Introduction

Aromatic diamidines and related dicationic molecules have been extensively studied for over 50 years as a consequence of the broad spectrum antimicrobial activity reported for these molecules.¹ Despite these efforts, pentamidine (1), first reported in 1942,² is the only compound belonging to this class that has found significant human use. Pentamidine is currently used against first-stage human African trypanosomiasis (HAT) and antimony-resistant leishmaniasis and also as a secondary drug for AIDS-related Pneumocystis jirovecii pneumonia (PCP).¹ An orally effective prodrug of furamidine (2) is currently in phase II clinical trials against malaria, HAT, and PCP and represents a different approach for administration of dicationic molecules.^1,3-6 A key step in the mode of action of these types of dicationic molecules is thought to be binding in the minor groove of DNA at AT-rich sites.¹ It has been postulated that minor groove binding leads to inhibition of DNA-dependent enzymes or possibly direct inhibition of transcription.^1,7-10

A fundamental element in the design of new potential therapeutics of this type has been that the molecular unit that bears the amidine groups should have a complementary shape to that of the curve of the minor groove of DNA.^11,12 Most of these crescent-shaped molecules consist of unfused aryl and heteroaryl rings linked by various connectors that may result in conjugated or nonconjugated systems (cf. furamidine and pentamidine in Figure 1). Nevertheless, not all minor groove binders have the more commonly encountered crescent shape. Recently, a more linear molecule, 3 (CGP 40215A), has been found to bind strongly to the minor groove of DNA and to exhibit excellent antitrypanosomal activity.^13-15 Thorough investigations of fused ring dicationic carbazoles, benzofurans, and benzothiophenes (4), some being more linear molecules, revealed several highly active antimicrobial agents.^16-18 Detailed biophysical studies demonstrated that these types of molecules bind in the minor groove at AT sites by a new type of minor groove binding complex.^19,20 As part of a program focused on the development of novel dicationic antiprotozoan agents, we have now investigated introduction of guanidine and "reversed amidine" cationic centers on some other fused ring frameworks. Earlier studies from these laboratories have demonstrated that the attachment of these cationic centers to classical groove binding frameworks, furamidine-like, can lead to effective antimicrobial agents.^21,22 Others have shown that benzguanidines connected by alkyl and related linkers also show promising antiprotozoal activity.²³

Figure 1 Structures of key dicationic antimicrobial agents.

The pK of aromatic amidines and guanidines is greater than 10, and as a result, these type molecules are charged at physiological pH.^24,25 The consequent lack of oral bioavailability of dicationic analogues is well documented.¹ Despite the complexity of metabolism sometimes encountered with prodrugs, they offer an approach to circumvent physicochemical problems inherent with certain classes of molecules.^26-28 Since, in general, an oral dosing regime is preferred over other routes of administration, we also report in this publication the exploration of N-alkoxy and carbamate prodrug approaches for the guanidine analogues. We have previously demonstrated that amidoximes and O-alkylamidoximes can function as effective prodrugs for aryl diamidines.^28-31 Similarly, carbamates were also shown to be effective prodrugs for diamidines.³² The study of potential prodrugs of guanidines is limited.^33-36 They are thought to be bioconverted by general esterases,^32-36 and as far as we have been able to determine, this is the first report of prodrugs for diguanidine analogues.

Chemistry

The synthesis of the diguanidino (9a-d and 12a-c) and reversed diamidino (13a,b) derivatives of a variety of fused ring systems, viz. fluorene, fluorenone, anthraquinone, and acridine, was achieved starting with the diamino derivatives of tricyclic fused ring pharmacophores (Schemes 1-3). These key precursors were either used directly as purchased or synthesized according to the literature methods by either catalytic or Zinin reduction of the nitro counterparts (Scheme 1).³⁷ The not previously described reduction (H₂/Pd/C) of 2,7-dinitro-9H-fluorene to the diamino derivative (7a) was achieved in high yield. As depicted in Scheme 1, synthesis of the guanidines (9a-d) was accomplished in two successive steps. First, the diamino derivatives (7a-d) were converted to the Boc-protected bisguanidines (8a-d) by the HgCl₂-promoted reaction with bis-Boc-protected S-methylpseudothiourea. The subsequent step was the HCl-assisted deprotection of the Boc-protected guanidines, providing the guanidine hydrochloride salts 9a-d.^38-40

Scheme 1. Guanidino Derivatives of Fused Ring Systems

Scheme 2 depicts the synthesis of N-substituted guanidines (12a-e), as well as the corresponding carbamate prodrugs (11a-g) of several of the synthesized DNA-binding dications. Starting with the appropriate diamine, reaction with ethyl isothiocyanatoformate gave the carbamoyl thioureas (10a,b), which were further reacted with various amines to furnish the carbamoyl guanidines (11a-g).^41-43 These synthetic intermediates (11a-g) may function as prodrugs for the final guanidines. The last step of decarbamoylation was performed under basic conditions to obtain the N-alkylguanidines (12a-c) as well as the N-alkoxyguanidines (12d) and (12e).

Scheme 2. Synthesis of Fused Ring System N-Substituted Dicationic Guanidines and Their Potential Prodrugs

The ¹H NMR and the ¹³C NMR data for the carbamoyl guanidine precursors of the N-alkoxyguanidines (11f,g) showed two sets of peaks for all the aliphatic and the aromatic functionalities present in the molecule, indicating the presence of a mixture of apparently two inseparable compounds that were otherwise pure. Mixtures were not detected for the other carbamates (11a-e). The apparent mixtures for 11f and 11g were directly subjected to alkaline hydrolysis yielding the corresponding N-alkoxyguanidines (12d,e) as single isomers, as demonstrated by their ¹H NMR and ¹³C NMR and other data. Thus, it can be concluded that the carbamoyl N-alkoxyguanidines (11f,g) likely exist in isomeric forms, possibly syn and anti rotamers arising from restricted rotation around the N-C bond of the carbamate (Figure 2).^44-46 Another plausible explanation is the presence of other tautomeric forms with the carbamoyl group and the alkoxy group on one or the other of the sp³ and sp² terminal nitrogens of the guanidino moiety. Similar isomeric mixtures for carbamoyl N-alkoxyguanidines have been observed in related examples.⁴⁵

Figure 2 Proposed structures for two possible carbamoyl N-alkoxyguanidine isomers.

Finally, reversed diamidino derivatives of 2,7-diaminofluorene were prepared according to the reported method^22,23,47 (Scheme 3) applying the reaction with the nonodoriferous reagent phenyl S-(2-naphthylmethyl)thioimidate or 2-pyridyl S-(2-naphthylmethyl)thioimidate, thus providing 13a or 13b, respectively. The corresponding reaction with the less basic diamines 7b-d failed. It is worth noting that the free bases of these compounds, being sparingly water-soluble, were converted to their hydrochloride salts to improve their aqueous solubility.

Scheme 3. 9H-Fluorene Reversed Diamidines

Biological Results

Relative Binding Affinity. T_m Measurements. T_m increases for compound complexes relative to uncomplexed DNA (T_m) at specific DNA sequences provide an excellent method for ranking compound binding affinities.¹ The T_m increases caused by the compounds in Table 1 have been determined for their complexes with an AT polymer. The charged compounds have T_m values in the range 6.1-22 C (Table 1). The uncharged prodrugs, as expected, do not increase the DNA T_m within experimental error. The T_m values for the fused ring dications are modest compared to that of furamidine but are generally in the range of that of pentamidine. Introduction of N-alkyl groups on the guanidine moiety (12a-c) resulted in a slight decrease in the T_m values, whereas introduction of aryl groups increased the values, as in the case of the reversed diamidines (13a,b). A similar effect was previously noted for N-substitution in unfused triaryl systems.^21,22 While the T_m value for the fluorene parent diguanidine (9a) is significantly lower than that of furamidine, it is similar to that of dicationic carbazoles, benzofurans, and benzothiophenes.^16-20 Comparison of the T_m values of 9a to those of the corresponding fluorenone 9b, anthraquinone 9c, and acridine 9d diguanidines shows a reduction in affinities as a result of the presence of a carbonyl group and six-membered rings in the central core of the fused ring system. The decline in T_m values may reflect steric clash of the central core of these other systems with the floor of the groove.

Binding Mode: Circular Dichroism (CD) Spectroscopy. Binding of 9a, 13a, 9b, and 12a to poly(d(A-T)₂) was characterized by CD spectroscopy in the wavelength range between 220 and 420 nm (Figure 3). The CD spectra monitor the asymmetric environment of the compounds when bound to DNA and therefore can be used to obtain information on the binding mode. The free compounds do not have CD spectra, but they have induced CD when bound to DNA. Minor groove binding is typically characterized by a strong positive CD signal at the maximum absorbance wavelength of the bound compound. Intercalation binding is generally characterized by weak CD signals that are usually negative but can be positive. Addition of 9a to DNA results in substantial positive CD signals between 270 and 335 nm, where the compound absorbs. Isoelliptic points are observed in the titration of 9a with DNA at 271 and 253 nm with large positive induced CD signals at 293 and 318 nm. A strong positive CD signal, centered at 330 nm, is also observed for the 13a-DNA complex with isoelliptic points at 288, 253, and 233 nm. A positive CD signal for the 9a complex is found at 263 nm where the compound absorbance overlaps with the DNA spectrum. An isoelliptic point is observed in the titration of 12a with DNA at 278 nm. Although the positive induced CD is characteristic of minor groove binding, the overlap of compound and DNA spectra prevents definitive conclusions for this compound. Strong positive CD signals arise at 280, 293, and 316 nm for 12a with isoelliptic points at 266, 253, and 224 nm. The large induced CD signals for the 9a-DNA, 13a-DNA, and 12a-DNA complexes suggest that they bind in the minor groove at AT DNA sequences. The results for 9b are consistent with minor groove binding, but spectral overlap complicates the interpretation from CD.

Figure 3 CD spectral titrations of 9a, 13a, 9b, and 12a with poly(d(A-T)2). Positive induced CD signals are seen for the compound-DNA complexes, above 300 nm for 9a, 13a, and 12a. The 9b spectrum overlaps with that of DNA near 260 nm. The small molecules exhibit no CD spectra but on binding to DNA show induced CD. A spectrum for DNA without compound is shown for reference and has no CD signal above 300 nm (red). The ratios of compounds to DNA base pairs are 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, and 0.50 as indicated by the arrow direction in the figures. The experiments were conducted in MES10, and the DNA concentration was 2 × 10-5 M in base pairs.

The preliminary biophysical studies with these dicationic molecules suggest that they bind in the DNA minor groove, similar to the carbazoles.^19,20 As has been previously noted for the prodrugs, these molecules are essentially uncharged (because of the lower pK) and do not bind significantly to DNA.^28,31

In Vitro Activities. Many of the diguanidines and the N-alkyl diguanidines show promising in vitro activity against both Trypanosoma brucei rhodesiense (9a,b and 12a-c) and Plasmodium falciparum (9a,c and 12a-12c) (Table 1) with IC₅₀ values ranging from 2 to 40 nM. The number of dicationic compounds^1,21,31 in the STI screen that have shown IC₅₀ values versus Plasmodium falciparum of less than 5 nM is quite small (approximately 30), which places 9a in the category of very active compounds. In the fluorene series, the addition of an N-alkyl group (12a-c) reduces the antiprotozoan activity and the activity declines with increasing alkyl size. Generally, the reversed diamidines in the fluorene series (13a,b) and the guanidines with a six-membered central ring (9c,d) show significantly diminished activity. The prodrugs, as expected, show essentially no in vitro activity. Selectivity for the parasites is generally quite good as judged from their low toxicity for L-6 cells (Table 1).

A general characteristic that has been noted for numerous dicationic minor groove binding systems is the lack of a direct correlation between DNA affinity and in vitro activities.^1,21,31 Some minimum threshold DNA affinity, which varies with class, seems to be required to observe activity because compounds that do not bind show negligible antimicrobial activity. High DNA affinity does not always lead to a correspondingly high antimicrobial activity; however, very low DNA affinity results in loss of biological activity.^1,21,31 A similar result is apparent for the fused ring dications.

In Vivo Activities. Given their high antiparasitic activity and low cell toxicity, several of the diguanidines were advanced to the rigorous STIB900 animal model for African trypanosomiasises (Table 2). Despite the quite good in vitro activity (IC₅₀ = 24 nM), the diguanidinofluorene 9a was inactive on ip dosage. In contrast, the diguanidinofluorenone 9b and the acridine 9d analogues showed promising activity, providing 2/4 cures in this model. The N-alkylfluorene compounds (12a-12c) exhibited excellent in vivo activity, providing 3/4, 5/7, and 4/4 cures, respectively. Interestingly 12b, while showing good activity, apparently was also toxic because one animal death was noted on the second dosing in two different experiments. The toxicity is acute, but the origin is unknown.

The utility of N-alkoxy analogues of diamidines as prodrugs has been quite clearly demonstrated,^28-31 and we prepared 12d and 12e to test this approach with diguanidines. When these two molecules were evaluated on oral dosing in the STIB900 mouse model, both were found to be ineffective. Further work is required to determine if this result is due to lack of uptake or lack of bioconversion to the parent drug.

The potential prodrug 11a was also evaluated in the STIB900 model to determine if carbamates are useful as a means to improve oral bioavailability of the diguanidino compounds. While the prodrugs 11a, 11d, and 11e failed to provide cures, they did exhibit a 3-fold or greater increase in survival time of the test animals compared to ip dosing of the parent molecules and thus showed that carbamates of diguanidino analogues can potentially serve as useful prodrugs. The value of carbamate prodrugs is more clearly demonstrated by the results for the N-methyl carbamate (11c), which gave 4/4 cures on oral administration in this model. Unfortunately, the compound did not yield cures in the chronic mouse model (central nervous system involvement) for African trypanosomiasis (data not presented).

In conclusion, this work demonstrates that diguanidino fused ring systems are potential candidates as antiprotozoan agents. We have shown, for the first time, that carbamate derivatives of diguanidines, which are inactive in vitro and effective in vivo, can function as prodrugs. Other carbamates are being explored with the goal of optimizing oral efficacy of these diguanidines.

Experimental Section

Absorbance Spectroscopy and Thermal Melting (T_m) Experiments. Experiments were done in MES10 buffer (0.01 M MES (2-(N-morpholino)ethanesulfonic acid), 0.001 M ethylenediaminetetraacetic acid (EDTA), and 0.1 M NaCl with the pH was adjusted to 6.25). DNA polymers were purchased from Pharmacia and characterized by their melting curves. Absorbance and thermal melting experiments were done using a Cary 300 Bio spectrophotometer with the software supplied with the instrument. For absorbance measurements, the buffer was scanned from 400 to 250 nm in 1 cm quartz cuvettes, aliquots of concentrated stock solutions of the compounds were titrated into the buffer, and the solutions were rescanned. The concentrations of the compounds were 1 × 10^-5, 2 × 10^-5, and 3 × 10^-5 M. For thermal melting the concentration of the DNA was about 1 × 10^-4 in bases and the ratio of the compound to DNA bases was 0.3.

Circular Dichroism. CD spectra were obtained on Jasco J-810 spectrometer in MES buffer. The software supplied by Jasco provided instrument control, data acquisition, and manipulation. DNA solutions in MES10 buffer were scanned in 1 cm quartz cuvettes. Aliquots of concentrated stock solutions of the compounds were titrated into DNA to give the desired ratio, and the complexes were rescanned.

Synthetic Protocols. Melting points were recorded using a Thomas-Hoover (Uni-Melt) capillary melting point apparatus and are uncorrected. TLC analysis was carried out on silica gel 60 F₂₅₄ precoated aluminum sheets and detected under UV light. ¹H and ¹³C NMR spectra were recorded employing a Varian GX400 or Varian Unity Plus 300 spectrometer, and chemical shifts () are in ppm relative to TMS as internal standard. Mass spectra were recorded on a VG analytical 70-SE spectrometer (EI) or a ThermoFinningan LCQ MSD (ESI). Elemental analyses were obtained from Atlantic Microlab Inc. (Norcross, GA). Some compounds were analyzed correctly for fractional moles of dichloromethane, water, and/or ethanol of solvation. In each case ¹H NMR showed the presence of the indicated solvent(s). All chemicals and solvents (including anhydrous solvents) were purchased from Aldrich Chemical Co. or Lancaster Synthesis and used as purchased. Acetonitrile and triethylamine were distilled from CaH₂. Synthesis of the bis-aminofluorenone (7b) and bis-aminoanthraquinone (7c) was achieved as described in Scheme 1 according to the literature.³⁷ S-(2-Naphthylmethyl)thioacetimidate was prepared by adapting the reported procedure.⁴⁶

2,7-Diamino-9H-fluorene (7a) (Scheme 1). To a suspension of 2,7-dinitro-9H-fluorene (5 g, 19.5 mmol) in EtOAc (50 mL) and EtOH (50 mL) was added Pd/C (1.25 g). The reaction mixture was shaken under hydrogen (55 psi) for 6 h, after which the reaction mixture was filtered through a pad of Celite. The filtrate was eveporated to dryness to give off-white shiny crystals that needed no further purification (3.82 g, quantitative): mp 159-60 C, ¹H NMR (DMSO-d₆) 3.56 (s, 2H), 4.89 (br s, 4H), 6.47 (d, J = 8.1 Hz, 2H), 6.67 (s, 2H), 7.24 (d, J = 8.1 Hz, 2H).

Preparation of Bis(N',N' '-di-Boc-guanidino) Derivatives (General Procedure) (Scheme 1). 2,7-Bis(N',N' '-di-Boc-guanidino)-9H-fluorene (8a). To a solution of 2,7-diaminofluorene (7a) (0.49 g, 2.5 mmol) in anhydrous DMF (15 mL) was added 1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea (1.52 g, 5.25 mmol), triethylamine (1.52 g, 15 mmol), and mercury(II) chloride (1.56 g, 5.75 mmol). The suspension was kept stirring at room temperature overnight. The reaction mixture was diluted with CH₂Cl₂, washed with Na₂CO₃ solution, and filtered through a pad of Celite. The organic layer was washed with water (3×) followed by brine and then dried over anhydrous Na₂SO₄. After evaporation of the solvent, the obtained residue was crystallized from CH₂Cl₂/MeOH, giving a light-yellow solid (1.15 g, 68%): mp >340 C; ¹H NMR (CDCl₃) 1.52, 1.54 (2s, 36H), 3.91 (s, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.90 (s, 2H), 10.43 (br s, 2H), 11.68 (br s, 2H); ¹³C NMR (CDCl₃) 163.6, 153.5, 153.4, 144.2, 138.1, 135.3, 120.9, 119.7, 118.9, 83.7, 79.6, 37.2, 28.2, 28.1. Anal. Calcd for C₃₅H₄₈N₆O₈ (680.79): C, 61.75; H, 7.11; N, 12.34. Found: C, 61.50; H, 7.11; N, 12.36.

2,7-Bis(N',N' '-di-Boc-guanidino)fluoren-9-one (8b). Orange solid (1.16 g, 65%), mp >340 C; ¹H NMR (CDCl₃) 1.51, 1.54 (2s, 36H), 7.42 (d, J = 8.4 Hz, 2H), 7.77 (s, 2H), 7.82 (d, J = 8.4 Hz, 2H), 10.46 (br s, 2H), 11.63 (br s, 2H); ¹³C NMR (CDCl₃) 192.7, 163.3, 153.5, 153.3, 140.4, 137.4, 135.2, 128.0, 120.6, 118.6, 84.0, 79.9, 28.1, 28.0. Anal. Calcd for C₃₅H₄₆N₆O₉· 0.1CH₂Cl₂ (703.26): C, 59.94; H, 6.62; N, 11.95. Found: C, 59.69; H, 6.67; N, 12.13.

2,7-Bis(N',N' '-di-Boc-guanidino)anthraquinone (8c). Yellow solid (1.24 g, 82%), mp >340 C; ¹H NMR (CDCl₃) 1.54, 1.56 (2 s, 36H), 8.24 (s, 2H), 8.30 (d, J = 8.7 Hz, 2H), 8.43 (d, J = 8.7 Hz, 2H), 10.82 (br s, 2H), 11.62 (br s, 2H); ¹³C NMR (CDCl₃) 182.5, 181.2, 163.1, 153.2, 142.4, 134.5, 129.5, 128.9, 126.7, 119.0, 84.4, 80.3, 28.1, 28.0. Anal. Calcd for C₃₆H₄₆N₆O₁₀· 0.5CH₂Cl₂ (765.25): C, 57.28; H, 6.19. Found: C, 57.30; H, 6.09.

3,6-Bis(N',N' '-di-Boc-guanidino)acridine (8d). Canary-yellow fluffy solid (0.88 g, 73%), mp >340 C; ¹H NMR (CDCl₃) 1.53, 1.55 (2s, 36H), 7.78 (dd, J = 9.0, 2.1 Hz, 2H), 7.9 (d, J = 9.0 Hz, 2H), 8.45 (d, J = 2.1 Hz, 2H), 8.6 (s, 1H), 10.69 (s, 2H), 11.68 (s, 2H); ¹³C NMR (CDCl₃) 163.5, 153.4, 153.3, 149.9, 138.5, 135.1, 128.8, 123.8, 122.3, 118.9, 83.9, 79.7, 28.2, 28.1. Anal. Calcd for C₃₅H₄₇N₇O₈·0.1CH₂Cl₂ (702.28): C, 60.02; H, 6.77; N, 13.96. Found: C, 59.97; H, 6.88; N, 13.90.

Deprotection of N',N' '-Di-Boc-guanidines (General Procedure) (Scheme 1). 2,7-Bis-guanidino-9H-fluorene Dihydrochloride (9a). The N',N' '-di-Boc-guanidine (8a) (0.25 g, 0.4 mmol) was dissolved in CH₂Cl₂ (10 mL) and diluted with dry EtOH (15 mL), and the chilled solution was saturated with dry HCl. The reaction mixture was then kept stirring at room temperature for 3 days (drying tube), where a precipitate of the product started forming over time. After evaporation of the solvent to dryness, the residue was washed with ether multiple times and was dried under reduced pressure at 50-60 C overnight to give a whitish-yellow solid of the bis-guanidine dihydrochloride (0.13 g): mp >340 C; ¹H NMR (DMSO-d₆) 3.95 (s, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.45 (s, 2H), 7.58 (br s, 8H), 7.95 (d, J = 8.4 Hz, 2H), 10.23 (br s, 2H); ¹³C NMR (DMSO-d₆) 156.1, 144.6, 138.6, 133.9, 123.4, 121.4, 120.9, 36.9; MS (EI) m/z (rel intens) 281 (M⁺ + 1, 5), 252 (100). Anal. Calcd for C₁₅H₁₆N₆·2HCl·0.25C₂H₅OH (364.76): C, 51.04; H, 5.39; N, 23.04, Cl, 19.44. Found: C, 50.74; H, 5.26; N, 22.99, Cl, 19.87.

2,7-Bis-guanidinofluoren-9-one Dihydrochloride (9b). Green solid (0.26 g), mp >340 C; ¹H NMR (DMSO-d₆) 7.43-7.46 (m, 4H), 7.68 (br s, 8H), 7.86 (d, J = 8.4 Hz, 2H), 10.25 (br s, 2H); ¹³C NMR (DMSO-d₆) 191.58, 156.0, 140.9, 136.5, 134.6, 130.8, 122.4, 119.9; MS (EI) m/z (rel intens) 295 (M⁺ + 1, 23), 278 (100). Anal. Calcd for C₁₅H₁₄N₆O·2HCl·0.35H₂O (373.53): C, 48.23; H, 4.51; N, 22.49, Cl, 18.95. Found: C, 48.51; H, 4.55; N, 22.13, Cl, 18.93.

2,7-Bis-guanidinoanthraquinone Dihydrochloride (9c). Orange-red solid (0.22 g), mp >340 C (dec); ¹H NMR (DMSO-d₆) 7.75 (d, J = 8.4 Hz, 2H), 7.97 (s, 2H), 8.06 (br s, 8H), 8.24 (d, J = 8.4 Hz, 2H), 10.87 (br s, 2H); ¹³C NMR (DMSO-d₆) 181.6, 180.4, 155.6, 142.1, 134.2, 129.3, 128.9, 127.6, 119.4; MS (EI) m/z (rel intens) 323 (M⁺ + 1, 100), 162 (59). Anal. Calcd for C₁₆H₁₄N₆O₂·2HCl·1.66H₂O (425.15): C, 45.20; H, 4.58; N, 19.77. Found: C, 45.24; H, 4.58; N, 19.47.

3,6-Bis-guanidinoacridine Trihydrochloride (9d). Orange solid (0.33 g), mp >340 C; ¹H NMR (DMSO-d₆) 7.75 (dd, J = 8.4, 2.1 Hz, 2H), 7.96 (d, J = 2.1 Hz, 2H), 8.04 (br s, 8H), 8.23 (d, J = 8.4 Hz, 2H), 8.41 (s, 1H), 10.83 (br s, 2H); ¹³C NMR (DMSO-d₆) 181.6, 180.5, 155.6, 142.1, 134.2, 129.3, 128.9, 127.7, 119.4. Anal. Calcd for C₁₅H₁₅N₇·3HCl·C₂H₅OH·0.33H₂O (454.72): C, 44.90; H, 5.46; N, 21.56, Cl, 23.39. Found: C, 45.08; H, 5.10; N, 21.48, Cl, 23.49.

Preparation of Carbamoyl Thiourea Derivatives (Scheme 2). 2,7-Bis(N'-ethoxycarbonylthiourea)-9H-fluorene (10a). A solution of 2,7-diamino-9H-flourene (7a) (1 g, 5.1 mmol) in CH₂Cl₂ (10 mL), to which was added ethyl isothiocyanatoformate (1.47 g, 11.2 mmol), was stirred at room temperature overnight. After flash chromatography, the reaction mixture was diluted with hexane and the precipitate formed was collected and dried to yield the bis-carbamoylthiourea as a light-brown solid (2.32 g, quantitative): mp >340 C (dec); ¹H NMR (DMSO-d₆) 1.27 (t, J = 6.9 Hz, 6H), 3.95 (s, 2H), 4.23 (q, J = 6.9 Hz, 4H), 7.75 (d, J = 8.4 Hz, 2H), 7.86-7.89 (m, 4H), 11.28 (s, 2H), 11.64 (s, 2H); ¹³C NMR (DMSO-d₆) 178.1, 153.4, 143.7, 138.2, 136.5, 123.4, 121.8, 119.7, 61.9, 36.8, 14.1; MS (EI) m/z (rel intens) 459 (M⁺ + 1, 18), 374.1 (8), 328 (100), 319 (8), 151 (12). Anal. Calcd for C₂₁H₂₂N₄O₄S₂ (458.56): C, 55.00; H, 4.83. Found: C, 55.21; H, 4.83.

2,7-Bis(N'-ethoxycarbonylthiourea)fluoren-9-one (10b). To a suspension of 2,7-diaminofluorenone (7b) (0.3 g, 1.4 mmol) in toluene (10 mL) was added ethyl isothocyanatoformate (0.41 g, 3.1 mmol). The reaction mixture was heated at reflux for 10 h. After cooling to room temperature, the reaction mixture was diluted with hexanes. The orange precipitate obtained was filtered off and crystallized from aqueous EtOH (0.66 g, 98%): mp >340 C (dec); ¹H NMR (DMSO-d₆) 1.26 (t, J = 7.2 Hz, 6H), 4.22 (q, J = 7.2 Hz, 4H), 7.69 (dd, J = 8.1, 1.8 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 1.8 Hz, 2H), 11.39 (s, 2H), 11.60 (s, 2H); ¹³C NMR (DMSO-d₆) 191.6, 178.7, 153.4, 140.8, 139.0, 133.8, 130.9, 121.2, 120.2, 62.0, 14.0. Anal. Calcd for C₂₁H₂₀N₄O₅S₂·H₂O (490.55): C, 51.41; H, 4.52. Found: C, 51.27; H, 4.54.

Preparation of N-Substituted Carbamoyl Guanidines (General Procedure) (Scheme 2). 2,7-Bis(N'-ethoxycarbonyl)guanidino-9H-fluorene (11a). A stirred solution of carbamoyl thiourea (10a) (0.58 g, 1.26 mmol), ammonia (0.5 M solution in dioxane) (10 mL, 5.05 mmol), and diisopropylethylamine (0.98 g, 7.56 mmol) in anhydrous CH₂Cl₂ (10 mL) was cooled to 0 C. EDCI (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride) (0.96 g, 5.05 mmol) was added, and the solution was stirred at room temperature overnight. The reaction mixture was washed with water (3×) followed by brine and dried over anhydrous Na₂SO₄. The residue remaining after removal of the solvent was crystallized from EtOH/water (0.45 g, 84%): mp >340 C; ¹H NMR (DMSO-d₆) 1.16 (t, J = 7.2 Hz, 6H), 3.87 (s, 2H), 3.98 (q, J = 7.2 Hz, 4H), 7.33 (d, J = 8.4 Hz, 2H), 7.54 (br s, 4H), 7.65 (s, 2H), 7.72 (d, J = 8.4 Hz, 2H), 9.13 (br s, 2H); ¹³C NMR (DMSO-d₆) 163.1, 159.0, 143.4, 136.9, 136.3, 120.4, 119.4, 118.4, 59.4, 36.4, 14.4. Anal. Calcd for C₂₁H₂₄N₆O₄·0.5C₂H₅OH (447.48): C, 59.04; H, 6.08; N, 18.78. Found: C, 58.96; H, 5.74; N, 18.88.

2,7-Bis(N'-ethoxycarbonyl)guanidinofluoren-9-one (11b). Brick-red solid (0.35 g, 75%), mp >340 C; ¹H NMR (DMSO-d₆) 1.17 (t, J = 6.9 Hz, 6H), 4.00 (q, J = 6.9 Hz, 4H), 7.47 (dd, J = 8.1, 1.8 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 1.8 Hz, 2H), 9.31 (br s, 2H); ¹³C NMR (DMSO-d₆) 192.9, 162.6, 158.6, 139.6, 138.4, 134.1, 126.5, 120.8, 116.7, 59.9, 14.6. Anal. Calcd for C₂₁H₂₂N₆O₅·0.15C₂H₅OH·0.5H₂O (454.35): C, 56.30; H, 5.30; N, 18.49. Found: C, 56.54; H, 5.10; N, 18.47.

2,7-Bis(N'-ethoxycarbonyl-N' '-methyl)guanidino-9H-fluorene (11c). With the same procedure for preparation of 11a, methylamine (2 M solution in THF) was used for the transformation of the thiourea compound (10a) into the N-substituted guanidine 11c. The reaction yielded an off-white solid (0.53 g, 93%): mp 157-8 C; ¹H NMR (DMSO-d₆) 1.15 (t, J = 7.2 Hz, 6H), 2.83 (s, 6H), 3.90 (s, 2H), 3.94 (q, J = 7.2 Hz, 4H), 7.32 (d, J = 8.1 Hz, 2H), 7.54 (s, 2H), 7.83 (d, J = 8.1 Hz, 2H); ¹³C NMR (DMSO-d₆) 161.9, 157.9, 143.7, 137.7, 135.9, 123.1, 121.1, 119.9, 59.9, 36.5, 28.3, 14.5; MS (ESI) m/z (rel intens) 453 (M⁺ + 1, 100), 407 (39), 323 (15). Anal. Calcd for C₂₃H₂₈N₆O₄·H₂O (470.52): C, 58.71; H, 6.42; N, 17.86. Found: C, 58.81; H, 6.39; N, 17.71.

2,7-Bis(N'-ethoxycarbonyl-N' '-ethyl)guanidino-9H-fluorene (11d). Following the general procedure, starting with 10a and utilizing ethylamine hydrochloride, the target compound was obtained as a beige solid (0.79 g, 96%): mp 220-2 C; ¹H NMR (DMSO-d₆) 1.12-1.19 (m, 12H), 3.32 (t, J = 6.9 Hz, 4H), 3.89 (s, 2H), 3.95 (q, J = 6.9 Hz, 4H), 7.31 (d, J = 8.1 Hz, 2H), 7.51 (s, 2H), 7.81 (d, J = 8.1 Hz, 2H); ¹³C NMR (DMSO-d₆) 163.3, 158.1, 143.7, 137.5, 136.0, 123.1, 121.1, 120.0, 59.7, 36.5, 35.7, 14.8, 14.6; MS (ESI) m/z (rel intens) 481 (M⁺, 80), 435 (20), 364 (10), 339 (12), 241 (100). Anal. Calcd for C₂₅H₃₂N₆O₄·0.25H₂O (485.06): C, 61.90; H, 6.75; N, 17.32. Found: C, 61.66; H, 6.79; N, 17.37.

2,7-Bis(N'-ethoxycarbonyl-N' '-isopropyl)guanidino-9H-fluorene (11e). With isopropylamine and the same synthetic steps used for preparing 11a, a beige solid was obtained (0.39 g, 88%): mp 142-4 C; ¹H NMR (DMSO-d₆) 0.84 (t, J = 7.2 Hz, 6H), 1.10-1.23 (m, 12H), 3.88 (s, 2H), 3.93 (q, J = 7.2 Hz, 4H), 4.10-4.21 (m, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.52 (s, 2H), 7.80 (d, J = 8.4 Hz, 2H); ¹³C NMR (DMSO-d₆) 163.42, 157.38, 143.54, 137.36, 136.25, 122.93, 120.93, 119.72, 59.60, 42.35, 36.47, 22.53, 14.57; MS (ESI) m/z (rel intens) 509 (M⁺ + 1, 19), 255 (100). Anal. Calcd for C₂₇H₃₆N₆O₄·0.25C₂H₅OH (520.13): C, 63.50; H, 7.26; N, 16.15. Found: C, 63.20; H, 7.06; N, 16.35.

2,7-Bis(N'-ethoxycarbonyl-N' '-methoxy)guanidino-9H-fluorene (11f). With the general procedure and O-methylhydroxylamine hydrochloride, a tan white solid was obtained (0.55 g, 87%): mp 180-2 C; ¹H NMR (DMSO-d₆) 0.92-0.95 (m, 6H), 1.20 (t, J = 6.9 Hz, 6H), 3.67 (s, 6H), 3.69 (s, 6H), 3.73-3.86 (m, 8H), 4.10 (q, J = 6.9 Hz, 4H), 6.99 (d, J = 8.1 Hz, 2H), 7.16 (s, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.55-7.62 (m, 6H), 8.34 (br s, 1H), 8.36 (br s, 1H), 8.70 (br s, 2H), 9.13 (br s, 2H), 9.15 (br s, 2H); MS (ESI) m/z (rel intens) 485 (M⁺ + 1, 100).

2,7-Bis(N'-ethoxycarbonyl-N' '-isobutoxy)guanidino-9H-fluorene (11g). Following the general procedure, O-isobutylhydroxylamine hydrochloride was used to prepare the target compound, which was obtained as creamy white crystals (0.8 g, 93%): mp 122-5 C; ¹H NMR (DMSO-d₆) 0.88-0.99 (m, 30H), 1.21 (t, J = 7.2 Hz, 6H), 1.92-2.02 (m, 4H), 3.65 (d, J = 6.6 Hz, 8H), 3.74-3.78 (m, 4H), 3.85 (q, J = 7.2 Hz, 4H), 4.11 (q, J = 7.2 Hz, 4H), 7.00 (d, J = 8.1 Hz, 2H), 7.17 (s, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.55-7.62 (m, 6H), 8.19 (br s, 1H), 8.21 (br s, 1H), 8.67 (br s, 1H), 8.68 (br s, 1H), 9.01 (br s, 1H), 9.02 (br s, 1H), 9.10 (br s, 1H), 9.11 (br s, 1H); ¹³C NMR (DMSO-d₆) 154.0, 153.9, 153.3, 143.4, 143.3, 143.1, 142.9, 142.9, 142.3, 142.2, 138.8, 138.6, 137.9, 137.7, 135.8, 135.6, 134.4, 134.2, 119.7, 119.4, 199.1, 118.8, 117.7, 117.6, 116.6, 114.5, 79.5, 79.3, 61.1, 60.4, 36.6, 36.5, 36.3, 27.3, 27.3, 19.3, 14.4; MS (ESI) m/z (rel intens) 569 (M⁺ + 1, 100).

Preparation of N-Substituted Guanidines (General Procedure) (Scheme 2). 2,7-Bis(N'-methyl)guanidino-9H-fluorene (12a). The substituted guanidine 11c (0.5 g, 1.1 mmol) was suspended in EtOH (5 mL), and 1 N KOH (11 mL, 11 mmol) was then added. The reaction mixture was kept stirring for 10 h, maintaining the temperature at 50 C. The solvent was evaporated, and the residue was washed multiple times with water and crystallized from aqueous EtOH to give a light-orange solid (0.24 g, 70%): mp 240-2 C (dec); ¹H NMR (DMSO-d₆) 2.66 (s, 6H), 3.69 (s, 2H), 4.96 (br s, 4H), 5.34 (br s, 2H), 6.70 (d, J = 8.1 Hz, 2H), 6.90 (s, 2H), 7.50 (d, J = 8.1 Hz, 2H).

For preparation of the HCl salt, a solution of the free base in dry EtOH (20 mL) chilled in an ice bath was treated with dry HCl gas for 10 min. The reaction mixture was concentrated under reduced pressure and then diluted with ether. The precipitate formed was collected by filtration to give an orange solid (0.16 g): mp 276-8 C; ¹H NMR (DMSO-d₆) 2.84 (s, 6H), 3.96 (s, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.46 (s, 2H), 7.77 (br s, 2H), 7.89 (br s, 2H), 7.95 (d, J = 8.4 Hz, 2H), 10.01 (br s, 2H); ¹³C NMR (DMSO-d₆) 155.7, 144.5, 138.6, 134.2, 123.5, 121.5, 120.9, 36.6, 28.3; MS (ESI) m/z (rel intens) 309 (M⁺ + 1, 100), 155 (9). Anal. Calcd for C₁₇H₂₀N₆·2HCl·0.25C₂H₅OH·0.75H₂O (406.33): C, 51.73; H, 6.20; N, 20.68. Found: C, 51.77; H, 6.24; N, 20.48.

2,7-Bis(N'-ethyl)guanidino-9H-fluorene (12b). Free Base. Starting with 11d and following the general procedure, a beige solid was obtained (0.24 g, 85%): mp 155-7 C (dec); ¹H NMR (DMSO-d₆) 1.07 (t, J = 7.2 Hz, 6H), 3.14 (q, J = 7.2 Hz, 4H), 3.68 (s, 2H), 5.00 (br s, 6H), 6.68 (d, J = 8.1 Hz, 2H), 6.88 (s, 2H), 7.49 (d, J = 8.1 Hz, 2H); ¹³C NMR (DMSO-d₆) 151.2, 148.9, 143.3, 134.3, 121.4, 119.6, 118.9, 36.2, 35.1, 15.0; MS (ESI) m/z (rel intens) 337 (M⁺ + 1, 90), 292 (60), 205 (50), 169 (100).

Dihydrochloride Salt. Bright-yellow solid, mp 193-5 C (dec); ¹H NMR (DMSO-d₆) 1.14 (t, J = 7.2 Hz, 6H), 3.28 (q, J = 7.2 Hz, 4H), 3.95 (s, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.44 (s, 2H), 7.74 (br s, 4H), 7.95 (d, J = 8.1 Hz, 2H), 8.03 (br s, 2H), 9.96 (br s, 2H). Anal. Calcd for C₁₉H₂₄N₆·2HCl·1.75H₂O·0.2C₂H₅OH (450.09): C, 51.76; H, 6.87; N, 18.67. Found: C, 51.86; H, 6.64; N, 18.68.

2,7-Bis(N'-isopropyl)guanidino-9H-fluorene (12c). Free Base. Starting with (11e) and following the general procedure, a salmon-orange solid was obtained (0.17 g, 79%): mp 247-9 C (dec); ¹H NMR (DMSO-d₆) 1.11 (d, J = 5.4 Hz, 12H), 3.68 (s, 2H), 3.83-3.87 (m, 2H), 4.93 (br s, 4H), 5.38 (br s, 2H), 6.69 (d, J = 8.1 Hz, 2H), 6.89 (s, 2H), 7.49 (d, J = 8.1 Hz, 2H).

Dihydrochloride Salt. Shiny yellow crystals, mp 308-9 C (dec); ¹H NMR (DMSO-d₆) 1.18 (d, J = 6.3 Hz, 12H), 3.85-3.94 (m, 4H), 7.23 (d, J = 8.4 Hz, 2H), 7.43 (s, 2H), 7.71 (br s, 4H), 7.93 (d, J = 8.4 Hz, 2H), 8.05 (br s, 1H), 8.07 (br s, 1H), 9.88 (br s, 2H); ¹³C NMR (DMSO-d₆) 153.9, 144.8, 138.9, 134.5, 123.7, 121.6, 121.1, 43.8, 22.3; HRMS calcd for MH + C₂₁H₂₈N₆: 365.2454. Observed 365.2467. Anal. Calcd for C₂₁H₂₈N₆·2HCl·1.4H₂O (462.63): C, 54.52; H, 7.14; N, 18.17. Found: C, 54.51; H, 6.74; N, 17.79.

2,7-Bis(N'-methoxy)guanidino-9H-fluorene (12d). Free Base. With 11f as a starting material, a pink solid was obtained (0.17 g, 79%): mp 200-2 C (dec); ¹H NMR (DMSO-d₆) 3.60 (s, 6H), 3.76 (s, 2H), 5.46 (br s, 4H), 7.20 (dd, J = 8.4 Hz, 2H), 7.51-7.54 (m, 4H), 7.90 (br s, 2H); ¹³C NMR (DMSO-d₆) 151.6, 143.1, 139.5, 133.7, 118.8, 116.1, 114.1, 60.5, 36.5; MS (ESI) m/z (rel intens) 341 (M⁺ + 1, 48), 168 (100).

Dihydrochloride Salt. Shiny tan-white solid, mp 248-9 C (dec); ¹H NMR (DMSO-d₆) 3.71 (s, 6H), 3.96 (s, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.49 (s, 2H), 7.96 (d, J = 8.1 Hz, 2H), 8.24 (br s, 4H), 10.43 (br s, 2H), 11.64 (br s, 2H); ¹³C NMR (DMSO-d₆) 156.1, 144.5, 138.8, 133.4, 123.5, 121.5, 120.9, 64.5, 36.6. Anal. Calcd for C₁₇H₂₀N₆O₂·2HCl·0.5C₂H₅OH (436.33): C, 49.64; H, 5.77; N, 19.26. Found: C, 49.95; H, 5.65; N, 19.40.

2,7-Bis(N'-isobutoxy)guanidino-9H-fluorene (12e). Free Base. Use of 11g and the general procedure provided a brick-red solid (0.16 g, 62%): mp 198-200 C; ¹H NMR (DMSO-d₆) 0.90 (d, J = 6.9 Hz, 12H), 1.93-2.02 (m, 2H), 3.55 (d, J = 6.9 Hz, 4H), 3.74 (s, 2H), 5.32 (br s, 4H), 7.21 (dd, J = 8.4, 1.8 Hz, 2H), 7.49-7.52 (m, 4H), 7.82 (br s, 2H); ¹³C NMR (DMSO-d₆) 151.5, 143.1, 139.4, 133.8, 118.8, 116.3, 114.3, 79.1, 36.5, 27.3, 19.3; MS (ESI) m/z (rel intens) 425 (M⁺ + 1, 100), 245 (10), 156 (56).

Hydrochloride Salt. Pink solid, mp 251-2 C (dec); ¹H NMR (DMSO-d₆) 0.91 (d, J = 6.9 Hz, 12H), 1.97-2.06 (m, 2H), 3.65 (d, J = 6.9 Hz, 4H), 3.95 (s, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.48 (s, 2H), 7.96 (d, J = 8.4 Hz, 2H), 8.16 (br s, 4H), 10.36 (br s, 2H), 11.65 (br s, 2H). Anal. Calcd for C₂₃H₃₂N₆O₂·2HCl (496.21): C, 55.53; H, 6.88; N, 16.89. Found: C, 55.45; H, 6.87; N, 16.70.

Preparation of Reversed Amidines (General Procedure) (Scheme 3). 2,7-Bis[4-(iminobenzylamino)]-9H-fluorene (13a). Free Base. A solution of 2,7-diamino-9H-fluorene (0.3 g, 1.5 mmol) in dry MeCN (10 mL) was diluted with dry EtOH (15 mL) and chilled in an ice-water bath. The solution was then treated with S-(2-naphthylmethyl)thiobenzimidate hydrobromide (1.13 g, 3.15 mmol). The reaction mixture was kept stirring at room temperature overnight, after which the solvent was evaporated to dryness leaving behind an oily residue that was triturated with ether to give a solid of the hydrobromide salt. The solid was then dissolved in EtOH and basified with 1 N NaOH, and the free base was extracted with EtOAc. After the sample was dried over Na₂SO₄, the solvent was evaporated to dryness, giving an off-white solid (0.45 g, 63%): mp 240-2 C; ¹H NMR (DMSO-d₆) 3.83 (s, 2H), 6.31 (br s, 4H), 6.83-6.85 (m, 2H), 7.05 (s, 2H), 7.39-7.46 (m, 6H), 7.71 (d, J = 8.4 Hz, 2H), 7.96-7.98 (m, 4H); ¹³C NMR (DMSO-d₆) 153.9, 148.5, 143.8, 135.9, 135.7, 129.9, 127.9, 126.9, 120.3, 119.7, 118.3, 36.5.

Hydrochloride Salt. An ice bath cold solution of the free base in dry EtOH was treated with HCl gas for 5-10 min, after which the mixture was concentrated to near dryness and the suspension was diluted with ether to furnish a yellow solid (0.28 g): mp 286-8 C; ¹H NMR (DMSO-d₆) 4.09 (s, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.65-7.70 (m, 4H), 7.75-7.79 (m, 4H), 7.95-7.97 (m, 2H), 8.16 (d, J = 8.4 Hz, 2H), 9.16 (br s, 2H), 9.86 (br s, 2H), 11.67 (br s, 2H); ¹³C NMR 163.1, 144.9, 140.1, 133.7, 133.6, 128.9, 128.8, 128.6, 124.5, 122.5, 121.6, 36.9; MS (EI) m/z (rel intens) 402 (M⁺, 100), 299 (38), 197 (9), 196 (60), 151 (7), 103 (32), 77 (10). Anal. Calcd for C₂₇H₂₂N₄·2HCl·0.25C₂H₅OH·H₂O (504.94): C, 65.41; H, 5.49; N, 11.09; Cl, 14.04. Found: C, 65.76; H, 5.40; N, 10.87; Cl, 14.09.

2,7-Bis[4-[imino-(2-pyridylmethyl)]amino]-9H-fluorene (13b). Free Base. The general procedure was used employing 2,7-diamino-9H-fluorene (0.5 g, 2.95 mmol) and S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide (2.22 g, 6.2 mmol) to give the reversed amidine as shiny yellow crystals (0.80 g, 67%): mp 230-2 C; ¹H NMR (DMSO-d₆) 3.84 (s, 2H), 6.56 (br s, 4H), 6.91 (d, J = 8.4 Hz, 2H), 7.11 (s, 2H), 7.53-7.57 (m, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.93-7.98 (m, 2H), 8.33 (d, J = 7.8 Hz, 2H), 8.63 (d, J = 7.8 Hz, 2H); ¹³C NMR (DMSO-d₆) 159.3, 149.7, 144.8, 144.5, 140.2, 138.2, 133.3, 128.5, 124.7, 124.5, 122.7, 121.5, 36.8.

Hydrochloride Salt. Yellow solid (0.33 g), mp 302-4 C; ¹H NMR (DMSO-d₆) 4.08 (s, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.74 (s, 2H), 7.83-7.87 (m, 2H), 8.14-8.24 (m, 4H), 8.58 (d, J = 8.1 Hz, 2H), 8.89 (d, J = 7.2 Hz, 2H), 9.39 (br s, 2H), 10.18 (br s, 2H); ¹³C NMR (DMSO-d₆) 159.6, 149.7, 144.0, 143.9, 140.4, 138.5, 133.5, 128.6, 124.9, 124.2, 122.9, 121.8, 36.8; MS (EI) m/z (rel intens) 404 (M⁺, 100), 300 (28), 283 (6), 196 (33), 152 (9), 105 (28), 78 (21). Anal. Calcd for C₂₅H₂₀N₆·3.5HCl·0.33C₂H₅OH·H₂O (565.29): C, 54.52; H, 4.89; N, 14.87; Cl, 21.95. Found: C, 54.57; H, 4.74; N, 14.97; Cl, 21.87.

Acknowledgment

The Bill and Melinda Gates Foundation supported this work.