Aminoalkyl Derivatives of Guanidine Diaromatic Minor Groove Binders

Article pubs.acs.org/jmc

Aminoalkyl Derivatives of Guanidine Diaromatic Minor Groove Binders with Antiprotozoal Activity Caitriona 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 binding observed for our previous series of diaromatic symmetric and asymmetric guanidinium and 2-aminoimidazolinium derivatives, we report now the synthesis of new aminoalkyl derivatives of diaromatic guanidines with potential as DNA minor groove binders and antiprotozoal activity. The preparation 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 was evaluated by means of DNA thermal denaturation experiments. Finally, the antiprotozoal activity of most of these aminoalkyl minor groove binders was evaluated in vitro against Trypanosoma brucei rhodesiense (8 compounds) and Plasmodium falciparum (18 compounds). The O-linked derivatives 13c and 14c showed 100 nM activities against P. falciparum, whereas for T. b. rhodesiense all compounds tested showed micromolar activity. Some of the derivatives prepared seem to exert the antimalarial activity by binding to the DNA minor groove whereas other sets of compounds could exert this antimalarial activity by inhibiting the parasite dihydrofolate reductase, for example.

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INTRODUCTION DNA is a key target for drugs aiming to interfere with normal cellular processes because of its involvement with how genes are expressed and how proteins are created. There are several ways in which drugs can target DNA, and one particular way is by binding in the minor groove of the double helix, which is the preferred site of interaction between DNA and proteins.1 This type of DNA ligand has been shown to exhibit different biological activities.2 In particular, it has been found that compounds that target the minor groove can be used for treating tropical diseases because they inhibit transcription directly or block the actions of DNA-dependent enzymes. Two well-known examples of minor groove binders (MGBs) are pentamidine and furamidine (Figure 1). Furamidine is a key aromatic diamidine active against Trypanosoma (T.) species in vitro. Pentamidine, an aliphatic analogue of furamidine,3 is orally active and effective in the treatment of human African trypanosomiasis (sleeping sickness) which is caused by bloodstream infections with parasitic protozoans of the subspecies T. brucei (T. b.) rhodesiense or T. b. gambiense. Recent studies show that bis-guanidine and bis-2-aminoimidazoline diphenyl derivatives, related to furamidine, display potent antitrypanosomal activity in vitro and in vivo against T. b. rhodesiense. In addition, a correlation between antitrypanosomal activity and DNA binding affinity has been observed, suggesting a possible mechanism of action for these compounds.4 The X-ray structure for the complex of © 2013 American Chemical Society

furamidine with the d(CGCGAATTCGCG)2 oligomer has been obtained,5 displaying the snug fit of this compound with the AATT sequence indicative of minor groove binding. This complex could be responsible for the inhibition of the microbial topoisomerase enzyme leading to anti-Pneumocystis carinii activity.6 In addition, the X-ray crystal structure of one of the bis-2-aminoimidazoline derivatives has been published, showing a very similar binding mode to that of furamidine.7 A large amount of evidence indicating that DNA minor groove binding is related to the antiprotozoal activity of compounds has been published;8−14 pentamidine has been shown to linearize kinetoplast DNA from trypanosomes,15 which could play an important role in the compound’s mechanism of action. Recently, some evidence has been published of minor groove binders with good antiprotozoal activity but poor DNA binding.16 Moreover, antimalarial drugs containing guanidine-like groups such as proguanyl or pyrimethamine were prepared in the past as selective inhibitors of the parasite dihydrofolate reductase thymidine synthetase (DHFR-TS).17−20 It is clear that further research in this area is required to deepen the understanding of the antiprotozoal mechanism of action of these derivatives. We have reported in the past the preparation of symmetric and asymmetric diaromatic guanidinium/2-aminoimidazoliReceived: June 4, 2012 Published: January 9, 2013 700

dx.doi.org/10.1021/jm301614w | J. Med. Chem. 2013, 56, 700−711

Journal of Medicinal Chemistry

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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

nium derivatives (Figure 1), studying the influence of the linker (X in Figure 1) and cations on minor groove binding by means of DNA denaturation experiments with both random sequence DNA (salmon sperm) and AT specific polynucleotides [poly(dA·dT)2 and poly(dA)·(dT)].4,21 In general, the increases in DNA denaturation temperature (ΔTm) obtained indicated strong binding to DNA, especially for those compounds with a NH or a CO group linking the phenyl rings. Moreover, in a different article, we determined the mode of binding and binding constants of these compounds by a number of biophysical techniques concluding that they strongly bind in the minor groove.22 In addition, we prepared monoguanidinium-like derivatives, leaving one of the aromatic amino groups unsubstituted and therefore yielding monocations. These compounds showed very poor affinity toward DNA probably due to their monocationic nature. Taking this into account and exploiting the binding activity of our MGBs, we intend to combine the aliphatic linker of pentamidine and the diaromatic structure of our MGBs by attaching aminoalkyl chains to one of the aromatic rings. This aminoalkyl group will be protonated at physiological pH and, moreover, the aliphatic chain will help to displace the spine of hydration present in the minor groove by hydrophobic interactions thereby increasing the strength of binding.

Hence, our aim is to explore the influence that such an aminoalkyl chain could have in the minor groove binding and antiprotozoal activity of these diaromatic compounds. Thus, in this article we present the synthesis, biophysical, and biochemical study of a series of aminoalkyl diaromatic MGB conjugates (aminoalkyl-MGBs, see Scheme 1) using as templates the compounds previously developed in our laboratory.

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RESULTS AND DISCUSSION Synthesis. The preparation of the aminoalkyl-MGBs proposed involves, first, the syntheses of the mono-guanidylated diaromatic systems, as previously described;8 this step is then followed by Boc-protection of the amino terminus of various amino acids of different lengths and, finally, the conjugation of both moieties by means of an amide functionality. Different aminoalkyl acids with three, four, seven, ten, and eleven methylene groups (4-aminobutanoic, 5-aminopentanoic, 8-aminooctanoic, 11-aminoundecanoic, and 12-aminododecanoic acids) were considered. The amino acid must be protected so that the amino group of the mono-guanidine MGB attacks at the carbonyl terminus leading to a successful coupling reaction. Different Boc-protection conditions were explored23−25 and, 701



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Table 1. Overall (Total) Yields and Thermal Denaturation Experiments’ Resultsa of the Aminoalkyl-MGB Conjugates (12a−e to 16a−e) and the Corresponding Bis- and Mono-guanidinium Unconjugated MGBs (17a−e and 18a−e) with Salmon Sperm DNA

compound

X

n

% yield

ΔTm

12a 12b 12c 12d 12e 13a 13b 13c 13d 13e 14a 14b 14c 14e 15a 15b 15c 15d 15e 16a 16b 16c 16d 16e 17a18 17d18 17e18 18a8 18b8 18c8 18d8 18e8 pentamidine

CH2 CH2CH2 O CO NH CH2 CH2CH2 O CO NH CH2 CH2CH2 O NH CH2 CH2CH2 O CO NH CH2 CH2CH2 O CO NH CH2 CO NH CH2 CH2CH2 O CO NH

1 1 1 1 1 2 2 2 2 2 5 5 5 5 8 8 8 8 8 9 9 9 9 9 − − − − − − − − −

30 28 51 45 21 61 5 21 26 3 51 40 7 3 24 60 85 44 19 35 61 14 13 3 − − − − − − − − 1

1.0 ± 0.0 5.0 ± 0.8 1.2 ± 0.8 5.1 ± 0.0 1.0 ± 0.0 4.0 ± 1.0 5.1 ± 0.6 6.0 ± 0.4 5.2 ± 1.0 6.1 ± 0.0 0.1 ± 0.0 2.0 ± 0.0 2.1 ± 0.8 2.0 ± 0.0 0.0 ± 0.0 1.1 ± 0.8 2.0 ± 0.0 2.0 ± 0.9 3.1 ± 0.0 0.1 ± 0.0 1.1 ± 0.4 2.0 ± 0.0 2.1 ± 0.0 5.0 ± 0.0 8.1 ± 0.0 4.2 ± 0.0 8.0 ± 0.0 3.0 ± 0.0 0.1 ± 0.4 2.0 ± 0.0 0.0 ± 0.0 2.1 ± 0.0 0.7;b 10.6;c11.1;d 20.6;d 12.8e

Melting temperature of salmon sperm DNA in phosphate buffer (10 mM) is 68 °C. Three measurements were carried out for each compound. Reference 31, calf thymus, phosphate buffer. cReference 32, calf thymus, MES buffer. dReference 33, poly(dA-dT) and calf thymus, Tris HCl buffer. e Reference 34, poly(dA-dT), PIPES buffer. a b

finally, methanol at 60 °C for 20 h in the presence of triethylamine was utilized, yielding the corresponding compounds 1−5 (Scheme 1). The Boc-protected mono-guanidine diaromatic derivatives 6a−e (Scheme 1) were prepared (as previously published by us8,26) by reaction of the commercial diamines (in excess) with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, triethylamine, and mercury(II) chloride in dichloromethane or dimethylformamide. To join the Boc-protected amino acid with the monoguanidine MGB derivatives, different coupling reactions were explored. Thus, we tried using the corresponding acid chloride of the amino acid and the mono-guanidine in acetonitrile in the presence of triethylamine,27 or coupling reagents such as N,N’-

dicyclohexylcarbodiimide 2 8 or O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU).29 This last method, which gave the best results, involved dissolving the Boc-protected amino acid, the Boc-protected mono-guanidine, TBTU, and N,N-diisopropylethylamine (DIEA) in acetonitrile at room temperature under argon. In that way, we prepared in good yields of compounds 7a−e, 8a− e, 9a−e, 10a−e, and 11a−e (Scheme 1) which were fully characterized. Finally, different deprotection methods for the Boc-protected intermediates were tested, and the most successful results were obtained with 4 M hydrochloric acid/dioxane in 2-propanol (IPA)/dichloromethane, leading to the direct formation of the hydrochloride salts (Scheme 1). Further purification by reverse 702



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compounds (see computational results) which could lead to a better fitting into the minor groove, occupying more space and displacing more of the water molecules within. Second, whereas the amino MGBs are monocations at physiological pH, the aminoalkyl-MGB conjugates are dicationic, which favors the interactions in the negatively charged environment of the minor groove. Third, the aminoalkyl chain may better direct the ammonium group to establish strong hydrogen bonds (HBs) and ionic interactions with the nucleic bases in the minor groove 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-, and CH2CH2-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 and an 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 NH linker. However, the corresponding amino MGB derivatives did not show strong binding to DNA, indicating that the aminoalkyl chain is playing a favorable role in the interaction with DNA. Regarding the aminoalkyl chain length, those compounds with four methylene groups (n = 2, compounds 13a−e) gave the best results and elongating the chain seemed to interfere with the binding except for those compounds with a NH group as a linker (15e, n = 8, and 16e, n = 9). Compounds 13b, 13c, and 13d showed ΔTm increases of 5, 4, and 5 °C when compared to the corresponding amino MGB derivatives, respectively. This could indicate that very short alkyl chains cannot contribute extra interactions with DNA to improve the binding, whereas alkyl chains longer than four CH2 groups are too long or too lipophilic to achieve an optimal interaction. Compounds 12b (X = CH2CH2) and 12d (X = CO) showed the strongest binding for the series where n = 1. This difference can be attributed to the distance between the cations in 12b being larger than that of 12a.8 This could result in the formation of optimum HBs with the DNA base pairs. Also in 12d the HB acceptor carbonyl linker could lead to stronger interactions between the molecule and the minor groove. To have a general idea of the distance between the guanidinium and the ammonium cations in these aminoalkylMGB conjugates, models for the X = NH derivatives with n = 1, 2, 5, 8, and 9 aminoalkyl chains were optimized using density functional theory (DFT) methods (B3LYP/6-31+G*,35 mimicking water solvation with PCM36). Considering the high flexibility that the aminoalkyl chains exhibit, several conformations would be possible; however, only that with the aminoalkyl 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

phase chromatography was required. The total overall yields obtained for each product in the coupling and deprotection steps are presented in Table 1. DNA Affinity. Biophysical studies were carried out to determine the affinity of these aminoalkyl-MGB conjugates toward DNA by means of DNA thermal denaturation experiments, which provide an easy and effective method to screen a large number of compounds. A solution of known concentration of unspecific wild type salmon sperm DNA (150 μM, which gives an absorbance of 1 au) was used, and the change in the thermal melting temperature (ΔTm) was monitored when adding the molecule to be tested at a known concentration. Thermal denaturation results obtained for the aminoalkylMGBs are presented in Table 1 and Figure 2. Previously, when

Figure 2. Graph showing the DNA thermal denaturation results of compounds 12b (X = CH2CH2, n = 1), 13c (X = O, n = 2), and 14c (X = O, n = 5).

studying the unconjugated MGBs, the best results were achieved for the symmetric bis-guanidinium derivatives (17a− e30 in Table 1); for that reason, the ΔTm values obtained for these bis-guanidiniums and for the corresponding monoguanidines (18a−e8) are also presented in Table 1 to allow a better structural comparison with the present conjugates. This will show the effect of the charged aminoalkyl chain on the affinity of the diaromatic mono-guanidiniums. It will also show the optimal length for these aminoalkyl chains in terms of DNA affinity. In Table 1 it can be observed that the introduction of an aminoalkyl chain, and in particular short chains, results in increased Tm. In general, most of the aminoalkyl-MGB conjugates showed moderately increased binding to DNA with ΔTm between 2 and 6. There are several differences between the amino mono-guanidines and the aminoalkyl-MGB conjugates that may lead to these increases in the DNA affinity of the latter. First, the total length of the aminoalkyl-MGB molecules is larger than that of the corresponding amino MGB

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). 703



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Table 2. Intercationic [C(+)−N(+)] Distances (Å) Computed for Compounds 12e, 13e, 14e, 15e, and 16e at B3LYP/6-31+G* Level and PCM−Water, for the “Extended” Conformationa

C(+)−N(+) ΔTm a

12e (n = 1)

13e (n = 2)

14e (n = 5)

15e (n = 8)

16e (n = 9)

17.33 1

18.40 6

22.08 2

26.23 3

27.24 5

Table 3. In Vitro Antiprotozoal Activities and Cytotoxicity of the Aminoalkyl-MGBsa

compound no.

The corresponding ΔTm are also included.

12a (X = CH2, n = 1) 12b (X = [CH2]2, n = 1) 12c (X = O, n = 1) 12e (X = NH, n = 1) 13a (X = CH2, n = 2) 13b (X = [CH2]2, n = 2) 13c (X = O, n = 2) 13d (X = CO, n = 2) 13e (X = NH, n = 2) 14c (X = O, n = 5) 15a (X = CH2, n = 8) 15b (X = [CH2]2, n = 8) 15c (X = O, n = 8) 15e (X = NH, n = 8) 16a (X = CH2, n = 9) 16c (X = O, n = 9) 16d (X = CO, n = 9) 16e (X = NH, n = 9) pentamidine

the aminoalkyl chain, and this is related to the ΔTm values with exception of compound 13e which shows a much better ΔTm than expected from the chain length. It seems that the optimal intercationic distance for binding should be around 18−19 Å; however, because of the enormous flexibility of the long chains and the hydrophobic favorable interactions that can be established within the minor groove by displacing the water spine, it cannot be discarded that aminoalkyl chains with seven methylene groups could adopt a conformation that would result in a good binding to DNA. Biological Results. Based on the DNA affinity results, the activity of several of the aminoalkyl-MGBs was evaluated in vitro against T. b. rhodesiense (STIB900 strain37) (compounds 12b, 13b−e, 14c, 15e, 16e), and against P. falciparum (NF54 strain38) and rat skeletal myoblasts (L639) cells (compounds 12a−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 reduction of resazurin. The 3H-hypoxanthine incorporation assay was used to measure the in vitro antimalarial activity. All the corresponding 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, upon lengthening the aliphatic chain, activity decreases with the exception of compound 16e which displays the best antitrypanosomal activity IC50= 4.06 μM) of the series. This could be due to the central NH linker that has good hydrogen bonding ability or to a fold of the long chain that results on a more efficient interaction with DNA. Unfortunately, this compound also displays some cytotoxicity on L6 cells and is therefore unselective (SI = 3). The compounds with CH2CH2 linkers (12b and 13b) display good activity against T. b. rhodesiense (IC50 13.3 and 20.2 μM, respectively), and they are relatively selective toward the parasite because of their high L6 cell’s IC50 values (133 and 109 μM). The electron-withdrawing CO linker derivative (13d) showed the poorest activity against T. b. rhodesiense; however, those compounds with electrondonating 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 shorter aminoalkyl chains that orient the amino group to a less efficient interaction with DNA. The isosteric replacement of the CH2 linker by an O linker (compounds 12c and 14c) did not lead either to an increased selectivity or an increased activity against T. b. rhodesiense. Compounds 12b and 13b showed the best selectivity (SI = 10 and 5.4, respectively) even though these values are rather poor. Although none of these compounds show an antitrypanosomal activity comparable to that of the control (melarsoprol, IC50 = 0.0055 μM), compounds 12b and 13b could be the base for further improvements.

IC50 L6 cellsb (μM)

IC50 T.b.r.c (μM)

selectivity index (SI)d

IC50 P. f.e (μM)

selectivity index (SI)f

207

−

−

2.62

79

133

13.3

10

1.14

116

173

−

−

0.880

197

127

−

−

1.20

106

105

−

−

0.589

179

109

20.2

5

1.06

103

13.1

3

0.106

316

1

2.55

55

33.5 140

121

223

57.7

4

1.46

153

136

31.9

4

0.149

913

30.3

−

−

2.02

15

70.6

−

−

5.79

12

62.3

−

−

1.41

44

85.4

2

3.79

41

23.8

−

−

2.32

10

22.4

−

−

1.71

13

10.62

−

−

1.98

5

11.9

4.06

3

2.71

4

0.027

56

157

1.51

0.002

755

a

All IC50 values were calculated from experiments in triplicate. bL6 cells. Control: podophyllotoxin, IC50 = 0.0145 μM. cT. brucei rhodesiense STIB900 strain. Control: melarsoprol, IC50 = 0.0055 μM. d Selectivity index = (IC50 L6-cells)/(IC50 T. b. rhodesiense). eP. falciparum NF54 strain. Control: chloroquine, IC50 = 0.0039 μM. f Selectivity index = (IC50 L6-cells)/(IC50 P. falciparum).

In the in vitro assays against P. falciparum, most of the compounds showed good activity with IC50 values in the nanoand micromolar range. The results, displayed in Table 3, show that, in general, lengthening the aminoalkyl chain does not correlate to IC50 values. Compounds 13c and 14c display the best activity of the series with IC50 values of 0.106 and 0.149 μM, respectively, and good to very good selectivity (SI = 316 and 900, respectively). Compounds 13c and 14c could be considered as interesting “leads” and would be good candidates for in vivo testing. The rest of the derivatives displayed moderate to poor activity against P. falciparum (IC50 values between 1 and 5) and poor SIs, and compounds 15b, 15e, 16e, and 12a showed the poorest activity. 704



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Structure−Activity Relationships Analysis. It is important to determine if there is any correlation between the antiprotozoal activity of all the compounds studied and their DNA binding ability. In principle, the compounds that have the strongest affinity for the minor groove should inhibit DNA replication most effectively and elicit the strongest antitrypanosomal or antiplasmodial activity. Analysis of the IC50 values obtained 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 was found in the case of the T. b. rhodesiense activity. Different families of guanidine and 2-aminoimidazoline analogue molecules (Figure 1), previously synthesized by us and by Dardonville, were tested in vitro against T. b. rhodesiense and P. falciparum.4 These dicationic diphenyl compounds displayed good antitripanosomal activities with IC50 values in the 100 nM range. The main difference between these families of compounds is that the present aminoalkyl-MGBs possess not only a guanidinium cation attached to an aromatic ring but also an ammonium cation attached to a flexible aliphatic chain of different lengths. The estimated pKa of these two different cations is more or less similar, being around 9−11 for aromatic guanidine-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 the middle NH group of 4,4′-bis(guanidino)diphenylamine (related to compounds 12e−16e) to be 2.8.40 Hence, all these molecules will be dicationic at physiological pH. In the mentioned previous work, we suggested that to obtain antitripanosomal activity in the 100 nM range, a dicationic molecule was essential. The aminoalkyl-MGBs presented here, even though dicationic, only showed IC50 values within the micromolar 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-like compounds, the IC50 values were much poorer for P. falciparum than for T. b. rhodesiense, whereas the aminoalkyl-MGBs compounds presented in this work exhibit 100 nM to low micromolar antimalarial activity. This seems to indicate that the presence of cations of different nature (a guanidinium and an alkylammonium cation) and the flexible aliphatic linker can favor the antimalarial activity versus the antitrypanosomal activity. 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 and DNA binding (Table 2), it seems that our compounds could exert 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 for compounds 12b, 13b, 13c, 13d, 13e, 15b, 15e, and 16e (ΔTm = −0.88 × IC50 + 6.49) with R2 = 0.87. This set of compounds includes a wide range of data, i.e., IC50 values ranging from 0.106 (13c, the best) to 5.79 μM and ΔTm values from 6 to 1. Hence, this correlation seems to indicate that this particular set of compounds, with aliphatic chains between 3 and 10 methylene groups and different linkers, exert their better or worse antimalarial activity by binding to the DNA minor groove in the same way that many other related compounds have been reported to do.8−14 In this set of compounds,

aminoalkyl chains with four methylene groups give the best results for binding to the DNA minor groove (compounds 13a−e) and, in general, they also show good antimalarial activity, 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 correlate to 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 between three and seven methylene groups show structural similarity with dihydrofolate and with some inhibitors of dihydrofolate reductase−thymidylate synthase (DHFR-TS) from P. falciparum 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 three compounds with tetrahedral linkers (X = O, CH2) and short aminoalkyl chains exert their antimalarial activity by inhibiting this particular enzymatic system. In any case, this hypothesis can only be fully proved by performing further enzymatic studies using DHFR-TS. We can conclude that, in general, aminoalkyl chains longer than seven methylene groups are good neither for antimalarial activity nor for DNA binding. Similarly, compounds with X = CO, which is a trigonal planar group, gave poor results in terms of antimalarial activity, despite being relatively good minor groove binders when carrying an aminoalkyl chain of four methylene groups (13d). Regarding the shortest aminoalkyl chain derivatives (n = 1, three methylene groups), they do not bind well to DNA except when X = CH2CH2 (longest linker); however, compound 12c (n = 1, X = O) shows an interesting antimalarial activity maybe because the tetrahedral nature of the linker can facilitate the interaction within the active site of enzymatic systems such as, for example, DHFR-TS. The case of compound 13c is an interesting one: its good antimalarial activity correlates well with a good DNA binding; however, we have found that, in our series, the tetrahedral linker X = O could be more associated to other mechanisms of action such as DHFR-TS inhibition. It could be the case that this compound, which is the best of the whole series, could exert its antimalarial activity by DNA binding and an additional mechanism of action. Hence, for future series, aminoalkyl chains between four and seven methylene groups should be used and the CO linker avoided. Regarding antimalarial activity, compounds 13c and 14c are really promising and in the future will be tested in vivo as recommended by the Swiss Tropical and Public Health Institute.

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CONCLUSIONS On the basis of the DNA binding results previously obtained by us,4,21,22 we have prepared a new series of aminoalkyl derivatives of guanidine diaromatic MGBs to explore the effect of the aminoalkyl chain on the DNA binding activity of the MGB 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 of unspecific sequence. The incorporation of aminoalkyl chains with four to seven methylene groups resulted in significant increases in the DNA melting temperature. The distance between cationic functionalities (guanidinium and ammonium) has been screened by DFT calculations; 705



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DCM (1:1) (0.5 mL) and stirred 4 h. The HCl/dioxane was then removed under vacuum. The residue was dissolved in H2O and washed with DCM (3 × 5 mL). Concentration of the aqueous layer followed by reverse phase column chromatography eluting with H2O/ acetonitrile yielded the required product. Examples of the Preparation of Aminoalkyl Boc-Protected Diaromatic Mono-guanidines. Preparation of 4-[2,3-Di(tertbutoxycarbonyl)guanidino]-4′-[4-(tert-butoxycarbonyl)aminopentanamido]diphenyl Ether (8c). Following method A, 8c was obtained as a yellow oil (438.5 mg, 68%); 1H NMR (CDCl3): δ 1.46 (s, 9H, (CH3)3), 1.53 (s, 9H, (CH3)3), 1.56 (s, 9H, (CH3)3), 1.61 (s, 2H, CH2CH2(CH2)2), 1.75−1.79 (m, 2H, (CH2)2CH2CH2), 2.42 (t, J = 7.5 Hz, 2H, CH2CO), 3.22 (t, J = 6.5 Hz, 2H, CH2NH), 4.69 (broad s, 1H, NHCO), 6.94−6.99 (m, 4H, Ar), 7.53 (d, J = 10.5 Hz, 4H, Ar), 7.55 (s, 1H, CONHPh), 10.33 (broad s, 1H, NH), 11.69 (broad s, 1H, NH); 13C NMR (CDCl3): δ 22.1, 27.6, 27.7, 27.9, 30.4, 36.1, 39.1, 78.9, 79.2, 83.3, 115.7, 117.3, 118.5, 120.1, 130.6, 132.2, 133.1, 142.1, 148.6, 154.7, 156.0, 162.0, 170.8; HRMS (EI) m/z [M + H]+ calcd for C33H48N5O8: 642.3503, found: 642.3508. Preparation of 4-[2,3-Di(tert-butoxycarbonyl)guanidino]-4′-[4(tert-butoxycarbonyl)aminooctanamido]diphenyl Ether (9c). Following method A, 9c was obtained as a yellow oil (148.4 mg, 22%); 1H NMR (CDCl3): δ 1.16−1.24 (m, 6H, (CH2)2(CH2)3(CH2)2), 1.41 (s, 9H, (CH3)3), 1.50 (s, 9H, (CH3)3), 1.57 (s, 9H, (CH3)3), 1.59−1.69 (m, 4H, CH2CH2(CH2)3CH2CH2), 2.18 (t, J = 7.2 Hz, 2H, CH2CO), 3.04 (t, J = 4.5 Hz, 2H, CH2NH), 4.81 (s, 1H, NHCO), 6.75 (d, J = 8.0 Hz, 2H, Ar), 6.88 (d, J = 8.0 Hz, 2H, Ar), 7.38 (d, J = 8.0 Hz, 2H, Ar), 7.54 (d, J = 8.0 Hz, 2H, Ar), 8.69 (s, 1H, CONHPh), 10.20 (s, 1H, NH), 11.67 (s, 1H, NH); 13C NMR (CDCl3): δ 26.1, 26.4, 27.5, 27.6, 27.9, 28.5, 28.7, 34.1, 36.6, 40.0, 78.4, 79.3, 83.3, 117.5, 119.2, 120.8, 124.0, 130.5, 134.3, 151.7, 152.8, 153.7, 154.8, 155.7, 162.9, 171.3; HRMS (EI) m/z [M + H]+ calcd for C36H54N5O8: 684.3972, found: 684.3966 Preparation of Hydrochloride Salts. Preparation of Dihydrochloride Salt of N-(4-(4′-Guanidinobenzyl)phenyl)-4-aminobutanamide (12a). Starting from 7a and following method B, 12a was obtained 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; purity data obtained by HPLC: retention time 22.21 min (95.4% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenylethyl)phenyl)-4-aminobutanamide (12b). Starting from 7b and following method B, 12b was obtained as a yellow oil (55 mg, 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 23.20 min (95.4% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenyl ether)-4-aminobutanamide (12c). Starting from 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 = 8.0 Hz, 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). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylmethanone)-4-aminobutanamide (12d). Starting from 7d and following method B, 12d was obtained as a yellow solid (53.5 mg, 99%). mp: 138−140 °C; 1H NMR (D2O): δ 1.46−1.52 (m, 2H, CH2CH2CH2), 2.25 (t, J = 8.0 Hz, 2H, CH2CO),

however, no clear conclusion can be derived considering the high 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 two different behaviors. In one set (eight compounds) a good activity/DNA binding correlation is found; however, in a second set (three compounds) no correlation is found even though a good antimalarial activity is observed, suggesting a different mechanism of action. The antiprotozoal activity and cytotoxicity of several of these compounds was evaluated. Thus, for T. b. rhodesiense (8 compounds tested), poor results were obtained. These derivatives proved to be more potent and selective toward P. falciparum (18 compounds tested) exhibiting IC50 within the low micromolar to 100 nM range. Compounds 13c and 14c displayed good in vitro antimalarial activity and good selectivity indices and will be tested in vivo in the future.

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EXPERIMENTAL SECTION

Synthesis. 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. Dry solvents were prepared using standard procedures, according to Vogel, with distillation prior to use. Chromatographic columns were run using a Biotage SP4 flash purification system with Biotage SNAP silica cartridges. Solvents for synthesis purposes were used at GPR grade. Analytical TLC was performed using Merck Kieselgel 60 F254 silica gel plates or Polygram Alox N/UV254 aluminum oxide plates. Visualization was by UV light (254 nm). NMR spectra were recorded in a Bruker DPX-400 Avance spectrometer, operating at 400.13 and 600.1 MHz for 1H NMR, and 100.6 and 150.9 MHz for 13C NMR. Shifts are referenced to the internal solvent signals. NMR data were processed using Bruker Win-NMR 5.0 software. HRMS spectra were measured on a Micromass LCT electrospray TOF instrument with a Waters 2690 autosampler with methanol as carrier solvent. Melting points were determined using a Stuart Scientific Melting Point SMP1 apparatus and are uncorrected. Purity was assessed using reverse phase HPLC with a diode-array detector scanning wavelengths from 200 to 950 nm. HLPC analysis was carried out using a Varian ProStar system equipped with a Varian Prostar 335 diode array detector and a manual injector (20 μL). Integration was performed at 245 nm, and peak purity was confirmed using a purity channel. The stationary phase consisted of an ACE 5 C18-AR column (150 × 4.6 mm). The method developed for this type of hydrochloride salt, which gave optimum retention 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 set for compounds to be tested pharmacologically. All mono-guanidine derivatives used in this work were prepared as previously reported by us.8 General Methods. Step A Method: General Method for the Preparation of the Aminoalkyl Derivatives of Boc-Protected Diaromatic Mono-guanidines. A solution of the corresponding Boc-protected amino acid (1.2 mmol) in MeCN (10 mL) was treated with DIEA (3.8 mmol), the mono-guanidine (1 mmol), and TBTU (1.2 mmol) under inert atmosphere. The reaction mixture was stirred at 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 flash chromatography 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 acid conjugate of the mono-guanidine derivative (1 mmol) was dissolved in 18 equiv of 4 M HCl/dioxane (4.5 mL) under argon to make up a 0.2 M solution. The mixture was made up to 5 mL solution with IPA/ 706



Article

P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylmethylene)-4-aminooctanamide (14a). Starting from 9a and following method B, 14a was obtained as a yellow 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 = 7.5 Hz, 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 for C22H32N5O: 382.2607, found: 382.2601; purity data obtained by HPLC: retention time 23.24 min (95.7% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylethylene)-4-aminooctanamide (14b). Starting from 9b and following method B, 14b was obtained as a yellow solid (45.9 mg, 97%). mp: 143−145 °C; 1H NMR (D2O): δ 1.18 (s, 6H, (CH2)2(CH2)3(CH2)2), 1.47 (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); 13 C 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). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenyl ether)-4-aminooctanamide (14c). Starting from 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(CH2)3(CH2)2), 1.55 (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 = 8.5 Hz, 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 for C21H30N5O2: 384.2400, found: 384.2409; purity data obtained by HPLC: retention time 24.07 min (98.8% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylamine)-4-aminooctanamide (14e). Starting 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, (CH2)2(CH2)3(CH2)2), 1.52−1.56 (m, 4H, 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 = 8.5 Hz, 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 for C21H31N6O: 383.2559, found: 383.2552; purity data obtained by HPLC: retention time 22.48 min (95.1% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylmethylene)-4-aminoundecanamide (15a). Starting from 10a and following method B, 15a was obtained as a white solid (152.5 mg, 58%). mp: 80−82 °C; 1H NMR (D2O): δ 1.17 (s, 12H, (CH2)2(CH2)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 = 8.5 Hz, 2H, Ar), 7.18 (d, J = 8.5 Hz, 2H, Ar), 7.21−7.25 (m, 4H, Ar); 13C NMR (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 27.69 min (95.5% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylethylene)-4-aminoundecanamide (15b). Starting from 10b and following method B, 15b was obtained as a yellow solid (222.3 mg, 90%). mp: 128−130 °C; 1H NMR (D2O): δ 1.17 (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

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; purity data obtained by HPLC: retention time 19.68 min (98.4% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylamine)-4-aminobutanamide (12e). Starting 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 = 7.5 Hz, 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 data obtained by HPLC: retention time 8.19 min (95.1% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylmethylene)-4-aminopentanamide (13a). Starting from 8a and following method B, 13a was obtained as a yellow solid (92.7 mg 88%). mp: 135−137 °C; 1H NMR (D2O): δ 1.62 (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 22.60 min (98.7% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenylethyl)phenyl)-4-aminopentanamide (13b). Starting from 8b and following method B, 13b was obtained as a yellow solid (26.9 mg, 8%). mp: 76−78 °C; 1H NMR (D2O): δ 1.62 (s, 4H, CH2(CH2)2CH2), 2.34 (t, J = 6.5 Hz, 2H, CH2CO), 2.85 (s, 4H, Ph(CH2)2Ph), 2.91 (t, J = 5.0 Hz, 2H, CH2NH), 7.08 (d, J = 8.0 Hz, 2H, Ar), 7.12 (d, J = 8.0 Hz, 2H, Ar), 7.19 (d, J = 8.0 Hz, 4H, Ar); 13C NMR (D2O): δ 26.0, 35.4, 35.8, 35.9, 39.0, 122.1, 125.8, 129.1, 130.0, 131.7, 134.5, 139.0, 141.8, 156.4, 174.7; HRMS (EI) m/z [M + H]+ calcd for C20H27N5O: 354.2292, found: 354.2294; purity data obtained by HPLC: retention time 23.59 min (98.1% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenyl ether)-4-aminopentanamide (13c). Starting from 8c and following method B, 13c was obtained as a brown solid (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 = 8.0 Hz, 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: retention time 21.84 min (95.4% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylmethanone)-4-aminopentanamide (13d). Starting from 8d and following method B, 13d was obtained as a yellow 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 = 8.0 Hz, 2H, Ar), 7.47 (d, J = 8.0 Hz, 2H, Ar), 7.52 (d, J = 8.0 Hz, 2H, Ar); 13 C 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 data obtained by HPLC: retention time 21.33 min (95.5% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylamine)-4-aminopentanamide (13e). Starting 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 = 4.5 Hz, 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 data obtained by HPLC: retention time 18.73 min (97.6% purity). 707



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yellow solid (64.2 mg, 36%). mp: 78−80 °C;1H NMR (D2O): δ 1.14 (s, 14H, (CH2)2(CH2)7(CH2)2), 1.48−1.54 (m, 4H, CH2CH2(CH2)7CH2CH2), 2.27 (t, J = 8.0 Hz, 2H,CH2CO), 2.83 (t, J = 8.0 Hz, 2H, CH2NH), 6.94−6.98 (m, 4H, Ar), 7.19 (d, J = 9.0 Hz, 2H, Ar), 7.30 (d, J = 8.5 Hz, 2H, Ar). 13C NMR (D2O): δ 25.2, 25.4, 26.5, 28.0, 28.2, 28.3, 28.3, 28.3, 28.4, 36.2, 39.4, 119.4, 119.7, 123.8, 128.0, 129.1, 132.8, 153.3, 156.5, 156.5, 176.0; HRMS (EI) m/z [M + H]+ calcd for C25H38N5O2: 440.3026, found: 440.3032; purity data obtained by HPLC: retention time 27.91 min (97.5% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylmethanone)-4-aminododecanamide (16d). Starting from 11d and following method B, 16d was obtained as a yellow solid (46.4 mg, 26%). mp: 60−62 °C; 1H NMR (D2O): δ 1.20 (s, 14H, (CH 2 ) 2 (CH 2 ) 7 (CH 2 ) 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: retention time 28.04 min (95.3% purity). P re pa r a ti o n o f D ih y d r o c h l o r i de Sa l t o f N - (4 - ( 4′ Guanidinophenyl)phenylamine)-4-aminododecanamide (16e). Starting from 11e and following method B, 16e was obtained as a black solid (29.3 mg, 10%). mp: 60−62 °C; 1H NMR (D2O): δ 1.22 (s, 14H, (CH2)2(CH2)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 obtained by HPLC: retention time 27.24 min (97.1% purity). DNA Thermal Denaturation Experiments. Thermal melting experiments were conducted with a Varian Cary 300 Bio spectrophotometer equipped with a 6 × 6 multicell temperaturecontrolled block. Temperature was monitored with a thermistor inserted into a 1 mL quartz cuvette containing the same volume of water as in the sample cells. Absorbance changes at 260 nm were monitored 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 sperm DNA was purchased from Sigma Aldrich (extinction coefficient ε260 = 6600 cm−1 M−1 base). Phosphate buffer solutions contained 10 mM Na2HPO4/NaH2PO4 adjusted to pH 7 were prepared using Millipore water. A quartz cell with a 1-cm path length was filled with a 1 mL solution of DNA polymer or DNA−compound complex. The DNA polymer (150 μM base) and the compound solution (15 μM) were prepared in the phosphate buffer, adjusted to pH 7) so that a compound to DNA base (P/D) ratio of 0.1 was obtained. The thermal melting temperatures of the DNA duplex or duplex−compound complex obtained from the first derivative of the melting curves are reported. Computational Methods. The systems have been optimized using the Gaussian0942 package at the B3LYP computational level with the 6-31+G* basis set. Frequency calculations have been performed at the same computational level to confirm that the resulting optimized structures are energetic minima (all positive frequencies). Effects of water solvation have been included by means of the SCFR-PCM approaches implemented in the Gaussian09 package including dispersion, repulsion, and cavitation energy terms of the solvent in the optimization. In Vitro Assays. Activity against T. b. rhodesiense STIB900 Strain. This stock was isolated in 1982 from a human patient in Tanzania and, after several mouse passages, cloned and adapted to axenic 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 added to each well of a 96-well microtiter plate. Serial drug dilutions of 11 3-

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). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenyl ether)-4-aminoundecanamide (15c). Starting from 10c and following method B, 15c was obtained as a yellow solid (59.5 mg, 99%). mp: 89−92 °C; 1H NMR (D2O): δ 1.18 (s, 12H, (CH2)2(CH2)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 = 8.5 Hz, 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 data obtained by HPLC: retention time 27.47 min (97.8% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylmethanone)-4-aminoundecanamide (15d). Starting from 10d and following method B, 15d was obtained as an orange solid (339.5 mg, 79%). mp: 71−73 °C. 1H NMR (D2O): δ 1.15 (s, 12H, (CH 2 ) 2 (CH 2 ) 6 (CH 2 ) 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 = 8.5 Hz, 2H, Ar), 7.64 (d, J = 8.5, 2H, Ar), 7.68 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (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 26.89 min (95.1% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylamino-4-aminoundecanamide (15e). Starting from 10e and following method B, 15e was obtained as a black oil (81.6 mg, 29%). 1H NMR (D2O): δ 1.25 (s, 12H, (CH2)2(CH2)6(CH2)2), 1.55−1.66 (m, 4H, 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; purity data obtained by HPLC: retention time 25.77 min (98.2% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylmethylene)-4-aminododecanamide (16a). Starting from 11a and following method B, 16a as a white solid (224.6 mg, 55%). mp: 114−116 °C; 1H NMR (D2O): δ 1.14 (s, 14H, (CH2)2(CH2)7(CH2)2), 1.47−1.55 (m, 4H, 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); 13C NMR (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 28.68 min (96.2% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenylethylene)-4-aminododecanamide (16b). Starting from 11b and following method B, 16b was obtained as a white solid (265.4 mg, 99%) mp: 116−117 °C; 1H NMR (D2O): δ 1.15 (s, 14H, (CH 2 ) 2 (CH 2 ) 7 (CH 2 ) 2 ), 1.50−1.54 (m, 4H, CH2CH2(CH2)7CH2CH2), 2.26 (t, J = 8.0 Hz, 2H, CH2CO), 2.84 (s, 6H, Ph(CH2)2Ph and CH2NH), 7.05−7.10 (m, 4H, Ar), 7.17 (d, J = 7.0 Hz, 4H, Ar); 13C NMR (D2O): δ 25.4, 25.6, 26.8, 28.2, 28.3, 28.5, 28.5, 28.5, 30.2, 35.9, 36.1, 36.4, 39.7, 122.0, 125.9, 129.2, 130.1, 131.9, 134.8, 138.8, 141.8, 156.5, 176.1; HRMS (EI) m/z [M + H]+ calcd for C27H42N5O: 452.3389, found: 452.3384; purity data obtained by HPLC: retention time 28.93 min (96.7% purity). P r e p ar a t i on of D i h y dr o c h l or i d e S a lt o f N - ( 4 - (4 ′ Guanidinophenyl)phenyl ether)-4-aminododecanamide (16c). Starting from 11c and following method B, 16c was obtained as a 708



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fold dilution steps covering a range from 100 to 0.002 μg mL−1 were prepared. Then 4 × 103 bloodstream forms of T. b. rhodesiense STIB900 in 50 μL were added to each well, and the plate was incubated at 37 °C under a 5% CO2 atmosphere for 70 h. A 10 μL resazurin solution (resazurin, 12.5 mg in 100 mL of double-distilled water) was then added to each well and incubation continued for a further 2−4 h.44 Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC50 values were calculated by linear regression45 from the sigmoidal dose−inhibition curves using SoftmaxPro software (Molecular Devices Cooperation). Melarsoprol was the reference drug used. Activity against P. falciparum NF54 Strain. In vitro activity against erythrocytic stages of P. falciparum was determined using a 3Hhypoxanthine incorporation assay,46,47 using the drug-sensitive NF54 strain (Schipol Airport, The Netherlands48) and the standard drug chloroquine (Sigma C6628). Compounds were dissolved in DMSO at 10 mg mL−1 and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/ L), NaHCO3 (2.1 g L−1), neomycin (100 U mL−1), Albumax (5 g L−1), and washed human red cells A+ at 2.5% hematocrit (0.3% parasitaemia). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg mL−1 were prepared. The 96well plates were incubated in a humidified atmosphere at 37 °C; 4% CO2, 3% O2, 93% N2. After 48 h, 50 μL of 3H-hypoxanthine (=0.5 μCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland), and the red blood cells were transferred onto a glass fiber filter and then washed with distilled water. The dried filters were inserted into a plastic foil with 10 mL of scintillation fluid and counted in a Betaplate liquid scintillation counter (Wallac). IC50 values were calculated from sigmoidal inhibition curves by linear regression24 using Microsoft Excel. Cytotoxicity with L6 Cells. Assays were performed in 96-well microtiter plates, each well containing 100 μL of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum, and 4000 L6 cells (a primary cell line derived from rat skeletal myoblasts).49,50 Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg mL−1 were prepared. After 70 h of incubation, the plates were inspected under an inverted microscope to ensure growth of the controls and sterile conditions. Ten microliters of resazurin solution was then added to each well, and the plates were incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC50 values were calculated by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxin was the reference drug used.

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ACKNOWLEDGMENTS

This work has been funded by the SFI−RFP grant CHE275, a Trinity College Dublin postgraduate award (C.M.K.), and a HEA PRTLI Cycle 4 grant (C.M.K.). We thank Dr. Padraic Nagle for his help with the biophysical experiments. We thank Monica Cal and Joelle Jourdan from the Swiss Tropical Institute, Socinstrasse, 57, CH-4002 Basel, Switzerland, for performing the biological tests.

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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,Ndiisopropylethylamine; HB, hydrogen bond; DFT, density functional theory; ΔTm, change in thermal melting temperature; P/D, phosphate to drug ratio; IC50, half-maximal inhibitory concentration; DHFR-TS, dihydrofolate reductase thymidine synthetase

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REFERENCES

(1) Bell, A.; Kittler, L.; Lober, G.; Zimmer, C. DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction. J. Mol. Recog. 1997, 10, 245−255. (2) Baraldi, P. G.; Bovero, A.; Fruttarolo, F.; Preti, D.; Tabrizi, M. A.; Pavani, M. G.; Romagnoli, R. DNA minor groove binders as potential antitumor and antimicrobial agents. Med. Res. Rev. 2004, 24, 475−528. (3) Lanteri, C. A.; Trumpower, B. L.; Tidwell, R. R.; Meshnick, S. R. DB75, a novel trypanocidal agent, disrupts mitochondrial function in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 2004, 48, 3968−3974. (4) Rodriguez, F.; Rozas, I.; Kaiser, M.; Brun, R.; Nguyen, B.; Wilson, W. D.; Nelson, G.; Dardonville, C. New bis(2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity. J. Med. Chem. 2008, 51, 909−923. (5) Laugthton, C. A.; Tanious, F.; Nunn, C. M.; Boykin, D. W.; Wilson, W. D.; Neidle, S. A Crystallographic and spectroscopic study of the complex between d(CGCGAATTCGCG)2 and 2,5-bis(4guanylphenyl)furan, an analogue of berenil. Structural origins of enhanced DNA-binding affinity. Biochemistry 1996, 35, 5655−5661. (6) (a) Boykin, D. W.; Kumar, A.; Xiao, G.; Wilson, W. D.; Bender, B. C.; McCurdy, D. R.; Hall, J. E.; Tidwell, R. R. 2,5-Bis[4-(Nalkylamidino)phenyl]furans as anti-Pneumocystis carinii agents. J. Med. Chem. 1998, 41, 124−129. (b) Boykin, D. W.; Kumar, A.; Spcychala, J.; Zhou, M.; Lombardy, R. J.; Wilson, W. D.; Dykstra, C. C.; Jones, S. K.; Hall, J. E. Dicationic diarylfurans as anti-Pneumocystis carinii agents. J. Med. Chem. 1995, 38, 912−916. (7) Glass, S. L.; Nguyen, B.; Goodwin, K. D.; Dardonville, C.; Wilson, W. D.; Long, E. C.; Georgiadis, M. M. Crystal structure of a trypanocidal 4,4′-bis(2-imidazolinylamino) diphenylamine bound to DNA. Biochemistry 2009, 48, 5943−5952. (8) Tidwell, R.; Boykin, D. Minor groove binders as antimicrobial agents. Small molecule DNA and RNA binder: synthesis to nucleic acid complexes; Wiley-VCH: New York, 2003; pp 416−460. (9) Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F. A.; Wilson, W. D.; Boykin, D. W. Synthesis and antiprotozoal activity of azaanalogues of furamidine. J. Med. Chem. 2003, 46, 4761−4769. (10) Arafa, R. K.; Brun, R.; Wenzler, T.; Tanious, F. A.; Wilson, W. D.; Stephens, C. E.; Boykin, D. W. Synthesis, DNA affinity, and antiprotozoal activity of fused ring dicationic compounds and their prodrugs. J. Med. Chem. 2005, 48, 5480−5488. (11) Dardonville, C.; Barrett, M. P.; Brun, R.; Kaiser, M.; Tanious, F.; Wilson, W. D. DNA binding affinity of bisguanidine and bis(2aminoimidazoline) derivatives with in vivo antitrypanosomal activity. J. Med. Chem. 2006, 49, 3748−3752.

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Preparation and spectroscopic data of Boc-protected monoguanidines, Boc-protected aminoalkyl carboxylic acids, aminoalkyl derivatives of Boc-protected diaromatic mono-guanidines, and spectroscopic data of final hydrochloride salts. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Aminoalkyl Derivatives of Guanidine Diaromatic Minor Groove Binders

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