values against T. b.
rhodesiense in the micromolar range (Table 3). In general, uponlengthening the aliphatic chain, activity decreases with theexception of compound 16e which displays the bestantitrypanosomal activity IC50= 4.06 μM) of the series. Thiscould be due to the central NH linker that has good hydrogenbonding ability or to a fold of the long chain that results on amore efficient interaction with DNA. Unfortunately, thiscompound also displays some cytotoxicity on L6 cells and istherefore unselective (SI = 3). The compounds with CH2CH2linkers (12b and 13b) display good activity against T. b.rhodesiense (IC50 13.3 and 20.2 μM, respectively), and they arerelatively selective toward the parasite because of their high L6cell’s IC50 values (133 and 109 μM). The electron-withdrawingCO linker derivative (13d) showed the poorest activity againstT. b. rhodesiense; however, those compounds with electron-donating linkers such as NH (16e), CH2CH2 (12b and 13b),or O (13c) showed IC50 values in the low micromolar range.Compounds 13e and 15e displayed a poor IC50 value against
T. b. rhodesiense compared to the other NH-linked derivative(16e), and this decrease in activity could be due to the shorteraminoalkyl chains that orient the amino group to a less efficientinteraction with DNA. The isosteric replacement of the CH2linker by an O linker (compounds 12c and 14c) did not leadeither to an increased selectivity or an increased activity againstT. b. rhodesiense. Compounds 12b and 13b showed the bestselectivity (SI = 10 and 5.4, respectively) even though thesevalues are rather poor. Although none of these compoundsshow an antitrypanosomal activity comparable to that of thecontrol (melarsoprol, IC50 = 0.0055 μM), compounds 12b and13b could be the base for further improvements.
In the in vitro assays against P. falciparum, most of thecompounds showed good activity with IC50 values in the nano-and micromolar range. The results, displayed in Table 3, showthat, in general, lengthening the aminoalkyl chain does notcorrelate to IC50 values. Compounds 13c and 14c display thebest activity of the series with IC50 values of 0.106 and 0.149μM, respectively, and good to very good selectivity (SI = 316and 900, respectively). Compounds 13c and 14c could beconsidered as interesting “leads” and would be good candidatesfor in vivo testing. The rest of the derivatives displayedmoderate to poor activity against P. falciparum (IC50 valuesbetween 1 and 5) and poor SIs, and compounds 15b, 15e, 16e,and 12a showed the poorest activity.
Table 2. Intercationic [C(+)−N(+)] Distances (Å)Computed for Compounds 12e, 13e, 14e, 15e, and 16e atB3LYP/6-31+G* Level and PCM−Water, for the “Extended”Conformationa
Structure−Activity Relationships Analysis. It is im-portant to determine if there is any correlation between theantiprotozoal activity of all the compounds studied and theirDNA binding ability. In principle, the compounds that have thestrongest affinity for the minor groove should inhibit DNAreplication most effectively and elicit the strongest antitrypa-nosomal or antiplasmodial activity. Analysis of the IC50 valuesobtained and the thermal denaturation results (Tables 2 and 3)can provide some evidence for this hypothesis.The potential correlation between both antiprotozoal
activities (as measured by their IC50) and DNA binding affinity(as measured by their ΔTm) was explored, but no relation wasfound in the case of the T. b. rhodesiense activity. Differentfamilies of guanidine and 2-aminoimidazoline analoguemolecules (Figure 1), previously synthesized by us and byDardonville, were tested in vitro against T. b. rhodesiense and P.falciparum.4 These dicationic diphenyl compounds displayedgood antitripanosomal activities with IC50 values in the 100 nMrange. The main difference between these families ofcompounds is that the present aminoalkyl-MGBs possess notonly a guanidinium cation attached to an aromatic ring but alsoan ammonium cation attached to a flexible aliphatic chain ofdifferent lengths. The estimated pKa of these two differentcations is more or less similar, being around 9−11 for aromaticguanidine-like groups40 and around 10−11 for aliphatic amines(i.e., 10.53 for propylamine, or 10.59 for butylamine).41
Additionally, we had previously measured the pKa of themiddle NH group of 4,4′-bis(guanidino)diphenylamine (re-lated to compounds 12e−16e) to be 2.8.40 Hence, all thesemolecules will be dicationic at physiological pH. In thementioned previous work, we suggested that to obtainantitripanosomal activity in the 100 nM range, a dicationicmolecule was essential. The aminoalkyl-MGBs presented here,even though dicationic, only showed IC50 values within themicromolar range.When comparing the previously prepared series and the
present aminoalkyl-MGBs regarding their activity against P.falciparum, we observe that in the bis-guanidinium-likecompounds, the IC50 values were much poorer for P. falciparumthan for T. b. rhodesiense, whereas the aminoalkyl-MGBscompounds presented in this work exhibit 100 nM to lowmicromolar antimalarial activity. This seems to indicate that thepresence of cations of different nature (a guanidinium and analkylammonium cation) and the flexible aliphatic linker canfavor the antimalarial activity versus the antitrypanosomalactivity.The results obtained for these 18 aminoalkyl-MGBs show a
wide range of potencies against P. falciparum (Table 3).Looking at all the data, both in terms of antimalarial activity andDNA binding (Table 2), it seems that our compounds couldexert their activity by different mechanisms of action.First, we have been able to find a linear correlation between
antimalarial activity and DNA thermal melting denaturation forcompounds 12b, 13b, 13c, 13d, 13e, 15b, 15e, and 16e (ΔTm= −0.88 × IC50 + 6.49) with R2 = 0.87. This set of compoundsincludes a wide range of data, i.e., IC50 values ranging from0.106 (13c, the best) to 5.79 μM and ΔTm values from 6 to 1.Hence, this correlation seems to indicate that this particular setof compounds, with aliphatic chains between 3 and 10methylene groups and different linkers, exert their better orworse antimalarial activity by binding to the DNA minorgroove in the same way that many other related compoundshave been reported to do.8−14 In this set of compounds,
aminoalkyl chains with four methylene groups give the bestresults for binding to the DNA minor groove (compounds13a−e) and, in general, they also show good antimalarialactivity, mostly for linkers X = CH2CH2 and NH.Second, there is a different set of data showing compounds
with very interesting antimalarial activity that does not correlateto their poor or nil DNA binding. Thus, compounds 14c, 13a,and 12c show IC50 values of 0.149 (the second best), 0.589,and 0.880 μM, and selectivity values of 913, 179, and 197,respectively. These compounds with aminoalkyl chains betweenthree and seven methylene groups show structural similaritywith dihydrofolate and with some inhibitors of dihydrofolatereductase−thymidylate synthase (DHFR-TS) from P. falcipa-rum such as proguanil, and 1,6-dihydro-6,6-dimethyl-1-[3-(2,4,5-trichlorophenoxy)propoxy]-1,3,5-triazine-2,4-diamine(WR9921018).17−19 It could be possible that these threecompounds with tetrahedral linkers (X = O, CH2) and shortaminoalkyl chains exert their antimalarial activity by inhibitingthis particular enzymatic system. In any case, this hypothesiscan only be fully proved by performing further enzymaticstudies using DHFR-TS.We can conclude that, in general, aminoalkyl chains longer
than seven methylene groups are good neither for antimalarialactivity nor for DNA binding. Similarly, compounds with X =CO, which is a trigonal planar group, gave poor results in termsof antimalarial activity, despite being relatively good minorgroove binders when carrying an aminoalkyl chain of fourmethylene groups (13d). Regarding the shortest aminoalkylchain derivatives (n = 1, three methylene groups), they do notbind well to DNA except when X = CH2CH2 (longest linker);however, compound 12c (n = 1, X = O) shows an interestingantimalarial activity maybe because the tetrahedral nature of thelinker can facilitate the interaction within the active site ofenzymatic systems such as, for example, DHFR-TS. The case ofcompound 13c is an interesting one: its good antimalarialactivity correlates well with a good DNA binding; however, wehave found that, in our series, the tetrahedral linker X = Ocould be more associated to other mechanisms of action such asDHFR-TS inhibition. It could be the case that this compound,which is the best of the whole series, could exert its antimalarialactivity by DNA binding and an additional mechanism ofaction.Hence, for future series, aminoalkyl chains between four and
seven methylene groups should be used and the CO linkeravoided. Regarding antimalarial activity, compounds 13c and14c are really promising and in the future will be tested in vivoas recommended by the Swiss Tropical and Public HealthInstitute.
■ CONCLUSIONS
On the basis of the DNA binding results previously obtained byus,4,21,22 we have prepared a new series of aminoalkylderivatives of guanidine diaromatic MGBs to explore the effectof the aminoalkyl chain on the DNA binding activity of theMGB moiety. In total, 24 hydrochloride salts were obtained.The affinity of these salts toward DNA was evaluated by
means of thermal denaturation experiments using DNA ofunspecific sequence. The incorporation of aminoalkyl chainswith four to seven methylene groups resulted in significantincreases in the DNA melting temperature.The distance between cationic functionalities (guanidinium
and ammonium) has been screened by DFT calculations;
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however, no clear conclusion can be derived considering thehigh flexibility of the aminoalkyl chains.No correlation was found between the antitrypanosomal
activity and the DNA binding affinity of the compounds tested.However, in the case of the antimalarial activity, we found twodifferent behaviors. In one set (eight compounds) a goodactivity/DNA binding correlation is found; however, in asecond set (three compounds) no correlation is found eventhough a good antimalarial activity is observed, suggesting adifferent mechanism of action.The antiprotozoal activity and cytotoxicity of several of these
compounds was evaluated. Thus, for T. b. rhodesiense (8compounds tested), poor results were obtained. Thesederivatives proved to be more potent and selective toward P.falciparum (18 compounds tested) exhibiting IC50 within thelow micromolar to 100 nM range. Compounds 13c and 14cdisplayed good in vitro antimalarial activity and good selectivityindices and will be tested in vivo in the future.
■ EXPERIMENTAL SECTIONSynthesis. All the commercial chemicals were obtained from
Sigma-Aldrich or Fluka and were used without further purification.Deuterated solvents for NMR use were purchased from Apollo. Drysolvents were prepared using standard procedures, according to Vogel,with distillation prior to use. Chromatographic columns were runusing a Biotage SP4 flash purification system with Biotage SNAP silicacartridges. Solvents for synthesis purposes were used at GPR grade.Analytical TLC was performed using Merck Kieselgel 60 F254 silicagel plates or Polygram Alox N/UV254 aluminum oxide plates.Visualization was by UV light (254 nm). NMR spectra were recordedin a Bruker DPX-400 Avance spectrometer, operating at 400.13 and600.1 MHz for 1H NMR, and 100.6 and 150.9 MHz for 13C NMR.Shifts are referenced to the internal solvent signals. NMR data wereprocessed using Bruker Win-NMR 5.0 software. HRMS spectra weremeasured on a Micromass LCT electrospray TOF instrument with aWaters 2690 autosampler with methanol as carrier solvent. Meltingpoints were determined using a Stuart Scientific Melting Point SMP1apparatus and are uncorrected. Purity was assessed using reverse phaseHPLC with a diode-array detector scanning wavelengths from 200 to950 nm. HLPC analysis was carried out using a Varian ProStar systemequipped with a Varian Prostar 335 diode array detector and a manualinjector (20 μL). Integration was performed at 245 nm, and peakpurity was confirmed using a purity channel. The stationary phaseconsisted of an ACE 5 C18-AR column (150 × 4.6 mm). The methoddeveloped for this type of hydrochloride salt, which gave optimumretention times, used a gradient from 100% aqueous formate buffer(30 mM, pH 3.0) to 85% formate buffered methanol (30 mM, pH 3.0)and 15% aqueous formate buffer. A minimum purity of 95.0% was setfor compounds to be tested pharmacologically. All mono-guanidinederivatives used in this work were prepared as previously reported byus.8
General Methods. Step A Method: General Method for thePreparation of the Aminoalkyl Derivatives of Boc-ProtectedDiaromatic Mono-guanidines. A solution of the correspondingBoc-protected amino acid (1.2 mmol) in MeCN (10 mL) was treatedwith DIEA (3.8 mmol), the mono-guanidine (1 mmol), and TBTU(1.2 mmol) under inert atmosphere. The reaction mixture was stirredat room temperature for 18 h and partitioned between brine (4 mL)and EtOAc (10 mL).The organic layer was washed with 0.1 M HCl (2× 5 mL) and 5% NaHCO3 (2 × 5 mL), dried over anhydrous Na2SO4,filtered, and concentrated under vacuum. Purification by flashchromatography with silica gel eluting with hexane/EtOAc (2:1)yielded the required product.Step B Method: General Method for Boc Deprotection and
Preparation of Hydrochloride Salts. The Boc-protected amino acidconjugate of the mono-guanidine derivative (1 mmol) was dissolved in18 equiv of 4 M HCl/dioxane (4.5 mL) under argon to make up a 0.2M solution. The mixture was made up to 5 mL solution with IPA/
DCM (1:1) (0.5 mL) and stirred 4 h. The HCl/dioxane was thenremoved under vacuum. The residue was dissolved in H2O andwashed with DCM (3 × 5 mL). Concentration of the aqueous layerfollowed by reverse phase column chromatography eluting with H2O/acetonitrile yielded the required product.
Preparation of Hydrochloride Salts. Preparation of Dihydro-chloride Salt of N-(4-(4′-Guanidinobenzyl)phenyl)-4-aminobutana-mide (12a). Starting from 7a and following method B, 12a wasobtained as a yellow solid (87 mg, 46%). mp: 58−60 °C; 1H NMR(D2O): δ 1.87−1.94 (m, 2H, CH2CH2CH2), 2.41 (t, J = 7.0 Hz, 2H,CH2CO), 2.96 (t, J = 8.0 Hz, 2H, CH2NH), 3.82 (s, 2H, PhCH2Ph),7.07 (d, J = 7.5 Hz, 2H, Ar), 7.14 (d, J = 7.5 Hz, 2H, Ar), 7.18−7.24(m, 4H, Ar); 13C NMR (D2O): δ 22.3, 32.5, 38.3, 39.6, 121.5, 125.6,128.8, 129.6, 131.5, 134.4, 138.0, 141.1, 155.8, 172.9; HRMS (EI) m/z[M + H]+ calcd for C18H24N5O: 326.1981, found: 326.1973; puritydata obtained by HPLC: retention time 22.21 min (95.4% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenylethyl)phenyl)-4-aminobutanamide (12b). Startingfrom 7b and following method B, 12b was obtained as a yellow oil (55mg, 39%); 1H NMR (D2O): δ 1.87−1.94 (m, 2H, CH2CH2CH2), 2.42(t, J = 8.0 Hz, 2H, CH2CO), 2.84 (s, 4H, Ph(CH2)2Ph), 2.95 (t, J =8.0 Hz, 2H, CH2NH), 7.07 (d, J = 7.0 Hz, 2H, Ar), 7.11 (d, J = 8.0 Hz,2H, Ar), 7.16−7.20 (m, 4H, Ar); 13C NMR (D2O): δ 30.1, 32.9, 35.8,35.9, 38.7, 121.8, 125.8, 129.2, 129.9, 131.6, 134.4, 138.9, 141.7, 156.3,173.3; HRMS (EI) m/z [M + H]+ calcd for C19H26N5O: 340.2137,found: 340.2140; purity data obtained by HPLC: retention time 23.20min (95.4% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenyl ether)-4-aminobutanamide (12c). Startingfrom 7c and following method B, 12c was obtained as a brown solid(153.5 mg, 85%). mp: 98−100 °C; 1H NMR (D2O): δ 1.89−1.97 (m,2H, CH2CH2CH2), 2.45 (t, J = 8.0 Hz, 2H, CH2CO), 2.98 (t, J = 8.0Hz, 2H, CH2NH), 6.98−7.01 (m, 4H, Ar), 7.21 (d, J = 8.5 Hz, 2H,Ar), 7.33 (d, J = 8.5 Hz, 2H, Ar); 13C NMR (D2O): δ 22.7, 32.8, 38.7,119.4, 119.7, 123.8, 124.6 128.0, 129.1, 132.6, 153.5, 156.5, 173.5;HRMS (EI) m/z [M + H]+ calcd for C17H22N5O2: 328.1774, found:328.1760; purity data obtained by HPLC: retention time 21.85 min(95.2% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylmethanone)-4-aminobutanamide (12d).Starting from 7d and following method B, 12d was obtained as ayellow solid (53.5 mg, 99%). mp: 138−140 °C; 1H NMR (D2O): δ1.46−1.52 (m, 2H, CH2CH2CH2), 2.25 (t, J = 8.0 Hz, 2H, CH2CO),
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm301614w | J. Med. Chem. 2013, 56, 700−711706
2.85 (t, J = 8.0 Hz, 2H, CH2NH), 7.33−7.38 (m, 4H, Ar), 7.74−7.79(m, 4H, Ar); 13C NMR (D2O): δ 29.7, 30.0, 38.3, 119.7, 123.7, 129.8,131.3, 131.4, 131.8, 132.1, 138.5, 155.4, 176.5, 197.3; HRMS (EI) m/z[M + H]+ calcd for C18H22N5O2: 340.1774, found: 340.1781; puritydata obtained by HPLC: retention time 19.68 min (98.4% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylamine)-4-aminobutanamide (12e). Start-ing from 7e and following method B, 12e was obtained as a black solid(134.1 mg, 41%). mp: 38−40 °C; 1H NMR (D2O): δ 1.82 (t, J = 7.5Hz, 2H, CH2CH2CH2), 2.39 (t, J = 8.0 Hz, 2H, CH2CO), 2.92 (t, J =7.5 Hz, 2H, CH2NH), 7.11 (d, J = 6.0 Hz, 6H, Ar), 7.20 (d, J = 8.0 Hz,2H, Ar); 13C NMR (D2O): δ 21.9, 30.5, 38.6, 118.7, 119.2, 122.6,124.0, 127.3, 127.6, 142.1, 143.3, 156.5, 176.8; HRMS (EI) m/z [M +H]+ calcd for C17H23N6O: 327.1933, found: 327.1940; purity dataobtained by HPLC: retention time 8.19 min (95.1% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylmethylene)-4-aminopentanamide (13a).Starting from 8a and following method B, 13a was obtained as ayellow solid (92.7 mg 88%). mp: 135−137 °C; 1H NMR (D2O): δ1.62 (t, J = 4.0 Hz, 4H, CH2(CH2)2CH2), 2.35 (t, J = 8.0 Hz,2H,CH2CO), 2.91 (t, J = 8.0 Hz, 2H, CH2NH), 3.89 (s, 2H,PhCH2Ph), 7.13 (d, J = 8.0 Hz, 2H, Ar), 7.19 (d, J = 8.5 Hz, 2H, Ar),7.22−7.26 (m, 4H, Ar); 13C NMR (D2O): δ 22.0, 26.1, 35.5, 39.1,40.1, 122.4, 126.3, 129.4, 130.2, 132.1, 134.9, 138.7, 141.7, 156.4,174.9; HRMS (EI) m/z [M + H]+ calcd for C19H26N5O: 340.2137,found: 340.2146; purity data obtained by HPLC: retention time 22.60min (98.7% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
calcd for C20H27N5O: 354.2292, found: 354.2294; purity data obtainedby HPLC: retention time 23.59 min (98.1% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenyl ether)-4-aminopentanamide (13c). Start-ing from 8c and following method B, 13c was obtained as a brownsolid (69.7 mg, 43%). mp: 52−55 °C; 1H NMR (D2O): δ 1.64 (t, J =3.5 Hz, 4H, CH2CH2(CH2)2), 2.37 (t, J = 6.5 Hz, 2H, CH2CO), 2.92(t, J = 8.0 Hz, 2H, CH2NH), 6.98−7.01 (m, 4H, Ar), 7.21 (d, J = 8.0Hz, 2H, Ar), 7.32 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (D2O): δ 22.0,26.1, 35.4, 39.0, 119.4, 119.6, 123.5, 127.8, 129.0, 132.8, 153.1, 156.3,156.4, 174.5; HRMS (EI) m/z [M + H]+ calcd for C18H24N5O2:342.1930, found: 342.1924; purity data obtained by HPLC: retentiontime 21.84 min (95.4% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylmethanone)-4-aminopentanamide (13d).Starting from 8d and following method B, 13d was obtained as ayellow solid (42.5 mg, 44%). mp: 40−42 °C; 1H NMR (D2O): δ 1.59(s, 4H, CH2(CH2)2CH2), 2.34 (t, J = 8.0 Hz, 2H, CH2CO), 2.91 (t, J= 8.0 Hz, 2H, CH2NH), 7.20 (d, J = 8.0 Hz, 2H, Ar), 7.39 (d, J = 8.0Hz, 2H, Ar), 7.47 (d, J = 8.0 Hz, 2H, Ar), 7.52 (d, J = 8.0 Hz, 2H, Ar);13C NMR (D2O): δ 21.4, 25.4, 35.8, 38.3, 119.6, 122.5, 131.2, 131.3,134.5, 136.9, 138.6, 141.6, 155.3, 174.3, 196.9; HRMS (EI) m/z [M +H]+ calcd for C19H24N5O2: 354.1930, found: 354.1944; purity dataobtained by HPLC: retention time 21.33 min (95.5% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylamine)-4-aminopentanamide (13e). Start-ing from 8e and following method B, 13e was obtained as a black solid(10.7 mg, 4%). mp: 75−77 °C; 1H NMR (D2O): δ 1.64 (s, 4H,CH2(CH2)2CH2), 2.39 (t, J = 4.0 Hz, 2H, CH2CO), 2.97 (t, J = 4.5Hz, 2H, CH2NH), 7.17−7.22 (m, 6H, Ar), 7.28 (d, J = 5.6 Hz, 2H,Ar); 13C NMR (D2O): δ 20.9, 26.0, 33.1, 39.0, 118.4, 118.9, 124.0,126.9, 127.3, 127.9, 142.6, 143.7, 155.6, 178.6; HRMS (EI) m/z [M +H]+ calcd for C18H25N6O: 341.2090, found: 341.2095; purity dataobtained by HPLC: retention time 18.73 min (97.6% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylmethylene)-4-aminooctanamide (14a).Starting from 9a and following method B, 14a was obtained as ayellow oil (183.3 mg, 96%); 1H NMR (D2O): δ 1.05 (s, 6H,(CH2)2(CH2)3(CH2)2), 1.36 (s, 4H, CH2CH2(CH2)2CH2CH2CH2),2.07 (t, J = 7.3 Hz, 2H, CH2CO), 2.71 (t, J = 7.0 Hz, 2H, CH2NH),3.50 (s, 2H, PhCH2Ph), 6.82 (d, J = 7.5 Hz, 4H, Ar), 6.90 (d, J = 7.5Hz, 2H, Ar), 7.10 (d, J = 7.5 Hz, 2H, Ar); 13C NMR (D2O): δ 25.2,25.4, 26.6, 27.9, 28.0, 36.4, 39.4, 40.0, 121.2, 125.5, 129.1, 130.0, 131.8,135.3, 137.6, 141.5, 155.9, 175.1; HRMS (EI) m/z [M + H]+ calcd forC22H32N5O: 382.2607, found: 382.2601; purity data obtained byHPLC: retention time 23.24 min (95.7% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylethylene)-4-aminooctanamide (14b).Starting from 9b and following method B, 14b was obtained as ayellow solid (45.9 mg, 97%). mp: 143−145 °C; 1H NMR (D2O): δ1 . 18 ( s , 6H , (CH2) 2 (CH 2 ) 3 (CH2 ) 2 ) , 1 . 4 7 ( s , 4H ,CH2CH2(CH2)3CH2CH2), 2.19 (t, J = 7.0 Hz, 2H, CH2CO), 2.63(s, 4H, Ph(CH2)2Ph), 2.82 (t, J = 7.5 Hz, 2H, CH2NH), 6.90−6.94(m, 4H, Ar) 7.00 (d, J = 7.5 Hz, 2H, Ar), 7.17 (d, J = 8.0 Hz, 2H, Ar);13C NMR (D2O): δ 25.3, 25.6, 26.5, 28.3, 28.7, 28.9, 33.4, 36.4, 39.0,118.7, 119.1, 121.3, 126.5, 128.3, 133.9, 151.6, 155.5, 156.0, 173.3;HRMS (EI) m/z [M + H]+ calcd for C23H34N5O: 396.2763, found:396.2768; purity data obtained by HPLC: retention time 28.12 min(98.4% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenyl ether)-4-aminooctanamide (14c). Startingfrom 9c and following method B, 14c was obtained as a brown solid(158.9 mg, 53%). mp: 50−52 °C; 1H NMR (D2O): δ 1.26 (s, 6H,(CH2) 2 (CH 2 ) 3 (CH2) 2 ) , 1 . 5 5 ( t , J = 8 . 0 Hz , 4H ,CH2CH2(CH2)3CH2CH2), 2.30 (t, J = 7.0 Hz, 2H, CH2CO), 2.86(t, J = 7.5 Hz, 2H, CH2NH), 6.98−7.02 (m, 4H, Ar), 7.21 (d, J = 8.5Hz, 2H, Ar), 7.30 (d, J = 8.5 Hz, 2H, Ar); 13C NMR (D2O): δ 25.2,26.5, 27.5, 27.7, 27.8, 36.1, 39.3, 119.4, 119.6, 124.0, 125.3, 128.0,129.1, 132.7, 153.4, 156.5, 175.9; HRMS (EI) m/z [M + H]+ calcd forC21H30N5O2: 384.2400, found: 384.2409; purity data obtained byHPLC: retention time 24.07 min (98.8% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylamine)-4-aminooctanamide (14e). Start-ing from 9e and following method B, 14e was obtained as a black solid(5.6 mg, 12%). mp: 52−54 °C. 1H NMR (D2O): δ 1.25 (s, 6H,( C H 2 ) 2 ( C H 2 ) 3 ( C H 2 ) 2 ) , 1 . 5 2 − 1 . 5 6 ( m , 4 H ,CH2CH2(CH2)3CH2CH2), 2.28 (t, J = 8.0 Hz, 2H, CH2CO), 2.85(t, J = 8.0 Hz, 2H, CH2NH), 7.01−7.05 (m, 4H, Ar), 7.09 (d, J = 8.5Hz, 2H, Ar), 7.21 (d, J = 8.5 Hz, 2H, Ar); 13C NMR (D2O): δ 25.0,25.2, 26.5, 27.6, 30.1, 36.1, 39.3, 117.8, 119.2, 123.8, 126.1, 127.8,130.5, 140.2, 143.7, 156.7, 176.0; HRMS (EI) m/z [M + H]+ calcd forC21H31N6O: 383.2559, found: 383.2552; purity data obtained byHPLC: retention time 22.48 min (95.1% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylmethylene)-4-aminoundecanamide (15a).Starting from 10a and following method B, 15a was obtained as awhite solid (152.5 mg, 58%). mp: 80−82 °C; 1H NMR (D2O): δ 1.17(s , 12H, (CH2)2(CH 2 ) 6(CH2)2) , 1 .46−1 .56 (m, 4H,CH2CH2(CH2)6CH2CH2), 2.27 (t, J = 8.0 Hz, 2H, CH2CO), 2.83(t, J = 8.0 Hz, 2H, CH2NH), 3.88 (s, 2H, PhCH2Ph), 7.13 (d, J = 8.5Hz, 2H, Ar), 7.18 (d, J = 8.5 Hz, 2H, Ar), 7.21−7.25 (m, 4H, Ar); 13CNMR (D2O): δ 25.1, 25.3, 26.5, 27.9, 28.0, 28.1, 28.2, 28.2, 36.3, 39.4,40.0, 122.3, 126.2, 129.2, 130.0, 132.0, 134.9, 138.5, 141.6, 156.3,176.1; HRMS (EI) m/z [M + H]+ calcd for C25H38N5O: 424.3076,found: 424.3073; purity data obtained by HPLC: retention time 27.69min (95.5% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylethylene)-4-aminoundecanamide (15b).Starting from 10b and following method B, 15b was obtained as ayellow solid (222.3 mg, 90%). mp: 128−130 °C; 1H NMR (D2O): δ1.17 (s, 12H, (CH2)2(CH2)6(CH2)2), 1.53 (t, J = 8.0 Hz, 4H,CH2CH2(CH2)6CH2CH2), 2.26 (t, J = 8.0 Hz, 2H, CH2CO), 2.85 (s,6H, Ph(CH2)2Ph and CH2NH), 7.07 (d, J = 7.5 Hz, 2H, Ar), 7.11 (d, J= 8.0 Hz, 2H, Ar), 7.18 (d, J = 7.5 Hz, 4H, Ar); 13C NMR (D2O): δ
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25.3, 25.5, 26.6, 28.0, 28.1, 28.2, 28.3, 28.4, 35.9, 36.0, 36.4, 39.5,122.1, 125.9, 129.2, 130.1, 131.8, 134.6, 138.9, 141.8, 160.4, 176.2;HRMS (EI) m/z [M + H]+ calcd for C26H40N5O: 438.3233, found:438.3226; purity data obtained by HPLC: retention time 27.83 min(99.0% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenyl ether)-4-aminoundecanamide (15c).Starting from 10c and following method B, 15c was obtained as ayellow solid (59.5 mg, 99%). mp: 89−92 °C; 1H NMR (D2O): δ 1.18(s , 12H, (CH2)2(CH 2 ) 6(CH2)2) , 1 .51−1 .56 (m, 4H,CH2CH2(CH2)6CH2CH2), 2.29 (t, J = 8.0 Hz, 2H, CH2CO), 2.84(t, J = 7.5 Hz, 2H, CH2NH), 6.96−7.00 (m, 4H, Ar) 7.20 (d, J = 8.5Hz, 2H, Ar), 7.30 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (D2O): δ 25.6,25.8, 26.8, 28.5, 28.6, 28.8, 28.8 28.8, 36.7, 39.5, 119.2, 119.6, 122.6,127.6, 129.0, 133.7, 152.6, 156.3, 156.5, 174.7; HRMS (EI) m/z [M +H]+ calcd for C24H36N5O2: 426.2869, found: 426.2860; purity dataobtained by HPLC: retention time 27.47 min (97.8% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylmethanone)-4-aminoundecanamide(15d). Starting from 10d and following method B, 15d was obtained asan orange solid (339.5 mg, 79%). mp: 71−73 °C. 1H NMR (D2O): δ1.15 (s, 12H, (CH2)2(CH2)6(CH2)2), 1.48−1.53 (m, 4H,CH2CH2(CH2)6CH2CH2), 2.30 (t, J = 8.0 Hz, 2H, CH2CO, 2.83 (t,J = 8.0 Hz, 2H, CH2NH), 7.32 (d, J = 8.0 Hz, 2H, Ar), 7.49 (d, J = 8.5Hz, 2H, Ar), 7.64 (d, J = 8.5, 2H, Ar), 7.68 (d, J = 8.0 Hz, 2H, Ar); 13CNMR (D2O): δ 25.0, 25.4, 26.5, 27.9, 28.0, 28.2, 28.3, 28.3, 36.6, 39.4,119.9, 123.9, 131.7, 131.7, 132.1, 135.0, 138.9, 142.1, 155.7, 176.1,197.9; HRMS (EI) m/z [M + H]+ calcd for C25H36N5O2: 438.2869,found: 438.2887; purity data obtained by HPLC: retention time 26.89min (95.1% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylamino-4-aminoundecanamide (15e).Starting from 10e and following method B, 15e was obtained as ablack oil (81.6 mg, 29%). 1H NMR (D2O): δ 1.25 (s, 12H,( C H 2 ) 2 ( C H 2 ) 6 ( C H 2 ) 2 ) , 1 . 5 5 − 1 . 6 6 ( m , 4 H ,CH2CH2(CH2)6CH2CH2), 2.35 (t, J = 6.4 Hz, 2H,CH2CO), 2.91 (t,J = 8.0 Hz, 2H, CH2NH), 7.11 (app t, 4H, Ar), 7.17 (d, J = 5.7 Hz, 2H,Ar), 7.28 (d, J = 5.7 Hz, 2H, Ar); 13C NMR (D2O) δ 25.3, 26.5, 27.9,28.0, 28.1, 28.2, 28.2, 34.2, 36.2, 39.4, 117.8, 119.2, 123.7, 126.1, 127.7,130.6, 140.1, 143.7, 156.6, 176.0 (q, CONH, C-11); HRMS (EI) m/z[M + H]+ calcd for C24H37N6O: 425.3029, found: 425.3033; puritydata obtained by HPLC: retention time 25.77 min (98.2% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenylmethylene)-4-aminododecanamide (16a).Starting from 11a and following method B, 16a as a white solid (224.6mg, 55%). mp: 114−116 °C; 1H NMR (D2O): δ 1.14 (s, 14H,( C H 2 ) 2 ( C H 2 ) 7 ( C H 2 ) 2 ) , 1 . 4 7 − 1 . 5 5 ( m , 4 H ,CH2CH2(CH2)7CH2CH2), 2.26 (t, J = 7.0 Hz, 2H, CH2CO), 2.82(t, J = 7.6, 2H, CH2NH), 3.87 (s, 2H, PhCH2Ph) 7.12 (d, J = 7.6 Hz,2H, Ar), 7.17 (d, J = 8.5 Hz, 2H, Ar), 7.21−7.25 (m, 4H, Ar); 13CNMR (D2O): δ 25.1, 25.4, 26.5, 28.0, 28.0, 28.1, 28.2, 28.3, 28.3, 36.3,39.4, 40.0, 122.2, 126.2, 129.2, 130.0, 132.0, 134.9, 138.4, 141.6, 156.3,176.1; HRMS (EI) m/z [M + H]+ calcd for C26H40N5O: 438.3233,found: 438.3241; purity data obtained by HPLC: retention time 28.68min (96.2% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
calcd for C27H42N5O: 452.3389, found: 452.3384; purity data obtainedby HPLC: retention time 28.93 min (96.7% purity).Preparat ion of Dihydrochlor ide Salt of N- (4- (4 ′ -
Guanidinophenyl)phenyl ether)-4-aminododecanamide (16c).Starting from 11c and following method B, 16c was obtained as a
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylmethanone)-4-aminododecanamide(16d). Starting from 11d and following method B, 16d was obtained asa yellow solid (46.4 mg, 26%). mp: 60−62 °C; 1H NMR (D2O): δ1.20 (s, 14H, (CH2)2(CH2)7(CH2)2), 1.55−1.62 (m, 4H,CH2CH2(CH2)7CH2CH2), 2.38 (t, J = 4.9 Hz, 2H, CH2CO), 2.90(t, J = 4.9 Hz, 2H,CH2NH), 7.40 (d, J = 5.8 Hz, 2H, Ar), 7.58 (d, J =5.8 Hz, 2H, Ar), 7.73 (d, J = 5.8 Hz, 2H, Ar), 7.77 (d, J = 5.8 Hz, 2H,Ar); 13C NMR (D2O): δ 25.0, 25.4, 26.5, 28.0, 28.0, 28.2, 28.3, 28.4,28.4, 36.6, 39.4, 120.0, 123.9, 131.7, 131.7, 132.2, 135.1, 138.9, 142.1,155.7, 176.2, 198.0; HRMS (EI) m/z [M + H]+ calcd for C26H38N5O2:452.3026, found: 452.3032; purity data obtained by HPLC: retentiontime 28.04 min (95.3% purity).
Preparat ion of Dihydrochlor ide Sal t of N- (4- (4 ′ -Guanidinophenyl)phenylamine)-4-aminododecanamide (16e).Starting from 11e and following method B, 16e was obtained as ablack solid (29.3 mg, 10%). mp: 60−62 °C; 1H NMR (D2O): δ 1.22(s , 14H, (CH2)2(CH 2 ) 7(CH2)2) , 1 .52−1 .63 (m, 4H,CH2CH2(CH2)7CH2CH2), 2.33 (t, J = 7.3 Hz, 2H,CH2CO), 2.89 (t,J = 8.0 Hz, 2H,CH2NH), 7.11 (m, 4H, Ar), 7.16 (d, J = 8.8 Hz, 2H,Ar), 7.28 (d, J = 8.8 Hz, 2H, Ar); 13C NMR (D2O): δ 25.3, 25.5, 26.7,28.1, 28.1, 28.2, 28.3, 28.4, 28.5, 36.3, 39.5, 117.9, 119.3, 123.8, 126.2,127.9, 130.7, 140.3, 143.9, 156.7, 176.2; HRMS (EI) m/z [M + H]+
calcd for C25H39N6O 452.3026, found: 439.3185; purity data obtainedby HPLC: retention time 27.24 min (97.1% purity).
DNA Thermal Denaturation Experiments. Thermal meltingexperiments were conducted with a Varian Cary 300 Biospectrophotometer equipped with a 6 × 6 multicell temperature-controlled block. Temperature was monitored with a thermistorinserted into a 1 mL quartz cuvette containing the same volume ofwater as in the sample cells. Absorbance changes at 260 nm weremonitored from a range of 30 °C to 90 °C with a heating rate of 1 °C/min and a data collection rate of five points per °C. The salmon spermDNA was purchased from Sigma Aldrich (extinction coefficient ε260 =6600 cm−1 M−1 base). Phosphate buffer solutions contained 10 mMNa2HPO4/NaH2PO4 adjusted to pH 7 were prepared using Milliporewater. A quartz cell with a 1-cm path length was filled with a 1 mLsolution of DNA polymer or DNA−compound complex. The DNApolymer (150 μM base) and the compound solution (15 μM) wereprepared in the phosphate buffer, adjusted to pH 7) so that acompound to DNA base (P/D) ratio of 0.1 was obtained. The thermalmelting temperatures of the DNA duplex or duplex−compoundcomplex obtained from the first derivative of the melting curves arereported.
Computational Methods. The systems have been optimizedusing the Gaussian0942 package at the B3LYP computational level withthe 6-31+G* basis set. Frequency calculations have been performed atthe same computational level to confirm that the resulting optimizedstructures are energetic minima (all positive frequencies). Effects ofwater solvation have been included by means of the SCFR-PCMapproaches implemented in the Gaussian09 package includingdispersion, repulsion, and cavitation energy terms of the solvent inthe optimization.
In Vitro Assays. Activity against T. b. rhodesiense STIB900Strain. This stock was isolated in 1982 from a human patient inTanzania and, after several mouse passages, cloned and adapted toaxenic culture conditions.43 Minimum Essential Medium (50 μL)supplemented with 25 mM HEPES, 1 g L−1 additional glucose, 1%MEM nonessential amino acids (100 × ), 0.2 mM 2-mercaptoethanol,1 mM Na-pyruvate, and 15% heat-inactivated horse serum was addedto each well of a 96-well microtiter plate. Serial drug dilutions of 11 3-
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fold dilution steps covering a range from 100 to 0.002 μg mL−1 wereprepared. Then 4 × 103 bloodstream forms of T. b. rhodesienseSTIB900 in 50 μL were added to each well, and the plate wasincubated at 37 °C under a 5% CO2 atmosphere for 70 h. A 10 μLresazurin solution (resazurin, 12.5 mg in 100 mL of double-distilledwater) was then added to each well and incubation continued for afurther 2−4 h.44 Then the plates were read with a Spectramax GeminiXS microplate fluorometer (Molecular Devices Cooperation, Sunny-vale, CA) using an excitation wavelength of 536 nm and an emissionwavelength of 588 nm. The IC50 values were calculated by linearregression45 from the sigmoidal dose−inhibition curves usingSoftmaxPro software (Molecular Devices Cooperation). Melarsoprolwas the reference drug used.Activity against P. falciparum NF54 Strain. In vitro activity against
erythrocytic stages of P. falciparum was determined using a 3H-hypoxanthine incorporation assay,46,47 using the drug-sensitive NF54strain (Schipol Airport, The Netherlands48) and the standard drugchloroquine (Sigma C6628). Compounds were dissolved in DMSO at10 mg mL−1 and added to parasite cultures incubated in RPMI 1640medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g L−1), neomycin (100 U mL−1), Albumax (5 gL−1), and washed human red cells A+ at 2.5% hematocrit (0.3%parasitaemia). Serial drug dilutions of eleven 3-fold dilution stepscovering a range from 100 to 0.002 μg mL−1 were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 °C; 4%CO2, 3% O2, 93% N2. After 48 h, 50 μL of 3H-hypoxanthine (=0.5μCi) was added to each well of the plate. The plates were incubatedfor a further 24 h under the same conditions. The plates were thenharvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland),and the red blood cells were transferred onto a glass fiber filter andthen washed with distilled water. The dried filters were inserted into aplastic foil with 10 mL of scintillation fluid and counted in a Betaplateliquid scintillation counter (Wallac). IC50 values were calculated fromsigmoidal inhibition curves by linear regression24 using MicrosoftExcel.Cytotoxicity with L6 Cells. Assays were performed in 96-well
microtiter plates, each well containing 100 μL of RPMI 1640 mediumsupplemented with 1% L-glutamine (200 mM) and 10% fetal bovineserum, and 4000 L6 cells (a primary cell line derived from rat skeletalmyoblasts).49,50 Serial drug dilutions of eleven 3-fold dilution stepscovering a range from 100 to 0.002 μg mL−1 were prepared. After 70 hof incubation, the plates were inspected under an inverted microscopeto ensure growth of the controls and sterile conditions. Ten microlitersof resazurin solution was then added to each well, and the plates wereincubated for another 2 h. Then the plates were read with aSpectramax Gemini XS microplate fluorometer (Molecular DevicesCorporation, Sunnyvale, CA, USA) using an excitation wavelength of536 nm and an emission wavelength of 588 nm. The IC50 values werecalculated by linear regression from the sigmoidal dose inhibitioncurves using SoftmaxPro software (Molecular Devices Cooperation,Sunnyvale, CA, USA). Podophyllotoxin was the reference drug used.
■ ASSOCIATED CONTENT
*S Supporting InformationPreparation and spectroscopic data of Boc-protected mono-guanidines, Boc-protected aminoalkyl carboxylic acids, amino-alkyl derivatives of Boc-protected diaromatic mono-guanidines,and spectroscopic data of final hydrochloride salts. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.
NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work has been funded by the SFI−RFP grant CHE275, aTrinity College Dublin postgraduate award (C.M.K.), and aHEA PRTLI Cycle 4 grant (C.M.K.). We thank Dr. PadraicNagle for his help with the biophysical experiments. We thankMonica Cal and Joelle Jourdan from the Swiss TropicalInstitute, Socinstrasse, 57, CH-4002 Basel, Switzerland, forperforming the biological tests.
■ ABBREVIATIONS USED
MGB, minor groove binder; DNA, deoxyribonucleic acid; AT,adenine-thymine base pair; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; DIEA, N,N-diisopropylethylamine; HB, hydrogen bond; DFT, densityfunctional theory; ΔTm, change in thermal melting temper-ature; P/D, phosphate to drug ratio; IC50, half-maximalinhibitory concentration; DHFR-TS, dihydrofolate reductasethymidine synthetase
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